IDENTIFYING THE ROLE OF CASPASE-MEDIATED PROTEOLYSIS ON INFLAMMATION

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。 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中被切割的蛋白质，以及负责的蛋白酶。蛋白水解酶的激活，尤其是炎性半胱氨酸酶，是先天免疫反应的标志，并调节对细菌感染、痛风、关节炎和疫苗接种的反应。令人惊讶的是，以前只有很少(不到10个)的蛋白质被确定为炎症半胱氨酸酶的底物。根据我们和其他人的工作，现在已知有超过500种蛋白质在细胞凋亡(受调控的细胞死亡)过程中被半胱氨酸酶切割。炎症性和凋亡性caspase具有非常相似的催化效率，并且炎症性caspase的底物特异性似乎没有受到更多限制。这两个家族所处理的蛋白质表观数量相差50倍的原因是什么？我们建议确定其他炎性caspase底物，并确定它们在炎症中的作用。我们已经开始使用一种新的、通用的基于质谱学的方法来解决这些问题，该方法用于对细胞中的蛋白质分解进行全球分析(我们称之为“降解”)。在与Burlingame实验室的合作中，我们开发了一种基于MS的方法来识别200多种新的凋亡caspase底物。对炎性半胱氨酸酶的初步蛋白质组学实验已经确定了许多(超过30个)促炎底物，其中几个在炎症中发挥已知作用。将使用定量质谱学来扩展这一列表并验证所识别的底物。选定的抗体将通过免疫印迹进一步确认，并使用基于酶联免疫吸附试验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.