Sheathing the swords of death: post-translational modulation of plant metacaspases

Abstract

Plant metacaspases (MCPs) are conserved cysteine proteases that have been postulated as regulators of programmed cell death (PCD). Although MCPs have been proven to have PCD relevant functions in multiple species ranging from fungi to plants, how these proteases are modulated in vivo remains unclear. Aside from demonstrating that these proteases are distinct from metazoan caspases due to their different target site specificities, how these proteases are used to tightly regulate cell death progression is a key question that remains to be resolved. Some recent studies on the biochemical characteristics of type-II MCP activities in Arabidopsis may begin to shed additional light on this aspect. The in vitro catalytic activities of recombinant AtMC4, AtMC5 and AtMC8 are found to be Ca(2+)-dependent while recombinant AtMC9 is active under mildly acidic conditions and not dependent on stimulation by Ca(2+). Alterations of cellular pH and Ca(2+) concentration commonly occur during various stresses and may help to orchestrate differential activation of latent MCPs under these conditions. Recent peptide mapping for recombinant AtMC4 (also called Metacaspase-2d) followed by site-specific mutagenesis studies have revealed multiple potential self-cleavage sites with the identification of a conserved lysine residue (Lys-225) as the key position for enzyme function both in vitro and in vivo. The multiple self-cleavage sites in MCPs may also facilitate desensitization of these proteases and can provide a means for fine-tuning their proteolytic activities in order to achieve more sensitive control of downstream processes.

Figure 1.

Calcium dependence of FSRase activities with 3 Type-II MCPs in crude bacteria lysate. (A) FSRase activities of rAtMC5 in crude BL21(DE3)pLysS lysate. Activity of rAtMC5 in crude lysate form compared in buffers with CaCl₂ varied between 0, 1, 10 and 50 mM. (B) Activity of rAtMC8 with different (0, 1, 10 and 50 mM) CaCl₂ concentrations in the assay buffer. (C) Activities of rAtMC9 in the presence of varying amounts (0, 1, 10 and 50 mM) of CaCl₂ in the assay buffer. For AtMC5 and AtMC8, the assay buffer consisted of 100 mM TRIS-HCl, pH7.5, 150 mM NaCl, 0.1 mM EDTA, 1 mM DTT and 100 μM FSR-MCA. Since, AtMC9 was previously shown to prefer acidic conditions, a different buffer and pH (100 mM MES, pH5.5) was used in place of TRIS-HCl. Due to much higher apparent specific activity of the recombinant protein, the enzymatic activity for rAtMC9 was assayed after a 5-fold dilution of the bacteria lysate. The relative amount of recombinant protein in each of the lysates were first normalized by comparing the expressed protein’s levels using protein gel blots with either anti-T7 antibodies or anti-AtMC4 polyclonal antibodies produced from rabbits. With FSR-MCA, there is less background activity observed with crude bacterial lysates than with GRR-MCA (data not shown). Thus, we opted to use this artificial substrate for our assay. RFU: relative fluorescence unit.

Figure 2.

Enhancement and sustained FSRase activity of AtMC4 in the presence of bacterial lysate. Activities of control lysate and lysates from bacteria expressing rAtMC4, rAtMC4^K225G, rAtMC4^C139A, and purified rAtMC4 are compared. In addition, purified rAtMC4 was also mixed with control lysate to test for enhancement of the activated FSRase activity. The reaction buffer contained 100 mM TRIS-HCl, pH7.5, 150 mM NaCl, 10 mM CaCl₂, 0.1 mM EDTA, 1 mM DTT and 100μM FSR-MCA. We constructed rAtMC4 expression vectors in two different vector backgrounds (pET23a and pET28a) to produce histine-tagged versions of this protease with the hexahistidines either at the N- (His-AtMC4) or C-terminus (T7-AtMC4-His), respectively. In crude lysates, these two different versions of rAtMC4 fusions behaved identically in terms of FSRase activity (data not shown). For purification of rAtMC4, the His-AtMC4 version of the fusion protein was used since the T7-AtMC4-His fusion gave very low yield in several attempts. For comparison with the two AtMC4 variants with point mutations at K225 and C139, the T7-AtMC4-His version of the fusion protein was shown in the figure since they were all constructed in the same expression vector background. In these cases, only crude lysates were used for comparison. RFU: relative fluorescence unit.