Apoptóza - arcomooio Apoptó; historie výzkumu Vogt mu-jriiäiäE cell dsälľi oacurs norniaiy dunng vatleHals dovekjpmonf [i] 0*1 ttM#i i* adh» process: fcfc/ifj ot ctevalopmerta] Bird honmnalN r*gulal&J cel **lh P5J__________ Obwvjnion oř ONA lnMara[fl]L |Lhkagti L.J '."Hall UašJhůnd DNA Üifiiil^lior. ;1;|. LHkaflS Ol apsfinnir; :inri HMA rfugradaliOn : 1ŕ]. /Programmed eeu deaur jUMIO rlflsnibstsi " diairs d j 1ha worm dedicated 1o cáimaeďh |13|| First :;ri DKI-'CŕU Mertllucalion <* DIABLO*«*« Bŕl kumdign mdlí kí prtcessed ;asKiee SJ iŕl! Inn&Mon of ca dorih cjfi křfid n autolmmuw fjljMj [83] UnfffcalianoICTLIdfcHi Únanzymt &) and aůc-ptos!* (Yfa caepasaa) [ď?l Fas arfdTTF k*ig bhKmdJb/ caspaee liHiblhr tOWA)[44JSj 1 -JllElf Ol l^tttre * O.** Wj-a^gggg^ C/t C ana dATf can act h a twnglgK m adície q^Macapsí luutáliuriĽj puiTim .jin'Fľ.'-" like protaln, AjktM m_____ Traislocallc-ue Invohwg IAP-2 and MLTrpzracaifasa in MALT lymphoma (75) Ca spase ;: Diŕud InlafacSon iĽou»iíiejit doma«» «í . uirec: line >[«Íj;ttndCEL>B|fi5-T1| E£S*.-1. a.6H3 orty pjnlcin csserilul Mr prog ram med cell dealt« h fie worm |74| -jMeUcn d BH3 wilj pnüw Bim |7jj]j lÜDllilÜilitAOl para-and m st sc as pass* [7SJ junction In arlmai stpaiinwdÉla[M| Apoptóza a vývoj jedince Figure 17-36. Molecular Biology of the Cell, 4th Edition. Apoptoza a onemocnení Table 3, Diseases with Dys regulated Ap op to si s Excessive Ap op to si s Deficient Apoptosis Degenerative neurological diseases (Al/heimer'sj Autoimmune lymp ho p rol iterative syndrome (Canale-Smith Huntington's, Parkinson's) syndrome) Aplastic anemia Graves' disease Acquired immunodeficiency syndrome Hypereosinophilia syndrome Hashimoto's thyroiditis Hashimoto's thyroiditis Lupus erythematosus Lupus erythematosus Liver Failure Lymphoma Multiple sclerosis Leukemia M ye lod ysp las ti c s y n d r o me Solid tumors Type I diabetes mellitus Type I diabetes mellitus Ulcerative colitis Osteoporosis Wilson's disease Developmental detects Chronic neutropenia Developmental defects Features of Apoptosis Vs Necrosis 1972 Kerr Wyllie Currie Apoptosis • Chromatin condensation • Cell Shrinkage • Preservation of Organelles and cell membranes • Rapid engulfment by neighboring cells preventing inflammation • Biochemical Hallmark: DNA FRAGMENTATION Necrosis * Nuclear swelling * Cell Swelling * Disruption of Organelles * Rupture of cell and release of cellular contents * Inflammatory response *mäá (A) ±^ä phagocytic cell engulfed dead cell Figure 17-37. Molecular Biology of the Cell, 4th Edition. Apoptosis Assays Apoptosis Chromatin condensation Cell Shrinkage ^ Preservation of Organelles and cell membranes Membrane Asymmetry lost and detected by Annexin V Transmission electron Micrograph Membrane BÍebbing =>r V ■ uinexin v 15 °-k......i^>y'» 10u 10 10 10 Ann-V FlTC Fragmentace jader Fragmentace cílových proteinů kaspázami PARP lamin B 113kDa (FL} 89 kDa (CF) 70 kDa (FL) 50 kDa (CF) Aktivace kaspáz MMC [li) U 6 12 24 36 Priii4i»|j-jsr-S ----- — < p35 Pruiit>|?ítn--6 ^ < ľ 11/42 — < p2G Pi<)taip;uf 7 < P20 PrncAxpAAe-fi Apoptosis -DN^ DNA Fragmentation DNA content analysis by flow. Apoptotic Cells Live cells m. (4 C O ü 1% o: dead ť*m™ř**Tum]"i rr r 2N 200 \jy^ww^"** S/G2/M I I 400 600 i ' ' ^ 800 PI ' Mitochondria and Apoptosis Membrane Potential^) Apoptotic Cells Live cells U fÜLFUU I UU l_J I l_J U U i_ I I II ,UUI_ CO : 1 -O Ü M2 4J Lo\v= dead O-. ^ mrn 10' 10' 10^ DI0C6 Apoptotic Cells Live cells o 4 J3 1 c 3 O ü v-* ■ 1-m *-*■ ^ ■ fc—^^ T Low= 10' 10 M 10 0I0C6 10 Apoptóza -modely Table 1. Evolutionary conservation of pro- and anti-apoptotic proteins3 Caenofiiabďttis pJegans Brosoph/la meJanagaster Mammalian Function Ref* CED-3 DREDDÍ.DCP-2) (l).DRONC(l). Caspases-2, -3.-9,-10,-12(1) Cysteine proteases are responsible ÍÍ.-1.I.'. Strica (Dream) (1) for cleavage oFcellular substrates: DCP-1 (II), Dries (II), DECAY (II). Caspases-3, -6,-7 (II) type lare initiator caspases and DAMM (II) contain long prodomains whereas type II are effector caspases and contain short prodomains DIAP-1 XIAP ML-IARclAP-1.clAP-2 Inlii bi tor oF apoptosis pro tei ns (lAPs) 5,66 DIAP-2. Deterin NAIFJsurvivin contain baculoviral IAP repeat (BIR) domains: DIAP-1, XIAP, ML-IAR clAP-1 and clAP-2 inlii bit caspases: su rvi vin appears to regulate cell-cycle progression Fteaper (AVAF). HID (AVPF), SM AC (DIABLO) (AVPI) Pro-apoptotic proteins prevent lAPsfrom 13.B7 Grim (AlAY) inhibiting caspases CED-4 DARK (DAPAF-1 or HAC-1) APAF-1 Adapter proteins oligomerize. bind and activate cysteine proteases 13.Ď4.5S CED-9 BCL-2 homolog? BCL-2(BH1-4), BCL-XlÍ.BHI-4). BCL-W(BH1-4). MCL-1 (EH1-4). Al (BH1-4), EOO (DIVA) (BH1.2.4) Anti-apoptotic proteins,: CED-9 directly inhibits CED-4: mammalian hornologs contain multiple BCL-2 homology (BH) domains and prevent activation oF APAF-1 by inhibiting the release oF cytochrome c From mitochondria 64,69 EGL-1 BIK(NBK)(BH3), Pro-apoptotic BH3-only proteins 28,64 ceBNIP-3b BAD (B H 3), BID(BH3), HRK(DPS) (BH3). BIM (BOD)(BH3). BLK (BH3).NIX(BH3). BNIP-3b(BH3), NOXA(BH3) heterodimeriie via the BH3 domain with anti-apoptotic CED-9 or BCL-2 proteins and inhibit theiranti-apoptotic Function DEBCL(DROB-1.DBOK BAX(BH1-3). Pro-apoptotic BAX-like proteins promote 69-71 orDBORG-1) BAKÍBH1-3), BOK(MTD)(BH1-3). BCL-XgtBHS^) cytochrome c release From mitochondria and activMtii m of ct.-.|i.tm:-. NUC-1 dCAD DFF (CAD), DNAse II, DNAse-?. NUC-1B. NUC-70 Nucleases mediate DNA fragmentation: DF F4 0 or CAD a ppea r to be the most important and are activated by caspase degradation oF associated inhibitors. DFF45orlCAD 72-74 ■APbreirlatlons: APAF-1, apoptotlc protease-actlvatlng factor 1; dAP-1 cellular Inhibitor or apoptoslsprotein 1 ; BH, BCL-Z homology domain; NAIP. neuronal apoptott Inhibitory protein; XIAP, X-llnKe.d IAP 'ceBNIP-1 contalnsa BH3domain butdlmerlzes through allernaledomalns. Caenorhabditis Elegans Why study worms? * Reproduce very rapidly, (3 week life span) * Easy to induce mutations with ethyl methylsulfonate (EMS) * Capable of reproducing as Hermaphrodites. * Simple organism with only 1090 somatic cells , * Development is invariant and has been mapped such that the fates of all cells are known. Caenorhabditis Elegans Apoptosis • 131 of 1090 somatic cells normally undergo PCD. • Death of these cells is not required for viability. • Special optics can be used to observe abnormal deaths in 1 i vine oreanisms. ĽM S Treat Examine for Characterize the ------------► ------------► Worms excess live cells mutant gene Dom-Recessive I. niquc'.'-Cloning These studies demonstrate "genetic" nature of PCD or apoptosis Molecular regulation of Apoptosis: C. Hlcgans: TRA-1A Transcriptional repression Hinds and inhibits CJed-9 E ill-1 Binds and inhibits Ced-4 Binds and Activates Ced-3 bv ■ oligonicrization Substrate cleavage and Death Caspase Caspase's JüC V 274 Píl 20049 • First identified as the enzyme which activates (converts) Interleukin Iß (ICC). • Cysteine protease which cleaves after Aspartic Acid. (Asp) • Activated by proteolysis (after Asp). • Substrates include themselves and other Caspase's • Thus amplification cascades are possible. • Apoptosis substrates are numerous (-40 and rising) and include PARP, DFF(ICAD), BID. Caspase Structure and Regulation Box I.General principles of caspase activation Caspases are cysteine proteases that cleave substrates after specific aspartate residues. The specificity of target sites seems to be determined by a four-amino-acid recognition motif, as well as by other aspects of the three-dimensional structure of the target protein. Caspases are synthesized as proenzymes that are activated through cleavage at internal aspartate residues by other caspases (Fig. I): however, caspases might also have weak catalytic activity in their unprocessed form. Proteins such asC gtegansCEDAcr its mammalian homolog Apaf-1 can bind to procaspases and can also multimerize. Multimerization might support cross-activation of adjacent caspase zymogens. Activated caspases consist of dimers of a large and a small subunit that, together, form the active site of the enzyme. Structures obtained by X-ray crystallography suggest that these heterodimers themselves dimerize to form an enzyme with two active sites. Procaspases are often divided into two classes: those with long M-terminal domains are termed initiator caspases. and those with short N-terminal domains are called executor caspases. Long prodomains can bind to activator molecules, such as Apaf-1. or adaptor molecules associated with membrane receptors, such as Fas. It is thought that long prodomain caspases activate short prodomain caspases: however, this assertion is only supported by a limited number of experiments. Cleavage site Prodomai N-tsrrninus ;l. Cleavage ails Large subunit Small subunit C-terminua t Catalytic cysteine TRSlDSin Ceti Bioiogy Figure I. caspase 9 ft 10 B 7 1 4 S 11 12 13 H other names ÍMCH S, MACH, FUČE] (MCIM) 3 1CPP3?. Apopain Yama] (UCH 2) (MCH-3. ICE-LAP3. CMH-1J dCEj (ICH-3. TX. IC^oill) (lO+O. Tr. ICFre,IH> {ERICE) (MICE) Caspases OSYt> 3É KM 1/FTfl ± ItALr ± IETD ± TPUÍ1 1 iriAii 3 zt wťHii i ťi\'Fjn i ± t V.'J'.-J ± i • n> ± preferred substrate DETU DEHD LÉHO LETO l FW hi vu VEHD DEVD WEHD (WL)EHD (WL)FHIl WEHD" wEHO* WEHD* apoptosis initiator apoptosis effector cytokine maturation Apoptosis Signaling: Annu Rev B i »ehem. Vít1) Pľ_ 217-24 >, 2ÜÜU (A) procaspase activation NH2 cleavage sites ;-----------r active caspase LI ■ COOH activatto by cieavage age i large subunit M small subunit inactive procaspase prodornain active caspase Figure 17-38 part 1 of 2. Molecular Biology of the Cell, 4th Edition. Table 2. Target Proteins of Caspases Cytoskeletal proteins Actin, ß-catenin> fodrin, gelsolin, gas2, keratins Nuclear proteins Lamms, Rb protein, Spl, IkB-ö, DNA-dependent protein kinase, poly(ADP)-ribosylating protein (PARP), Mdm2, Ul-70 kD subunit of small nuclear ribonucleoprotein, topoisomerases I and II, h Í stone Hl, h n RP CI and C2, differentiation specific element binding protein (DSEB)/ RF-C140, dentatorubral-pallidoluysian atrophy gene protein (DRPLA), sterol regulatory element binding protein (SREBP) Regulatory proteins Procaspases, focal adhesion kinase (FAK), protein kinase cô, prese nil Í n 1 and 2, rabaptÍn-5, MAPK/ERK kinase kinase 1 (MEKKl), PAK2/hPAK65, PITSLRE protein kinase, Huntington, D4-GDI (GDP dissociation inhibitor), phospholipase A2, DNA fragmentation factor (DFF-45) or inhibitor of caspase activated Dnase (ICAD), Bel-2, Bcl-xL, p28 Bap31 ICAD - příklad substrátu Factor deprivation Death factor Tf-ray Caspase3 genotoxic anti-cancer drugs Cleavage of many death substrates CAD ICAD t ICAD /: nuclaus ICAD CAD m RNA Degradation of DNA CAD ■u bs (rates APOPTOSIS -* Cell membrane *N------? Death receptor Cytoplasm Cell death D Cell death protein in C. elegans. Drosophita and mammals D Cell death protein in Dfosophila and mammals □ Cell death protein only in Dfosophila n Cell death protein in C. elegans and mammals 3 Cell death protein only in mammals TRENDS in Celí Bioíogy Figure Z Pathways that reg u I ate c as pa ses .This figure summarizes three major pathways leading to caspase activation as gleaned from studies in mammals, Drosophita and C. elegans. The evidence used to draw this figure comprises both genetic epistasis studies and biochemical experiments. Membrane receptor complexes, such as Fas or TNF receptor complexes, can aclivatecaspases directly following receptor aggregation. Mitochondrial proteins, including members of the Bci-2 family, control caspase activity by regulating caspase activators such as the C elegans protein CED-4 or its mammalian homolog Apaf-1. CEĽMand Apaf-1 promote caspase activation by acting as scaffoids, thereby allowing cross-activation of adjacent caspase zymogens [61. IAP (inhibitor of apoptosis) proteins inhibit apoptosis by binding to and inactivating mature caspases. Molecular regulation of Apoptosis: C. Hlcgans Vs Mammals Core pathway Activators Bcl-2 & Ced-4 family Ces-1 Ces-2 Caspase .Ced-9. X V Egl-1 —► Ced-4 Ced-3 Apoptotic Death C. Elegans FAS/TNF Cxc í tot oxi city Growth Factoi Deprivation Radiation Chemotherapy APAľ-l Cytochrome C M i loc hond rial Dvs funel i on Caspase Activation Apoptotic Death Mammals 'Non Apoptotic" Death Bcl-2: Structure and Function • 1988: Bcl-2 acts by inhibiting apoptosis and I synergistic \\ ith c-myc in cancer development, I • Has transmembrane domain v\ hich targets | predominantly to Mitochondria. • Shown to inhibit cell death with little or no I stimulation of cell growth. Background: Overview of Be 1-2 Family Members _________________________________________________Q|_y_________li] 13 Rl 11 lil 12TV1 Mammals Anti-Apoptotic C. Eleüans Bcl-2 Bcl-Xj Bd-w Md-1 Al NR-13 Ced-9 U—■- U—Ľh- U—D-----D-Q -Q U—D- Pro-Apoptotic Bax Full Member Bak Bok Mammals Bcl-2 Homoloj • 1993-Bcl-2 IP identified Binding Partner-Bax • Bax Homologous to Bcl-2 • Had the opposite activity when overexpressed. ue discovered Bcl-2 Bcl-2 Survival A *t 1 T Bcl-2 (V *t ( BaxY BaxJ Death Pro-apoptotic Bcl-2 Family members Full Member BH3 Only Bax Bak Bok Bad Bik Bid Bim Noxa P04 _l_ P04 _l_ Selective regulation of Pro-apoptotic Bcl-2 family members • Bax Dimerizes and Translocates to Mitochondria • Bad is Phosphorylated and inactivated by 14-3-3 sequestration • Bid is activated by caspase 8 cleavage and induces Cyto C release Bim interacts with cyto skel e ton IF«*-L,THF) Hecepto' (Fm.TNF.H1) \ ■ ŕ *3 -ŕnltwtur ■ L- f ~\ "efíecfor' vP^ ca$pasb-a ffTFl pír ť.rflss et a!, fitnes & Development VIJ ľ£ mv Selective function of BH3 only family members Ena bier Block Bcl-2 Noxa Bad Bik P04 _l_ P04 _L Activators Bid Directly activates Bax/BAK Bim Bcl-2: Proposed Mechanisms of Action Binds and inhibits Proapoptotic Family Members Regulates ion flux across the Mitochondria and stabilizes the membrane potential (PTP) Regulates cytochrome C release. Binds and inactivates APAF1 ROS inhibition Many others: Ca Homeostasis, RAFl interaction Regulates VDAC and thus ATP/ADP ratio Regulační síť proteinu Bcl-2 rodiny DRL fBimf Bmf cytoskeleton APOPTOSIS CELL CYCLE ARREST WD repeats CI............ľ Substrate proteolysis Cellular collapse "PH? Fig. S.Cytochrome c promotes assembly oľ the apoptosome. Binding or cytochrome c toApaM promotes ollgomerlHtlonof ine latter and recmltmentorcaEpiase-g Into a mjltlmerlc Apa r-l-caspase-9 complex rnatresultslnnaspHSH-B activation, several heat-shock proteins (Hspsj might I nterfenewlth assembly or me apoptcsome, either through Interaction with cytochrome c, or through Interaction with Apa M. Inhibitor or apoptoslsi proteins (lAPsfl might Inter rere with caspase activation events downstream orapcptceome assembly bydlrecUy binding tocertalncaspases. SmadDlablo, which Is alsoreleased rrom mitochondria during apoptosls, might racllltate caspase activation In this pamway by neutralizing IAP function. T he modular structure oľApaM is Indicated wl lni n the Insert. • !•!• Další proteiny odílející se na regulaci kaspáz a apoptóz« Cpi AP Cpi AP 275 Ol API DIAF2 defiUCE 3ctcr n Drosophila HH- Mammalian "ŕHIHl ĽCEIR2 G eJ^gíms 303 Spi AP Sel AP Tri AP SflAP Yeast Lepidopteran l DIR RING ^GARD HEUBG lAPs - inhibitors of apoptosis Extrinsic pathway Intrinsic pathway Chemotherapy Figure 2 | The intrinsic and extrinsic cell-death pathways. In this simplified scheme, receptor-mediated apoptosis is initiated with the recruitment and activation of caspase-8. Caspase-8 can directly cleave caspase-3. The intrinsic pathway involves the translocation to mitochondria of pro-apoptotic Bcl-2 family members such as Bax, which results in the release of cytochrome c into the cytosol, oligomerization of Apaf-1 in a complex with caspase-9 (the apoptosome), and the subsequent activation of caspase-3. In some cases, receptor-initiated signals can be transduced through the mitochondrial pathway; for example, through the cleavage and activation of Bid. FADD, Fas-associated death domain protein; UV, ultraviolet light; XIAR X-linked IAR Smac/DIABLO Plasma membrane Extrinsic pathway I n tri n&l c pathway Bel proteins y—-,_________J\ ŕ Caspase-9 / L XX Oml S mac IAP Bid tBId I 1 Caspasfe-S \ Caspase-3 TÍES Death receptors and adaptor proteins CD95L TNF Apo3L TNFR1 RAF2 NF-kB \ Apoptosis I- Fig. 1. Apoptosis signaling by CD95, DD, death domain; DED, death effector domain. Fig. 2. Proapoptotic and antiapoptotíc signaling by TNFRl and DR3. FLIP no FLIP CDS5 CD05L F ADD C10 low FLIP FLIPL ""V if* 01 OfflO Bid pro-C3 P Lectin etc sctin te. apoptosis hi FLIP RIP c-FLIPL C-FLIPL ca í t proliferation? Figure 1 Model of the different functions of c-FLIPL Shorn is Ihe CD95 DISC at differentcancenlralioris oľc-FLIPL. In the absence ol c-FLIPt (no FLIP), bolh procaspase-B(Ca) and pracaspase-1ü [C 10) are recruited to the DISC Ihrough binding lo the adaptor molecule FADD. This recruilment causes processing and aclivalion ol the iniliator caspases through homadimeriiation, release oľ Ihe acLive enzymes (heterotelrameric structures). subsequent cleavage ot various intracellular caspase substrates and apoplosis. When c-FLIPL is expressed aJ low levels (low FLIP), activation ol caspase-&'-10 is accelerated due lo Lhe abilily at c-FLIPl to associate with caspase-EV-10 and i1s acLivity lo form haLenodimers more eflicienlly 1han caspases-SMO to form homodimers. AL high concenlralions ol c-FLIPi. caspase-9/-1ü are slili activated, bul are not released any longer from the DISC. According üg the model, DISC-tethered caspase-ÍMO has the same subslralespecilicily as aclivecaspase subunils released inlo Lhe cytosol. However, owing to their DlSC-proximal Iccalion, Ihesa incompletely processed, bul fully aclive, caspases cleave a dilterent set of substrates such as themselves. RIP and c-FLIPl. These cleavage events may be important in regulating apoplosis-indepenrJenl processes such as prolilieralion. The inactive aclive site in Ihe caspase domain of c-FUPL is labelled X. Decoy receptors huDRSs ■u bs (rates APOPTOSIS -* Indukce apoptózy nebo přežití buňky je vždy důsledkem integrace mechanismů regulujících apoptózu, proliferaci a diferenciaci. Rozhodující je působení vnéjších signálů (ostatní buňky v populaci, buňky imunitního systému, ECM) a vnitřních kontrolních mechanismů buňky (kontrola integrity DNA, checkpointy apod.) nebo genetického programu v dané buň. populaci.