ER-aminopeptidases: Conformational regulation and antigen presentation function

项目总结/摘要 ERAP 1、ERAP 2和IRAP是M1家族锌氨肽酶，在调节抗原性方面具有重要作用 肽前体用于装载到MHC-I蛋白上。erap 1基因的常见多态性与 对自身免疫性疾病的易感性增加，包括强直性脊柱炎、银屑病、白塞病 糖尿病、鸟射性视网膜病、某些癌症易感性增加和原发性高血压。 多态性残基位于酶活性位点的远端，其作用机制 酶活性是未知的。ERAP 2多态性不太常见，与以下疾病的相关性较弱： 自身免疫性疾病比ERAP 1。关于ER氨基肽酶的关键问题包括它们的作用机制， 作用，特别是独特的长度依赖性切割活性的机制基础， 多态性变异与自身免疫性疾病的联系，以及对ERAP 1的机制性见解的程度 可以扩展到催产素酶亚家族ERAP 2和IRAP的其他成员。一个总体 这一建议的假设是，大规模的构象变化提供了一个机械基础， ER氨肽酶多态性对酶活性和疾病相关性的影响， 构象平衡通过与内质网中其它蛋白质的相互作用来调节。一 拟议研究的具体目的是了解ER氨肽酶结构域之间的相互作用 调节酶的活性。将开发ERAP 1催化机制的详细机理模型， 测试.该模型将肽底物的N-和C-末端附近的ERAP 1结合相互作用与 稳定关键活性位点残基的催化活性构型的大规模结构域闭合运动。 使用已知改变ERAP 1构象动力学的盐桥突变，以及 改变ERAP 1构象平衡，我们将测试疾病相关的多态性是否通过 开放和封闭构象的微分稳定，我们将确定ERAP 2和IRAP是否 类似地利用大规模结构域闭合运动来调节酶活性。第二个具体目标是 确定ERp 44介导的内质网滞留的结构基础和功能后果 ERAP 1和ERAP 2。我们的目标是确定ERp 44与ERAP 1和ERAP 2的复合物的结构， 表征ERp 44相互作用对ERAP 1和ERAP 2加工的影响，并评估ERp 44在ERAP 1和ERAP 2加工中的作用。 ERAP 1-ERp 44在ER应激下产生新表位的相互作用。第三个具体目标是评估 ER分子伴侣tapsin和TAPBPR对ERAP 1修饰表位前体的影响， 与MHC-I结合。 1

PROJECT SUMMARY/ABSTRACT ERAP1, ERAP2, and IRAP are M1-family zinc aminopeptidases with important roles in trimming antigenic peptide precursors for loading onto MHC-I proteins. Common polymorphisms in the erap1 gene are associated with increased susceptibility to autoimmune diseases including ankylosing spondylitis, psoriasis, Behçet's disease, and birdshot retinopathy, increased susceptibility to certain kinds of cancer, and essential hypertension. Polymorphic residues are located distal to the enzyme active site, and the mechanism underlying their effects on enzymatic activity is unknown. ERAP2 polymorphisms are less common and more weakly associated with autoimmune diseases than for ERAP1. Key questions about ER aminopeptidases include their mechanism of action, in particular the mechanistic basis for the unique length-dependent cleavage activity, the nature of the linkage of polymorphic variants with autoimmune disease, and to what degree mechanistic insights about ERAP1 can be extended to the other members of the oxytocinase subfamily ERAP2 and IRAP. An overarching hypothesis of this proposal is that large-scale conformational alterations provide a mechanistic underpinning for the effects of ER aminopeptidase polymorphisms on enzymatic activity and disease association, and that the conformational equilibria are modulated by interactions with other proteins in the endoplasmic reticulum. One specific aim of the proposed research is to understand how interactions between ER aminopeptidase domains regulate enzyme activity. A detailed mechanistic model for ERAP1 catalytic mechanism will be developed and tested. The model couples ERAP1 binding interactions near the N- and C-termini of peptide substrates with large-scale domain closure motions that stabilize the catalytically active configuration of key active site residues. Using salt-bridge mutations known to alter ERAP1 conformational dynamics, and small-molecule inhibitors that alter ERAP1 conformational equilibria, we will test whether disease-associated polymorphisms act through differential stabilization of open and closed conformers, and we will determine whether ERAP2 and IRAP similarly utilize large-scale domain closure motions to regulate enzymatic activity. A second specific aim is to determine the structural basis and functional consequences of ERp44-mediated endoplasmic reticulum retention of ERAP1 and ERAP2. We aim to determine structures of complexes of ERp44 with ERAP1 and ERAP2, to characterize the effect of ERp44 interaction on ERAP1 and ERAP2 processing, and to evaluate the role of ERAP1-ERp44 interactions in generating new epitopes under ER stress. A third specific aim is to evaluate the influence of the ER chaperones tapsin and TAPBPR on ERAP1 trimming of epitope precursors while they are bound to MHC-I. 1