Propeptide Mediation of Immunoproteasome Assembly

免疫蛋白酶体组装的前肽介导

• 批准号: 6739021
• 负责人: THOMAS A GRIFFIN
• 金额: $ 11.96万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-15 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6739021
• 关键词: MHC class I antigen; SDS polyacrylamide gel electrophoresis; antigen presentation; chemical fingerprinting; enzyme activity; immunoprecipitation; molecular assembly /self assembly; proteasome; protein biosynthesis; protein protein interaction; protein structure function; western blottings; yeast two hybrid system

This proposal outlines a research career development plan for a physician-scientist interested in the molecular biology of immunoproteasomes. It relies on a supportive research environment at Cincinnati Children's Hospital Medical Center (CCHMC). The sponsor, Dr. Robert Colbert, has related interests in antigen processing as it applies to the pathogenesis of HLA-B27-associated autoimmune disease. This proposal will provide an expanded knowledge base in molecular immunology via lab meetings, journal clubs, and research seminars, and it includes a new collaboration with Dr. Jeff Molkentin in the Dvision of Molecular Cardiovascular Biology at CCHMC. Immunoproteasomes are specialized for optimal MHC class I antigen processing. By virtue of their role in processing self-antigens, immunoproteasomes are potential targets in the treatment of autoimmune disease. Cooperative assembly of immunoproteasomes ensures their homogeneity in cells that also express constitutive proteasomes that serve many housekeeping functions. Cooperative assembly is dependent on differences between the propeptides of immunosubunits vs. constitutive subunits. We hypothesize that these propeptides mediate their effects through differential propeptide-protein interactions that are the focus of this project. In Aim 1, functionally important sequence differences between homologous propeptides will be identified by testing chimeric propeptides in whole cell assembly assays. In Aim 2, differential interactions between homologous propeptides and an assembly chaperone, proteassemblin, will be characterized using a yeast two-hybrid interaction trap assay. In Aim 3, a novel protein component of early pre-immunoproteasomes will be identified by peptide fingerprinting and characterized by co-immunoprecipitation. Characterizing the mechanisms of cooperative immunoproteasome assembly will serve a long-term goal of identifying targets for manipulating the assembly of immunoproteasomes while leaving vital constitutive proteasome assembly intact.

该提案为对以下领域感兴趣的内科科学家勾勒出了研究生涯发展计划 免疫蛋白酶体的分子生物学。它依赖于支持性的研究环境， 辛辛那提儿童医院医疗中心。赞助商罗伯特·科尔伯特博士 抗原处理在人类白细胞抗原B27相关致病机制中的相关研究 自身免疫性疾病。这项提议将提供一个扩展的分子知识库 免疫学通过实验室会议、杂志俱乐部和研究研讨会，它包括一个新的 与Jeff Molkentin博士在CCHMC的分子心血管生物学方面的合作。免疫蛋白酶体专用于MHC-I类抗原的最佳处理。由于其在处理自身抗原方面的作用，免疫蛋白酶体是治疗自身免疫性疾病的潜在靶点。免疫蛋白酶体的协同组装确保了它们在细胞中的同质性，这些细胞也表达起到许多管家功能的结构性蛋白酶体。协同组装依赖于免疫亚基和构成亚基的前肽之间的差异。我们假设这些前肽通过不同的前肽-蛋白质相互作用来调节它们的作用，这是本项目的重点。在目标1中，将通过在全细胞组装试验中测试嵌合前肽来鉴定同源前肽之间的功能重要序列差异。在目标2中，将使用酵母双杂交相互作用陷阱试验来表征同源前肽和组装伴侣蛋白组装蛋白之间的差异相互作用。在目标3中，将通过多肽指纹图谱和免疫共沉淀鉴定早期免疫前蛋白酶体的一个新的蛋白质组分。确定免疫蛋白酶体协同组装的机制将有助于确定操纵免疫蛋白酶体组装的靶标，同时保持重要的结构性蛋白酶体组装的完整性。