**7. Metacaspase targets**

**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.

34 Enzyme Inhibitors and Activators

Little is known about the metacaspase natural substrates, to the present date. This lack of information makes hard to construct a PCD pathway as was done for caspases. Despite this, the efforts aiming at the elucidation of this question are growing.

The first biological substrate discovered for a metacaspase is the Tudor Staphylococcal Nuclease (TSN), a protein involved on gene expression regulation, highly conserved phylogenetically. The cleavage of this protein prevents its function and is important for the execution of apoptosis; also, the protein is known to be a part of the human caspase 3 degradome. TSN was shown to be cleaved by the metacaspase McIIPa (type II metacaspase of *P. abies*) on an *in*  *vitro* assay. *In vivo,* the McIIPa activity was shown to be simultaneous to the decrease of TSN activity, what was deduced from TUNEL positive embryonic cells [81].

Another substrate found for a metacaspase was the already mentioned GAPDH. This protein was detected as a digest product of an YCA1 metacaspase‐enriched extract from *S. cere‐ visiae* cells subjected to H<sup>2</sup> O2 treatment. As recombinant YCA1 was shown to cleave GAPDH *in vitro*, the *in vivo* evaluation of the GAPDH performed during a comparison between the wild‐type and a mutant yeast strain, disrupted for YCA1, both under PCD triggering condition, showed a reduction of the enzyme levels on the wild‐type (resistant to PCD). The GAPDH is also a caspase substrate, but the cleavage again happens on different sites of those targeted by the metacaspases. Yet, this is another evidence for the existence of conserved molecular members of a PCD pathway in metazoan and in metacaspase‐bearing organisms [56].

Recently, as a remarkable effort, a proteome‐wide‐level study of *A. thaliana* seedlings, focusing on the identification of physiologic substrates of metacaspase 9, has been performed employing a digestome analysis strategy. Important features of the target proteins were prospected, and it was possible to map the frequencies of the amino acids sitting at the neighbourhood of the Arg or Lys P1‐specific cleavage sites. Along with other interesting features, the enzyme has shown a strong tendency to prefer acid residues, as Asp and Glu at the P1' position. Among the identified substrates, was phosphoenolpyruvate carboxykinase 1 (PEPCK1), a gluconeogenesis enzyme. This protein was shown to be cleaved *in vivo* in such a manner that its activity was enhanced, and thus, the glucose *de novo* synthesis pathway may be stimulated during PCD [82].
