PDF(Pubmed)
Abstract：
Human CLCA2 regulates store-operated calcium entry (SOCE) by interacting with Orai1 and STIM1. It is expressed as a 943aa type I transmembrane protein that is cleaved at amino acid 708 to produce a diffusible 100 kDa product. The N-terminal ectodomain contains a hydrolase-like subdomain with a conserved HEXXH zinc-binding motif that is proposed to cleave the precursor autoproteolytically. Here, we tested this hypothesis and its link to SOCE. We first studied the conditions for autocleavage in isolated membranes and then in a purified protein system. Cleavage was zinc-dependent and abolished by mutation of the E in the HEXXH motif to Q, E165Q. Cleavage efficiency increased with CLCA2 concentration, implying that it occurs in trans. Accordingly, the E165Q mutant was cleaved by co-transfected wildtype CLCA2. Moreover, CLCA2 precursors with different epitope tags co-immunoprecipitated. In a membrane-free system utilizing immunopurified protease and target, no cleavage occurred unless the target was first denatured, implying that membranes provide essential structural or conformational cues. Unexpectedly, cleavage caused a conformational shift: an N-terminal antibody that immunoprecipitated the precursor failed to precipitate the N-terminal product unless the product was first denatured with an ionic detergent. The E165Q mutation abolished the stimulation of SOCE caused by wildtype CLCA2, establishing that the metalloprotease activity is required for this regulatory function.
摘要：
人类CLCA2通过与Orai1和STIM1相互作用来调节储存操作的钙进入（SOCE）。其表达为943aa型I跨膜蛋白,其在氨基酸708处被切割以产生可扩散的100kDa产物。N末端胞外域包含具有保守的HEXXH锌结合基序的水解酶样亚结构域，该基序被提议以自身蛋白水解方式切割前体。这里,我们检验了这一假设及其与SOCE的联系。我们首先研究了在分离的膜中，然后在纯化的蛋白质系统中进行自切割的条件。切割是锌依赖性的，并通过将HEXXH基序中的E突变为Q而废除，E165Q.切割效率随着CLCA2浓度的增加而增加，暗示它发生在反式。因此，通过共转染的野生型CLCA2切割E165Q突变体。此外，具有不同表位标签的CLCA2前体共免疫沉淀。在利用免疫纯化蛋白酶和靶标的无膜系统中，除非靶标首先变性，否则不会发生切割，暗示膜提供必要的结构或构象线索。出乎意料的是,裂解引起构象改变:免疫沉淀前体的N-末端抗体不能沉淀N-末端产物,除非产物首先用离子去污剂变性。E165Q突变消除了由野生型CLCA2引起的SOCE的刺激,确立了金属蛋白酶活性是该调节功能所必需的。
