PROTEOMIC ANALYSIS OF PROTEIN PALMITOYLATION IN YEAST

酵母蛋白质棕榈酰化的蛋白质组学分析

• 批准号: 7957729
• 负责人: NICHOLAS G DAVIS
• 金额: $ 0.33万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7957729
• 关键词: Biology; Biotin; Biotinylation; Chemicals; Computer Retrieval of Information on Scientific Projects Database; Cysteine-Rich Domain; Drops; Enzymes; Funding; Fungal Genome; Genes; Grant; Hydroxylamine; Institution; Link; Mediating; Membrane Proteins; Methodology; Methods; Modification; Protein Analysis; Protein Family; Proteins; Proteome; Proteomics; Protocols documentation; Reagent; Research; Research Personnel; Resources; Source; Specificity; Sulfhydryl Compounds; Transferase; United States National Institutes of Health; Work; Yeasts; Zinc Fingers; palmitoylation; protein complex

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have developed a proteomic protocol that allows the purification of the palmitoylated subset of proteins from complex protein extracts. The crux of the new methodology is a chemical exchange of biotin for the attached palmitoyl modifications. Extract proteins are first treated exhaustively with NEM to block free thiols. Next, the attached palmitoyl groups are released with hydroxylamine, exposing new thiols. Then, the newly-exposed thiols are biotinylated with a thiol-specific biotinylation reagent. The method works well, being quite specific for palmitoylation. Using this methodology, a first project will be a comprehensive characterization of the yeast palmitoyl-proteome. A second project will link identified palmitoyl-proteins to the enzymes that mediate their palmitoylation, that is to their cognate protein acyl transferase (PAT). Our prior results have pointed towards the DHHC protein family, a set of polytopic membrane proteins with zinc finger-like DHHC cysteine-rich domains, as likely being a family of PAT specificities. The yeast genome encodes seven DHHC proteins. Strains deleted for different DHHC genes either singly or in combination will have their palmitoyl-proteomes profiled as described above. Palmitoyl-proteins dropping out of particular DHHC deletion strains profiles will be further characterized as potential substrates for the deleted PAT enzyme.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我们开发了一种蛋白质组学方案，可以从复杂的蛋白质提取物中纯化棕榈酰化的蛋白质子集。 新方法的关键是生物素与所附棕榈酰修饰的化学交换。 首先用 NEM 彻底处理提取蛋白以封闭游离硫醇。 接下来，连接的棕榈酰基与羟胺一起释放，暴露出新的硫醇。 然后，用硫醇特异性生物素化试剂对新暴露的硫醇进行生物素化。 该方法效果很好，对于棕榈酰化非常具体。 使用这种方法，第一个项目将是酵母棕榈酰蛋白质组的全面表征。 第二个项目将把已鉴定的棕榈酰蛋白与介导其棕榈酰化的酶（即其同源蛋白酰基转移酶（PAT））联系起来。 我们之前的结果表明 DHHC 蛋白家族是一组多胞膜蛋白，具有锌指状 DHHC 富含半胱氨酸结构域，很可能是 PAT 特异性家族。 酵母基因组编码七种 DHHC 蛋白。 单独或组合删除不同 DHHC 基因的菌株将具有如上所述的棕榈酰蛋白质组图谱。 从特定 DHHC 删除菌株谱中剔除的棕榈酰蛋白将被进一步表征为删除的 PAT 酶的潜在底物。