Calpain-Catalyzed Proteolysis of Human dUTPase Specifically Removes the Nuclear Localization Signal Peptide

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.【背景】钙蛋白酶（Calpain）可经钙离子诱导活化后，通过对多种底物进行特异性蛋白水解，驱动细胞内信号转导过程。近期研究在果蝇细胞中鉴定出dUTP酶（dUTPase）——一种维持基因组完整性所必需的酶——为钙蛋白酶的生理底物。本研究旨在探究人类dUTP酶经钙蛋白酶活化后发生蛋白水解的潜在结构与功能意义。 【方法与主要结果】本研究在有或无钙蛋白酶或dUTP酶同源配体的条件下，对哺乳动物m-钙蛋白酶（m-calpain）介导的人类dUTP酶有限蛋白水解过程进行了考察。仅在Ca(II)离子存在、钙蛋白酶被激活的情况下，方可观测到显著的蛋白水解现象。dUTP类似物α,β-亚氨基-dUTP的存在与否，对钙离子激活的钙蛋白酶切割人类dUTP酶的过程无显著影响。钙蛋白酶切割并未改变dUTP酶的催化速率常数。凝胶电泳分析显示，钙离子激活的钙蛋白酶切割人类dUTP酶可产生多个可明确辨识的dUTP酶片段。通过质谱对钙蛋白酶切割片段进行鉴定，共确定了三个钙蛋白酶切割位点，分别位于残基4S-E5、7T-P8以及31L-S32之间。其中，31L-S32肽键处的切割可特异性移除柔性N端核定位信号，而该信号对于dUTP酶的同源定位不可或缺。 【结论与意义】本研究结果支持如下调控机制：钙离子激活的钙蛋白酶可通过调控dUTP酶的核内可用性与降解过程，实现对其功能的调节。