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APOPTOSE Professora: Maria Marta Figueiredo Email: mariamartafigueiredo@gmail.com A APOPTOSE é um processo ativo cuja marca registrada é a autodigestão controlada dos constituintes celulares, devido à ativação de proteases endógenas e pode ser comparada metaforicamente a um “suicídio celular”. A célula morre de forma ordenada sem prejudicar células vizinhas. APOPTOSE APOPTOSE Morte Fisiológica e Patológica Depende de ATP Não há autólise Ativação de Enzimas Celulares, como Caspases e Endonucleases APOPTOSE Núcleo Contração Condensação Fragmentação Onde ocorre APOPTOSE? Na EMBRIOGÊNESE (separação de dedos dos membros, ...) Na IMUNOGÊNESE maturação de células T Na REGULAÇÃO HORMONAL prolactina leva à involução de glândulas mamárias; endométrio na menstruação. Modelos e Mecanismos Nas DOENÇAS - NEOPLÁSICAS ( apoptose) - INFECÇÃO VIRAL (eliminação de células T – HIV) Na DOENÇA NEURODEGENERATIVA Morte dos neurônios Modelos e Mecanismos APOPTOSE – Causas Patológicas Linfócitos T CD8+ citotóxicos – Célula Alvo em Apoptose APOPTOSE – Causas Patológicas Célula Alvo em Apoptose - Linfócitos T CD8+ citotóxicos Características Atinge células individualmente; A célula se encolhe e o citoplasma fica denso; A cromatina se torna condensada e disposta em grumos colados à carioteca; Características O núcleo se fragmenta; A membrana citoplasmática emite projeções e forma brotamentos que contêm fragmentos do núcleo; Características Fragmentação das células em múltiplos brotos (corpos apoptóticos). Características Características APOPTOSE CARACTERÍSTICAS MICROSCÓPICAS – H&E Características Importância Regulação celular Crescimento celular para prevenir o câncer por supressão da morte celular Deleção de células anormais danificadas, por radiação, toxinas ou outros estímulos Indução pode ocorrer através: • Sinais extrínsecos: - ligantes a receptores; - radiação ionizante; - infecção viral ou fatores químicos; • Linfócito-T citotóxico; • Privação de fatores de crescimento; • Estímulos internos. Mecanismos APOPTOSE – Características bioquímicas Clivagem de proteínas do citoesqueleto caspases Aparecimento de corpos apoptóticos Quebras típicas do DNA por endonucleases 200 pb (gel em “escada”) Reconhecimento fagocítico exposição de fosfatidilserina Bioquímica da apoptose: Receptores de Morte - Iniciam as cascatas de Caspases Pertencem à superfamília TNF (tumor necrosis family): - CD95 (ou Fas) - TNF R1 (TNF Receptor 1) - Linfócitos T - DR4 e DR5 Mecanismos Caspases Estão presentes no citosol sob a forma de pró-enzimas inativas, tornando-se ativas após clivagem proteolítica. Quando ativas clivam proteínas chaves: - Proteínas que inativam a DNAase - Na manutenção da integridade da estrutura celular. Reação de amplificação - cascata Mecanismos Receptor CD95 (Fas) Mecanismos Via extrínseca Mecanismos Via intrínseca Mitocôndria – alteração da permeabilidade da membrana Liberação de Citocromo c Ativação de caspases. Proteínas reguladoras intracelulares • Bcl-2, Bcl-XL, Bcl-w, Bcl-1 e A1 - proteínas anti-apoptóticas • Bax, Bad, Bid proteínas pró-apoptóticas IAP- Liga às caspases, inibindo sua atividade ou ativação. Proteínas que controlam o sinal apoptótico e estabelecem conexão ao programa de execução Bcl2 Bax Bad- Bcl2 Bcl2 Apaf1 Mecanismos Gene p53 – crítico - envolvido no controle da apoptose. Governa a resposta celular a danos ao DNA. Defeito - células deixam de morrer e proliferam acumulando danos genéticos que podem gerar o câncer Mecanismos Integração Sinais extracelulares regulam os sinais intracelulares apoptóticos (ativa ou inibe). Ação – regulam os níveis ou atividades dos membros das famílias Bcl2 e IAP. Se há danos DNA, há interrupção da fase antes de ir para fase S. O sinal de parada em G1 é dado pela p53 (níveis intracelulares aumentam). G1/S-ciclina –replicação DNA S-ciclina- inicio da replicação M-ciclina – eventos da mitose As cdks são inibidas por sinais (- ), CC para Integridade Celular: o poro de transição de permeabilidade mitocondrial se mantém habitualmente fechado. APOPTOSE Depleção de ATP NECROSE expressão p53 Expressão de Bax Abertura do poro mitocondrial Lesão do DNA Ativação das caspases (liberação do citocromo c) Abertura do poro de algumas mitocôndrias Mecanismos Mecanismos NECROSE E APOPTOSE CARACTERÍSTICAS NECROSE APOPTOSE Estímulo Patológico Fisológico ou Patológico Ocorrência Grupo de Células Células Individuais Reversibilidade Irreversível Irreversível Ativação de Endonucleases Não Sim Liberação de Enzimas Lisossomais Sim Não Inflamação Exsudativa Presente Ausente Alterações Nucleares Presente Presente Morfologia Lise Celular Corpos Apoptóticos NECROSE E APOPTOSE Mecanismos Caspases are a family of cysteine proteases that act in concert in a cascade triggered by apoptosis signaling. The culmination of this cascade is the cleavage of a number of proteins in the cell, followed by cell disassembly, cell death, and, ultimately, the phagocytosis and removal of the cell debris. The Caspase cascade is activated by two distinct routes: one from cell surface and the other from mitochondria (Ref.1). The pathway leading to Caspase activation varies according to the apoptotic stimulus. Initiator Caspases (including 8, 9, 10 and 12) are closely coupled to pro- apototic signals. Pro-apoptotic stimuli include the FasL (Fas Ligand), TNF (Tumor Necrosis Factor), Granzyme-B, GRB (Growth Factor Receptor-Bound Protein), DNA damage, Ca2+ (Calcium) channels and ER (Endoplasmic Reticulum) stress. Once activated, these Caspases cleave and activate downstream effector Caspases (including 3, 6 and 7). Caspase8 cleaves BID (BH3 Interacting Death Domain). tBID (Truncated BID) disrupts the outer mitochondrial membrane to cause release of the pro- apoptotic factors CytoC (Cytochrome-C) which is crucial for activating pro-Caspase9. CytoC that is released from the intermembrane space binds to APAF1 (Apoptotic Protease Activating Factor-1), which recruits Caspase9 and in turn can proteolytically activate Caspase3. SMAC (Second Mitochondria- Derived Activator of Caspase)/DIABLO is also released from the mitochondria along with CytoC during apoptosis, and it functions to promote caspase activation by inhibiting IAP (Inhibitor of A representative signaling cascade of the mitochondria-mediated apoptosis. X-ray irradiation causes double-strand DNA breaks. Via an unknown mechanism, the linker histone H1.2 translocates from the nucleus to the mitochondria, where it activates Bak to release cytochrome c and other pro-apoptotic proteins such as Smac/DIABLO. Cytochrome c induces the formation of apoptosome and subsequent activation of caspase-9 whereas Smac/DIABLO removes IAP-mediated inhibition of caspases. In this diagram, the signaling steps prior to and after mitochondria are colored blue and orange, respectively. Although not shown, the anti-apoptotic Bcl-2 and Bcl-xL also reside in the outer membrane of mitochondria. Cytochrome c is anchored tothe outer surface of the mitochondrial inner membrane by electrostatic and hydrophobic interactions with cardiolipin. During the early phase of apoptosis, mitochondrial ROS production is stimulated, and cardiolipin is oxidized by a peroxidase function of the cardiolipin–cytochrome c complex. The hemoprotein is then detached from the mitochondrial inner membrane and can be extruded into the soluble cytoplasm through pores in the outer membrane. Cardiolipin also serves as a mitochondrial target to the C-terminal cleavage product of the Bcl-2 protein, Bid, which promotes pore formation in the outer membrane by Bax or Bak, a process that is inhibited by Bcl-2 or Bcl-XL. Finally, permeabilization of the outer membrane is further enhanced by cardiolipin hydroperoxides, which stimulate the release into the cytoplasm of cytochrome c and Smac/Diablo. Cardiolipin-ox, oxidized cardiolipin. c-MYC sensitizes cells to a wide range of pro-apoptotic stimuli. During apoptosis, c-MYC induces release of cytochrome c from the mitochondria into the cytosol, possibly through activation of the pro-apoptotic molecule BAX (a). Activated BAX within the mitochondrial membrane leads to creation or alteration of membrane pores, resulting in mitochondrial-outer-membrane permeabilization (MOMP). Once released into the cytosol, cytochrome c associates with apoptotic protease-activating factor 1 (APAF1) protein and procaspase-9 to form the apoptosome ('wheel of death'). In the presence of ATP, caspase-9 is activated, leading to activation of downstream effector caspases, including caspase-3, which ultimately leads to the degradation of cell components and the demise of the cell. Other pathways that involve c-MYC-induced cytochrome c release and apoptosis include indirect activation of the p53 tumour suppressor via ARF, leading to transcription of BAX (b). Ligation of the death receptor CD95/FAS triggers the association of the intracellular adaptor protein FADD (FAS-associated death domain) with the CD95 receptor (c). FADD then recruits procaspase-8, resulting in auto-activation of the procaspase, which cleaves and activates executioner caspases. Caspase-8 might also activate the pro-apoptotic protein BID, which might promote MOMP. Survival signals that serve to block c-MYC-induced apoptosis (d) include signalling via the IGF1 receptor or activated RAS, which can lead to the activation of AKT serine/threonine kinase and subsequent phosphorylation of the pro-apoptotic protein BAD. Phosphorylated BAD is sequestered and inactivated by cytosolic 14-3-3 proteins. Anti-apoptotic proteins, such as BCL2 and BCL-XL, reside in the outer mitochondrial membrane and block cytochrome c release, possibly through the sequestration of BAX. Mitochondria-mediated caspase activation at the apoptosome. A. Apoptotic stimuli trigger the release of apoptogenic factors from the mitochondrial intermembrane space to the cytosol, such as cytochrome c which induces the formation of the apoptosome and the activation of procaspase-9. B. By the action of cytochrome c (Cyto C) and dATP the Apaf-1 protein adopts a conformation that allows the formation of a heptameric, wheel-like structure, the apoptosome. Procaspase-9 molecules can bind to the inner “hub” region of the apoptosome and are activated by dimer formation. Active caspase-9 dimers further mediate activation of effector caspases [Acehan, 2002]. Following DNA damage and p53 activation, the mitochondrial outer membrane is permeabilized to allow the release of cytochrome c into the cytosol. Here, it causes the oligomerization of apoptosis activating factor 1 (APAF1) and binds procaspase-9 to form the apoptosome. Procaspase-9 is cleaved to generate activated caspase-9, which subsequently activates caspase-3 — an effector caspase. This, in turn, cleaves cellular substrates, leading to apoptosis. Anti-apoptotic members of the BCL2 family can inhibit release of cytochrome c from the mitochondrion by blocking voltage-dependent anion channels. Legenda da figura anterior. Mitochondrial apoptotic pathway. Death can be induced by the binding of ligands (such as FasL) to specific receptors (such as FAS) located at the cell surface. FAS contains a cytoplasmic death domain where FADD (Fas-associated death domain) can bind in presence of FasL, and recruit Pro-caspase 8 for subsequent activation in caspase 8. This induces caspase 3 activation. Caspase 3 cleaves I-CAD, the inhibitor of CAD (Caspase-activated DNase), which is released to enter the nucleus and cleaves DNA. In addition caspase 8 cleaves Bid protein, resulting in a truncated Bid (tBid) that, upon dimerisation of Bax or Bad, causes the release of cytochrome c from mitochondria. The mechanisms by which Bax leads to mitochondrial membrane permeabilisation and subsequent release of pro-apoptotic factors still remain unclear. It is proposed that Bax could interact with the permeability transition pore, or form channels by self- oligomerization. This leads to the mitochondrial release of cytochrome c and Smac/Diablo (Smac: second mitochondrial- derived activator of caspase; Diablo: direct IAP-binding protein with low pI), AIF (apoptosis inducing factor) and various procaspases. Bcl2 inhibits the release of cytochrome C and AIF in the cytoplasm and prevents the variation of the permeability transition pore. In the cytosol, cytochrome c binds to Apaf-1 (apoptosis- protease- activating factor). Both proteins form the apoptosome, which converts procaspase 9 in caspase 9. This results in activation of downstream effector caspases. Smac/DIABLO binds to IAP (Inhibitors of apoptosis) and prevent them from inhibition of the caspase 9 and caspase 3 activation. AIF has an indirect role in chromosome degradation as it activates endonuclease G, a DNase that moves from the mitochondria to the nucleus during apoptosis. Interestingly to note, the mtDNA is not fragmented during apoptosis (93).
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