Apoptóza - apoptosis Apoptó; historie výzkumu Vntjf racogrissE cell death oacura nomaiv úvmg VBrtaHíils davebpmonf [i] HnJ *í*iii*ihíůF*i8iaas;íf*iTeo1 devslopntartal Bird Itoriranaliy r*sulal«f cet«*1h [íj_ Obserúcu af DNA toddafa jď]}. Li-ifcjyij iA Dali daalh&nd DNA ctegrasallon | LUkajfl til Hpoplosi* and DMA rJugruilaliDii 'morsammad oell deam' J USSfl 10 rlflvrihů tpf " ijtäiri dJiin; Irwsťletrale y Fas-FasL |4Í] SlrUdUfC 0[ Ml.! ii J-.!i.n.li::ri [731 i 63-^1 Idemhralion -i' H laRl.CVRnuii: [ pmcaÉ—d a*9tm 8 [61 j UrtrfcttanolCTLklUng iHianlytne B) and ipůpíásl* lva caapases) [J7| Fas arid TTF kJ*nq caspase irfih; (t[gjiandCEL>B|65-Ti| Efil-1. a.6H3 orly pjnlein raxenl&J Mr programmed«11 d sainlilrw *onn |74|| —|Daie1loinH BH3 ôňfr prrJíim Blm |7j]j pani-and i(ia1siaspassa[79J C^spaSĽ ii4iftijúr& liMKtJon In animal sepaiimffllÉla [M| Apoptóza a vývoj jedince 1 mm Figure 17-35. Molecular Biology of the Cell, 4th Edition. Table 3, Diseases with Dys regulated Ap op to sis Excessive Ap op to sis Deficient Apoptosis Degenerative neurological diseases (Alzheimer's, H u nt i ng, to n1 s, P a r ki n s on 's) Aplastic anemia Acquired immunodeficiency syndrome H a s h i mot o5 s t h v ro id it i s ■ Lupus erythematosus Liver failure Multiple sclerosis Mye lodysp las ti c syn d ro me Type I diabetes mellitus Ulcerative colitis Wilson's disease Chronic neutropenia Developmental defects Aut o immii ne 1 y mp ho p r ol i fe ra t i ve sy nd ro me (Can ale- S m it h syndrome) Graves' disease Hype r eo s i no ph i li a sy nd r o me Hashimoto's thyroiditis Lupus erythematosus Lymphoma Leukemia Solid tumors Type I diabetes mellitus Osteoporosis 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 Apoptosis Assays Apoptosis Chromatin condensation Cell Shrinkage Preservation of Organelles and cell membranes Membrane Asymmetry lost and detected by Annexin V Trims miss ion electron Micrograph Membrane Blebbtng 10 10 10 Ann-V FlTC Fragmentace jader Fragmentace cílových proteinů kaspázami PARP lamin B 4— 113 kDa (FLJ 4— 89 kDa [CF) 4— /O kDa (FL) 4— 50 kDa (CF) Aktivace kaspáz MMC [10 0 6 1Z 24 36 P]mui|P4Jir:-S Prótaí|jjir-S Procaipa&e 7 Apoptosis -DNA fragmentation Assay DNA Fragmentation DNA content analysis by flow. Apoptotic Cells Live cells y^ijyyij i i_j i— t■ i i ^vu.uul .iilfll 0.5 - *!!! } 1% 2N j dead S/G2/M 1 (—» Ill 1 I 1—1—'-1-1-r™^^^^ 200 400 600 eoo 1000 Apoptotic Cells - 1000 PI PI Mitochondria and Apoptosis Membrane Potential^) Apoptóza -modely Table 1. Evolutionary conservation of pro-andanti-apoptotic proteins Caenortiabditis elegans Drosophtia m eJan oga slp-r Mammalian Function Ref* CED-3 □ REDD (DCP-2) (1). DRONC (1), Caspases-2, -3. -5,-10. -1 2 (1) Cysteine proteases are responsible i Í.-1.ľ. Strica (Dream) (1) for cleavage oFcellular substrates: □CP-1 (II), Dries (||), DECAY (II). Caspases-3, -6, -7 (II) type 1 are initiator caspases and DAMM (II) contain long prodomains whereas type II are eFTec rorcaspases and contain short prodomains □ IAP-1 XIAfi ML-IAR clAP-ljClAP^ Inhi bi tor oF apoptosis pro tei ns (lAPs) 5,06 DIAP-2. Deterin NAIR survi vin contain baculoviral IAP repeat (BIR) domains: DIAP-1, XIAP, ML-IAR clAP-1 and c IAP-2 inhibit caspases: survi vin appears to regulate cell-cycle progression Reaper (AVAF). HID (AVPF). SMAC (DIABLO) (AVPI) Pro-apoptotic proteins prevent lAPs From 13,67 Grim (AIAY) inhibiting caspases CED-4 DARK (DAPAF-1 orHAC-1) APAF-1 Adapter proteins oligomerize. bind and 1S.54.6S activate cysteine proteases CED-B BCL-2 homology BCL-2 (BH1-4), An ti-apoptotic proteins: CED-0 directly 54 69 BCL-Xl (BH1-4). inhibits CED-4: mammalian homologs BCL-W(BH1-4). contain multiple BCL-2 homology (BH) MCL-1 (BH1-4). domains and prevent activation oF AI (BH1-4), APAF-1 by inhibiting the release oF BOO (DIVA) (BH 1.2.4) cytochrome cfrom mitochondria EGL-1 - 111 K (NBK) (BH3), Pro-apoptotic BH3-only proteins 28,64 ceENIP-3b BAD (BH3). heterodimerize via the BH3 domain BID (BH3), with anti-apoptotic CED-9 or BCL-2 HRK(DPS) (BH3). proteins and inhibit theiranti-apoptotic BIM (BOD) (BH3). Function BLK (BH3).NIX(BH3>. BNIP-3b(BH3). NOXA(BH3) DEBCL (DROB-1. DBOK BAX(BH1-3). Pro-apoptotic BAX-like proteins promote 69-71 or DBORG-1) BAK (BH1-3). cytochrome c release From mitochondria BOK (MTD) (BH1-3). and a c ti vat ion oF caspases BCL-Xg (BH3.4) NUC-1 dCAD DFF (CAD). DNAse II. DNAse-?. Nucleases mediate DNA Fragmentation: 72-74 NUC-13. NUC-70 DFF40 or CAD appear to be the most important and are activated by caspase degradation oF associated inhibitors. DFF45 orlCAD 'Abbre'vlallons: APAF-1. apoptotic protease-actlvatlng factor 1; clAP-1, cellular InnIrjlLor or a poptosls protein 1; BH, BCL-2 homology domain; NAIP. neuronal apoptott Inhibitory protein; X1AP. K-l nUBd IAR 'ceBNIP-3 contalnsa FJH3 domain butdlmerlres through alternate domains. 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 livins organisms. *__ <__ EMS Treat Examine for Characterize the -► -► Worms excess live cells mutant gene Dom-Recessive I nKjLie?-( loning These studies demonstrate "genetic" nature of PCD or apoptosis Molecular regulation of Apoptosis: C. Elcaans: TRA-1A I nstriptional repression Hinds and inhibits Ccd-9 Ed-1 Binds and inhibits (ed-4 Binds and Activates Ced-3 ■ oli"onir rization Caspase Substrate cleavage and Death Caspase's J BO V 274 Pii 2004') First identified as the enzyme which activates mf (converts) Interleukin 1 [3 (ICC). Cysteine protease which cleaves after Aspartic Acid. (Asp) Activated by proteolysis (after Asp). Substrates include themselves and other Caspase Thus amplification cascades are possible. Apoptosis substrates are numerous (-40 and rising) and include PARP, DFF(ICAD), BID. Caspase Structure and Regulation Box 1. General principles of caspase activation Caspases are cystine 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 slogans CED-4 or 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. CI 9av3g 9 sito Cleavage aitB Prodomain N-terrninus Large subunit Small subunit C-terminus Catalytic cysteine ~RZ\'DS in Ce>" Biology Figuře I. Caspases caspase 9 a lü 6 7 I A 5 II 12 Vi u other names Cod-3 {iCH-i.NBdd a) fMCH e. ICE-LA PC J [MCHS, MACH, FLfCEj ^ (MCI I-4) -E CARD 3 Apopain Yanw] (MCH-3) (MCI1-3. ICE-LAP3. CMhM) (fCE) IICH ?. TX. IC^eill) (ICH-3. TV. ICE^III) CARD {EHICE) (MICE) DSVO V FTFl IEAD ± IL EU 3T J ± ± preferred substrate DETU DEHD L£HD LETD I I KIE3 DEVD VEND DEVD WE HD (VVLJEHD (Wl )EHD WEHD-WEKr WE HD' YVEHD* Lipo ptOSiS initiator a po ptosis effector cytokine maturation Apoptosis Signaling: Annu Rev Biuchem. v69 Pg. 217-24>_ 200Ö (A) procaspase activation NH2 cleavage sites active caspase □ activation by cleavage COOH inactive procaspase large subunit M small subunit prodomain active caspase Table 2. Target Proteins of Caspases Cytoskeletal proteins Act in, jS-catenin, tod r in, gelsolin, gas 2, keratins Nuclear proteins Lamins, Rb protein, Spi, IkB-o, DNA-dependent protein kinase, poly(ADP)-ribosylating protein (PARP), Mdm2, Ul-70 kD subunit of small nuclear ribonucleoprotein, topoisomerases I and II, histone HI, hnRP CI and C2, differentiation specific element binding protein (DSEB)/ RF-C140, dentatorubral-pall idol uysian atrophy gene protein (DRPLA), sterol regulatory element binding protein (SREBP) Regulatory proteins Procaspases, focal adhesion kinase (FAK), protein kinase cS, prese nil in 1 and 2, rabaptin-5, MAPK/ERK kinase kinasel (MEKK1), 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 ^ Mitochondrial pathway} subsists* APOPTOSIS -* *N-7 Death Cell membrane receptor Cell death ! ] Cell death protein in Celegans. Drosophila and mammals _| Cell death protein in Drosophila and mammals —Cell death protein only in Drosophila ] Cell death protein in Celegans and mammals ~~\ Cell death protein only in mammals TREWS in Celt Biology FigureZ. Pathways that regulatecaspases.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 acli vatecaspases 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 homoiog Apaf-1. CE&4 and Apaf-1 promote caspase activation by acting as scaffolds, thereby allowing cross-activation of adjacent caspase zymogens |"6K 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 Caspase Ces-1 Ces-2 Ced-4 Ced- FAS/TNF Excitotoxicity Growth Factor Deprivation Radiation Chemotherapy APAF-1 Cytochrome C Mitochondrial Dysfunction Apoptotic Death C. Elegans Caspase^^^ Apoptotic Activation Death Mammals "Non Apoptotic" Death ■ Bcl-2: Structure and Function |..«* BC-2 actS ,v inhibiti„g aP0Pt0sis a„d I synergistic with c-myc in cancer development. I • Has transmembrane domain which targets I 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 KM4 BID Bill Bl I2TM Mammals Anti-Apoptotic C. Ele^ans i. Bcl-2 Bcl-x Bcl-vv Mcl-1 Al NR-13 Ced-9 ■a—o- ■a—o- a—■ -a—□—q-q Pro-Apoptotic Bax Full Member Bak Bok Mammals BH3 Only Bcl-xs Bad Bik Bid Bim Noxa p£!_ _5 1-1 -□—a -D -D- O ■D-D- Bcl-2 Homologue discovered • 1993-Bcl-2 IP identified Binding Partner-Bax Bcl-2 Bcl-2 Survival It Bax Homologous to Bcl-2 Bcl-2 Had the opposite activity when over expressed. (^Bax^ t Death Pro-apoptotic Bcl-2 Family members Full Member BH3 Only Bax Bak Bok Bad Bik Bid B N mi oxa PG4 I P04 I I I Cr-Q 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 Cylo C release Bim interacts with cy to skeleton Gross el a!, Clencs & Development VIJ 1"« WW Selective function of BH3 only family members Ena bier Block Bcl-2 Noxa Bad Bik P0<1 I K>4 _L Ac Li valors Bid Direcllv 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, RAF1 interaction • Regulates VDAC and thus ATP/ADP ratio Regulační síť proteinů Bcl-2 rodiny DRL ÁPOPTOS1S CĽLL CYCLĽ Á K REST WD repeats Hsp-70 Substrate proteolysis Cellular collapse "PIT? Dalsi proteiny podilejici se na regulaci kaspaz a apoptozi fig. 3. cytochrome c promotes assembly or the apoptosome. bl ndlng of cytochrome c to apar-1 promotes oligomer izallon of me latter and recrullmenlorcas.pase-9 into a mjlllmerlc apari-caspase-9 complex mat results i ncaspase-9 activation. several hart-shcx* proteins (hspsj might i n terrene with assembly or ine apoptosome, either through interaction with cytochrome c, or ihrough interaction wlthapar-l. inhibitor or apoptoslsproteins(laps) might interfere with caspase activation events (lovriistrearn orapcptosome assembly bydlreclly binding locertalncaspases. smacjdiablo, which is alsoreleased rrom mitochondria during apoptosls, might racllltatecaspase activation in this palflway by neutralizing iap function. the modular structure of apam is indlcaled within the insert. QplAF 01 API DIAF2 dBRUCE 3ctcr n CeGIRl CcSIRS SplAP SclAF TrlAP Drosophiia lAPs - inhibitors of apoptosis i predicted) Mammalian MS Yeast 937 9Hd Lepidopteran - — 373 377 □IR MRIN3 W/KARD HEUBG 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 Bel-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 Death receptors and adaptor proteins CD35L TNF A|x>3L TNFR1 DR3/ Apo3/ W&I1 TRADD FADD .TRADD fRAF2 V NIK IKK NF-kB \ Apoptosis I- I-kB/NF-kB - NF-kB Fig. 1, Apoptosis signaling by CD95. DD. death domain; DED. death effector domain. Fig. 2. Proap opt otic and antiapoptotic signaling by TNFR1 and DR3. no FLIP low FLIP hi FLIP CD95 CD85L FADD C10 FLIPL;^ *f oi OfflO Bid PFO-C3 Pfectfn etc ■ttin I it. ± { RIP C-FLIPl apoptosis proliferation? Figure 1 Model of the different functions of c-FLIPL Shorn is Ihe CD95 DISC al ditfenenl cancenlraliona oi c-FLIPL. In the absence otc-FLIPL (no FLIP), bolh procaspase-6(Ca) and procaspase-10 [C1Q) aie recruited Id the DISC Ihrough bindino lo the adaptor molecule FADD. This recruitment causes processing and aclivalion oi the iniliator caspases through homadimeriation, release of Ihe active enzymes (heterotelrameric shuchjres), subsequent cleavage nt various intracellular caspase substrates and apophysis. When c-FLIPL is expressed aJ lo* levels (lew FLIP), activation ol caspase-BMO is accelerated due lo the ability at c-FLIPl Id associate with caspase-R'-IG and i1s activity lo form haterodimers more ellicienlly 1han caspases-SMO lo form homodimers. At high concenlralions ol c-FLIPl, caspase-uMO are sfill activated, butare not released any longer from the DISC. According Id the model, DlSC-tethened caspase-SMQ has the same sutelrale specificity as active caspase subunils released inlo the cytosol. However, owing to Iheir DlSC-proximal localion, Ifiesa incompletely processed, bul fully aclive, caspases cleave a diherent set of substrates such as themselves, RIP and c-FLIPl. These cleavage events may be important in regulaling apoplosis-independenl processes such as proliferalion. The inactive aclive site in Ihe caspase domain of c-FLIPL is labelled X. Decoy receptors ;RD2 CRD3 DD hu DR5s 1 CRD2 CRD3 1 CRD2 CRD3 DD DD hu DR5I hu DR4 CRD2 CRD3 i ii iii iv V hu DcR1 CRD2 CRD3 1---- DD hu DcR2 1 CRD2 CRD3 T M- DD ■ mu DR4/5 1 CRD2 CRD3 c;HD4 : huOPG Smae 0mi APAF-1 ID ^ Mitochondrial pathway} subsists* APOPTOSIS -* Indukce apoptózy nebo přežití buňky je vždy důsledkem integrace mechanismů regulujících apoptózu, proliferaci a diferenciaci. Rozhodující je působení vnějších 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.