**4. Metacaspases**

The importance of this cascade can be seen when disturbance of its regulation occurs on the cells, what causes immunodeficiency, carcinogenesis and other troubles related to aberrant

**Figure 1.** Representation of caspase, metacaspase and paracaspase major domains. IC: initiator caspase; EC: executor caspase; MI: type I metacaspase; MII: type II metacaspase; PC: paracaspase. Red: p20 domain; blue: p10 domain; orange: prodomain/recruitment domain; purple: death domain; green: immunoglobulin‐like domain; yellow: paracaspase

The cascade overview reveals two functional groups of caspases, concerning their position on the sequence of proteolytic events (**Figure 1**). The first caspases to be activated are the initiators which become active by the "induced proximity mechanism", often triggered by recruitment of adaptor protein complexes, such as recruitment domain of membrane receptors or by a huge protein complex called apoptosome. This induced proximity leads to the oligomerization of caspase molecules which form a heterodimer that becomes able to trigger the activation of the effector caspases by cleavage of a pro‐domain, since these enzymes are synthetized as zymogens. After this cleavage, the effectors themselves also stay organized as heterodimer, and cleave the apoptotic substrates, leading to a typical cellular morphology [8]. Despite the positional and functional differences, all caspases belong to a group named ICE family (interleukin‐1β‐converting enzyme/caspase 1 family), since caspase 1 was the first

About the phylogeny, these molecules are unique and well distributed among the Animalia Kingdom. Their presence ranges from vertebrate organisms such as *Homo sapiens* and *Danio rerio* to invertebrates such *C. elegans* and *Drosophila melanogaster* [32]. Also, their involvement

Alongside caspases, on clan CD, there are caspase relatives called paracaspases which are found in metazoans and in slime molds [4, 32] (**Figure 1**). In humans, paracaspase/MALT1 is associated to the lymphocyte activation by the NF‐kB pathway [33, 34]. In *Dictyostelium*, the paracaspase gene disruption does not affect the developmental cell death process, what leads to the suggestion that the enzyme is not required for the phenomenon [35]. A function for human paracaspase MALT 1 on caspase‐8 modulation is reinforced by Kawadler et al. [36], whose data show that MALT1 activates caspase‐8 during TCR signalling but reduces the

About its structural architecture, the human MALT 1 monomer has an apparent molecular mass of 41 kDa and the dimer, about 84 kDa and the overall predicted structure shows an N‐terminal death domain, two immunoglobulin domains, a paracaspase domain and another immunoglobulin‐like domain. The paracaspase domain is folded in a similar way to that of caspases and exhibits the ability to bind on substrate. Also, the enzyme, contrary to caspases,

cleavage of caspase‐3, avoiding apoptosis and inducing cell proliferation.

seems not to require cleavage of loop 4 to become active [37, 38].

PCD [29–31].

28 Enzyme Inhibitors and Activators

C‐terminal region. Based on Vercammen et al. [8].

characterized member.

with cell death processes is also well documented.

#### **4.1. Plant metacaspases**

After the first finding of caspase‐like activities on plants [41], as well as on other non‐metazoan organisms, an extensive search for the enzymes responsible for these activities was performed [32]. For the model plant, *Arabidopsis thaliana*, for example, nine metacaspases encoded by its genome were found: 3, of type I and 6, of type II metacaspases [42]. The list is being extended, among other examples are: soybean (*Glycine max*) with 16 metacaspases (10, type I and 6, type II); wheat (*Zea mays*) with 12 metacaspases (8, type I and 4, type II) and *Oryza sativa* sp japonica with eight metacaspases (4, type I and 4, type II) [43].

The first observations using cell extracts suggested that metacaspases could be responsible for the caspase‐like activities found in plants [44]. Nevertheless, recorded reports show that plant metacaspases are unable to cleave caspase synthetic substrates [45–47]. The caspases cleave their substrates after aspartate residues and metacaspases cleave after Asp or Lys residues at P1 position [43]. So, appears that the metacaspases are not directly responsible for the caspase‐like activities found in plants.

Although metacaspases do not have caspase‐like activities, many works suggest that they have a role in PCD [48]. The inhibition of a type II metacaspase (McIIPa) suppressed PCD in suspensor cells from an embryonic culture from *Picea abies*. During *P. abies* embryogenesis, it was shown that McIIPa is transported from cytosol to nuclei, where its presence was correlated to DNA fragmentation detection. Also, through experiments with a cell free system in the presence of a mutant form of this protein on its active site, the nuclear alterations were not detected [44]. These data reinforce that McIIPa is directly involved on a pathway which generates nuclear degradation, an event present on most eukaryotic PCD programmes. In this pathway, the protease can play a role on the cleavage of nuclear proteins, similar to what happens to the metazoan effector caspases [46].

The heterologous expression of the *A. thaliana* metacaspases MCP1b and MCP2b on a *Saccharomyces cerevisiae* strain, disrupted for its metacaspase YCA1 gene, was able to trigger an apoptosis‐like phenotype under hydrogen peroxide treatment, and this effect was seen to be abolished on the presence of the pancaspase inhibitor based on the sequence VAD. These findings reinforce a metacaspase role on PCD, as well as the requirement of proteins with caspase‐like activity [42].

The involvement of metacaspases on PCD is also suggested by works with plants under pathogen attack. Some examples are given by the detection of gene expression for MCA1 on *A. thaliana* leaves infected by *Pseudomonas syringae* [49], and by the increasing levels of a type II metacaspase on tomato leaves under *Botrytis cynerea* infection, with the detection of cell death phenotype [50].

It cannot be excluded, though, that metacaspases could not be directly involved on PCD regulation, but indirectly involved on signalling cascades that leads to PCD [48].

In face of this, the role of metacaspases are still under discussion, as well as their classification, since there are evidences which favour, and others contrary, to their groupment together with caspases [51, 52]. A cascade mechanism comparable to that of caspases was proposed for vegetable systems concerning cysteine proteases on senescent leaves and seeds on maturation. Bozhkov et al. [46] reinforced the idea that the execution of PCD in plants is controlled by two groups of enzymes with separated cellular localization. One of them is accumulated on lytic compartments and vacuoles, and the other has cytoplasmic‐nuclear localization, as in the case of MCIIPa.

#### **4.2. Yeast metacaspases**

The first report of a metacaspase on yeast was made by Madeo et al. [53], where the overexpression of the protein codified by theYor197w gene stimulated PCD associated‐caspase‐ like activity on *S. cerevisiae* cells, under H<sup>2</sup> O2 stress. The authors proposed the name Yeast Caspase‐1 (YCA1) for the protein. Also, a *S. cerevisiae* mutant strain with an inactive gene encoding a deubiquitinating enzyme (UBP10) showed an apoptosis‐related phenotype which appears to be related to the YCA1 presence, in a manner similar to that observed when the cells were submitted to external stimuli, such as treatment with H<sup>2</sup> O2 . The overexpression of YCA1 in cells lacking UBP10 resulted on a decrease of about 53% in viability [54].

A study with frataxin‐deficient yeast cells (Δ*yfh 1*), sensible to pro‐oxidant chemicals, showed that YCA1 is induced under H<sup>2</sup> O2 stress. When performance of metacaspase‐deficient yeasts (Δ*yca1*) was evaluated under oxidative stressing condition, a resistance to cell death, reflected by higher glutathione concentrations than the wild‐type, was detected, probably caused by the absence of the metacaspase [55].

By using an original approach of combining the techniques of a digestome analysis (an *in vitro* assay aimed to search putative specific substrates of proteases), cleavage of recombinant GAPDH by metacaspase and evaluation of protein levels *in vivo*, wild‐type and YCA1 overexpressing yeast cells upon H<sup>2</sup> O2 ‐induced apoptosis were evaluated. Under these oxidative conditions, the enzyme glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), was shown, by mass spectrometric analysis, to be a specific target of metacaspase. This result suggests a link between programmed cell death and metabolism [56]. Besides the potential role in MCP, this metacaspase seems to have involvement on cell cycle regulation. Deficient *yca1* yeast revealed an increased level of proteins related to the vacuolar catabolism, including six peptidases, as well as chaperones involved on stress response and protein aggregates elimination. YCA1 was also copurified with chaperones that respond to protein aggregation and with a cell‐cycle control protein; thus, a role on clearance of protein aggregates during mitosis and aging was proposed [57]. This potential on protein clearance is dependent on the presence of Hsp40 and Hsp104 chaperones, and is reduced by deletion of the proteasome regulator Rnp4, which reduces the proteasome levels. When overproduced, YCA1 extended the life span of the cells by 46–56% [58].

Metacaspase studies are also being performed with other fungi, as in the case of *Candida albicans* metacaspase 1 (CaMCA1). Mutant strains for this gene showed a lack of PCD under oxidative stress, as well as a decrease of energy metabolism intensity followed by accumulation of trehalose and increase on expression of genes related to trehalose biosynthesis, what is a well‐known protective mechanism against oxidative stresses [59]. Other example is the study of two metacaspases from *Aspergillus fumigates*, whose deficiency was associated with loss of membrane phospholipid asymmetry and an increased growth under conditions of endoplasmic reticulum stress [60].

#### **4.3. Metacaspases of protozoa**

generates nuclear degradation, an event present on most eukaryotic PCD programmes. In this pathway, the protease can play a role on the cleavage of nuclear proteins, similar to what

The heterologous expression of the *A. thaliana* metacaspases MCP1b and MCP2b on a *Saccharomyces cerevisiae* strain, disrupted for its metacaspase YCA1 gene, was able to trigger an apoptosis‐like phenotype under hydrogen peroxide treatment, and this effect was seen to be abolished on the presence of the pancaspase inhibitor based on the sequence VAD. These findings reinforce a metacaspase role on PCD, as well as the requirement of proteins with caspase‐like activity [42]. The involvement of metacaspases on PCD is also suggested by works with plants under pathogen attack. Some examples are given by the detection of gene expression for MCA1 on *A. thaliana* leaves infected by *Pseudomonas syringae* [49], and by the increasing levels of a type II metacaspase on tomato leaves under *Botrytis cynerea* infection, with the detection of cell death phenotype [50]. It cannot be excluded, though, that metacaspases could not be directly involved on PCD regu-

In face of this, the role of metacaspases are still under discussion, as well as their classification, since there are evidences which favour, and others contrary, to their groupment together with caspases [51, 52]. A cascade mechanism comparable to that of caspases was proposed for vegetable systems concerning cysteine proteases on senescent leaves and seeds on maturation. Bozhkov et al. [46] reinforced the idea that the execution of PCD in plants is controlled by two groups of enzymes with separated cellular localization. One of them is accumulated on lytic compartments and vacuoles, and the other has cytoplasmic‐nuclear localization, as in the case of MCIIPa.

The first report of a metacaspase on yeast was made by Madeo et al. [53], where the overexpression of the protein codified by theYor197w gene stimulated PCD associated‐caspase‐

Caspase‐1 (YCA1) for the protein. Also, a *S. cerevisiae* mutant strain with an inactive gene encoding a deubiquitinating enzyme (UBP10) showed an apoptosis‐related phenotype which appears to be related to the YCA1 presence, in a manner similar to that observed when the

A study with frataxin‐deficient yeast cells (Δ*yfh 1*), sensible to pro‐oxidant chemicals, showed

(Δ*yca1*) was evaluated under oxidative stressing condition, a resistance to cell death, reflected by higher glutathione concentrations than the wild‐type, was detected, probably caused by

By using an original approach of combining the techniques of a digestome analysis (an *in vitro* assay aimed to search putative specific substrates of proteases), cleavage of recombinant GAPDH by metacaspase and evaluation of protein levels *in vivo*, wild‐type and YCA1 overex-

tions, the enzyme glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), was shown, by mass

stress. The authors proposed the name Yeast

O2

stress. When performance of metacaspase‐deficient yeasts

‐induced apoptosis were evaluated. Under these oxidative condi-

. The overexpression of

O2

YCA1 in cells lacking UBP10 resulted on a decrease of about 53% in viability [54].

cells were submitted to external stimuli, such as treatment with H<sup>2</sup>

O2

O2

lation, but indirectly involved on signalling cascades that leads to PCD [48].

happens to the metazoan effector caspases [46].

30 Enzyme Inhibitors and Activators

**4.2. Yeast metacaspases**

like activity on *S. cerevisiae* cells, under H<sup>2</sup>

that YCA1 is induced under H<sup>2</sup>

pressing yeast cells upon H<sup>2</sup>

the absence of the metacaspase [55].

Among the first reports concerning metacaspases on protists was the work of Szallies et al. [61]. According to this, *Trypanosoma brucei* encodes five putative metacaspases. The expression of the *T. brucei* metacaspase MCA4 in *S. cerevisiae* caused a PCD‐related phenotype, and the protein was found to be located on nuclei, alongside the yeast metacaspase MCA1. When overexpressed, MCA4 induced PCD.

Two metacaspases (LdMC1 and LdMC2) of *Leishmania donovani* were also characterized and displayed high homology among each other. The proteins were seen to be capable of cleaving trypsin substrates and were inhibited by classical trypsin inhibitors such as leupeptin and antipain. Despite not being able to cleave caspase substrates, when their genes were overexpressed in the parasite cells, a higher sensibility to PCD was observed [62]. The metacaspases MCA2, MCA3 and MCA 5 were detected *in vivo* in their full molecular form and seem not to suffer processing to become active in the parasite cells. They were found to be associated with recycling endosomes that contain recycling receptors and PCD‐independent functions were proposed for this association with endosomes [63].

The capability to induce PCD was also investigated for the *Leishmania major* single metacaspase (LmjMCA), whose heterologous expression in a *S. cerevisiae* defective strain was successful on the induction of PCD by oxidative stress. The protease originated from the metacaspase gene revealed an arginine‐specific activity and through approaches of site‐directed mutagenesis, it was possible to determine that its activity is dependent of H147 and C202 [64]. This metacaspase was suggested to be essential for the parasite, since the tentative to obtain a null mutant was unsuccessful. The protein was detected in variable levels in different compartments at different stages of the cell cycle, in association with the dividing kinetoplast and the nuclear spindle, what may suggests a role on DNA segregation. Also, it was seen that the protein was associated with cytoskeleton filaments. A role on differentiation and proliferation has been additionally proposed [65].

The activity of a *Plasmodium falciparum* metacaspase 1 (PfMCA‐1) increased under high concentrations of calcium and induced PCD under stress conditions [66]. The authors have also presented evidences of the ability of PfMCA1 of triggering a downstream enzyme that is sensible to the pancaspase inhibitor z‐VAD‐fmk.
