**2.1. Initiation of apoptosis**

The activation of apoptosis requires the assembly of an intricate web of intracellular signaling pathways that occurs in three phases: initiation or activation, execution, and cellular demolition that are triggered in three different ways: the extrinsic pathway, the intrinsic pathway (subdivided in mitochondrial-induced apoptosis and endoplasmic reticulum stress-induced apoptosis) and the caspase-independent pathway [5–9].

#### *2.1.1. The extrinsic pathway*

This pathway is activated through extracellular stress signals that are detected and amplified by transmembrane receptors called death receptors [10–12]. Some of these receptors include the Tumor Necrosis Factor receptor (TNFR), Fas receptor (CD95), DR3/WSL, and Apo-2L (TRAIL-R1/DR4, TRAIL-R2/DR) [13, 14], which are characterized for the presence of intracellular domains called death domains (DD), which include the TNFR or TRADD and Fas or FADD death domains [15]. Once receptors become engaged with their respective ligands, activating proteins such as RIPK1, FADD, c-FLIP, c-IPAs, and ubiquitin ligase E3 are recruited [16–21], and in consequence, a supramolecular complex is formed by the activating protein-receptor domain that is recognized as a Death-Inducing Signaling Complex (DISC), which activates procaspase 8, the precursor of caspase 8 [16, 18–22]. In some cases, the extrinsic pathway can be triggered without a ligand as is the case of DCC and UNC5B receptors where, in the absence of a ligand, DCC interacts with cytoplasmic adapting protein DRAL to assemble an activation platform for caspase 9 [23]. In a similar manner, the UNC5B receptor, in the absence of netrins, recruits a molecular complex composed of PP2A and Death Associated Protein Kinase 1 (DAPK1) [24]. In both cases, caspase 8 is activated to initiate cell death via apoptosis.

#### *2.1.2. The intrinsic pathway*

embryogenesis, metamorphosis, and constant cellular changes, being of upmost importance for the maintenance of homeostasis in all tissues [2]. The term apoptosis was coined since 1972 by Kerr, to define a type of programmed cell death with morphological and molecular characteristics different from other types of cell death. These characteristics include retraction of pseudopods with the consequent reduction of cellular volume and rounding of the cell, nuclear volume reduction (pyknosis) and fragmentation (karyorrhexis), structural modification of organelles followed by the formation of vesicles due to blebbing of the plasma membrane [3, 4]. Apart from being a fundamental process of cells for the maintenance of homesostasis, apoptotic cell death represents an important defense mechanism against intracellular pathogens. Against it, a wide spectrum of microorganisms has developed diverse strategies to inhibit apoptosis of their host cells. These strategies involve different signaling pathways that are hijacked by pathogens to achieve their goal of inhibiting apoptosis and persist inside cells. The purpose of this chapter is to better understand the signaling pathways that are targeted by protozoan

The activation of apoptosis requires the assembly of an intricate web of intracellular signaling pathways that occurs in three phases: initiation or activation, execution, and cellular demolition that are triggered in three different ways: the extrinsic pathway, the intrinsic pathway (subdivided in mitochondrial-induced apoptosis and endoplasmic reticulum stress-induced

This pathway is activated through extracellular stress signals that are detected and amplified by transmembrane receptors called death receptors [10–12]. Some of these receptors include the Tumor Necrosis Factor receptor (TNFR), Fas receptor (CD95), DR3/WSL, and Apo-2L (TRAIL-R1/DR4, TRAIL-R2/DR) [13, 14], which are characterized for the presence of intracellular domains called death domains (DD), which include the TNFR or TRADD and Fas or FADD death domains [15]. Once receptors become engaged with their respective ligands, activating proteins such as RIPK1, FADD, c-FLIP, c-IPAs, and ubiquitin ligase E3 are recruited [16–21], and in consequence, a supramolecular complex is formed by the activating protein-receptor domain that is recognized as a Death-Inducing Signaling Complex (DISC), which activates procaspase 8, the precursor of caspase 8 [16, 18–22]. In some cases, the extrinsic pathway can be triggered without a ligand as is the case of DCC and UNC5B receptors where, in the absence of a ligand, DCC interacts with cytoplasmic adapting protein DRAL to assemble an activation platform for caspase 9 [23]. In a similar manner, the UNC5B receptor, in the absence of netrins, recruits a molecular complex composed of PP2A and Death Associated Protein Kinase

1 (DAPK1) [24]. In both cases, caspase 8 is activated to initiate cell death via apoptosis.

parasites in order to evade the defense mechanism of apoptosis.

apoptosis) and the caspase-independent pathway [5–9].

**2. Generalities of apoptosis**

96 Current Understanding of Apoptosis - Programmed Cell Death

**2.1. Initiation of apoptosis**

*2.1.1. The extrinsic pathway*

The mitochondrial intrinsic pathway can be initiated by different intracellular stimuli such as irreversible genotoxic damage, increase in the cytoplasmic calcium (Ca+ ) concentration, oxidative stress, among others [15]. In this pathway, a family of proteins called Bcl-2, characterized for having from 1 to 4 conserved domains that share homology with Bcl-2 or BH [6], has a leading role. This family is composed of proapoptotic proteins that, according to the BH domains that possess, are divided into Bax and "BH3 only" subfamilies. The members of the Bax subfamily are Bak, Bax, Bok, and Mtd and possess three BH domains (BH1-BH3), while the "BH3 only" subfamily, as denoted by its name, possesses a single BH3 domain and is composed of Bid, Bad, Bim, Bik, Blk, Hrk, NOXA or PUMA. On the other hand, the antiapoptotic proteins family present four BH domains (BH1-BH4) and is composed of Bcl-2, Bcl-XL, Bcl-W, Bfl-1, and Mcl-1 [6]. The BH1 and BH2 domains are structurally similar to the diphtheric toxin [25, 26]. The antiapoptotic proteins Bcl-2 and Bcl-xL are located in the outer mitochondrial membrane and prevent the release of cytochrome c, while the proapoptotic proteins Bad, Bid, Bax, and Bim are located in the cytosol and under certain stimuli are translocated to the mitochondria, where they induce the release of cytochrome c [25, 26]. Additionally, caspase 8 may take part in the intrinsic pathway through Bid proteolysis, turning it into tBid, which also translocates to the mitochondria and activates Bcl-2, Bax, and Bak [27]. Once Bax and Bak have been translocated to the mitochondrial membrane, a molecular complex referred to as PTPC is activated and induces the Mitochondrial Transition Permeability (MTP) phenomenon [28, 29]. These events culminate in the permeabilization of the outer mitochondrial membrane or MOMP, which is the rate-limiting step in apoptosis that conducts to an energetic and metabolic damage and the cell faces irreversible apoptotic cell death. The release of cytochrome c from the mitochondrion permits its association with the Apoptosis Activation Factor (Apaf-1) thus forming a structure to which procaspase 9 is incorporated, originating a molecular complex referred to as the apoptosome. As procaspase 9 is activated, it recruits executor caspases 3 and 7, which causes a proteolytic effect inducing cell death [27]. As mentioned earlier, the intrinsic pathway can also be activated via endoplasmic reticulum stress whose main stimulus is the misfolding of proteins and their subsequent accumulation in the endoplasmic reticulum (ER). Once misfolded proteins reach a critical concentration, they activate ER membrane sensors [30].

The induction of apoptosis conducts hopelessly to the activation of caspases; nevertheless, the damage to the mitochondria can, in some cases, provoke the release of some molecules with proapoptotic capacities such as HTRA2, AIF and ENDOG that have the ability to induce apoptosis without the intervention of caspases. HTRA2 has the ability to attack proteolytically the cytoskeleton, while AIF and ENDOG can enzymatically attack DNA [31].

#### **2.2. Caspases**

Caspases (**C**ysteine-dependent, **ASP**artate-specific peptid**ASE**) owe their name to the fact that their proteolytic functions lie specifically in an aspartate residue and require the presence of cysteine to perform their catalytic activity [6]. There are many types of caspases and are classified according to their function in initiation caspases: 2, 8, 9, and 10; executor caspases: 3, 6, and 7; and inflammatory caspases: 1, 4, and 5. In addition, there are other caspases that perform diverse functions such as caspase 11, which regulates cytokines during septic shock, caspase 12 that is associated with endoplasmic reticulum stress apoptosis, and caspase 14, which has only been isolated in embryonic tissue, specifically in keratinocytes. Caspases are found in cells in an inactive state called zymogens or procaspases that possess three distinct regions: a prodomain located in the N-terminal end, a minor subunit close to the C-terminal end and, in between, the major subunit. Procaspases are activated through autoactivation or activation by another caspase or molecule that cause the excision in two sites of the aspartate residues, the first one between the prodomain region and the major subunit, and the second between the major and minor subunits [32]. The activation of caspases starts with an initiator caspase that requires the formation of a multimeric adaptor protein complex called apoptosome whose formation is mediated by Apaf-1, an inactive monomer in nonapoptotic cells [31]. MOMP-mediated release of cytochrome c triggers the formation of the apoptosome through the binding of cytochrome c to Apaf-1 on the WDR domain, following by the conversion of ADP into dATP/ATP in the NOD domain [33–35]. Finally, procaspase 9 binds to Apaf-1 through a homotypical interaction with the CARD domains [36]. The apoptosome catalyzes the autoproteolytic action of procaspase 9, and its active form, caspase 9, remains active and bound to Apaf-1 as a holoenzyme [36].

These mitogen-activated protein kinases, as denoted by their name, are activated not only by mitogens but also by other physical and chemical stimuli, such as growth factors, UV radiation, genotoxic agents, oxidative stress, inflammatory signals, and cytokines. Once activated, MAPK go through three secuencial phosphorylation steps [52], carried out by three groups of enzymes: (1) MAPK kinase kinase (MAPKKK or MAP3K), for example ASK1; MAPK kinase (MAPKK), for example MEK 1 through 7; MAPK such as ERK 1/2, JNK, and p38. MAPKs belong to the serine-/threonine-type kinases [53, 54] and possess tyrosine (Tyr) and threonine (Thr) conserved double phosphorylation domains [52]. They are further divided in three subfamilies according

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**1.** The p38-MAPK subfamily features glycine between the two phosphorylation sites (Thr-Gly-Tyr) and is activated through stress signals, growth, and differentiation factors. This subfamily is composed of the p38-MAPKα, p38-MAPKβ, p38-MAPKγ, and p38-MAPKδ isoforms that share a 12-amino acid activation loop and differ in affinity for the activating protein, tissue expression, and downstream effect. The p38α isoform, commonly referred to as p38, as well as the p38β isoform are ubiquitous being present in almost every tissue, while p38γ and p38δ isoforms have a more restricted localization. When p38 is activated, it initiates the three rounds of phosphorylation that culminate in the phosphorylation of p38 specifically at Thr180 and Tyr182 sites. This phosphorylation process produces conformational changes that lead to the enzyme binding with ATP and the acceptor substrate of the phosphate [56]. This subfamily participates in the regulation of certain growth factors, kinases and phosphatases, as well as in the regulation of ATF-2, MEF2, MAPKAPK, CDC25 or MSK1/2 and their activa-

tion triggers cellular proliferation, differentiation, apoptosis, among others [57, 58].

phosphorylate p53, while JNK1 can only modify it post-transcriptionally [67, 68].

**3.** The ERK subfamily features glutamic acid between the two phosphorylation sites (Thr-Glu-Tyr) and is composed of ERK1, also known as MAPK3, and ERK2, also known as MAPK1 or p42MAPK [62, 69]. These kinases are activated by growth factors, hormones,

**2.** The JNK subfamily features proline between the two phosphorylation sites (Thr-Pro-Tyr) and is composed by the JNK1, JNK2, and JNK3 isoforms. These proteins are also known as stress-associated MAPKs or SAPKS (stress-activated protein kinases) and participate in cellular growth, differentiation, and apoptosis [59, 60] as a response to diverse stress signals, such as UV or gamma radiation, protein synthesis inhibitors (anisomycin), hyperosmolarity, toxins, ischemic damage, thermal shock, antineoplastic drugs, peroxides, and inflammatory cytokines, among others [59]. Stress signals initiate the three cycles of phosphorylation with the activation of MAP3K, ASK1 and ASK2, among others, which in turn activate MEK4 and MEK7 through phosphorylation of two specific serine and threonine residues. Finally, MEK4 and MEK7, also known as MKK4 (SEK1/JNKK1) or MKK7 (SEK2/JNKK2) phosphorylate JNK in threonine-proline-tyrosine (Thr-Pro-Tyr) specific residues [59, 61, 62]. Interestingly, the biological roles of JNK isoforms are similar [63], although they are physically different and also differ in tissue localization. JNK1 and JNK2 are expressed in all tissues, while JNK3 isoform is found predominantly in nervous tissue, and to a lesser extent in the heart and sperm [64–66]. Although JNK1, JNK2, and JNK3 can all induce apoptosis, there is evidence suggesting that each protein induces apoptosis through a different pathway. It has been demonstrated that all of them associate with p53, a nuclear transcription factor that activates proapoptotic gene expression, such as BAX or PUMA, but interestingly their expression varies with respect to p53. In the case of JNK1, its expression is inversely proportional to p53, contrary to JNK2 expression, which is directly proportional to p53. Both JNK2 and JNK3 can

to the amino acid present in both phosphorylation sites (Thr-XXX-Tyr) [53–55]:

#### **2.3. Cellular demolition**

Once apoptosis is triggered through one of the different pathways just explained, the activation of caspase 9 unchains a cascade of executioner caspases [6, 15], whose proteolytic action is directed to multiple substrates that finally culminate in the demolition of the cell. One central substrate targeted by caspases is ROCK1, an actin cytoskeleton activity regulator that upon activation loses its C-terminal end, subsequent phosphorylation, and thus activation of the myosin for is subsequently phosphorylated, and thus activates the myosin light chain, which generates actin contraction that in turn triggers several phenomena such as phosphatidylserine translocation, cellular rounding and retraction, as well as vesicle formation or blebbing and loss of intercellular unions due to the proteolytical attack of desmosomes or other forms of cell to cell junctions. It also affects nuclear membrane integrity and provokes further fragmentation of DNA and degradation of proteins associated with transcription and translation [6, 37–49]. Other targets attacked by caspases are, for example, the caspase-activated DNase (CAD), whose activation culminates in DNA degradation at internucleosomal sites [49] or Golgi reassembly and stacking proteins (GRASP)that participate in Golgi apparatus conformation, cistern formation and connections leading to Golgi fragmentation and disintegration [6, 50]. Continuing with the demolition events, the mitochondrial proteins Bax and Bak are activated due to BH3 action, which in turn generate pores in the mitochondrial membranes and release of their contents. Also, the p75 subunit of the electron transport chain complex 1 is proteolytically degraded [6, 50]. One of the final acts of apoptosis is the release of chemotactic cytokines and other molecules, as well as the formation of union sites for phagocytic cells indispensable for the elimination of cellular remains by phagocytes for these cells [6, 51].
