Regulated proteolysis in developmental signaling

1. 我们正在进行的γ -分泌酶的研究取得了进一步的进展，γ -分泌酶是一种蛋白质水解复合物，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过表达其四种已知成分蛋白在功能上重建（Hu和Fortini， 2003）。在这些条件下，升高的γ分泌酶活性导致Notch受体的切割增加，Notch受体是γ分泌酶复合物的底物之一。我们的发现意义重大，因为它们定义了γ -分泌酶组装和功能的最小蛋白质集，并且该复合物的活性对于许多参与癌细胞信号传导的蛋白质的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γ分泌酶切割受体完成的。其他几种癌症相关蛋白，包括ErbB4酪氨酸激酶和CD44蛋白，也需要γ -分泌酶的功能来调节其切割。我们对γ -分泌酶的重构研究表明，一组最小的四种蛋白（早老素、Nicastrin、Aph-1和Pen-2）定义了γ -分泌酶的活性，因此它们是开发靶向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物。利用与γ -分泌酶单个组分的逐步共表达方法，我们已经获得了复合物Nicastrin和Aph-1的两个组分形成亚复合物的证据。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见解，每个步骤都可能适用于不同药物化合物的靶向。我们的研究结果还表明，Nicastrin和Aph-1明显发挥非催化作用，这与早老素成分对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，除了早老素（迄今为止是γ -分泌酶抑制药物最常见的靶点）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，如癌症和阿尔茨海默病。我们继续对果蝇的tau基因进行分析。最近在实验室进行了大规模的基因筛选，目的是分离新的化学诱导的tau等位基因。这一筛选成功地恢复了一个新的假定的tau等位基因，这是基于我们现有的基于缺失的tau突变的基因测试。目前正在对这个新突变体进行表型分析。我们希望这些研究将为tau和相关微管相关蛋白（MAPs）的正常功能提供见解，这些蛋白已广泛涉及细胞骨架功能，神经元形态发生和人类疾病。

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.