Type II Metacaspases Atmc4 and Atmc9 of Arabidopsis thaliana Cleave Substrates after Arginine and Lysine

Abstract

Nine potential caspase counterparts, designated metacaspases, were identified in the Arabidopsis thaliana genome. Sequence analysis revealed two types of metacaspases, one with (type I) and one without (type II) a proline-or glutamine-rich N-terminal extension, possibly representing a prodomain. Production of recombinant Arabidopsis type II metacaspases in Escherichia coli resulted in cysteine-dependent autocatalytic processing of the proform into large and small subunits, in analogy to animal caspases. A detailed biochemical characterization with a broad range of synthetic oligopeptides and several protease inhibitors of purified recombinant proteins of both metacaspase 4 and 9 showed that both metacaspases are arginine/lysine-specific cysteine proteases and did not cleave caspase-specific synthetic substrates. These findings suggest that type II metacaspases are not directly responsible for earlier reported caspase-like activities in plants.

Antagonic activities of Trypanosoma cruzi metacaspases affect the balance between cell proliferation, death and differentiation

“…Plant type II, but not type I, metacaspases were clearly shown to be autocatalytically activated in vitro by a cleavage event at the p20-p10 boundary. 6 Among trypanosomatids, LmjMCA has been reported to undergo self-proteolytic processing in yeast cells overexpressing LmjMCA, but not its inactive mutants. 30 However, neither the processing sites were clearly identified nor further analysis of the recombinant protein was performed to correlate maturation with activity.…”

“…5 Their substrate specificity is for basic residues at P1 position, making them unable to cleave caspase substrates. 6 Evidences suggest that metacaspases modulate cell death, [7][8][9][10][11] cell cycle progression [12][13][14] and protein aggregation, 15 but there is still controversy about their functions and their relation with caspases. [16][17][18] The fact that metacaspases are not present in humans and fulfil important roles in trypanosomatids make them attractive drug targets, and a first series of inhibitors with trypanocidal activity has been developed recently.…”

Caspase substrates

“…This deep, highly basic pocket ( Figure 1) is perfectly shaped to accommodate an Asp side chain, accounting for the up to four orders of magnitude lower catalytic efficiency for cleavage of peptides with a P 1 Glu residue in most caspases, 4 with the notable exception of the Drosophila caspase Dronc, which seems to have an equal specificity for Asp and Glu. 5 The caspases are members of a clan, or structurally related group, known as cysteine protease clan CD that also contains the plant metacaspases, 6 and mammalian and plant proteases of the legumain family (involved in specific processing events), the eucaryotic protease separase (required for sister chromatid separation during mitosis), and the bacterial proteases clostripain and gingipain. 7 Common to all these proteases is a shared stringent specificity for one type of amino acid side chain at the primary (P 1 ) specificity position in substrates (Arg or Lys for metacaspases, clostripain and gingipains; Asn for legumains; and Arg for separase).…”

Recognition and signal transduction in the expression of Phytophthora resistance in soybean [Glycine max (L) Merr]

“…RIP13 has a coding region of 1275 bp and encodes a protein of 424 amino acids. Based on its sequence alignment with nine metacaspases from Arabidopsis and the recently characterized mcII-Pa from Norway spruce we conlude that RIP13 encodes a type II metacaspase (Bozhkov et al, 2005;Vercammen et al, 2004). As in mcII-Pa, the Arg residue at position 187 (R 187 ) separates a p20 caspase-like subunit and a type II metacaspase-specific linker in RIP13.…”

“…After the linker sequence a Lys residue (K 271 ) separates the linker from the C-terminal p10 caspase-like subunit ( Figure 5). The sequence context of the catalytic histidine and cystein residues are conserved (Vercammen et al, 2004).…”