**2. Apoptosis: type 1 program cell death**

Apoptosis (will be referred to as PCD) as the name suggest is the targeted killing of cells that occurs as a part of homeostasis, development, and pathogenic processes throughout life. PCD is well-operated in the physiological processes in an orchestrated manner to eliminate any defect in cells or tissues [2, 4]. The three well-recognized apoptotic cell disassembly are (i) cell shrinkage, (ii) membrane blebbing, and (iii) the formation of membrane protrusions and fragmentation [2]. Apoptosis can be triggered via two ways; i) the extrinsic pathway where the cell death receptors such as Fas, TNFαR, and Death receptors (DR3, DR4, and DR5) bind to their ligands ii) the intrinsic pathway when Bcl-2 family pro-apoptotic proteins lead to permeabilization of the mitochondrial outer membrane. Both extrinsic and intrinsic pathways are converged on the activation of the caspase family, which is responsible for the removal of the damaged cell [5, 6]. The stimulation of apoptosis via the extrinsic pathway requires classic ligand-cell-surface receptor interaction. Death-receptors-induced extrinsic cell death is mainly critical for the function of the immune system [7]. The intrinsic apoptotic cell death starts in a cell-autonomous manner. Cellular defects and stresses, such as DNA damage (due to toxic agents or defect s in the cell-cycle process) or endoplasmic reticulum (ER) stress (caused by accumulated unfolded proteins), leads to apoptosis when the damage is further than repair [8].

#### **2.1 The activation of caspases, functions, and regulation**

The initiation of apoptosis requires the activation of caspases, which then promote a cascade of events culminating in the death of a cell. Caspases are a family of cysteine proteases, very well conserved proteins playing many vital roles. Caspases can be divided into two categories (i) involved in immunity and (ii) the ones facilitating apoptotic cell death. These enzymes are expressed inactively inside the cell and activated to cleave for specific target substrates [9]. Pro-apoptotic caspases cleave a much broader subset of intracellular proteins to intercede cell death via apoptosis, on contrary, the caspases those involved in immunity can remove the cells via other non-apoptotic mechanisms. Pro-apoptotic caspases are fundamental for the development, and maintenance of tissue homeostasis [10]. Although many different caspases are found in mammals, here attention will be given to those which are very well characterized. During program cell death, the activation of caspases leads to biochemical and structural changes such as condensation of chromatin, mitochondria permeabilization, and membrane blebbing which are characteristic of apoptotic cell bodies. These essential processes display two important functions, firstly, the promotion of cell death and secondly, the elimination of damaged cells. Caspase-dependent program cell death generally ends with cell death [11]. On the contrary, the failure of apoptosis or non-complete process cause many disorders, most importantly, cancer. Nonetheless, cells have many other regulators that positively or negatively maintaining the activation and/or function of caspases [12]. The expression of caspases differs with stimuli type and expression level. While some caspases are expressed following a death trigger, the others are expressed constantly but being in an inactive state [13, 14]. Once the cell senses a death signal and/or being damaged the initiator caspases are being activated and recruited to the plasma membrane or in the cytoplasm. Upon the cleavage of

initiator caspases, they activate the destructor caspases. The activation of destructor caspases in turn cleave important cellular substrates during program cell death. The initiator caspases (caspase −8, −9, and − 10) and destructor caspases (caspase −3, −6, and − 7) prefer different cleavage sites [15]. Many of the destructor caspases include a cleavage site that is consistent with the peptide substrate specificity. Even though, caspase −3 and − 7 contain identical peptide substrate preferences they cleave different substrates. Such as Bid, Caspase −6 and − 9, Bid, XIAP, and gelsolin are cleaved by caspase −3, while p23 is cleaved by caspase-7 [16]. The destructor caspases can cleave more than thousands of substrates to enable program cell death. Also, the initiator caspases have many additional important mediators for program cell death. The activation of initiator caspase-8 is through the extrinsic program cell death pathway which is mediated via death receptors [17]. The other initiator caspase-9 is activated by the intrinsic program cell death pathway which is regulated by the apoptosome complex. The active initiator caspases (both −8, and − 9) can cleave downstream destructor caspases (caspase −3, −6, and − 7) [18]. The activation of initiator caspases can be via several cell-death signaling complexes. These different signaling complexes respond to many intra- and extracellular events, which induce the recruitment of initiator caspases for the activation process [19, 20]. It is important to mention that the interactions of caspase-activation platforms and those caspases indicate a death-fold domain in the proteins. These sorts of forms are existing in caspases and adaptor proteins: the caspase-recruitment domain (CARD) and death effector domain (DED). There are also different other death domains, such as the death domain (DD), and pyrin domain (PYR), which are included in some of the activations of caspases but absent on caspases [21]. Indeed, death fold domains generally intervene in interactions between proteins via homotypic interaction. For example, some extracellular ligands are tumor-necrosis factor (TNF) family, TNF-related apoptosis-inducing ligand (TRAIL), and FasL. These specific ligands bind to different TNF receptor (TNFR) family, so, upon their binding stimulating conformational changes. The specific signaling complex that is linked with Fas is DISC (death-inducing signaling complex) and contains FADD (Fas-associated death domain), which is a small adaptor protein. TNFR induces the activation of several cell-death signaling complexes [22]. Complex IIa involves FADD and TRADD, and the other complex IIb involves FADD, and RIPK1, RIPK3 (receptor-interacting serine–threonine kinases). Both complexes can activate caspase-8 and program cell death [23]. Once caspase-9 is activated, the cell death stimuli induce Bcl-2 family proteins Bax or Bak and lead to mitochondrial outer membrane permeabilization (MOMP) [24]. Later, MOMP is freeing cytochrome-c (Cyt-c) and other pro-death factors from the mitochondrial membrane to the cytosol [25]. The electrons are carried by Cyt-c in the respiratory chain of healthy cells, which function as a co-factor of Apaf-1 in the apoptotic cell's cytosol. The Cyt-c and ATP bind to Apaf-1 to form the apoptosome complex, which results in the recruitment and activation of caspase-9 [26]. The active caspase-9 cleaves, and in turn, induces the activation of caspase-3. Importantly, this intrinsic program cell death pathway is related to a death-receptor pathway through Bcl-2 family member, Bid. The cleavage of Bid through death-receptor activated caspase-8 leads to truncated Bid, which in turn causes the activation of Bax/Bak-induced MOMP [27]. Hence, result in the activation of caspase-9. Caspase-2 activation induced by the PIDDosome protein complex involves the Rip-associated protein with a death domain (RAIDD) and p53-induced protein with a death domain (PIDD) [28]. The activation of caspase-1 in turn cleaves pro-IL-1b to cause inflammation [23]. Different inflammasomes form particular responses to different stimuli such as Nod-like receptors (NLRs) including NLRP1, NLRP3, and NLRC4 [29–31]. The NLR inflammasome protein contains an adapter binding domain and adaptor

apoptosis-associated speck-like protein (adaptor ASC) containing CARD. Once ASC oligomerizes, it, in turn, leads to the recruitment of caspase-1 and activates caspase-1 [23]. The activation process of caspases has many violent consequences. Thus, the regulation is tightly managed. The cFLIP (cellular FLICE inhibitory protein), which is caspase-8's inactive mimic, binds to signaling complexes that are associated with death receptors to repress caspase-8 [32]. XIAP (X-linked inhibitor of apoptosis) is the only active site inhibitor of caspases and contains three BIR (baculovirus IAP repeat) motifs. The region BIR2 inhibits the activation of destructor caspases, caspase-3 and -7 via two different mechanisms. The first one, by binding the catalytic side of caspase-3 and -7, and the second one is by binding the newly cleaved protein terminus [33]. Also, the BIR3 motif inhibits the caspase-9 and blocks the dimerization of caspase-9. Of note, the regulation of caspases can be post-transcriptionally by phosphorylation. Several studies reported to inhibit the activity of caspase-2, −8, and − 9 [34]. Besides, caspases can play roles in diverse signaling pathways by their constant activation, for example, some of the specific morphological features of apoptotic cells are due to caspase-intervened activation of some actin cytoskeleton modulators such as p21-activated kinase 2 (PAK2), Rhoassociated kinase I (ROCKI) and gelsolin [35–37] (**Figure 1**).
