**6. Metacaspase activators and regulators**

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

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

Caspases, metacaspases and paracaspases have a conserved pattern of tridimensional organization and are then considered as structural homologues. The degree of this conservation is variable, but the overall structure is related to a conformation named caspase fold that is characterized by a core formed by a contiguous six‐stranded β‐sheet (β1–β4, β7 and β8) and helices α1–α5 region, present in every caspase structure. Also, the presence of three well‐

As it was early discussed, metacaspases are divided into two groups based on the presence of a pro domain or a linker region. Type I metacaspases possess an N‐terminal prodomain with length of about 80–120 amino acids [32], with two CXXC‐type LSD1‐like zinc finger structures as well as a proline/glutamine rich region [45, 68]. The type II metacaspases do not have prodomains but, otherwise, contain a large loop (linker region) between the p10 and p20

The p10 and p20 domains are present in all of these proteins. For p10, there is a SGCXDXQTSADV consensus sequence, as well as other conserved short sequences [40]. The p20 domain contains the conserved catalytic dyad histidine/cysteine as a remarkable feature, where the two amino acids are distant from each other in about 29–47 amino acids. There are also other conserved regions/amino acids that together give about 80% of consensus on the entire sequence [32]. A noticeable signature is the motif DSCHSG in the surroundings of the

Differently of metacaspases, all caspases contain a conserved QACXG (where X can be R, Q, or G) pentapeptide active‐site motif. The catalytic residues histidine 237 and cysteine 285, and those involved in forming the P1 carboxylate binding pocket on caspase 1 (Arg‐179, Gln‐283, Arg‐341 and Ser‐347), are also conserved in all other caspases, except for the conservative substitution of the threonine for the serine 347 in caspase 8. This explains the requirement for an aspartate in the substrate P1 position. The residues that form the P2–P4 binding pocket are not well conserved, suggesting that they may determine the substrate specificities of the different caspases [71]. The metacaspases do not have these features and present cleavage specificity for lysine/arginine on the P1 position on the substrate, so their binding residues seem to be of

The type II metacaspases present autoprocessing sites, whose cleavage seems to be necessary for their full activation: the residues Lys 260 and Arg 214, on the wheat type II metacaspase

catalytic Cys, which is highly conserved among all type II plant metacaspases [70].

has been additionally proposed [65].

32 Enzyme Inhibitors and Activators

sensible to the pancaspase inhibitor z‐VAD‐fmk.

**5. The caspase fold of metacaspases**

ordered loops (L1, L2 and L4) is well characterized [67].

domains which ranges about 90–150 amino acids [32, 69].

opposite chemical nature from those of caspases.

Despite the growing knowledge about the structure and function of CD clan members of cysteine proteases, the molecules involved on the control of their activities are still to be more unravelled. As discussed before, one of the main activation mechanisms of the caspases is its cleavage on specific sites, promoted by other caspases. This processing results on conformational changes and dimerization that enhance the substrate cleavage activity of the target caspase.

For this to happen, it is necessary the assemblage of huge protein complexes, which function as activation platforms. Examples of these are the Fas death‐induced signalling complex (DISC), whose association is required for the caspase 8 (pro‐caspase 8) self‐activation and the apoptosome, which binds to caspase 9 (pro‐caspase 9) prior to its activation [11, 28, 31]. It is not clear, if in the case of metacaspases, the auto processing and dimerization are always required for their activation as well as the formation of protein complexes [8].

Until now, biochemical studies have demonstrated that only type II metacaspases undergo autocatalytic activation, similar to the phenomenon observed for caspases. Contrary to that,

**Figure 2.** Proposed cascade model of plant proteolytic events triggered by a death inductor signal. The type II metacaspase inhibitors (red arrow) and stimulators (green arrow) are displayed, as well as possible substrates.

no proteolytic activation was observed for type I metacaspases. So a scenery, where type I prometacaspases become active by the action of death signals coupled to the activation of type II prometacaspases, which by their turn become active and able to cleave other proteases and trigger the degradation of cellular components, was proposed as a probable signal transduction pathway during PCD proteolysis in plants [69] (**Figure 2**).

Alongside with the proteolysis processing requirements, there are reports which show calcium dependence for type II metacaspases. By *in vitro* experiments, a type II recombinant metacaspase from *A. thaliana* exhibited Ca2+ dependence (10 mM) for its activation, on a pattern indicative of auto‐processing [76]. A similar Ca2+ dependence was detected on *in vitro* assays with the *T. brucei* MCA2, whose activity peaks on the presence of 10 mM of Ca2+, although the enzyme does not require autoprocessing [77]. This characteristic was shown to be valid for both *L. major* and *P. falciparum* metacaspases [66].

One candidate to be a metacaspase regulator is a serine protease inhibitor called Serpin. Serpin1 from *A. thaliana* was shown to have a potent cleavage activity towards the reactive centre loop of metacaspase 9, besides the ability of covalent binding to the target protein on *in vitro* assays. This study has also demonstrated that both proteins were localized on extracellular space, suggesting they could interact under *in vivo* conditions as well [78].

Other control mechanism proposed is the S‐nitrosylation, that is used as a regulation strategy of certain proteins under basal NO levels. The S‐nitrosylation of the catalytic cysteine (Cys‐147) on *A. thaliana* metacaspase 9 (AtMC9) on its mature processed form does not affect its activity. This happens because other cysteine (Cys‐29) residue can act as alternative nucleophile. Despite this, the enzyme can be kept inactive through S‐nitrosylation, and otherwise, become active only under conditions of disturbance on cellular redox balance [79].

The effect of zinc on metacaspase activity was also investigated. The supplementation of plant embryos with extra zinc suppressed the terminal differentiation and death of the suspensors, delaying the embryo maturing, and also reducing the intensity of metacaspase activity between 96 and 168 h of development, which is the period when the suspensor death occurs [80]. These data, alongside the work of Bozhkov et al. [46], suggest that zinc may be part of a mechanism of posttranslational regulation of metacaspases, still to be further examined.
