Extrinsic and intrinsic pathways
Cysteine aspartic proteases (caspases) play critical roles in regulating apoptosis and inflammation. Caspases involved in apoptosis are classified into distinct subsets based on function, effector caspases (caspases-3, -6 and -7) and initiator caspases (caspases-2, -8, -9 and -10). Caspases-1, -4, -5, -11 and -12 are involved in inflammation.
All caspases are synthesized in inactive forms, referred to as pro-caspases, that require dimerization/oligomerization and subsequent cleavage for activation. Extrinsic or intrinsic death signals mediate caspase activation and apoptosis via two distinct pathways (see image 1). The extrinsic pathway is triggered by ligand binding to cell surface death receptors, and the intrinsic pathway is activated by factors such as DNA damage, growth factor withdrawal or loss of extracellular matrix contact and is mediated through the mitochondria.
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Image 1. Interactive diagram of the intrinsic and extrinsic apoptosis pathways.
Death receptors are cell surface receptors that transmit apoptotic signals initiated by specific ligands such as Fas ligand and tumor necrosis factor (TNF)-alpha. They are characterized by the presence of an 80 amino acid death domain at their cytoplasmic tail. The death receptors involved in the extrinsic pathway belong to the TNF superfamily and include TNF receptor 1 (TNFR1), CD95 (also called Fas and APO-1), death receptor 3 (DR3), TNF-related apoptosis inducing ligand receptor-1 (TRAIL-R1/DR4) and TRAIL-R2 (DR5 in humans) (Guicciardi and Gores 2009). Death receptor dimerization/oligomerization, upon ligand binding, results in receptor activation. This is followed by recruitment of adaptor proteins such as FADD, which contributes to the recruitment of pro-caspase-8 to the death inducing signaling complex (DISC) at the cytoplasmic tail of the death receptor. This leads to oligomerization and subsequent cleavage and activation of caspase-8, which activates the caspase cascade. In some cells however, extrinsic activation of caspase-8 can also lead to activation of the intrinsic pathway through caspase-8 mediated cleavage of the Bcl-2 family member Bid (Mcllwain et al. 2013, Kantari and Walczak 2011).
Intrinsic apoptosis is also called mitochondrial apoptosis because it is mediated by the release of signaling proteins from the mitochondria. The process is triggered by apoptotic stimuli such as DNA damage. The key event leading to the activation of this pathway is the induction of mitochondrial outer membrane permeabilization (MOMP) mediated by the Bcl-2 family members Bax and Bak (Giansanti and Scovassi 2008). Upon activation by interaction with the pro-apoptotic factor Bid, these proteins insert themselves into the outer mitochondrial membrane as homo or heterodimers. They eventually form a mitochondrial apoptosis-induced channel, which is a large pore that facilitates the release of pro-apoptotic factors into the cytoplasm, including cytochrome c. Induction of MOMP leads to a decrease in mitochondrial membrane potential, which is an early signal of apoptosis. Formation of the Bax/Bak complex can be inhibited by the anti-apoptotic proteins Bcl-2 and Bcl-xL (Giansanti and Scovassi 2008).
Upon its release to the cytoplasm, cytochrome c interacts with the adaptor protein apoptotic protease-activating factor-1 (APAF-1). The nucleotide deoxy-ATP (dATP) binds to APAF-1 to mediate its activation. APAF-1 then recruits pro-caspase-9, which collectively form a complex called the apoptosome. Formation of the apoptosome triggers cleavage and activation of caspase-9, which leads to a caspase activation feedback loop. The feedback loop is initiated by caspase-9 mediated activation of caspase-3 and -7, which then activate effector caspases downstream (see image 1). This ultimately leads to apoptosis induced by key signaling pathways, cellular and structural proteins and DNA repair molecules (Mcllwain et al. 2013). In cells, apoptosis is manifested phenotypically as membrane blebbing, DNA fragmentation and a condensed nucleus. The table below summarizes the main caspases involved in the intrinsic and extrinsic apoptosis pathways.
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Table 1. Molecular weight of apoptotic caspases and examples of their inhibitors
*1 Molecular weight data are based on western blot analysis and information from the universal protein resource database (UniProt)
*2 The procaspase form is a monomer
*3 FMK is the abbreviation for fluoromethylketone. The addition of FMK to the peptide sequence results in irreversible linkage and permanent inactivation of caspases
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