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）小鼠中的自身免疫性糖尿病均由T细胞介导的产生胰岛素的胰腺β细胞的自身免疫性破坏引起。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细胞都参与其中。然而，T细胞受体（TCR）限于识别由三聚体I类分子呈递的自身抗原肽，所述三聚体I类分子由MHC重链、β 2-微球蛋白（β 2 m）和肽组成，这是NOD小鼠中疾病起始所绝对需要的。自身免疫性糖尿病是一种多基因疾病，特定的MHC单倍型提供了人类和NOD小鼠易感性的主要遗传成分。最近，（beta2 m）被确定为NOD小鼠中的糖尿病易感基因，这是第一个被确定的在MHC区域之外的基因，位于染色体2上的Idd 13基因座内。两个β 2 m等位基因在常见的实验室小鼠品系中广泛分布。当NOD小鼠中发现的a2 ma等位基因被a2 mb等位基因取代时，糖尿病的发展被阻止。这两种等位基因蛋白质之间的氨基酸序列差异在于位置85处的Asp（“a”同种型）与Ala（“B”同种型）的单一交换。许多以前的血清学和T细胞识别研究，使用多种MHC等位基因产物和来自不同物种的α 2 m蛋白，已经表明依赖于复合物中存在的特定β 2 m的I类分子的显著构象灵活性。基于这些发现，可以假设与含有β 2 mb的那些相比，含有β 2 ma的I类MHC分子可能表现出改变的构象。这种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。该提案的总体目标是通过系统地检查含有两种β 2 m亚型的疾病相关MHC-肽复合物的结构、生化和生物学特性来验证这一假设。提出了四个具体目标：（1）分离的等位基因β 2 m蛋白的结构，热力学和动力学表征，使用X射线衍射分析，化学和热变性，和酰胺质子交换：（2）类似的糖尿病相关的MHC/肽复合物的表征，包括β 2 m和肽交换率的测量;（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对含有β 2 m两种异构体的糖尿病相关TCR/MHC-肽复合物进行结构、生化和动力学表征;和（4）使用含有两种不同β 2 m同种型的MHC-肽复合物对T细胞识别和TCR停留时间的细胞分析。完成拟定的目标后，应能够确定导致β 2 m依赖性易感性或疾病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.