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的膜内蛋白分解和选择的跨膜转录因子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分解产生淀粉样β-多肽，是阿尔茨海默病发病机制中的一个中心事件。目前认为，γ-分泌酶是一种多聚体、高相对分子质量的复合体，至少含有四种膜蛋白：早老素、尼古丁、APH-1和PEN-2。这四种蛋白质的确切作用尚不清楚，也不清楚这种不寻常的伽马分泌酶是如何在疏水环境中特异地切割选定的膜蛋白的多肽键的。也不清楚没有序列同源性的伽马分泌酶底物是如何被选择并招募到伽马分泌酶复合体进行切割的。我们假设，伽马分泌酶复合体的组装和酶活性是由四种蛋白质之间的分子间相互作用控制的，而底物特异性是由尼古丁胞外结构域控制的，这两者都可能是由额外的未知分子辅助的。该模型将在以下四个假设驱动的具体目标中进行检验。首先，将建立研究伽马分泌酶及其单个组分的体外实验系统。其次，将通过定义尼卡斯汀、早老素、APH-1和PEN-2的直接结合伙伴和对接位置，以及通过测定这四种蛋白质的复合体的大小、化学计量比和酶活性来检查伽马分泌酶复合体的组装和蛋白分解活性。第三，尼古丁是否是伽马分泌酶底物特异性的分子传感器，将通过检测尼古丁是否选择和招募伽马分泌底物，以及通过识别尼古丁胞外结构域的潜在配体来测试。第四，将确定与尼古丁、APH-1和PEN-2相关的其他蛋白质，并表征它们对伽马分泌酶活性的影响。这些研究应该为伽马分泌酶复合体的分子性质和生化机制提供重要的见解，并应该揭示为阿尔茨海默病和相关疾病设计更好的治疗策略的新靶点和途径。

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.