Identification and Characterization of Wolframin, the Product of the Wolfram Syndrome Gene (WFS1), as a Novel Calmodulin-Binding Protein

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• 作者：Saki Yurimoto ; Naoya Hatano ; Mitsumasa Tsuchiya ; Kiyohito Kato ; Tomohito Fujimoto ; Tsutomu Masaki ; Ryoji Kobayashi ; Hiroshi Tokumitsu
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：May 12, 2009
• 年：2009
• 卷：48
• 期：18
• 页码：3946-3955
• 全文大小：989K
• 年卷期：v.48,no.18(May 12, 2009)
• ISSN：1520-4995

文摘

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 μM. 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.