3. Apoptosis

 

3.1.Apoptosis Machinery

 

The apoptosis mechanisms are very complicated and include two main pathways (Elmore S., 2007):

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1. Extrinsic or death receptor pathway

2. Intrinsic or mitochondrial pathway

 

Recently, it was discovered that the components of one pathway could have an effect on the other pathway and these pathways are inter-linked (Igney F H and Krammer P H., 2002 as cited in Elmore S., 2007) (Elmore S., 2007).

 

The T-cell mediated cytotoxicity and perforin-granzyme-dependent destroying of the cell, were further discovered as an added apoptosis pathways. The latter pathway can promote apoptosis through either granzyme A or granzyme B (Elmore S., 2007).

 

All three apoptosis pathways i.e., intrinsic, extrinsic and granzyme B congregate on the analogous lethal or killing pathway (Elmore S., 2007). According to Elmore S., 2007 “ This pathway is initiated by the cleavage of caspase-3 and results in DNA fragmentation, degradation of cytoskeletal and nuclear proteins, cross-linking of proteins, formation of apoptotic bodies, expression of  ligands for phagocytic cell receptors and finally uptake by phagocytic cells ” (Elmore S., 2007). 

 

Additionally, the impairment of single stranded DNA causes induction of an analogous granzyme- A promoted pathway i.e., a caspase-independent apoptosis pathway (Martinvalet D et al., 2005) (Elmore S., 2007).

 

Apoptosis Machinery References

 1.       Elmore, S. Apoptosis: A Review of Programmed Cell Death. Toxicol. Pathol. 35, 495–516 (2007).

2.        Igney, F. H. & Krammer, P. H. Death and anti-death: tumour resistance to apoptosis. Nat. Rev. Cancer 2, 277–288 (2002).

3.        Martinvalet, D., Zhu, P. & Lieberman, J. Granzyme A Induces Caspase-Independent Mitochondrial Damage, a Required First Step for Apoptosis. Immunity 22, 355–370 (2005).

 

3.2.Biochemical Characteristics

 

Induction of caspases results in facilitating the commence of a protease cascade after initially triggering other procaspases (Elmore S., 2007). However, caspases in the majority of cells are extensively uttered in the shape of inactive proenzymes (Elmore S., 2007). Also, there is a possibility of accumulation and auto-stimulation of some procaspases (Elmore S., 2007). The cell death process speeds up and apoptotic signalling pathways are augmented in the proteolytic cascade, where a caspase stimulating other caspases (Elmore S., 2007). Also, there is an irretrievable guarantee for apoptosis when caspases start to be stimulated (Elmore S., 2007).

 

Regardless of the range of specificities of various caspases for the identification of different adjacent amino acids, the proteolytic function of caspases enables them to cut proteins at aspartic acid residues (Elmore S., 2007). 

 

According to Elmore S., 2007 “ To date, ten major caspases have been identified and broadly catogorised into initiators (caspase-2, -8,-9, -10), effectors or executioners (caspase -3, -6, -7) and inflammatory caspases (caspase-1,-4,-5)” (Cohen G M., 1997) (Rai N K et al., 2005 as cited in Elmore S., 2007) (Elmore S., 2007). 

 

Also, according to Elmore S., 2007 “there are other caspases like caspase 11, which is known for its role in the control of apoptosis and cytokine development throughout septic shock. The other caspase, known as caspase 12, facilitates endoplasmic specific apoptosis and cytotoxicity by amyloid-β. Caspase 13 is a bovine gene and caspase 14 is mainly uttered in embryonic tissues and diminished in adult tissues” (Hu S et al., 1998) (Nakagawa T et al., 2000 as cited in Elmore S., 2007) (Koenig U et al., 2001 as cited in Elmore S., 2007) (Kang S J et al., 2002 as cited in Elmore S., 2007) (Elmore S., 2007).

 

Biomedical Characteristics References 

 

1.        COHEN, G. M. Caspases: the executioners of apoptosis. Biochem. J. 326, 1–16 (1997).

2.        Elmore, S. Apoptosis: A Review of Programmed Cell Death. Toxicol. Pathol. 35, 495–516 (2007).

3.        Hu, S., Snipas, S. J., Vincenz, C., Salvesen, G. & Dixit, V. M. Caspase-14 Is a Novel Developmentally Regulated Protease. J. Biol. Chem. 273, 29648–29653 (1998).

4.        Kang, S. J., Wang, S., Kuida, K. & Yuan, J. Distinct downstream pathways of caspase-11 in regulating apoptosis and cytokine maturation during septic shock response. Cell Death Differ. 9, 1115–1125 (2002).

5.        Koenig, U., Eckhart, L. & Tschachler, E. Evidence That Caspase-13 Is Not a Human but a Bovine Gene. Biochem. Biophys. Res. Commun. 285, 1150–1154 (2001).

6.        Nakagawa, T. et al. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-β. Nature 403, 98–103 (2000).

7.        Rai, N. K., Tripathi, K., Sharma, D. & Shukla, V. K. Apoptosis: A Basic Physiologic Process in Wound Healing. Int. J. Low. Extrem. Wounds 4, 138–144 (2005).

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3.3. Extrinsic Pathway

 

Transmembrane receptor mediated communications play a vital role in extrinsic signaling pathways that induce apoptosis and include death receptors, which are members of tumor necrosis factor (TNF) receptor gene superfamily (Locksley R M et al., 2001) (Elmore S., 2007) 

 

Members of TNF receptor gene superfamily contain the “death domain”, which includes the cytoplasmic domain encompassing 80 amino acids and also cysteine rich extracellular domains (Ashkenazi A and Dixit V M., 1998 as cited in Elmore S., 2007) (Elmore S., 2007) (Fulda S and Debatin K M., 2006). The death domain plays a significant role in transducing death signals intracellulary from cell membrane (Elmore S., 2007). The ligands that bind death receptors consists of Fasl/FasR, TNFα/TNFR1, Apo3L/DR3, Apo2L/DR4 and Apo2L/DR5 (Chicheportiche Y et al., 1997) (Ashkenazi M and Dixit V M., 1998 as cited in Elmore S., 2007) (Peter M E and Krammer P H., 1998 as cited in Elmore S., 2007) (Suliman A et al., 2001 as cited in Elmore S., 2007) (Rubio-Moscardo F et al., 2005) (Elmore S., 2007).

 

The FasL/FasR and TNFα/TNFR1 models most importantly classify the extrinsic stage of apoptosis (Elmore S., 2007). In these models, the binding of a specific ligand to the corresponding receptor takes place (Elmore S., 2007). Deployment of cytoplasmic adaptor proteins that display the matching death domains, which bind with the receptors, occurs due to the induction by aforementioned ligand-receptor binding (Elmore S., 2007). The binding of FasL to Fas R and TNFα toTNFR1 results in the binding of adaptor protein FADD and TRADD, respectively (Hsu H et al., 1995) (Wajant H., 2002 as cited in Elmore S., 2007) (Elmore S., 2007). The auto-catalytic induction of procaspase-8 and consequently death inducing signaling complex (DISC) configuration takes place as FADD links to procaspase-8 through dimerisation of the death effector domain (Kischkel F C et al., 1995) (Elmore S., 2007). Prompting of caspase-8 induces the execution stage of apoptosis (Elmore S., 2007). On the other hand, C-Flip, is a protein that can inactivate the death receptor initiated apoptosis through binding to FADD and caspase 8 and causing their ineptness (Kataoka T et al., 1998) (Scaffidi C et al., 1999) (Elmore S., 2007).

 

Also, it is suggested that there is another point of apoptosis regulation, which occurs due to Toso protein inactivating the caspase 8 processing mechanism, a procedure that is responsible for hindering of Fas-activated apoptosis in T cells (Hitoshi Y et al., 1999) (Elmore S., 2007).

 

Extrinsic Pathway References

 

1.        Ashkenazi, A. & Dixit, V. M. Death Receptors: Signaling and Modulation. Science (80-. ). 281, 1305–1308 (1998).

2.        Chicheportiche, Y. et al. TWEAK, a New Secreted Ligand in the Tumor Necrosis Factor Family That Weakly Induces Apoptosis. J. Biol. Chem. 272, 32401–32410 (1997).

3.        Elmore, S. Apoptosis: A Review of Programmed Cell Death. Toxicol. Pathol. 35, 495–516 (2007).

4.        Fulda, S. & Debatin, K.-M. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25, 4798–4811 (2006).

5.        Hitoshi, Y. et al. Toso, a Cell Surface, Specific Regulator of Fas-Induced Apoptosis in T Cells. Immunity 8, 461–471 (1998).

6.        Kataoka, T. et al. FLIP prevents apoptosis induced by death receptors but not by perforin/granzyme B, chemotherapeutic drugs, and gamma irradiation. J. Immunol. 161, 3936–42 (1998).

7.        Kischkel, F. C. et al. Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J. 14, 5579–88 (1995).

8.        Locksley, R. M., Killeen, N. & Lenardo, M. J. The TNF and TNF Receptor Superfamilies. Cell 104, (2001).

9.        Rubio-Moscardo, F. Characterization of 8p21.3 chromosomal deletions in B-cell lymphoma: TRAIL-R1 and TRAIL-R2 as candidate dosage-dependent tumor suppressor genes. Blood 106, 3214–3222 (2005).

10.     Scaffidi, C., Schmitz, I., Krammer, P. H. & Peter, M. E. The Role of c-FLIP in Modulation of CD95-induced Apoptosis. J. Biol. Chem. 274, 1541–1548 (1999).

11.     Suliman, A., Lam, A., Datta, R. & Srivastava, R. K. Intracellular mechanisms of TRAIL: apoptosis through mitochondrial-dependent and -independent pathways. Oncogene 20, 2122–2133 (2001).

12.     Wajant, H. The Fas Signaling Pathway: More Than a Paradigm. Science (80-. ). 296, 1635–1636 (2002).

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3.4.Granzyme/Perforin Pathway

 

The preponderate way of apoptosis in which execution of marked cells takes place through the extrinsic pathway and FasL/FasR collaboration, is caused by cytotoxic T lymphocytes (CTLs) (Brunner T et al., 2003 as cited in Elmore S., 2007) (Elmore S., 2007). Additionally, a novel pathway that entails perforin (a transmembrane pore-forming molecule) excretion followed by the exophytic release of cytoplasmic particles via the stoma and into the target cell, is achieved by cytotoxic T lymphocytes (CTLs), which is a way for CTLs to enforce their cytotoxic influence on tumor cells and virus infected cells (Trapani J A and Smyth M J., 2002 as cited in Elmore S., 2007) (Elmore S., 2007). It is vital to mention that the most critical parts within the granules are serine proteases, granzyme A and granzyme B (Elmore S., 2007).

 

Granzyme B

 

Cleavage of proteins at aspartate residues and induction of procaspase-10 are achieved by granzyme B. Granzyme B is also capable of cleavage of factors such as ICAD (Inhibitor of Caspase Activated DNAse) (Sakahira H et al., 1998 as cited in Elmore S., 2007) (Elmore S., 2007).

 

Also, cleavage of Bid and activation of the release of cytochrome c can intensify the death signal, which is done by usage of a mitochondrial pathway by granzyme B (Barry M and Bleackley R C., 2002 as cited in Elmore S., 2007) (Russell J H and Ley T J., 2002 as cited in Elmore S., 2007) (Elmore S., 2007).

 

Furthermore, direct induction of caspase 3 can be achieved by granzyme B, which can result in direct activation of the execution phase of apoptosis and circumvent the upstream signaling pathway (Elmore S., 2007).

 

It is essential to highlight that the vital steps for execution, carried out by granzyme B, include both the mitochondrial pathway and caspase 3 induction (Goping I S et al., 2003). Furthermore, it is highlighted that regulating the mechanism of T cell development of type 2 helper T (Th2) cells is dependent on cytotoxicity achieved by granzyme B in the aforementioned way (Devadas S et al., 2006) (Elmore S., 2007).

 

Granzyme A

 

Granzyme A is critical in activating apoptosis via cytotoxic T cells and inducing the caspase independent pathway (Elmore S., 2007).

 

Granzyme A is known to induce DNA nicking through DNAse NM23-H1 in the cell, which is an artifact of a tumor suppressor gene (Fan Z et al., 2003) (Elmore S., 2007). This DNase plays a vital role in enhancing immunity and avoiding cancer by activating the apoptosis of tumor cells (Elmore S., 2007).

 

Apoptotic DNA degradation occurs as cleavage of the nucleosome assembly protein (SET) complex takes place by Granzyme A protease, which results in dismissing NM23-H1 inhibition (Elmore S., 2007). Furthermore, chromatin and DNA structure are guarded by the proteins that assemble the (SET, Ape, Pp32 and HMG2) complex acting together (Lieberman J and Fan Z., 2003 as cited in Elmore S., 2007) (Elmore S., 2007). Thus granzyme A plays role in occurrence of apoptosis as it inhibits the above complex, the process that results in preventing the conservation of DNA and chromatin structure substances (Elmore S., 2007).

 

Granzyme/Perforin Pathway References

 

1.        Barry, M. & Bleackley, R. C. Cytotoxic T lymphocytes: all roads lead to death. Nat. Rev. Immunol. 2, 401–409 (2002).

2.        Brunner, T. et al. Fas (CD95/Apo-1) ligand regulation in T cell homeostasis, cell-mediated cytotoxicity and immune pathology. Semin. Immunol. 15, (2003).

3.        Devadas, S. et al. Granzyme B Is Critical for T Cell Receptor-Induced Cell Death of Type 2 Helper T Cells. Immunity 25, 237–247 (2006).

4.        Elmore, S. Apoptosis: A Review of Programmed Cell Death. Toxicol. Pathol. 35, 495–516 (2007).

5.        Fan, Z., Beresford, P. J., Oh, D. Y., Zhang, D. & Lieberman, J. Tumor Suppressor NM23-H1 Is a Granzyme A-Activated DNase during CTL-Mediated Apoptosis, and the Nucleosome Assembly Protein SET Is Its Inhibitor. Cell 112, 659–672 (2003).

6.        Goping, I. S. et al. Granzyme B-Induced Apoptosis Requires Both Direct Caspase Activation and Relief of Caspase Inhibition. Immunity 18, 355–365 (2003).

7.        Lieberman, J. & Fan, Z. Nuclear war: the granzyme A-bomb. Curr. Opin. Immunol. 15, 553–559 (2003).

8.        Russell, J. H. & Ley, T. J. Lymphocyte-Mediated Cytotoxicity. Annu. Rev. Immunol. 20, 323–370 (2002).

9.        Sakahira, H., Enari, M. & Nagata, S. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391, 96–99 (1998).

10.     Trapani, J. A. & Smyth, M. J. Functional significance of the perforin/granzyme cell death pathway. Nat. Rev. Immunol. 2, 735–747 (2002).

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3.5. Intrinsic Pathway

 

The intrinsic signaling pathways that lead to apoptosis are mitochondrial-initiated procedures, which consist of intracellular signals that work exactly on cellular components (Elmore S., 2007). These signals are mediated by various ranges of stimuli, which are formed by non-receptor constituents (Elmore S., 2007).

 

The induction of the intrinsic pathway through stimuli leads to either positive or negative intracellular signals (Elmore S., 2007).

 

The negative stimuli, which lead to the breakdown of prevention of death programs and initiation of apoptosis, usually caused by missing of specific growth factors, hormones and cytokines (Elmore S., 2007).

 

The positive stimuli, on the other hand, include radiation, toxins, hypoxia, viral infection, hyperthermia and free radicals (Elmore S., 2007).

 

The caspase dependent or independent death effectors play a role in furthering cell death via apoptosis related protein within the cytosol (Saelens X et al., 2004 as cited in Fulda S and Debatin K M., 2006) (Fulda S and Debatin K M., 2006).

 

Caspase 9 plays a role in the induction and regulation of intrinsic apoptosis. This caspase 9 role is achieved as it reveals its Caspase Recruitment Domain (CARD domain) and attaches to Apoptotic Protease Activating Factor 1 (APAF1) (Fulda S and Debatin K M., 2006) (D’ Arcy M S., 2019). In the non-apoptotic cell, procaspase 9, including its CARD domain, cannot attach APAF1’s CARD domain due to obstruction of this domain on APAF1 (D’ Arcy M S., 2019). On the other hand, apoptosis induction results in variation in the mitochondrial membrane and opening of Mitochondrial Permeability Transition (MPT) pore, which in turn initiate apoptosis as pro- apoptotic proteins such as cytochrome c, Smac/Diablo and HtrA2/Omi, escape from mitochondria into cytoplasm (Cain K et al., 2002 as cited in D’ Arcy M S., 2019) (D’ Arcy M S., 2019).

 

Furthermore, conformational variation in APAF1 occurs as cytochrome c attaches to the WD domain of APAF1 monomer, which results in uncovering a nucleotide binding and oligomerisation domain of APAF1, capable of attaching to dATP and ultimately triggering apoptosis (D’ Arcy M S., 2019). Following this event then  further conformational change in APAF1 occurs, which permits assembly of several APAF1s into a complex identified as apoptosome, which is by itself an outcome of uncovering APAF1’s both CARD and oligomerisation domain (Acehan D et al., 2002) (D’ Arcy M S., 2019). Number of procaspase 9 proteins are deployed and induced by numerous uncovered CARD domains in the apoptosome open centre.  Thus, killer procaspase 3 that activate apoptosis when it is in the shape of caspase 3, is triggered by those prompted caspase 9 enzymes, which are responsible for the occurrence of this process (Cain K et al., 2002 as cited in D’ Arcy M S., 2019) (D’ Arcy M S., 2019).

 

The intrinsic signaling pathway also occurs through the Bcl-2 family of proteins (Cory S and Adam J M., 2002 as cited in Fulda S and Debatin K M., 2006) (Fulda S and Debatin K M., 2006).

 

In this pathway, the permeabilisation of the outer mitochondrial membrane is associated with caspase induction. This process takes place through proapoptotic members of the Bcl family of proteins (Green D R and Kroemer G., 2004 as cited in Fulda S and Debatin K M., 2006) (Fulda S and Debatin K M., 2006).

 

As the outer mitochondrial membrane is disordered, in between the outer and inner mitochondrial membranes, some proteins such as cytochrome c, AIF, endonuclease G, Smac/DIAB and Omi/HtrA2 can be discovered (Saelens X et al., 2004 as cited in Fulda S and Debatin K M., 2006) (Fulda S and Debatin K M., 2006).

 

According to Belkacemi L., 2018 "The Bcl2 family of proteins split into three groups according to their conserved BH (Bcl-2 Homology) regions (Danial N N et al., 2004) (Belkacemi L., 2018). These groups are divided into anti-apoptotic proteins such as Bcl-2, Bcl-XL, Bcl-XS, Bcl-W and Mcl-1, which include all four BH regions, and pro-apoptotic proteins that itself split into two groups, given their number and activity of BH. The first group of pro-apoptotic proteins with common BH 1-3 regions are Bax, Bak and Bok. The second group of pro-apoptotic proteins are the ones with homology only in their BH3 regions and include Bad, Bim, Bid, Noxa, Pumas, Bik/Blk and Bmf"(Shamas-Din A et al., 2011) (Belkacemi L., 2018).

 

Furthermore, according to the method of binding in the Bcl-2 family of proteins, then two models, namely direct and indirect models, have been established (O’Neill J W et al., 2006 as cited in Belkacemi L., 2018) (Belkacemi L., 2018). In the direct model, the cell’s susceptibility to a large number of apoptotic incentives is established through the Bcl-2/Bax ratio, as BH3 only contains a group of proteins, namely Bid, Bim and PUMA, directly attaching to bax and bak (Belkacemi L., 2018). On the other hand, in the indirect model, as BH3 only proteins expatriate Bax and Bak from pro-survival proteins, then these pro-apoptotic proteins become stimulated (Belkacemi L., 2018). 

 

As Bax and Bak oligomerises are induced, then the mitochondrial pathway becomes activated and apoptosis occurs as Bax and Bak produce apoptotic pore in the mitochondrial outer membrane (Vela L et al., 2013) (Belkacemi L., 2018). Moreover, as Bcl-XL or Bcl-2 heterodimerise with Bad, then their shielding influences are defused, resulting in activation of apoptosis (Yang E et al., 1995) (Belkacemi L., 2018).

 

Also, regulation of induction of caspase proteases such as caspase 9 by anti-apoptotic proteins such as Bcl-2 and Bcl- XL blocks apoptosis, for example, as Bcl-XL attaches to APAF1, then induction of caspase 9 is repressed and hindered (Newmeyer D D et al., 2000 as cited in Belkacemi L., 2018) (Hu Y et al., 1998) (Belkacemi L., 2018).

 

Intrinsic Pathway References

 

1.        Acehan, D. et al. Three-Dimensional Structure of the Apoptosome. Mol. Cell 9, 423–432 (2002).

2.        Belkacemi, L. Exploiting the Extrinsic and the Intrinsic Apoptotic Pathways for Cancer Therapeutics. J. Cancer Cure (2018).

3.        Cain, K., Bratton, S. B. & Cohen, G. M. The Apaf-1 apoptosome: a large caspase-activating complex. Biochimie84, 203–214 (2002).

4.        Cory, S. & Adams, J. M. The Bcl2 family: regulators of the cellular life-or-death switch. Nat. Rev. Cancer 2, 647–656 (2002).

5.        D’Arcy, M. S. Cell death: a review of the major forms of apoptosis, necrosis and autophagy. Cell Biol. Int. 43, 582–592 (2019).

6.        Danial, N. N. & Korsmeyer, S. J. Cell Death. Cell 116, 205–219 (2004).

7.        Elmore, S. Apoptosis: A Review of Programmed Cell Death. Toxicol. Pathol. 35, 495–516 (2007).

8.        Fulda, S. & Debatin, K.-M. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25, 4798–4811 (2006).

9.        Green, D. R. & Kroemer, G. The Pathophysiology of Mitochondrial Cell Death. Science (80-. ). 305, 626–629 (2004).

10.     Hu, Y., Benedict, M. A., Wu, D., Inohara, N. & Núñez, G. Bcl-X L interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation. Proc. Natl. Acad. Sci. 95, 4386–4391 (1998).

11.     Newmeyer, D. D. et al. Bcl-xL does not inhibit the function of Apaf-1. Cell Death Differ. 7, 402–407 (2000).

12.     O’Neill, J. W., Manion, M. K., Maguire, B. & Hockenbery, D. M. BCL-XL Dimerization by Three-dimensional Domain Swapping. J. Mol. Biol. 356, 367–381 (2006).

13.     Saelens, X. et al. Toxic proteins released from mitochondria in cell death. Oncogene 23, 2861–2874 (2004).

14.     Shamas-Din, A., Brahmbhatt, H., Leber, B. & Andrews, D. W. BH3-only proteins: Orchestrators of apoptosis. Biochim. Biophys. Acta - Mol. Cell Res. 1813, 508–520 (2011).

15.     Vela, L., Gonzalo, O., Naval, J. & Marzo, I. Direct Interaction of Bax and Bak Proteins with Bcl-2 Homology Domain 3 (BH3)-only Proteins in Living Cells Revealed by Fluorescence Complementation. J. Biol. Chem. 288, 4935–4946 (2013).

16.     Yang, E. et al. Bad, a heterodimeric partner for Bcl-xL and Bcl-2, displaces bax and promotes cell death. Cell80, 285–291 (1995).

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