Type III metacaspases: calcium‐dependent activity proposes new function for the p10 domain

Abstract

Metacaspases are a subgroup of caspase homologues represented in bacteria, algae and plants. Although type I and type II metacaspases are present in plants, recently discovered and uncharacterized type III metacaspases can only be found in algae which have undergone secondary endosymbiosis. We analysed the expression levels of all 13 caspase homologues in the cryptophyte Guillardia theta in vivo and biochemically characterized its only type III metacaspase, GtMC2, in vitro. Type III metacaspase GtMC2 was shown to be an endopeptidase with a preference for basic amino acids in the P1 position, which exhibited specific N-terminal proteolytic cleavage for full catalytic efficiency. Autolytic processing, as well as the activity of the mature enzyme, required the presence of calcium ions in low millimolar concentrations. In GtMC2, two calcium-binding sites were identified, one with a dissociation constant at low and the other at high micromolar concentrations. We show high functional relatedness of type III metacaspases to type I metacaspases. Moreover, our data suggest that the low-affinity calcium-binding site is located in the p10 domain, which contains a well-conserved N-terminal region. This region can only be found in type I/II/III metacaspases, but is absent in calcium-independent caspase homologues.

Metacaspase involvement in programmed cell death of the marine cyanobacterium Trichodesmium

“…A large number of metacaspases‐like genes are common in diazotrophic filamentous cyanobacteria. Nine metacaspases are found in Anabaena variabilis ATCC 29413, nine in Nostoc punctiforme while in the T. erythraeum IMS101 genome 12 TeMC have been identified (Jiang et al ., ; Asplund‐Samuelsson, ; Klemenčič and Funk, ); cumulatively representing 0.22% of total encoded proteome (Jiang et al ., ). We analysed the domain structures of the 12 TeMC proteins provide to additional context to their secondary structure and obtain possible clues for their cellular function.…”

“…The highest number of metacaspases from all cyanobacteria analysed to date are found in the marine diazotrophic Trichodesmium genome which contains 12 metacaspases (Jiang et al ., ; Asplund‐Samuelsson et al ., ; ). Due to the lack of the P10 domain in their sequence, metacaspases in prokaryotes are termed metacaspase‐like proteins or at other times orthocaspases (Choi and Berges, ; Klemenčič et al ., ; Klemenčič and Funk, ). Here, we use the term TeMC to describe Trichodesmium metacaspase‐like (TeMC) proteases.…”

Domain swap between two type‐II metacaspases defines key elements for their biochemical properties

“…As expected, caspases are regulated through the post‐translational regulation of their activity as many of these genes are constitutively expressed (Fuentes‐Prior and Salvesen, ). When whole‐genome sequences became available over the past two decades, it was apparent that caspases have evolved specifically in the animal kingdom, and structurally related proteases called metacaspases (MCs) exist in other phyla that extend back to single‐cell eukaryotes, such as microalgae and fungi (Tsiatsiani et al ., ; Klemencic and Funk, ). Related genes encoding metacaspase‐like proteases with a conserved p20 domain that has catalytic motifs related to MCs have also been identified in sequenced genomes of many eubacteria and archaea, thus implicating their evolution from prokaryotic ancestors (Asplund‐Samuelsson et al ., ).…”

“…Although the results from loss‐of‐function analysis with several MC‐encoding genes in yeast and plants are consistent with their role in regulating PCD and stress responses, analogous to the case of caspases in animals, there are also interesting distinctions found between them. One key difference is that all three types of MCs that have been identified and characterized prefer target sites with a basic amino acid residue (arginine or lysine) at the P1 position instead of an aspartate, as in the case for caspases (Vercammen et al ., ; Zhang and Lam, ; Klemencic and Funk, ). Structural studies of type‐I MCs from yeast and protozoa also demonstrated that these enzymes function as monomers instead of dimers in the case of caspases (McLuskey et al ., ; Wong et al ., ).…”

Unravelling the mode of action of plant proteases

“…As the regulatory modes of type I and II metacaspases are uncovered, the development of sequencing technologies gives access to genes encoding other types of metacaspases in other organisms. For example, through a bioinformatic analysis, Klemenčič & Funk (in this issue, pp. 1179–1191) discuss a type III metacaspase, named GtMC2, in the cryptophyte Guillardia theta .…”