Abstract
BackgroundCalpain proteases drive intracellular signal transduction via specific proteolysis of multiple substrates upon Ca2+-induced activation. Recently, dUTPase, an enzyme essential to maintain genomic integrity, was identified as a physiological calpain substrate in Drosophila cells. Here we investigate the potential structural/functional significance of calpain-activated proteolysis of human dUTPase.Methodology/Principal FindingsLimited proteolysis of human dUTPase by mammalian m-calpain was investigated in the presence and absence of cognate ligands of either calpain or dUTPase. Significant proteolysis was observed only in the presence of Ca(II) ions, inducing calpain action. The presence or absence of the dUTP-analogue α,β-imido-dUTP did not show any effect on Ca2+-calpain-induced cleavage of human dUTPase. The catalytic rate constant of dUTPase was unaffected by calpain cleavage. Gel electrophoretic analysis showed that Ca2+-calpain-induced cleavage of human dUTPase resulted in several distinctly observable dUTPase fragments. Mass spectrometric identification of the calpain-cleaved fragments identified three calpain cleavage sites (between residues 4SE5; 7TP8; and 31LS32). The cleavage between the 31LS32 peptide bond specifically removes the flexible N-terminal nuclear localization signal, indispensable for cognate localization.Conclusions/SignificanceResults argue for a mechanism where Ca2+-calpain may regulate nuclear availability and degradation of dUTPase.
Highlights
Calpains, the intracellular calcium activated cysteine proteases, play an important role in calcium-dependent signal transduction
To decide if human dUTPase may be susceptible to calpaincatalyzed proteolysis, as we reported it in Drosophila [8], we prepared an in vitro digestion assay with the human recombinant DUT-N and rat m-calpain
We have recently identified in vivo calpain substrates in a Drosophila system, providing a list of proteins, not yet described as calpain substrates, which belonged to several different cellular pathways, reflecting the well-known promiscuous calpain character [8]
Summary
The intracellular calcium activated cysteine proteases, play an important role in calcium-dependent signal transduction. The potential role of calpain function in apoptotic pathways has been recently addressed in several laboratories [1,2,3,4]. Identification of in vivo cellular substrates of calpain was attempted in a proteomic approach in Drosophila Schneider 2 cells, wherein we provided a list of putative bona fide targets [8]. One of these target proteins is dUTPase, lack of which is involved in inducing cell death [9,10]. Calpain proteases drive intracellular signal transduction via specific proteolysis of multiple substrates upon Ca2+-induced activation. We investigate the potential structural/functional significance of calpain-activated proteolysis of human dUTPase
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