Propeptide Mediation of Immunoproteasome Assembly

免疫蛋白酶体组装的前肽介导

• 批准号: 6597811
• 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/6597811
• 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.

该建议概述了对有兴趣的医师科学家的研究职业发展计划 免疫蛋白酶体的分子生物学。它依赖于支持性研究环境 辛辛那提儿童医院医疗中心（CCHMC）。赞助商罗伯特·科尔伯特（Robert Colbert）博士 抗原加工的相关兴趣适用于HLA-B27相关的发病机理 自身免疫性疾病。该提案将提供分子扩展的知识基础 通过实验室会议，期刊俱乐部和研究研讨会的免疫学，其中包括一个新的 与杰夫·莫尔肯丁（Jeff Molkentin）博士在CCHMC上的分子心血管生物学的合作。 免疫蛋白酶体专门用于最佳的MHC I类抗原处理。由于它们在加工自我抗原中的作用，免疫蛋白酶体是治疗自身免疫性疾病的潜在靶标。免疫蛋白酶体的合作组装可确保它们在细胞中的同质性，这些细胞也表达了构成蛋白酶体的蛋白酶体，这些蛋白酶体可起到许多管家功能。 合作组装取决于免疫支出与本构亚基的丙肽之间的差异。我们假设这些丙肽通过差异性丙肽蛋白相互作用是该项目的重点。在AIM 1中，通过在整个细胞组装分析中测试嵌合丙肽，可以鉴定出在功能上重要的序列差异。在AIM 2中，使用酵母双杂交相互作用陷阱测定法对同源丙肽和组装伴侣蛋白酶膜之间的差异相互作用将进行表征。在AIM 3中，将通过肽指纹识别早期免疫原子膜体的新型蛋白质成分，并以共免疫沉淀为特征。表征合作免疫蛋白酶体组件的机制将实现一个长期目标，即确定操纵免疫蛋白酶体组装的目标，同时使重要的组成型蛋白酶体组装完整。