Identification and Characterization of Wolframin, the Product of the Wolfram Syndrome Gene (WFS1), as a Novel Calmodulin-Binding Protein
Identification and Characterization of Wolframin, the Product of the Wolfram Syndrome Gene (WFS1), as a Novel Calmodulin-Binding Protein
复制标题
DOI：
10.1021/bi900260y
复制
发表时间：
2009-05-12
期刊：
影响因子：
2.9
通讯作者：
Tokumitsu, Hiroshi
中科院分区：
文献类型：
--
作者：
Yurimoto, Saki;Hatano, Naoya;Tokumitsu, Hiroshi
为了寻找钙调蛋白 (CaM) 靶标，我们使用与 GST 融合的 CaM 作为亲和配体对大鼠脑提取物进行亲和层析纯化。然后进行蛋白质组分析以鉴定 CaM 结合蛋白。除了鉴定出 36 种已知的 CaM 结合蛋白（包括 CaM 激酶、钙调神经磷酸酶、nNOS、IP3 受体和 Ca2+-ATP 酶）之外，我们还鉴定了一种内质网跨膜蛋白 Wolframin（Wolfram 综合征基因 (WFS1) 的产物）具有相互作用。 CaM 叠加和免疫沉淀测定表明，Wolframin 能够在体外和转染细胞中结合 Ca2+/CaM 复合物。表面等离子体共振分析和零长度交联表明，钨胺的N端胞质结构域（残基2-285）以Ca2+依赖性方式与等摩尔单位的CaM结合，CaM的K-D为0.15μM。各种截短和缺失突变体表明，钨胺中的Ca2+/CaM结合区域位于Glu90至Trp186。此外，我们证明与 Wolfram 综合征相关的三种突变（Ala127Thr、Ala134Thr 和 Arg178Pro）完全废除了 Wolframin 的 CaM 结合。这一观察结果可能表明 CaM 结合对于 Wolframin 功能很重要，并且突变对这种相互作用的损害导致了 Wolfram 综合征的病理学。
To search for calmodulin (CaM) targets, we performed affinity chromatography purification of a rat brain extract using CaM fused with GST as the affinity ligand. Proteomic analysis was then carried out to identify CaM-binding proteins. In addition to identifying 36 known CaM-binding proteins, including CaM kinases, calcineurin, nNOS, the IP3 receptor, and Ca2+-ATPase, we identified an ER transmembrane protein, wolframin [the product of the Wolfram syndrome gene (WFS1)] as interacting. A CaM overlay and an immunoprecipitation assay revealed that wolframin is capable of binding the Ca2+/CaM complex in vitro and in transfected cells. Surface plasmon resonance analysis and zero-length cross-linking showed that the N-terminal cytoplasmic domain (residues 2-285) of wolframin binds to an equimolar unit of CaM in a Ca2+-dependent manner with a K-D for CaM of 0.15 mu M. Various truncation and deletion mutants showed that the Ca2+/CaM binding region in wolframin is located from Glu90 to Trp186. Furthermore, we demonstrated that three mutations (Ala127Thr, Ala134Thr, and Arg178Pro) associated with Wolfram syndrome completely abolished CaM binding of wolframin. This observation may indicate that CaM binding is important for wolframin function and that impairment of this interaction by mutation contributes to the pathology seen in Wolfram syndrome.