Elucidation of the molecular mechanisms underlying metabolic disorders using protemomics analysis

IRS蛋白被激活的胰岛素受体在多个酪氨酸残基上磷酸化。我们用~（32）P-ATP标记的IRS-1探针筛选cDNA文库，并在小鼠肝脏中用myc和FLAG标记的IRS-1过表达的免疫沉淀物中鉴定Pin 1，从而鉴定Pin 1与IRS-1相关。Pin 1与pSer/Pro或pThr/Pro结合，转化脯氨酸残基的顺-反构象变化，从而引起目的蛋白的构象变化。Pin 1与IRS-1的关联不仅在使用Sf-9和HepG 2细胞的过表达实验中观察到，而且在小鼠肝脏中内源性地观察到。在HepG 2细胞中Pin 1和IRS-1之间的关联被冈田酸处理显著增加。GST-pin 1对Sf-9细胞产生的IRS-1有牵拉作用，而单独GST对IRS-1无牵拉作用，而碱性磷酸酶对IRS-1无牵拉作用。使用缺失突变和点突变的Pin 1和IRS-1构建体的分析显示，WW d ...更多信息 位于Pin 1 N端的omain和IRS-1 SAIN结构域的Ser 434参与了它们的结合。Pin 1在HepG 2中的过表达显著增强胰岛素诱导的IRS-1磷酸化及其下游事件; PI 3-激酶与IRS-1的结合，Akt磷酸化和GSK 3 β磷酸化。相反，用Pin 1抑制剂Juglone处理HepG 2细胞抑制了它们。此外，Pin 1基因在ob/ob小鼠肝脏中的过表达增加了IRS-1介导的胰岛素信号通路，而Pin 1基因敲除小鼠则表现出胰岛素抵抗和葡萄糖耐受不良。高脂饲料喂养2周后，在肝脏和肌肉中，Pin 1蛋白表达和与Pin 1相关的IRS-1增加了数倍。还显示Pin 1表达在禁食条件下较低，并且通过再喂食而增加。综上所述，我们得出结论，Pin 1的表达受营养条件的调控，并根据营养条件增强IRS-1介导的胰岛素作用发挥重要作用。少

IRS-proteins are phosphorylated on multiple tyrosine residues by the activated insulin receptor. We identified Pin1 to be associated with IRS-1 by screening a cDNA library using 32P-ATP labeled IRS-1 probe and identifying Pin1 in the immunoprecipitates of overexpressed IRS-1.with myc and FLAG tags in mouse livers. Pin1 reportedly binds to pSer/Pro or pThr/Pro containing motif and converts the cis-trans conformational change of praline residue, which induce the conformational change of target protein. The association of Pin1 with IRS-1 was observed in not only overexpression experiments using Sf-9 and HepG2 cells, but also endogenously in the mouse liver. The association between Pin1 and IRS-1 in HepG2 cells was significantly increased by the treatment of okadaic acid. IRS-1 produced in Sf-9 cells was pulldowned by GST-pin1 but not GST alone, while IRS-1 treated with alkaline phosphatase was not. The analysis using deletion- and point-mutated Pin1 and IRS-1 constructs revealed that WW d … More omain located in the N-terminus of Pin1 and Ser 434 in the SAIN domain of IRS-1 is involved in their association.Next, we investigated the role of Pin1 on IRS-1 mediating insulin signaling. The overexpression of Pin1 in HepG2 markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events; PI 3-kinase binding with IRS-1, Akt phosphorylation and GSK3beta phosphorylation. In contrast, the treatment of HepG2 cells with Pin1 inhibitor Juglone suppressed them. In addition, overxpression of Pin1 in ob/ob mouse liver elevated IRS-1 mediating insulin signaling, while Pin1 KO mice exhibited insulin resistance and glucose intolerance.Finally, the regulation of Pin1 expression in the mouse liver and muscle was investigated. After 2 weeks high-fat diet feeding, in both liver and muscle, Pin1 protein expression and IRS-1 associated with Pin1 was increased by several folds. It was also shown that Pin1 expression is low in the fasted condition, and increased by re-feeding. Taken all results together, we conclude that Pin1 expression is regulated by the nutrient conditions, and play an important role on enhancing IRS-1 mediating insulin actions in accordance with the nutrient conditions. Less