Regulated proteolysis in developmental signaling

发育信号中的调节蛋白水解

• 批准号: 6952132
• 负责人: Michael Lee
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6952132
• 关键词: Alzheimer's disease; Drosophilidae; biological signal transduction; chemical cleavage; drug discovery /isolation; endopeptidases; enzyme activity; enzyme complex; enzyme inhibitors; enzyme substrate; gene expression; microtubule associated protein; molecular assembly /self assembly; neoplasm /cancer pharmacology; presenilin; protein structure function; proteolysis; receptor binding; tau proteins

1. We have made further progress in our ongoing studies of gamma-secretase, the proteolytic complex responsible for cleavage of many integral membrane protein substrates. Our recent work has demonstrated that g-secretase activity can be functionally reconstituted through overexpression of its four known component proteins (Hu and Fortini, 2003). Under these conditions, elevated gamma-secretase activity results in increased cleavage of the Notch receptor, one of the substrates for the gamma-secretase complex. Our findings are significant because they define the minimal set of proteins for gamma-secretase assembly and function, and activity of this complex is needed for proper posttranscriptional regulation of many proteins involved in cancer cell signaling. The Notch receptor, for instance, is mutated in T-cell acute lymphoblastic leukemia, and its biochemical activation is accomplished by gamma-secretase cleavage of the receptor. Several other cancer-related proteins, including the ErbB4 tyrosine kinase and the CD44 protein, also require gamma-secretase function for their regulated cleavage. Our reconstitution studies on gamma-secretase suggest that a minimal set of four proteins (Presenilin, Nicastrin, Aph-1, and Pen-2) define gamma-secretase activity, and are therefore the main candidates to consider in developing small compound inhibitors to target gamma-secretase for therapeutic intervention strategies. 2. Using a stepwise co-expression approach with the individual components of gamma-secretase, we have obtained evidence that two components of the complex, Nicastrin and Aph-1, form a subcomplex. Our data suggest that this subcomplex forms independently of other gamma-secretase components, and is likely to be a transient intermediate in the gamma-secretase assembly pathway (Hu and Fortini, 2003). These studies provide insight into the different steps of gamma-secretase complex formation, each of which might be amenable to targeting by different drug compounds. Our results also indicate that Nicastrin and Aph-1 apparently perform noncatalytic roles that are distinct from the presumed enzymatic (protease) activity of the Presenilin component towards gamma-secretase substrate molecules. Our findings therefore indicate that in addition to Presenilin, which has so far been the most common target for gamma-secretase inhibitory drugs, Nicastrin and Aph-1 are also attractive targets for the development of novel approaches to modulate gamma-secretase activity in diseases such as cancer and Alzheimer's disease. 3. We have continued to pursue our analysis of the Drosophila tau gene. A large-scale genetic screen in the lab was performed recently with the aim of isolating new chemical-induced alleles of tau. This screen led to the successful recovery of one new putative tau allele, based on genetic tests with our existing set of deletion-based tau mutations. Phenotypic analysis of this new mutant is currently underway. We are hopeful that these studies will provide insights into the normal function of tau and related microtubule-associated proteins (MAPs), which have been widely implicated in cytoskeletal function, neuronal morphogenesis, and human disease.

1. 我们正在进行的γ-分泌酶研究取得了进一步进展，γ-分泌酶是一种蛋白水解复合物，负责切割许多完整的膜蛋白底物。我们最近的工作表明，g 分泌酶活性可以通过其四种已知成分蛋白的过度表达进行功能重建（Hu 和 Fortini，2003）。在这些条件下，γ-分泌酶活性升高导致Notch受体（γ-分泌酶复合物的底物之一）的裂解增加。我们的发现具有重要意义，因为它们定义了γ-分泌酶组装和功能的最小蛋白质组，并且该复合物的活性对于涉及癌细胞信号转导的许多蛋白质的适当转录后调节是必需的。例如，Notch 受体在 T 细胞急性淋巴细胞白血病中发生突变，其生化激活是通过受体的 γ 分泌酶裂解来完成的。其他几种与癌症相关的蛋白质，包括 ErbB4 酪氨酸激酶和 CD44 蛋白质，也需要 γ 分泌酶功能来调节裂解。我们对 γ 分泌酶的重构研究表明，最少的四种蛋白质（早老素、尼卡斯特林、Aph-1 和 Pen-2）定义了 γ 分泌酶活性，因此是开发靶向小化合物抑制剂时要考虑的主要候选者。 γ-分泌酶用于治疗干预策略。 2.使用与γ-分泌酶的各个组分的逐步共表达方法，我们获得了复合物的两个组分Nicastrin和Aph-1形成亚复合物的证据。我们的数据表明，该子复合物的形成独立于其他γ-分泌酶成分，并且可能是γ-分泌酶组装途径中的短暂中间体（Hu和Fortini，2003）。这些研究提供了对γ-分泌酶复合物形成的不同步骤的深入了解，每个步骤都可能适合不同药物化合物的靶向。我们的结果还表明，Nicastrin 和 Aph-1 显然发挥非催化作用，这与推测的早老素成分对 γ-分泌酶底物分子的酶（蛋白酶）活性不同。因此，我们的研究结果表明，除了迄今为止最常见的 γ 分泌酶抑制药物靶点 Presenilin 之外，Nicastrin 和 Aph-1 也是开发调节 γ 分泌酶活性的新方法的有吸引力的靶点，例如如癌症和阿尔茨海默病。 3. 我们继续对果蝇 tau 基因进行分析。最近在实验室进行了大规模遗传筛选，目的是分离新的化学诱导的 tau 等位基因。根据我们现有的一组基于缺失的 tau 突变的基因测试，该筛选成功恢复了一个新的推定 tau 等位基因。目前正在进行这种新突变体的表型分析。我们希望这些研究能够深入了解 tau 和相关微管相关蛋白 (MAP) 的正常功能，这些蛋白与细胞骨架功能、神经元形态发生和人类疾病广泛相关。