LMP POLYMORPHISMS IN B27-ASSOCIATED JUVENILE ARTHRITIS

幼年类风湿性关节炎是一种异质性自身免疫性疾病 疾病可能是由多种遗传和环境因素引起的 各种因素。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)，与迟发有关 特别是JRA，是人类白细胞抗原B27。在初步研究中，我们已经确定 LMP2基因B等位基因纯合性研究 蛋白酶体亚单位基因(LMP2和LMP7)，作为一种附加基因 人类白细胞抗原B27阳性儿童关节炎易感性的危险因素。 因为蛋白酶体参与了所呈现的多肽的产生 通过像B27这样的MHC 1类分子，我们假设 LMP7基因的多态性也可能影响JRA的易感性。 此外，LMP2、LMP7及其等位基因变异可能会影响 人类白细胞抗原B27呈递的多肽表位的产生 影响这些人的疾病易感性。这项建议 概述职业发展计划，旨在完成 对对分子机制感兴趣的内科科学家进行培训 研究人类白细胞抗原B27相关自身免疫性疾病的发病机制。它依赖于 霍华德·休斯医学院的优势和潜力 由约翰·摩纳哥博士领导的研究实验室将进一步提供 现代分子免疫学技术培训，特别是 关于蛋白酶体的功能，以及强烈的分裂 由大卫·格拉斯博士领导的风湿科，具有长远利益 以及在JRA的遗传关联方面的专业知识。除了基本的 研究部分，培训计划将为应聘者提供 通过参与以下活动扩大免疫学知识基础 实验室会议、杂志俱乐部和研究研讨会。在特定目标中1 我们建议使用分子基因分型来确认和扩展我们的 LMP2的初步观察，并确定LMP7是否 基因多态与JRA的亚群相关。以特定的目标 2我们讨论了LMP亚基在影响切割位点中的作用。 蛋白酶体通过消化含已知的寡肽的特异性 人类白细胞抗原I类限制性多肽表位。将确定表位，并 使用质谱学(MS)和 细胞毒性T淋巴细胞(CTL)检测。在特定的目标3中，我们将测试 假设LMP2和LMP7基因多态导致功能性 蛋白酶体活性的差异。体外诱变技术将用于 模拟LMP2和LMP7中自然发生的多态，以及 含有这些等位基因变异的蛋白酶体的切割位点特异性 将使用MS和CTL识别分析进行评估。这些研究 将定义LMP等位基因和JRA之间的关联，以及 有助于我们理解MHC编码的功能 蛋白酶体亚基及其等位基因变异的意义。 此外，它们将对我们确定多态程度至关重要 涉及I类抗原不同部位的基因产物 加工途径可能相互作用，使某些个体易于患上 杰拉。

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.