**3. Signal transduction pathways in apoptosis**

#### **3.1. MAPK family**

For apoptosis to be carried out an orchestrated array of signal transduction pathways needs to be put into action among which mitogen-activated protein kinases (MAPK) play a leading role. 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 to the amino acid present in both phosphorylation sites (Thr-XXX-Tyr) [53–55]:

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].

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].

For apoptosis to be carried out an orchestrated array of signal transduction pathways needs to be put into action among which mitogen-activated protein kinases (MAPK) play a leading role.

**2.3. Cellular demolition**

98 Current Understanding of Apoptosis - Programmed Cell Death

**3.1. MAPK family**

**3. Signal transduction pathways in apoptosis**


and neurotransmitters through binding to different G-protein coupled receptors, tyrosinekinase receptors, and ion channels. Then, signal transduction continues with an adaptor protein that transmits the signal to a MAP3K of which several have been described for ERK such as Raf-1B-Raf, A-Raf, and TPL2 [62]. Following the described phosphorylation pattern (MAPKKK → MAPKK → MAPK), the stimulus activates MAPKKK (i.e., Raf-1), which in turn phosphorylates MEK1 and MEK2 (both MAPKK) and these finally phosphorylate and activate ERK1 and ERK2 [62].

[83, 84]. PIP3

inducing apoptosis.

interacts with the pleckstrine homology (PH) domain, located in the N-terminal

Signaling Pathways Targeted by Protozoan Parasites to Inhibit Apoptosis

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region of the serine/threonine kinase Akt or PKB, with the final result of the kinase being recruited to the plasma membrane [85–87]. Furthermore, PDK1 phosphorylates Akt/PKB producing a conformational change that facilitates a second phosphorylation by the rictormTOR1 complex [88]. Finally, the PI3K/Akt pathway leads to diverse effects associated with cellular proliferation and survival [89, 90]. Specifically, it produces the inactivation of many proapoptotic signals, such as BAD, procaspase-9, and FKHR (Forkhead) transcription factors [21, 91]. It also promotes the activation of CREB, NF-κB, and HIF-1α transcription factors,

Apoptosis constitutes a very important defense mechanism against intracellular microorganisms [95], whom in order to survive inside cells need to inhibit the induction of apoptosis. It has been demonstrated that diverse intracellular pathogens including virus [96], bacteria [97], and protozoan parasites [98] have developed mechanisms to persist within host cells without

*Leishmania* is an obligate intracellular parasite that infects a variety of cells such as neutrophils, macrophages (Mφ) and dendritic cells (DC). *Leishmania* has developed diverse mechanisms to manipulate host cells in order to evade the immune response and survive inside cells. Some of these strategies are the evasion of the phagosome-lysosome fusion and the inhibition of apoptosis. Studies have demonstrated that monocytes, macrophages, and dendritic cells grown in apoptogenic conditions and infected with different species of *Leishmania* present an inhibition of normal apoptosis. Also, *Leishmania* infection prevents natural apoptosis of neutrophils. The first demonstration of the inhibition of apoptosis by *Leishmania* was performed by Moore and Matlashewski in 1994 who demonstrated that the infection of bone marrow derived-macrophages (BMM) with *Leishmania donovani* promastigotes or the stimulus with LPG inhibited apoptosis induced by the deprivation of M-CSF. Interestingly, the culture supernatant of infected BMM was able to inhibit apoptosis suggesting that the effect could be due to soluble mediators [99]. A later study showed that cellular activation increased the production of TNF-α, TGF-β, IL-6, and GM-CSF, while the secretion of M-CSF and IL-1β diminished [100]. Studies performed later with another species, *Leishmania major,* showed that the infection of macrophages grown in the absence of M-CSF or in the presence of staurosporine inhibited the release of mitochondrial cytochrome c, thus delaying apoptosis. It was observed that the infection of BMM with *L. major* promastigotes inhibited caspase-3 activation owed to a decrease in MOMP and subsequent release of cytochrome c, which was not associated to NF-κB activation since the use of specific inhibitors did not affect the capacity of L. major to inhibit macrophage apoptosis. It was also demonstrated that the infection of BMM obtained from BALB/c o C57BL/6 mice with *L. major* promastigotes preserved the phenomenom of apoptosis inhibition despite the genetic background of the host or type of immune response (Th2 or Th1, respectively) [101]. Also, studies performed with cell lines reported

which in turn activate the expression of antiapoptotic genes [92–94].

**5. Apoptosis inhibition and infection**

**5.1. Inhibition of apoptosis by** *Leishmania*

#### **3.2. MAPK participation in apoptosis**

One of the upmost actions of MAPK is the activation of transcription factors, which regulate the expression of genes that lead to crucial molecular events in the cell regarding growth, proliferation, inflammatory cytokine production, and apoptotic cell death [56]. In relation to apoptosis, a key participant is JNK that plays its role through two different mechanisms. The first one is related to nuclear events in which JNK is translocated to the nucleus and activates c-Jun and other transcription factors that promote proapoptotic gene expression, through p53/73 or c-Jun/AP1-dependent mechanisms [70, 71]. The second mechanism relates to JNK activation and translocation to the mitochondria, where it promotes the phosphorylation of protein 14–3-3, a protein that normally inhibits Bax by being bound to it. As protein 14–3-3 is phosphorylated, Bax is released and translocates to the interior of the mitochondria where it oligomerizes and forms pores in the mitochondrial membrane with the subsequent release of cytochrome c and apoptosis induction through the intrinsic pathway. Apart from these two mechanisms, JNK can also phosphorylate "BH3-only" family members, whose antiapoptotic effect inhibits Bcl-2 and Bcl-xL and is also involved in the posttranslational modifications of Bid and Bim, both of which induce Bad and Bax activity [70, 71]. Another MAPK deeply involved in apoptosis is p38, which in many times is simultaneously activated with JNK [72]. p38 exerts its central role in apoptosis through the activation of proapoptotic proteins, mainly BimEL, BAD, and Bax [73–77] and simultaneously induces the inhibition of ERK and Akt antiapoptotic pathways [76, 77]. Also, p38 and JNK participate in TLR signaling pathways. These key participants of the innate immune response function as regulatory sensors of both apoptosis signaling through the induction of MAPK p38 and JNK [78, 79] and survival signals through PI3K and some Bcl-2 family members [80–82].
