7.1. Molecular mechanisms of apoptosis signalling pathway
Apoptosis can be initiated by various stimuli from outside or inside the cell, e.g. by specific ligand of cell surface receptors, by DNA damage, failure of DNA repair mechanisms, certain cytotoxic drugs or irradiation of cell, by a lack of survival signals.
Now LET'S TALK about why they called as caspases and what is the role of caspases. Caspases are endoproteases have an active site Cys(C) and which cleave at the C- terminal side of Asp residues ( asp ) and hence are known as caspases. their catalytical activity depends on a critical cysteine-residue within a highly conserved active-site consist of pentapeptide Glutamine, Alanine, Cystein, Arginine Glycine (QACRG). So far, 7 different caspases have been identified in Drosophila, and 14 in mammals, with caspase-11 and caspase–12 only identified in the mouse. These caspases form a cascade of proteases which are activated in this process. Caspases are expressed as zymogenes (procaspases), which are activated by different apoptosis inducers.
Phases of Apoptosis
Apoptosis responds to the initial stimulus by a cascade of events. Hence , it can be divided into 3 phases:
- The initiation phase(or signaling phase): The activation of surface death receptors(Tumour necrosis receptor, Fas) are involved. The mitochondrial pathway or initiation of apoptosis by other stimuli.
- The signal transduction phase(or preparation phase): The activation of initiator caspases and certain kinases/ phosphatases are involved.
- The execution phase(or death phase): The activation of effector caspases are involved.
The process of apoptosis is controlled by cell signals, these cell signals are either extracellular (extrinsic inducers) or intracellular (intrinsic inducers).
Extracellular signals may have hormones, growth factors, nitric oxide, cytokines and toxins. Intracellular signals are cell stress, cell suicide, heat, radiation, glucocorticoids, nutrient deprivation, viral infection, hypoxia and increased calcium concentration. These signals may positively (i.e., triger) or negatively (i.e., repressor, inhibitor,) apoptosis. Apoptosis is also regulated by a number of cellular components, such as poly ADP ribose polymerase.
Mechanisms of Apoptosis
Now LET'S TALK about how cell activates the caspases
The mechanisms of apoptosis are highly complex and regulated, involving an energy-dependent cascade biochemical events. There are three main apoptotic pathways:
- The extrinsic or death receptor pathway
- The intrinsic or mitochondrial pathway
- The perforin/granzyme pathway(induce apoptosis via granzyme B)
The extrinsic, intrinsic and granzyme pathway are co-related on the execution pathway. Execution pathway initiated by the cleavage of caspase-3. This pathway results in DNA fragmentation, degradation of cytoskeletal and nuclear proteins, cross-linker of proteins, formation of apoptotic bodies, expression of ligand for phagocytic cell receptors and finally uptake by phagocytic cells.
7.1.1. Extrinsic apoptosis
The extrinsic pathway begins outside the cell through activation of death receptors bind with death ligands. The extracellular signal molecular induce apoptosis. Death receptors belong to the tumor necrosis factor receptor (TNFR) superfamily. one typical example is a Fas receptor. The member of the TNFR family have cyteine-rich extracellular domains and death domain which is 80-amino-acid-long region. This death domain transfer the signals from cell surface to intracellular signaling pathway. The most studied death receptors are Fas (CD95/Apo-1), TNFR1, TRAIL-R1 (DR4) and TRAIL-R2 (DR5/Killer/TRICK2). The ligands (FasL, TNFα and TRAIL) that bind to the death receptors.
The ligand FasL is, a homotrimeric transmembrane protein, carried out in killer lymphocyte. When FasR bind with FasL, an adaptor protein called Fas-Associated protein with death domain(FADD). FADD forms Death-Inducing signaling complex(DISC). FADD binds to Fas causing the aggregation of procaspase-8 results in the proteolytic cleavage of procaspase-8 to active form of caspase-8. The activated caspase-8 activate caspase-3 to induce apoptosis. Death receptor mediated apoptosis inhibited by a protein known as c-FLIP that bind to FADD and caspases-8.
A cellular FLICE-inhibitory protein (c-FLIP) regulate the Activation of caspase-8 and -10. Many FLIP variants are generated by alternative splicing of the mRNA. All isoforms are recruited to the DISC through DED-DED interactions. The c-FLIP inhibit the apoptosis as it contains caspase-like domain but lacks catalytic cysteine and therefore has no protease activity.
TRAIL-induced non-apoptotic signaling
TRAIL can activate multiple signal transduction pathways such as MAPKs (extracellular signal regulated kinase (ERK), JUN N-terminal kinase (JNK) and p38, PI3-kinase-Akt and NF-kB. TRAIL-induced apoptosis has also been shown to use both mitochondrial dependent and independent pathways.
Regulation of apoptosis by PI3-kinase-Akt-pathway
The phosphatidyl-inositol-3-kinase (PI3K)-Akt signaling pathway is cell survival pathway as its regulates fundamental cellular functions such as transcription, translation, proliferation, growth hence survival. Over activation of PI3K-Akt pathway leads to cancer, increase the survival and resistance of cell to chemotherapy in solid tumors.
Now LET'S TALK about how Akt prevent the apoptosis
Activated Akt phosphorylating BH3-domain conatining protein Bad at Ser-136. Phosphorylation of Bad causes binding of the 14-3-3 protein family. this bounded Bad cannot promote apoptosis.
The proapoptotic genes like Fas ligands, Bim and Puma are transcribed by a FOXO, a member of forkhead family of transcription factor present in the nucleus . That means in order to block the apoptosis FOXO must export out of nucleus.
Akt Phosphorylates FOXO. phosphorylated FOXO is exported out of the nucleus. In cytoplasm the phosphorylated FOXO binds with 14-3-3 proteins and later it is degraded by Ubiquitin proteasome pathway.
Akt phosphorylates procaspase-9. This causes decrease in the protease activation of caspase-9.
Akt phosphorylates and stabilizes XIAP (X-linked inhibitor of apoptosis protein). XIAP blocks apoptosis.
Akt can phosphorylate and activate IkB kinase a (IKKa), The substrate of IKKa kinase phosphorylates IkB. The phosphorylated IkB is degraded by Ubiquitin proteasome pathway. This leads to the activation and transport of NFkB to nucleus.
The transcription of antiapoptotic genes, such as Bcl-2 and Mcl-1 is increased by cyclic AMP-response element-binding (CREB) protein. Akt phosphorylates and activates the cyclic AMP-response element-binding (CREB) protein.
Akt causes phosphorylation and nuclear translocation of Mdm2 (Hdm2 in humans) which is a E3 ligase for P53. Mdm2 causes degradation of p53. By way of regulating mdm2 akt regulate the p53.
They lack transmembrane and intracellular domains and may be the products of splice isoforms or obtained by proteolytic cleavage. Decoy receptors TRAIL-R3 and TRAIL-R4 can bind TRAIL but lack intracellular death domain and therefore not able to mediate apoptosis.
Glucocorticoids (GCs) are steroid hormones secreted from adrenal cortex and play a role as anti-inflammatory and immune-suppression. The GCs induces cell cycle arrest and apoptosis in lymphoid cells hence use in the treatment of lymphoid malignancies.
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.