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Apoptosis pathways

Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s P rogrammes at the University of Pécs and at the University of Debrecen Identification number : TÁMOP-4.1.2-08/1/A-2009-0011.

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Apoptosis pathways

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  1. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identificationnumber: TÁMOP-4.1.2-08/1/A-2009-0011

  2. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Tímea Berki and Ferenc Boldizsár Signaltransduction Apoptosispathways

  3. „The process of naturaldeath” • The word „apoptosis” (Greekspelling of apoptosis) is usedinGreektodescribethe „droppingoff” or „fallingoff” of petalsfromflowers, orleavesfromtrees • Professor James Cormack of theDepartmentofGreek, University of Aberdeen, suggestedthistermfortheprocessofprogrammedcelldeathin 1972

  4. Role of apoptosis • Apoptosis, ingeneral, confersadvantagesduring an organism's life cycle: oneappropriateresponseto a signal is forthecelltocommitsuicide –presumablyforthegood of theorganism • Between50 and 70 billioncells die eachdayduetoapoptosisintheaverage human adult • Programmedcelldeath is encodedinthegenome • Apoptosisdoesnotrequirenewtranscriptionortranslation, suggestingthatthemolecularmachineryrequiredforcelldeathlaydormantinthecell, and justrequiresappropriateactivation.

  5. Whenwouldit be advantageoustotheorganism? • To „sculpt” an organismduringdevelopmentsuchasduringembryodevelopment, metamorphosis and tissueatrophy • Regulatethetotalnumber of cells • Defend and removeunwantedordangerouscellslike tumor cells, virallyinfectedcells, orimmunecellsthatrecognizeself • Is requiredintheimmunesystemforthematuration, selection of lymphocytes

  6. The actualstepsincelldeathrequire • Condensing of thecellnucleus and breakingitintopieces • Condensing and fragmenting of cytoplasmintomembraneboundapoptoticbodies • Breakingchromosomesintofragmentscontainingmultiplenumber of nucleosomes (a nucleosomeladder)

  7. Apoptosissignals • Extracellular: • A hormone - suchasthyroxinewhichcausesapoptosisintadpoletails • Lack of a „survival” signal (whichinhibitsapoptosis) suchasagrowthfactor • Cell-cellcontactfrom an adjacentcell • Toxins, nitricoxide, cytokines • Increasedintracellularcalcium→calpainproduction (calciumbindingprotease) • Intracellular: • Ionizingradiation, heat, deprivation of nutrients • Virusinfection • Oxidativedamagefrom free radicals, hypoxia • Glucocorticoids

  8. Mechanism of apoptosis: caspases • A wholefamily of proteases (about 10 inhumans) calledcaspasesarerequiredforprogrammedcell • Caspases:cyscontaining-aspspecificproteases • Theyareendoproteaseshaving an active site Cys (C) and cleaveattheC-terminalside of Aspresidues (asp) • Theyarefirstsynthesizedasinactivepro-caspases • Theseproteasesarefoundinthecellininactiveformwhich must undergolimited proteolysisforactivation • Thesecaspasesform a cascade

  9. Initiatorcaspases • Initiatorcaspasecan be activatediftheyaggregateto a criticalconcentration • The prodomain of theinitiatorcaspasescontaindomainssuchas a CARD domain (e.g. caspases-2 and -9) or a deathdomain (DED) (caspases-8 and -10) thatenablesthecaspasestointeractwithothermoleculesthatregulatetheiractivation • The activeinitiatorcaspaseactivatetheeffectorcaspases

  10. The caspasecascadecan be activatedby • Granzyme B: a serin protease (releasedbycytotoxic T lymphocytes and NK cells), which is knowntoactivatecaspase-3and -7 • Deathreceptors:Fas, TRAIL receptors and TNF receptors, whichcanactivatecaspase-8 and -10 • Apoptosome: is regulatedbycytochrome-c and the Bcl-2 family, whichactivates caspase-9

  11. Apoptosispathways Kinase Enzyme EXTRINSIC INTRINSIC Phosphatase Deathligands (FasL, TRAIL, TNF) Stimuli (Cytokine deprivation, viral infection, DNA damage, irradiation, cell stress) Caspase Deathreceptors (FasL, TRAIL, TNF) Pro-survival Cyclin, pro-apoptotic BH3 only molecules FADD DISC Anti apoptopic Bcl-2 familymembers FLIP Transcription factor Pro- Caspase-8 Bax Bak GAP/GEF Smac GTP-ase Activated Caspase-8 Mitochondria Cytc XIAP cIAP-1 cIAP-2 Survivin Apaf-1 Apoptosome IAPs Effector Caspases Caspase-9 Apoptosis

  12. Intrinsicapoptoticpathway • 1Involvement ofmitochondria: openingof a channelcalledanonspecificinnermembranepermeabilitytransitionpore • 2Collapse of theelectrochemicalpotentialacrosstheinnermembrane • 3Cytochrome C, Smac/DIABLO, Omi/HtrA2, AIF and endonuclease G leaks out of theintermembranespace and bindsto a cytoplasmic protein calledApaf-1 (apoptoticproteaseactivatingfactor-1) • 4Thisthenactivates an initiator caspase-9 inthecytoplasm

  13. Mitochondrialapoptosispathway P Bad P P Apoptoticsignals Bad P Bcl-2 Bcl-2 Bcl-2 Bcl-2 Bax Bax Bad Bax P P Cytc Apaf-1 Mitochondrion Caspase-9 Cytc Apoptosome PT Pore Caspasecascade

  14. Permeabilitytransitionpore • Outermembrane protein (porin, thevoltage-gatedanion channel - VDAC) • Innermembrane protein (adeninenucleotidetranslocator – ant) • Thischannelpassesanythingsmallerthanmolecularweight 1500.   Collapsingthe proton gradientuncouplesoxidation and phosphorylationinthemitochondria

  15. Apoptosome Cytc Apaf-1 Apaf-1 Cytc Apaf-1 Apaf-1 Cytc Cytc Apoptosomeformation Recruitment of Procaspase-9 Apaf-1 Apaf-1 Apaf-11 Cytc Pro caspase-9 Cytc Cytc Caspaseactivation

  16. Bcl-family Anti-Apoptotic Bcl-2, Bcl-XL Mcl1, CED9 BH4 BH3 BH1 BH2 TM A1, Bfl-1 BH4 BH3 BH1 BH2 Pro-Apoptotic Bax, Bak Diva BH3 BH1 BH2 TM TM BH4 BH3 BH1 BH2 Bcl-Xs TM BH4 BH3 Bik, Bim BH3 TM BH3 Bad, Bid, Egl-1

  17. Whatcausesallthesechangesinthemitochondria? • Disruption of ox-phos. and electrontransport, causedbyirradiationandcertainsecondmessengerssuchasceramide • Changesincellredoxpotential and generation of reactiveoxygenspecies (ROS) • Damageto DNA causedbyradiation, ROS, etc.  A protein called p53 is oftenexpressedincellswith DNA damage.  Expression of this protein resultsininhibition of celldivision, orapoptosis, bothofwhichwouldkeepthedamagedcellfrombecoming a tumor cell.  Hence the p53 gene is a tumor suppressorgene.  It is inactivatedbymutationinapproximately 50% of all human tumor cellsstudied.  p53 caninducegeneexpression.  Of the 14 differentgeneswhoseexpressionaresignificantly altered by p53, manyseemto be usedbycellstogenerateorrespondtooxidativestress.   Cellsundergo p53 apoptosisthroughoxidativedamage. • Increasesinintracellularcalciumionsthroughsignaltransduction

  18. Apoptosispathwaysinactivated T cells

  19. Extrinsicapoptoticpathway:deathreceptors • Activatedimmunecellsstart expressingFas a fewdaysafteractivation, targetingthemforelimination • SomecellswhichhavebeenstressedexpressbothFas and Fasligandandkillthemselves • Variouscellsexpress CD95 (Fas), butCD95L (Fas-Ligand) is expressedpredominatelybyactivated T cells

  20. Role of deathreceptors: Fas • FAS receptor (alsoknownas Apo-1 or CD95): • FADD (Fas-associateddeathdomain)  bindstotheaggregatedcytoplasmicdomain (thedeathdomain) of CD95 • Recruitsinactivecaspase-8 and 10tothe site → death-inducingsignalingcomplex(DISC)

  21. TNF receptor mediatedapoptosisI FasL TNF TNF Fas/ CD95 TNFR-1 TNFR2 Daxx DAPK c-IAP1/2 TRADD FADD TRAF2 ASK1 RAIDD Caspase-8,-10 FADD RIP RIP TRAF2 Caspase-8,-10 ASK1 APO-3L/TWEAK APO-2L/TRAIL DR3 APO-3 DR4/5 FADD TRADD Caspase-8,-10 FADD RIP Caspase-8,-10 TRAF2

  22. TNF receptor mediatedapoptosisII FasL TNF- TNF- APO-3L/TWEAK APO-2L/TRAIL Fas/ CD95 TNFR-1 TNFR2 DR3 APO-3 DR4/5 Daxx c-IAP1/2 DAPK TRAF2 TRADD TRADD FADD FADD ASK1 RAIDD Caspase-8,-10 Caspase-8,-10 FADD FADD RIP RIP UB RIP UB TRAF2 TRAF2 ASK1 NIK FLIP Caspase- independent celldeath Bid IKK Smac lB tBid MKK7 NFB HtrA2 NFB FLIPs Bcl-2 JNK Cytc Bcl-2 xIAPs Apaf-1 Caspase-9 Caspase-6 Caspase-3 Caspase-7 Lamin A Actin Fodrin Gas2 Rock-1 Acinus ICAD PARP CAD Cell shrinkage Membrane blebbing Chromatin condensation DNA fragmentation DNA repair Apoptosis

  23. TNFR signaling • TNF-R1 is expressedin most tissues→soluble and membranebound TNF • TNF-R2is foundonlyincells of theimmunesystem→onlymembranebound TNF • Effects: • IKK → IkB →NFkB→Transcription of proteinsinvolvedincellsurvival and proliferation, inflammation, andanti-apoptoticfactors • MKK7 →JNK→Ap-1 →Celldifferentiation, proliferation, pro-apoptotic • Caspase-8→ Caspase 3 →Apoptosisinduction • Caspase-8 →Bid→Apoptosisinduction

  24. Controllingapoptosis • Apoptosisinhibitors: Bcl-2 and Bcl-X • Theyhave a hydrophobictail and bindtotheoutsidesurface of mitochondria and otherorganelleslikethenucleusandendoplasmicreticulum • Bcl-2 canalsobindto Apaf-1 and inhibititsactivation of initiator caspase-9 • Overexpression of Bcl-2 cancause a celltobecomea tumor cell. SomevirusmakeIAP’s (Inhibitors of APoptosis) • Bcl-xLinhibitstheformation of thesuper-molecularholesbyBax, Bak, Bid and cardiolipin. • Anothermember of thefamily, BAX and BADbindtomitochondria and facilitateapoptosisbystimulatingcytochrome C release

  25. BID a bridgebetweentheextarcellular and mitochondrialapoptosispathways • Activatedcaspase 8 causesthecleavage of theaminoterminalportionofthecytosolic protein Bidtogeneratet-Bidthat is translocatedintomitochondriaduringapoptosis • Bid = BH3 interactingdomaindeathagonist, is a pro-apoptoticmember of the Bcl-2 protein family • BidinteractswithBaxleadingtotheinsertion of Baxintotheoutermitochondrialmembrane • Bax is believedtointeractwith, and inducetheopening of themitochondrialvoltage-dependent anion channel, VDAC • The anti-apoptotic Bcl-2 proteinsmayinhibitapoptosisbysequestering BID, leadingtoreducedBaxactivation

  26. Effectormolecules • Caspaseactivation→DNA endonucleaseactivation→DNAdamage • Caspase 3 cleavesgelsolin→ cleavesactinfilaments→membranechanges • Whencellsundergoapoptosis, Phosphatidyl-serinenormallyfoundonlyintheinnerleaftlet, is exposedtotheoutside→ Itcanthenbindtoreceptorsonphagocyticcells • Caspase 3 activatesp21-activated kinase 2 (PAK-2)→ formation of apoptoticbodies

  27. Membranelipidtransportwithscramblases • Scramblasesaremembers of thegeneralfamilyoftransmembranelipidtransportersknownasflippases, theycantransport (scramble) thenegatively-chargedphospholipidsfromtheinner-leaflettotheouter-leaflet, and vice versa • Phosphatidyl-serine is translocatedtotheoutermembrane→ providing a phagocyticsignaltothemacrophagesthatengulf and cleartheapoptoticcells

  28. PS labellingwithAnnexin V Apoptosis Annexin V Annexin V binding Ca2+ Ca2+ Ca2+ Ca2+ Cytoplasmic membrane Phosphatidyl serine Normalcell Apoptoticcell

  29. Efferocytosis • The effect of efferocytosis is that dead cells are removed before their membrane integrity is breached and their contents leak into the surrounding tissue. • This prevents exposure of tissue to toxic enzymes, oxidantsand other intracellular components such as proteases and caspase. • Mediated by macrophages, DC, fibroblasts, and epithelial cells

  30. Cellsurfaceeventsalsocaninhibitapoptosis • Binding of "survival" factors (likegrowthfactors) tocellsurfacereceptorscanshutofapoptoticpathwaysinthecells • TheyarecoupledtoPI-3-kinase(phosphoinositol-3-kinase) throughthe G protein ras(p21) →produces PI-3,4-P2 and PI-3,4,5-P3, whichactivatesAkt, a Ser/Thr protein kinase→ phosphorylatestheproapoptotic-protein BAD, whichthenbecomesinactive • ActiveAktphosphorylatesprocapse →whichwillnotinteractwithcytochrome C, hence inhibitingapoptosis

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