Mechanisms of beta2-microglubin in Autoimmune Diabetes

描述(由申请人提供)：人类和非肥胖糖尿病(NOD)小鼠的自身免疫性糖尿病是由T细胞介导的产生胰岛素的胰岛β细胞的自身免疫破坏造成的。I类主要组织相容性复合体(MHC)限制性T细胞和II类MHC限制性T细胞均参与其中。然而，T细胞受体(TCR)仅限于识别由三聚体I类分子呈递的自身抗原肽，由MHC重链、β2-微球蛋白(β2M)和多肽组成，是NOD小鼠发病所必需的。自身免疫性糖尿病是一种多基因疾病，特殊的MHC单倍型是人类和NOD小鼠易感性的主要遗传成分。最近，(Beta2m)被鉴定为NOD小鼠的糖尿病易感基因，这是第一个定位于MHC区外的基因，位于2号染色体上的Idd13基因座。两个Beta2m等位基因广泛存在于常见的实验室小鼠品系中。当在NOD小鼠中发现的a2ma等位基因被a2mb等位基因取代时，糖尿病的发展就被阻止了。这两个等位蛋白之间的氨基酸序列差异在于第85位的Asp(“a”亚型)与Ala(“b”亚型)的单一交换。以前的许多血清学和T细胞识别研究，使用了来自不同物种的各种MHC等位基因产物和a2m蛋白，表明I类分子的显著构象灵活性取决于复合体中存在的特定的β2m。基于这些发现，可以推测，与含有Beta2mb的MHC分子相比，含有Beta2mA的I类MHC分子可能呈现出不同的构象。这些变化可能对自身反应性T细胞的选择和自身抗原肽的呈递产生影响。这项提议的总体目标是通过系统地检查包含两种β2m异构体的与疾病相关的MHC-肽复合体的结构、生化和生物学特性来检验这一假说。提出了四个具体目标：(1)利用X射线衍射分析、化学和热变性以及酰胺质子交换，对分离的等位基因β_2m蛋白进行结构、热力学和动力学表征；(2)对糖尿病相关的MHC/肽复合体进行相似的表征，包括测量β_2m和肽交换率；(3)利用X射线衍射分析、酰胺质子交换和表面等离子体共振技术，对含有两种β_2m异构体的糖尿病相关TCR/MHC-肽复合体的结构、生化和动力学特征进行表征；(4)用含有两种不同β-2m亚型的MHC-肽复合体进行T细胞识别和TCR停留时间的细胞分析。完成拟议的目标应该能够识别导致β2M依赖的易感性或对疾病的保护的分子和原子决定因素。

DESCRIPTION (provided by applicant): Autoimmune diabetes in both humans and nonobese diabetic (NOD) mice results from T cell-mediated autoimmune destruction of insulin-producing pancreatic beta cells. Both class I major histocompatibility complex (MHC)-restricted and class II MHC-restricted T cells are involved. However, T cell receptors (TCRs) restricted to recognition of autoantigenic peptides presented by the trimeric class I molecule, consisting of MHC heavy chain, beta2-microglobulin (beta2m), and peptide, are absolutely required for the initiation of disease in the NOD mouse. Autoimmune diabetes is a polygenic disease, with particular MHC haplotypes providing the primary genetic component of susceptibility in both humans and NOD mice. Recently, (beta2m) was identified as a diabetes susceptibility gene in NOD mice, the first such gene to be identified that maps outside of the MHC region, lying within the Idd13 locus on Chromosome 2. Two beta2m alleles are widespread throughout the common laboratory mouse strains. When the a2ma allele found in NOD mice is replaced by the a2mb allele, development of diabetes is prevented. The amino acid sequence difference between these two allelic proteins resides in a single exchange of Asp ("a" isoform) for Ala ("b" isoform) at position 85. A number of previous serological and T cell recognition studies, using a variety of MHC allelic products and a2m proteins from different species, have suggested significant conformational flexibility of the class I molecule dependent on the particular beta2m present in the complex. Based on these findings, it can be hypothesized that class I MHC molecules containing beta2ma might exhibit an altered conformation as compared to those containing beta2mb. Such changes could exert effects on both selection of autoreactive T cells and presentation of autoantigenic peptides. The overall goal of this proposal is to test this hypothesis by systematically examining the structural, biochemical, and biological properties of disease-relevant MHC-peptide complexes containing the two beta2m isoforms. Four Specific Aims are proposed: (1) Structural, thermodynamic, and dynamic characterization of the isolated allelic beta2m proteins, using X-ray diffraction analysis, chemical and thermal denaturation, and amide proton exchange; (2) Similar characterization of the diabetes-related MHC/peptide complexes, including measurements of beta2m and peptide exchange rates; (3) Structural, biochemical, and dynamic characterization of the diabetes-related TCR/MHC-peptide complexes containing the two isoforms Of beta2m, using X ray diffraction analysis, amide proton exchange, and surface plasmon resonance; and (4) Cellular analysis of T Cell recognition and TCR dwell time using MHC-peptide complexes containing the two different beta2m isoforms. Completion of the proposed Aims should allow identification of the molecular and atomic determinants that result in beta2m-dependent susceptibility or protection against disease.