IDENTIFYING THE ROLE OF CASPASE-MEDIATED PROTEOLYSIS ON INFLAMMATION

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 这项建议的长期目标是阐明参与先天免疫的蛋白酶信号通路。 我们建议确定在先天免疫反应过程中被切割的蛋白质，以及负责的蛋白酶。 蛋白酶的激活，特别是炎症性半胱天冬酶，是先天免疫应答的标志，并调节对细菌感染、痛风、关节炎和疫苗接种的应答。 令人惊讶的是，很少有（少于10）蛋白质先前已被确定为炎症半胱天冬酶的底物。 从我们的工作和其他人的工作，现在已知有超过500种蛋白质在细胞凋亡（调节性细胞死亡）过程中被半胱天冬酶切割。 炎性和凋亡半胱天冬酶具有非常相似的催化效率，并且炎性半胱天冬酶的底物特异性似乎没有受到更多的限制。 是什么原因导致这两个家族所加工的蛋白质数量相差50倍？我们建议确定额外的炎症caspase底物，并确定其在炎症中的作用。 我们已经开始使用一种新的和通用的基于质谱的方法来解决这些问题，用于细胞中蛋白质水解的全局分析（我们称之为“降解组学”）。 与Burlingame实验室合作，我们开发了一种基于MS的方法来鉴定超过200种新的凋亡caspase底物。 初步的蛋白质组学实验与炎症半胱天冬酶已经确定了许多（超过30）促炎底物，其中几个发挥已知的作用，在炎症。 定量质谱法将用于扩展该列表并验证所识别的底物。 将通过蛋白质印迹进一步确认所选命中，并使用基于ELISA的方法分析功能意义。

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. The long term goal of this proposal is to elucidate the protease signaling pathways involved in innate immunity. We propose to identify the proteins that are cleaved during the innate immune response, and the proteases responsible. Activation of proteases, notable the inflammatory caspases, is a hallmark of the innate immune response, and regulates responses to bacterial infection, gout, arthritis, and vaccinations. Surprisingly very few (less than 10) proteins have previously been identified as substrates of the inflammatory caspases. From our work and that of others, there are now known to be greater than 500 proteins cleaved by caspases during apoptosis (regulated cell death). The inflammatory and apoptotic caspases have very similar catalytic efficiencies and the substrate specificities of the inflammatory caspases do not appear more restricted. What accounts for the 50-fold difference in the apparent number of proteins known to be processed by these two families? We propose to identify additional inflammatory caspase substrates and identify their roles in inflammation. We have begun to address these questions using a new and general mass spectrometry-based method for global profiling of proteolysis in cells (we call "degradomics"). In collaboration with the Burlingame lab we have developed an MS-based method to identify greater than 200 new apoptotic caspase substrates. Preliminary proteomic experiments with the inflammatory caspases have identified numerous (greater than 30) pro-inflammatory substrates, several of which play known roles in inflammation. Quantitative mass-spectrometry will be used to expand this list and validate the identified substrates. Selected hits will be further confirmed via western blot and analyzed for functional significance using ELISA-based methods.