PROTEOMIC ANALYSIS OF APOPTOTIC PROTEIN CLEAVAGES IN DROSOPHILA MELANOGASTER

果蝇凋亡蛋白裂解的蛋白质组学分析

• 批准号: 8363826
• 负责人: JAMES A WELLS
• 金额: $ 0.39万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363826
• 关键词: Address; Apoptosis; Apoptotic; Aspartic Endopeptidases; Caenorhabditis elegans; Caspase; Cell Death; Cells; Cysteine; Drosophila melanogaster; Enzymes; Event; Funding; Grant; Human; In Vitro; Individual; Label; Mass Spectrum Analysis; Mus; N-terminal; National Center for Research Resources; Peptide Hydrolases; Principal Investigator; Process; Proteolysis; Proteome; Proteomics; Regulatory Element; Research; Research Infrastructure; Resources; Source; United States National Institutes of Health; cost

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.

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。 半胱氨酸天冬氨酸蛋白酶（半胱氨酸蛋白酶）是负责细胞凋亡的细胞重塑的关键酶，但它们的许多切割靶点仍未确定。 威尔斯实验室已经进行了凋亡重塑过程的蛋白质组规模的体外研究，并致力于发现新的细胞死亡调控元件。 虽然已经以这种方式鉴定了数百种胱天蛋白酶底物，但每个单独的切割事件的功能相关性仍然未知。 可能的是，一些裂解对于细胞凋亡的安全和有效进行是至关重要的，而另一些对细胞凋亡过程几乎没有影响，并且实际上可能是“旁观者”裂解。 我们建议区分这两种类型的卵裂，通过评估其跨物种的保护。 我们将首先研究黑腹果蝇的细胞含有与人类半胱氨酸天冬氨酸蛋白酶高度序列相似的半胱氨酸天冬氨酸蛋白酶，并经历与人类细胞凋亡形态相似的凋亡。我们还将检测小鼠细胞和C.线虫全虫提取物。我们将使用Mahrus等人（2008）描述的N-末端标记方法富集蛋白酶底物，并通过质谱法对其进行鉴定。 通过这种方法，我们朝着大规模解决半胱天冬酶切割的功能相关性问题迈出了关键一步。