Apoptosis Proteins


 Apoptosis Proteins Background

The mechanism of programmed cell death to remove unwanted and damaged cells in a multi-cellular organism is said to be apoptosis. Apoptosis may start with various morphological and physiological changes such as cell shrinkage, chromatin condensation, chromosomal fragmentation or nuclear fragmentation leading to the removal of dead cells by phagocytosis. Activated cascade signals at the outer surface of apoptotic cells leads to phagocytosis initiation and these signals are activated by caspases, a family of cysteine proteinases. Excess apoptosis may lead to atrophy while lack of apoptosis leads to uncontrolled growth of tumor cells causing cancer. Development of cancer may be due to the disregulation of factors such as Nuclear Kappa B (NFkB), p53 or phosphatidylinositol 3-kinases/Akt. Most of the anticancer agents available today destroy cancer cells through apoptosis. Therefore the necessity of understanding the apoptotic pathway mechanisms is increasing to develop anti-cancer drugs specifically targeting apoptotic genes. The two major apoptotic pathways accountable for handling stress signals and achieving cell death in mammalian cells are the extrinsic and the intrinsic pathways. The intrinsic and extrinsic pathways are activated via the death-receptor pathway and the mitochondrial pathway respectively, which both lead to the activation of caspase-3 and caspase-7.

Extrinsic Pathway
The extrinsic pathway is initiated by the activation of death receptors at the extracellular surface of cells by toxins, hormones and growth factors which lead to initiation of pathways involving caspases.
In the extrinsic apoptotic pathway, apoptosis of cells is due to the binding to cell surface death receptors. All death receptors have two domains. One domain is an extracellular cysteine rich domain for ligand binding and the other is an intracellular domain used for transmitting apoptotic signals with the help of recruitment of effector caspases. Proapoptotic ligands belonging to the cytokine tumor necrosis factor (TNF) super family play a key role in the extrinsic pathway and may exist on the cell surface or be discharged into the extracellular space. A death inducing signaling complex (DISC) is formed by binding these proapoptotic ligands to the TNF receptor, where FAS and FADD assist in formation of DISC which then activates caspase 8 and 10 through proteolysis of their zymogens. Release of activated caspase 8 and 10 into the cytoplasm promotes activation of the effector caspases 3, 7 and 6 which leads to cell death.
The extrinsic pathway results in two sub pathways during activation by binding of CD95L/FasL to CD95/Fas. In sub pathway 1, amplification of the mitochondrial signal responsible for the intrinsic pathway is not necessary due to the sufficient amount of production of caspase 8 at the DISC complex to activate the downstream effector caspases 3, 6 and 7. In sub type two, mitochondrial amplification of CD95 signal is necessary due to the low levels of caspase 8 bound to the DISC.

Intrinsic Pathway
The intrinsic pathway includes intracellular activity initiated as a response to stress signals such as heat, nutrient deprivation, glucocorticoids, viral infection and hypoxia, all of which leads to the activation of caspases 9, 3 and 7.
The intrinsic pathway is mostly managed by the Bcl2 family of proteins and the mitochondria. Factors such as DNA damage, non-interfered oncogenes and growth factor appropriation activate the intrinsic pathway. DNA damage activates the tumor suppressor p53 which transmits death signals by inducing the expression of BH3. BH3 activates Bax and Bak resulting in the formation of mitochondrial apoptogenic proteins such as RNA/DNA endonuclease, apoptosis inducing factor and cytochrome C. Cytochrome C released from the mitochondria joins with Apaf-1 to form a complex called apoptosome. Apoptosome initiates the oligomerization of ATP to Apaf-1 which activates procaspase-9 through protease cleavage to caspase-9. Activation of caspase 9 is facilitated by SMAC, an antagonizing member of the inhibitors of apoptosis protein (IAP). Apoptosis inducing factor and endonuclease G promote DNA degradation. Apoptosome formation requires growth factors such as ATPase-activating proteins or nucleotide exchange factors and leads to activation of downstream effectors caspase 3, 6 and 7.

ER stress Induced Apoptosis
Besides the traditional apoptotic pathways, recent studies discovered alternative pathways such as ER stress induction plays a key role in apoptosis through inflammation. Accumulation of unfolded proteins inside the ER leads to ER stress with the involvement of transcriptional and translational machinery necessary to induce the expression of factors involved in ER stress mediated cell death. Cells maintain homeostasis by removing the unfolded proteins formed through intracellular stress induced by hypoxia, DNA damage or reactive oxygen species through activation of mechanisms such as ubiquitination and degradation by the proteasome. But when the accumulation of unfolded proteins is massive, homeostasis can’t be achieved resulting in the transferring of unfolded proteins into the ER where cells activate a recovery mechanism through which chaperones such as the Heat shock protein 70 (HSP70) could mediate folding. Key players of ER stress such as Inositol-requiring enzyme 1 alpha (IRE1α), Protein kinase RNA-like ER kinase (PERK) and Activating transcription factor 6 alpha (ATF6α) were physiologically controlled by Binding Immunoglobulin heavy chain protein (BIP). Aggregation of unfolded proteins can be prevented by the dissociation of BIP from IRE1α, PERK and ATF6α under stress conditions.
ER stress also induces up-regulation of apoptotic factors Bcl-2-interacting killer (BIK) and Bcl-2- interacting mediator of cell death (BIM) and downregulation of anti-apoptotic members of Bcl-2 with consequent involvement of mitochondria and activation of the intrinsic apoptotic pathway managed by caspase 9 which terminally activates caspase 3 and 7. Alternative caspases such as an increase in caspase 4 play crucial roles in the cell killing mechanism through ER stress induction by unfolded protein response.