第十一章 蛋白质相互作用.pptVIP

第十一章 蛋白质相互作用 The Wnt pathway Interactions between proteins in Wnt signaling and approximate sites of binding 蛋白质相互作用包括三个方面： ⑴ 多亚基蛋白质的形成； ⑵ 多成分蛋白质的相互作用； ⑶ 瞬时的蛋白质相互作用，控制着一些重要的生命活动。 生物传感器(biosensor) 　　由固定化的生物敏感材料作识别元件与适当的理化换能器及信号放大装置构成的分析工具或系统。 特点： ① 采用固定化生物活性物质作催化剂；价值昂贵的试剂可多次重复使用，克服了过去酶法分析试剂费用高和化学分析繁琐复杂的缺点； ② 专一性强，只对特定的底物起反应，而且不受颜色、浊度的影响； ③ 分析速度快，可以在一分钟得到结果； ④ 准确度高，一般相对误差可以达到1％； ⑤ 操作系统比较简单，容易实现自动分析； ⑥ 成本低，在连续使用时，每例测定仅需要几分钱人民币； ⑦ 有的生物传感器能可靠地指示微生物培养系统内的供氧状况和副产物的产生，能得到许多复杂的物理化学传感器综合作用才能获得的信息，同时还可指明增加产物得率的方向。 酵母双杂交系统 (yeast two-hybrid system) 噬菌体展示 (Phage display techniques, PDT) 串联亲和纯化(tandem affinity purification, TAP) * * Resting state of the Wnt/Wingless signalling pathway Active Stage of the WNT/Wingless Signalling Pathway 蛋白质亲和层析 荧光共振能量转移 噬菌体三维结构 C-端和N-端TAP 标签简图 * * * In the presence of a Wnt ligand, if not inhibited by secreted antagonists, the Wnt ligand binds a frizzled (Fz)/low density lipoprotein receptor related protein (LRP) complex, activating the cytoplasmic protein dishevelled (Dsh in Drosophila and Dvl in vertebrates). Precisely how Dsh/Dvl is activated is not fully understood, but phosphorylation by casein kinase 1 (CK1) and casein kinase 2 (CK2) have been suggested to be partly responsible (Willert et al., 1997; Sakanaka et al., 1999; Amit et al., 2002).Dsh/Dvl then inhibits the activity of the multiprotein complex (b-catenin-Axin-adenomatous polyposis coli (APC)-glycogen synthase kinase (GSK)-3b), which targets b-catenin by phosphorylation for degradation by the proteasome. Dsh/Dvl is suggested to bind CK1 and thereby inhibiting priming of b-catenin and indirectly preventing GSK-3b phosphorylation of b-catenin (Amit et al., 2002). Upon Wnt stimulation, Dvl has also been shown to recruit GSK-3 binding protein (GBP) to the multiprotein complex. GBP might titrate GSK-3b from Axin and in this way inhibits phosphorylation of b-catenin. Finally, sequestration of Axin at the cell membrane by LRP has been described (Mao et al., 2001b). The overall res