mechanics of oxidative stress and protein in c2c12 myotubes · alex armstrong, jessica terrill,...

Mechanics of Oxidative Stress and Protein Turnover in C2C12 Myotubes

Ya‐tzu Chang BSc

School of Chemistry and Biochemistry School of Anatomy, Physiology and Human Biology

This thesis is presented for the degree of Master of Philosophy in Biochemistry of The University of Western Australia

2016

i

Abstract

Skeletalmuscle is amajor component of bodymass, and not only controls voluntary

movementsbutalsoservesasamajorsourceofbodyheat.Alossofmusclemass,also

described as muscle wasting or atrophy, can be detrimental to overall health. An

imbalance between protein synthesis and degradation is thought to contribute to

musclewasting.

One of the key causes of muscle wasting is oxidative stress caused by a mismatch

betweenreactiveoxygenspecies(ROS)generationanddegradation.Asthemechanisms

thatunderlyoxidativestressandmusclewastingarestillunknown,itwashypothesized

thatanincreaseinoxidativestressdecreasestherateofproteinsynthesisleadingtoa

netlossoftotalprotein.

Thefirstpartofthethesisinvestigatedtheeffectsofoxidativestressontheleveloftotal

proteincontent.UsingaC2C12myotubeculturemodel,levelsofhydrogenperoxidewere

modulated with catalase and glucose oxidase. The treated myotubes were harvested

withtrichloroaceticacid(TCA)andlevelsoftotalproteincontentweremeasuredusing

themicroBCAassay.Asexpected,theleveloftotalproteinwassignificantlyincreased

withcatalasetreatmentanddecreasedwithglucoseoxidasetreatment.

Since the level of total protein was regulated with the balance between protein

synthesis and degradation, the level of protein synthesis and the rate of protein

degradation were then measured. Unexpectedly, the level of protein synthesis was

significantly decreased with catalase treatment and not significantly affected with

glucoseoxidasetreatment.Therateofproteindegradationwassignificantlydecreased

with catalase treatment but still not significantly affected with glucose oxidase

treatment.To explainhow the levels of protein synthesiswereaffected, the signaling

pathwayswerethenobserved.

Previous studies have found the 4EBP1 signaling pathway and eIF2α pathway areaffectedbyoxidativestress.Whentherateofphosphorylationof4EBP1decreases,the

levelofproteinsynthesisisreducedbysuppressingtheactivityofeIF4E.Inthepresent

study, the rate of phosphorylation of 4EBP1 was not affected by either catalase or

glucoseoxidase.TheeIF2α pathwaywasthen investigated.While thephosphorylation

ii

ofeIF2αincreases,thelevelofproteinsynthesisisreducedbysuppressingtheactivity

ofeIF2B.Asexpected,theratesofphosphorylationofeIF2αwereincreasedafterboth

catalase and glucose oxidase treatment. This suggested that the eIF2α signaling

pathway is involved in inducing the changes in protein synthesis in myotubes in

responsetooxidativestress.

To further explore the effect of ROS on muscle proteins, the C2C12 myotubes were

treatedwithcatalaseandglucoseoxidaseandthioloxidationassessed.Thiolgroupsare

potentially powerful antioxidants which react to oxidative stress. They contain

sulfhydryl (‐SH) groups that are readily oxidized to form stable disulphide bonds.

Previousstudieshaveshownthatthestructureofthiolsinvariouscelltypesarealtered

in response to oxidative stress. In the present study, 2 tag method developed for

labelingmusclesamples(Armstrongetal.2011)wasadaptedtolabeltheC2C12culture

samples.Usingthismethod,thioloxidationinC2C12myotubeswasobservedforthefirst

time. However, unexpectedly, total thiol oxidation and thiol oxidation of specific

proteins were not found to be significantly changed by catalase or glucose oxidase

treatment.

iii

Acknowledgement

IwouldliketoextendmymostsinceregratitudetomysupervisorsA/ProfessorPeterG.

Arthur, A/Professor Tea Shavlakadze and Professor Miranda D. Grounds for their

invaluable guidance, support, advice and supervision throughout this challenging but

rewardingexperience. Iwouldalso liketoextendmymostsinceregratitudetoschool

staffDr.JoanneEdmondson,LouseWedlockandSatoJuniperfortheirinvaluableadvice

and support throughout this challenging process of composing this thesis. All their

immenseknowledge,scientificingenuity,constantenthusiasmandseeminglyunlimited

patiencearegratefullyacknowledged.

I would like to thank my dearest family and friends. It was a tough journey as an

internationalstudent.Thankstoalltheirlove,supportandencouragementthatgaveme

strengthtogetthroughthisjourney.Thankyousomuch,andloveyouall.

To themembersofGroundsandPGA labs, thankyou for all your support andadvice

throughout my entires study. I would like to especially thank Tinashe Chinzou for

gettingmefamiliarwiththe labswhileI firstarrivedthe lab, thecountry. Ialsothank

AlexArmstrong,JessicaTerrill,PearlTan,RuthChai,StevenKho,SumiiHaleemandZoe

Soffefortheirhelpandguidanceinlaboratorywork.

MysincerethankstoMrGregCozensforhisexpertadviceonmolecularbiologywork

andalwayskeepingthelaboratorywellorganized.

IalsowanttoacknowledgetheNationalHealthandMedicalResearchCouncil(NHMRC)

grantwhichsupportedtheworkofthisthesis.

Lastbutnotleast,IamreallyappreciativeoftheScholarshipsforInternationalResearch

Fees(SIRF)thatawardedbyUWAthatsupportedmystudiesinUWA.

Thankyou.

iv

Declaration of Contributions

Alltheworkpresentedinthisthesiswasperformedsolelybytheauthor.

Thisthesiswaswrittensolelybytheauthor.

I hereby declare that thework containedwithin this thesis is entirelymy ownwork,

whichhasbeencontributedisclearlystated.

Ya‐tzuChang

May2016

v

Table of Contents

Abstract .................................................................................................................................i

Acknowledgement .............................................................................................................. iii

Declaration of Contributions ............................................................................................. iv

Table of Contents ................................................................................................................ v

List of Figures ..................................................................................................................... ix

List of Tables ...................................................................................................................... xi

List of abbreviations .......................................................................................................... xii

Chapter 1 Literature review ................................................................................................ 1

1.1 Introduction ............................................................................................................................. 1

1.2 Oxidative stress ...................................................................................................................... 1

1.2.1 Overview ............................................................................................................................ 1

1.2.2 Hydrogen peroxide, catalase and glucose oxidase ........................................................... 3

1.2.3 Thiol oxidation .................................................................................................................... 4

1.2.4 Effects of oxidative stress in muscle and pathology........................................................... 5

1.3 Skeletal muscle biology ......................................................................................................... 6

1.3.1 Overview ............................................................................................................................ 6

1.3.2 Main proteins in skeletal muscle ........................................................................................ 7

1.3.3 Muscle differentiation and C2C12 myotubes model ............................................................. 8

1.3.4 Muscle wasting ................................................................................................................ 10

1.4 Protein turnover .................................................................................................................... 11

1.4.1 Overview .......................................................................................................................... 11

1.4.2 Protein degradation .......................................................................................................... 12

Ubiquitin-proteasome system (UPS) .................................................................................................... 12

Lysosomal-autophagy (LA) system ...................................................................................................... 14

1.4.3 Protein synthesis .............................................................................................................. 15

Initiation ................................................................................................................................................ 15

Elongation ............................................................................................................................................. 18

Termination ........................................................................................................................................... 19

1.5 Signalling pathway ............................................................................................................... 20

1.5.1 Overview .......................................................................................................................... 20

1.5.2 mTOR/4EBP1 pathway .................................................................................................... 21

1.5.3 PERK/eIF2α pathway ....................................................................................................... 23

Aim ...................................................................................................................................... 26

vi

Chapter 2 Material and Methods ...................................................................................... 27

2.1 Cell Culture ............................................................................................................................ 27

2.1.1 Proliferation ...................................................................................................................... 27

2.1.2 Trypsinization and seeding of myoblasts ......................................................................... 28

2.1.4 Treatment conditions ....................................................................................................... 29

2.2 Protein extraction ................................................................................................................. 29

2.2.1 TCA acetone extraction ................................................................................................... 29

2.2.2 Phospho-safe extraction .................................................................................................. 30

2.3 Protein quantification ........................................................................................................... 31

2.3.1 Bradford assay ................................................................................................................. 31

2.3.2 Micro BCA assay ............................................................................................................. 31

2.3.3 Detergent compatible (DC) protein assay ........................................................................ 32

2.4 Measurement of protein synthesis ...................................................................................... 32

2.4.1 Incorporation of radioactive leucine ................................................................................. 32

2.4.2 Harvest ............................................................................................................................. 33

2.4.3 Radiation analysis ............................................................................................................ 33

2.5 Measurements of protein degradation ................................................................................ 34

2.5.1 Incorporation of radioactive leucine ................................................................................. 34

2.5.2 Harvest ............................................................................................................................. 34

2.5.3 Radiation analysis ............................................................................................................ 35

2.6 Western Blot .......................................................................................................................... 35

2.6.1 SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) .................................................. 36

2.6.2 Bio-Rad system ................................................................................................................ 38

2.6.3 Densitometry analysis ...................................................................................................... 39

2.7 Measurements of thiol oxidation-2 tag labeling ................................................................. 39

2.7.1 Preparation of protein samples ........................................................................................ 40

2.7.2 Dual labeling of protein thiols with fluorescent tags ......................................................... 40

2.7.3 Fluorescence measurement using a plate reader ............................................................ 41

2.7.4 SDS-PAGE ...................................................................................................................... 42

2.8 Statistics ................................................................................................................................ 43

Chapter 3: Development of methods for the study of protein content in C2C12

myotubes in response to treatment with catalase and glucose oxidase ...................... 44

3.1 Introduction ........................................................................................................................... 44

3.2 Methods ................................................................................................................................. 46

Myotube cultures .................................................................................................................................. 46

Protein extraction .................................................................................................................................. 46

Protein quantification- micro BCA assay .............................................................................................. 47

3.3 Results ................................................................................................................................... 47

vii

3.3.1 Modifying the extraction method to extract proteins from C2C12 myotubes ...................... 47

3.3.2 Method to quantify protein content in C2C12 myotubes .................................................... 49

3.3.3 Measuring the level of protein content in C2C12 myotubes in response to catalase and

glucose oxidase ........................................................................................................................ 51

3.4 Discussion ............................................................................................................................. 52

Chapter 4: Development of methods for the measurement of protein synthesis and

degradation in C2C12 myotubes in response to treatment with catalase and glucose

oxidase ............................................................................................................................... 54

4.1 Introduction ........................................................................................................................... 54

4.2 Methods ................................................................................................................................. 55


Protein synthesis .................................................................................................................................. 55

Protein degradation .............................................................................................................................. 56

4.3 Results ................................................................................................................................... 56

4.3.1 Establishment of method for measuring protein synthesis in C2C12 myotubes ................ 56

4.3.2 Establishment of method for measuring protein degradation in C2C12 myotubes ............ 57

4.3.3 Measuring protein synthesis in C2C12 myotubes .............................................................. 59

4.3.4 Measuring protein degradation in C2C12 myotubes .......................................................... 60

4.4 Discussion ............................................................................................................................. 61

Chapter 5: Development of methods for the study of signaling pathway on protein

synthesis in C2C12 myotubes in response to treatment with catalase and glucose

oxidase ............................................................................................................................... 63

5.1 Introduction ........................................................................................................................... 63

5.2 Methods ................................................................................................................................. 64



Protein quantification ............................................................................................................................ 64

Western Blot- SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) system ................................... 65

Western Blot- Bio-Rad system ............................................................................................................. 66

Antibodies ............................................................................................................................................. 66

5.3 Results ................................................................................................................................... 67

5.3.1 Optimization of method for measuring 4EBP1 phosphorylation in C2C12 myotubes ........ 67

5.3.2 Optimization of method for measuring eIF2αphopsphorylation in C2C12 myotubes ........ 68

5.3.3 Measuring the rate of phosphorylation on 4EBP1 in C2C12 myotubes ............................. 71

5.3.4 Measuring eIF2α phosphorylation in C2C12 myotubes ..................................................... 71

5.4 Discussion ............................................................................................................................. 72

viii

Chapter 6: Development of a method to measure thiol oxidation in C2C12 myotubes in

response to treatment with catalase and glucose oxidase ............................................ 74

6.1 Introduction ........................................................................................................................... 74

6.2 Methods ................................................................................................................................. 74



Protein quantification- micro BCA assay .............................................................................................. 75

2 tag labeling ........................................................................................................................................ 75

Protein quantification-DC assay ........................................................................................................... 77

FLm and TRm quantification ................................................................................................................ 77

6.3 Results ................................................................................................................................... 78

6.3.1 Optimize the 2 tag method for C2C12 myotubes model .................................................... 78

6.3.2 Measuring total thiol oxidation in fluorescently labeled C2C12 myotubes using a

fluorescent plate reader ............................................................................................................ 79

6.3.3 Measuring thiol oxidation in C2C12 myotubes on actin and myosin by gel electrophoresis

.................................................................................................................................................. 80

6.4 Discussion ............................................................................................................................. 82

Chapter 7: General discussion ......................................................................................... 83

7.1 Introduction ........................................................................................................................... 83

7.2 Muscle wasting ..................................................................................................................... 83

7.3 Protein turnover .................................................................................................................... 84

7.4 Signalling pathways ............................................................................................................. 85

7.5 Thiol oxidation ...................................................................................................................... 86

7.6 Future studies ....................................................................................................................... 86

Bibliography ....................................................................................................................... 88

Appendices ...................................................................................................................... 113

ix

List of Figures

Figure 1.1 The sources and cellular responses to ROS ................................................... 2

Figure 1.2 Catalase decomposition of hydrogen peroxide .............................................. 3

Figure 1.3 Glucose oxidase composition of hydrogen peroxide..................................... 3

Figure 1.4 Structure of skeletal muscle ............................................................................. 7

Figure 1.5 Myosin structure and contractile apparatus in muscle (Richfield 2014) ....... 8

Figure 1.6 Changes in C2C12 cell morphology in response to myogenic differentiation

.............................................................................................................................................. 9

Figure 1.7 Changes in muscle mass with age ................................................................. 11

Figure 1.8 Ubiquitin-proteasome system (UPS) .............................................................. 13

Figure 1.9 Lysosomal digestion ....................................................................................... 14

Figure 1.10 Cyclical process of translation ..................................................................... 17

Figure 1.11 Translation elongation in bacteria ............................................................... 19

Figure 1.12 Translation termination ................................................................................. 20

Figure 1.13 Inhibition of translation under different types of stress ............................. 20

Figure 1.14 Regulating cap-dependent translation initiation ......................................... 21

Figure 1.15 mTORC1 pathway and ageing ...................................................................... 22

Figure 1.16 Effects of ROS on mTOR/4EBP1 pathway ................................................... 23

Figure 1.17 The relationship between ER stress and ROS ............................................ 25

Figure 2.1 Haemocytometer .............................................................................................. 28

Figure 2.2 The assemble of transfer cassette ................................................................. 37

Figure 3.1 Changes in muscle mass accompanying cancer ......................................... 44

Figure 3.2 The total protein content ................................................................................. 48

Figure 3.3 The total protein content ................................................................................. 49

Figure 3.4 The standard curve of DC assay in Tris buffer ............................................. 50

Figure 3.5 Standard curve for micro BCA assay using BSA in various buffers .......... 51

x

Figure 3.6 Total protein levels in C2C12 myotubes in response to catalase and glucose

oxidase treatment .............................................................................................................. 52

Figure 4.1 Leucine incorporation in C2C12 myotbes treated with catalase ................... 57

Figure 4.2 Radioactive leucine release from C2C12 myotubes treated with catalase and

TNF ...................................................................................................................................... 58

Figure 4.3 Radioactive leucine release from pre-labeled C2C12 myotubes with various

treatments .......................................................................................................................... 59

Figure 4.4 Protein synthesis in C2C12 Myotubes with catalase and glucose oxidase

treatment ............................................................................................................................ 60

Figure 4.5 Radioactive leucine release from C2C12 myotubes with various treatments

............................................................................................................................................ 61

Figure 5.1 Detection of phosphorylated 4EBP1 and total 4EBP1 .................................. 67

Figure 5.2 Detection of total eIF2α ................................................................................... 68

Figure 5.3 Detection of phosphorylated eIF2α ................................................................ 69



Figure 5.6 4EBP1 phosphorylation in C2C12 myotubes after catalase and glucose


Figure 5.7 eIF2α phosphorylation in C2C12 myotubes after catalase and glucose


Figure 6.1 Total protein levels after precipitation with ethanol and acetone ............... 79

Figure 6.2 Thiol oxidation in C2C12 myotubes in response to catalase and glucose


Figure 6.3 Thiol oxidation on myosin in C2C12 myotubes in response to catalase and

glucose oxidase treatment ............................................................................................... 81

Figure 6.4 Thiol oxidation on actin in C2C12 myotubes in response to catalase and

glucose oxidase treatment ............................................................................................... 81

xi

List of Tables

Table 1.1 Formation and properties of different forms of ROS ....................................... 2

Table 1.2 Redox-regulated proteins and complexes ........................................................ 4

Table 1.3 Effectiveness of antioxidant treatment to skeletal muscle wasting ............... 5

Table 1.4 Metabolic consequences of sarcopenia and cachexia .................................. 10

Table 1.5 Eukaryotic initiation factors ............................................................................. 16

Table 2.1 Composition of resolving and stacking gel .................................................... 36

Table 2.2 Primary antibodies ............................................................................................ 38

Table 2.3 Chemiluminescence substrate solution used for protein detection ............. 38

Table 2.4 Dilution for FLm and TRm standards .............................................................. 42

Table 2.5 In-gel FLm/TRm standards ............................................................................... 42

xii

List of abbreviations

3’UTR 3’untranslatedregion

4EBP1 Eukaryotictranslationinitiationfactor4E‐bindingprotein1

5’UTR 5’untranslatedregion

ABCE1 ATP‐bindingcassettesubfamilyEmember1

AIDS AcquiredImmuneDeficiencySyndrome

Akt ProteinkinaseB

A‐site Aminoacyl‐site

ATCC AmericanTypeCultureCollection

ATF4 Activatingtranscriptionfactor4

ATP Adenosinetriphosphate

BCAassay Bicinchoninicacidassay

BSA Bovineserumalbumin

Cat. Catalase

ddi Doubledeionized

DCassay Detergentcompatibleassay

Deptor DEP‐domain‐containingmTOR‐interactingprotein

DEX Dexamethasone

DMEM Dulbecco’smodifiedEaglemedium

DMSO Dimethylsulfoxide

xiii

DTT Dithiothreitol

EDTA Ethylenediaminetetraaceticacid

eEF1A Eukaryotictranslationelongationfactor1A

eEF2 Eukaryotictranslationelongationfactor2

EF‐G ElongationfactorG

EF‐Tu Elongationfactorthermounstable

eIFs Eukaryotictranslationinitiationfactors

eIF1 Eukaryotictranslationinitiationfactor1

eIF1A Eukaryotictranslationinitiationfactor1A


eIF2α α subunitofeIF2

eIF2γ γsubunitofeIF2

eIF2B Eukaryotictranslationinitiationfactor2B


eIF3 j subunitofeIF3


eIF4E Eukaryotictranslationinitiationfactor4E

eIF4F Eukaryotictranslationinitiationfactor4F

eIF4G Eukaryotictranslationinitiationfactor4G


xiv

ER Endoplasmicreticulum

eRF1 Eukaryotictranslationterminationfactor1

eRF3 Eukaryotictranslationterminationfactor3

E‐site Exit‐site

FBS Fetalbovineserum

FLm BODIPYFL‐N‐(2‐aminoethyl)maleimide

GAP GTPase‐activatingprotein

GCN2 eIF2αkinase4

GDP Guanosinediphosphate

GEF Guaninenucleotideexchangefactor

GluO. Glucoseoxidase

GPx Glutathioneperoxidase

GSH Glutathione

GTP Guanosine‐5’‐triphosphate

GTPase SingularGTPase

H+ Hydrogenion

HRI Heme‐regulatedinhibitorkinase

HS Horseserum

IGF Insulin‐growthfactor‐1

Met Methionine

xv

Mg2+ Magnesiumion

mLST8 MammalianlethalwithSec13protein8

mRNA MessengerRNA

mTOR Mammaliantargetofrapamycin

NAC N‐acetylcysteine

NADPH Nicotinamideadeninedinucleotidephosphate

NPSH Intracellularnon‐proteinthiols

O2 Dioxygen

•O2‐ Superoxideanions

•OH Hydroxylradical

p4EBP1 Phosphorylated4EBP1atThr37/46

PABP Poly(A)‐bindingprotein

PBS PhosphateBufferedSaline

peIF2α PhosphorylatedeIF2αatSer51

PERK PKR‐likeER‐localizedeIF2αkinase

PGC‐1α Peroxisomeproliferator‐activated‐receptor‐gamma‐coactivator‐1α

PI3K Phosphoinositide‐3‐kinase

PIKKs PI3K‐relatedkinases

PKR ProteinkinaseR

post‐TCs Post‐terminationribosomalcomplexes

xvi

PP Proteinphosphatase

PRAS40 Proline‐richAKTsubstrate40kDa

P‐site Peptidyl‐site

PVDF Polyvinylidenedifluoride

RO• Alkaoxyl

RO2• Peroxyl

ROS Reactiveoxygenspecies

rRNA RibosomalRNA

SDS‐PAGE Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis

‐SH Sulfhydryl

SOD Superoxidedismutase

TBS TrisBufferedSaline

TBS‐T TBSbufferwithTween‐20

TCA Trichloroaceticacid

TCEP Tris(2‐carboxyethyl)phosphinehydrochloride

TNF Tumournecrosisfactor

Trp Tryptophan

TRm TEXASRED‐C2‐malemide

TRx Thioredoxinreductase

tTNA TransferRNA

xvii

UCP Uncouplingproteins

UPP Ubiquitin‐proteasomepathway

UPR Unfoldedproteinresponse

UV Ultraviolet

1

Chapter 1 Literature review

1.1 Introduction

Oxidative stress occurs when there is an imbalance between oxidant generation and

degradationanditcanresultinthewastingofmusclemassindifferentconditions.This

reviewdescribestheroleofhydrogenperoxide,catalaseandglucoseoxidaseonprotein

levels,proteinturnover,signallingpathwaysandthioloxidationinskeletalmusclecells.

Thereviewbeginswithadescriptionoftheroleofoxidativestress,thioloxidation,and

effects of oxidative stress on muscle. This is followed by the description of skeletal

muscle biology, skeletal muscle models, muscular proteins and two types of muscle

wasting,sarcopeniaandcachexia.Thedescriptionsofproteindegradationandprotein

synthesisthatareinvolvedintheprocessofmusclewastingarethendiscussed.Finally,

theroleofthemTOR/4EBP1andPERK/eIF2αsignallingpathwaysthatmayinvolvein

themodulationofproteinsynthesis inmusclewastingarediscussed.Fivehypotheses

are proposed to demonstrate the possible interactions between oxidative stress and

C2C12myotubesunderdifferentlevelsofhydrogenperoxide.

1.2 Oxidative stress

1.2.1 Overview

Reactive oxygen species (ROS) is a collective term that broadly describes a group of

reactivecompoundsderivedfromoxygen(Table1.1)(Halliwelletal.1994;Circuetal.

2010). The oxidants are generated continuously as a consequence of aerobic

metabolisminorganellessuchasmitochondriaandperoxisomes(Doriaetal.2012),or

anumberofexternalagentssuchasultraviolet(UV)light(Fig.1.1)(Barbierietal.2012;

Terrilletal.2013).

2

Table1.1FormationandpropertiesofdifferentformsofROSDifferentformsofROScanbederivedfromsuperoxidebythesequentialadditionofelectrons,andeachofthemhasdifferentpropertiesinthecell.ReprintedbypermissionfromMacmillanPublishersLtd:[CurrentOpinioninClinicalNutritionandMetabolicCare](3971721443198),copyright(Arthuretal.2008).

O2+e‐ O2‐+e‐ H2O2 +e‐ •OH+e‐ H2O

Superoxide Hydrogenperoxide Hydroxylradical

Negativelychargedradicalion

Uncharged,non‐radical,relatively

stable

Highlyreactiveradical

Propertiesofinterest

Reactswithnitricoxidetoformperoxinitrite

Causeformationofdisulfidebonds

Primaryagentofprotein,DNAandlipiddamage

Duringphysiologicalhomeostasis, anoveralloxidativebalance ismaintained in tissue

bymatching theproductionofROSvia the removal actionbyavarietyof antioxidant

systems.Inthisenvironment,ROSservesasasignallingmoleculetostimulateoractasa

secondarymessengerinvarietyofsignallingtransductionpathways(Arthuretal.2008;

Barbierietal.2012;Terrilletal.2013).LowingROS levelsbelowthehomeostaticset

pointmay interrupt the physiological function in cellular proliferative responses and

host defenses. Oxidative stress also occurs when the action of antioxidants are

outweighed by the generation of ROS (Terrill et al. 2013). Oxidative stress has been

implicated in numerous conditions including ageing, inflammatory disorders, cancer,

musclewasting andmusculardystrophies (Fig. 1.1) (Finkel et al. 2000;Tidball 2005;

Barbierietal.2012;Terrilletal.2013).

Figure1.1ThesourcesandcellularresponsestoROS

3

Reprinted by permission from Macmillan Publishers Ltd: [Nature] (3971711505027), copyright(Finkeletal.2000).

1.2.2 Hydrogen peroxide, catalase and glucose oxidase

Hydrogenperoxide,anon‐radical,weakoxidantwitharelativelylonghalf‐life,isoneof

theseROS.Thissmallandstablemoleculecandiffusereadilywithincellsandacrosscell

membranesandalsoactasasignallingmoleculetoactivatealargenumberofsignalling

pathways(Bienertetal.2006;Rhee2006;Vealetal.2007;Arthuretal.2008;Paulsenet

al.2010;Barbierietal.2012).

Catalaseisanenzymethatcanremovehydrogenperoxidefromthecelltodecreasethe

levelofoxidativestress.Catalasedecomposeshydrogenperoxidetowaterandoxygen

bysuccessivereductionofthecatalaseironbyhydrogenperoxideanditsre‐oxidation

byO2(Fig1.2)(Keilinetal.1938;Jonesetal.1968).

Figure1.2Catalasedecompositionofhydrogenperoxide

Incontrast,glucoseoxidaseisanenzymethatcangeneratehydrogenperoxidethrough

the glucose/glucose oxidase pathway (Fig 1.3) (Weiss et al. 1981; Starkebaum et al.

1986; Salazar et al. 1997). This can result in the steady accumulation of hydrogen

peroxideinthecell(Starkebaumetal.1986;Dayetal.1997;Salazaretal.1997),which

canincreaseoxidativedamageandstress.

Figure1.3Glucoseoxidasecompositionofhydrogenperoxide

DifferentkindsofROSareinvolvedinavarietyofsignalingpathways.Theyalsoimpact

onawidearrayofproteinssuchaskinases,phosphatesandtranscription factors that

contain reduction‐oxidation (redox) sensitive residue (Table 1.2) (Arthur et al. 2008;

Paulsenetal.2010).

4Fe3‐+2H2O2=4Fe2‐+4H‐+2O24Fe2‐+4H‐+O2=4Fe3‐+2H2O

2H2O2=2H2O+O2

β‐D‐glucose+O2 Glucoseoxidase

D‐gluconolactone+H2O2

4

Table1.2Redox‐regulatedproteinsandcomplexesReprintedbypermissionfromMacmillanPublishersLtd:[AmericanChemicalSociety],copyright(Paulsenetal.2010).

Stimulation Organism(A) ROSsource(B) EffectofstimulationEpidermalgrowthfactor

(EGF)Hs,M,R NOX(C) Proliferation

Platelet‐derivedgrowthfactor(PDGF)

Hs,M,R NOX Proliferation/migration

Basicfibroblastgrowthfactor(bFGF)

B NOX Proliferation

Vascularendothelialgrowthfactor(VEGF)

P L Angiogenesis/proliferation

Granulocyte‐macrophagecolony‐stimulatingfactor

(GM‐CSF)

H ND Proliferation/migration

Insulin M,R NOX,Cytokines Glucoseuptake/transportLipopolysaccharide

(LPS)M NOX Inductionofimmuneresponse

Interleukin‐1β(IL‐1β) Hs,M NOX,L InductionofimmuneresponseInterleukin‐3(IL‐3) Hs ND InductionofimmuneresponseInterleukin‐4(IL‐4) Hs NOX InductionofimmuneresponseCD28stimulation Hs L Inductionofimmune

response/proliferationTumournecrosisfactor

α(TNF‐α)B,M,Hs NOX Apoptosis

Transforminggrowthfactor‐β1(TGF‐β1)

M NDAgonistofGPCRs(D)

Cellcyclearrest

AngiotensinII(AngII) R NOX HypertrophyLysophosphatidicacid

(LPA) Hs NOX,L Proliferation

Thrombin Hs NOX ProliferationSerotonin Ha NOX,

Otherstimulants

Proliferation

Wounding Z NOX LeukocyterecruitmentOxidativeStress D MT Differentiation

Reoxygenationafterhypoxia

R MT O2•‐burst

(A) B, bovine; D, Drosophila melanogaster; Ha, hamster; Hs, human; M, mouse; Z, zebrafish. (B) NOX, NADPH oxidase; M, mitochondria; L, lipoxygenase; ND, not determined. (C) For many of these cases, the specific NOX isoform activated is unknown. Each NOX isoform demonstrates disparate tissue expression, and continued studies will be required to elucidate the regulation of each NOX isoform in response to diverse external signals. (D) GPCRs= guanosine triphosphate (GTP)-binding protein (G protein)-coupled receptors.

1.2.3 Thiol oxidation

Hydrogenperoxidemodifiesproteinfunctionbyoxidizingthethiolgroupofthetarget

proteins to formdisulfide bonds (Bienert et al. 2006; Rhee 2006; Arthur et al. 2008). Thiols

areorganicsulfurderivatives,identifiedbythepresenceofsulfhydrylresidues(‐SH)at

theiractivesite.Biological thiols include low‐molecular‐weight free thiolsandprotein

thiols, the functional group of the amino acid cysteine are important in cellular

antioxidantdefencesandredoxsignalling (Batyetal.2005;Terrilletal.2013). In the

5

presence of ROS, ‐SH residues of thiol proteins (such as cysteine) may undergo

reversible structural modifications, whereby the ‐SH bonds are broken and disulfide

bondsareformed.

Themodificationonthiols isoneof themajorcellularconsequencesofROSexposure,

includinghydrogenperoxideexposure.Oxidationofthiolproteinsiscrucialtocellsasit

affectsvariouscellfunctionsincludingproteinstructure,proteintoproteininteractions,

catalysis, electron transfer, ion channel modulation, signalling pathway, post‐

translational protein modifications, and transcription activation (Baty et al. 2005;

Arthur et al. 2008; Paulsen et al. 2010; Terrill et al. 2013). Hydrogen peroxide, for

example,canaffectnumeroussignallingpathwaysbyoxidizingthethiolgroupsof the

targetproteins (Nealetal.1998;Rhee2006;Arthuretal.2008;Barbierietal.2012).

However,thismodificationcouldbereversedbacktothethiolformsviathiol/disulfide

exchangethroughtheactionofantioxidantmoleculesorenzymessuchasglutaredoxin

orperoxiredoxins(Bienert et al. 2006; Rhee 2006; Arthur et al. 2008; Terrill et al. 2013).

1.2.4 Effects of oxidative stress in muscle and pathology

Oxidative stresshasbeen implicated in thepathologyofnumerousmusculardiseases

suchasmusculardystrophies(Terrilletal.2013)thatarecharacterizedbyprogressive

skeletalmusclewastinganddegeneration.Ithasbeenshownthatmusclefunctioncan

be improved after treatment with antioxidants in different muscle wasting models

(Table1.3)(Bonettoetal.2009;Terrilletal.2013).

Table1.3EffectivenessofantioxidanttreatmenttoskeletalmusclewastingReprintedbypermissionfromMacmillanPublishersLtd:[FreeRadicalBiologyandMedicine](3971721188561),copyright(Bonettoetal.2009).

Antioxidanttreatment Effectivedisease IneffectiveoruncertainVitaminE Diabetes Ageing,ALSVitaminC Diabetes Ageing,ALSResveratrol Ageing,diabetes Cancercachexia

Dehydroepiandrosterone Diabetes, cancercachexia ALSOrnithine,cysteine,NAC Cancercachexia,DMD ALS

Carnitine Cancercachexia,ageing,ALS,diabetes

‐

Epigallocatechingallate DMD ‐Low‐intensitytraining Ageing,diabetes,cancer

cachexia,DMD‐

ALS= amyotrophic lateral sclerosisDMD= Duchenne muscular dystrophy

6

Duchenne muscular dystrophy (DMD) is one of the muscular diseases and the

relationshipbetweenDMDandoxidativestresshasbeenextensivelyinvestigated.The

severityofdystropathology invivocanbe reducedwith thiol‐reducingantioxidantN–

acetylcysteine (NAC) ,asmeasuredbydecreased levelsofplasmacreatinekinaseand

reduced myonecrosis, and the thiol oxidation can also be reduced with NAC in

dystrophicmuscle(Terrilletal.2013).

1.3 Skeletal muscle biology

1.3.1 Overview

In skeletal muscle, contractile proteins (such as troponin, tropomyosin, myosin and

actin)containthiolsidechainsthataresensitivetooxidation,andthesemodifications

may alter excitation/contraction coupling and cross‐bridge cycling, and therefore

modulatemusclecontraction(Terrilletal.2013).

About 40% of human bodymass consists of skeletalmuscle and there are over 600

individual skeletalmuscles are related in daily life such as breathing, eating, posture,

walking and reflexes (Shavlakadze et al. 2006; Saladin 2011). Muscle is highly

metabolically active,with the restingmetabolic rateof skeletalmuscle accounting for

about20‐30%ofrestingwhole‐bodyoxygenconsumptionandalsoservesasamajor

sourceofbodyheat(Zurloetal.1990).

Skeletalmuscle(Fig.1.4) iscomposedofbundlesofmuscle fibrescalled fascicles.The

cell membrane surrounding the muscle cell is called sarcolemma, and beneath the

sarcolemma is called sarcoplasm that contains the cellular proteins, organelles, and

myofibrils. The myofibrils are composed of contractile units called sarcomeres that

consist of thickmyosin filaments and thin actin filaments. The arrangement of these

filaments gives skeletal muscle its striated appearance, and the muscle contract by

slidingthethickandthinfilamentsalongeachothersotheskeletalmuscleiscapableof

remarkableadaptationsinresponsetoalteredactivity(Sakumaetal.2015).Thereare

threekindsofmuscle connective tissue: theepimysium,covers thewholemuscle; the

perimysium,coversthebundlesofmusclefibres;theendomysium,coverseachmuscle

fibre.

7

Figure1.4StructureofskeletalmuscleSkeletal muscle is made up of muscle fibres that are composed by myofibrils that consist thickmyosin filaments and thin actin filaments. There are three connective tissues: epimysium,perimysium,andendomysiumthatconnectsallmuscletissuestogether(IvyRose2003;Gebski2009;MedicaLook 2012). Reprinted by permission fromMacmillan Publishers Ltd: [Muscle and Nerve](3972350046350),copyright(Gilliesetal.2011)

1.3.2 Main proteins in skeletal muscle

Thetwomainproteinsfoundinmusclearemyosinandactinandbothareinvolvedin

muscularfunction.Myosins(Fig.1.5)areakeypartofthecontractileproteinsofmuscle

and play an important role in signal transduction and the establishment of polarity

(Bähler 2000). They also act as actin‐based motors that play a fundamental role in

differentformsofeukaryoticmotilityincludingcellcrawling,cytokinesis,phagocytosis,

growth cone extension, maintenance of cell shape, and organelle/particle trafficking

(Berg et al. 2001).

Membersofthemyosinsuperfamilyaredefinedbythepresenceofaheavychainwitha

conserved~80kDacatalyticdomain.Inmostmyosins,thecatalyticdomainisfollowed

by an α‐helical light chain‐binding region consisting of one or more IQ motifs. Most

myosinsalsohaveaC‐terminal tail and/oranN‐terminalextension thought toconfer

class‐specific properties such as membrane binding or kinase activity (Hodge et al.

2000;Bergetal.2001).

8

Figure1.5Myosinstructureandcontractileapparatusinmuscle(Richfield2014)

Actinplaysakeyroleinmusclecontraction,cellmobility,andothercellprocessesand

functions including cell division, endocytosis, secretion, signal transduction, the

regulation of enzyme activity, and themaintenance of cell shape.Actin has also been

shown to regulate the activity ofmembranes and participate in transcription,mRNA

transportandtranslationandsynaptictransmission(Khaitlina 2001).

Actin has α, β, and γ‐isoforms that have been classified according to differences in

mobilities (Storti et al. 1976; Whalen et al. 1976; Rubenstein et al. 1977; Khaitlina

2001).Theseisoformscannotsubstituteforeachother,andthehigh‐levelsynthesisof

exogenous actins lead to changes in cell organization andmorphology. This suggests

that actins are functionally specialized for the tissues in which they predominate

(Khaitlina 2001).

1.3.3 Muscle differentiation and C2C12 myotubes model

Thedevelopmentofskeletalmuscleinvivoandthedifferentiationofmyoblastsinvitro

areaccompaniedbyachangeinisoactinpatterns.Onlycytoplasmicβ‐andγ‐actinsare

participatedinearlymuscledevelopmentandinpre‐fusedculturedmyoblasts(Stortiet

al. 1976; Whalen et al. 1976). During development, the relative amount of α‐actin

increasesuntilitbecomesthepredominantactinspecies.Thisincreasehappensbyday

9

20ofembryonicdevelopment inchicken thighmuscle (Stortietal.1976). Incultured

chickenembryonicmyoblasts,thisincreasebeginsatabout44hrafterplating.At96hr,

when fusion is complete and the myotubes begin to spontaneously contract, α‐actin

becomesthemajorcomponentintheculture(Rubensteinetal.1977).

Themurineimmortalizedcellcellline,C2C12,isaninvitromodelusedforstudiesofthe

molecularbasisofskeletalmusclecelldifferentiation(Kislingeretal.2005;Montesano

etal.2013).C2C12cellswereoriginallyobtainedfromthethighmuscleofC3Hmiceafter

crush injury and are capable of differentiation (Yaffe et al. 1977). In this model,

undifferentiated myoblasts are recognized as flat, fusiform or star‐shaped cells,

scatteredonthesubstrateandrigorouslymono‐nucleated.Afterreachingconfluence,or

24hrafter serumexchange from20% fetalbovineserum(FBS) to2%horseserum

(HS),theorientation,length,andthickeningofthesemyoblastsareconsideredtobeat

anearlystageofdifferentiationat thispoint.Later, thesecellsbegin to fuseand form

multi‐nucleatedmyotubes(Fig.1.6)(Kislingeretal.2005;Koetal.2006;Montesanoet

al.2013).

Figure1.6ChangesinC2C12cellmorphologyinresponsetomyogenicdifferentiation

Light microscopy‐based images of undifferentiated proliferating C2C12 myoblasts (myoblast) anddifferentiatingcellsatvarioustimepoints(2,4,6‐day‐old)followingserumstarvation.Bar,450μmReprinted by permission from Macmillan Publishers Ltd: [Molecular & Cellular Proteomics],copyright(Kislingeretal.2005).

Myoblast 2-day-old

4-day-old 6-day-old

10

1.3.4 Muscle wasting

Loss ofmusclemass ormusclewasting, is related to a poor quality of life, increased

morbidity and mortality, and affected metabolic functions. Two common forms of

musclewastingaresarcopeniaandcachexia(Table1.4).

Table1.4MetabolicconsequencesofsarcopeniaandcachexiaReprintedbypermissionfromMacmillanPublishersLtd:[AnnualReviewofMedicine],copyright(Dodsonetal.2011)

Metaboliccondition Sarcopenia CachexiaMuscleproteinsynthesis Decreased DecreasedMuscleproteindegradation Nochange IncreasedMusclemass,strength,and

functionDecreased Decreased

Fatmass Increased NochangeordecreasedBasalmetabolicrateandtotal

energyexpenditureDecreased Increased

Inflammation Nochange IncreasedInsulinresistance Increased Increased

Severemusclewastingisknownascachexiaandoftenaccompaniesdiseasestatessuch

as cancer, immunodeficiency diseases, HIV/AIDS, rheumatoid arthritis, chronic renal

insufficiency and chronic uremia (Thomas 2007; Tazi et al. 2010;White et al. 2011;

Palusetal.2014).Cachexiaaffectsup to80%ofpatientswithadvancedcancersand

also accounts for nearly 30%of cancer‐related deaths (Glass et al. 2010; Zhou et al.

2010). Although the mechanism of cancer cachexia is poorly understood, multiple

biological pathways and factors such as tumour‐specific proteolysis‐inducing factor

(PIF) and tumournecrosis factorα (TNF‐α) are thought to be involved. In particular,

TNF‐αhasbeenshowntohaveadirectcataboliceffectonskeletalmuscleandleadsto

musclewastingthroughtheubiquitin‐proteasomesystem(UPS).Oxidativestressisalso

thoughttoplayakeyroleincachexiabystimulatingtheUPS(Lenketal.2010;Silverio

etal.2011;Sakumaetal.2012).

Sarcopeniaisassociatedwithaprogressivedeclineofmusclemass,qualityandstrength

asaresultofageingandisassociatedwithlossofmusclefibres,especiallythetypeIIa

fibres.Estimatesoftheprevalenceofsarcopeniarangefrom13%to24%inadultsover

60yearsofagetomorethan50%inadultsaged80yearsandolder(Fig.1.7).Thisloss

ofmusclemass ismostnotable in the lower limbmusclegroups,withvastus lateralis

beingmostaffected.Sarcopeniaisinvolvedinamultifactorialprocessincludesphysical

11

activity,nutritionalintake,oxidativestressandhormonalchanges(Thomas2007;Lenk

etal.2010;Sakumaetal.2012;Sakumaetal.2015).

Figure1.7ChangesinmusclemasswithageComputedtomography(CT)scanof theupper leg(midthigh level) ina25(A)and81yearold(B)male,matched forbodymass andheight.Decreasedmuscle area, increased subcutaneous fat, andincreased fat and connective tissue infiltration into themuscle canbe seen in the elderly subject.ReprintedbypermissionfromMacmillanPublishersLtd:[JournalofAppliedPhysiology],copyright(Koopmanetal.2009).

Loss ofmusclemass can result frommyofibre death or from the reduction in size of

individualmyofibresduetoanetlossofproteincontentresultingfromtheunbalanced

protein degradation and synthesis (Balagopal et al. 1997; Smith et al. 1999; Tisdale

2001;Jackmanetal.2004;Khaletal.2005;Shavlakadzeetal.2006;Moylanetal.2007;

Thomas2007;Arthuretal.2008;Bonettoetal.2009;Evans2010;Pennaetal.2010;

Sakumaetal.2015).ROSispresumedtodelaythedifferentiationprocessofmyoblastto

myotubesbyoxidativelydamagingthecell(Arthuretal.2008;Barbierietal.2012).An

imbalanceinproteindegradationandsynthesishasbeenobservedinmusclecellsafter

hydrogenperoxidetreatment(Jackmanetal.2004).

1.4 Protein turnover

1.4.1 Overview

The turnover of protein in muscle is controlled by protein degradation and protein

synthesis mechanism, and an imbalance in these two can result in muscle wasting.

A. B.

12

Proteindegradation involves therapidmodulationofcellular functionandremovalof

damaged molecules by two systems: the ubiquitin‐proteasome system (UPS) and

lysosomalautophagy(LA)system.ProteinsaretargetedfordestructionbytheUPSviaa

series of enzymatic reactions that tag themwith ubiquitin. In contrast to UPS, LA is

restricted to the cytoplasm but is capable of degrading a much wider spectrum of

substrates, which tend to be long‐lived proteins (Korolchuk et al. 2010). Protein

synthesis involves three main processes: initiation, elongation and termination.

Initiation, in particular, is involved with different kinds of initiation factors that are

regulatedbyvarioussignallingpathwaysthatmaybeaffectedbyoxidativestress.

1.4.2 Protein degradation

Ubiquitin‐proteasome system (UPS)

In eukaryotic cells, the ATP‐dependent UPS is essential for regulating protein

degradation in the cytosol and nucleus, includingmuscle proteins (Palus et al. 2014;

Sakumaetal.2015).Proteinsaretargetedforthisdegradationbyaseriesofenzymatic

reactions that label themwithubiquitin (a76aminoacid residue) inaprocesscalled

poly‐ubiquitylation (Ciechanover et al. 1980; Hershko et al. 1980; Korolchuk et al.

2010).Thismarksthetargetprotein for transportationtothe26Sproteasome,where

the protein is degraded into oligopeptides and then released into the cytoplasm or

nucleoplasmforfurtherdigestionintoaminoacidbypeptidases(Korolchuketal.2010;

Sakumaetal.2015).

The specificity and selectivity of the ubiquitylation process is controlled by three

enzymes,E1,E2andE3(Fig.1.8).E1enzymesactivateubiquitinfunctiontoattackthe

substrateaminogroup.TheactivatedubiquitinisthentransferredfromE1enzymesto

E2enzymeswhicharealsocalledubiquitin‐carriersorconjugatingproteins.Themost

remarkablefeatureofubiquitylationistheextraordinarydiversityofitstargetprotein

substrateandE3enzymes, theuniqueubiquitin ligase,are thekey in this recognition

process. The activated ubiquitin is transferred on the onto the lysine residues of the

target protein substrate by E3 enzymes after the recognition process (Pickart et al.

2004;Leckeretal.2006;Ciechanover2010;Korolchuketal.2010).Thetargetprotein

becomesmono‐ubiquitylatedinoneormoreplaceswiththisprocess,however, this is

insufficientforproteasometargetingasthistargetingrequirespoly‐ubiquitylationofat

least fourubiquitins.Thesepoly‐ubiquitinchainsare formed in subsequent roundsat

13

the lysine residues of ubiquitin (position 6, 11,27, 31, 33, 48 and 68). All these sites

couldbeanacceptorofanotherubiquitin.(Fushmanetal.2010;Korolchuketal.2010;

Sakumaetal.2015).

Figure1.8Ubiquitin‐proteasomesystem(UPS)Theareseveralsteps involved intheUPSprocess. (1)ubiquitin(Ub) isactivatedby theubiquitin‐activatingenzyme,E1enzyme; (2)activatedubiquitin is transferred toaubiquitin‐carrierprotein,E2enzyme;(3)E2enzymetransferstheactivatedubiquitintothetargetproteinsubstratewhichisbound specifically to a unique ubiquitin ligase, E3 enzyme; (4) the transfer of activated ubiquitincouldbedoneviaanadditionalthiol‐esterintermediateonE3enzyme;(5)successiveconjugationofubiquitin to one another generates a poly‐ubiquitin chain; (6) poly‐ubiquitin chain serves as thebindinganddegradationsignal for thedownstream26Sproteasome, theproteinsubstrate is thendegraded into short peptides; (7) free and reusable ubiquitin is released by de‐ubiquitinatingenzymes(DUBs)forfutureuse.ReprintedbypermissionfromMacmillanPublishersLtd:[RambamMaimonidesMedicalJournal],copyright(Ciechanover2010;Ciechanover2012).

Poly‐ubiquitylation marks the target protein for transportation to a barrel‐shaped

organelle, 26S proteasome which consists of a 20S central complex and two 19S lid

complexes.The19Scomplexescontroltheentrybyremovingthepoly‐ubiquitinchain

andunfoldingthetargetproteinbeforeenteringthe20Scomplexthroughthenarrow

catalyticpore(Nandietal.2006;Korolchuketal.2010;Sakumaetal.2015).Onceinside

the 20S complex, the proteins are exposed to trypsin‐, chymotrypsin‐ and peptidyl‐

glutamylpeptide‐hydrolyzing‐likeactivitiesoftheproteasome(Heinemeyeretal.1997;

Korolchuk et al. 2010).After thewholeUPSprocess, short peptidesderived from the

targetproteinandreusableubiquitinarethenreleased(Glickmanetal.2002).

14

Lysosomal‐autophagy (LA) system

In addition to the UPS, the degradation ofmost long‐lived proteins,macromolecules,

biological membranes, and whole organelles, including the mitochondria, ribosomes,

the endoplasmic reticulum, and peroxisomes also occurs by autophagy which is

associated with lysosomes (Sakuma et al. 2015). The various hydrolytic enzymes in

lysosome areworking optimally at an acidic environment, therefore, the lysosome is

surrounded by a membrane to protect cellular contents from enzymatic actions

(Ciechanover2010).

The digest action of lysosome is dynamic and it targets substrates specifically in

numerouswayswhichinclude:(1)receptor‐mediatedendocytosisandpinocytosis;(2)

phagocytosis also known as herterophagy; and (3) microautophagy and

macroautophagyinthelysosomallumen(Fig.1.9)(Mortimoreetal.1987;Ciechanover

2005; Ciechanover 2010). For example, mitochondria, endoplasmic reticulum (ER)

membranes, glycogenbodies andother cytoplasmic entities are degradedby lysosme

under extreme conditions bymacroautophagy (Ashford et al. 1962; Mortimore et al.

1987;Ciechanover2010).

Figure1.9LysosomaldigestionReprinted by permission fromMacmillan Publishers Ltd: [RambamMaimonidesMedical Journal],copyright(Ciechanover2010;Ciechanover2012).

15

1.4.3 Protein synthesis

Initiation

Translation is a cyclicalprocess (Fig.1.10)and ribosomal subunits thatparticipate in

initiation are recycled from post‐termination ribosomal complexes (post‐TCs) that

consistofan80SribosomeboundtomRNA,aP‐sitedeacylatedtRNAandat leastone

eukaryoticreleasefactor1(eRF1).Thesepost‐TCsaredisassembledbyreleasingthese

factors and dissociating the ribosomes into subunits before the process of initiation

(Jacksonetal.2010).

Tostartinitiation,eukaryoticinitiationfactors(eIFs)(Table1.5)suchaseIF2withthe

anticodon loop of an initiator tRNA (Met‐ tRNAMeti) and recycled 40S ribosomal unit

fromdisassembledpost‐TCsareattached together to form43Spre‐initiation complex

for the attachment tomRNA (Unbehaun et al. 2004; Fraser et al. 2007; Jackson et al.

2010).However,beforethisattachment,thesecondarystructureofmRNAneedstobe

sufficiently unwound to allow the loading of the 43S pre‐initiation complex. This

processrequires theworkofeIF4Fcomplex (consistsofeIF4E,eIF4GandeIF4A)and

eIF4Btounwindthe5’cap‐proximalregionofmRNAinanATP‐dependentmannerto

prepareit forribosomalattachment.Oncethe43Spre‐initiationcomplexbindstothis

unwoundmRNA,itstartsthescanningfortheinitiationcodonwhichisusuallythefirst

AUGtriplet(—GCC(A/G)CCAUGG—,withapurineatthe‐3andaGatthe+4position)

from5’ to3’direction(Kozak1991;Pestovaetal.2002; Jacksonetal.2010).The48S

pre‐initiationcomplexisthenformedafterthebindingofinitiatortRNA(Met‐tRNAMeti)

totheinitiationcodon.The60Sribosomalunitthebindstothis48Scomplexwiththe

release of the rest initiation factors to form 80S initiation complex to achieve next

progressoftranslation,theelongationofthepeptide(Pisarevetal.2006;Yuetal.2009;

Jacksonetal.2010).

16

Table1.5EukaryoticinitiationfactorsReprintedbypermissionfromMacmillanPublishersLtd:[NatureReviewsMolecularCellBiology](3971781259397),copyright(Jacksonetal.2010)

Name FunctioneIF2 FormsaneIF2‐GTP‐Met‐tRNAMeti ternarycomplexthatbindstothe40Ssubunit,thus

mediatingribosomalrecruitmentofMet‐tRNAMetieIF3 Binds40Ssubunits,eIF1,eIF4GandeIF5;promotesattachmentof43Scomplexesto

mRNAandsubsequentscanning;andpossessesribosomedissociationandanti‐associationactivities,preventingjoiningof40Sand60Ssubunits

eIF1 Ensuresthefidelityofinitiationcodonselection;promotesribosomalscanning;stimulatesbindingofeIF2‐GTP‐Met‐tRNAMetito40Ssubunits

eIF1A StimulatesbindingofeIF2‐GTP‐Met‐ tRNAMeti to40SsubunitsandcooperateswitheIF1inpromotingribosomalscanningandinitiationcodonselection

eIF4E Bindstothe5’cap‐proximalregionofmRNAeIF4A(1) DEAD‐boxATPaseandATP‐dependentRNAhelicaseeIF4G(2) BindseIF4E,eIF4A,eIF3,PABP,andmRNAandenhancesthehelicaseactivityofelF4AeIF4F Acap‐bindingcomplex,comprisingeIF4E,eIF4AandeIF4G;unwindsthe5′

cap‐proximalregionofmRNAandmediatestheattachmentof43Scomplexestoit;andassistsribosomalcomplexesduringscanning

eIF4B AnRNA‐bindingproteinthatenhancesthehelicaseactivityofeIF4AeIF4H AnRNA‐bindingproteinthatenhancesthehelicaseactivityofeIF4Aandishomologous

toafragmentofeIF4BeIF5 AGTPase‐activatingprotein,specificforGTP‐boundeIF2,thatinduceshydrolysisof

eIF2‐boundGTPonrecognitionoftheinitiationcodoneIF5B Aribosome‐dependentGTPasethatmediatesribosomalsubunitjoiningeIF2B AguanosinenucleotideexchangefactorthatpromotesGDP–GTPexchangeoneIF2

(1) Two paralogues (eIF4AI and eIF4AII), encoded by different genes, are functionally indistinguishable, but eIF4AIII has no activity as an eIF. (2) Two paralogues (eIF4GI and eIF4GII), encoded by different genes, are functionally similar but show some selectivity towards different mRNAs. eIF4GI is generally the more abundant.

17

Figure1.10Cyclicalprocessoftranslation(1)Recyclingofpost‐terminationcomplexes(post‐TCs)toseparate40Sand60Sribosomalsubunits;(2) formation of eIF2‐GTP‐Met‐tRNAMeti; (3) formation of 43S preinitiation complex with a 40Sribosomalsubunit,eIF1,eIF1A,eIF3,eIF2‐GTP‐Met‐tRNAMeti,andeIF5;(4)activationofmRNAbytheATP‐dependentmannerofeIF4FandeIF4B;(5)attachmentof43SpreinitiationcomplextomRNAregion;(6)scanningofthestartcodon(AUG);(7)recognitionoftheinitiationcodon;(8)joiningof60Sribosomalsubunitto48ScomplexandconcomitantdisplacementofeIF2‐GDPandotherfactors(eIF1,eIF3,eIF4B,eIF4F,andeIF5)mediatedbyeIF5B;(9)releaseofeIF1AwitheIF5Bfollowedbythe assembly of elongation‐competent 80S ribosome. Reprinted by permission from MacmillanPublishersLtd:[NatureReviewsMolecularCellBiology](3971781259397),copyright(Jacksonetal.2010).

(2)(1)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

18

Elongation

The peptide elongation process (Fig. 1.11) is highly conserved across eukaryotes,

prokaryotesandthearchaea(Spahnetal.2001;Ramakrishnan2002;Kappetal.2004).

Tostartthisprocess,apeptidyltRNAsitsintheribosomalP‐siteandanaminoacyltRNA

isbrought to the ribosomalA‐siteasa ternarycomplexwith theelongation factor1A

(eEF1A; EF‐Tu in bacteria) and GTP. tRNA, the anticodon, corresponds to the three

basesof thecodononthemRNA.Whencorrectcodon‐anticodonpairingoccurs, three

basesofrRNAinthe40Sribosomalsubunitinducetheswingoutandinteractionwith

the resulting mRNA‐tRNA duplex to stabilize the tRNA binding and prevent other

aminoacyl tRNA binding via hydrolysis of GTP by eEF1A‐GTPase (Ogle et al. 2001;

Rodninaetal.2001;Ramakrishnan2002;Kappetal.2004).TheaminoacyltRNAinthe

A‐site then swings to the peptidyl transferase site to formpeptide bond in a process

calledaccommodationafterthereleaseofeEF1A‐GDP(Ramakrishnan2002;Kappetal.

2004).

The P‐site tRNA is then deacylated and the peptide chain is transferred to the A‐site

tRNAtoformpeptidechain.TheA‐sitetRNAwithpeptidechainisthentranslocatedto

P‐siteviahydrolysisofGTPbyfactor2(eEF2;EF‐Ginbacteria).Thiscycleisrepeated

untilastopcodonisencounteredandterminationbegins(Ramakrishnan2002;Moore

etal.2003;Kappetal.2004;Peskeetal.2004).

19

Figure1.11TranslationelongationinbacteriaTheprocessofelongationishighlyconservedacrossthethreekingdomsoflife.Thisdiagramshowstheprocessofelongation inbacteriawhich issimilartoeukaryotes.ReprintedbypermissionfromMacmillanPublishersLtd:[Cell](3971790172892),copyright(Ramakrishnan2002).

Termination

Terminationoftranslation(Fig.1.12)occursinresponsetoastopcodon(5’‐UAG‐3’,5’‐

UGA‐3’,or5’‐UAA‐3’)intheribosomalA‐site(Bertrametal.2001;Ramakrishnan2002;

Kappetal.2004).UnlikeothercodonswhichisrecognizedbyaminoacyltRNA,thethree

stop codons are recognized by eukaryotic release factors (eRFs). In eukaryotes, two

releasefactors(eRF1andeRF3)functionasaterminationcomplex(Zhouravlevaetal.

1995;Keelingetal.2011).TheeRF1recognizesandbindstoallthreestopcodonsinthe

ribosomalA‐siteandmediatesthereleaseofthenascentpolypeptidefromtheribosome

(Songetal.2000;Bertrametal.2001;Kappetal.2004;Keelingetal.2011).TheeRF3

actsasaGTPasetoassisteRF1instopcodonrecognitionandreleaseofthepolypeptide

(Bertrametal.2001;Salas‐Marcoetal.2004;Keelingetal.2011).

TerminationendswithreleaseofthecompletedpolypeptidefromtheP‐sitetRNAwhich

isbelievedto involvepeptidyl transferaseat thecentreof theribosome(Caskeyetal.

1971;Arkovetal.1998;Seit‐Nebietal.2001;Zavialovetal.2002;Kappetal.2004).

20

Figure1.12TranslationterminationReprinted by permission fromMacmillan Publishers Ltd: [WILEY INTERDISCIPLINARY REVIEWS:RNA](3971790698161),copyright(Keelingetal.2011)

1.5 Signalling pathway

1.5.1 Overview

Duringtranslationandtheotherprocessesinvolvedinproteinproduction,asubstantial

amount of energy and cellular material is consumed. For these reasons, mammalian

cellshaveevolvedelaboratemechanismstoregulatetranslationinresponsetovarious

stimulithatindicatesdown‐regulationisrequitedforcellsurvival.Thesestimuliinclude

changes in nutrient availability, cellular energy, stress, hormones and growth factors

(Fig.1.13)(Maetal.2009).

Figure1.13InhibitionoftranslationunderdifferenttypesofstressReprinted by permission from Macmillan Publishers Ltd: [Molecular Cell] (3971790974348),copyright(Spriggsetal.2010)

21

Inskeletalmuscle,arangeofextracellularanabolicorcatabolicstimulusareinvolvedin

dynamicregulationofproductioninmusclefibres,andthisregulationoccursprimarily

attheinitiationoftranslation(Syntichakietal.2006;Maetal.2009;Tisdale2009).The

initiation factors eIF4E and eIF2B, in particular, from the mTOR1/4EBP1 and

PERK/eIF2αsignallingpathways,playimportantrolesinthisregulation.

1.5.2 mTOR/4EBP1 pathway

In eukaryotes, the eIF4F complex (comprised of eIF4E, eIF4G and eIF4A) plays a key

role in initiation.TheeIF4Esubunit, inparticular, isoneof themainregulatorsof the

assembly of the eIF4F complex (Duncan et al. 1987; Powers et al. 2011) and is

controlled by its reversible association with the 4E‐binding proteins such as 4EBP1

(Kimballetal.2006;Spriggsetal.2010;Powersetal.2011).The4EBP1canblockeIF4F

assemblythoughcompetitionwitheIF4GforeIF4Ebinding(Kimballetal.2006;Powers

et al. 2011). When 4EBP1 is phosphorylated via the mTOR pathway, 4EBP1 is

dissociatedfromeIF4Etoallowtranslationtoproceed(Phametal.2000;Kimballetal.

2006; Powers et al. 2011). Conversely, dephosphorylation of 4EBP1 by a protein

phosphataseresultsinincreasedassociationof4EBP1witheIF4Eandinhibitionofthe

formation of the eIF4F complex (Fig. 1.14) which leads to a decrease in translation

(Phametal.2000;Powersetal.2011).

Figure1.14Regulatingcap‐dependenttranslationinitiationTherecruitmentof the40Sribosomalsubunit to the5′endofmRNA isacrucialandrate‐limitingstep during cap‐dependent translation. A number of translation initiation factors, including the 5′cap‐bindingprotein eukaryotic translation initiation factor 4E (eIF4E), have essential roles in thisprocess. Hypophosphorylated 4E‐BPs bind tightly to eIF4E, thereby prevents its interaction witheIF4Gandthusinhibitsproteinsynthesis.ThemTORC1‐mediatedphosphorylationof4e‐BPsreleasethe 4E‐BPs from eIF4E, resulting in the recruitment of eIF4G to the 5′ cap, and thereby allowingtranslation initiation to proceed. Reprinted by permission fromMacmillan Publishers Ltd: [NatureReviewsMolecularCellBiology](3971791370596),copyright(Maetal.2009).

22

MammalianTOR(mTOR)existsintwodistinctcomplexescalledcomplex1(mTORC1)

and complex 2 (mTORC2) (Guertin et al. 2007). mTOR responds to various stresses

including genotoxic, nutrient, energy and oxidative stress (Sengupta et al. 2010) and

playsacriticalroleindiabetesandageing(Zoncuetal.2011).Studieshaveshownthe

level of protein synthesis decreases in old age due to decreased phosphorylation of

4EBP1(Fig.1.15)(Drummondetal.2008).IthasalsobeenshownthatinsulinandIGF‐1

activationofthePI3K/Akt/mTORpathwayleadstoanincreaseinproteinsynthesisand

adecrease inproteindegradation resulting inhypertrophyof themuscle (Palusetal.

2014).

Figure1.15mTORC1pathwayandageingmTOR regulated the process of ageing via different factors. With a depression in translation viaeIF4E/4EBP1 pathway, ageing was then generated. Reprinted by permission from MacmillanPublishersLtd:[Aging(AlbanyNY)],copyright(Handsetal.2009).

Many studies suggest that oxidants depress protein synthesis by decreasing

phosphorylationof4EBP1,therebyinhibitinginitiationoftranslation(Fig.1.16)(Pham

et al. 2000; Shenton et al. 2006; Zhang et al. 2009; Powers et al. 2011). Hydrogen

peroxidehasbeenfoundtostimulatedephosphorylationof4EBP1byincreasingprotein

phosphatase (PP1/PP2A) activity and resulting in an increase in the association

23

between 4EBP1 with eIF4E, and a decrease in protein synthesis (Pham et al. 2000;

Powersetal.2011).

Figure1.16EffectsofROSonmTOR/4EBP1pathwayAdaptedfrom(Powersetal.2011).

1.5.3 PERK/eIF2α pathway

eIF2α is assumed to be anothermechanism involved in the regulation of translation

inanition by phosphorylation (Spriggs et al. 2010). eIF2 consists of three subunits

(α,β,γ) and is one of the key initiation factors that carries the initiator tRNA (Met‐

tRNAMeti) with GTP to form the 43S pre‐initiation complex. During the process of

initiation,eIF2istransformedfromtheGTPformtoaGDPformbutitcanberecycled

for the next translation process by eIF2B to progress the GTP‑exchange reaction.

However,phosphorylationofeIF2α atresidueSer51preventsthisreactionbyinhibiting

the dissociation of eIF2 from eIF2B (Deng et al. 2002;DangDo et al. 2009;Ma et al.

2009;Powleyetal.2009;Spriggsetal.2010).

24

TherearefourkinasesinvolvedinthephosphorylationofeIF2αinresponsetoarange

of external stresses. These includeGCN2, PERK,HRI, andPKR (Fig. 1.13) (Deng et al.

2002;Hardingetal.2003;Cullinanetal.2006;DangDoetal.2009;Spriggsetal.2010;

Emara et al. 2012). In the mice that bear the cachexia‐inducing MAC 16 tumour,

phosphorylationofeIF2αandPKRhavebeenshowntoincreasewithoutchangesinthe

amountof eIF2α andPKR.Thesemicealso showadecrease inweightandmyosinas

phosphorylationof eIF2α increases (Eley et al. 2007).Thishas alsobeenobserved in

vitrostudiesofMCF7andMCF12Acells(Kimetal.2000).

The PERK/eIF2α pathway is also involved in the responds of endoplasmic reticulum

(ER)tostress.Tomaintainhomeostasisineukaryoticcells,ERsensesandresponsesto

cellular stresses in a range of ways including the unfolded protein response (UPR)

(Schroderetal.2005;Cullinanetal.2006;Backetal.2009;Changetal.2010).TheUPR

is reduces ER stress by clearing misfiled proteins in the ER though PERK/eIF2α

pathway(Hardingetal.2001;Ozcanetal.2004;Cullinanetal.2006;Liangetal.2006;

Shentonetal.2006;Rutkowskietal.2007;Scheuneretal.2008;Backetal.2009).This

pathway leads to a reduction in protein synthesis, which reduces protein folding

demands and allows for the clearance of misfolded proteins (Cullinan et al. 2006;

Rutkowskietal.2007;Backetal.2009).

Recentevidencesuggests that there isacloserelationshipbetweenERstress,protein

misfolding, and oxidative stress (Fig. 1.17). In this relationship, ROS leads to the

accumulationofmisfoldedproteinsintheER,creatingacycleofERstressandoxidative

stress(Teraietal.2005;Scheuneretal.2008;Backetal.2009;Changetal.2010).As

thePERK/eIF2αpathwayhasbeenshowntoplayarole in theclearanceofmisfolded

proteins, the PERK/eIF2α pathway is also likely to be involved in the response to

oxidativestress.

25

Figure1.17TherelationshipbetweenERstressandROSProtein folding within the ER lumen was ushered by a family of oxidoreductase that catalyzeddisulfidebondformationandisomerization.UnderERstress,therewasanincreaseintheformationof incorrect intermolecular and/or intramolecular disulfide bonds that leaded to the formation ofROS. Inturn,ROScouldalsocauseERstressthroughmodificationofproteinsand lipidsthatwerenecessary to maintain ER homeostasis. Reprinted by permission from Macmillan Publishers Ltd:[EndocrineReviews](3971800940503),copyright(Scheuneretal.2008).

26

Aim

An increase in oxidative stress has been seen to occur alongsidemusclewasting and

changes in protein turnover in various conditions and disease such as cancer, type 2

diabetes,chronic inflammationandageing(Sohaletal.1996;Klaunigetal.1998;Wei

1998;Finkeletal.2000;Atalayetal.2002;Evansetal.2002;Weietal.2002;Maritimet

al.2003;Robertson2004;Khaletal.2005;Phillipsetal.2005;Roloetal.2006;Valkoet

al.2006;Parketal.2007;Thomas2007;Chenetal.2008;Bonettoetal.2009;Parketal.

2009;Evans2010;Reuteretal.2010;Terrilletal.2013).Adecreaseinoxidativestress

with antioxidant has also been shown to improve muscle pathology and decrease

necrosis(Terrilletal.2013).Thissuggeststhatoxidativestressmayimpactonmuscle

wasting.Whilethesignallingpathwayinvolvedinproteinsynthesisandoxidativestress

isnotyetclear,themTOR/4EBP1andPERK/eIF2αpathwaysareconsideredthemost

likelypathwaysaffectedbyoxidativestress.

This study uses the skeletal muscle culture system of C2C12 myotubes to study the

effectsofhydrogenperoxidemediatedoxidativestress,specificallytheeffectofcatalase

andglucoseoxidaseon(i)totalproteinlevels,(ii)proteinsynthesislevelsandprotein

degradation rates, (iii) phosphorylation rates of 4EBP1 and eIF2α, and (iv) thiol

oxidation of whole protein, actin, and myosin. As direct application of hydrogen

peroxideintothemediumwouldlikelybecytotoxic(Halliwelletal.2000),catalaseand

glucoseoxidasewereapplied to theC2C12 culturemedia.Glucoseoxidase isknown to

generateendogenoushydrogenperoxidecontinuously(Boverisetal.1972;Gruneetal.

1995;Gruneetal.1997).

27

Chapter 2 Material and Methods

2.1 Cell Culture

Inthisstudy,immortalizedcultureswereusedasinvitromodelsforinvivomyofibres.

The source of the cell cultures and their preparation are described below. The C2C12

culture techniques developed in our laboratory were adapted in this study (Gebski

2009).Inthisstudy,allmyotubesweretreatedunderserum‐starvedconditiontoavoid

theanypossibleeffectofenzymesintheserum.

2.1.1 Proliferation

C2C12mousemyoblastsoriginatedfromthethighmuscleofC3Hmiceaftercrushinjury

(Yaffe et al. 1977). This cell line was purchased from the American Type Culture

Collection (ATCC, Manassas, USA). In this study, all experiments were performed on

cellsatpassage4.

TheC2C12myoblastswerestoredascryopreservedstocksinliquidnitrogen.Theywere

frozen at a concentrationof approximately0.5‐1.5× 106 cells/ml in freezingmedium

consisting of Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, 11965‐118)

supplementedwith1%(v/v)ofpenicillin/streptomycin(Invitrogen,15070‐063),20%

Fetal Bovine Serum (FBS; Invitrogen, 16000044) and 10% (v/v) dimethyl sulfoxide

(DMSO;Sigma,D2650).

Cellswerethawedundersterileconditionsinalaminarflowhoodbyadding1mlofpre‐

warmed(37°C)proliferationmediumconsistingofDMEMsupplementedwith1%(v/v)

ofpenicillin/streptomycinand20%ofFBS.Thecellswerethawedslowlybyaspirating

with1mlofproliferationmedium.Itwasthentransferredtoafalcontubewith5mlof

proliferation medium. The cells were centrifuged at 1500 rpm for 5 min at room

temperature in a centrifuge (Eppendorf , 5702). The supernatantwas then discarded

andthecellpelletwasresuspendedin1mlofproliferationmediumandtransferredtoa

T‐75cm2flask(Falcon)with10mlofproliferationmedium.Thecellswereincubatedat

37°Cin5%CO2and95%air.

28

2.1.2 Trypsinization and seeding of myoblasts

Themyoblaststookapproximately5‐7daystoreach80‐90%confluenceinculture.For

seeding, theproliferationmediumwas firstremovedandthecellswerewashedtwice

with 5 ml phosphate‐buffered saline (PBS, Medicago, 09‐8912‐100) then 7 ml of

Trypsin/EDTAsolution(Sigma,T4049)wasaddedtotheflask.Afterincubationat37°C

for5min,celldetachmentwasconfirmedbymicroscopicexaminationoftheflask.The

trypsin reactionwas stopped by the applying 5ml of proliferationmedium. The cell

suspensionwasthentransferredtoa15mlfalcontubeandcentrifugedat1500rpmfor

5 min at room temperature. The supernatant was discarded and the cell pellet was

resuspended in1mlofproliferationmedium.10µlof cell suspensionwasdiluted20‐

timeswithTrypanBluesolution(Sigma,T8154) ina0.6mlmicrocentrifuge tubeand

transferred to a haemocytometer (Brand, 717805) to determine the total cell count

underthelightmicroscope.Fourquadrants(Fig.2.1)werecountedseparatelyandthe

totalcountwasaveraged.

Figure2.1HaemocytometerCellswerecounted inthefour largesquares(A,B,C,andD),whicharefurthersubdivided into16smallersquares.Theaveragenumberofcellswastakenandmultipliedby1×104andtheTrypanbluedilutionfactor(20).

29

Theconcentrationofthecellsuspensionwascalculatedusingthemultiplicationfactor

(104)derivedfromthevolumeofeachofthefourcountedsquares(0.1mmdeepand1

× 1mm square)which equates to a volume of 0.1mm3 or 0.0001ml (10‐4ml). The

formulausedtodeterminethecellcontentrationwas:

Thecell suspensionwas thendiluted inproliferationmediumtoa final concentration

dependingontheintendeduseofthecellsandthesizeoftheculturedishtobeseeded.

2.1.4 Treatment conditions

Catalaseandglucoseoxidasewereusedtomodulatethelevelofhydrogenperoxidein

themyotubes.Catalase is ananti‐oxidase that reduces the levelofhydrogenperoxide

andglucoseoxidaseisanoxidasethatincreasesthelevelofhydrogenperoxide.IGFand

dexamethasone (DEX) were used as controls in the experiments focused on protein

turnover.

Alltreatmentsstartedatday7offusion.Catalase(Sigma‐Aldrich,C3155)wasappliedat

3000units/ml, glucose oxidase (Sigma‐Aldrich, G7141)was applied at 10munits/ml,

IGF (Sigma‐Aldrich, Australia) was applied at 20 ng/ml, and DEX (Sigma‐Aldrich,

Australia)wasapplied.All treatmentswererefreshedevery24hruntil thecellswere

harvested.

2.2 Protein extraction

Proteins frommyotubes were extracted using either TCA acetone or a phospho‐safe

methoddependingontheintendeduseofthecells.

2.2.1 TCA acetone extraction

Followingtreatment(Section2.1.4),myotubecultureswerewashedtwicewithPBSand

lysedwith 20% (w/v) trichloroacetic acid (TCA) in acetone to denature, precipitate

proteinsandprevent furtherredoxreactions(Armstrongetal.2011).Asobservedby

30

Armstrong and coworkers (2011), application of the TCA turned the protein pellet

whitebutnotthesupernatantconfirmingtheproteinswerepresentinthepelletnotthe

supernatant.

Afterwashing,700µlof20%TCA/acetonewasaddedtoeachdishandthemyotubes

were detached using a cell scraper (SARSTEDT, 831830). The supernatant and the

resultingpelletthatcontainthemyotubesweretransferredtoa1.5mlmicrocentrifuge

tube and then centrifuged at 10000rpm for 5 min (4°C) in a centrifuge (Eppendorf,

5417R).ToremovetheremainingTCAinthesamples,thesupernatantwasdischarged

andtheproteinpelletswerewashedtwicewith1mlofacetone.Theproteinpelletwas

thenresuspendedin300µlofTrisbuffer(50mMTriswith0.5%SDS).

Tocompletelyresuspendtheproteins,thesamplewassonicatedat40%ampfor2min

onice,followingby30minofvortexing.Thesamplewasthentransferredtoanew1.5

mlmicrocentrifugetubeforproteinquantification(Section2.3.2).

2.2.2 Phospho‐safe extraction

Forwesternblotting,theproteinsfrommyotubeswereextractedusingaphospho‐safe

method.Followingtreatment(Section2.1.4),myotubecultureswerewashedtwicewith

PBS and lysed with a phospho‐safe extraction cocktail consisting of 2.5 ml of

PhosphoSafeExtractionBuffer(Novagen,71296)andaquarterofaproteaseinhibitor

tablet(Roche,04693159001).

After washing with 1 ml of PBS (4°C) twice, 100 µl of cold phospho‐safe extraction

cocktailwasaddedintoeachdishandmyotubesweredetachedusingacellscraper.The

supernatantandtheresultingpelletthatcontainthemyotubesweretransferredtoa1.5

ml microcentrifuge tube and incubated on ice for 20 min. Halfway through the

incubation,thesamplewasvortexedforapproximately5sec.Aftertheincubation,the

samplewasvortexedagainfollowedbycentrifugationat12000gfor10min(4°C)ina

centrifuge (Eppendorf, 5417R). 97 µl of supernatant was transferred to a 0.6 ml

microcentrifuge tube forwesternblottingandremainingsupernatantwas transferred

toanother0.6mlmicrocentrifugetubeforproteinquantification(Section2.3.1).

31

2.3 Protein quantification

Theproteinsampleswerequantifiedbydifferentmethodsdependingonthe intended

useofcells.

2.3.1 Bradford assay

Forwesternblotting, theproteinsampleswerequantifiedusing theBradfordmethod

(Bradford 1976). Following harvesting (Section 2.2.2), the Bio‐Rad Protein Assay Kit

(Bio‐Rad, 500‐0001)was used to quantify the concentration of each sample. Protein

samples were quantified with reference to a Bovine serum albumin (BSA) standard

absorbancecurve.BSAstandardsofknownconcentrations(0,100,200,300,400,and

500µg/ml)preparedbyserialdilutionofBSA(stockconcentration1mg/ml)in0.01M

PBS.

Theproteinsampleswere firstdiluted20‐foldwith0.01MPBSand then10µlof the

dilutedproteinsampleandstandardsweretransferredintriplicatetothewellsofa96‐

well plate. 200 µl of Bio‐Rad reagent was then added to each well. The plate was

incubatedfor10minwithgentleshakingonashakeratroomtemperatureandatthe

endofthisincubationperiod,theabsorbanceofeachwellwasmeasuredat595nmina

platereader(BioTekPowerwaveXSSpectrophotometerwithKC4ver.3.4program).The

concentrationoftheproteinsampleswasextrapolatedfromtheBSAstandardcurve.

2.3.2 Micro BCA assay

ThemicroBCAassaykit(Sigma,QPBCA‐1KT)wasusedtoaccesstotalproteincontent.

Afterharvestingandresuspension(Section2.2.1),proteinsampleswerequantifiedwith

referencetoaBovineSerumAlbumin(BSA)standardabsorbancecurve.BSAstandards

ofknownconcentrations(0,5,10,20,30,and40µg/ml)preparedbyserialdilutionof

BSA (stock concentration 40µg/ml) in Tris buffer (2mM Triswith 0.5% SDS). The

workingreagentwascomposedwith25partsofreagentA,25partsofreagentB,and1

partofreagentC.

1µlofproteinsamplewasdiluted25‐foldwith0.5%SDSandafurther10‐folddilution

withTrisbuffer(2mMTriswith0.5%SDS)ina1.5mlmicrocentrifugetube.Thiswas

32

followed by adding 250 µl of working reagent added into each tube. 200 µl of each

standardwastransferredto1.5mlmicrocentrifugetubesand200µlofworkingreagent

was then added into each tube. Thesemicrocentrifuge tubeswere vortexed and then

incubatedfor1hratat60°C.Themicrocentrifugetubeswerevortexedagainand100

µlofincubatedsolutionwasthenaliquotedintriplicatetoeachwellofa384‐wellplate.

Theabsorbanceofeachwellwasmeasuredat562nmintheBioTekplatereader.

2.3.3 Detergent compatible (DC) protein assay

For 2‐tag labeling, the protein samples were quantified using the DC assay method.

After dual‐labeling (Section 2.7.2), a DC assay kit (Bio‐Rad, 500‐0112) was used to

quantify the concentrations of each sample. Protein samples were quantified with

referencetoaBovineSerumAlbumin(BSA)standardabsorbancecurve.BSAstandards

ofknownconcentrations(0,0.1,0.2,0.3,0.4,0.6,0.8,and1mg/ml)preparedbyserial

dilutionofBSA(stockconcentration1mg/ml)inassaybufferconsistingofTrisbuffer

(0.5MTriswith0.5%SDS)diluted1:1withdistilleddoubledeionized(ddi)water.

Beforeperformingthequantification,thereagentA’wasmadewith1mlofreagentA,

20µlof reagentC, and1.02mlofddiwater.7.5µlofproteinsamplewasdiluted1:1

withTrisbuffer(0.5MTriswith0.5%SDS)andfollowedwithanother1:1dilutionwith

ddiwaterina1.5mlmicrocentrifugetube.30µlofeachstandardwastransferredtoa

1.5mlmicrocentrifuge tube. 105 µl of reagent A’ and 255 µl of reagent Bwere then

added to each microcentrifuge tube. The tubes were vortexed for 5 min and then

incubated for 10 min at room temperature. 100 µl of incubated solution was then

aliquotedintriplicatetoeachwellofa384‐wellplate.Theabsorbanceofeachwellwas

measuredat750nmintheBioTekplatereader.

2.4 Measurement of protein synthesis

Thelevelofproteinsynthesisinmyotubeswasmeasuredusingradioactiveleucine.

2.4.1 Incorporation of radioactive leucine

7‐day‐oldC2C12myotubesweretreatedwithtreatments(catalase,glucoseoxidaseand

IGF)for24hrunderserum‐starvedconditions.Thefollowingday,1µCiofradioactive

33

leucine (leucine,L‐[3,4,5‐3H(N)],PerKinElmer,NET‐460)with treatmentswasapplied

tothesemyotubesfor24hr.

2.4.2 Harvest

After radioactive leucine incorporation and treatment, themyotubeswere harvested.

Prior to harvesting, the medium was removed from each dish and transferred to a

centrifugetube for laterdeterminationofradioactivity.700µlofTCA/acetone(20%)

was added to each dish and myotubes were detached using a cell scraper. The

supernatantandtheresultingpelletthatcontainthemyotubesweretransferredtoa1.5

mlmicrocentrifuge tube centrifuged at 10000 gat room temperature in a centrifuge

(Eppendorf,5415C).Thesupernatantwasdiscardedwithoutdisturbingthepelletand

thepelletwasthenwashedtwicewith500µlofleucinesolution(1mML‐leucinein0.6

M HClO4, Sigma, L8000). The samplewas then resuspended in 300 µl of NaOH (300

mM).Thesamplewasheatedto40°Candvortexeduntilfullysolubilized.

2.4.3 Radiation analysis

100µlofsupernatantandproteinsampleweretransferredintotubescontaining2mlof

scintillation cocktail solution and vortexed for 5 sec. The radioactivity was then

measured in a scintillation counter.The incorporation rateof radioactive leucinewas

obtainedasfollows:

The amount of incorporated radioactive leucine was then obtained by timing the

incorporationrateofradioactiveleucineasfollows:

The amount of non‐radioactive leucine in DMEM was obtained from supplier. The

concentrationofL‐leucineinDMEMwas0.802mMand2mlofDMEMwasusedineach

dish.Therefore,theamountofnon‐radioactiveleucineineachdishwas1.604µmol.The

34

ratio of radioactive leucine in total leucine could thenbe calculatedwith the valueof

non‐radioactiveleucineandradioactiveleucineasfollows:

The ratio of radioactive leucine was then used to covert the amount of radioactive

leucineincorporationtototalleucineincorporationasfollows:

2.5 Measurements of protein degradation

Therateofproteindegradationinmyotubeswasmeasuredusingradioactiveleucine.

2.5.1 Incorporation of radioactive leucine

Radioactivelecuine(1µCi)wasappliedtothe6‐day‐oldC2C12myotubesunderserum‐

containedconditions.Themediumwasremovedthenextdayandtreatments(catalase,

glucoseoxidase, IGF,andDEX)wereappliedtothese labeledmyotubes for48hr.The

treatmentwasrefreshedevery24hrandtheradioactivity levels inthespentmedium

weredetermined.

2.5.2 Harvest

After radioactive leucine incorporation and treatment, themyotubeswere harvested.

Prior to harvesting, the medium was removed from each dish and transferred to a

centrifugetube forradioactivityanalysis.700µlofTCA/acetone(20%)wasaddedto

eachdishandmyotubesweredetachedusinga cell scraper.The supernatantand the

resultingpelletthatcontainthemyotubesweretransferredtoa1.5mlmicrocentrifuge

tube centrifugedat10000gat room temperature ina centrifuge (Eppendorf,5415C).

The supernatantwasdiscardedwithout disturbing thepellet and thepelletwas then

35

washed twicewith500µl of leucine solution (1mML‐leucine in0.6MHClO4, Sigma,

L8000).The samplewas then resuspended in300µl ofNaOH (300mM).The sample

washeatedto40°Candvortexeduntilfullysolubilized.

2.5.3 Radiation analysis

100µlofsupernatantandproteinsampleweretransferredintotubescontaining2mlof

scintillation cocktail solution and vortexed for 5 sec. The radioactivity was then

measured in a scintillation counter. The ratio of radioactive leucine release was as

obtainedasfollows:

UnlikeDMEM, the concentration of leucine in serumwasnot able to bemeasured or

obtained. Therefore, the ratio of radioactive leucine releasewas taken as the rate of

proteindegradation.

For data presentation, the rates of protein degradation of all other treatment groups

werenormalizedtotherateofproteindegradationofuntreatedculturesasfollows:

2.6 Western Blot

Western blotting was used to detect the specific proteins and signaling pathways

affected by catalase and glucose oxidase treatment. Two types of western blotting

procedureswereused.TheSodiumdodecylsulfatepolyacrylamidegelelectrophoresis

(SDS‐PAGE) was used to establish themethod. The Bio‐Rad systemwas used for all

subsequentexperiments.

36

2.6.1 SDS Polyacrylamide Gel Electrophoresis (SDS‐PAGE)

After treatment, themyotubeswere harvested using a phospho‐safemethod (Section

2.2.2)andquantifiedusingtheBradfordassay(Section2.3.1).Thesequantifiedproteins

wereseparatedbySDS‐PAGEon12%(resolving)polyacrylamidegelunderdenaturing

conditions. Protein samples were prepared by adding 3× protein loading buffer

(consisting of 0.19M Tris pH6.8, 6% (w/v) SDS, 30% (v/v) glycerol, 0.03%(w/v)

BromophenolBlueand0.3MDTT)totheproteinsamplesandthenheat‐denaturedat

95°Cfor5min.

SDS‐PAGEgelswerepreparedpriortoelectrophoresis.Theresolvinggel(seeTable2.1)

waspouredintotheglassplateassembly,overlaidwithddiwaterandlefttopolymerise

forapproximately20minatroomtemperature.Followingremovaloftheddiwater,the

stacking gel (see Table 2.1) was then poured on top of the set resolving gel and gel

combswereinsertedimmediately.APSandTEMEDwereaddedtobothsolutionsprior

topouring.

Table2.1CompositionofresolvingandstackinggelReagent 12%Resolvinggel 4%Stackinggel

1.5MTris(pH8.8) 7.5ml

0.5MTris(pH6.8) 3.75ml

10%SDS 300µl 150µl

30%Acrylamide/Bissolution,37:5:1 12ml 1.95ml

ddiwater 9.87ml 8.25ml

10%APS 300µl 75µl

TEMED 30µl 15µl

Oncethestackinggelhadpolymerized,thegelcastingchamberwasthentransferredto

ageltankcontainingcold1×electrodebufferconsistingof6.06gTris,28.83gGlycine,

2gSDSandmadeup to1Lwithddiwater.Thegelcombswereremovedandall the

remainingun‐polymerizedpolyacrylamidewasflushedoutof thewellswithelectrode

buffer. Prepared protein samples and 5 µl of Precision Plus Protein™ Kaleidoscope

Standards (Bio‐Rad, 161‐0375) were loaded in the wells and the gel was

electrophoresedforapproximately2hrat120Vat4°C.

37

While the gel was electrophoresing, polyvinylidene difluoride (PVDF) membrane

(Amersham)wassoakedinmethanolfor5sec.Thesoakedmembranewithfilterpapers

and spongepadswerepre‐soaked in transferbuffer (consistingof3.03 gTris, 14.4 g

Glycine,100mlMethanolandmadeupto1Lwithddiwater)at4°C.

After electrophoresis, the gel was assembled in the transfer cassette (Fig. 2.2) and

placed in the electroblotting tank filled with cold transfer buffer. The proteins were

transferredfromgeltothePVDFmembraneviaelectrophoresisat100Vfor90minat

roomtemperature.

Figure2.2TheassembleoftransfercassetteThepolyacrylamidegelandmembraneweresandwichedbetweenthepre‐soakedspongesandfilterpapers in transfer buffer. Proteins on the gelwere transferred to themembrane from cathode toanode.

After transfer, thePVDFmembranewasremovedandwashedbriefly inTrisBuffered

Saline(TBSpH7.5;consistingof12.1gTris,9gNaCl,madeupto1Lwithddiwaterand

pH with HCl) with 0.1 % (v/v) Tween‐20 (TBS‐T). The washed membrane was

38

incubated in blocking buffer (5% skimmilk in TBS‐T) for 1 hr at room temperature

withgentleshaking.ThiswasfollowedbywashingwithTBS‐Ttwice,10mineachtime,

and incubated with diluted primary antibody at 4� overnight with gentle shaking.

Primaryantibodies(Table2.2)werediluted1:1000inTBS‐Tcontaining5%BSA.The

previouslyoptimizedprotocolsprovidedbysupplier(CellSignaling)wereadopted.

Table2.2PrimaryantibodiesAntibody CatLog No.

Phospho‐4EBP1(Thr37/46)RabbitmAb 2855

Phospho‐eIF2α(Ser51)XP® RabbitmAb 5199

4EBP1Rabbit 9452

eIF2αRabbit 9722

AktRabbit 9272

After the incubation, themembranewaswashedwithTBS‐T twice,10mineach time,

and incubated with secondary antibody (Thermo, 31460) diluted 1:5000 in TBS‐T

containing 5 % skim milk for 1 hr at room temperature with gentle shaking. The

membrane was washed briefly twice with TBS‐T and then incubated with

chemiluminescent substrate solution (Table 2.3) for 5min at room temperature. The

signalwasexposedtofilmandthefilmwasdevelopedinadarkroom.

Table2.3Chemiluminescencesubstratesolutionusedforproteindetection

Product ProviderProtein

abundanceonmembrane

SuperSignalWestPicoChemiluminescentSubstrate

ThermoScientific

High

WesternLightingUltra PerkinElmer Low

LuminataCrescendoWesternHRPsubstrate

Millipore Medium

2.6.2 Bio‐Rad system

Western Blot analysis using Bio‐Rad system was adopted for all subsequent

experiments.

After treatment, themyotubeswere harvested using a phospho‐safemethod (Section

2.2.2)andquantifiedusingtheBradfordassay(Section2.3.1).Thesequantifiedproteins

samples were separated by precast gradient gels (Bio‐Rad, 456‐1086). The gel was

placed in a gel tank containing 1× electrode buffer (diluted 1 in 10 from 10 × Tris/

39

Glycine/SDS,Bio‐Rad,161‐0772)andthegelcombswereremoved.10µgoftheprotein

sampleand4µlofPrecisionPlusProtein™Kaleidoscope™Standardswerethenloaded

inthewellsandthesamplewereelectrophoresedat150Vforapproximately1.5hrat

roomtemperature.

Protein transferwas carriedoutusing theTrans‐Blot®Turbo™Transfer System (Bio‐

Rad, 170‐4155), which involves semi‐dry protein transfer. The gel was sandwiched

between thenitrocellulosemembrane and filter papers from theTrans‐Blot®Turbo™

Mini Nitrocellulose Transfer Packs (Bio‐Rad) and transfer was performed using the

3minproteintransferprogram(turbosetting).

After transfer, themembranewaswashedwithTBS‐Tandblocked inblockingbuffer.

Afterblotting,themembranewaswashedwithTBS‐Tandincubatedindilutedprimary

antibodyat4°Covernightwithgentle shaking.Themembranewas thenwashedwith

TBS‐Tand incubated insecondaryantibodyfor1hratroomtemperaturewithgentle

shaking.ThemembranewaswashedbrieflytwicewithTBS‐Tandthenincubatedwith

chemiluminescencesubstratesolution(Table2.3)for5minatroomtemperature.The

signalwasdetectedandcapturedbyusingChemiDoc™MPSystem(Bio‐Rad,170‐8280)

andImageLab™SoftwareVersion4.0(Bio‐Rad).

2.6.3 Densitometry analysis

Densitometrywasperformedonthewesternblottingresultimages(Sections2.6.1and

2.6.2) using the NIH Image freeware program Image Processing and Analysis in Java

(Image J) (http://rsb.info.nih.gov/ij/). The phosphorylation level of a specific protein

was expressed as the densitometry ratio of the phosphorylated protein to the total

amountofthatprotein,forexample(phosphorylatedeIF2α/totaleIF2α).TotalAktwas

usedasaloadingcontrol.

2.7 Measurements of thiol oxidation‐2 tag labeling

General oxidation and oxidation of specific proteinswere assessed by 2 tag labeling.

This method was developed in our laboratory for labeling animal protein samples

(Armstrongetal.2011)andwasadaptedheretolabelcellculturesamples.

40

2.7.1 Preparation of protein samples

Three dishes of myotubes from each culture batch were treated with catalase and

glucoseoxidase.Aftertreatment,themyotubeswereharvestedwith20%TCA/acetone

(Section 2.2.1) but only one dish was resuspended in Tris buffer for protein

quantification(Section2.3.2).TheremainingdisheswerekeptinTCA/acetonefor2tag

labeling(duallabeling).

2.7.2 Dual labeling of protein thiols with fluorescent tags

ThemyotubesinTCA/acetoneweresonicatedat40%Ampsfor2minonice.Thiswas

followedbytransferring100µgoftheproteinpellettoa1.5mlmicrocentrifugetube.

The tubes were then centrifuged at 10000rpm for 5 min (4°C) in a centrifuge

(Eppendorf, 5417R). The supernatant was discharged and the protein pellet was

washed with cold acetone (150 µl, 4°C). The centrifugation and washing steps were

repeatedtoremoveanyresidualTCAbeforesuspensionandlabeling.

After removal of TCA, the reduced protein thiols in protein samplewere labeled. To

perform this labeling, theproteinpelletwas suspended in50µl ofTris buffer (0.5M

Triswith0.5%SDS,pH7.3)and5µlof5mMBODIPYFL‐N‐(2‐aminoethyl)maleimide

(FLm, Invitrogen, B10250). To fully suspend the protein pellet, the samples were

sonicatedat40%Ampsfor1minonicefollowedbyvortexingandincubationatroom

temperaturefor30minindark.ToremovetheexcessFLm,thesampleswereapplied

andmixedwith200µlofcoldacetoneandthenincubatedovernightat‐20°Cforprotein

precipitation.Theproteinsamplewascentrifugedat10000rpmfor10min(4°C)next

day and resulted protein pellet was washed with 200 µl of cold acetone to remove

unboundFLm.Thiswasfollowedbyanincubationovernightat‐20°C.Onthenextday,

the protein samplewas centrifuged at 10000rpm for 10min (4°C) and the resulting

protein pellet was resuspended in 50 µl of Tris buffer (0.5 M Tris with 0.5 % SDS,

pH7.0). 21 µl of the suspended protein sample was transferred to a 0.6 ml

microcentrifugetube.

As oxidized thiols needed to be reduced before labeling, 4 µl of 25 mM Tris(2‐

carboxyethyl) phosphine hydrochloride (TCEP, Sigma, C4706) was added and mixed

41

with the 21 µl of FLm‐labeled protein sample to give a final TCEP concentration of

4mM.Thiswasfollowedbyincubationfor1hrinthedarkatroomtemperature.

Afterreduction,theproteinsamplewasmixedwith25µlofTrisbuffer(0.5MTriswith

0.5%SDS,pH7.0)and5µlof5mMTEXASRED‐C2‐malemide(TRm,Invitrogen,T6008)

tolabeltheoxidizedthiols.Afterincubationfor1hrinthedarkatroomtemperature,

excessTRmdyewas removedbymixing thesamplewith220µlof coldacetone then

incubated overnight at ‐20°C for protein precipitation. On the next day, the protein

samplewascentrifugedat10000rpmfor10min(4°C)andtheresultingproteinpellet

was resuspended in25µlofTrisbuffer (0.5MTriswith0.5%SDS,pH7.0)and then

mixedwith100µlofcoldacetone.Thesamplewasspundownwithmini‐centrifuging

andthenincubatedovernightat‐20°Cforproteinprecipitation.Theproteinsamplewas

centrifuged at 10000rpm for 10min (4°C) next day and resulting protein pelletwas

resuspendedandincubatedagainovernightat‐20°Cforproteinprecipitation.Afterthe

finalcentrifugation,theresultingproteinpelletwasresuspendedin50µlofTrisbuffer

(0.5MTriswith0.5%SDS,pH7.0).7.5µlofdual‐labeledproteinsamplewastakento

measure the levelofproteincontent (Section2.3.3)and therestwaskept toquantify

theleveloffluorescence(Sections2.7.3and2.7.4).

2.7.3 Fluorescence measurement using a plate reader

Fluorescence measurements of FLm and TRm for the protein samples were

standardized to FLm and TRm standard curves (Table 2.4). To prepare the standard

curves, eachdyewasdiluted from5mMto1.5mMbymixing6µlof5mMdyewith

14 µl of DMSO. The 60 µM dye/ovalbumin stock solution was consisting of 16 µl of

1.5mMdye,160µlof2mMovalbumin(Sigma,A5378)and224µlofTrisbuffer(0.5M

Triswith0.5%SDS,pH7.0).Thissolutionwasincubatedindarkfor30minbeforeuse.

For the TRm standard curve, the dye/ovalbumin stock solution was further diluted

8‐fold.

Afterincubation,allstandardswerediluted10‐foldwith0.1MNaOH,andeachprotein

samplewasdiluted32‐foldwith0.1MNaOH.Alldilutedstandardsandproteinsamples

were aliquoted in triplicate (100 μl/ well) to each well of a 384‐well plate. The

fluorescence of each sample was then measured using a fluorescent plate reader

42

(FluostarOptima)withwavelengthssetat485nmexcitationand520nmemissionfor

FLmand595nmexcitationand610nmemissionforTRm.

Table2.4DilutionforFLmandTRmstandardsFLm(µM) TRm(µM) Stocksolution(µl) Trisbuffer(µl)

0 0 0 100

6 0.75 10 90

12 1.5 20 80

24 3.0 40 60

36 4.5 60 40

48 6.0 80 20

60 7.5 120 0

2.7.4 SDS‐PAGE

After the protein quantification (Section 2.3.3), dual‐labeled protein samples were

separatedwithprecast gradient gel (Section2.6.2). Toquantify reduced andoxidized

thiolsofaspecificproteinbands, in‐gelFLmandTRmstandardcurveswereprepared

(Section 2.7.2) with several modifications. Firstly, the stock solution was made by

mixing4µlof60µMFLm/ovalbumin,1µlof60µMTRm/ovalbuminand95µlof2mM

ovalbuminthatgavea finalconcentrationof2.4µMFLmwith0.6µMTRm.Theblank

ovalbuminwasconsistingof3µlofTrisbuffer(0.5MTriswith0.5%SDS,pH7.0)with

97 µl of 2 mM ovalbumin. The in‐gel standards were then prepared by a range of

dilutionswiththisovalbuminsolution(Table2.5).

Table2.5In‐gelFLm/TRmstandardsFLm/TRm(nmol) Stocksolution(µl) Ovalbumin(µl)

0/0 0 10

0.0048/0.0012 2 8

0.0096/0.0024 4 6

0.0144/0.0036 6 4

0.0192/0.0048 8 2

0.024/0.006 10 0

Afterpreparingthein‐gelstandards,3×proteinloadingbufferwasaddedtotheprotein

samplesandstandardsandthenheat‐denaturedat95°Cfor5min.3µgofproteinand

10 µl of each standard were loaded and the gel was electrophoresed at 150 V for

43

approximately1.5hratroomtemperature.Thefluorescenceofeachlanewasmeasured

using a typhoon gel scanner (GE Healthcare Life Science, Typhoon Trio) with

wavelengthssetat520nmforFLmand610nmforTRm.Followinggelanalysisusing

theImageJsoftware,theamountofreducedandoxidizedthiolsinspecificproteinwas

determinedwithreferencetotheFLm/TRmstandardcurves.

2.8 Statistics

All data were analyzed with one‐way ANOVA with post‐hoc tests (unstacked) on

Statplus(AnalystSoft,U.S.A.).Thep‐valuewasobtainedusingFisher’sLeastSignificant

Difference (Fisher LSD). The stats only performed when experiments were repeated

morethanthreetimes.

44

Chapter 3: Development of methods for the study of protein content in C2C12 myotubes in response to treatment with catalase and glucose oxidase

3.1 Introduction

Sarcopeniaandcachexiaaretwotypesofmusclewasting.Sarcopeniaisareductionin

musclemassandstrengththatoccurswithageingandisassociatedwithareductionin

motorunitnumberandatrophyofmusclefibers,especiallytypeIIafibers.Thelossof

musclemassisclinicallyimportantbecauseitleadstodiminishedstrengthandexercise

capacity as a result of the loss of 5 % of muscle mass per decade of life from 40s

onwardsandmoreafter theageof65 (Lenk et al. 2010). Cachexia iswidely recognized

asseverewastingaccompanyingdiseasestatessuchascancer(Tazi et al. 2010)(Fig.3.1)

or immunodeficiency (Thomas 2007). About 80 % of all cancer patients suffer from

cachexiawhich leads to impairedmobility and accounts directly for around 20% of

cancer‐related deaths (Glass et al. 2010; Mathew 2011; Silverio et al. 2011; Wang et al.

2011; Wysong et al. 2011).

Figure3.1ChangesinmusclemassaccompanyingcancerComparedwithahealthymouse (A), thehindlimbofamousebearingC26coloncarcinoma(B) isseverelyatrophiedat3weeks following transplantation.Reprintedbypermission fromMacmillanPublishersLtd:[BMCCancer],copyright(Aulinoetal.2010;Coletti2013).

Previousstudiesof sarcopeniaandcachexiahaveshown thatan increase inoxidative

stress can induce a decrease in muscle size. In sarcopenia patients, an increase in

A. B.

45

oxidative stresswith age resulting in changes tomitochondrial function is thought to

playanimportantroleinthedeclineofphysiologicfunction.Mitochondrialproduction

of superoxide anions has been proposed to be the primary source of this oxidative

stress (Mansouri et al. 2006) and the oxidative damage of mitochondria has been

demonstrated to increase the generation of hydrogen peroxide in cells (Lass et al.

1998).The increasedgenerationof these reactiveoxygen species (ROS) is thought to

inducetheoxidationofmuscleproteinsandsubsequentlossofmusclemass(Capeletal.

2005).

In cachexia, tumour‐bearinganimals show lossofmuscleweight forup to twoweeks

aftertumourimplantation(Guarnieretal.2010).Thislossofmusclemassisthoughtto

result from an increased rate of protein degradation as regulated by the ubiquitin‐

proteasomeproteolyticpathway(Pennaetal.2010;Eddinsetal.2011;Mathew2011;

Wang et al. 2011). Loss of muscle mass is also thought to be linked to increased

oxidative stress resulting from decreased antioxidase activity and high levels of ROS

generation(Mantovanietal.2002;Mantovanietal.2002;Lenketal.2010;Silverioetal.

2011). Further evidence to support a role of the attenuation of muscle wasting in

tumour‐bearingmiceaftertreatmentwiththeantioxidantEGCGderivedfromgreentea

isfurtherevidencetosupportaroleforoxidativestressinmusclewasting(Wangetal.

2011).

Whileoxidativestress is thought tobeakeycauseofmusclewasting incachexiaand

sarcopenia,itisnotcertainhowthisoxidativestresseffectsproteindegradation.Inthe

C2C12 murine model of muscle wasting, oxidative stress of these myotubes has been

shown to decrease protein levels. When these myotubes were treated with 100 μM

hydrogen peroxide, the rate of ubiquitin conjugation by the ubiquitin‐proteasome

proteolyticpathway increased leading toproteindegradation (Lietal.2003).Further

understanding of the mechanism underlying these myotube changes in response to

changes in hydrogen peroxide induced by catalase and glucose oxidase, however, is

required. This includes the role of protein turnover, signaling pathways, and thiol

oxidation.Tobetterunderstand thesemechanisms,methodsneed tobedeveloped to

measure thesechanges in theC2C12model.This chapterdescribes theoptimizationof

existingmethodstoharvestandquantifyproteinlevelsintheC2C12modelinresponse

tocatalaseandglucoseoxidasetreatment.

46

3.2 Methods

AllmethodsaredescribedindetailinChapter2.

Myotubecultures

The myotubes were cultured in 35mm petri dishes. In the preliminary experiments

which aimed to optimize protein extraction, a single untreated or catalase‐treated

culture sample was used. In the preliminary experiments which aimed to optimize

proteinquantification,differentconcentrationsofbovineserumalbumin(BSA) inTris

bufferwereused. Inallsubsequentexperiments,proteinextractionandquantification

was performed for up to three petri dishes per treatment for every treatment group

(untreated,catalase,andglucoseoxidase).Theseexperimentswererepeateduptofive

timesusingfreshC2C12cultures.

Proteinextraction

Trichloroaceticacid(TCA)wasusedtoprotonateallthiolsandtoprecipitatethecellular

proteins to prevent their subsequent oxidation (Aslund et al. 1999; Delaunay et al. 2000).

To extract proteins from themyotubes, the petri dishes containingmyotube cultures

were washed briefly twice with 1ml phosphate buffered saline (PBS). 20 %

TCA/acetone (w/v) (700 µl) was then added to the petri dishes and the cells were

harvestedwithacellscraper.TheTCA/acetoneandcellsweretransferredtoa1.5ml

microcentrifugetubeandthetubeswerethencentrifugedat10000rpmfor5min(4°C).

Thesupernatantwasdischargedandtheproteinpelletwaswashedwithcoldacetone(1

ml).ThecentrifugationandwashingstepswererepeatedtoremoveanyresidualTCA.

Theproteinpelletwas thensuspended inTrisbuffer (300µl,50mMTriswith0.5%

SDS,pH7.0)andquantifiedusingthemicroBCAassay.

47

Proteinquantification‐microBCAassay

ThecommercialkitfromSigma(QuantiPro™BCAAssayKit,QPBCA‐1KT)wasusedto

quantifythetotalproteincontentforeachtreatmentgroup.1µloftheproteinsample

wasdiluted25‐foldwithSDSbuffer(0.5%SDS), followedbyanother10‐folddilution

withTrisbuffer(2mMTriswith0.5%SDS,pH7.0)ina1.5mlmicrocentrifugetube.250

µl of the cocktail reagent (A:B:C=25:25:1) was added into each diluted sample and

incubated for1hrat60°C.The incubatedsolution (100µl)was then transferred toa

384‐wellplateandtheabsorbancewasanalyzedat562nminaplatereader(BioTek,

PowerWaveHT).

3.3 Results

3.3.1 Modifying the extraction method to extract proteins from C2C12 myotubes

An existing method developed in our laboratory for the extraction of protein from

muscle(Armstrongetal.2011)wasadaptedtosuitC2C12tissueculturesamples.Inthe

existingmuscle tissueprotocol,1mlof20%TCA/acetone isused toextractproteins

from20mgofskeletalmuscle.Sincetheamountofproteinineachtissueculturedishis

much less than in the skeletal muscle samples used in the existing method, the

myotubeswereharvestedwitharangeof20%TCA/acetonevolumes(20µl,40µl,100

µl, 300 µl, 500 µl, and 700 µl). Using this range of TCA/acetone volumes, 100 µl of

TCA/acetonewasfoundtobetheminimumvolumerequiredtocoverthesurfaceofthe

dish.Although300µl and500µlwere sufficient to cover the surfaceof thedish, the

acetonerapidlyevaporatedattheselowvolumes,makingextractionmoredifficultand

reducing protein yields (Fig. 3.2). Therefore, 700 µl TCA/acetone was used in all


48

Figure3.2ThetotalproteincontentMyotubes (7‐day‐old) were harvested with different volumes of 20 % TCA/acetone. The proteinpelletwaswashedwithacetoneandjustresuspendedwithTrisbuffer.TheproteincontentwasthenmeasuredusingthemicroBCAassay.

AfterresuspensionwithTrisbuffer,mostoftheproteinpelletremainedunsuspended.

Therefore, we presumed that sonication was needed for full suspension. A range of

sonicationtimesweretestedfrom30secto2min.Overthisrangeofsonicationtimes,

theproteinpelletwasfoundtobefullyresuspendedaftersonicationat40%ampfor2

min and protein recovery was increased substantially (Fig. 3.3). Therefore, in all

subsequentexperiments,theproteinpelletsweresonicatedusingtheseconditions.

0

5

10

15

20

25

30

35

700 500 300

Total protein/ dish

(µg)

20% TCA/acetone (µl)

49

Figure3.3ThetotalproteincontentMyotubes (7‐day‐old) were harvested with different volumes of TCA/acetone and washed withacetone.TheproteincontentwasthenmeasuredusingthemicroBCAassay.Theproteinpelletwasresuspendedandsonicatedat40%ampfor2minoniceinTrisbuffer.

3.3.2 Method to quantify protein content in C2C12 myotubes

In the existing method for the extraction of protein frommuscle taken from animal

models,thedetergentcompatibleproteinassay(DCassay,Bio‐rad500‐0112)wasused

to assay the skeletal muscle samples. However, as tissue culture samples generally

produce lower protein levels than animal models, it was predicted that this method

would not be sensitive enough to detect changes in protein levels at these low

concentrations. Preliminary experiments using a range of low concentration bovine

serum albumin (BSA) solutions prepared using Tris buffer were undertaken to

determine the suitability of theDC assay and protein assays for the quantification of

proteinlevelsinC2C12myotubesinresponsetochangesinoxidativestress.

The DC assay is designed for samples suspended in detergent‐based solution. To

determineifthisassayissuitablefortheC2C12cultures,BSAwasfirstdissolvedinTris

buffer (50mM Tris with 0.5% SDS) at 0‐0.4mg/ml and then assayed using the DC

method. As expected and as evident in standard curve, this assay was not sensitive

enough todetect low levelsofBSA(Fig.3.4)and thereforeunlikely tobe thesuitable

methodfortheC2C12tissueculturesamplesinthisstudy.

0

100

200

300

400

500

600

700

800

700 500

Total protein/dish (µg)

20% TCA/acetone (µl)

50

Figure3.4ThestandardcurveofDCassayinTrisbufferBSAinTrisbufferfrom0to0.4mg/mlwasusedtoperformDCassay.Theabsorbancewasanalyzedat750nminaplatereader(BioTek,PowerWaveHT).

TheBradford assay (Bio‐rad, 500‐0006) is another commonmethodused toquantify

theconcentrationofproteinsamples.Toperformthisassay,BSAwasfirstdissolvedin

Tris buffer (50 mM Tris with 0.5 % SDS) from 0‐0.4 mg/ml. After mixing with the

workingreagent,theSDSintheTrisbufferreactedwiththeworkingreagenttoproduce

aprecipitatethatpreventedaccuratemeasurementofabsorbance.Therefore,thisassay

wasnotusedforfurtheranalysisoftheC2C12model.

The micro BCA assay was then tested for protein detection sensitivity using a

commercialKit(Sigma,QuantiPro™BCAAssayKit,QPBCA‐1KT).Thiskitisdesignedto

givealinearresponsefrom0.5to30µg/mlofprotein.Toassessthesuitabilityofthis

kit toproteindetection from tissue culture samples,BSAwasdissolved inTrisbuffer

(50mMTriswith0.5%SDS)from0‐0.04mg/mlandassayed.Asevidentfromstandard

curve (Fig. 3.5‐A), this assay was not sensitive enough to analyze low concentration

proteinsamplesundertheseconditions.

According to the product technical bulletin for the micro BCA assay, the maximum

allowableTris concentration for theassay is50mMand themaximumallowableSDS

concentrationis5%.TotestifTrisorthecombinationofTrisplusSDSwasinterfering

withtheBSAquantification,0‐0.04mg/mlBSAstandardswerethenpreparedinin0.5

y = -0.1122x2 + 0.0926x + 0.063 R² = 0.98001

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0 0.1 0.2 0.3 0.4 0.5

Abs

orb

ance

BSA (mg/ml)

0.5

51

%SDS solution and 2mMTris bufferwith 0.5% SDS. As evident from the standard

curves (Fig. 3.5‐B and Fig. 3.5‐C), a reduction on the Tris concentration allowed low

concentration protein samples to be detected. In all subsequent experiments, the

proteinlevelsofC2C12myotubeswerequantifiedwiththemicroBCAassaywith2mM

Trisand0.5%SDS.

Figure3.5StandardcurveformicroBCAassayusingBSAinvariousbuffersBSA (0 to 0.04 mg/ml) in different buffers assayed using the micro BCA assay. (A) HighconcentrationTrisbuffer(50mMTriswith0.5%SDS)(B)0.5%SDSsolution(C)LowconcentrationTris buffer (2 mM Tris with 0.5 % SDS). Absorbance was analyzed at 750 nm in a plate reader(BioTek,PowerWaveHT).

3.3.3 Measuring the level of protein content in C2C12 myotubes in response to catalase and glucose oxidase

To investigate the changes in protein levels in response to changes in hydrogen

peroxide and oxidative stress levels, the myotubes were treated with catalase and

glucoseoxidaseinserum‐starvedconditionsfor48hrand72hr,withtreatmentsbeing

refreshedevery24hr.Thismyotubeswerethenharvestedwith20%TCA/acetoneand

theprotein levelsquantifiedusingthemicroBCAassay.Asexpected, the levelof total

protein was significantly increased after treatment with catalase for 72 hr and

significantly decreased after treatment with glucose oxidase for 48 hr (Fig. 3.6).

Evidenceofmyotubedeathwasapparentinthe72hsampleslikelyduetotheageofthe

cultures. To investigate changes in protein turnover, signaling pathways, and thiol

oxidation in response to catalase and glucose oxidase treatment, all subsequent

experimentswereanalyzedafter48hroftreatment.

y = -6.8722x2 + 1.0375x + 0.084 R² = 0.98945

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0 0.01 0.02 0.03 0.04 0.05

Ab

sorb

ance

BSA (mg/ml)

y = -60.682x2 + 13.766x + 0.0873 R² = 0.99883

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.01 0.02 0.03 0.04 0.05

Abs

orb

anc

e

BSA (mg/ml)

y = -72.267x2 + 24.693x + 0.1025 R² = 0.99874

0

0.2

0.4

0.6

0.8

1

1.2

0 0.01 0.02 0.03 0.04 0.05

Abs

orba

nce

BSA (mg/ml)

A. B. C.

52

Figure3.6TotalproteinlevelsinC2C12myotubesinresponsetocatalaseandglucoseoxidasetreatment

Myotubes(7‐day‐old)wereleftuntreatedortreatedwithcatalase(3000units/ml;Cat.),orglucoseoxidase(10munits/ml;GluO.)for48hrinserum‐starvedconditions.Proteinsampleswerecollectedas described in Chapter 2.2.1 and quantified as described in Chapter 2.3.2. (A) This data was anaverageoffiveexperimentsforeachtreatmentgroup(2‐3dishes/treatmentgroup)(B)Thisdatawasanaverageoffourexperimentsforeachtreatmentgroup(2‐3dishes/treatmentgroup).72hrdataisnotshownbecausemyotubedeathwasevidentatthistime‐point.Dataisshownasmean±SEM.

3.4 Discussion

In the present chapter, the impact of catalase and glucose oxidase on total protein

content was examined using protein extraction and quantification methods. These

methodswerefirstlyoptimizedformyotubeculturesamplesandthisisthefirstreport

of changes in total protein content in C2C12 myotubes in response to catalase and

glucoseoxidasetreatment.

Usingproteinextractionandquantificationmethods, theleveloftotalprotein inC2C12

myotubes was found to be increased after treatment with catalase for 72 hr and

decreased after treatment with glucose oxidase for 48 hr. This finding is similar to

previousstudiesofpatientswithmuscleloss(Thomas2007;Lenketal.2010;Tazietal.

2010).Whileoxidativestressisthoughttoinducetheselossesinmusclemass(Lasset

al.1998;Mantovanietal.2002;Mantovanietal.2002;Capeletal.2005;Mansourietal.

2006;Lenketal.2010;Silverioetal.2011), theexactmechanismsthatunderly these

0

200

400

600

800

1000

1200

1400

1600

1800

2000

Total p

rotein

(µg/dish)

0

200

400

600

800

1000

1200

1400

1600

1800

2000

Total p

rotein

(µg/dish)

*

* A. B.

53

losses are not clear yet but an imbalance between the level of protein synthesis and

degradationisthoughttobeinvolved(Balagopaletal.1997).InChapter4,methodsto

investigate changes in protein turnover bymeasure the changes in protein synthesis

anddegradationinC2C12myotubesaredeveloped.

54

Chapter 4: Development of methods for the measurement of protein synthesis and degradation in C2C12 myotubes in response to treatment with catalase and glucose oxidase

4.1 Introduction

Thebalancebetweenproteinsynthesisanddegradationcontrolsproteinlevelswithina

cell organism (Bassell et al. 1997). Whenmusclewasting occurs, there is a decrease in

proteinsynthesisand/oranincreaseinproteindegradation,whichleadstoadecrease

in total protein content. Oxidative stress is thought to be a key intermediary in

promoting muscle wasting (Muller et al. 2006; Arthur et al. 2008). In this study, an

inducer of oxidative stress, hydrogen peroxide, is used to investigate the effects of

oxidativestressinmyotubes.

InChapter3,anincreaseinthetotalproteincontentwasobservedinmyotubestreated

with catalase, which reduces cellular hydrogen peroxide levels (Jones et al. 1968;

Boverisetal.1972;Orretal.1994;Dayetal.1997).Adecreaseintotalproteincontent

inmyotubestreatedwithglucoseoxidasewasalsoobserved.Glucoseoxidaseincreases

the level of hydrogen peroxide in cells (Weiss et al. 1981; Starkebaum et al. 1986;

Salazaretal.1997).Whilethemechanismsthatinducedthesechangesintotalprotein

in these myotubes are not clear, hydrogen peroxide mediated changes in protein

synthesishavebeenreportedinotherstudies.

Inyeastcells,thelevelofproteinsynthesishasbeenshowntodecreaseinresponseto

up to 2mMhydrogen peroxide (Shenton et al. 2003; Shenton et al. 2006). In Clone9 cell, a

cell culturemodel of normal liver epithelial cells, the rate of protein degradation has

been shown to increase in response up to 1mM hydrogen peroxide or a continuous

hydrogenperoxidefluxgeneratedbytheglucose/glucoseoxidasereaction(Gruneetal.

1995;Gruneetal.1997).Whenhemoglobinispre‐treatedwith0.5to50mMhydrogen

peroxide,therateofproteindegradationhasalsobeenfoundtoincrease(Fligieletal.

1984).Inmusclecells,proteinsynthesishasalsobeenshowntodecreaseinresponseto

100 µM hydrogen peroxide (Orzechowski et al. 2002) and the expression of

arogin1/MAFbx, the ubiquitin ligase gene that mediates muscle atrophy, is also

enhancedinresponsetohydrogenperoxide(Lietal.2005).

55

Given the apparent importance of hydrogen peroxide in protein synthesis and

degradation, the present study set out to investigate changes in the level of protein

synthesis and the rate of protein degradation inmyotubes treatedwith catalase and

glucoseoxidasebyadaptingestablishedmethods(Pollard1996;Reinheckeletal.2000;

Casey et al. 2002; Catalgol et al. 2009). As insulin growth factor (IGF) is known to

increase the level of protein synthesis and decrease the rate of protein degradation

(Pham et al. 2000; Brink et al. 2001; Li et al. 2004; Sacheck et al. 2004; Zhao et al. 2007;

McGilchrist et al. 2011; Chen et al. 2012; Clemmons 2012), IGFwas used as the control for

theproteinsynthesisanddegradationexperiements.Astumournecrosisfactor(TNF)is

knowntoincreaseproteindegradationrates(Lietal.2000;Lietal.2005;Leckeretal.

2006),itwasusedasacontrolfortheinitialproteindegradationstudies.TNFwaslater

replaced by dexamethasone (DEX) which is known to increase protein degradation

rates(Sacheck et al. 2004; Sandri et al. 2004).

4.2 Methods


Myotubecultures


whichaimedtooptimizethetimepointoftheincorporationofradioactivity,threepetri

dishes per treatment for every treatment group (untreated, catalase, TNF) from one

single C2C12 culture was used. In all subsequent experiments, two petri dishes per

treatment for every treatment group (untreated, catalase, glucose oxidase, IGF, and

dexamethasone).TheseexperimentswererepeateduptoeighttimesusingfreshC2C12

cultures. In this study, all myotubes were treated under serum‐starved condition to

avoidtheanypossibleeffectofenzymesintheserum.

Proteinsynthesis

Protein synthesiswasassessedbymeasuring the incorporationof radioactive leucine

(leucine, L‐[3,4,5‐3H(N)] into protein. The experimentswere carried out in 7‐day‐old

C2C12myotubecultures(Section2.4) treatedwithcatalase,glucoseoxidase,or IGF for

24hr.Therefreshedtreatmentplusradioactiveleucine(0.5µCi/ml)werethenapplied

totheculturesforafurther24hr.Thelevelofradioactiveleucineintheproteinpellet

56

and supernatant was then analyzed using a scintillation counter. Protein synthesis

results are expressed as the total leucine incorporation per dish per 24 hr

(µmol/dish/24hr)andstatisticalanalysiswasperformedusingStatPlus.

Proteindegradation

Protein degradation was assessed by measuring the release of radioactive leucine

(leucine,L‐[3,4,5‐3H(N)]fromcells.Theexperimentswerecarriedoutin6‐day‐oldC2C12

myotube cultures (Section 2.5) pre‐labeled for 24 hr with radioactive leucine (0.5

µCi/ml, leucine (L‐[3,4,5‐3H(N)]). The pre‐labeled myotubes were then treated with

catalase,glucoseoxidase,IGF,orDEXfor48hr,andtreatmentwasrefreshedevery24

hr.Thelevelofradioactiveleucineinproteinpelletandsupernatantwasthenanalyzed

using a scintillation counter. Protein degradation results are presented as the

percentage of the radioactive leucine release per dish per 24 hr (%/dish/24hr) and

statisticalanalysiswasperformedusingStatPlus.

4.3 Results

4.3.1 Establishment of method for measuring protein synthesis in C2C12 myotubes

TomeasurethelevelofproteinsynthesisinC2C12myotubes,theamountofradioactive

leucine (leucine, L‐ [3,4,5‐3H(N)]) incorporated into newly synthesized proteins was

assessed.Toestablishthismethod,apreliminaryexperimentwasundertakenapplying

radioactiveleucine(1µCi/ml,initialactivity)andcatalaseto7‐day‐oldmyotubesfor24

hr. The protein pellets and supernatantwere collected and the leucine incorporation

wasassessed.

This preliminary experiment showed there was no significant changes in protein

synthesis in the catalase‐treated myotubes compared to the untreated cultures,

however, the incorporationofradioactive leucinewithin thecatalase treatmentgroup

was highly variable (Fig 4.1‐A). This high level of variability in leucine incorporation

suggeststhatthemeasurementmightbeaffectedbysomemetabolicdisturbancesinthe

catalasetreatmentgroup.

To minimize metabolic disturbance (Pollard 1996), all myotubes were subsequently

labeledinpre‐conditionedmediapriortotheadditionofradioactiveleucinebytreating

57

the7‐day‐oldmyotubeswith/withouttreatmentfor24hrinserum‐starvedconditions.

Themediawith/withouttreatmentwasthenrefreshedandthemyotubeswereexposed

toradioactiveleucine(0.5µCi/ml,initialactivity)forafurther24hr.Thisresultedina

reductioninthevariabilityofleucineincorporationandanincreaseinleucineuptake,in

boththecatalase‐treatedgroupanduntreatedgroup(Fig.4.1‐B).

Figure4.1LeucineincorporationinC2C12myotbestreatedwithcatalase(A) Myotubes (7‐day‐old) were left untreated or treated with catalase (3000 units/ml; +Cat.)together with radioactive leucine (1 µCi/ml, initial activity) for 24 hr under serum‐starvedconditions.(B)Myotubes(7‐day‐old)werepre‐conditionedwithcatalase(3000units/ml;+Cat.),orinsulingrowthfactor(30ng/ml;+IGF)for24hrunderserum‐starvedconditionsRadioactiveleucine(0.5µCi/ml,initialactivity)andfreshcatalasewerethenappliedtothesepre‐conditionedmyotubesfor a further 24 hr under serum‐starved conditions. The total leucine incorporation in eachtreatmentgroupwasexpressedasmean±SEM(n=3/treatmentgroup).ThesupernatantandproteinpelletwerecollectedandassayedsimultaneouslyforradioactivityasdescribedinChapter2,section2.4.3.

All subsequent protein synthesis measurements used this pre‐conditionedmedia. As

levels of unincorporated radioactive leucine were also high, the initial levels of

radioactiveleucineweredecreasedfrom1µCi/mlto0.5µCi/mlforallsubsequenttests.

4.3.2 Establishment of method for measuring protein degradation in C2C12 myotubes

Tomeasure therateofproteindegradation in theC2C12myotubes, thereleaserateof

radioactive leucine (leucine,L‐ [3,4,5‐3H(N)])wasassessed.Toestablish thismethod,

0

5

10

15

20

25

Untreated +Cat. +IGF

Leucine

inco

rpora

tion

(µmol/d

ish/24

hr)

0.0

0.5

1.0

1.5

2.0

Untreated +Cat.

Leucine incorporation (µmol/dish/24hr)

A. B.

58

catalaseortumournecrosisfactor(TNF)wasappliedto7‐day‐oldmyotubesfor24hr.

Radioactive leucine (0.5µCi/ml, initial activity) and fresh catalase or TNFwere then

applied to these pre‐treated myotubes for another 48 hr, with refreshment of these

treatmentsafterthefirst24hr.Theproteinpelletsandsupernatantwerecollectedand

thereleaseof radioactive leucinewasassessed.Therewasnosignificantdifference in

protein degradation in either the catalase‐ or TNF‐treated group compared to the

untreatedthegroup(Fig.4.2).

Figure4.2RadioactiveleucinereleasefromC2C12myotubestreatedwithcatalaseandTNF

Myotubes (7‐day‐old) were left untreated or treated with catalase (3000 units/ml; +Cat.) andtumournecrosisfactor(20ng/ml;+TNF)for24hr.FreshcatalaseorTNFwithradioactiveleucine(0.5µCi/ml,initialactivity)wereappliedtothesemyotubesforafurther24hr.FreshcatalaseorTNFwereappliedtotheselabeledandtreatedmyotubesforanother24hr.Thepercentageofthereleaseof radioactive leucine fromeach treatment groupwas expressed asmean± SEM (n=3 /treatmentgroup). The supernatant and protein pellets were collected and assayed simultaneously forradioactivity as described in Chapter2, section 2.5.3. The myotubes were cultured under serum‐starvedconditions.

As shown in Fig. 4.1‐B, the amount of leucine incorporation was different between

treatments,whichmayhaveimpactedontheaccuratemeasurementofleucinerelease.

Toequalizetheamountofincorporatedleucinepriortomeasurerelease,the6‐day‐old

myotubeswerepre‐labeledwithradioactiveleucine(0.5µCi/ml, initialactivity)under

serum‐containedconditionfor24hrpriortoanytreatment.Thesemyotubeswerethen

treated with IGF, DEX, and TNF for 48 hr under serum‐starved conditions, with the

0

10

20

30

40

50

60

Untreated +Cat. +TNF

Radioactive leucine

release (%/dish/24hr)

59

treatmentsrefreshedafterthefirst24hr.AsevidentinFig.4.3,undertheseconditions,

changeswereobservedintheIGFandDEXtreatmentgroupsandthevariabilitywithin

treatmentsgroupswaslow.However,afterseveralroundsofexperiments,nochangein

protein degradationwas observed in the TNF‐treatedmyotubes. Therefore, DEX and

IGFwereusedascontrolsinsteadofTNFinallsubsequentexperiments.

Figure4.3Radioactiveleucinereleasefrompre‐labeledC2C12myotubeswithvarioustreatments

Myotubes (6‐day‐old) were pre‐labeled with radioactive leucine (0.5 µCi/ml, initial activity) inserum‐containing medium (2 % horse serum) for 24 hr. These labeled myotubes were then leftuntreatedortreatedwithinsulingrowthfactor(30ng/ml;+IGF),dexamethasone(40ng/ml;+DEX),ortumournecrosisfactor(20ng/ml;+TNF)inserum‐freemediumfor48hr.Freshtreatmentswerereplacedafter24hr.Thepercentage releaseof radioactive leuine fromeach treatmentgroupwasnormalizedwithuntreatedgroupandexpressedasmean±SEM[n=4culturebatches(+IGF,+DEX);n=5culturebatches(+TNF)](unpublisheddata).ThesupernatantandproteinpelletswerecollectedandassayedsimultaneouslyforradioactivityasdescribedinChapter2,Section2.5.3.*p<0.05

4.3.3 Measuring protein synthesis in C2C12 myotubes

Usingtheconditionsestablishedinsection4.1,themyotubeswerepre‐conditionedand

thentreatedwithcatalase,glucoseoxidaseandIGF.Thelevelofproteinsynthesiswas

significantlydecreasedaftercatalasetreatment(Fig.4.4‐A).Whileasignificantdecrease

inproteinsynthesiswasnotobservedafterglucoseoxidasetreatment(Fig.4.4‐B),there

was a significant increase with IGF treatment, indicating the system is capable of

detectingchangesofproteinsynthesisinC2C12myotubes.

0

20

40

60

80

100

120

Radioactive leucine

release (%/dish/24hr)

*

*

60

Figure4.4ProteinsynthesisinC2C12Myotubeswithcatalaseandglucoseoxidasetreatment

(A)Myotubes(7‐day‐old)wereleftuntreatedortreatedwithcatalase(3000units/ml;+Cat.)orIGF(30ng/ml;+IGF)for24hr.Radioactiveleucine(0.5µCi/ml,initialactivity)withfreshcatalaseorIGFtogetherwerethenappliedtothesepre‐conditionedmyotubesforafurther24hr.(B)Myotubes(7‐day‐old) were left untreated or treated with glucose oxidase (10 munits/ml; +GluO.) for 24 hr.Radioactive leucine (0.5 µCi/ml, initial activity) with fresh glucose oxidase together were thenapplied to these pre‐conditionedmyotubes for a further 24 hr. The total leucine incorporation ineach treatment group was expressed as mean ± SEM [n=8 culture batches (+Cat.); n=4 culturebatches (+IGF, +GluO.)]. The supernatant and protein pellets were collected and assayedsimultaneouslyforradioactivityasdescribedinChapter2,Section2.4.3.Themyotubesin(A)and(B)wereculturedunderserum‐starvedconditions.*p<0.05,**p<0.005

4.3.4 Measuring protein degradation in C2C12 myotubes

Usingtheconditionsestablishedinsection4.2,themyotubeswerepreconditionedand

then treated with catalase, glucose oxidase, IGF and DEX. As expected, the rate of

protein degradation was significantly decreased after catalase treatment. While a

significantincreaseinproteindegradationratewasnotobservedafterglucoseoxidase

treatment(Fig.4.5).TheIGFsignificantlydecreasedtherateofproteindegradationand

theDEXsignificantlyincreasedtherateofproteindegradation,indicatingthissystemis

capableofdetectingchangesofproteindegradationinC2C12myotubes.

0

5

10

15

20

25

Untreated +Cat. +IGF

Leu

cin

e in

corp

ora

tio

n (

µm

ol/2

4h

r)

0

5

10

15

20

25

Untreated +GluO. L

eu

cin

e in

co

rpo

rati

on

(µ

mo

l/24h

r)

*

* *A. B.

61

Figure4.5RadioactiveleucinereleasefromC2C12myotubeswithvarioustreatments

Myotubes (6‐day‐old) were pre‐labeled with radioactive leucine (0.5 µCi/ml, initial activity) inserum‐contained medium (with 2 % horse serum) for 24 hr. These labeled myotubes were leftuntreatedortreatedwithcatalase(3000units/ml;+Cat.),glucoseoxidase(10munits/ml;+GluO.),insulingrowthfactor(30ng/ml;+IGF),ordexamethasone(40ng/ml;+DEX)inserum‐freemediumfor48hr.Freshtreatmentswererefreshedatthefirst24hr.Thepercentagereleaseofradioactiveleuine fromeachtreatmentgroupwasnormalizedwithuntreatedgroupandexpressedasmean±SEM [n=5culturebatches (+Cat.); n=4 culturebatches (+GluO,+IGF, +DEX)].The supernatant andproteinpelletswerecollectedandassayedsimultaneouslyforradioactivityasdescribedinChapter2,Section2.5.3.*p<0.05

4.4 Discussion

Incorporation of radioactive amino acids into proteins is frequently used tomeasure

changes in protein synthesis and degradation (Ratan et al. 1994; Grune et al. 1995;

Pollard1996;Reinheckeletal.2000;Siwiketal.2001;Caseyetal.2002;Shentonetal.

2003; Shenton et al. 2006; Catalgol et al. 2009). To measure changes in protein

synthesis and degradation in C2C12myotubes, twonovel controlswere used: IGF and

DEX. Using these controls, the methods developed in this study were found to be

effectiveinmeasuringproteinturnoverinC2C12myotubes.

0

20

40

60

80

100

120

140

Untre

ated

+Cat

+Glu

O

+IG

F

+DEX R

adioac

tive

leucine

degrad

atio

n (%

/24hr)

*

*

*

62

Thesemethodswerethenusedtoshow,forthefirsttime,howproteinturnoverinC2C12

myotubesisaffectedbycatalaseandglucoseoxidasetreatment.Whenproteinturnover

in C2C12 myotubes was measured after expose to catalase, there was a significant

decreaseinproteinsynthesisanddegradation.Thisisconsistentwiththeknownaction

ofcatalase,whichdecreasesoxidativestress incellsbydecreasinghydrogenperoxide

levels,andtheincreaseintotalnetproteinobservedinthesemyotubesafterexposure

to catalase in Chapter 3. However, in response to glucose oxidase treatment, protein

synthesisanddegradationintheC2C12myotubeswasnotchangedsignificantlydespitea

significant decrease in total net protein observed in response to this treatment in

Chapter3.Eventhoughthechangesinproteinturnoverafterglucoseoxidasetreatment

werenotsignificant,decreasedproteinsynthesisandincreasedproteindegradationis

often indicative of a decrease in protein levels. According to Finkel and coworkers, a

decrease in oxidative stress might in itself stress to the cell, resulting in decreased

proteinsynthesis.

Arangeofconflictingresultshavebeenobservedinotherstudies focusingonprotein

turnoverindifferentcelltypesexposedtohydrogenperoxide.Insomestudies,protein

synthesis decreased and protein degradation increased in response to hydrogen

peroxide (Grune et al. 1997; Orzechowski et al. 2002; Shenton et al. 2003; Shenton et al.

2006).Inastudyofcardiacfibroblasts,collagensynthesislevelswereshowntodecrease

after exposure to hydrogen peroxide, but the level of total protein synthesis did not

change(Siwik et al. 2001).

Furtherstudiesofthesignalingpathwaythatinducechangesinproteinsynthesismay

provideabetterunderstandingofhowmusclecellsresponsetohydrogenperoxide.In

skeletalmuscle,regulationofproteinsynthesisoccursprimarilyattheinitiationphase

of protein translation, which involves at least 13 initiation factors, many of which

assemble from numerous subunits (Syntichaki et al. 2006; Tisdale 2009). As these

eventsarecoordinatedbyinitiationfactorseIF4EandeIF2B(Syntichakietal.2006),the

mechanismbywhichcatalaseandglucoseoxidaseaffecttheseinitiationfactorsinC2C12

myotubeswasaddressedinChapter5.

63

Chapter 5: Development of methods for the study of signaling pathway on protein synthesis in C2C12 myotubes in response to treatment with catalase and glucose oxidase

5.1 Introduction

Changes in total protein content (Chapter 3) and protein turnover (Chapter 4) were

observed in C2C12 myotubes after catalase and glucose oxidase treatment. The

mechanism that induces these changes inprotein synthesis, however, isnot clearbut

mayinvolvechangestosignalingpathwaysinresponsetohydrogenperoxideexposure.

Previousstudiessuggestthe4EBP1signalingpathwaymaybeinvolvedinmediationof

proteinsynthesis.Whenphosphorylationofeukaryotictranslationinitiationfactor4E‐

bindingprotein1(4EBP1)decreases,activityofeukaryoticinitiationfactor4E(eIF4E)

is suppressed leading to a reduction in protein synthesis. In PC12 cells from

phaeochromocytomaoftheratadrenalmedulla,phosphorylationof4EBP1decreasesin

responsetoupto2mMhydrogenperoxideinadose‐dependentmanner.Moreover,this

reduction can be attenuated by pre‐treating the cells with 5 mM of reactive oxygen

species (ROS) scavenger, N‐acetyl‐cysteine (NAC) (Chen et al. 2010). A decrease in

phosphorylationof4EBP1inresponsetoelevatedlevelofhydrogenperoxidehasalso

been observed in a range of cell types, such as aged muscle cells, human lung

adenocarcinomacells,mouseembryonic fibroblastsandcardiacmyocytes (Pateletal.

2002;Zhangetal.2009;Wuetal.2010;Emaraetal.2012).

Otherstudieshaveshownthatanothersignalingpathway,theeIF2αpathway,mayalso

be involved in the mediation of protein synthesis. When phosphorylation on the α

subunit of eukaryotic initiation factor 2 (eIF2α) increases, activity of the eukaryotic

initiationfactor2B(eIF2B)isattenuatedleadingtoareductioninproteinsynthesis.In

PC12 cells, phosphorylation of eIF2α increases in response to 1‐3 mM of hydrogen

peroxideinadose‐dependentmanner.Moreover,thisinductioncouldbeabolishedby

pre‐treatingthecellswith10mMofNAC(O'Loghlenetal.2003).ThisincreaseineIF2α

phosphorylationhasalsobeenobservedinarangeofcelltypes,suchasyeastcellsand

agedskeletalmuscle(Shenton et al. 2006; Mascarenhas et al. 2008; Wu et al. 2010).

64

Giventheapparentimportanceofhydrogenperoxideinphosphorylationof4EBP1and

eIF2α,thepresentstudysetouttoinvestigatethechangesinphosphorylationof4EBP1

and eIF2α with catalase and glucose oxidase. Methods are developed and used to

determinetheeffectsinC2C12myotubes.

5.2 Methods


Myotubecultures


whichaimedtoestablishwesternblotmethod,twotothreeuntreatedortreatedculture

sampleswereused. Inall subsequentexperiments, twopetridishesper treatment for

every treatment group (untreated, catalase, and glucose oxidase). These experiments

wererepeateduptothreetimesusingfreshC2C12cultures.

Proteinextraction

Phospho‐safeextractioncocktailwasusedtoextractallproteinsandtopreventtheloss

of phosphorylated protein during the process of extraction. To extract proteins from

myotubecultures,thepetridishescontainingthemyotubecultureswereplacedonice

andwashedbrieflytwicewith1mlofcold(4°C)phosphatebufferedsaline(PBS).100µl

of phospho‐safe extraction cocktail was then added to the petri dishes and the cells

wereharvestedwithacellscraper.Thephospho‐safeextractioncocktailandcellswere

transferredtoa1.5mlmicrocentrifugetube.Thetubeswereplacedonicefor20min

andvortexedfor5secevery10min.Thetubeswerethencentrifugedat10000rpmfor

5min(4°C).Themajorityofthesupernatant(96µl)waskeptforelectrophoresis,and

theremainingsupernatantwaskeptforproteinquantification.

Proteinquantification

TheBio‐RadProteinAssayKitwasusedtoquantifytheproteinineachmyotubesample.

Protein sampleswere quantifiedwith reference to a BSA standard absorbance curve.

65

BSAstandardsofknownconcentrations (0,100,200,300,400,and500µg/ml)were

preparedbyserialdilutionofBSA(stockconcentration1mg/ml)in0.01MPBS.

Theprotein sampleswere first diluted20‐foldwith0.01MPBS and then10µl of the

dilutedproteinsampleandstandardsweretransferredintriplicatetothewellsofa96‐

well plate. 200 µl of Bio‐Rad reagent was then added to each well. The plate was

incubatedfor10minwithgentleshakingonashakeratroomtemperatureandatthe

endofthisincubationperiod,theabsorbanceofeachwellwasmeasuredat595nmina

plate reader. After protein quantification, the concentration of each sample was

calculatedandtheproteinswerethenseparatedbyelectrophoresis.

WesternBlot‐SDSPolyacrylamideGelElectrophoresis(SDS‐PAGE)system

Self‐made 12 %, 1.5 mm thick acrylamide gels were used in the preliminary

experiments. 20 µg of the protein sample and 4 µl of Precision Plus Protein™

Kaleidoscope™Standards(Bio‐Rad,161‐0375)wereloadedinthewellsandthesample

wereelectrophoresedforapproximately2.5hrat120Vat4°C.Theseparatedsamples

werethentransferredat4°CfromthegeltoaPVDFmembraneovernightat30mA.

Themembranewas thenwashed briefly twicewithTBS‐T buffer and incubatedwith

blockingbuffer (5% skimmilk inTBS‐T) for1hr at room temperature.Theblocked

membranewas thenwashed twice,10mineach time,withTBS‐Tand incubatedwith

dilutedprimaryantibodyat4°Covernightwithgentleshaking.Themembranewasthen

washed again with TBS‐T twice, 10 min each time, and incubated with diluted

secondary antibody for 1 hr at room temperature with shaking. Themembranewas

then briefly washed twice with TBS‐T and then incubated with chemiluminescence

substratesolutionfor5minatroomtemperature.Thesignalwasexposedtofilmand

thefilmwasthendevelopedinadarkroom.

66

WesternBlot‐Bio‐Radsystem

Precastgradientgels(Bio‐Rad,456‐1086)wereusedinallsubsequentexperiments.10

µgoftheproteinsampleand4µlofPrecisionPlusProtein™Kaleidoscope™Standards

(Bio‐Rad,161‐0375)wereloadedinthewellsandthesamplewereelectrophoresedfor

approximately 1.5 hr at room temperature. The separated samples were then

transferredfor7minfromthegeltoamembrane(Bio‐Rad,170‐4158)withTrans‐Blot®

Turbo™ Transfer System (Bio‐Rad, 170‐4155) on turbo setting. The membrane was

thenwashedbriefly twicewithTBS‐Tbufferand incubatedwithblockingbuffer(5%

skimmilk in TBS‐T) for 1 hr at room temperature. The blockedmembranewas then

washed twice, 10 min each time, with TBS‐T and incubated with diluted primary

antibodyat4°Covernightwithgentleshaking.Themembranewasthenwashedagain

withTBS‐Ttwice,10mineachtime,andincubatedwithdilutedsecondaryantibodyfor

1hratroomtemperaturewithshaking.Themembranewasthenbrieflywashedtwice

withTBS‐Tandthenincubatedwithchemiluminescencesubstratesolutionfor5minat

room temperature. The signal was detected and captured by using ChemiDoc™ MP

System(Bio‐Rad,170‐8280)andImageLab™SoftwareVersion4.0(Bio‐Rad).Imagesof

thesemembraneswerethenanalyzedusingImageJ.

Aktwasusedas the loading control and is commonlyusedbyour laboratory for this

purpose(Tan2013;Tanetal.2015).

Antibodies

The primary antibodies used in this research (p‐eIF2α at Ser51 (5199), p‐4EBP1 at

Thr37/46 (2855), eIF2α (9722), 4EBP1 (9452), andAkt (9272))were obtained from

Cell Signaling (Danvers, U.S.A). All primary antibodieswere diluted 1:1000with 5%

BSAinTBS‐T.Thesecondaryanti‐rabbitantibody(Thermo,31460)wasdiluted1:5000

with5%skimmilkinTBS‐T.

67

5.3 Results

5.3.1 Optimization of method for measuring 4EBP1 phosphorylation in C2C12 myotubes

Tomeasurephosphorylationon4EBP1inC2C12myotubes,westernblottingwasusedto

detect phosphorylated 4EBP1 (p4EBP1) and total 4EBP1 (4EBP1). Preliminary

experimentswereperformedtodeterminetherequiredantibodydilutionfactorsandto

optimizetheblottingmethodforthemyotubes.

20µgofproteinsamples fromthemyotubecultureswereseparatedandthesignalof

p4EBP1 and 4EBP1 was detected using western blot‐SDS‐PAGE system. The dilution

factor of primary antibodies was 1:1000 with 5 % BSA in TBS‐T, and 1:10000 for

secondaryantibodywith5%skimmilk inTBS‐T.Themembranewas incubatedwith

chemiluminescencesubstrate(Millipore,WBLUR0100)for5minatroomtemperature.

Using thisprotocol, thep4EBP1and4EBP1bandswere clearly visible and therewas

little background (Fig. 5.1). As this protocol was shown to be suitable for the

measurement of phosphorylation of 4EBP1 in C2C12 myotubes it was used for all


Figure5.1Detectionofphosphorylated4EBP1andtotal4EBP1Myotubes(7‐day‐old)were left inserum‐starvedconditions(Untreated24hr)orserum‐containedconditions(+2%HS24hr)for24hr.ProteinsampleswerecollectedasdescribedinChapter2.2.2andquantifiedasdescribedinChapter2.3.1.Westernblotwasperformedwith20µgC2C12myotubessamplesasdescribedaboveand60µganimalsample (muscle frommice,obtained fromAssociateProfessorTeaShavlakadze)todetectthesignalofp4EBP1and4EBP1.Theexposuretimeofp4EBP1was1min,and4EBP1was4min.TheprocessofwesternblotwasasdescribedinChapter2.6.1.

p4EBP1

4EBP1

68

5.3.2 Optimization of method for measuring eIF2αphopsphorylation in C2C12 myotubes

TomeasurephosphorylationoneIF2αinC2C12myotubes,westernblottingwasusedto

detect phosphorylated eIF2α (peIF2α) and total eIF2α (eIF2α). As with the previous

4EBP1detectionmethod, theantibodydilution factorsandblottingconditionsneeded

tobeoptimized,soaseriesofpreliminaryexperimentswereconducted.

Usingthesamedilutionfactorsandconditionsfordetectionof4EBP1,westernblotwas

performed for peIF2α and eIF2α. For eIF2α, the bands could be detected using this

protocol (Fig. 5.2). However, these conditions were not suitable for peIF2α as the

backgroundstainingoftheblotobscuredthepeIF2αbands(datanotshown).Toreduce

thebackground stainingof thepeIF2α blot, thepeIF2α primaryantibodywasdiluted

with5%skimmilk inTBS‐T.However, thisdidnot reduce thebackground(datanot

shown).

Figure5.2DetectionoftotaleIF2αMyotubes(7‐day‐old)werecollectedorleftindifferentconditionsthatmyotubeswereincubatedinserum‐starved conditions (Untreated), serum‐starved conditions with catalase (3000 units/ml;+Cat.), serum‐starved conditions with glucose oxidase (10 munits/ml; +GluO.), serum‐containingconditions(+2%HS),orserum‐containingconditionswithcatalase(3000units/ml;+2%HS+Cat.)for48hr.ProteinsampleswerecollectedasdescribedinChapter2.2.2andquantifiedasdescribedinChapter2.3.1.Westernblotwasperformedwith20µgC2C12myotubessamplesasdescribedabovetodetectthesignalofeIF2α.TheeIF2αprimaryantibodywasdiluted1:1000with5%BSAinTBS‐T.Thefilmwasexposedtosignalfor5secanddevelopedfor5sec.TheprocessofwesternblotwasasdescribedinChapter2.6.1.

eIF2α

69

Low affinity between the antibody and peIF2α on the membrane may have been

responsible for the high level of background staining. To increase affinity of this

antibody, the primary antibody was used at a higher concentration by reducing the

dilution factor. The blotting process was repeated with a range of primary antibody

dilutions (1:250 to 1:1000)with 5% skimmilk in TBS‐T. High levels of background

stainingwerestillpresentandthepeIF2αbandswerebarelydistinguishableontheblot

evenatthedilutionfactorof1:250(Fig.5.3).

Figure5.3DetectionofphosphorylatedeIF2αMyotubes(7‐day‐old)werecollectedorleftindifferentconditionsthatmyotubeswereincubatedinserum‐starved conditions (Untreated), serum‐starved conditions with catalase (3000 units/ml;+Cat.), serum‐starved conditions with glucose oxidase (10 munits/ml; +GluO.), serum‐containingconditions(+2%HS),orserum‐containingconditionswithcatalase(3000units/ml;+2%HS+Cat.)for48hr.ProteinsampleswerecollectedasdescribedinChapter2.2.2andquantifiedasdescribedinChapter2.3.1.Westernblotwasperformedwith20µgC2C12myotubessamplesasdescribedabovetodetectthesignalofpeIF2α.ThepeIF2αprimaryantibodywasdiluted1:250with5%skimmilkinTBS‐T.Thefilmwasexposedtosignalfor5minanddevelopedfor5sec.TheprocessofwesternblotwasasdescribedinChapter2.6.1.

To reduce the background staining, 5 % BSA in TBS‐T was used as blocking buffer

insteadof5%skimmilk inTBS‐T.Asshown inFig.5.4, thebackgroundstainingwas

reducedbutwasstillhigh.

peIF2α

70

Figure5.4DetectionofphosphorylatedeIF2αMyotubes(7‐day‐old)werecollectedorleftindifferentconditionsthatmyotubeswereincubatedinserum‐starved conditions (Untreated), serum‐starved conditions with catalase (3000 units/ml;+Cat.), serum‐starved conditions with glucose oxidase (10 munits/ml; +GluO.), serum‐containingconditions(+2%HS),orserum‐containingconditionswithcatalase(3000units/ml;+2%HS+Cat.)for48hr.ProteinsampleswerecollectedasdescribedinChapter2.2.2andquantifiedasdescribedinChapter2.3.1.20µgC2C12myotubessamplesasdescribedabovewereusedtoperformwesternblotasdescribed inChapter2.6.1butwith5%BSA inTBS‐Tasblockingbuffer todetect thesignalofpeIF2α.ThepeIF2αprimaryantibodywasdiluted1:250with5%skimmilkinTBS‐T.Thefilmwasexposedtosignalfor5minanddevelopedfor5sec.

Tosolvethe lowdilutionandbackgroundstainingissue, thepeIF2αprimaryantibody

was replaced with a hypersensitive antibody (5199, Cell Signaling) and the blotting

processassection5.1wasrepeated.Asshown inFig.5.5,using thisantibody,peIF2α

bandswere clear and the background stainingwas greatly reduced.Measurement of

phosphorylation of eIF2α in C2C12myotubes in all subsequent experiments used this

antibodythatdilutedwith5%BSAinTBS‐Tand5%skimmilkinTBS‐Twasusedas

blockingbuffer.

Figure5.5DetectionofphosphorylatedeIF2αMyotubes(7‐day‐old)wereleftuntreatedortreatedwithcatalase(3000units/ml;+Cat.)for48hrinserum‐starved conditions. Protein samples were collected as described in Chapter 2.2.2 andquantified asdescribed inChapter 2.3.1.Westernblotwasperformedwith20µgC2C12myotubessamplesasdescribedabovetodetectthesignalofpeIF2αbyusingChemiDoc™MPSystem(Bio‐Rad,170‐8280)andtheexposuretimewas1sec.TheprocessofwesternblotwasasdescribedinChapter2.6.1.

peIF2α

peIF2α

71

5.3.3 Measuring the rate of phosphorylation on 4EBP1 in C2C12 myotubes

InChapter3and4, thechanges intotalproteincontentandproteinturnoverinC2C12

myotubes in response to catalase and glucose oxidase treatment suggest the

phosphorylation on 4EBP1 may change in response to different levels of hydrogen

peroxide.Totestthis,thesemyotubesweretreatedwithcatalaseandglucoseoxidasein

serum‐starvedconditions for48hrand the levelsofphosphorylated4EBP1, the total

4EBP1, and the total Akt were assessed. The phosphorylation on 4EBP1 was not

significantlychangedineitherthecatalase‐treatedorglucoseoxidase‐treatedmyotubes

(Fig.5.6).

Figure5.64EBP1phosphorylationinC2C12myotubesaftercatalaseandglucoseoxidasetreatment

Myotubes(7‐day‐old)wereleftuntreatedortreatedwithcatalase(3000units/ml;+Cat.),orglucoseoxidase (10 munits/ml; +GluO.) for 48 hr in serum‐starved conditions. Protein samples werecollectedasdescribedinChapter2.2.2,quantifiedasdescribedinChapter2.3.1,andtheprocessofwestern blot was as described in Chapter 2.6.2. (A) Phosphorylated 4EBP1(Thr37/46) and total4EBP1with totalAktusedas the loadingcontrol.Themembrane imagehadbeencroppedtoonlyshow the relevant treatments. (B)Phosphorylation levels.Thisdata representsanaverageof fourexperimentsforeachtreatmentgroup(twodishes/treatmentgroup)andisshownasmean±SEM.

5.3.4 Measuring eIF2α phosphorylation in C2C12 myotubes

As the eIF2α pathway is another pathway that may modulates the rate of protein

synthesis, the myotubes were treated with catalase and glucose oxidase in serum‐

starved conditions for 48 hr, then harvested, and quantified. Western blotting was

undertaken forphosphorylatedeIF2α, totaleIF2α, and totalAkt.Thephosphorylation

0

2

4

6

8

10

12

14

16

18A

B

72

on eIF2αwas significantly increased in these C2C12myotubes in response to catalase

andglucoseoxidasetreatment(Fig.5.7).

Figure5.7eIF2αphosphorylationinC2C12myotubesaftercatalaseandglucoseoxidasetreatment

Myotubes(7‐day‐old)wereleftuntreatedortreatedwithcatalase(3000units/ml;+Cat.),orglucoseoxidase (10 munits/ml; +GluO.) for 48 hr in serum‐starved conditions. Protein samples werecollectedasdescribedinChapter2.2.2,quantifiedasdescribedinChapter2.3.1,andtheprocessofwestern blotwas as described in Chapter 2.6.2. (A) Phosphorylated eIF2α(Ser51) and total eIF2αwith total Akt as the loading control. The membrane image had been cropped to only show therelevanttreatments.(B)Phosphorylationlevels.Thisdatarepresentsanaverageoffourexperimentsforeachtreatmentgroup(twodishes/treatmentgroup)andisshownasmean±SEM.

5.4 Discussion

Although protein synthesis is known to be modulated by signaling pathways, how

catalaseandglucoseoxidasemodulatechangesinproteinsynthesisisunknown.Inthe

present chapter, the impact of catalase and glucose oxidase on signaling pathways in

C2C12myotubeswasexaminedwithwesternblotting.

Phosphorylation of 4EBP1 was not found to be significantly changed by catalase or

glucose oxidase treatment. This suggests that 4EBP1 might not be involved in the

changesinproteinsynthesisinducedbycatalaseandglucoseoxidaseinC2C12myotubes.

This isoncontrasttopreviousstudiesinothercelltypesthathaveshownthat4EBP1

phosphorylation decreases in response to increased hydrogen peroxide (Patel et al.

2002; Zhang et al. 2009; Chen et al. 2010; Wu et al. 2010; Emara et al. 2012).

73

OtherstudiessuggestthattheeIF2αpathwayisresponsibleforthechangesinducedby

hydrogen peroxide in various cells (O'Loghlen et al. 2003; Shenton et al. 2006;

Mascarenhas et al. 2008; Wu et al. 2010). In C2C12 myotubes, we found the rate of

phosphorylation on eIF2α was increased significantly in response to catalase and

glucose oxidase. This may account for the changes in protein synthesis in C2C12

myotubesinresponsetocatalaseandglucoseoxidaseinChapter4.

In addition to the impact that hydrogen peroxide has on protein content, protein

turnoverandsignallingpathways, thisROShasbeenshown toenhanceor inhibit the

formation of disulfide bonds in select proteins in a dose‐dependent manner. These

disulfide bonds are formed while the thiol (–SH) groups of redox sensitive cysteine

residuesareoxidizedand leads to changes inprotein function (Bienert et al. 2006; Rhee

2006),however,howthioloxidationchangesinresponsetocatalaseandglucoseoxidase

treatmentisunknown.InChapter6,toinvestigatechangesinthioloxidation,amethod

tomeasurechangesinthiolgroupsinC2C12myotubesisdeveloped.

74

Chapter 6: Development of a method to measure thiol oxidation in C2C12 myotubes in response to treatment with catalase and glucose oxidase

6.1 Introduction

Changes in total protein content (Chapter 3), protein turnover (Chapter 4) and the

eIF2αpathway(Chapter5)wereobservedinC2C12myotubesaftercatalaseandglucose

oxidasetreatment.Themechanismsthatinducedthesechanges,however,arenotclear

butmayinvolvethioloxidationinresponsivetohydrogenperoxideexposure(Baty et al.

2005).

Thiols contain sulfhydryl (‐SH) groups that are readily oxidized to form stable

disulphide bonds and are important in cellular antioxidant defences and redox

signalling (Mulier et al. 1998; Baty et al. 2005; Terrill et al. 2013). Previous studies suggest

these thiol groups may be oxidized in response to hydrogen peroxide. In Jurkat T‐

lymphocytecells,structuralchangesinsomethiolgroup‐containingproteinshavebeen

observed in response to 200 µM hydrogen peroxide (Baty et al. 2005). Similar results

havebeenfoundinarangeofcells,suchasyeastcells(Delaunay et al. 2000; Imlay 2008).

Giventheapparentimportanceofhydrogenperoxideincellfunction,thepresentstudy

set out to adapt amethod established to investigate changes in the thiol oxidation of

total protein, myosin and actin in muscle tissue (Armstrong et al. 2011) to C2C12

culturedcells.

6.2 Methods


Myotubecultures


which aimed to optimize precipitation efficiencywith ethanol or acetone, onedish of

untreated and catalase‐treated myotubes were used. In all subsequent experiments,

three petri dishes per treatment for every treatment group (untreated, catalase, and

glucose oxidase) were tested. One dish was used for protein quantification and the

75

remainingtwodisheswereusedfor2taglabeling.Theseexperimentswererepeatedup

tofourtimesusingfreshC2C12cultures.

Proteinextraction

Trichloroaceticacid(TCA)wasusedtoprotonateallthiolsandtoprecipitatethecellular

proteins to prevent their subsequent oxidation (Aslund et al. 1999; Delaunay et al. 2000).

To extract proteins from themyotubes, the petri dishes containingmyotube cultures

were washed briefly twice with 1ml phosphate buffered saline (PBS). 20 %

TCA/acetone (w/v) (700 µl) was then added to the petri dishes and the cells were

harvestedwithacellscraper.TheTCA/acetoneandcellsweretransferredtoa1.5ml

microcentrifugetube.Onesamplewasthencentrifugedat10000rpmfor5min(4°C)in

preparation for protein quantification. The supernatantwas then discharged and the

protein pelletwaswashedwith cold acetone (1ml). The centrifugation andwashing

steps were repeated to remove any residual TCA. The protein pellet was then

suspended in Tris buffer (300µl, 50mMTriswith 0.5% SDS, pH7.0) and quantified

usingthemicroBCAassay.Thesamplesfor2taglabelingwereleftinTCA/acetone.

Proteinquantification‐microBCAassay

ThecommercialkitfromSigma(QuantiPro™BCAAssayKit,QPBCA‐1KT)wasusedto

quantifythetotalproteincontentforeachtreatmentgroup.1µloftheproteinsample

wasdiluted25‐foldwithSDSbuffer(0.5%SDS), followedbyanother10‐folddilution

withTrisbuffer(2mMTriswith0.5%SDS,pH7.0)ina1.5mlmicrocentrifugetube.250

µl of the cocktail reagent (A:B:C=25:25:1) was added into each diluted sample and

incubated for1hrat60°C.The incubatedsolution (100µl)was then transferred toa

384‐wellplateandtheabsorbancewasanalyzedat562nminaplatereader(BioTek,

PowerWaveHT).

2taglabeling

76

Samplesfor2taglabelingwereleftin20%TCA/acetone,andsonicatedat40%Amps

for 2 min on ice. 100 µg of the protein pellet was then transferred to a 1.5 ml

microcentrifugetube.TheexcessTCAwasremovedfortheprocessoflabelling.

AfterremovalofTCA,theproteinsampleswerelabelledwithfirsttag,BODIPYFL‐N‐(2‐

aminoethyl)maleimide(FLm,Invitrogen,B10250).Thistaglabelsthereducedformof

inproteinsamples.Toperformthislabelling,50µlofTrisbuffer(0.5MTriswith0.5%

SDS,pH7.3)with5µlofFLm(5mM)wasaddedtotheproteinpellet,whichwasthen

got sonicatedat40%Amps for1minon ice.The resulting labelledsamplewas then

vortexed and incubated at room temperature for 30 min in dark. Excess FLm was

removed by two rounds of precipitation. Each time, 200 µl of ice‐cold acetone was

addedto thesample followedby incubationovernightat ‐20°Candcentrifugation the

followingmorning.Theresultingproteinpelletwas thenresuspended in50µlofTris

buffer(0.5MTriswith0.5%SDS,pH7.0).

Theoxidizedthiolswerethenlabelledwiththesecondtag,Texasredmaleimide(TRm,

Invitrogin,T6008).Toperformthislabelling,21µlofFLm‐labelledproteinsamplewas

then taken andmixed with 4 µl of TCEP (25 mM) in a 0.6 ml microcentrifuge tube,

followedbyincubationatroomtemperaturefor1hrinthedark.Thesampleswerethen

mixedwith25µlTrisbuffer(0.5MTriswith0.5%SDS,pH7.0)and5µlofTRm(5mM),

vortexedbrieflyand incubatedatroomtemperature for1hr in thedark.ExcessTRm

wasremovedbytheapplicationof220µlice‐coldacetoneandincubationovernightat‐

20°C.Theproteinpelletwascentrifuged,resuspendedwith25µlTrisbuffer(0.5MTris

with0.5%SDS,pH7.0)thenprecipitatedagainovernightwith100µlofice‐coldacetone

at‐20°.Thiscentrifugation,resuspension,andprecipitationstepwasrepeatedandthe

resulting2taglabelledproteinpelletwasthenresuspendedin50µlofTrisbuffer(0.5

MTriswith0.5%SDS,pH7.0).

77

Proteinquantification‐DCassay

TheDCassaykit(Bio‐Rad,500‐0112)wasusedtoquantifytheproteincontentofthe2

taglabelledproteinsamples.Trisbuffer(0.25MTris,0.25%SDS,pH7.0)wasusedas

theassaybuffer.7.5µlofeachproteinsamplewasdilutedtwo‐foldwithTrisbuffer(0.5

MTriswith 0.5% SDS, pH7.0) followed by a further two‐fold dilutionwithDDi. The

sampleswerethentreatedaccordingtothekitinstructionsusingthesuppliedworking

reagents. The final product was aliquoted in triplicate (100 µl/well) into a 384‐well

plateandtheabsorbancewasanalyzedat750nminaplatereader(BioTek,PowerWave

HT).

FLmandTRmquantification

The rate of thiol oxidation was measured using a fluorescent assay and gel

electrophoresis.

Theanalysisof fluorescentmeasurementwasstandardizedtoFLmandTRmstandard

curves. The standard curve for FLm was prepared from 0 to 60 µM with 60 µM

FLm/ovalbumin stock solution. For TRm, it was prepared from 0 to 7.5 µM with

TRm/ovalbuminstocksolution.Allpreparedstandardswerediluted10‐foldwith0.1M

NaOHand10µlofproteinsampleswerediluted32‐foldwith0.1MNaOH.Thediluted

standards and protein sampleswere aliquoted in triplicate (100µl/well) into a 384‐

wellplate.Thefluorescenceofeachsamplewasthenmeasuredusingafluorescentplate

reader (Fluostar Optima) with wavelengths set at 485 nm excitation and 520 nm

emissionforFLmand595nmexcitationand610nmemissionforTRm.

ThegelanalysiswasperformedbyelectrophoresiswithprecastgelsfromBio‐Rad.To

quantify the reduced and oxidized thiols of specific protein, in‐gel Flm and TRm

standardswerepreparedas for the fluorescentplate readermeasurement.The in‐gel

FLmstandardswerepreparedfrom0to0.024nmol,and0to0.006nmolforin‐gelTRm

standards. After electrophoresis, the fluorescence of each lanewasmeasured using a

typhoongelscanner(GEHealthcareLifeScience,TyphoonTrio)withwavelengthssetat

520nmforFLmand610nmforTRm.FollowinggelanalysisusingtheImageJsoftware,

78

the amount of reduced and oxidized thiols in specific protein was determined with

referencetotheFLm/TRmstandardcurves.

6.3 Results

6.3.1 Optimize the 2 tag method for C2C12 myotubes model

An existing method developed in our laboratory for 2 tag labelling in muscle tissue

(Armstrong et al. 2011)wasadaptedtosuittheC2C12tissueculturesamples.Thismethod

requires100µgofmyotubeprotein.Whilemusclesamplescanbeeasilyweighed,itis

difficulttoweighmyotubetissueculturesamples.Astheproteinlevelswithinthesame

treatmentgroupswerefoundtobesimilarinpreviousexperiments(seeChapter3),it

wasassumedthatproteinamountswouldbeapproximatelythesamewithinthesame

treatmentgroupsand the samples couldbeweighed inTCA/acetone.Therefore, after

protein quantification of one representative myotube sample, the remainder of the

samplesweresonicatedinTCA/acetoneat40%Ampsfor2minonicethenthevolume

representing100µgofsamplewasaliquotedintoafreshtube.ResidualTCAwasthen

removedfromthis100µgsamplebywashingtwicewith300µlice‐coldacetone.

Accordingtotheoriginalprotocol,theproteinpelletshouldbeprecipitatedrepeatedly

withice‐cold(‐20°C)ethanolafterFLmandTRmlabelling.Totestiftheproteinsamples

fromthemyotubescouldbeprecipitatedusinganethanolsolvent, twoseparated100

µgproteinaliquotsweretakenfromonesample,centrifuged,resuspendedwith50µlof

Trisbuffer(0.5MTriswith0.5%SDS,pH7.3)andthenprecipitatedwitheitherice‐cold

(‐20°C) ethanol or acetone. As shown in Fig. 6.1, therewas a 50%protein losswith

ethanolprecipitationbut little losswithacetoneprecipitation.Therefore,acetonewas

usedforprecipitationinallsubsequentexperiments.Ashigherproteinconcentrations

were believed to increase precipitation efficiency based on precipitation kinetics

(Devidal et al. 1997), the protein pellet was resuspended in lower amounts of Tris

buffer than original protocol recommended. The labelled myotube protein samples

were suspended with 25 µl of Tris buffer during the removal of excess TRm and

resuspendedwith50µlofTrisbufferforthefinalanalysis.

79

Figure6.1Totalproteinlevelsafterprecipitationwithethanolandacetone100µgsamplesfromC2C12myotubesweresuspendedin50µlofTrisbuffer(0.5MTriswith0.5%SDS,pH7.3),sonicated,andprecipitatedwithice‐coldethanolorice‐coldacetoneovernightat‐20°C.ProteincontentwasthenmeasuredusingthemicroBCAassay.

6.3.2 Measuring total thiol oxidation in fluorescently labeled C2C12 myotubes using a fluorescent plate reader

InChapter3 to5, the changes inC2C12myotubes in response to catalase andglucose

oxidasetreatmentsuggestthatthioloxidationmaybechangedinresponsetodifferent

levelsofhydrogenperoxide.Totestthis,thesemyotubesweretreatedwithcatalaseand

glucose oxidase in serum‐starved conditions for 48 hr and the fluorescence of the

labelledsampleswasassessed.Thethioloxidationintotalproteinwasnotsignificantly

changedineitherthecatalase‐treatedorglucoseoxidase‐treatedmyotubes(Fig.6.2).

0

20

40

60

80

100

Ethanol Acetone

Total protein

(µg/sample)

Precipitation solvent

80

Figure6.2ThioloxidationinC2C12myotubesinresponsetocatalaseandglucoseoxidasetreatment

Myotubes(7‐day‐old)wereleftuntreatedortreatedwithcatalase(3000units/ml;+Cat.),orglucoseoxidase (10 munits/ml; +GluO.) for 48 hr in serum‐starved conditions. The fluorescence wasmeasuredafterlabellingthethiolsintheproteinsamples.Thisdatawasanaverageof4experimentsfor each treatment group (2 dishes/treatment group) and was shown as mean ± SEM. Proteinsampleswerecollectedasdescribed inChapter2.2.1and labelledasdescribed inChapter2.7.Thelabelled protein content was quantified as described in Chapter 2.3.3 and fluorescence wasmeasuredasdescribedinChapter2.7.3.

6.3.3 Measuring thiol oxidation in C2C12 myotubes on actin and myosin by gel electrophoresis

Althoughthethioloxidationwasnotsignificantlychangedintotalprotein,itislikelyto

havesignificantchangesinspecificproteinssuchasmyosinandactin.Totestthis,the

labelledproteinsampleswereseparatedbyelectrophoresisandthegelswerescanned

usingatyphoonfluorescencescanner.AsshowninFig.6.3and6.4,thethioloxidations

of myosin and actin were not significantly changed in either the catalase‐treated or

glucoseoxidase‐treatedmyotubes.

26.5

27.0

27.5

28.0

28.5

29.0

29.5

30.0

30.5

31.0

31.5

32.0

Untreated +Cat. +GluO.

Thiol oxidat

ion

(%)

81

Figure6.3ThioloxidationonmyosininC2C12myotubesinresponsetocatalaseandglucoseoxidasetreatment

Myotubes(7‐day‐old)wereleftuntreatedortreatedwithcatalase(3000units/ml;+Cat.),orglucoseoxidase (10 munits/ml; +GluO.) for 48 hr in serum‐starved conditions. Protein samples werecollectedasdescribedinChapter2.2.1andlabelledasdescribedinChapter2.7.3µglabelledproteinwasloadedtothegelandthefluorescencewasthendetectedwithtyphoonfluorescencescanner.(A)Imageof scanned gel. (B)Average thiol oxidationonmyosin of 4 experiments for each treatmentgroup (2 dishes/treatment group) represented asmean ± SEM. The labelled protein contentwasquantified as described in Chapter 2.3.3 and fluorescencewasmeasured as described in Chapter2.7.4

Figure6.4ThioloxidationonactininC2C12myotubesinresponsetocatalaseandglucoseoxidasetreatment

Myotubes(7‐day‐old)wereleftuntreatedortreatedwithcatalase(3000units/ml;+Cat.),orglucoseoxidase (10 munits/ml; +GluO.) for 48 hr in serum‐starved conditions. Protein samples werecollectedasdescribedinChapter2.2.1andlabelledasdescribedinChapter2.7.3µglabelledproteinwasloadedtothegelandthefluorescencewasthendetectedwithtyphoonfluorescencescanner.(A)Imageof scanned gel. (B)Average thiol oxidationonmyosin of 4 experiments for each treatmentgroup (2 dishes/treatment group) represented asmean ± SEM. The labelled protein contentwasquantified as described in Chapter 2.3.3 and fluorescencewasmeasured as described in Chapter2.7.4

BA

0

5

10

15

20

25

30

35

40

45

50


Thiol o

xida

tion

(%)

BA

19.0

19.5

20.0

20.5

21.0

21.5

22.0

22.5

23.0


Thiol o

xida

tion

(%)

82

6.4 Discussion

Afteradaptingamethod tomeasure thiol oxidation inmuscle tissue tomeasure thiol

oxidationinC2C12cellcultures,itwasfoundthattotalthioloxidationandthioloxidation

of myosin and actin were not significantly changed by catalase or glucose oxidase

treatmentof thesemyotubes.This is incontrast topreviousstudiesof increasedthiol

oxidation of proteins from yeast cells and human umbilical vein endothelial cells in

responsetohydrogenperoxidetreatment(Delaunayetal.2000;Imlay2008).However,

anotherstudythatsubsequentlyusedthismethodhasbeenabletodetectanincreasein

thioloxidationinC2C12myotubesinresponsetooxidanttreatmentusingdiamide(Tan

et al. 2015), suggesting this system is capable of measuring thiol oxidation in C2C12

myotubes.

Thedurationofoxidanttreatmentisthoughttobethepossiblereasonfortheabsence

ofthioloxidationobservedinpresentstudy.Inyeastcells,thioloxidationwasobserved

aftertreatingthecellswithhydrogenperoxidefor2.5min,butwasnotevidentafter1

hr hydrogen peroxide treatment (Delaunay et al. 2000). In A548 cells treated with 0.1

mMhydrogen peroxide, an initial decrease in intracellular non‐protein thiols (NPSH)

wasobservedfrom0‐2hrandfollowedbyasubsequentrecoveryby8hrofhydrogen

peroxidetreatment.ThesechangesinNPSHwereattributedtothioloxidationsincethe

levelofNPSHincreasedwithantioxidanttreatment(2mMNAC)forupto2hr(Mulier et

al. 1998).

Inthepresentstudy,themyotubesweretreatedwithcatalaseandglucoseoxidasefora

period of 48 hr to investigate longer term down‐stream effects, like changes in the

signallingpathways.Astheeffectsofthesetreatmentsonthioloxidationmayhavebeen

transient and early in treatment, this study may not have been able to detect them.

StudiesofC57BL/6Jfemalemicehaveshownthatage‐relatedthioloxidationcannotbe

detectedingastrocnemiusmusclesofthesemicefrom3‐29monthsofage(Tohmaetal.

2014),furthersuggestingthattheeffectsofhydrogenperoxideonmuscleproteinthiols

maybyearlyand transientandunlikely tobedetected in longer‐termmodels.Future

studies could focus on assessing thiol oxidation in these myotubes over a range of

shorter,morefrequenttimepoints.

83

Chapter 7: General discussion

7.1 Introduction

Oxidativestressisdefinedasanimbalancebetweenthegenerationofreactiveoxygen

species (ROS) and a reduction in protective mechanism such as antioxidase activity.

This imbalance leads to damage in biomoleculeswith potential impact on thewhole

organism(Reuteretal.2010).ProteinsinparticularareeasilyattackedbyROSresulting

in changes in structure and enzyme activity. Oxidative stress can also affect the

activation of transaction factors, can alter signalling pathways and can damage

membranes(Klaunigetal.1998).

Oxidative stress and oxidative damage to tissues are thought to play a key role in a

range of chronic diseases and conditions including cancer, diabetes, and ageing

(Jefferson1980;Rooyackersetal.1996;Aruoma1998;Wei1998;Baynesetal.1999;

Brownetal.2001;Atalayetal.2002;Ryazanovetal.2002;Martinez‐Vicenteetal.2005;

Valko et al. 2006). In cancer cells, an increase in the generation of ROS enhances

metabolicstressandproliferativecapacity(Chenetal.2008;Reuteretal.2010).Inboth

typesofdiabetes,elevatedglucose levels induce thegenerationofmitochondrialROS,

nonenzymatic glycation of protein, and glucose autooxidation (Evans et al. 2002;

Maritim et al. 2003; Robertson 2004; Rolo et al. 2006; Scheuner et al. 2008). The

generationrateofhydrogenperoxidefrommitochondriaisincreased(Sohaletal.1996;

Wei et al. 2002) inducing the accumulation of irreversibly modified proteins

(Tavernarakis 2008). These oxidized proteins have been implicated in a number of

agingprocessesanddiseases,mostnotablyAlzheimer’sdisease(Klaunigetal.2010).

Inthisstudy,theresponsesofC2C12myotubestocatalaseandglucoseoxidasetreatment

wereassessed to investigate themechanismsofmusclewasting inducedbyoxidative

stress.

7.2 Muscle wasting

Musclewasting involves lossofmuscleproteinmass and function.Cachexia iswidely

recognized as severe wasting accompanying disease states. Up to 80% of advanced

cancer patients have cachexia and 20 % of cancer‐related deaths are thought to be

linkedtothiscondition(Glassetal.2010;Tazietal.2010;Mathew2011;Silverioetal.

84

2011;Wang et al. 2011;Wysong et al. 2011). In elderlywithundiagnosed/diagnosed

diabetes,declines inmusclemassandtotalbodymasshavealsobeenassociatedwith

cachexia(Parketal.2007;Parketal.2009).Sarcopenia,isthelossofmusclemasswith

ageingandcausesdiminishedstrengthandexercisecapacity (Phillips et al. 2005; Lenk et

al. 2010).

Sinceoxidativestressisthoughttoinducetheselossesinmusclemass(Lassetal.1998;

Mantovanietal.2002;Mantovanietal.2002;Capeletal.2005;Mansourietal.2006;

Lenketal.2010;Silverioetal.2011),itwashypothesizedthatthetotalproteincontent

in C2C12myotubeswould change in response to changes in hydrogen peroxide levels

modulatedbycatalaseandglucoseoxidasetreatment.Overall,thetotalproteincontent

was increasedwith catalase treatment for 72 hr and decreasedwith glucose oxidase

treatment for 48 hr (Fig. 3.6). These findings validated the use of C2C12model for

investigatingthemechanismsthatunderlymyotubeproteinresponsestocatalaseand

glucoseoxidasetreatment.

7.3 Protein turnover

Animbalancebetweenproteinsynthesisandproteindegradationleadstoadecreasein

totalproteincontentandresultsinmusclewasting(Balagopaletal.1997;Evans2010).

Incachexiapatients,abnormalitiesinproteinmetabolismhavebeenlinkedtodecreases

in protein synthesis and increases in protein degradation in skeletalmuscle (Tisdale

2001).Otherstudiesofcancerpatientswithcachexiahavefoundthatproteinsynthesis

issignificantlydecreased(Emeryetal.1984;Smithetal.1999)andthishasalsobeen

observedindiabetespatientsandelderlypatientswithmusclewasting(Millwardetal.

1976;Jefferson1980;Gelfandetal.1987;Rooyackersetal.1996;Balagopaletal.1997;

Anthony et al. 2002). Increases in protein degradation are also observed in cachexia

patientswithcancerordiabetes(Gelfandetal.1987;Smithetal.1999;Tisdale2001;

Bachetal.2005),andactinandmyosinhavebeenfoundtobeselectivelytargetedfor

degradationincachexiapatients(Evans2010).

Basedon theseprevious studiesand the changesof totalproteincontentobserved in

this study in response tocatalaseandglucoseoxidase treatment, itwashypothesized

that catalasewould increaseprotein synthesis anddecreaseproteindegradation, and

glucoseoxidasewoulddecreaseproteinsynthesisandincreaseproteindegradationin

85

C2C12myotubes.However,asdescribedinChapter4,thelevelofproteinsynthesiswas

significantlydecreasedinresponsetocatalasetreatmentandtherewaslittlechangein

proteinsynthesisafterglucoseoxidasetreatment(Fig.4.4).Inaddition,whiletherateof

proteindegradationwassignificantlydecreasedinresponsetocatalasetreatment,there

waslittlechangeafterglucoseoxidasetreatment(Fig.4.5).

7.4 Signalling pathways

Previousstudieshaveshownthattheregulationofproteinsynthesisinskeletalmuscle

occurs primary at the initiation phase of protein translation and the initiation factor

eIF4E and eIF2 are involved in this regulation (Syntichaki et al. 2006; Tisdale 2009).

eIF4E, as described in Chapter 1, regulates protein synthesis via its reversible

associationwith4E‐bindingproteins,including4EBP1,throughthemTORpathway.The

protein synthesis is increased when 4EBP1 is phosphorylated, and a decrease in

phosphorylationof4EBP1hasbeen linked toageing, canceranddiabetes (Shahet al.

2000;Syntichaki et al.2006;Armengol et al. 2007;Eleyet al. 2007;Drummondet al.

2008;Handsetal.2009).

Basedonthesestudies,thechangesintotalprotein(Chapter3)andinproteinsynthesis

(Chapter4)observedinthisstudy,itwashypothesizedthatphosphorylationof4EBP1

would decreasewith both catalase and glucose oxidase treatment in C2C12myotubes.

However, phosphorylation of 4EBP1 was not found to be significantly changed with

eithercatalaseorglucoseoxidasetreatment(Fig.5.6),suggestingthat4EBP1mightnot

beinvolvedinproteinsynthesischangesinresponsetocatalaseandglucoseoxidase.

eIF2,asdescribedinChapter1,decreasesproteinphosphorylationbyphosphorylation

of its α subunit through the PERK/eIF2α pathway. As a link between eIF2α

phosphorylationandweightlosshasbeenobservedincanceranddiabetesstudies(Kim

etal.2000;Hardingetal.2001;Ozcanetal.2004;Eleyetal.2007;Scheuneretal.2008),

itwashypothesizedthateIF2αphosphorylationinC2C12myotubeswouldincreaseafter

catalase and glucose oxidase treatment. As expected, increases in eIF2α

phosphorylationwereobservedforbothtreatments(Fig.5.7),suggestingthateIF2α is

involvedintheregulationofproteinsynthesisinC2C12myotubes.

86

7.5 Thiol oxidation

Proteins can respond to ROS in different ways, including the formation of disulfide

bonds from thiol groups containing cysteine residues (Atalay et al. 2002; Poon et al.

2004;Valkoetal.2006).The formationof thesedisulfidebondsare thought toplaya

key role in cancer (Toyokuni et al. 1995;Klaunig et al. 1998;Kumar et al. 2008) and

bothtypesofdiabetes(Maritimetal.2003;Robertson2004;Roloetal.2006).

As significant changes on level of total protein content, protein turnover, and eIF2α

pathwaywereobservedinC2C12myotubesinresponsetocatalaseandglucoseoxidase

treatmentinthepresentstudy,itwashypothesizedthatthioloxidationwoulddecrease

with catalase treatment and increasewith glucose oxidase treatment. However, thiol

oxidation was not found to be significantly changed by either catalase or glucose

oxidase. Subsequent studies undertaken in this laboratory support this observation

(Tohmaetal.2014;Tanetal.2015).

7.6 Future studies

Overall, thisstudyshowsthattheC2C12myotubemodelcanbeusedtoinvestigatethe

relationship between oxidative stress andmuscle wasting. Changes in protein levels,

protein synthesis, protein degradation and eIF2α phosphorylation were observed in

responsetocatalaseandglucoseoxidasetreatmentinthesemyotubes,butnochanges

werefoundinthioloxidationinthecurrentstudy.Theextendeddurationoftreatment,

chosentoexplorethelong‐termdown‐streameffectsofchangesinhydrogenperoxide

levels, might be the reason why thiol oxidation was not detected. Further studies

focused on earlier and more frequent sampling of the cultures may give a better

indicationoftheroleofthioloxidationinthisprocess.

While it is unknown if exogenous enzymes can be taken up by C2C12 myotubes, the

changes observed in total protein levels, protein turnover and signalling pathway

observed in this study suggest glucose oxidase and catalase mediate these changes,

probablyaftercellularuptakefromthemedia.Futurestudiescouldalsobeundertaken

tocompareintracellularandextracellularlevelsofhydrogenperoxidefollowingglucose

oxidase and catalase treatment to confirm the changes observed were a result of

intracellularhydrogenperoxideproduction(Beersetal.1952;Picketal.1980;Picket

87

al. 1981). Hydrogen peroxide could potentially be used as a control, although direct

application is likely to cause significant cell death. Alternatively, uptake of these

enzymescouldbemonitoredbypackagingtheminfluorescentlylabellednanoparticles

suchasliposomes.However,previousresearchinourlaboratoryandothershasshown

the activity of enzymesdecreases in a time‐dependentmanner (Tse et al. 1987;Kho

2010). Therefore, in addition of being time‐consuming and difficult, the expected

reduction in enzyme activity may also make the results of this potential approach

difficulttointerpret.

Further studiesmay also focuson theotherpossibleprotein target sites thatmaybe

affectedbyoxidativestress,suchasthebackboneandothersidechains.Ultimately,itis

hoped that a better understanding of the mechanisms underlying muscle wasting

inducedbyoxidativestresswillleadtonewtherapiesformusclewasting.

88

Bibliography

Anthony, J.C.,A.K.Reiter,T.G.Anthony, S. J. Crozier, C.H. Lang,D.A.MacLean, S.R.

Kimball and L. S. Jefferson (2002). "Orally administered leucine enhances protein

synthesis in skeletalmuscle of diabetic rats in the absence of increases in 4E‐BP1 or

S6K1phosphorylation."Diabetes51(4):928‐936.

Arkov, A. L., D. V. Freistroffer, M. Ehrenberg and E. J. Murgola (1998). "Mutations in

RNAsofbothribosomalsubunitscausedefects intranslationtermination."TheEMBO

Journal17(5):1507‐1514.

Armengol,G., F.Rojo, J. Castellvi, C. Iglesias,M. Cuatrecasas,B. Pons, J. Baselga andS.

RamonyCajal(2007)."4E‐bindingprotein1:akeymolecular"funnelfactor"inhuman

cancerwithclinicalimplications."CancerResarch67(16):7551‐7555.

Armstrong,A.E.,R.Zerbes,P.A.FournierandP.G.Arthur(2011)."Afluorescentdual

labeling technique for the quantitativemeasurement of reduced andoxidizedprotein

thiolsintissuesamples."Freeradicalbiology&medicine50(4):510‐517.

Arthur,P.G.,M.D.GroundsandS.Tea(2008)."Oxidativestressasatherapeutictarget

during muscle wasting: considering the complex interactions." Current Opinion in

ClinicalNutrition&MetabolicCare11(4):408‐416.

Aruoma,O. (1998). "Free radicals, oxidative stress, and antioxidants in humanhealth

anddisease."JournaloftheAmericanOilChemists'Society75(2):199‐212.

Ashford, T. P. and K. R. Porter (1962). "Cytoplasmic components in hepatic cell

lysosomes."TheJournalofCellBiology12:198‐202.

Aslund, F., M. Zheng, J. Beckwith and G. Storz (1999). "Regulation of the OxyR

transcription factor by hydrogen peroxide and the cellular thiol‐disulfide status."

Proceedings of the National Academy of Sciences of the United States of America

96(11):6161‐6165.

89

Atalay,M.andD.E.Laaksonen(2002)."Diabetes,oxidativestressandphysicalexercise."

JournalofSportsScienceandMedicine1(1):1‐14.

Aulino,P.,E.Berardi,V.M.Cardillo,E.Rizzuto,B.Perniconi,C.Ramina,F.Padula,E.P.

Spugnini, A. Baldi, F. Faiola, S. Adamo and D. Coletti (2010). "Molecular, cellular and

physiological characterizationof thecancercachexia‐inducingC26coloncarcinoma in

mouse."BMCCancer10:363.

Bach,D.,D.Naon,S.Pich,F.X.Soriano,N.Vega,J.Rieusset,M.Laville,C.Guillet,Y.Boirie,

H.Wallberg‐Henriksson,M.Manco,M.Calvani,M.Castagneto,M.Palacin,G.Mingrone,J.

R. Zierath, H. Vidal and A. Zorzano (2005). "Expression of Mfn2, the Charcot‐Marie‐

Toothneuropathy type2Agene, inhuman skeletalmuscle: effectsof type2diabetes,

obesity, weight loss, and the regulatory role of tumor necrosis factor alpha and

interleukin‐6."Diabetes54(9):2685‐2693.

Back, S. H., D. Scheuner, J. Han, B. Song, M. Ribick, J. Wang, R. D. Gildersleeve, S.

Pennathur and R. J. Kaufman (2009). "Translation Attenuation through eIF2α

PhosphorylationPreventsOxidativeStressandMaintains theDifferentiatedState inβ

Cells."Cellmetabolism10(1):13‐26.

Bähler,M.(2000)."AreclassIIIandclassIXmyosinsmotorizedsignallingmolecules?"

BiochimicaetBiophysicaActa(BBA)‐MolecularCellResearch1496(1):52‐59.

Balagopal,P.,O.E.Rooyackers,D.B.Adey,P.A.AdesandK.S.Nair(1997)."Effectsof

aging on in vivo synthesis of skeletal muscle myosin heavy‐chain and sarcoplasmic

proteininhumans."TheAmericanJornalofPhysiology273(4Pt1):E790‐800.

Barbieri,E.andP.Sestili(2012)."Reactiveoxygenspeciesinskeletalmusclesignaling."

JournalofSignalTransduction2012:982794.

Bassell,G.andR.H.Singer(1997)."mRNAandcytoskeletalfilaments."Currentopinion

incellbiology9(1):109‐115.

90

Baty, J. W., M. B. Hampton and C. C. Winterbourn (2005). "Proteomic detection of

hydrogen peroxide‐sensitive thiol proteins in Jurkat cells." The Biochemical Journal

389(Pt3):785‐795.

Baynes,J.W.andS.R.Thorpe(1999)."Roleofoxidativestressindiabeticcomplications:

anewperspectiveonanoldparadigm."Diabetes48(1):1‐9.

Beers,R.F.,Jr.andI.W.Sizer(1952)."Aspectrophotometricmethodformeasuringthe

breakdown of hydrogen peroxide by catalase." The Journal of Biological Chemistry

195(1):133‐140.

Berg,J.S.,B.C.PowellandR.E.Cheney(2001)."Amillennialmyosincensus."Molecular

BiologyofTheCell12(4):780‐794.

Bertram, G., S. Innes, O. Minella, J. Richardson and I. Stansfield (2001). "Endless

possibilities:translationterminationandstopcodonrecognition."Microbiology147(Pt

2):255‐269.

Bienert,G.P.,J.K.SchjoerringandT.P.Jahn(2006)."Membranetransportofhydrogen

peroxide."Biochimicaetbiophysicaacta1758(8):994‐1003.

Bonetto,A.,F.Penna,M.Muscaritoli,V.G.Minero,F.RossiFanelli,F.M.BaccinoandP.

Costelli (2009). "Are antioxidants useful for treating skeletal muscle atrophy?" Free

radicalbiology&medicine47(7):906‐916.

Boveris, A., N. Oshino and B. Chance (1972). "The cellular production of hydrogen

peroxide."TheBiochemicalJournal128(3):617‐630.

Bradford,M.M.(1976)."Arapidandsensitivemethodforthequantitationofmicrogram

quantities of protein utilizing the principle of protein‐dye binding." Analytical

Biochemistry72:248‐254.

Brink,M., S. R. Price, J. Chrast, J. L. Bailey, A. Anwar,W. E.Mitch and P. Delafontaine

(2001). "Angiotensin II Induces Skeletal Muscle Wasting through Enhanced Protein

Degradation and Down‐Regulates Autocrine Insulin‐Like Growth Factor I."

Endocrinology142(4):1489‐1496.

91

Brown, N. S. and R. Bicknell (2001). "Hypoxia and oxidative stress in breast cancer.

Oxidativestress:itseffectsonthegrowth,metastaticpotentialandresponsetotherapy

ofbreastcancer."BreastCancerResearch3(5):323‐327.

Capel,F.,L.Demaison,F.Maskouri,A.Diot,C.Buffiere,P.PatureauMirandandL.Mosoni

(2005)."Calciumoverloadincreasesoxidativestressinoldratgastrocnemiusmuscle."

JournalofPhysiologyandPharmacology56(3):369‐380.

Casey, T. M., J. L. Pakay, M. Guppy and P. G. Arthur (2002). "Hypoxia causes

downregulation of protein and RNA synthesis in noncontracting Mammalian

cardiomyocytes."CirculationResearch90(7):777‐783.

Caskey,C.T.,A.L.Beaudet,E.M.ScolnickandM.Rosman(1971)."Hydrolysisof fMet‐

tRNAbypeptidyltransferase."ProceedingsoftheNationalAcademyofSciences68(12):

3163‐3167.

Catalgol,B.andT.Grune(2009)."Turnoverofoxidativelymodifiedproteins:theusage

ofinvitroandmetaboliclabeling."FreeRadicalBiologyandMedicine46(1):8‐13.

Chang,Y.C.andL.M.Chuang(2010)."Theroleofoxidativestressinthepathogenesisof

type2diabetes:frommolecularmechanismtoclinicalimplication."Americanjournalof

TranslationalResearch2(3):316‐331.

Chen, L., B. Xu, L. Liu, Y. Luo, J. Yin, H. Zhou,W. Chen, T. Shen, X. Han and S. Huang

(2010). "Hydrogen peroxide inhibits mTOR signaling by activation of AMPKalpha

leadingtoapoptosisofneuronalcells."Laboratoryinvestigation;a journaloftechnical

methodsandpathology90(5):762‐773.

Chen, Y., J. Ke, X. Long, Q. Meng, M. Deng, W. Fang, J. Li, H. Cai and S. Chen (2012).

"Insulin‐likegrowthfactor‐1booststhedevelopingprocessofcondylarhyperplasiaby

stimulatingchondrocytesproliferation."OsteoarthritisandCartilage20(4):279‐287.

Chen,Y.,E.McMillan‐Ward,J.Kong,S.J.IsraelsandS.B.Gibson(2008)."Oxidativestress

induces autophagic cell death independent of apoptosis in transformed and cancer

cells."Celldeathanddifferentiation15(1):171‐182.

92

Ciechanover, A. (2005). "Proteolysis: from the lysosome to ubiquitin and the

proteasome."Naturereviews.Molecularcellbiology6(1):79‐87.

Ciechanover,A. (2010). "Intracellularproteindegradation: fromavague idea through

thelysosomeandtheubiquitin‐proteasomesystemandontohumandiseasesanddrug

targeting."Medicina(BAires)70(2):105‐119.

Ciechanover,A.(2012)."IntracellularProteinDegradation:FromaVagueIdeathrough

the Lysosome and the Ubiquitin‐Proteasome System and onto Human Diseases and

DrugTargeting."RambamMaimonidesMedicalJournal3(1):e0001.

Ciechanover,A.,H.Heller, S.Elias,A.L.HaasandA.Hershko (1980). "ATP‐dependent

conjugation of reticulocyte proteins with the polypeptide required for protein

degradation."ProceedingsoftheNationalAcademyofSciences77(3):1365‐1368.

Circu,M.L.andT.Y.Aw(2010)."Reactiveoxygenspecies,cellularredoxsystems,and

apoptosis."FreeRadicalBiology&Medicine48(6):749‐762.

Clemmons, D. R. (2012). "Metabolic actions of insulin‐like growth factor‐I in normal

physiology and diabetes." Endocrinology and metabolism clinics of North America

41(2):425‐443.

Coletti, D. (2013). "RESEARCH INTERESTS: Cellular and molecular mechanisms

underlyingmusclewasting."fromhttp://dariocolettithescientist.blogspot.tw/.

Cullinan,S.B.andJ.A.Diehl(2006)."CoordinationofERandoxidativestresssignaling:

the PERK/Nrf2 signaling pathway." The International Journal of Biochemistry & Cell

Biology38(3):317‐332.

DangDo,A.N.,S.R.Kimball,D.R.CavenerandL.S.Jefferson(2009)."eIF2alphakinases

GCN2 and PERKmodulate transcription and translation of distinct sets of mRNAs in

mouseliver."PhysiologicalGenomics38(3):328‐341.

Day, B. J., I. Fridovich and J. D. Crapo (1997). "Manganic porphyrins possess catalase

activity and protect endothelial cells against hydrogen peroxide‐mediated injury."

ArchivesofBiochemistryandBiophysics347(2):256‐262.

93

Delaunay,A.,A.D.IsnardandM.B.Toledano(2000)."H2O2sensingthroughoxidation

oftheYap1transcriptionfactor."TheEMBOJournal19(19):5157‐5166.

Deng,J.,H.P.Harding,B.Raught,A.C.Gingras,J.J.Berlanga,D.Scheuner,R.J.Kaufman,

D. Ron and N. Sonenberg (2002). "Activation of GCN2 in UV‐irradiated cells inhibits

translation."CurrentBiology:CB12(15):1279‐1286.

Devidal, J.‐L., J.SchottandJ.‐L.Dandurand(1997)."Anexperimentalstudyofkaolinite

dissolution and precipitation kinetics as a function of chemical affinity and solution

composition at 150°C, 40 bars, and pH 2, 6.8, and 7.8." Geochimica et Cosmochimica

Acta61(24):5165‐5186.

Dodson, S., V. E. Baracos, A. Jatoi,W. J. Evans, D. Cella, J. T. Dalton andM. S. Steiner

(2011). "Muscle wasting in cancer cachexia: clinical implications, diagnosis, and

emergingtreatmentstrategies."AnnualReviewofMedicine62:265‐279.

Doria, E., D. Buonocore, A. Focarelli and F. Marzatico (2012). "Relationship between

human agingmuscle andoxidative systempathway."OxidativeMedicine andCellular

Longevity2012:830257.

Drummond,M.J.,H.C.Dreyer,B.Pennings,C.S.Fry,S.Dhanani,E.L.Dillon,M.Sheffield‐

Moore,E.VolpiandB.B.Rasmussen(2008)."Skeletalmuscleproteinanabolicresponse

to resistance exercise and essential amino acids is delayed with aging." Journal of

AppliedPhysiology104(5):1452‐1461.

Duncan,R.,S.C.MilburnandJ.W.Hershey(1987)."Regulatedphosphorylationandlow

abundanceofHeLa cell initiation factor eIF‐4F suggest a role in translational control.

HeatshockeffectsoneIF‐4F."TheJournalofBiologicalChemistry262(1):380‐388.

Eddins, M. J., J. G. Marblestone, K. G. Suresh Kumar, C. A. Leach, D. E. Sterner, M. R.

MatternandB.Nicholson (2011). "Targeting theubiquitinE3 ligaseMuRF1 to inhibit

muscleatrophy."CellBiochemistryandBiophysics60(1‐2):113‐118.

Eley,H.L.,S.T.RussellandM.J.Tisdale(2007)."Effectofbranched‐chainaminoacids

onmuscleatrophyincancercachexia."TheBiochemicalJournal407(1):113‐120.

94

Eley,H.L.andM.J.Tisdale(2007)."SkeletalMuscleAtrophy,aLinkbetweenDepression

of Protein Synthesis and Increase in Degradation." Journal of Biological Chemistry

282(10):7087‐7097

Emara, M. M., K. Fujimura, D. Sciaranghella, V. Ivanova, P. Ivanov and P. Anderson

(2012)."HydrogenperoxideinducesstressgranuleformationindependentofeIF2alpha

phosphorylation."BiochemicalandBiophysicalResearchCommunications423(4):763‐

769.

Emery,P.W.,R.H.Edwards,M.J.Rennie,R.L.SouhamiandD.Halliday(1984)."Protein

synthesisinmusclemeasuredinvivoincachecticpatientswithcancer."BritishMedical

Journal(ClinicalResearchEd.)289(6445):584‐586.

Evans,J.L.,I.D.Goldfine,B.A.MadduxandG.M.Grodsky(2002)."Oxidativestressand

stress‐activated signaling pathways: a unifying hypothesis of type 2 diabetes."

Endocrinereviews23(5):599‐622.

Evans, W. J. (2010). "Skeletal muscle loss: cachexia, sarcopenia, and inactivity." The

AmericanJournalofClinicalNutrition91(4):1123S‐1127S.

Finkel, T. and N. J. Holbrook (2000). "Oxidants, oxidative stress and the biology of

ageing."Nature408(6809):239‐247.

Fligiel,S.E.,E.C.Lee,J.P.McCoy,K.J.JohnsonandJ.Varani(1984)."Proteindegradation

following treatmentwith hydrogenperoxide."American Journal of Pathology115(3):

418‐425.

Fraser,C.S.,K.E.Berry,J.W.B.HersheyandJ.A.Doudna(2007)."eIF3jIsLocatedinthe

DecodingCenteroftheHuman40SRibosomalSubunit."MolecularCell26(6):811‐819.

Fushman,D.andO.Walker(2010)."ExploringtheLinkageDependenceofPolyubiquitin

ConformationsUsingMolecularModeling." Journal ofMolecularBiology395(4): 803‐

814.

Gebski,B.(2009).InvestigatingTNFinhibitionofIGF‐1signallingviaJNKincellculture

modelsofskeletalmuscleatrophy.PhD,TheUniversityofWesternAustralia.

95

Gelfand,R.A.andE.J.Barrett(1987)."Effectofphysiologichyperinsulinemiaonskeletal

muscleproteinsynthesisandbreakdowninman."TheJournalofClinicalInvestigation

80(1):1‐6.

Gillies, A. R. and R. L. Lieber (2011). "Structure and function of the skeletal muscle

extracellularmatrix."Muscle&Nerve44(3):318‐331.

Glass, D. and R. Roubenoff (2010). "Recent advances in the biology and therapy of

musclewasting."AnnalsoftheNewYorkAcademyofSciences1211:25‐36.

Glickman, M. H. and A. Ciechanover (2002). "The ubiquitin‐proteasome proteolytic

pathway:destruction for thesakeof construction."PhysiologicalReviews82(2):373‐

428.

Grune, T., T. Reinheckel andK. J. Davies (1997). "Degradation of oxidized proteins in

mammaliancells."TheFASEBJournal11(7):526‐534.

Grune,T.,T.Reinheckel,M. JoshiandK. J.Davies(1995)."Proteolysis inculturedliver

epithelial cells during oxidative stress. Role of themulticatalytic proteinase complex,

proteasome."JournalofBiologicalChemistry270(5):2344‐2351.

Guarnier,F.A.,A.L.Cecchini,A.A.Suzukawa,A.L.Maragno,A.N.Simao,M.D.Gomes

andR.Cecchini(2010)."Timecourseofskeletalmusclelossandoxidativestressinrats

withWalker256solidtumor."Muscle&Nerve42(6):950‐958.

Guertin,D.A.andD.M.Sabatini(2007)."DefiningtheroleofmTORincancer."Cancer

Cell12(1):9‐22.

Halliwell,B.,M.V.Clement, J.RamalingamandL.H.Long(2000)."Hydrogenperoxide.

Ubiquitousincellcultureandinvivo?"IUBMBLife50(4‐5):251‐257.

Halliwell, B. andC. E. Cross (1994). "Oxygen‐derived species: their relation to human

diseaseandenvironmentalstress."EnvironmentalHealthPerspectives102(Suppl10):

5‐12.

96

Hands, S. L., C. G. Proud and A.Wyttenbach (2009). "mTOR's role in ageing: protein

synthesisorautophagy?"Aging(AlbanyNY)1(7):586‐597.

Harding,H.P.,H.Zeng,Y.Zhang,R.Jungries,P.Chung,H.Plesken,D.D.SabatiniandD.

Ron (2001). "DiabetesMellitus and Exocrine PancreaticDysfunction in Perk−/−Mice

RevealsaRoleforTranslationalControlinSecretoryCellSurvival."MolecularCell7(6):

1153‐1163.

Harding,H.P.,Y.Zhang,H.Zeng,I.Novoa,P.D.Lu,M.Calfon,N.Sadri,C.Yun,B.Popko,R.

Paules,D.F.Stojdl,J.C.Bell,T.Hettmann,J.M.LeidenandD.Ron(2003)."Anintegrated

stress response regulates amino acidmetabolism and resistance to oxidative stress."

Molecularcell11(3):619‐633.

Heinemeyer,W.,M.Fischer,T.Krimmer,U.StachonandD.H.Wolf(1997)."Theactive

sites of the eukaryotic 20 S proteasome and their involvement in subunit precursor

processing."TheJournalofBiologicalChemistry272(40):25200‐25209.

Hershko,A.,A.Ciechanover,H.Heller,A.L.HaasandI.A.Rose(1980)."Proposedroleof

ATP in protein breakdown: conjugation of protein with multiple chains of the

polypeptide of ATP‐dependent proteolysis." Proceedings of the National Academy of

Sciences77(4):1783‐1786.

Hodge,T.andM.J.Cope(2000)."Amyosinfamilytree."JournalofCellScience113Pt

19:3353‐3354.

Imlay, J. A. (2008). "Cellular defenses against superoxide and hydrogen peroxide."

AnnualReviewofBiochemistry77:755‐776.

IvyRose. (2003). "Structure of Muscle and associated connective tissues." from

http://www.ivyroses.com/HumanBody/Muscles/Muscle_Structure.php.

Jackman, R. W. and S. C. Kandarian (2004). "The molecular basis of skeletal muscle

atrophy."AmericanJournalofPhysiology.CellPhysiology287(4):C834‐843.

97

Jackson, R. J., C. U. Hellen and T. V. Pestova (2010). "The mechanism of eukaryotic

translation initiation and principles of its regulation."Nature Reviews.Molecular Cell

Biology11(2):113‐127.

Jefferson, L. S. (1980). "Lilly Lecture1979: role of insulin in the regulationofprotein

synthesis."Diabetes29(6):487‐496.

Jones,P.andA.Suggett(1968)."Thecatalase‐hydrogenperoxidesystem.Atheoretical

appraisalof themechanismof catalaseaction."TheBiochemical Journal110(4):621‐

629.

Kapp,L.D.andJ.R.Lorsch(2004)."Themolecularmechanicsofeukaryotictranslation."

AnnualReviewofBiochemistry73:657‐704.

Keeling, K. M. and D. M. Bedwell (2011). "Suppression of nonsense mutations as a

therapeutic approach to treatgeneticdiseases."Wiley InterdisciplinaryReviews:RNA

2(6):837‐852.

Keilin, D. and E. F. Hartree (1938). "On the Mechanism of the Decomposition of

HydrogenPeroxidebyCatalase."ProceedingsofTheRoyalSocietyofLondon.SeriesB,

BiologicalSciences124(837):397‐405.

Khaitlina,S.Y.(2001)."FunctionalSpecificityofActinIsoforms."InternationalReviewof

Cytology202:35‐98.

Khal,J.,S.M.Wyke,S.T.Russell,A.V.HineandM.J.Tisdale(2005)."Expressionofthe

ubiquitin‐proteasome pathway and muscle loss in experimental cancer cachexia."

BritishJournalofCancer93(7):774‐780.

Kho,S.(2010).HowDoesOxidativeStressAffectProteinLossinSarcopenia?Honours,

TheUniversityofWesternAustralia.

Kim,S.H.,A.P.Forman,M.B.MathewsandS.Gunnery(2000). "Humanbreastcancer

cellscontainelevatedlevelsandactivityoftheproteinkinase,PKR."Oncogene19(27):

3086‐3094.

98

Kimball,S.R.andL.S.Jefferson(2006)."Newfunctionsforaminoacids:effectsongene

transcriptionandtranslation."TheAmericanJournalofClinicalNutrition83(2):500S‐

507S.

Kislinger,T.,A.O.Gramolini,Y.Pan,K.Rahman,D.H.MacLennanandA.Emili(2005).

"Proteome dynamics during C2C12 myoblast differentiation." Molecular & Cellular

Proteomics4(7):887‐901.

Klaunig,J.E.,L.M.KamendulisandB.A.Hocevar(2010)."Oxidativestressandoxidative

damageincarcinogenesis."ToxicologicPathology38(1):96‐109.

Klaunig,J.E.,Y.Xu,J.S.Isenberg,S.Bachowski,K.L.Kolaja,J.Jiang,D.E.Stevensonand

E. F. Walborg, Jr. (1998). "The role of oxidative stress in chemical carcinogenesis."

EnvironmentalHealthPerspectives106Suppl1:289‐295.

Ko,J.A.,Y.Kimura,K.Matsuura,H.Yamamoto,T.GondoandM.Inui(2006)."PDZRN3

(LNX3, SEMCAP3) is required for the differentiation of C2C12 myoblasts into

myotubes."JournalofCellScience119(Pt24):5106‐5113.

Koopman, R. and L. J. van Loon (2009). "Aging, exercise, and muscle protein

metabolism."JournalofAppliedPhysiology(Bethesda,Md.:1985)106(6):2040‐2048.

Korolchuk,V.I.,F.M.MenziesandD.C.Rubinsztein(2010)."Mechanismsofcross‐talk

between the ubiquitin‐proteasome and autophagy‐lysosome systems." FEBS Letters

584(7):1393‐1398.

Kozak,M.(1991)."StructuralfeaturesineukaryoticmRNAsthatmodulatetheinitiation

oftranslation."JournalofBiologicalChemistry266(30):19867‐19870.

Kumar,B.,S.Koul,L.Khandrika,R.B.MeachamandH.K.Koul(2008)."OxidativeStress

IsInherentinProstateCancerCellsandIsRequiredforAggressivePhenotype."Cancer

Research68(6):1777‐1785.

Lass, A., B. H. Sohal, R. Weindruch, M. J. Forster and R. S. Sohal (1998). "Caloric

restriction prevents age‐associated accrual of oxidative damage to mouse skeletal

musclemitochondria."FreeRadicalBiology&Medicine25(9):1089‐1097.

99

Lecker, S. H., A. L. Goldberg and W. E. Mitch (2006). "Protein degradation by the

ubiquitin‐proteasomepathway innormalanddiseasestates." Journalof theAmerican

SocietyofNephrology17(7):1807‐1819.

Lenk, K., G. Schuler and V. Adams (2010). "Skeletal muscle wasting in cachexia and

sarcopenia: molecular pathophysiology and impact of exercise training." Journal of

Cachexia,SarcopeniaandMuscle1(1):9‐21.

Lenk, K., G. Schuler and V. Adams (2010). "Skeletal muscle wasting in cachexia and

sarcopenia: molecular pathophysiology and impact of exercise training." J Cachexia

SarcopeniaMuscle1(1):9‐21.

Li, B. G., P. O. Hasselgren, C. H. Fang and G. D. Warden (2004). "Insulin‐like growth

factor‐I blocks dexamethasone‐induced protein degradation in culturedmyotubes by

inhibitingmultipleproteolyticpathways:2002ABApaper."TheJournalofBurnCare&

Rehabilitation25(1):112‐118.

Li,Y.P.,Y.Chen,J.John,J.Moylan,B.Jin,D.L.MannandM.B.Reid(2005)."TNF‐alpha

acts via p38MAPK to stimulate expressionof the ubiquitin ligase atrogin1/MAFbx in

skeletalmuscle."TheFASEBJournal19(3):362‐370.

Li,Y.P.,Y.Chen,A.S.LiandM.B.Reid(2003)."Hydrogenperoxidestimulatesubiquitin‐

conjugatingactivityandexpressionofgenesforspecificE2andE3proteinsinskeletal

musclemyotubes."Americanjournalofphysiology.Cellphysiology285(4):C806‐812.

Li,Y.P.andM.B.Reid(2000)."NF‐kappaBmediatestheproteinlossinducedbyTNF‐

alpha in differentiated skeletal muscle myotubes." American Journal of Physiology ‐

Regulatory,IntegrativeandComparativePhysiology279(4):R1165‐1170.

Liang, S. H., W. Zhang, B. C. McGrath, P. Zhang and D. R. Cavener (2006). "PERK

(eIF2alpha kinase) is required to activate the stress‐activatedMAPKs and induce the

expressionofimmediate‐earlygenesupondisruptionofERcalciumhomoeostasis."The

BiochemicalJournal393(Pt1):201‐209.

Ma,X.M.andJ.Blenis(2009)."MolecularmechanismsofmTOR‐mediatedtranslational

control."NatureReviewsMolecularCellBiology10(5):307‐318.

100

Mansouri, A., F. L.Muller, Y. Liu, R. Ng, J. Faulkner,M. Hamilton, A. Richardson, T. T.

Huang,C.J.EpsteinandH.VanRemmen(2006)."Alterationsinmitochondrialfunction,

hydrogen peroxide release and oxidative damage inmouse hind‐limb skeletalmuscle

duringaging."MechanismsofAgeingandDevelopment127(3):298‐306.

Mantovani,G.,A.Maccio,C.Madeddu,L.Mura,G.Gramignano,M.R.Lusso,C.Mulas,M.

C.Mudu,V.Murgia,P.Camboni,E.Massa,L.Ferreli,P.Contu,A.Rinaldi,E.Sanjust,D.

Atzei and B. Elsener (2002). "Quantitative evaluation of oxidative stress, chronic

inflammatory indices and leptin in cancer patients: correlation with stage and

performancestatus."InternationalJournalofCancer98(1):84‐91.

Mantovani,G.,A.Maccio,C.Madeddu,L.Mura,E.Massa,G.Gramignano,M.R.Lusso,V.

Murgia, P. Camboni and L. Ferreli (2002). "Reactive oxygen species, antioxidant

mechanisms and serum cytokine levels in cancer patients: impact of an antioxidant

treatment."JournalofCellularandMolecularMedicine6(4):570‐582.

Maritim,A.C.,R.A. Sandersand J.B.Watkins,3rd (2003). "Diabetes,oxidative stress,

andantioxidants:areview."JournalofBiochemicalandMolecularToxicology17(1):24‐

38.

Martinez‐Vicente, M., G. Sovak and A. M. Cuervo (2005). "Protein degradation and

aging."ExperimentalGerontology40(8‐9):622‐633.

Mascarenhas,C.,L.C.Edwards‐Ingram,L.Zeef,D.Shenton,M.P.AsheandC.M.Grant

(2008). "Gcn4 is required for the response to peroxide stress in the yeast

Saccharomycescerevisiae."MolecularBiologyofTheCell19(7):2995‐3007.

Mathew, S. J. (2011). "InACTIVatINg cancer cachexia." DiseaseModels &Mechanisms

4(3):283‐285.

McGilchrist,P.,D.W.Pethick,S.P.F.Bonny,P.L.GreenwoodandG.E.Gardner(2011).

"Whole body insulin responsiveness is higher in beef steers selected for increased

muscling."Animal5(10):1579‐1586.

MedicaLook(2012).SKELETALMUSCLESANATOMY.

101

Millward,D. J.,P. J.Garlick,D.O.Nnanyelugoand J.C.Waterlow(1976). "Therelative

importance of muscle protein synthesis and breakdown in the regulation of muscle

mass."BiochemicalJournal156(1):185‐188.

Montesano, A., L. Luzi, P. Senesi, N. Mazzocchi and I. Terruzzi (2013). "Resveratrol

promotesmyogenesisandhypertrophy inmurinemyoblasts." JournalofTranslational

Medicine11:310.

Moore,P.B.andT.A.Steitz(2003)."Aftertheribosomestructures:howdoespeptidyl

transferasework?"RNA9(2):155‐159.

Mortimore,G.E.andA.R.Poso(1987)."Intracellularproteincatabolismanditscontrol

duringnutrientdeprivationandsupply."AnnualReviewofNutrition7:539‐564.

Moylan, J. S. and M. B. Reid (2007). "Oxidative stress, chronic disease, and muscle

wasting."Muscle&Nerve35(4):411‐429.

Mulier,B., I.Rahman,T.Watchorn,K.Donaldson,W.MacNeeandP.K. Jeffery (1998).

"Hydrogen peroxide‐induced epithelial injury: the protective role of intracellular

nonproteinthiols(NPSH)."EuropeanRespiratoryJournal11(2):384‐391.

Muller, F. L.,W. Song, Y. Liu, A. Chaudhuri, S. Pieke‐Dahl, R. Strong, T. T. Huang, C. J.

Epstein,L.J.Roberts,2nd,M.Csete,J.A.FaulknerandH.VanRemmen(2006)."Absence

ofCuZnsuperoxidedismutaseleadstoelevatedoxidativestressandaccelerationofage‐

dependent skeletal muscle atrophy." Free Radical Biology & Medicine 40(11): 1993‐

2004.

Nandi, D., P. Tahiliani, A. Kumar and D. Chandu (2006). "The ubiquitin‐proteasome

system."JournalofBiosciences31(1):137‐155.

Neal,R.,K.Cooper,H.GurerandN.Ercal (1998). "EffectsofN‐acetylcysteineand2,3‐

dimercaptosuccinic acid on lead induced oxidative stress in rat lenses." Toxicology

130(2‐3):167‐174.

O'Loghlen, A., M. I. Perez‐Morgado, M. Salinas and M. E. Martin (2003). "Reversible

inhibitionof theproteinphosphatase1byhydrogenperoxide. Potential regulationof

102

eIF2alphaphosphorylationindifferentiatedPC12cells."ArchivesofBiochemistryand

Biophysics417(2):194‐202.

Ogle, J. M., D. E. Brodersen, W. M. Clemons, Jr., M. J. Tarry, A. P. Carter and V.

Ramakrishnan (2001). "Recognition of cognate transfer RNA by the 30S ribosomal

subunit."Science292(5518):897‐902.

Orr, W. C. and R. S. Sohal (1994). "Extension of life‐span by overexpression of

superoxide dismutase and catalase in Drosophila melanogaster." Science 263(5150):

1128‐1130.

Orzechowski, A., J. Grizard, Jank Micha, B. Gajkowska, M. Lokociejewska, M. Zaron‐

TeperekandM.Godlewskia(2002)."Dexamethasone‐mediatedregulationofdeathand

differentiation of muscle cells. Is hydrogen peroxide involved in the process?"

ReproductionNutritionDevelopment42(3):197‐216.

Ozcan,U.,Q.Cao,E.Yilmaz,A.H.Lee,N.N.Iwakoshi,E.Ozdelen,G.Tuncman,C.Gorgun,

L.H.GlimcherandG.S.Hotamisligil(2004)."Endoplasmicreticulumstresslinksobesity,

insulinaction,andtype2diabetes."Science306(5695):457‐461.

Palus,S.,S.vonHaehlingandJ.Springer(2014)."Musclewasting:anoverviewofrecent

developmentsinbasicresearch."JournalofCachexia,SarcopeniaandMuscle5(3):193‐

198.

Park,S.W.,B.H.Goodpaster, J.S.Lee,L.H.Kuller,R.Boudreau,N.deRekeneire,T.B.

Harris, S. Kritchevsky, F. A. Tylavsky,M.Nevitt, Y.W. Cho andA. B. Newman (2009).

"Excessive lossofskeletalmusclemass inolderadultswithtype2diabetes."Diabetes

Care32(11):1993‐1997.

Park,S.W.,B.H.Goodpaster,E.S.Strotmeyer,L.H.Kuller,R.Broudeau,C.Kammerer,N.

deRekeneire,T.B.Harris,A.V.Schwartz,F.A.Tylavsky,Y.W.ChoandA.B.Newman

(2007). "Accelerated loss of skeletal muscle strength in older adults with type 2

diabetes: the health, aging, andbody composition study."Diabetes Care30(6): 1507‐

1512.

103

Patel,S.,J.VanDerKaayandC.Sutherland(2002)."Insulinregulationofhepaticinsulin‐

likegrowthfactor‐bindingprotein‐1(IGFBP‐1)geneexpressionandmammaliantarget

of rapamycin (mTOR) signalling is impaired by the presence of hydrogen peroxide."


Paulsen,C.E.andK.S.Carroll(2010)."Orchestratingredoxsignalingnetworksthrough

regulatorycysteineswitches."ACSChemicalBiology5(1):47‐62.

Penna, F., D. Costamagna, A. Fanzani, G. Bonelli, F.M. Baccino and P. Costelli (2010).

"MusclewastingandimpairedmyogenesisintumorbearingmicearepreventedbyERK

inhibition."PloSone5(10):e13604.

Peske, F., A. Savelsbergh, V. I. Katunin, M. V. Rodnina and W. Wintermeyer (2004).

"Conformational changes of the small ribosomal subunit during elongation factor G‐

dependent tRNA‐mRNA translocation." Journal of Molecular Biology 343(5): 1183‐

1194.

Pestova,T.V.andV.G.Kolupaeva(2002)."Therolesofindividualeukaryotictranslation

initiation factors in ribosomal scanning and initiation codon selection." Genes &

Development16(22):2906‐2922.

Pham,F.H.,P.H.SugdenandA.Clerk(2000)."RegulationofproteinkinaseBand4E‐

BP1byoxidativestressincardiacmyocytes."CirculationResearch86(12):1252‐1258.

Phillips,T.andC.Leeuwenburgh(2005)."MusclefiberspecificapoptosisandTNF‐alpha

signaling in sarcopenia are attenuated by life‐long calorie restriction." FASEB Journal

19(6):668‐670.

Pick,E. andY.Keisari (1980). "A simple colorimetricmethod for themeasurementof

hydrogen peroxide produced by cells in culture." Journal of lmmunological Methods

38(1‐2):161‐170.

Pick,E.andD.Mizel(1981)."Rapidmicroassaysforthemeasurementofsuperoxideand

hydrogenperoxideproductionbymacrophages incultureusinganautomaticenzyme

immunoassayreader."JournaloflmmunologicalMethods46(2):211‐226.

104

Pickart, C.M. andM. J. Eddins (2004). "Ubiquitin: structures, functions,mechanisms."

BiochimicaetBiophysicaActa1695(1‐3):55‐72.

Pisarev, A. V., V. G. Kolupaeva, V. P. Pisareva,W. C.Merrick, C. U. T. Hellen and T. V.

Pestova (2006). "Specific functional interactions of nucleotides at key ‐3 and +4

positions flanking the initiation codon with components of the mammalian 48S

translationinitiationcomplex."Genes&Development20(5):624‐636.

Pollard, J. (1996). Radioisotopic Labeling of Proteins for Polyacrylamide Gel

Electrophoresis.TheProteinProtocolsHandbook.J.Walker,HumanaPress:121‐126.

Poon,H.F.,V.Calabrese,G.ScapagniniandD.A.Butterfield(2004)."Freeradicals:keyto

brainagingandhemeoxygenaseasacellularresponsetooxidativestress."TheJournals

ofGerontology.SeriesA,BiologicalSciencesandMedicalSciences59(5):478‐493.

Powers, S. K., A. J. Smuder and D. S. Criswell (2011). "Mechanistic Links Between

OxidativeStressandDisuseMuscleAtrophy."Criswell.Antioxidants&RedoxSignaling

15(9):2519‐2528.

Powley,I.R.,A.Kondrashov,L.A.Young,H.C.Dobbyn,K.Hill,I.G.Cannell,M.Stoneley,

Y.‐W. Kong, J. A. Cotes, G. C.M. Smith, R.Wek, C. Hayes, T.W. Gant, K. A. Spriggs,M.

BushellandA.E.Willis(2009)."TranslationalreprogrammingfollowingUVBirradiation

ismediatedbyDNA‐PKcsandallowsselectiverecruitmenttothepolysomesofmRNAs

encodingDNArepairenzymes."Genes&Development23(10):1207‐1220

Ramakrishnan,V.(2002)."Ribosomestructureandthemechanismoftranslation."Cell

108(4):557‐572.

Ratan, R. R., T. H. Murphy and J. M. Baraban (1994). "Macromolecular synthesis

inhibitorspreventoxidativestress‐inducedapoptosisinembryoniccorticalneuronsby

shuntingcysteinefromproteinsynthesistoglutathione."JournalofNeuroscience14(7):

4385‐4392.

Reinheckel, T., T. Grune and K. A. Davies (2000). The Measurement of Protein

Degradation inResponse toOxidativeStress. StressResponse. J.WalkerandS.Keyse,

HumanaPress.99:49‐60.

105

Reuter, S., S. C. Gupta,M.M. Chaturvedi andB. B. Aggarwal (2010). "Oxidative stress,

inflammation, and cancer: how are they linked?" Free Radical Biology & Medicine

49(11):1603‐1616.

Rhee, S. G. (2006). "Cell signaling. H2O2, a necessary evil for cell signaling." Science

312(5782):1882‐1883.

Richfield, D. (2014). Medical gallery of David Richfield 2014. Wikiversity Journal of

Medicine.1.

Robertson,R. P. (2004). "Chronic oxidative stress as a centralmechanism for glucose

toxicity in pancreatic islet beta cells in diabetes." The Journal of Biological Chemistry

279(41):42351‐42354.

Rodnina,M. V. andW.Wintermeyer (2001). "Fidelity of aminoacyl‐tRNA selection on

theribosome:kineticandstructuralmechanisms."AnnualReviewofBiochemistry70:

415‐435.

Rolo, A. P. and C. M. Palmeira (2006). "Diabetes and mitochondrial function: role of

hyperglycemia and oxidative stress." Toxicology and Applied Pharmacology 212(2):

167‐178.

Rooyackers,O.E.,D.B.Adey,P.A.AdesandK.S.Nair(1996)."Effectofageoninvivo

ratesofmitochondrialproteinsynthesisinhumanskeletalmuscle."Proceedingsofthe

NationalAcademyofSciences93(26):15364‐15369.

Rubenstein,P.A.and J.A.Spudich(1977). "Actinmicroheterogeneity inchickembryo

fibroblasts."ProceedingsoftheNationalAcademyofSciences74(1):120‐123.

Rutkowski, D. T. and R. J. Kaufman (2007). "That which does not kill me makes me

stronger:adaptingtochronicERstress."Trends inBiochemicalSciences32(10):469‐

476.

Ryazanov,A. G. andB. S.Nefsky (2002). "Protein turnover plays a key role in aging."

MechanismsofAgeingandDevelopment123(2‐3):207‐213.

106

Sacheck, J. M., A. Ohtsuka, S. C. McLary and A. L. Goldberg (2004). "IGF‐I stimulates

muscle growth by suppressing protein breakdown and expression of atrophy‐related

ubiquitinligases,atrogin‐1andMuRF1."AmericanJournalofPhysiology.Endocrinology

andMetabolism287(4):E591‐601.

Sakuma, K., W. Aoi and A. Yamaguchi (2015). "Current understanding of sarcopenia:

possible candidates modulatingmuscle mass." Pflügers Archiv ‐ European Journal of

Physiology467(2):213‐229.

Sakuma, K. and A. Yamaguchi (2012). "Sarcopenia and cachexia: the adaptations of

negative regulators of skeletal muscle mass." Journal of Cachexia, Sarcopenia and

Muscle3(2):77‐94.

Saladin,K.(2011).Anatomy&Physiology:TheUnityofFormandFunction.

Salas‐Marco,J.andD.M.Bedwell(2004)."GTPhydrolysisbyeRF3facilitatesstopcodon

decoding during eukaryotic translation termination." Molecular and Cellular Biology

24(17):7769‐7778.

Salazar, J. J.andB.VanHouten(1997)."PreferentialmitochondrialDNAinjurycaused

byglucoseoxidaseasasteadygeneratorofhydrogenperoxide inhuman fibroblasts."

MutationResearch385(2):139‐149.

Sandri,M.,C.Sandri,A.Gilbert,C.Skurk,E.Calabria,A.Picard,K.Walsh,S.Schiaffino,S.

H. Lecker and A. L. Goldberg (2004). "Foxo transcription factors induce the atrophy‐

relatedubiquitinligaseatrogin‐1andcauseskeletalmuscleatrophy."Cell117(3):399‐

412.

Scheuner, D. and R. J. Kaufman (2008). "The Unfolded Protein Response: A Pathway

ThatLinksInsulinDemandwithβ‐CellFailureandDiabetes."EndocrineReviews29(3):

317‐333.

Schroder,M.andR.J.Kaufman(2005)."ERstressandtheunfoldedproteinresponse."

MutationResearch569(1‐2):29‐63.

107

Seit‐Nebi, A., L. Frolova, J. Justesen and L. Kisselev (2001). "Class‐1 translation

termination factors: invariant GGQ minidomain is essential for release activity and

ribosomebindingbutnot forstopcodonrecognition."NucleicAcidsResearch29(19):

3982‐3987.

Sengupta,S.,T.R.PetersonandD.M.Sabatini(2010)."RegulationofthemTORcomplex

1pathwaybynutrients,growthfactors,andstress."MolecularCell40(2):310‐322.

Shah, O. J., J. C. Anthony, S. R. Kimball and L. S. Jefferson (2000). "4E‐BP1 and S6K1:

translational integration sites for nutritional and hormonal information in muscle."

AmericanJournalofPhysiology‐EndocrinologyandMetabolism279(4):E715‐729.

Shavlakadze, T. and M. Grounds (2006). "Of bears, frogs, meat, mice and men:

complexity of factors affecting skeletalmusclemass and fat." BioEssays28(10): 994‐

1009.

Shenton,D.andC.M.Grant(2003)."ProteinS‐thiolationtargetsglycolysisandprotein

synthesis in response to oxidative stress in the yeast Saccharomyces cerevisiae."


Shenton,D., J.B.Smirnova, J.N.Selley,K.Carroll,S. J.Hubbard,G.D.Pavitt,M.P.Ashe

and C. M. Grant (2006). "Global Translational Responses to Oxidative Stress Impact

upon Multiple Levels of Protein Synthesis." Journal of Biological Chemistry 281(39):

29011‐29021.

Silverio,R.,A.Laviano,F.RossiFanelliandM.Seelaender(2011)."l‐carnitineandcancer

cachexia:Clinicalandexperimentalaspects."JournalofCachexia,SarcopeniaandMuscle

2(1):37‐44.

Siwik,D.A.,P. J.PaganoandW.S.Colucci (2001). "Oxidativestressregulatescollagen

synthesis and matrix metalloproteinase activity in cardiac fibroblasts." American

JournalofPhysiology‐CellPhysiology280(1):C53‐60.

Smith,H. J.,M. J.LoriteandM.J.Tisdale(1999)."Effectofacancercachecticfactoron

protein synthesis/degradation in murine C2C12 myoblasts: modulation by

eicosapentaenoicacid."CancerResearch59(21):5507‐5513.

108

Sohal,R.S.andR.Weindruch(1996). "Oxidativestress,caloricrestriction,andaging."

Science273(5271):59‐63.

Song,H.,P.Mugnier,A.K.Das,H.M.Webb,D.R.Evans,M.F.Tuite,B.A.Hemmingsand

D. Barford (2000). "The crystal structure of human eukaryotic release factor eRF1‐‐

mechanismofstopcodonrecognitionandpeptidyl‐tRNAhydrolysis."Cell100(3):311‐

321.

Spahn,C.M.T.,R.Beckmann,N.Eswar,P.A.Penczek,A.Sali,G.n.Blobeland J.Frank

(2001)."Structureofthe80SRibosomefromSaccharomycescerevisiaetRNA‐Ribosome

andSubunit‐SubunitInteractions."Cell107(3):373‐386.

Spriggs, K. A., M. Bushell and A. E. Willis (2010). "Translational regulation of gene

expressionduringconditionsofcellstress."MolecularCell40(2):228‐237.

Starkebaum,G.andJ.M.Harlan(1986)."Endothelialcellinjuryduetocopper‐catalyzed

hydrogenperoxidegenerationfromhomocysteine."TheJournalofClinicalInvestigation

77(4):1370‐1376.

Storti, R. V., D. M. Coen and A. Rich (1976). "Tissue‐specific forms of actin in the

developingchick."Cell8(4):521‐527.

Storti,R.V.andA.Rich(1976)."Chickcytoplasmicactinandmuscleactinhavedifferent

structuralgenes."ProceedingsofTheNationalAcademyofSciences73(7):2346‐2350.

Syntichaki, P. and N. Tavernarakis (2006). "Signaling pathways regulating protein

synthesisduringageing."ExperimentalGerontology41(10):1020‐1025.

Tan, P. (2013). The Effects of Reactive Oxygen Species (ROS) on Insulin‐like Growth

Factor1(IGF‐1)Signaling:ImplicationinAgeRelatedSkeletalMuscleWasting.PhD,The

UniversityofWesternAustralia.

Tan, P. L., T. Shavlakadze, M. D. Grounds and P. G. Arthur (2015). "Differential thiol

oxidation of the signaling proteins Akt, PTEN or PP2A determines whether Akt

phosphorylation is enhanced or inhibited by oxidative stress in C2C12 myotubes

109

derivedfromskeletalmuscle."TheInternationalJournalofBiochemistry&CellBiology

62:72‐79.

Tavernarakis, N. (2008). "Ageing and the regulation of protein synthesis: a balancing

act?"TrendsinCellBiology18(5):228‐235.

Tazi,E.andH.Errihani(2010)."Treatmentofcachexia inoncology." IndianJournalof

PalliativeCare16(3):129‐137.

Terai, K., Y. Hiramoto, M. Masaki, S. Sugiyama, T. Kuroda, M. Hori, I. Kawase and H.

Hirota(2005)."AMP‐ActivatedProteinKinaseProtectsCardiomyocytesagainstHypoxic

Injury through Attenuation of Endoplasmic Reticulum Stress."Molecular and Cellular

Biology25(21):9554‐9575.

Terrill, J. R., H. G. Radley‐Crabb, T. Iwasaki, F. A. Lemckert, P. G. Arthur and M. D.

Grounds (2013). "Oxidative stress and pathology in muscular dystrophies: focus on

proteinthioloxidationanddysferlinopathies."FEBSJournal280(17):4149‐4164.

Thomas,D.R.(2007)."Lossofskeletalmusclemassinaging:examiningtherelationship

ofstarvation,sarcopeniaandcachexia."ClinicalNutrition26(4):389‐399.

Tidball, J. G. (2005). "Inflammatory processes inmuscle injury and repair." American

Journal of Physiology. Regulatory, Integrative and Comparative Physiology 288(2):

R345‐353.

Tisdale, M. J. (2001). "Loss of skeletal muscle in cancer: biochemical mechanisms."

FrontiersinBioscience6:D164‐174.

Tisdale, M. J. (2009). "Mechanisms of cancer cachexia." Physiological Reviews 89(2):

381‐410.

Tohma, H., A. F. El‐Shafey, K. Croft, T. Shavlakadze, M. D. Grounds and P. G. Arthur

(2014). "Protein thiol oxidation does not change in skeletal muscles of aging female

mice."Biogerontology15(1):87‐98.

110

Toyokuni, S., K. Okamoto, J. Yodoi and H. Hiai (1995). "Persistent oxidative stress in

cancer."FEBSLetters358(1):1‐3.

Tse,P.H.andD.A.Gough(1987)."Time‐dependentinactivationofimmobilizedglucose

oxidaseandcatalase."BiotechnologyandBioengineering29(6):705‐713.

Unbehaun, A., S. I. Borukhov, C. U. T. Hellen and T. V. Pestova (2004). "Release of

initiation factors from 48S complexes during ribosomal subunit joining and the link

betweenestablishmentofcodon‐anticodonbase‐pairingandhydrolysisofeIF2‐bound

GTP."Genes&Development18(24):3078‐3093.

Valko,M.,C.J.Rhodes,J.Moncol,M.IzakovicandM.Mazur(2006)."Freeradicals,metals

and antioxidants in oxidative stress‐induced cancer." Chemico‐Biological Interactions

160(1):1‐40.

Veal, E. A., A. M. Day and B. A. Morgan (2007). "Hydrogen peroxide sensing and

signaling."MolecularCell26(1):1‐14.

Wang,H., Y. J. Lai, Y. L. Chan, T. L. Li and C. J.Wu (2011). "Epigallocatechin‐3‐gallate

effectively attenuates skeletal muscle atrophy caused by cancer cachexia." Cancer

Letters305(1):40‐49.

Wei,Y.H.(1998)."OxidativestressandmitochondrialDNAmutationsinhumanaging."

ProceedingsofTheSocietyforExperimentalBiologyandMedicine217(1):53‐63.

Wei, Y. H. and H. C. Lee (2002). "Oxidative stress, mitochondrial DNAmutation, and

impairment of antioxidant enzymes in aging." Experimental Biology and Medicine

227(9):671‐682.

Weiss,S.J.,J.Young,A.F.LoBuglio,A.SlivkaandN.F.Nimeh(1981)."Roleofhydrogen

peroxideinneutrophil‐mediateddestructionofculturedendothelialcells."TheJournal

ofClinicalInvestigation68(3):714‐721.

Whalen, R. G., G. S. Butler‐Browne and F. Gros (1976). "Protein synthesis and actin

heterogeneity in calfmuscle cells in culture."Proceedingsof theNationalAcademyof

Sciences73(6):2018‐2022.

111

White, J. P.,K.A.Baltgalvis,M. J. Puppa, S. Sato, J.W.Baynes and J.A. Carson (2011).

"MuscleoxidativecapacityduringIL‐6‐dependentcancercachexia."AmericanJournalof

Physiology.Regulatory,IntegrativeandComparativePhysiology300(2):R201‐211.

Wu,M.,H.Liu,J.Fannin,A.Katta,Y.Wang,R.K.Arvapalli,S.Paturi,S.K.Karkala,K.M.

RiceandE.R.Blough(2010)."Acetaminophenimprovesproteintranslationalsignaling

inagedskeletalmuscle."RejuvenationResearch13(5):571‐579.

Wysong, A., M. Couch, S. Shadfar, L. Li, J. E. Rodriguez, S. Asher, X. Yin, M. Gore, A.

Baldwin, C. Patterson andM. S.Willis (2011). "NF‐kappaB inhibition protects against

tumor‐induced cardiac atrophy in vivo." The American Journal of Pathology 178(3):

1059‐1068.

Yaffe, D. and O. Saxel (1977). "Serial passaging and differentiation of myogenic cells

isolatedfromdystrophicmousemuscle."Nature270(5639):725‐727.

Yu,Y.,A.Marintchev,V.G.Kolupaeva,A.Unbehaun,T.Veryasova,S.C.Lai,P.Hong,G.

Wagner, C. U. Hellen and T. V. Pestova (2009). "Position of eukaryotic translation

initiation factor eIF1A on the 40S ribosomal subunit mapped by directed hydroxyl

radicalprobing."NucleicAcidsResearch37(15):5167‐5182.

Zavialov,A.V.,L.Mora,R.H.BuckinghamandM.Ehrenberg(2002)."Releaseofpeptide

promotedby theGGQmotif of class1 release factors regulates theGTPaseactivityof

RF3."MolecularCell10(4):789‐798.

Zhang,L.,S.R.Kimball,L.S.JeffersonandJ.S.Shenberger(2009)."Hydrogenperoxide

impairs insulin‐stimulated assembly of mTORC1." Free Radical Biology & Medicine

46(11):1500‐1509.

Zhao, J., J. J. Brault, A. Schild, P. Cao, M. Sandri, S. Schiaffino, S. H. Lecker and A. L.

Goldberg (2007). "FoxO3 coordinately activates protein degradation by the

autophagic/lysosomal and proteasomal pathways in atrophying muscle cells." Cell

Metabolism6(6):472‐483.

Zhou,X.,J.L.Wang,J.Lu,Y.Song,K.S.Kwak,Q.Jiao,R.Rosenfeld,Q.Chen,T.Boone,W.

S.Simonet,D.L.Lacey,A.L.GoldbergandH.Q.Han(2010)."ReversalofCancerCachexia

112

andMuscleWastingbyActRIIBAntagonismLeadstoProlongedSurvival."Cell142(4):

531‐543.

Zhouravleva,G.,L.Frolova,X.LeGoff,R.LeGuellec,S.Inge‐Vechtomov,L.Kisselevand

M. Philippe (1995). "Termination of translation in eukaryotes is governed by two

interacting polypeptide chain release factors, eRF1 and eRF3." The EMBO Journal

14(16):4065‐4072.

Zoncu,R.,A.EfeyanandD.M.Sabatini(2011)."mTOR:fromgrowthsignalintegrationto

cancer,diabetesandageing."NatureReviewsMolecularCellBiology12(1):21‐35.

Zurlo,F.,K.Larson,C.BogardusandE.Ravussin(1990)."Skeletalmusclemetabolismis

a major determinant of resting energy expenditure." The Journal of Clinical

Investigation86:1423‐1427.

113

Appendices

Posterpresentedat21stAnnualCombinedBiologicalSciencesMeetings,TheUniversity

Club, University of Western Australia, 26th August 2011.

114

PosterpresentedatJointAuPS/ASCEPT/HBPRCAMeeting,PerthConventionExhibition

Centre(PCEC),4th‐7thDecember2011.

115

Poster presented at Development, Function and Repair of the Muscle Cell, Kimmel

Center, New York University, New York, NY, USA, 4th ‐8th June 2012.

mechanics of oxidative stress and protein in c2c12 myotubes · alex armstrong, jessica terrill,...

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