PROTEOMIC ANALYSIS OF APOPTOTIC PROTEIN CLEAVAGES IN DROSOPHILA MELANOGASTER

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 半胱氨酸天冬氨酸蛋白酶(Caspase)是导致细胞凋亡重塑的关键酶，但它们的许多切割靶点仍未确定。威尔斯实验室对细胞凋亡重塑过程进行了蛋白质组规模的体外研究，并朝着发现新的细胞死亡调控元件的方向发展。虽然已经以这种方式鉴定了数百种caspase底物，但每个单独的切割事件的功能相关性仍然未知。很可能有些裂解对细胞凋亡的安全和有效进行是至关重要的，而另一些裂解对细胞凋亡过程几乎没有影响，实际上可能是“旁观者”裂解。我们建议通过评估它们在不同物种中的保守性来区分这两种类型的卵裂。我们将首先研究果蝇细胞中含有与人类caspase序列高度相似的caspase，并经历与人类细胞形态相似的凋亡。我们还将检测小鼠细胞和线虫全虫提取物中caspase的裂解情况。我们将使用Mahrus等人描述的N-末端标记法。(2008)对蛋白酶底物进行富集化，并通过质谱学对其进行鉴定。通过这种方法，我们正在朝着大规模解决caspase裂解的功能相关性问题迈出关键一步。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Caspases (cysteine aspartyl proteases) are key enzymes responsible for the cellular remodeling of apoptosis, yet many of their cleavage targets remain unidentified. The Wells lab has undertaken proteome-scale in vitro studies of apoptotic remodeling process, and towards discovery of new cell death regulatory elements. While hundreds of caspase substrates have been identified in this manner, the functional relevance of each individual cleavage event remains unknown. It is likely that some cleavages are vital for apoptosis to proceed safely and efficiently, while others have little effect on the process of apoptosis and may in fact be "bystander" cleavages. We propose to distinguish between these two types of cleavages by evaluating their conservation across species. We will first study cells of Drosophila melanogaster contain caspases with high sequence similarity to human caspases, and undergo apoptosis that is morphologically similar to human cell apoptosis. We will also examine caspase cleavages in mouse cells, and in C. elegans whole worm extract. We will use the N-terminal labeling approach described by Mahrus et al. (2008) to enrich for protease substrates and identify them by mass spectrometry. With this approach, we are taking a key step towards addressing the question of the functional relevance of caspase cleavages on a large scale.