Propeptide Mediation of Immunoproteasome Assembly

该提案概述了对以下方面感兴趣的医生-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 抗原处理的相关利益，因为它适用于HLA-B27相关的发病机制， 自身免疫性疾病。这一建议将提供一个扩大的知识基础，在分子 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部门合作。 免疫蛋白酶体专门用于最佳的MHC I类抗原加工。由于它们在处理自身抗原中的作用，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，所述细胞还表达提供许多管家功能的组成型蛋白酶体。 协同组装依赖于免疫亚基与组成型亚基的前肽之间的差异。我们假设这些前肽通过差异前肽-蛋白质相互作用介导其效应，这是本项目的重点。在目的1中，同源前肽之间的功能上重要的序列差异将通过在全细胞组装测定中测试嵌合前肽来鉴定。在目标2中，同源前肽和组装分子伴侣蛋白，蛋白组装蛋白之间的差异相互作用，其特征在于使用酵母双杂交相互作用陷阱测定。在目标3中，一种新的蛋白质组分的早期前免疫蛋白酶体将被确定肽指纹图谱和免疫共沉淀的特点。表征合作免疫蛋白酶体组装的机制将服务于一个长期的目标，即确定操纵免疫蛋白酶体组装的目标，同时保持重要的组成性蛋白酶体组装完整。

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.