Apoptóza - a7io7iToaia Apoptóza -historie výzkum u VotjlTLiiiyriiMS cell deartli oocura "cuiifir;,1 1B4Z flliKksrTvínrfs ií iwiví úľ tál dearth durkig '-ariebrato davaloproenl [ŕ] Karťa fmt paper op mil i ■ Otf chuti i* flťJhřft piBfaas; inUig ort develop™rial Bird hormonaliy rsgulalcrJ ^e* c(55)- ISutalkU Ci mammafcin CECm I protein, ^ ..i : 1^53_ . ■ i .' .n i cATiJ can aaM in MALT lymphoma (75) MerllhKalisn flí [>l*8L{VSmíic [77.7S] Bft kneiadion moiir ifi pi (.ietsf ä casrŕaas 8 [B1i _Ed-1, a 6H3 arly pjnlein ĽsaEfilul far |prijgrammed «II deamínlrw worm |74| —|DalertbMm< BHS vn^'pmim Bim |7j]j ldaiŕi1iCŠ*Ľíi Ql para-arid iiialíjiíisfiďse j [7ä] CaspiSĽ iiÉiil.jrS liMKtJon In animal jHpii»rtiŕJrii[M| Apoptóza a vývoj jedince Apoptóza a onemocnění Table 3. Di se ti ses with Dys regulated Apoptosis Excessive Apoptosis Deficient Apoptosií Degenerative neurological diseases (Alzheimer's, H u nt i ng to n' 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 lody sp las ti c syn d ro me Type I diabetes mellitus Ulcerative colitis Wilson's disease Chronic neutropenia Developmental defects Aut o im mu ne ly nip ho p rol i re ra t i ve sy nd i o me (Ca n 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 e ryt hem a tosus 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 * Transmission electron Micrograph Membrane Bf ebbing 101 10£ 103 Ann-V FITC Fragmentace jader 0 Fragmentace cílových proteinů kaspázami PARP 113 kDa (FL} 89 kDa [CF} — —, lamin B 70 kDa [FL] 50 kDa [CF) Aktivace kaspáz MMC [li) U ů 12 Z4 36 P]«f41i|3iilP-S PrůLjis|lair-fl Proca&pa&e 7 KriJť ,n p .i a o i"i Apoptosis -DNA fragmentation Assay DNA Fragmentation DNA content analysis by flow. Apoptotic Cells Live cells *J^WKt\J I I— I y i- tl I 1 "JUL S/G2/M T t i i I i r^^^r eoo PI PI Mitochondria and Apoptosis Membrane Potential^) Apoptotic Cells Live cells Apoptotic Cells Live cells DI0C6 Apoptóza -modely Table 1. Evolutionary conservation of pro-and anti-apopt otic proteins' Cae-nofiiabditis e-legans Drosaph/la m elan ocu ster IVldrnm alien Function ReFi CED-3 □ REDD (DCP-2) (1). DRONC [1), Caspases-2. -3. -8,-10, -12 (1) Cysteine proteases are responsible ■> (An'. Strica (Dream) (1) for cleavage of cellular substrates: DCP-1 (II). Dries (II), DECAY (II). Caspases-3. -tW (.11) type 1 are initiator caspases and IW.1M (II) contain long prodornains whereas type II are effee forcaspases and contain short prodomains DIAP-1 XIAP, ML-IAR clAP-1, clAP-2 Inhibitor oFapoptosis proteins (lAPs) 5,88 DIAP-2, Deterin NAIR survivin contain baculoviral IAP repeat (BIR) domains: DIAP-1, XIAR ML-IAR clAP-1 and clAP-2 inhibit caspases: survivin appears to regulate cell-cycle progression Reaper (AVAF), HID (AVPF). SMAC (DIABLO) (AVPIj 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 I&.64 Oi activate cysteine proteases CED-9 BCL-2 homology BCL-2 (BH1-4). Anti-apoptotic proteins.: CED-fJ directly i,i eg BCL-X,_ (BH1-4). inhibits CED-4: mammalian homology ECL-W (BH1-4), contain multiple BCL-2 homology (BH) MCL-1 (BH1-4). domains and prevent activation oF A1 (BH1-4), APAF-1 by inhibiting the release oF BOO (DIVA) (BH 1.2,4) cytochrome c From mitochondria EG M - BIK (NBK) (BH3), Pro-apoptotic BH3-only proteins 28.64 ceBNIP-3b BAD (BH3). heterodimerize via the BH3 domain BID (BH3), with anti-apoptotic CED-9 or BCL-2 HRK (DPS) (BH3). proteins and inhibit thoiranti-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 activation oFcaspases BCL-Xg(BH3.4) NUC-1 dCAD DFF (CAD). DNAse II. DNAse-y. Nuc leases medi a te D NA Fragmen rat ion: ?Z 74 NUC-1B, NUC-70 DFF40 or CAD appear to be the most important and are active ted by caspase degradation oF associated inhibitors. DFF45 or ICAD ■ADtjrevlatlons: APAF-1, apoplolt protease activating raclor 1; dAP-1, cellular InMIbllor or apoptoslsprotein 1; BH, HCL-2 homology domain; NAIP, neuronal apoptott Inhibitory protein; XIAR K-l nKed IAP. 'iceBNIP-3 contalnsa BHl domain butdlmerlies Ihrough alternate domains. Caenorhabditis Elegans Why studv worms? • Reproduce very rapidly, (3 week life span) • Easy to induce mutations with ethyl methylsulfbnate (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 living organisms. EMS Treat Examine for Characterize the -+■ -> Worms excess live cells mutant gene Dom-Recessive I nique'.'-C loning These studies demonstrate "genetic" nature of PCD or apoptosis Molecular regulation of Apoptosis: C. Hlcgans: TRA-l A Trim ri phonal rep rcssion Hinds and inhibits Ctd-9 Binds and inhibits ( od-4 Binds and Activates Ced-3 ■ oligonicrization Caspasi? Substrate cleavage and Death Caspase's JBC \ 274 Pa 20049 • First identified as the enzyme which activates (converts) Interleukin 1 (3 (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 1, GEneral principe of caspase activation Caspases arecysteina proteases that cleave substrates after specific aspartate residues. The specificity of target sites seems to be determined by a Four-a mi no-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 efegans CED-4 or its mammalian homolog Apaf-1 can bind to procaspases and can also multimerize. Multimerization might support cross-act i vat ion 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 N-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 Prodomain N-terrninus Clea^ge site Large subunit 4, Small subunit C-terminus t Catalytic cysteine TRB-'DS in CeHBiaiagy Figure I. Caspases caspase 10 6 7 I A 5 II 12 13 14 other names Ced 3 {ICH-l.ilBdd 2) fMCH 6. ICE LA PC J [MCH b, MACH, FIXE) (MCI 1-4} CARD CARD 3 Ap&psin Yaihal (MCH 2) (MCJI-3. ICF-LAP3. CMI+1} IICH 2. TX. IC^ajll) (ICH-3. TY. ICE^jlll) CARD (MICE) rasvr> 3i LAU ± IL EU it 3F WUHU I ± ± IV II* ± preferred substrate DETD DEHD LE.HD LFTD 11 m I IS -IJ VEHC □EVD WE HO (WLJEHD (Wl JE HD WE HD" WE HD* WE HD* WEHD* apoptosis initiator apoptosis effector cytokine maturation Apoptosis Signaling: Anim Rev Biochein. V69 Pg. 217-1.45, 2000 (A) procaspase activation NH2 I cleavage sites active caspase large subunit activation by cleavage T small subunit COOH inactive procaspase prodomain active caspase Ftgure 17-38 part 1 of 2. Molecular Biology of the Cellr 4th Edition. Table 2. Target Proteins of Caspases Cytoskeletal proteins Act in, jS-catenin, fodrin, gelsolin, gas2, keratins Nuclear proteins LamÍns, Rb protein, Spi, IkB-o, DNA-dependent protein kinase, poly(ADP)-rÍbosylatÍng 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-pallidoluysian atrophy gene protein (DRPLA), sterol regulatory element binding protein (SREBP) Regulatory proteins Procaspases, focal adhesion kinase (FAK), protein kinase c6, p resen i lín 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 ^ Mitochondrial pathway} subsists* APOPTOSIS -* I I Cell death protein in C.elegans. Drosophila and mammals [~| Cell death protein in Drosophila and mammals Q Cell death protein only in Drosophila I I Cell death protein in Cl eiegansanó mammals Q Cell death protein only in mammals TRENDS in Celt Biology Figure2L Pathways that regulatecaspases.This figure summarizes three major pathways leading to caspase activation as gleaned from studies in mammals, Drosophila and C. etegans. The evidence used to draw this figure comprises both genetic epfstasfs studies and biochemical experiments. Membrane receptor complexes, such as Fas or TNF receptor complexes, can activatecaspases directly following receptor aggregation. Mitochondrial proteins, including members of the Bcl-Z family, control caspase activity by regulating caspase activators such as the C. elegans protein CED-4 or its mammalian homolog Apaf-1. CED-4 and Apaf-1 promote caspase activation by acting as scaffolds, thereby allowing cross-activation of adjacent caspase zymogens [6]. IAP (inhibitor of apoptosis) proteins inhibit apoptosis by binding to and inactivating mature caspases. Molecular regulation of Apoptosis: C. Elcgans Vs Mammals Core pathway Activators Bcl-2 & Ced-4 family Ces-1 Ces-2 Caspase Eel-1 Ced-4 Ced-3 Apoptotic Death C. Elegans PAS/TNP Excitotoxicity Growth Factor Deprivation Radiation Chemotherapy APAr-i Cytochrome C Mitochondrial Dysfunction CaspaseApoptotic Activation Death Mammal ;tNon Apoptotic" Death ■ Bcl-2: Structure and Function . 19BS: BC,2aetsbyinhibitingaP0Pt0sisand I synergistic with c-myc in cancer development. I * Has transmembrane domain which targets I predominantly to Mitochondria. I * Shown to inhibit cell death with little or no I stimulation of cell growth. Background: Overview of Be 1-2 Family Members 1J]]3 EiEM BN2TM Mammals Anti-Apoptotic Bcl-2 Bcl-xL Bcl-w Mcl-1 Al KR-13 ■a—o- -Q {J—□-Ch-0 ■a—□— -D-Q C. Ele^ans Ced-9 ■a—□—q-q Pro-Apoptotic Bax Full Member Bak Bok Mammals BH3 Only Bcl-x Bad Bik Bid Bim Noxa ■a—o- -D -Q -D -0 Bcl-2 Homolosue discovered • 1993-Bcl-2 IP identified Binding Partner-Bax Bcl-2 Bcl-2 Survival Bax Homologous to Bcl-2 Bcl-2 Had the opposite activity when overexpressed. t Death Pro-apoptotic Bcl-2 Family members Full Member BH3 Only Bax Bak Bok Bad Bik Bid Bim Noxa P04 r< >4 0-» O-Q Selective regulation of Pro-apoptotic Bcl-2 family members. Bax Dimerizes and Translocates to Mitochondria Bad is Phosphory lated and inactivated by 14-3-3 sequestration Bid is activated by caspase 8 cleavage and induces Cyto C release Bim interacts with cyto skeleton (F*=-L, TNF) Haceptw (FaE,"fflF.R1) \ 'mit«tor' \ p22 BID easpasa-3 JOffip17 Other rmmh Subacute* (rrnsifl ill. Cecity & Devt>lfl|H]n>i]l VL3 I'fc Selective function of BH3 only family members Ena bier Block Bcl-2 Noxa Bad Bik P04 I P04 _l_ Activators 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 • Manv others: Ca Homeostasis, RAF1 interaction • Regulates VDAC and thus ATP/ADP ratio Regulační síť proteinů Bcl-2 rodiny DRL diimagĽ AťOPTOSIS CELL CVCLĽ ARKĽSľ WD repeats CED-4 Hsp-70 Substrate proteolysis Cellular collapse Dalsi proteiny podilejici se na regulaci kaspaz a apoptoze Fig. 3. Cytochrome c promotes assembly or the apoptosome. Bl ndlng of cytochrome c to Acar-1 promotes oligomer izaLlon or melallerandrecrullmenlorcaspase-9lnloa rriJlLlmeNc Apar-l-caspase-9 complex that results I ncaspase-9 actuation. Several haat-shn* proteins (Hspsj might I nterf enewlth assembly or Ihe apoplcraorne, either through Interaction with cytochrome c, or through Interaction wlthApar-l. Inhibitor or apoptoslsproteins(lAPs) might Interrerewlm caspaseactlvallonevenLs downstream orapoptosorne assembly bydlreclly binding bocertalncaspases. SmadDlablo, which Is alsoreleased rrom mitochondria during apoptcsls, might facllltatecaspase activation In this palhY/ay byneutrallilnglAPrunotlon.TnemodularstnictureorApari Is Indicated wl thin the Insert. 0IAP1 DIAP2 deftucE [Jeter n Drosophiia 4$e lAPs - inhibitors of apoptosis i predicted) CcSIR2 SplAP SclAP TnlAP Mammalian H '-Wr^rq HHubg 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-1 inked I AR Smac/DIABLO Extrinsic pathway Death receptors and adaptor proteins CD95L TNF A|X>3L Fig. 1. Apoptosis signaling by CD95. DD, death domain; DED. death effector domain. Fig. 2. Proapoptotic and antiapoptotic signaling by TNFR'I and DP3. FLIP Figure 1 Model of the different functions of :-FLIPL Shown is the CD95 DISC at different concentrations of c-FLIPL. In the absence ol c-FLIPL (no FLIP). bolh procaspase-B (CH) and procaspase-IO (C1D) are recruited Id trie DISC through binding lo the adaptor molecule FADD. This recruibnenl causes processing and activation ol the initiator caspases through homodimerization, release of Ihe active enzymes (heterotelrameric structures), subsequent cleavage ol various intracellular caspase substrates and apoplosis. When c-FLIPL is expressed at tow levels (lew FLIP), activalion ol caspase-BZ-IO is accelerated due lo the ability of c-FLIPl Id associate wilh caspase-E/-10 and ils activity to form heterodimers more efficiently lhan caspases-aV-10 to form bomodimers. At high concentrations ol c-FLIFi. caspase-aV-10 are slill activated, bul are not released any longer from the DISC. According lo the model, DISC-tethered caspase-flMO has the same substratespecilicity as aclivecaspase subunils released inlo the cytosol. However, crying to Iheir DISC-proximal location. Ihese incompletely processed, bul fully active, caspases cleave a dilferenl sel of substrates such as themselves. RIP and c-FLIPi. These cleavage evenls may be important in regulating apoptosis-independent processes such as proliferation. The inactive active site in Ihe caspase domain of c-FLIPL is labelled X. Decoy receptors ;RD2 CRD3 DD hu DR5s - 1 CRD2 CRD3 1 CRD2 CRMS DD DD hu DR- hu DR4 2H CRD2 CRD3 i ii iii iv V hu DcR1 CRD2 CRD3 1---- DD hu DcR2 ;RD2 CRD3 DD rnu DR4/5 1 CRD2 CRD3 CRD4 : huOPG ^ 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.