ER-aminopeptidases: Conformational regulation and antigen presentation function

项目摘要/摘要 ERAP1、ERAP2和IRAP是M1家族的锌氨肽酶，具有重要的抗原性修剪作用 用于装载到MHC-I蛋白上的多肽前体。ERAP1基因常见的多态是相关的 自身免疫性疾病的易感性增加，包括强直性脊柱炎、牛皮癣、Behçet‘s 疾病，鸟弹视网膜病，增加对某些类型癌症的易感性，以及高血压。 多态残基位于酶活性部位的远端，以及它们作用的机制 对酶活性的影响尚不清楚。ERAP2基因多态不太常见，与 自身免疫性疾病多于ERAP1。内质网氨基肽酶的关键问题包括其作用机制 作用，特别是独特的依赖于长度的切割活动的机制基础， 多态变异与自身免疫性疾病的联系，以及对ERAP1的机制洞察到什么程度 可以延伸到催产素酶亚家族ERAP2和IRAP的其他成员。最重要的是 这一提议的假设是，大范围的构象变化为 内质网氨基肽酶基因多态性对酶活性和疾病关联性的影响 构象平衡受到与内质网中其他蛋白质相互作用的调节。一 拟议研究的具体目的是了解内质网氨基肽酶结构域之间的相互作用 调节酶的活性。将开发一个详细的ERAP1催化机理模型，并 测试过。该模型将多肽底物N-末端和C-末端附近的ERAP1结合作用与 稳定关键活性中心残基的催化活性构型的大规模结构域闭合运动。 使用已知的改变ERAP1构象动力学的盐桥突变和小分子抑制剂 在ERAP1构象平衡之后，我们将测试疾病相关的多态是否通过 开放和闭合构象的微分稳定化，我们将确定ERAP2和IRAP 同样，利用大规模的结构域闭合运动来调节酶的活性。第二个具体目标是 确定ERp44介导的内质网滞留的结构基础和功能后果 ERAP1和ERAP2。我们的目标是确定ERp44与ERAP1和ERAP2的复合物的结构，以 鉴定ERp44相互作用对ERAP1和ERAP2加工的影响，并评估ERAP44的作用 ERAP1-ERp44在内质网应激下产生新表位的相互作用。第三个具体目标是评估 内质网伴侣蛋白Tapsin和TAPBPR对表位前体ERAP1修剪的影响 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