Intracellular Role of Apoptosis in In-Vivo Reactions Occurring in Multi-Cellular Organisms

There are two well-known methods of cell execution: Apoptosis and Necrosis. Necrosis is regarded as a violent form of cell death, whereas apoptosis, on the other hand, is a more benign, modulated, and rational mode of killing cells. This process involves making well-informed decisions about selected cells to be terminated for the greater good of the host organisms. The advantages associated with the use of apoptosis as a regulated manner of programmed cell execution are well documented in literature. These include not exhibiting inflammatory responses, providing insights for drug designs in the pharmaceutical sector, being used for cancer treatments in health institutions, and being utilized as courier agents in the form of apoptotic bodies capable of material transport to selected cell recipients.

The self-destruct mode of action of apoptosis is mainly channeled through a variety of serine proteases called caspases. The caspases, otherwise described as cell death executors, are activated by death signals relayed via signaling pathways. In terms of initiation, both internal and external stimuli can kick-start the apoptotic process through different pathways. After commencement, these two divergent stimuli may coalesce together to form apoptotic bodies, which are subsequently consumed by phagocytes via efferocytosis. Some schools of thought view this process as the last stage in a cell's life (i.e., efferocytosis is regarded as the concluding portion of apoptosis), whereas others argue that a cell's useful lifecycle goes beyond this stage to include reuse, recycle, and transfer of materials earlier formed in apoptotic bodies (Peter et al., 2008).

In apoptotic cells, some of the noticeable changes in their morphology include nuclear fragmentation, cell blebbing and shrinkage, the formation of apoptotic bodies, condensation and fragmentation of chromatin, and nucleosomal DNA as genetic materials. At the commencement of apoptosis, cell shrinkage and chromatin condensation (pyknosis) occur side by side. These two processes are closely followed by nucleus fragmentation (karyorrhexis) and the blebbing of the plasma membrane into smaller apoptotic bodies which are subsequently consumed by phagocytes. In other words, phagocytosis serves the dual purpose of securing the external environment from accidental spillage of harmful materials present in apoptotic bodies, as well as ensuring the final dissociation of target cells (Bortner et al., 2008).

Considering the rather complicated biological mechanism of apoptosis, which involves an interwoven web-like design of proteins, signal transducers and cascades of signalling pathways, it is no surprise that different mechanistic pathways exist. These can be categorized under intrinsic and extrinsic apoptosis pathways. The extrinsic pathway utilizes membrane receptors (used in transmitting death signals) classified under the tumour necrosis factor (TNF) receptor superfamily and activated by two major ligands, TNF and Fas. Furthermore, while moving through the TNF pathway, TNF and its two receptors (TNFR-1 and TNFR-2) transmit death signals using two co-opted agents, the TNF receptor-associated death domain (TRADD) and the Fas-associated death domain protein (FADD), effectively carrying out premeditated cell execution via these adaptor proteins (caspases). In addition, a third extrinsic apoptotic pathway, known as the TNF-related apoptosis inducing ligand (TRAIL) pathway, is also feasible. It usually undergoes proteolytic cleavage by protease to transform into a soluble ligand (often existing as trimers: trimeric TRAIL), which attaches to DR4 and DR5 membrane receptors to initiate an intracellular signalling cascade akin to the Fas pathway. On the other hand, the intrinsic pathway of apoptosis is kick-started by non-receptors that serve as mediators, further backed by the regulatory action of mitochondria. Their transmembranes are disrupted by stimuli to make them more permeable, which also brings about the production of mitochondrial permeability transition pore on the outer membrane, whose function is to direct pro-apoptotic factors into the cytosol (Nagaraj et al., 2013; Leung et al., 2015).

There are two obvious choices available to a cell in response to changes in its internal or external conditions. These choices are either to live by adopting survival tactics or to die via rational or irrational means. The cellular health status in consonance with the cell's genome makeup determines whether a cell is due for execution or should have its lifespan extended. In other words, the instantaneous condition of a cell's metabolome when placed under duress influences the molecular decision to either live or die (Thorenoor et al., 200).

Role of Apoptosis in Disease Mediation

The role of apoptosis in disease mediation has been investigated over time by various studies. Results indicate that in most cases, significant changes brought about by a distortion of tissue homeostatic function tend to lead to cell death via varied forms. This premeditated cell death is often a biological phenomenon aimed at correcting the distorted pathological and hormonal imbalance in selected areas of the human immune system. Thus, apoptosis intervention has been noted to play a major role in selected ailments such as neurological disorders, cancers, cardiovascular disorders and other immune imbalance related diseases (Kawai et al., 2009; Nagaraj et al., 2013).

There are several sequences that have been documented in literature as avenues through which cells can initiate the apoptotic process (programmed cell death). This can be in response to either an increase or decrease in an existing cellular precondition ranging from hormonal balance and homeostasis to cell pathology. Case studies of four such established sequences (E.coli mediated apoptosis, enzyme mediated apoptosis, mitochondria mediated apoptosis, cytochrome c mediated apoptosis) will be briefly discussed to support this point.

Escherichia coli (E. coli) as a multi-cellular organism has been shown to undergo programmed cell death when impacted by the death effector domain (DED) of the mammalian apoptosis mediator, called the Fas-associated death domain protein (FADD). A recent study investigated the biological events leading up to the FADD-DED induced cell death in E.coli, which was found to be similar to the bactericidal-antibiotic induced cell murder (Thorenoor et al., 2010).

Through a myriad of experimental methods ranging from proteomic approaches to co-expression studies, the researchers were able to prove that the down-regulation of proteins, which play a key role in energy production and conversion, were attributed to the FADD-DED expression. In other words, the FADD-DED cell expression was in direct response to suppressing the reactive oxygen species (ROS) effect. Furthermore, overwhelming proof of the analogous inference to Bax-mediated apoptosis in mammalian cells was found, brought about by the localization of FADD-DED in membranes, specifically the mitochondrial membrane. In addition, their results showed that membrane localization of FADD-DED typically triggered the FADD-DED induced ROS generation, causing target cells to enter an excited state that consequently stimulated the ROS effect, ultimately resulting in cell death. In summary, the above steps illustrate a mechanistic pathway for ROS-dependent cell execution at unicellular and multi-cellular levels (Thorenoor et al., 2010).

Cases of enzyme-mediated apoptosis have been studied previously, with a prominently featured study involving the elucidation of the functions of Thioredoxin 1 (Trx1) and glutaredoxin 1 (Grx1) as redox enzymes. These not only play significant roles in redox homeostasis but also engage in other diverse functions such as stress sensing, inflammation activation, DNA synthesis enhancement, and suppression of apoptosis. Thus, as a direct consequence of this phenomenon, Trx1 and Grx1 have also been involved in cancer cell lines and patient tumours, as well as executing a monitoring role in protein-protein interaction, e.g., with a specified protein such as the apoptosis signal-regulating kinase 1 (Ask1). In other words, apoptosis can be suppressed in cancer cells by inhibition of Ask1 by these ubiquitous redox enzymes (Kekulandara et al., 2018).

Cytochrome c (Cyt c) is described as a multifunctional mitochondrial associated with significant roles in electron transfer and apoptosis. When acting in conjunction with cardiolipin (CL), cytochrome c tends to carry out a principal role during the early stages of apoptosis. During these initial stages, cytochrome c usually orchestrates its release from mitochondria by taking on peroxidase activity, which enables it to effectively catalyse cardiolipin peroxidation. In other words, studies have shown that after binding to cardiolipin, cytochrome c usually acquires peroxidase activity which results in the peroxidation of cardiolipin, detachment or disentanglement of cytochrome c from the inner mitochondrial membrane, transfer into the cytosol, and subsequently, the induction of apoptosis through caspase activation.

Furthermore, three distinguishable sites on cytochrome c’s surface that have been noted as possible regions of interaction with cardiolipin include site A (formed by Lys [72, 73, 86 and 87]), site C (situated close to Asn52), and site L (requires Lys [22, 25, 27] and His [26, 33]) which takes place under reduced pH. In addition, an N-site (focused on residues [Phe36, Gly37, Thr58, Trp59, and Lys60]) has recently been discovered as a new cardiolipin binding site through the characterisation of the cytochrome c-cardiolipin interaction in reverse micelle encapsulation. Hence, in summary, the emergence of peroxidase activity in cytochrome c can be credited to the partial unfolding of the protein followed by the breaking of the bond between heme iron and its axial ligand (Met80), and consequently, the generation of alternative non-native conformers (Millazo et al., 2017).

Mitochondria occupy a prominent role in cell apoptosis by virtue of the fact that they contain apoptogenic protein procaspases, apoptosis-inducing factor, and cytochrome c as releasing factors that can be discharged into the cytosol in order to participate in the breakdown phase of apoptosis. These apoptogenic proteins tend to be released as a triggered response to alternative stimuli (such as cytokine withdrawal) or ceramide treatment, both of which heighten the deregulation of intracellular pH as a constituent step towards the initiation of cell death.

Within the eukaryotic cellular environment, cytosolic pH is normally monitored via proton pumps, channels, and ion transporters, which push H+/H+ equivalents and HCO3- ions in and out of the cell. Thus, disentangling cytochrome c from the inner mitochondrial membrane tends to kick-start cytosol events that eventually lead to apoptosis, thereby connoting the existence of a reversible interaction between the target protein and the lipid bilayer. These associations with lipid bilayers are made possible through the existence of alternative cytochrome c sites (A and C). In other words, while site A is an electrostatically interactive site composed of basic residues in cytochrome c (Lys72, Lys73), site C tends to take the form of a different cytochrome c lipid-binding site with a high appetite for protonated acidic phospholipids through its engagement in hydrogen bonding with Asn 52. On the other hand, the external surface of the inner mitochondrial membrane hosts the clusters of positively charged amino acid side chains of site "A", which are touted to be an imperative ingredient for the recognition and binding of cytochrome c to reductase and oxidase.

Furthermore, the presence of an additional site (site L) for the electrostatic interaction of cytochrome c with acidic phospholipids that makes use of amino acid residues [(Lys22, 25, 27) and His (26, 33)] has been discovered. Attributes of site L include that it maintains a controlling hold on pH-dependent binding of horse cytochrome c to membranes and it also maintains a pKa of 7.0. This has therefore led to the further hypothesis that cytochrome c’s interaction with the inner mitochondrial membrane may be regulated by the mitochondrial transmembrane potential. Therefore, the elevated pH status existing within the intermembrane space as a result of the decimation of the transmembrane potential may lead to the deprotonation of site L and consequently contribute to the disentanglement of cytochrome c from the mitochondrial membrane, hence, heralding the onset of apoptosis in cytosol (Kawai et al., 2009).

The foregoing discussions and case studies have illustrated the fact that apoptosis can be a veritable tool for premeditated cell execution as a direct response to either a perceived increase or decrease in erstwhile optimal conditions within a target cellular environment. As a bio-analytical tool, programmed cell death has beneficial applications, especially with regard to the killing of cancerous cells. However, it must also be used with caution in order to prevent unwarranted or unplanned execution of healthy cells carrying out important functions in non-diseased regions of the immune system."

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