Localization of the voltage-dependent anion channel-1 Ca2+-binding sites
Localization of the voltage-dependent anion channel-1 Ca2+-binding sites
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DOI：
10.1016/j.ceca.2006.06.005
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发表时间：
2007-03-01
期刊：
影响因子：
4
通讯作者：
Shoshan-Barmatz, Varda
中科院分区：
文献类型：
--
作者：
Israelson, Adrian;Abu-Hamad, Salah;Shoshan-Barmatz, Varda
近些年来，光反应叠氮Ru(Azru)被证明能与钙结合蛋白特异地相互作用，并强烈抑制它们的钙依赖活性。在紫外线照射下，Azru可以共价结合到这些蛋白质上。在这项研究中，Azru被用来定位和表征电压依赖性阴离子通道(VDAC)中的钙结合位点。Azru降低了重组成双层的VDAC的电导，而在I-M-NaCl存在下，Ca~(2+)阻止了这一作用，而不是在Mg~(2+)存在下。Azru对突变的E72Q-或E202Q-VDAC1电导没有影响，[Ru-103]Azru标记的是天然的，但不是E72Q-VDAC1，这表明这些残基是Azru与VDAC钙结合位点(S)相互作用所必需的。在内源性hVDAC1缺失的T-Rex-293细胞中，Azru对天然的但不是E72Q-或E202Q-小鼠VDAC1的过度表达诱导的细胞凋亡具有保护作用。胰凝乳酶和胰酶消化Azru标记的VDAC，然后进行MALDI-TOF分析，发现两个Azru结合的多肽，分别对应于E72和E202-序列。这些结果表明，VDAC的钙结合位点包括E72和E202，根据提出的VDAC1拓扑模型，它们位于两个不同的胞质环上。此外，Azru对细胞凋亡的保护涉及与这些残基的相互作用。光反应Azru是鉴定新的钙结合蛋白和定位其钙结合位点的重要工具。(C)2006爱思唯尔有限公司。保留所有权利。
Photoreactive azido ruthenium (AzRu) has been recently shown to specifically interact with Ca2+-binding proteins and to strongly inhibit their Ca2+-dependent activities. Upon UV irradiation, AzRu can bind covalently to such proteins. In this study, AzRu was used to localize and characterize Ca2+-binding sites in the voltage-dependent anion channel (VDAC). AzRu decreased the conductance of VDAC reconstituted into a bilayer while Ca2+, in the presence of I M NaCl, but not Mg2+, prevented this effect. AzRu had no effect on mutated E72Q- or E202Q-VDAC1 conductance, and [Ru-103]AzRu labeled native but not E72Q-VDAC1, suggesting that these residues are required for AzRu interaction with the VDAC Ca2+-binding site(s). AzRu protected against apoptosis induced by over-expression of native but not E72Q- or E202Q- murine VDAC1 in T-REx-293 cells depleted of endogenous hVDAC1. Chymotrypsin and trypsin digestion of AzRu-labeled VDAC followed by MALDI-TOF analysis revealed two AzRu-bound peptides corresponding to E72- and E202-containing sequences. These results suggest that the VDAC Ca2+-binding site includes E72 and E202, located, according to a proposed VDAC1 topology model, on two distinct cytosolic loops. Furthermore, AzRu protection against apoptosis involves interaction with these residues. Photoreactive AzRu represents an important tool for identifying novel Ca2+-binding proteins and localizing their Ca2+-binding sites. (c) 2006 Elsevier Ltd. All rights reserved.