Redox homeostasis in theEndoplasmic Reticulum

Dmitri FomenkoDmitri Fomenko

Redox Biology CenterRedox Biology CenterUniversity of Nebraska University of Nebraska -- LincolnLincoln

Dmitri FomenkoDmitri Fomenko

Endoplasmic reticulum

The endoplasmic reticulum (ER) is organelle thatforms an interconnected network of tubules, vesicles,and cisternae within cells.

Rough endoplasmic reticulum is responsible forproteins synthesis.

Smooth endoplasmic reticulum is responsible forsynthesize lipids and steroids, metabolizecarbohydrates, regulate calcium concentration and drugdetoxification.detoxification.

ER is a major place of protein folding in the cell

ER retention C-terminal peptides KDEL/HDEL

Outline

1. Oxidative protein folding

2. Redox control of protein glycosylation

3. Glycoprotein folding quality control – link to redox processes 3. Glycoprotein folding quality control – link to redox processes

4. Redox control of ER to cytosol protein translocation

The Saccharomyces cerevisiae thiol redoxome. This thiol redoxome has 45 proteins

Homo sapiens Thiol Redoxome (135 proteins Cys-110 Sec-25)

Protein Number Localization Phosducin 6

Cytosol (42) Cys (34) Sec (8)

Arsenic methyltransferase 1

Glutathione S-transferase 2

MoeB-like 5

Peroxiredoxin 3

Rhodanese-like 1

Methionine sulfoxide reductase A 1

Gutaredoxin-like 3

Thioredoxin-like 10

Glutathione reductase 1

Peroxiredoxin 1

Thioredoxin reductase 2

Selenoprotein R 1

Selenoprotein W 1

Selenoprotein W 1 Selenoprotein W 1

Glutathione peroxidase 3

ERO1 2

ER (33) Cys (26) Sec (7)

Glutathione peroxidase 2

Methionine R sulfoxide reductase 1

Anterior gradient protein 3

Protein disulfide isomerase 9

OST complex thiol oxidoreductases 2

Thioredoxin-like 6

Ouiescin Q6 sulfhydryl oxidase 1

15kDa Selenoprotein 1

Selenoprotein M 1

Deiodinase 1

Selenorprotein N 1

Selenoprotein K 1

Selenoprotein S 1

Selenoprotein T 1

Thioredoxin (ββββ−−−−αααα−−−−ββββ−−−−αααα−−−−ββββ−−−−ββββ−−−−αααα))))

Major representatives:

ThioredoxinsGlutharedoxinsPeroxiredoxinsGluthatione peroxidasesProtein disulfide isomerases (PDI)

More then 60% of known thiol oxidoreductases are thioredoxin fold proteinsare thioredoxin fold proteins

Oxidative protein folding – disulfide bonds formation in the ER

Protein

S S PDIEro1 O2

PDI Erv2 O2

QSOX O2

Ero1 is present in all eukaryotesErv2 is typical for fungi QSOX is typical metazoans, plants and protists

Oxidative protein folding – disulfide bonds formation in the ER

Ero1 and Erv2 2 R-SH + O2 R-S-S-R + H2O2

Flavine dependant enzyme Ero1 provides oxidizing

equivalents for disulfide bond formation in the ER by relaying

the oxidizing power from molecular oxygen to the reduced

PDI. Ero1 directly oxidizes the active site of PDI by thiol-

disulfide exchange reactions with the oxidized shuttle Cys

pair, the reduced form of which is reoxidized by the active-

site Cys pair.

Ero1 is ER-localized disulphide-generating oxidase

Ero1 activity can be regulated by intramolecular disulphide switches, toprevent cellular hyperoxidation.

Humans have two Ero1 homologues, Ero1α and Ero1β. Both Ero1isoforms are transcriptionally upregulated under conditions of ER stress.Ero1α protein is expressed in most cell types, detectable protein levels ofEro1β are found only in several tissues

Grey – likely structural cysteines, black - active site or red - regulatoryfunction. The thick orange line at Cys166 indicates the connection to alikely (but unidentified) disulphide partner.

Mechanism of Humans Ero1α redox control

Appenzeller-Herzog, Nature 2008

Quiescin sulfhydryl oxidases

The Quiescin-sulfhydryl oxidase is family of flavoenzymes catalyzes the direct of

disulfide bonds into unfolded reduced proteins with concomitant reduction of oxygen

to hydrogen peroxide.

QSOX 2 R-SH + O2 R-S-S-R + H2O2

Heckler et al, Biochim Biophys Acta. 2008

ER environment is strongly oxidizing

1. Each disulfide bond formation is associated

with H2O2 production

2. GSSG/GSH ratio is 5-100x higher compare to cytosol

3. ER reducing equivalent provider is not known

NADPH GRGSH

TRGSH GPXs,

?GSH Protein

S S

OST

N33, IAP

Redox control of N-linked protein glycosylation

Sep15, SelM

SelS, SelK

ERCytosol

The enzyme that catalyzes this process is a multimeric

membrane-complex, called Oligosaccharyl transferase (OST).

OST3/6- like proteins are membrane linked thiol

oxidoreductases characterized by thioredoxin-fold and CxxC

redox motifs in the active site.

OST

N33, IAP

Protein glycosylation

Chaperones

Folding

UGT, Sep15, SelM

Quality control

RetrotranslocationCytosolic Degradation

ER

N33 and IAP - Role of thiol/disulfide oxidoreductases in protein glycosylation

Export Secretory pathway

Homozygous deletion of N33 correlates with metastatic prostate

cancer and its allelic deletion is associated with human colorectal

and pancreatic cancers. This observation suggests a possible tumor

suppressor function of N33.

There are two known cases of a natural knockout of N33 in humans

which are associated with nonsyndromic mental retardation.

Deletion of corresponding genes in yeast system cause ER-stress and

General properties of OST3/6 proteins

Deletion of corresponding genes in yeast system cause ER-stress and

strongly affect synthesis of glycosylated transmembrane proteins

These proteins behave as strong reductases – redox potential is in

range -280 -300mV

OST

N33, IAP

Glycoprotein folding quality control link to redox processes

Sep15, SelM

SelS, SelK

ERCytosol

Human 15 kDa selenoprotein (ER protein)MAAGQGGWLRPALGLRLLLATAFQAASALGAEFASEACRELGFSSNLLCSSCDLLGQFNLLPLDPVCRGC

CQEEAQFETKKLYAGAILEVCGUKLGRFPQVQAFVRSDKPKLFRGLQIKYVRGSDPVLKLLDDNGNIAEE

LSILKWNTDSVEEFLSEKLERI

Human SelM protein (ER protein)MSILLSPPSLLLLLAALVAPATSTTNYRPDWNRLRGLARGRVETCGGUQLNRLKEVKAFVTEDIQLYHNLVMK

Signal PeptideSignal Peptide

UGTUGT--bindingbinding

domaindomain Redox-domain

CGUActive site

MSILLSPPSLLLLLAALVAPATSTTNYRPDWNRLRGLARGRVETCGGUQLNRLKEVKAFVTEDIQLYHNLVMK

HLPGADPELVLLSRNYQELERIPLSQMTRDEINALVQELGFYRKSAPEAQVPPEYLWAPAKPPEEASEHDDL

Signal PeptideSignal Peptide Redox- domain

CGGUActive site

LEVCG-UKLGRFPQVQAFVRSDKPKLFRGLQIKYVRGSDPVLKLLDDNGNIAEELSILKWNTDSVEEFLSE

+E CG C+L R +V+AFV D +L+ L +K++ G+DP L LL N E + + + D + + E

VETCGGUQLNRLKEVKAFVTED-IQLYHNLVMKHLPGADPELVLLSRNYQELERIPLSQMTRDEINALVQE

Function of UDP-Glc:glycoprotein glucosyltransferase (UGGT)

Freeze et al, Essentials of Glycobiology 2009

Proposed roles of Sep15 in the quality control mechanismof protein folding.

1) Sep15 may function as a thiol-disulfide oxidoreductase that catalyzes

reduction, isomerization, or oxidation of disulfide bonds in misfolded proteins

recognized by UGT;

2) Sep15 may be required for recognition of glycoprotein substrates of UGT with

incorrectly formed disulfide bonds and may influence the enzymatic activity of

UGT towards substrates with incorrectly formed disulfide bonds.

Selenoprotein M (ββββ−−−−αααα−−−−ββββ−−−−ββββ−−−−ββββ−−−−αααα)

MSILLSPPSLLLLLAALVAPATSTTNYRPDWNRLRGLARGRVETCGGUQLNRLKEVKAFVTEDIQLYHNLVMKHLPGADPELVLLSRNYQELERIPLSQMTRDEINALVQELGFYRKS

________________________________________EEEEE________HHHHH___________EEEE_______EEEEEE______EEEEEE___HHHHHHHHH________

15 kDa Protein (ββββ−−−−αααα−−−−ββββ−−−−ββββ−−−−ββββ−−−−αααα)

LDQQPAAQRTYAKAILEVCTUKFRAYPQIQAFIQSGRPAKFPNLQIKYVRGLDPVVKLLDASGKVQETLSITKWNTDTVEEFFETHLAKDGAGKNS

____________EEEEEEE_______HHHHHHHHH_______EEEEEEE_____EEEEEE_____EEEE________HHHHHHHHH__________

Trx SelM Sep15

Many glycoproteins expressed as oligomers and quality control system

should monitor folding and oligomerisation status of multimerizing proteins.

Thiol retention is quality control system that monitoring the redox state of

specific cysteine residues in proteins. This system is also involved in

retaining unassembled proteins in the ER until specific cysteines formed

intersubunits disulfide bonds. Exact mechanism of thiol retention remain

elusive. Thiol retention was discovered in studies of the assembly of complex

oligomeric glycoproteins- immunoglobulin M.

Thiol retention and ER processes

OST

N33, IAP

Redox control of ER - cytosol protein translocation

Sep15, SelM

SelS, SelK

ERCytosol

>gi|34783186|gb|AAH13162.2| Selenoprotein K [Homo sapiens]

MVYISNGQVLDSRSQSPWRLSLITDFFWGIAEFVVLFFKTLLQQDVKKRRSYGNSSDSRY

DDGRGPPGNPPRRMGRINHLRGPSPPPMAGGUGR

>gi|45439349|ref|NP_982298.1| Selenoprotein S [Homo sapiens]

MERQEESLSARPALETEGLRFLHTTVGSLLATYGWYIVFSCILLYVVFQKLSARLRALRQ

RQLDRAAAAVEPDVVVKRQEALAAARLKMQEELNAQVEKHKEKLKQLEEEKRRQKIEMWD

SMQEGKSYKGNAKKPQEEDSPGPSTSSVLKRKSDRKPLRGGGYNPLSGEGGGACSWRPGR

Selenoprotein K and Selenoprotein S

SMQEGKSYKGNAKKPQEEDSPGPSTSSVLKRKSDRKPLRGGGYNPLSGEGGGACSWRPGR

RGPSSGGUG

Proline and glycine reach proteins involved in translocation of

misfolded proteins form ER to cytosol for proteosomal

degradation

Shchedrina et al, ARS 2010

SelS is a component of the ER-Associated Protein Degradation (ERAD) system .

ERAD is a pathway which protects cells from accumulation of misfolded proteins

by transferring these proteins from the ER to cytosol for subsequent ubiquitination

and proteasomal degradation. SelS was proposed as a mediator in the interaction

of p97 ATPase and the ER membrane integral protein Derlin-1 forming a new type

of retrotranslocation channel

Selenoprotein T – ER protein with unknown function

>gi|18803851|SelT Homo sapiens

MRLLLLLLVAASAMVRSEASANLGGVPSKRLKMQYATGPLLKFQ

ICVSUGYRRVFEEYMRVISQRYPDIRIEGENYLPQPIYRHIASF

LSVFKLVLIGLIIVGKDPFAFFGMQAPSIWQWGQENKVYACMMV

FFLSNMIENQCMSTGAFEITLNDVPVWSKLESGHLPSMQQLVQIFFLSNMIENQCMSTGAFEITLNDVPVWSKLESGHLPSMQQLVQI

LDNEMKLNVHMDSIPHHRS

SelT deficiency alters cell adhesion and enhances the expression of

oxidoreductase genes, while decreasing the expression of genes involved in

cell structure organization, suggesting the involvement of SelT in redox

regulation. Furthermore, loss of SelT elevates expression of another

selenoprotein - selenoprotein W

Selenoprotein W EEEEEEE HHHHHHHHHHHHHH EEEEEEE EEEEE EEEEEE HHHHHHHHHHHH

2FA8 AGROBACTERIUM TUMEFACIENS PRIAIRYCTQCN-WLLRAGWMAQEILQTFASDIGEVSLIPST--GGLFEITVD-----GTIIWERKRDG-----GFPG----PKELKQRIRDLID

SelW PSEUDOMONAS FLUORESCENS PEIVITYCTQCQ-WLLRAAWLAQELLSTFGDDLGKVSLVPGT--GGIFHITCN-----DVQIWERKADG-----GFPE----AKVLKQRVRDQID

SelW VIBRIO CHOLERAE AQIEIYYCRQCN-WMLRSAWLSQELLHTFSEEIEYVALHPDT--GGRFEIFCN-----GVQIWERKQEG-----GFPE----AKVLKQRVRDLID

SelW BOS TAURUS VVVRVVYCGAUG-YKPKYLQLKKKLEDEFPSR-LDICGEGTPQVTGFFEVFVA-----GKLVHSKKGGD-----GYVDTESKFLKLVAAIKAALA

SelW MUS MUSCULUS LAVRVVYCGAUG-YKPKYLQLKEKLEHEFPGC-LDICGEGTPQVTGFFEVTVA-----GKLVHSKKRGD-----GYVDTESKFRKLVTAIKAALA

SelW SEA URCHIN VIVKVIYCGGUG-YGPRYRRLKQELKDEFGDD-VDMAGESTPGTTGWLEVXVN-----GKLIHSKKNGD-----GYIDSESKLKKIVNAVSAAM-

Selenoprotein V

SelV MUS MUSCULUS ILIRVMYCGLUS-YGLRYIILKRTLEHQFPNL-LEFEEERATQVTGEFEVFVD-----GKLIHSKKKGD-----GFVD-ESGLKKLVGAIDEEIK

SelV RATTUS NORVEGICUS ILIRVMYCGLUS-YGLRYILLKKTLEHQFPNL-LEFEEERATQVTGEFEVFVD-----GKLIHSKKKGD-----GFVD-ETSLKKLVGAIDEEIK

SelV HOMO SAPIENS VLIRVTYCGLUS-YSLRYILLKKSLEQQFPNH-LLFEEDRAAQATGEFEVFVN-----GRLVHSKKRGD-----GFVN-ESRLQKIVSVIDEEIK

Selenoprotein H

SelH MUS MUSCULUS ATVVIEHCTSURVYGRHAAALSQALQLEAPE--LPVQVNPSKPRRGSFEVTLLRSDNSRVELWTGIKKGPPRKLKFPE----PQEVVEELKKYLS

SelH HOMO SAPIENS ATVVIEHCTSURVYGRNAAALSQALRLEAPE--LPVKVNPTKPRRGSFEVTLLRPDGSSAELWTGIKKGPPRKLKFPE----PQEVVEELKKYLS

SelH BOS TAURUS PSVVIEHCTSURVYGRNAAALSQALRLQAPE--LTVKVNPARPRRGSFEVTLLRADGSSAELWTGLKKGPPRKLKFPE----PHVVLEELKKYLS

SelH DANIO RERIO LRVVIEHCKSURVYGRNAVVVREALADSHPE--LKVMINPHNPRRNSFEITLMDG-ERADVLWSGIKKGPPRKLKFPE----PAEVVTALKQALE

Rdx12

Rdx12 SUS SCROFA VRIVVEYCEPCG-FEATYLELASAVKEQYPG--IEIESRLGG--TGAFEIEIN-----GQLVFSKLENG-----GFPY----EKDFIEAIRRASN

Rdx12 HOMO SAPIENS VRIVVEYCEPCG-FEATYLELASAVKEQYPG--IEIESRLGG--TGAFEIEIN-----GQLVFSKLENG-----GFPY----EKDLIEAIRRASN

Rdx12 GALLUS GALLUS VHIMVEYCEPCG-FGATYEELASAVREEYPD--IEIESRLGG--TGAFEIEIN-----GQLVFSKLENG-----GFPY----EKDLIEAIRRARN

Rdx12 DANIO RERIO 2A VQIKVEYCGGUG-YEPRYQELKRVVTAEFTD--ADVSGFVGR--QGSFEIEIN-----GQLIFSKLETS-----GFPY----EDDIMGVIQRAYD

Rdx12 DANIO RERIO 2 VKVKIEYCGAUG-YEPRFQELKREICGNCPD--AEVSGFVGR--RGCFEIQIN-----DFLVFSKLESG-----GFPY----SEDIIEAVVKAKD

Selenoprotein T EEEEEEEEE HHHHHHHHHHHHHH EEEEEE EEEEE EEEEE HHHHHHHHHHH

SelT BOS TAURUS PLLKFQICVSUG-YRRVFEEYMRVISQRYPD--IRIEGENYL TGAFEITLN-----DVPVWSKLESG-----HLPS----MQQLVQILDNEMK

SelT SUS SCROFA PLLKFQICVSUG-YRRVFEEYMRVISQRYPD--IRIEGENYL TGAFEITLN-----DVPVWSKLESG-----HLPS----MQQLVQILDNEMK

SelT MUS MUSCULUS PLLKFQICVSUG-YRRVFEEYMRVISQRYPD--IRIEGENYL TGAFEITLN-----DVPVWSKLESG-----HLPS----MQQLVQILDNEMK

SelT ANOPHELES GAMBIAE ATMTFLYCYSCG-YRKAFDDYHNLILEKYPE--ITIRGSNYD SGAFEITLN-----DVPVWSKLETG-----RFPA----PQEMFQIIDNHLQ

SelT SOLANUM TUBEROSUM NTVTIDFCSSCS-YRGTAVTMKNMLDNQFPG--IHVVLANYP SGAFEVYCN-----GELVFSKLKEN-----RFPG----ELELKDLVGRKIA

SelT MEDICAGO TRUNCATULA NTVSIDFCTSCS-YKGNAVSVKNTLESLFPG--INVVLANYP SGAFEVYFN-----GELVFSKLKEN-----RFPG----EFELKELIGRRIG

HHHHHHHHHHHHHHHHHHHHHHH HHHH HHHHH HHHHHHHHHHHHHHHHHHHHHHH

SelT BOS TAURUS PQPIYRHIASFLSVFKLVLIGLIIVGKDPFAFFGMQ-APSIWQWG-QENKVYACMMVFFLSNMIENQCMS

SelT SUS SCROFA PQPIYRHIASFLSVFKLVLIGLIIVGKDPFAFFGMQ-APSIWQWG-QENKVYACMMVFFLSNMIENQCMS

SelT MUS MUSCULUS PQPIYRHIASFLSVFKLVLIGLIIVGKDPFAFFGMQ-APSIWQWG-QENKVYACMMVFFLSNMIENQCMS

SelT ANOPHELES GAMBIAE PSGVNMLLSKVLLVTKLLLIAALMSNYDIGRYIGNP-FAGWWQWC-FNNKLYASMMIFFLGNTLEAQLIS

SelT SOLANUM TUBEROSUM PPLPKRLLGKVVPVFQFGVIGLVMAGEQIFPRLGIAVPPPWFYQL-RANRFGTMATTWLLGNFFQSMLQS

SelT MEDICAGO TRUNCATULA PPLPKRALSKVVPVLQTGAIIAITAGDQIFPRLGVT-PPQLYYSL-RANKFGSIASIWLLSNFVQSFLQS

>gi|172044683|SELN_HUMANMGRARPGQRGPPSPGPAAQPPAPPRRRARSLALLGALLAAAAAAAVRVCARHAEAQAAARQELALKTLGT

DGLFLFSSLDTDGDMYISPEEFKPIAEKLTGSCSVTQTGVQWCSHSSLQPQLPWLNUSSCLSLLRSTPAA

SCEEEELPPDPSEETLTIEARFQPLLPETMTKSKDGFLGVSRLALSGLRNWTAAASPSAVFATRHFQPFL

PPPGQELGEPWWIIPSELSMFTGYLSNNRFYPPPPKGKEVIIHRLLSMFHPRPFVKTRFAPQGAVACLTA

ISDFYYTVMFRIHAEFQLSEPPDFPFWFSPAQFTGHIILSKDATHVRDFRLFVPNHRSLNVDMEWLYGAS

ESSNMEVDIGYIPQMELEATGPSVPSVILDEDGSMIDSHLPSGEPLQFVFEEIKWQQELSWEEAARRLEV

AMYPFKKVSYLPFTEAFDRAKAENKLVHSILLWGALDDQSCUGSGRTLRETVLESSPILTLLNESFISTW

SLVKELEELQNKQENSSHQKLAGLHLEKYSFPVEMMICLPNGTVVHHINANYFLDITSVKPEEIESNLFS

Selenoprotein N

SLVKELEELQNKQENSSHQKLAGLHLEKYSFPVEMMICLPNGTVVHHINANYFLDITSVKPEEIESNLFS

FSSTFEDPSTATYMQFLKEGLRRGLPLLQP

SelN defficiensy is assosiated with classical form of rigid

spine muscular dystrophy. Recent studies have identified

SelN as a key protein in cell protection against oxidative

stress and redox-related calcium homeostasis. SelN

work reductase in the regulation of ryanodine receptor

calcium channel activity.

>gi|25123199|gb|AAH40053.1| MSRB3 protein [Homo sapiens]

MSAFNLLHLVTKSQPVALRACGLPSGSCRDKKNCKVVFSQQELRKRLTPLQYHVTQEKGTESAFEGEYTH

HKDPGIYKCVVCGTPLFKSETKFDSGSGWPSFHDVINSEAITFTDDFSYGMHRVETSCSQCGAHLGHIFD

DGPRPTGKRYCINSAALSFTPADSSGTAEGGSGVASPAQADKAEL

Methionine sulfoxide reductase – MsrB3

Methionine sulfoxide reduction is an important process, by which cells regulate

biological processes and cope with oxidative stress. MsrA and MsrB are

enzymes involved in the reduction of methionine sulfoxides in proteins

gi|5453541|ref|NP_006399.1| anterior gradient protein 2

MEKIPVSAFLLLVALSYTLARDTTVKPGAKKDTKDSRPKLPQTLSRGWGDQLIWTQTYEEALYKSKT

SNKPLMIIHHLDECPHSQALKKVFAENKEIQKLAEQFVLLNLVYETTDKHLSPDGQYVPRIMFVDPS

LTVRADITGRYSNRLYAYEPADTALLLDNMKKALKLLKTEL

>gi|19068182|gb|AAL55402.1| anterior gradient protein 3

MMLHSALGLCLLLVTVSSNLAIAIKKEKRPPQTLSRGWGDDITWVQTYEEGLFYAQKSKKPLMVIHH

LEDCQYSQALKKVFAQNEEIQEMAQNKFIMLNLMHETTDKNLSPDGQYVPRIMFVDPSLTVRADIAG

RYSNRLYTYEPRDLPLLIENMKKALRLIQSEL

Anterior gradient proteins

AGR2 is present within the ER of intestinal secretory epithelial cells and is

essential for in vivo production of the intestinal mucin MUC2, a large, cysteine-rich

glycoprotein that forms the protective mucus gel lining the intestine. A cysteine

residue within the AGR2 thioredoxin-like domain forms mixed disulfide bonds with

MUC2, indicating a direct role for AGR2 in mucin processing. Anterior Gradient

family proteins are implicated in the formation of the cement gland and the

induction of forebrain fate. The human homologs, hAGR2 and hAGR3, are

proteins associated with estrogen-positive breast tumors. hAGR2 has also been

implicated in prostate cancer.

Summary

• ER is a second cell compartment with largest number of thioloxidoreductases and intensive thiol related processes

• ER environment is strongly oxidizing; however, exact oxidizing status is unknown

• Reductive processes in the ER are remain unclear. Existing ROS sensors are not suitable for measurement of ER ROS level

• Function of the 30% of ER thiol oxidoreductases is unknown