IDENTIFICATION OF CASPASE-2 SUBSTRATES

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。 Caspase-2是不同物种中最保守的Caspase。据报道，Caspase-2参与了卵母细胞死亡、DNA损伤、热休克诱导的死亡和有丝分裂灾难。然而，由于缺乏对caspase-2底物的了解，caspase-2在这些生物学事件中的确切作用尚不清楚。我的项目是使用已建立的“降解”技术来鉴定caspase-2底物。首先，我会用重组有活性的caspase-2来处理健康的Jurkat提取物。样品中的蛋白质N末端将被标记、浓缩和质谱分析，以鉴定caspase-2的裂解。其次，我将使用caspase-2诱导系统来诱导内源底物切割。细胞提取液也将被标记、浓缩并通过质谱学进行测序。来自两个实验的数据将被比较和整合，以确定caspase-2底物。

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. Caspase-2 is the most conserved caspase across different species. It has been reported that Caspase-2 is involved in oocyte death, DNA damage, heat shock-induced death and mitotic catastrophe. However, the exact role of caspase-2 in these biological events is not clear yet due to the lack of knowledge of the substrates of caspase-2. My project is to identify caspase-2 substrates using the established "degradomics" techniques. First, I will treat healthy Jurkat extracts with recombinant active caspase-2. Protein N-terminus in the sample will be labeled, enriched and sequenced by mass spectrometry to identify caspase-2 cleavage. Second, I will use a caspase-2 inducible system to induce endogenous substrate cleavage. Cell extracts will also be labeled, enriched and sequenced by mass spectrometry. Data from both experiments will be compared and integrated to identify caspase-2 substrates.