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）决定了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蛋白。

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.