Regulated proteolysis in developmental signaling

发育信号中的调节蛋白水解

• 批准号: 7061024
• 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/7061024
• 关键词: Alzheimer' s disease; Drosophilidae; aspartic endopeptidases; biological signal transduction; chemical cleavage; drug discovery /isolation; 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.我们已经取得了进一步的进展，在我们正在进行的研究γ-分泌酶，蛋白水解复合物负责切割许多完整的膜蛋白底物。我们最近的工作已经证明，g-分泌酶活性可以通过其四种已知组分蛋白的过表达而在功能上重建（Hu和Fortini，2003）。在这些条件下，升高的γ-分泌酶活性导致Notch受体（γ-分泌酶复合物的底物之一）的裂解增加。我们的研究结果是重要的，因为他们定义了最小的一组蛋白质的γ-分泌酶的组装和功能，这种复合物的活性是需要适当的转录后调控的许多蛋白质参与癌细胞信号传导。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γ-分泌酶切割受体完成的。其他几种癌症相关蛋白，包括ErbB 4酪氨酸激酶和CD 44蛋白，也需要γ-分泌酶功能来调节其切割。我们对γ-分泌酶的重建研究表明，最小的四种蛋白质（早老素，Nicastrin，Aph-1和Pen-2）定义了γ-分泌酶活性，因此是开发靶向γ-分泌酶的小化合物抑制剂用于治疗干预策略的主要候选者。使用逐步共表达的方法与单个组件的γ-分泌酶，我们已经获得的证据表明，两个组件的复杂，Nicastrin和Aph-1，形成一个子复合物。我们的数据表明，该亚复合物的形成独立于其他γ-分泌酶组分，并且可能是γ-分泌酶组装途径中的瞬时中间体（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，其中每一个步骤可能适合于由不同的药物化合物靶向。我们的研究结果还表明，Nicastrin和Aph-1显然执行noncatalytic作用，这是不同的推定的酶（蛋白酶）活性的早老素组分对γ-分泌酶底物分子。因此，我们的研究结果表明，除了早老素，这是迄今为止最常见的目标γ-分泌酶抑制药物，Nicastrin和Aph-1也是有吸引力的目标，用于开发新的方法来调节γ-分泌酶活性的疾病，如癌症和阿尔茨海默病。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的遗传筛选，目的是分离新的化学诱导的tau等位基因。基于对我们现有的一组基于缺失的tau突变进行的遗传测试，该筛选成功地恢复了一个新的推定的tau等位基因。目前正在对这种新突变体进行表型分析。我们希望这些研究将为tau蛋白和相关微管相关蛋白（MAP）的正常功能提供见解，这些蛋白已广泛参与细胞骨架功能，神经元形态发生和人类疾病。