Apoptose: Morte Celular Controlada

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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).