THE INTRINSIC OR MITOCHONDRIAL PATHWAY OF MOLECULAR MECHANISMS OF APOPTOSIS PATHWAY IN APOPTOSIS
7.1.2. The intrinsic or mitochondrial pathway
Intrinsic or mitochondrial apoptotic pathway is triggered by multiple stimuli including DNA damage or cytotoxic drugs. In intrinsic pathway mitochondria plays important role by releasing cytochrome C. The Cytochrome C binds Apaf-1(Apoptotic proteases Activating Factor), dATP, and procaspase -9 to create a complex known as the Apoptosome. The procaspase-9 cleaves into caspase-9 to generate caspase-3, which causes apoptosis.
Mitrochondrial proteins known as SMACs (small mitochondrial-derived activator of caspases) are released in cytosol following increase in permeability. The permeability transition pore (PTP) induces translocation and mutimerization of proapoptotic protein Bax. The box helps in permeabilization of inner mitochondrial membrane resulting in the leakage of cytochrome c and others proteins that gives signals for apoptosis. The activator of caspases (Smac/DIABLO ) is mitochondrial protein that inhibits the inhibitor of apoptosis protein (IAP). Inhibitor of apoptosis protein block the processing of caspases 3 and Caspases-9. The HtrA2 and Omi also inhibits inhibitor of apoptosis protein (IAP). Several elements of (Reactive oxygen species) ROS-induced apoptosis are also included, such as the formation of highly toxic nitrogen reactive species (NRS). Finally endonucleases such as CAD, are activated by caspases-3.
Mitochondrial outer membrane permeabilization (MOMP)
MOMP is regarded as the “point of no return” during apoptosis. The increase in permeability release numerous apoptotic proteins that are present in the peri-mitochondrial space. MOMP cause collapse of mitochondrial membrane potential (Δm). Depending on the stimuli, the loss of Δym can occur before, during or after MOMP. Inner mitochondrial membrane (IMM) can also induced the MOMP through permeability transition pore (PTP) formation. Permeability transition pore (PTP) is a complex composed of voltage dependent anion channel (VDAC) in the outer mitochondrial membrane (OMM). The adenine nucleotide translocator (ANT) in the IMM and cyclophilin D (cypD) in the matrix. The opening of permeability transition pore (PTP) leads to an influx of ions into the mitochondrial matrix causing the loss of Δm, and swelling of the matrix as water enters leading to rupture of OMM and MOMP.
Bcl-2 family proteins in the regulation of MOMP
The Bcl-2 family of proteins is divided into three groups based on their function and the number of BCL-2 homology (BH) domains present. Bcl2 family is homologs of ced-9 in C.elegans.
- The anti-apoptotic members : Present in OMM and have four BH domains (BH1-BH4). eg Bcl-2, Bcl-XL, Bcl-w, Mcl- 1, A1 and Bcl-B. Bcl2 and Bcl-x prevents the pore formation on OMM.
- The pro-apoptotic members are divided into two groups.
2a Bax-like multidomain contain three BH domain (BH1-BH3) apoptotic proteins ( eg Bax, Bak, Bak) and 2b BH3-only proteins (Bik, Bid, Bad, Puma, Noxa, Bim, Hrk, Bmf). Bax and Bak form pore on OMM that trigger apoptosis.
Activation of BH3-only proteins
Activity of BH3-only proteins is regulated both at the transcription level and at the post-translational level . Hrk Puma, Noxa and Bim are actively transcribed during apoptotic stimuli. Bad, Bim and Bik are regulated by phosphorylation. The phosphorylation causes decrease in proapoptotic potential. The proteolytic cleavage product of Bid is truncated Bid (tBid) which is targeted to mitochondria.
Bax and Bak activation
Bax and Bak are constitutively expressed and induce MOMP following apoptotic stimuli. Bax is present in monomeric form in cytoplasm and sometimes associated with OMM. During apoptotic stimuli it is inserted into OMM.
Inhibitor of apoptosis proteins (IAPs)
The IAP family of proteins consists of eight human analogues, including cellular IAP1 (c-IAP1), cellular IAP2 (c-IAP2), X-linked inhibitor of apoptosis (XIAP), survivin, Apollon (also known as Bruce), melanoma IAP (ML-IAP, also known as Livin) and IAP-like protein 2 (ILP-2). All IAP proteins contain BIR (baculoviral IAP repeat) domain which inhibit the caspase acativity. In addition to the BIR domain, all IAPs except survivin contains RING domain, which possesses E3 ubiquitin ligase activity.
The linker region that precedes the BIR-domain binds to the active site of caspase-3 or 7. This binding prevents substrate binding.
Glycogen synthase kinase-3
Glycogen synthase kinase-3 (GSK3) is a serine/threonine kinase which is ubiquitously expressed in mammalian cells. GSK3 is a kinase that phosphorylates glycogen synthase and make it inactive. Akt phosphorylates GSK3 on these serine residues. Overexpression of GSK3 promotes mitochondrial intrinsic apoptosis. GSK3 is present in mitochondria and mitochondrial GSK3 activity is increased during DNA damage or ER stress, that leads to apoptosis. GSK3 inhibits extrinsic apoptosis by inhibiting the death-receptor-mediated Apoptosis.
7.1.3. The perforin-granzyme pathway
Granzyme pathway is additional pathway. The Granzyme pathway induce apoptosis via either granzyme A or granzyme B. Granzymes are serine proteases that are released by cytoplasmic granules within cytotoxic T-cells and nature killer cells. This pathway activates caspase-independent cell death pathway via single standard DNA damage. This mechanism used for to kill tumor cells and virus infected cells. Cytotoxic T-cells release perforin and granzyme proteins. Perforin protein pore in the membrane of infected cells, allowing the entry of granzyme A and granzyme B. granzyme B induces activation of caspase 10 and caspase 9. Caspase 9 activates especially caspase 3. Caspase-3 acfivates CAD. CAD proceeds for DNA fragmentation and associates with apoptosis. Granzyme A form a SET complex which also induce DNA cleavage and apoptosis.
7.2. Regulation of Apoptosis :
- The IAP(inhibitors of apoptosis proteins) proteins is the most important regulators of apoptosis.
- The mammalian Bcl-2 family of anti-apoptotic genes, the homologs of ced-9 gene found in C. elegans. Bcl-2 proteins inhibit apoptosis. Bax and bak form the pore , while Bcl2, Bcl-X inhibit its formation.
- Apoptosis regulates by Hsp that is induced by stress. HSP(Hsp27, Hsp70, Hsp 90) inhibits apoptosis by inhibiting caspase-3.HSP-90 blocks the oligomerization of the Apaf-1 complex, while Hsp27 and Hsp70 block the signaling pathway from TNF-α or fas receptors.HSP-60/10 helps the activation of caspases-3.
- Apoptosis inhibit by Nitric oxide(NO) through up-regulation of kinases, like Akt. NO is combined with superoxides forming nitrogen peroxides,which are efficient initiators of the apoptotic response.Hsp90 is overexpressed by N2O2 and regulate apoptosis.
7.3. Apoptois in C. elegans
C. elegans is used as model organism to study the process of apoptosis. 131 cells of C. elegans undergo apoptosis out of 1090 cell in the embryonic stage.
The steps of apoptosis can be summarized as follows :-
- Activation of the cysteine protease ced-3 by oligomerization with ced-4.
- Apoptosis inhibitor ced-9 and the apoptosis inducer egl-1 regulate the activity of ced-3/ced-4 complex
- Ced-4 counterpart in the mammalian apoptosis is apoptotic protease activating factor 1, Apaf-1.
- Ced-3 is the single member of cysteine proteases family in C. elegans.
- Egl-1 and ced-9 are members of the Bcl-2 family of pro- or antiapoptotic proteins, respectively.