Genetics, molecular and cell biology of cell death 

Ágnes Czibula, Roberta Fajka-BojaInstitute of GeneticsDecember 14, 2016

„Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities”TÁMOP-4.1.1.C-13/1/KONV-2014-0001

Morphological classification of cell deaths:apoptosis and necrosis

Apoptosis:- Membrane blebbing- Cytosolic condensation, cell shrinkage- Protein degradation - Nuclear condensation - Fragmentation of nuclear DNA- Formation of apoptotic bodies - Genetically regulated- The apoptotic bodies are phagocytosed,no inflammatory response!

Necrosis:- Cell swelling, round up- Rapid loss of membrane integrity- No nuclear condensation - No DNA degradation - Spilling of cellular content- Accidental, tissue injury- The release of cytoplasmic contents triggers inflammatory response.

• Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972, 26(4):239-57

• Apoptosis occurs in all tissues as part of normal cellular turnover.

• Apoptosis also occurs during embryogenesis in which particular cells are ‘programmed’ to die, and hence the term ‘programmed cell death’ is used to describe this process.

• Intrinsic program for removal of damaged cells.

• Examples:- Embryogenesis, development - Regulation of organ size and morphology - Immunohomeostasis - DNS-damage, stress, hypoxia

1. Initiation: cells receive death signals-lack of obligatory survival factors-shortage of metabolic supply-physical sock: heat, irradiation, oxidative stress-subnecrotic damage by toxins, chemotherapeutics-ligation of death-signal transmitting receptors

2. Effector phase:-the death signals are translated into metabolic reactions and the cells become committed to death.-early changes in the membrane structure-activation of catabolic enzymes (caspases)

3. Degradation phase:-massive protein degradation and DNA fragmentation -encapsulation of the fragments into apoptotic bodies-clearance of the apoptotic bodies by the adjacent cells

Phases of apoptosis

The molecular mechanism

Death receptor

Death ligand

Death inducing signaling complex

Iniciator caspase-

8/10

Effectorcaspase-3/6/7

Extrinsic / Death receptor-mediated Intrinsic / Mitochondrial pathways

CelldeathCell

death

Cellular stress

Mitochondrial disfunction

Cytochrome C

Bcl-2 family

Iniciator caspase-9

Apoptosome

Death receptors belong to the Tumor Necrosis Factor Receptor (TNFR) Superfamily.

TNFR:- Induce both activating and death signaling pathways- Play role in cell metabolism, proliferation, differentiation, survival and apoptosis- Most members are expressed by immune cells- 8 members have Death Domains, responsible for transmitting apoptotic signal! - DR1 (TNFR1), DR2 (CD95, Fas), DR3, DR4 (TRAILR1), DR5 (TRAILR2), DR6, EDAR, NGFR

I. Apoptosis induction by death receptors

- ‘decoy receptors’ (DcR1, DcR2, DcR3) are soluble, or having no intracellular part –inhibitors.

Death domain

Transmembrane domain

Ligand binding domain

Extracellular Intracellular

TNF:- Membrane bound or soluble- Act as trimers, induce trimerization of the receptors - Most members are expressed by immune cells- One ligand can bind to more receptors and one receptor can have more ligands

Ligands of the Death receptors belong to the Tumor Necrosis Factor (TNF) Superfamily.

CD95

DcR3

TNFR1 TNFR2

DR4 DR5 DcR1 DcR2

CD95L TNF TRAILTL1A LT

DR1 DR2

TRAILR1 TRAILR2

Signals emerging from the Death receptor-death ligand complexes can: (1) Induce apoptosis via activation of the protease, caspase-8 (or caspase-10);(2) Stimulate adaptive, non-apoptotic signaling pathways mediated by activation of the transcription factor nuclear factor-κB (NF-B) and the mitogen activated protein kinases (MAPKs), which regulate developmental and inflammatory processes.

Signaling trough death receptorsTrimeric ligand → trimerization of the receptor → recruitment of adaptor proteins that bind to the Death Domain of the receptor → binding platform for signal-transducing proteins. Death ligand

Death receptor

NF-B pathwayMAPK/JNK

Stress response

Survival/cell death

Differentiation Inflammation

Survival

Caspase activation

Differentiation apoptosis

Survival Cell death

Fas (CD95) – FasL (CD95L)

K.O. mice: lymphadenopathia, splenomegalia, autoimmunity- Fas-/- lpr (lymphoproliferation)- FasL-/- gld (generalized lymphoproliferative disease)- FADD-/- not viable

FADD = Fas Associated Death Domain - adapter proteinDD = Death DomainDED = Death Effector DomainForm homotypic interactions, recruit procasp-8 and-10

DISC = Death Inducing Signaling ComplexFasL+Fas+FADD+procasp-8

procasp-8 dimerization and intramol. cleavage

FLIP = FLICE-like Inhibitory Protein, without protease activity

CD95L/FasL/APO-1L

activecaspase-8/10

CD95/Fas/APO-1

FADD

Procaspase-8/-10 c-FLIP

TRAF = Tumor Necrosis Factor Receptor Associated Factor - adaptor proteinRIP = Receptor Interacting Protein - kinaseTRADD = TNFR Associated Death Domain - adaptor protein

TNF - TNFR

TNFR1

TNF

Complex I

procaspase-8/10

TRADD

TRAF2RIP1

NF-B

survival

Endocytosis

TRADD

Caspase activation

apoptosis

TNFR1

TNF

Complex IIFADD

IAPs

c-FLIP

TRAIL - TRAILR

•Tumor cells: TRAILR1 and TRAILR2, FADD association - apoptosis•Healthy cells: TRAILR3 and TRAILR4 decoy receptors, TRADD and TRAF association, NF-B activation

Healthy cells are resistant to TRAIL-induced apoptosis – is it good for fighting tumors?

TRAILR4

TRAIL

procaspase-8/10

TRADD

TRAF2NIK

NF-B

survival

TRADD

FADD

Caspase activation

apoptosis

TRAIL

TRAILR1,R2

II. Apoptosis by mitochondrial pathwaySignals:- lack of growth factors- detachment from the tissue, lack of cell-cell interaction – anoikis- destruction of the cytoskeleton – amorphosis- DNA-damage by UV-light or drugs- reactive oxygen species, hypoxia

Steps:- Change in the balance and localization of anti-apoptotic and pro-apoptotic Bcl-2 family proteins- Opening of the mitochondrial permeability transition pore- Decrease of mitochondrial membrane potential- Release of apoptotic factors from mitochondria, i.e. cytochrome C, Smac/DIABLO, Omi/HtrA2, etc.

Cytochrome C

Anti-apoptotic (Bcl-2)

Pro-apoptotic (Bax, Bak)

Stress signal

Bcl-2 family1. Anti-apoptotic Bcl-2:- Inhibit the apoptosis- Maintain the integrity of the mitochondrial membrane- Bcl-2, Bcl-w, Bcl-xL, A1, MCL-1.- Multiregion proteins, BH1-BH4, BH: Bcl-2 homology domain, MBR: membrane binding region.

3. Executioner proteins – Bax group:- Multiregion proteins, integrate into the outer membrane of the mitochondria in response to apoptotic stimuli, form pores - Bax and Bak expressed in every cells.

2. Sensors of apoptotic signals - BH3-only:- they are needed for initiation of mitochondrial pathway, but not sufficient for execution- direct activators: Bid, Bim, Puma – they bind directly to Bax and Bak.- sensitizers/de-repressors: Bad, Bmf, Bik, Hrk, Noxa –they bind directly to anti-apoptotic Bcl-2 proteins.

MBRBH1BH4 BH2BH3 Bcl-2

MBRBH3 Bim

BH3-only proteins are the sensors of the apoptotic signal

BH3-only proteins inhibit the anti-apoptotic ones, and/or activate Bax and Bak

Bax and Bak integrate into the outer mitochondrial membrane and induce the

opening of mitochondrial pores and release of cytochrome C

Cytochrome C

Bax,Bak

Stress signal

BH3sen

BH3act

Bcl-2,Bcl-XL

Regulation of BH3-only proteinsAs BH3-proteins are the switch of the mitochondrial pathway of apoptosis, their expression level and localization are tightly regulated!!!

• Bim and Bmf are anchored to the cytoskeleton – released in anoikis or amorphosis• Bad is phosphorylated by growth factor receptor signaling route, then sequestered by 14-3-3 • Bid is cleaved by casp-8 or granzyme B – the link between the death receptors and mitochondria•The transcription of Hrk, Noxa, PUMA is induced by p53 – DNA damage

Loss of contact Cytokine deprivation DNA damage UV irradiation

hypoxia

Death receptors

p53

Bim, Bmf Bad Bid

Hrk,Noxa, PUMA Bim

Crosstalk between death receptor and mitochondrial pathway

Death receptor

Death ligand

Death inducing signaling complex

Iniciator caspase-

8/10

Effectorcaspase-3/6/7

CelldeathCell

death

Cellular stress

Cytochrome C

Bcl-2 family

Iniciator caspase-9

Apoptosome

Bid

• The BH3-only protein Bid is cleaved by casp-8 or granzyme B – the link between the death receptors and mitochondria

Caspase-9 activation downstream of mitochondria

(Apaf-1: apoptotic protease-activating factor-1)

Apoptosome=cytC+Apaf-1+ATP+procaspase 9

Cytochrome C

Bax,Bak

Stress signal

BH3

caspase-3/6/7

Apoptosome

Caspases• cysteinyl aspartate proteinases= cystein proteases- they have Cys in their active center, and cleave after aspartic acid

• They are synthesized in inactive form

• Activation: dimerization and proteolysis

• ICE: Interleukin-1-converting enzyme = caspase-1 (1992)ced-3 : Caenorhabditis elegans (1993)

• 12 human members of the family

Active heterodimer

Intramolecular cleavage

p20p10p10p20

Large SmallProdomain

Zymogen

Large SmallProdomain Dimerization

Classification by structure and function:

Classification by substrate specificity and function:I. group: inflammatory caspases-1, -4, -5, ( -11 mouse), substrate: WEHD,II. group: initiator caspases

-2, -8, -9, -10, -12 (mouse, Africa), (LV)EXD,III. group: effector caspases-3, -6, -7,-14 (keratinocyte) DEXD.

Large SmallCARD

Large SmallDEDDED Caspase-8/-10

Caspase-2/-9

Large Small Caspase-3/-6/-7

Initiator caspases – with large prodomain

Effector caspases – without prodomain

Activation of initiator caspases

1/ DISC: Procasp-8 and -10 activationHomotypic association between DD and DED domains.

“Induced proximity” – dimerization is required for activation and subsequent autocatalytic cleavage of DED prodomain and the large and small domains

2/ Apoptosome: procasp-9 activationheptamer Apaf-1 + cyt C + ATP + procasp-9

Death receptor

FADD

Procaspase-8/-10

ATP

heptamerCyt C Apaf-1

procaspase-9 active caspase-9

Activation of effector caspasesProcasp -3, -6, -7: they are homodimers in zymogen form, the cleavage by initiator caspases stabilizes the conformation of the active center.

procaspase-3zymogen

Proteolysis

caspase-3active

Substratein active center

Regulation of caspase activity• The first inhibitor of apoptosis (IAP) protein was identified from a baculovirus strain in 1993 by Miller et al. • IAPs contain BIR-domain (baculoviral IAP repeat)• type I BIR domains (BIR1) interact with tumour necrosis factor receptor-associated factor 1 (TRAF1) and TRAF2, leading to NF-B activation and survival• type II BIR domains (BIR2 and BIR3) interact with caspases:

- BIR3 domains form dimers with the cleaved and active casp-9 ,- occupy the active center of effector caspases casp-3 and -7.

• IAPs contain ubiquitin (Ub)-binding UBA domain and a carboxy-terminal RING domain required for Ub ligase activity, and docking site for ubiquitin conjugating enzymes (E2s).• XIAP can polyubiquitinate the large subunit of active caspase-9 in vitro, but not the inactive procaspase-9.

RINGBIR2BIR1 BIR3 UBAXIAP

NF-B polyubiquitination

caspase-3,-7 caspase-9

Inhibition of inhibitors:

- Smac/Diablo and HtrA2/Omi are released from mitochondria (with cyt C):They can bind the BIR2 and BIR3 domains of IAPs with their N-terminal IAP-binding motif (IBM), thereby blocking their association with caspases.

Cytochrome CSmac

caspase-3,-7

Apoptosomecaspase-9

IAPs

• The activation of effector caspases leads to the destruction of the cells. • There is consensus recognition sequence of 3-4 amino acids, with aspartic acid at the position P1. • Several hundred possible substrates according to this sequence• Limited proteolysis!

Caspase substrates

W- Trp D- Asp X- any AAY- Tyr E- Glu - Gly, Ala, Thr, Ser, AsnI- Ile V- ValL- Leu

P4 P3 P2 P1 P1’

W/Y E X D caspase -1,-4,-5,-14

I/L E X D caspase-8,-9,-10

D E X D caspase-3,-7

V E X D caspase-6

- Inactivation or activation of signaling proteins, enzymes (kinases, phosphatases, phospholipases, Bcl-2 family proteins) – amplification of apoptotic signaling.

- Desintegration of cytoskeletal elements (actin, gelsolin, lamin A,B) – morphological changes.

- Cleavage of nuclear proteins (DNA polimerases, inhibitor of DNase) – stopping DNA replication and repair, internucleosomal fragmentation of DNA (see the practical demonstration).

Consequences of caspase activation:

Steps of clearance of apoptotic cells:

1/ Recruitment of phagocytes – “Find-me” chemotactic signals, i.e. lysophosphatidyl-coline (LPC), product of the caspase-activated phospholipaseA2

2/ Recognition of apoptotic cells - “Eat-me” signals for initiation phagocytosis, i.e. LPC, modified sugars, phosphatidyl-serine. Phagocytes have scavenger receptors for these: Annexin I, CD14, CD36, thrombospondin-1…

3/ Cytosceletal rearrangement and engulfment

4/ Post-engulfment steps- Processing of corpse and release of anti-inflammatory cytokines (IL-10, TGF-).

Clearing of apoptotic cellsApoptotic cells and the membrane bound apoptotic bodies are cleared/phagocytosed by macrophages, dendritic cells and other cells of the tissue.

Phagocyte Apoptotic cell

“Find me” signals

“Eat me” signals

Cytokine release

Engulfment

Phosphatidylserine (PS) is an “eat me”-signal exposed on the cell surface of apoptotic cells:- Healthy cells: (PS) is in the inner lipid layer of the plasma membrane- Apoptotic: Ca++-dependent activation of scramblase increases the flip-flop of lipids - PS in inner and outer lipid layer

Hoechst Annexin V-FITC

Plasma membrane of healthy cells

Apoptosis

Plasma membrane of apoptotic cells

EC

ICPS

Annexin V

Annexin V is a lipid-binding protein, binds to PS - detection of apoptotic cells (see the practical demonstration).

7 October 2002The Nobel Assembly at Karolinska Institutet has today decided to

award The Nobel Prize in Physiology or Medicine for 2002jointly to

Sydney Brenner, H. Robert Horvitz and John E. Sulstonfor their discoveries concerning

"genetic regulation of organ development and programmed cell death"

Apoptosis is C.elegans

959 cells in adults 1090 cells in larval stadium

131 cells die with programmed cell death

Genetic screens in C.elegans:

EGL-1EGL-1Ced 3Ced 4Ced 9

Ced 9

Gain of function

Gain of functionLoss of function

Loss of functionLoss of functionLoss of function

+++--

-

Apoptosis

+++

-Ced 3: the only caspase in C. elegans

Large SmallCARD

EGL1

BH3

Ced 9

Bcl-2

Ced 4

Apaf-1

Ced 3

Caspase

C. elegans

Mammals

Caspase activation in D. melanogaster

IAPs (inhibitors of caspases): binding and ubiquitination of caspases – inactivation or degradation

Inhibitors of IAPs:RHG proteins: Rpr, Hid, Grim, functional homology with Smac, competitors for IAPs.

Large SmallCARD

Large SmallDEDDED Dredd – ro role in apoptosis

Dronc

Large Small DriceDcp-1

Effector caspases

Initiator caspases

RINGBIR2BIR1

Comparison of mitochondrial apoptotic pathways is different species

- Cyt C plays role in caspase activation only in mammals!- The role of Bcl-2 family in D.melanogaster is not clear- Death receptors are only in mammals

EGL1

Ced 9

Ced 4

Ced 3

BH3

Bcl-2

Apaf-1

Caspase

C.elegans Mammals D. melanogaster

Bid, Bim

Bcl-2

Apaf-1

Casp3

Bax, Bak

CytC

Casp9

Smac

IAPs

Apaf-1

Drice

Dronc

ReaperHid

Grim

DIAP

Malfunctioning of apoptosisSuppressed apoptosis Excessive apoptosis

• CancerLymphomasGastricColorectalLungNeuroblastomaetc.

• Autoimmun disordersAutoimmune lymphoproliferativesyndrome (ALPS)Systemic lupus erymatosusMyastenia gravis

• Frequent infectionsViral infections

• Neurodegenerative diseasesAlzheimer’s diseaseParkinson’s diseaseHuntington’s diseaseStrokeAmyotrophoc lateral sclerosisetc.

• Cardiovascular diseasesHeart failureMyocardial infarction

• Other disordersInflammationSepsistypeI DiabetesAIDS

CASP6 1.48% of 14 different types tumors

CASP3

2% of esophageal carcinoma3% of head/neck carcinoma

CASP7

2% of gastric or colon cancer2% of head/neck carcinoma

Somatic mutations of caspases

5% of invasive carcinoma13% of hepatocellular carcinoma10.7% of gastric cancer

CASP8

CASP10 14.5% of non-Hodgkin’s lymphoma4.3% of colon cancer

Frequency of somatic mutations ofcaspases are relatively low in tumors andmutations frequently occur together withother mutations in apoptotic pathways

Overexpression of cFLIPs in several tumor types has been shown and it is implicated in chemotherapy resistance

Elevated level of IAPs in certain tumor types has been reported and it has been correlated with tumor survival.

RINGBIR2BIR1 BIR3 UBA

Caspases and their inhibitors in tumors

TP53 – tumor suppressor gene

p53RE Target gene

p53

p73

p63

RRRCW WGYYY________RRRCW WGYYY

Consensus motif

R, purineW, adenine or thymineY, pyrimidine

P53 is a key transcription factor in apoptosis regulation

Acts as homo- or heterotetramer with p63 and p73

>100 p53 target genes have been identified

Role of p53 in apoptosis regulation

Nucleus

p53

DNA damage,oxidative stress

Hrk,Noxa, PUMA

p53

Bcl-XL

p53U

Cyt C

Bcl-2Bcl-XL

BaxBak

apoptosis

Caspaseactivation

Bax

MDM2 p53

UUUUUU

degradationpolyubiquitination

monoubiquitination

phosphorylationacetylation

methylation

Normal cell condition Stressed cell condition

Stress signal

Regulators

Mutations and SNPs of TP53

Mutations of TP53

Somatic mutations ap. 50% of tumors

Germline mutationsLi-Fraumeni syndrome

Polymorphisms of TP53

Transactivationdomains

Proline-richdomain

DNA-bindingdomain

Tetra-merization

domain

Basicdomain

R47

S

R72

P

Exonic polymorphism

Non-synonymouspolymorphism

Polymorphismwith funtional change

P PP P PP PP PP P P PP

Somatic mutations

P Phosphorylation

A new regulatory level - microRNAs

p53

p53 p53

p53

miR-25miR-30dmiR-214

miR-125bmiR-380miR-504

miR-1246miR-1204 cluster

miR-200b-200a-429miR-224

miR-34 familymiR-205

miR-200c-141miR-199a-3p

miR-107let-7

miR-17-92 clusterp53

p72

p68

DGCR8

DROSHA

Maturation of miR-15a, miR-16-1, miR-143,

miR-145, mirR-199a-3p, miR-122

MDM2

miR-192miR-194miR-215miR-605

http://AtlasGeneticsOncology.org/Anomalies/t1418ID2006.html

Bcl-2 – proto-oncogene

B cell lymphoma

Enhancer CJh

Bcl-2

Bcl-2 E C

Translocation t(14;18)

Ch: 18

Ch: 14

Bcl-2 overexpression

Reduced apoptosis

Bax Bcl-2

Ig heavy chain coding region

bcl-2 gene 3. exonic region

Pathological role of apoptosisin neurodegenerative disorders

• Inheritent autosomal dominant disordercaused by mutation in huntingtin’scoding gene

• Caspase-6 cleavage of mutant htt isresponsible for neuropathologicalsymptoms

• Hippi-hip complex activates caspase-8

Alzheimer’s disease

Huntington’s disease

• APP cleavage by caspase-3 resulting-peptide accumulation

• Caspase-6 depend axon degeneration• Oxidative stress caused by -peptide

trigger FASL expression in neuronsand glia

Parkinson’s disease

• PINK1 mutation is responsible forintrinsic/mitochondria-depend apoptoticpathway upregulation

• Death receptors (FAS, TNFRs)expression is eleveted in neurons

Amyotrophic lateralsclerosis (ALS)

• In spinal cord of ALS mouse modelcaspase-1 , caspase-3 , caspase-9 areactivated

References

2/ Chipuk et al. The BCL-2 family reunion. Mol Cell. 2010, 37(3):299-310.

4/ Crawford ED, Wells JA. Caspase substrates and cellular remodeling. Annu Rev Biochem. 2011, 80:1055-87.

7/ Kepp et al. Cell death assays for drug discovery. Nat Rev Drug Discov. 2011, 10(3):221-37.

5/ Gyrd-Hansen M, Meier P. IAPs: from caspase inhibitors to modulators of NF-kappaB, inflammation and cancer. Nat Rev Cancer. 2010, 10(8):561-74.

6/ Ravichandran KS. Find-me and eat-me signals in apoptotic cell clearance: progress and conundrums. J Exp Med. 2010, 207(9):1807-17.

1/ Sessler et al. Structural determinants of DISC function: new insights into death receptor-mediated apoptosis signalling. Pharmacol Ther. 2013, 140(2):186-99.

3/ Riedl SJ, Shi Y. Molecular mechanisms of caspase regulation during apoptosis. Nat Rev Mol Cell Biol. 2004, 5(11):897-907.

8/ Favaloro B et al. Role of apoptosis in disease. Aging (Albany NY). 2012, 4(5):330-49.

9/ Whibley C, Pharoah PD, Hollstein M. p53 polymorphisms: cancer implications. Nat Rev Cancer. 2009, 9(2):95-107.

Thank you for your attention!

This work is supported by the European Union, co-financed by the European Social Fund, within the framework of " Practice-

oriented, student-friendly modernization of the biomedical education for strengthening the international

competitiveness of the rural Hungarian universities " TÁMOP-4.1.1.C-13/1/KONV-2014-0001 project.