LMP POLYMORPHISMS IN B27-ASSOCIATED JUVENILE ARTHRITIS

幼年型类风湿性关节炎（JRA）是一种异质性自身免疫性疾病， 可能由多种遗传和环境因素引起的疾病 因素一个遗传因子编码在主要的I类区域， 组织相容性复合体（MHC），并与迟发性 特别是JRA，是HLA-B27。在初步研究中，我们发现 LMP 2的B等位基因的纯合性，LMP 2是两个MHC编码的 蛋白酶体亚基基因（LMP 2和LMP 7），作为额外的遗传 HLA-B27阳性儿童关节炎易感性的危险因素 由于蛋白酶体参与肽的产生， 由MHC 1类分子如B27，我们假设， LMP 7的多态性也可能影响对JRA的易感性。 此外，LMP 2、LMP 7和它们的等位基因变体可能影响 产生由HLA-B27呈递的肽表位，因此 影响这些个体的疾病易感性。 这项建议 概述了职业发展计划，旨在完成 培养对分子机制感兴趣的医生和科学家 HLA-B27相关的自身免疫性疾病的发病机制。它要依赖 霍华德休斯医学研究所的优势和潜力 由John Monaco博士领导的研究实验室， 现代分子免疫学技术培训，特别是 关于蛋白酶体的功能，和强大的司 以大卫格拉斯博士为首的流变学，长期兴趣 和JRA遗传关联方面的专业知识。除了基本的 研究部分，培训计划将提供候选人 通过参与以下活动扩大免疫学知识基础： 实验室会议、期刊俱乐部和研究研讨会。 具体目标1 我们建议使用分子基因分型来确认和扩展我们的 LMP 2的初步观察，并确定LMP 7是否 多态性与JRA的亚组相关。具体目标 2我们解决了LMP亚基在影响切割位点中的作用， 蛋白酶体的特异性，通过消化含有已知的 HLA I类限制性肽表位。将鉴定表位， 使用质谱法（MS）评估其相对量， 细胞毒性T淋巴细胞（CTL）测定。在第三章中，我们将测试 假设LMP 2和LMP 7多态性导致功能性 蛋白酶体活性的差异。 体外诱变将用于 模拟LMP 2和LMP 7中天然存在的多态性， 含有这些等位基因变体的蛋白酶体的切割位点特异性 将使用MS和CTL识别试验进行评估。这些研究 将定义LMP等位基因和JRA之间的关联， 有助于我们理解MHC编码的 蛋白酶体亚基及其等位变异的意义。 此外，它们对于我们确定多态性 涉及I类抗原不同位点的基因产物 处理途径可能相互作用，使某些个体倾向于 JRA。

Juvenile rheumatoid arthritis (JRA) is a heterogeneous autoimmune disease likely to result from multiple genetic and environmental factors. One genetic factor encoded in the class I region of the major histocompatibility complex (MHC), and associated with late onset particular JRA, is HLA-B27. In preliminary studies we have identified homozygosity for the B allele of LMP2, one of two MHC-encoded proteasome subunit genes (LMP2 and LMP7), as an additional genetic risk factor for susceptibility to arthritis in HLA-B27-positive children. Since proteasomes ar involved in the generation of peptides presented by MHC class 1 molecules like B27, we hypothesize that polymorphisms in LMP7 may also affect susceptibility to JRA. Furthermore, LMP2, LMP7, and their allelic variants may influence the production of peptide epitopes presented by HLA-B27, thus influencing disease susceptibility in these individuals. This proposal outlines a career development program designed to complete the training of a physician-scientist interested in the molecular mechanisms of HLA-B27-associated autoimmune disease pathogenesis. It relies on the strengths and potential of a Howard Hughes Medical Institute research laboratory headed by Dr. John Monaco to provide further training in modern techniques of molecular immunology, particularly with regard to proteasome function, and strong Division of Rheumatology headed by Dr. David Glass, with long-term interests and expertise in genetic associations in JRA. In addition to the basic research component, the training program will provide the candidate an expanded knowledge base in immunology through participation in lab meetings, journal clubs, and research seminars. In Specific Aim 1 we propose to use molecular genotyping to confirm and extend our initial observations wit LMP2, and determine whether LMP7 polymorphisms are associated with subgroups of JRA. In Specific Aim 2 we address the role of LMP subunits in influencing the cleavage site specificity of proteasomes by digesting oligopeptides containing known HLA class I-restricted peptide epitopes. Epitopes will be identified and their relative quantities assessed, using mass spectroscopy (MS) and cytotoxic T lymphocyte (CTL) assays. In Specific Aim 3 we will test hypothesis that LMP2 and LMP7 polymorphisms result in functional differences in proteasome activity. In vitro mutagenesis will be used to mimic naturally-occurring polymorphisms in LMP2 and LMP7, and cleavage site specificity of proteasomes containing these allelic variants will be assessed using MS and CTL recognition assays. These studies will define the association between LMP alleles and JRA, and contribute to our understanding of the function of MHC-encoded proteasome subunits and the significance of their allelic variation. Furthermore, they will essential to our determining how polymorphic gene products involved at different sites in the class I antigen processing pathway might interact to predispose certain individuals to JRA.