Abstract
Nine aminoacyl-tRNA synthetases (aaRSs) and three scaffold proteins form a super multiple aminoacyl-tRNA synthetase complex (MSC) in the human cytoplasm. Domains that have been added progressively to MSC components during evolution are linked by unstructured flexible peptides, producing an elongated and multiarmed MSC structure that is easily attacked by proteases in vivo. A yeast two-hybrid screen for proteins interacting with LeuRS, a representative MSC member, identified calpain 2, a calcium-activated neutral cysteine protease. Calpain 2 and calpain 1 could partially hydrolyze most MSC components to generate specific fragments that resembled those reported previously. The cleavage sites of calpain in ArgRS, GlnRS, and p43 were precisely mapped. After cleavage, their N-terminal regions were removed. Sixty-three amino acid residues were removed from the N terminus of ArgRS to form ArgRSΔN63; GlnRS formed GlnRSΔN198, and p43 formed p43ΔN106. GlnRSΔN198 had a much weaker affinity for its substrates, tRNA(Gln) and glutamine. p43ΔN106 was the same as the previously reported p43-derived apoptosis-released factor. The formation of p43ΔN106 by calpain depended on Ca(2+) and could be specifically inhibited by calpeptin and by RNAi of the regulatory subunit of calpain in vivo. These results showed, for the first time, that calpain plays an essential role in dissociating the MSC and might regulate the canonical and non-canonical functions of certain components of the MSC.
Highlights
The human multiple aminoacyl-tRNA synthetase complex (MSC) has an elongated and multiarmed structure, which might be sensitive to proteases such as calpain in vivo
Our results suggested that the calpain family is involved in dissociation of the MSC and might regulate the functions of MSC components and connect their canonical and non-canonical functions
We used the supernatant of Jurkat extracts (Jurkat S100) containing calpain 2 in vitro to assay hydrolysis of recombinant LeuRS (N terminus His6-tagged) and found that LeuRS was cleaved under Ca2ϩ stimulation as shown by Western blotting with the His6 antibody (Fig. 1B)
Summary
The human multiple aminoacyl-tRNA synthetase complex (MSC) has an elongated and multiarmed structure, which might be sensitive to proteases such as calpain in vivo. The formation of p43⌬N106 by calpain depended on Ca2؉ and could be inhibited by calpeptin and by RNAi of the regulatory subunit of calpain in vivo These results showed, for the first time, that calpain plays an essential role in dissociating the MSC and might regulate the canonical and noncanonical functions of certain components of the MSC. Proteolysis of the Multiple tRNA Synthetase Complex enough space for accessibility of all its components and a large protein surface area for interaction with additional factors, suggesting that the MSC might be attacked by proteases in vivo [8]. Calpain cleaved other MSC components, which affected their aminoacylation activity and released their specific fragments with potential new functions. Our results suggested that the calpain family is involved in dissociation of the MSC and might regulate the functions of MSC components and connect their canonical and non-canonical functions
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