Biochemistry of Nicastrin in the Gamma-Secretase Complex

描述（由申请人提供）： γ-分泌酶介导的淀粉样前体蛋白、Notch和选择性跨膜转录因子的膜内蛋白水解代表了一种新的信号转导机制。淀粉样前体蛋白的异常蛋白水解产生淀粉样β-肽，是阿尔茨海默病发病机制中的中心事件。目前认为γ-分泌酶是一种多聚体、高分子量复合物，含有至少四种膜蛋白：早老素、nicastrin、APH-1和PEN-2。这四种蛋白质的确切作用尚不清楚，也不知道这种不寻常的γ-分泌酶如何组装以特异性地切割疏水环境中选择的膜蛋白的肽键。同样不清楚的是，不共享序列同源性的γ-分泌酶底物如何被选择并募集到γ-分泌酶复合物中进行切割。我们推测，γ-分泌酶复合物的组装和酶活性由四种蛋白质之间的分子间相互作用控制，而底物特异性由nicastrin胞外域控制，这两者都可能由额外的未知分子辅助。这一模式将在以下四个假设驱动的具体目标中得到检验。首先，将建立研究γ-分泌酶及其单个组分的体外实验系统。其次，γ-分泌酶复合物的组装和蛋白水解活性将通过定义nicastrin、早老素、APH-1和PEN-2的直接结合伴侣和对接位点，以及通过确定四种蛋白质复合物的大小、化学计量和酶活性来检查。第三，通过检查nicastrin是否选择和募集γ-分泌酶底物，并通过鉴定nicastrin胞外域的潜在配体，来测试nicastrin是否是γ-分泌酶底物特异性的分子传感器。第四，将鉴定与nicastrin、APH-1和PEN-2相关的其他蛋白质，并表征它们对γ-分泌酶活性的影响。这些研究应该为γ-分泌酶复合物的分子性质和生化机制提供重要的见解，并应该揭示新的目标和途径，为阿尔茨海默病和相关疾病设计更好的治疗策略。

DESCRIPTION (provided by applicant): Gamma-secretase-mediated intramembrane proteolysis of the amyloid precursor protein, Notch, and select transmembrane transcription factors represents a novel mechanism of signal transduction. Aberrant proteolysis of the amyloid precursor protein, which gives rise to amyloid beta-peptides, is a central event in the pathogenesis of Alzheimer's disease. It is now believed that gamma-secretase is a multimeric, high-molecular-weight complex that contains at least four membrane proteins: presenilin, nicastrin, APH-1, and PEN-2. The precise roles of these four proteins are not known, nor is how the unusual gamma-secretase assembles to specifically cleave peptide bonds of select membrane proteins within a hydrophobic environment. It is also not clear how gamma-secretase substrates, which share no sequence homology, are selected and recruited to the gamma-secretase complex for cleavage. We hypothesize that assembly and enzymatic activity of the gamma-secretase complex is governed by intermolecular interactions among the four proteins, while substrate specificity is controlled by the nicastrin ectodomain, both of which are likely aided by additional unknown molecules. This model will be tested in the following four hypothesis-driven specific aims. First, in vitro experimental systems for studying gamma-secretase and its individual components will be established. Second, the assembly and proteolytic activity of the gamma-secretase complex will be examined by defining the direct binding partners and the docking sites of nicastrin, presenilin, APH-1, and PEN-2; and by determining the size, stoichiometry, and enzymatic activity of the complex of the four proteins. Third, whether nicastrin is a molecular sensor for gamma-secretase substrate specificity will be tested by examining whether nicastrin selects and recruits gamma-secretase substrates, and by identifying potential ligands for the ectodomain of nicastrin. Fourth, additional proteins that associate with nicastrin, APH-1, and PEN-2 will be identified and their effects on gamma-secretase activity will be characterized. These studies should provide crucial insights into the molecular nature and biochemical mechanism of the gamma-secretase complex, and should reveal new targets and avenues for designing better therapeutic strategies for Alzheimer's disease and related disorders.