Regulated proteolysis in developmental signaling

1.我们正在进行的伽马分泌酶的研究取得了进一步的进展，伽马分泌酶是一种蛋白质分解复合体，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个已知组成蛋白的过度表达而在功能上重新构建(Hu和Fortini，2003)。在这种情况下，伽马分泌酶活性的提高会导致Notch受体的切割增加，Notch受体是伽玛分泌酶复合体的底物之一。我们的发现意义重大，因为它们定义了伽马分泌酶组装和功能的最小蛋白质集，并且这种复合体的活性是正确转录后调节参与癌细胞信号传递的许多蛋白质所必需的。例如，Notch受体在T细胞急性淋巴细胞性白血病中发生突变，其生化激活是通过该受体的伽马分泌酶裂解完成的。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，也需要伽马分泌酶功能来调节它们的切割。我们对伽玛分泌酶的重组研究表明，最小的四种蛋白质(早老素、尼卡斯特素、APH-1和PEN-2)定义了伽玛分泌酶的活性，因此是开发针对伽玛分泌酶的小分子化合物抑制剂用于治疗干预策略的主要候选药物。使用逐步共表达的方法和单独的伽马分泌酶成分，我们已经获得了证据，该复合体的两个组成部分，尼古丁和APH-1，形成一个亚复合体。我们的数据表明，这个亚复合体的形成独立于其他伽马分泌酶成分，并且很可能是伽马分泌酶组装途径中的一个瞬时中间体(Hu和Fortini，2003)。这些研究为伽马分泌酶复合体形成的不同步骤提供了洞察力，每一步都可能受到不同药物化合物的靶向。我们的结果还表明，尼古丁和APH-1显然发挥着不同于假定的早老素成分对伽马分泌酶底物分子的酶(蛋白酶)活性的非催化作用。因此，我们的发现表明，除了到目前为止最常见的伽马分泌抑制药物靶标早老素外，尼古丁和APH-1也是开发调节癌症和阿尔茨海默病等疾病的伽玛分泌酶活性的新方法的有吸引力的靶标。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。根据我们现有的一组基于缺失的tau突变的基因测试，这一筛选导致成功地恢复了一个新的假定tau等位基因。对这个新突变体的表型分析目前正在进行中。我们希望这些研究将对tau和相关的微管相关蛋白(MAP)的正常功能提供深入的见解，这些蛋白广泛地与细胞骨架功能、神经元形态发生和人类疾病有关。

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.