ER-aminopeptidases: Conformational regulation and antigen presentation function

ER-氨基肽酶：构象调节和抗原呈递功能

• 批准号: 10045425
• 负责人: Lawrence J. Stern
• 金额: $ 50.25万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-07 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10045425
• 关键词: Active Sites; Affect; Aminopeptidase; Ankylosing spondylitis; Antigen Presentation; Autoimmune Diseases; Autoimmunity; Behavior; Behcet Syndrome; Binding; Binding Proteins; Biochemical; Catalysis; Cellular Assay; Chromosome 5; Complex; Couples; Cross Presentation; Cryoelectron Microscopy; Crystallization; Data; Dependence; Development; Disease; Distal; ERAP1 gene; Endoplasmic Reticulum; Enzymatic Biochemistry; Enzymes; Epitopes; Equilibrium; Essential Hypertension; Estrogen receptor positive; Family; Family member; Gene Cluster; Genes; Genetic Polymorphism; Human; Human Chromosomes; Hypertension; Immune response; Infectious Agent; Inflammation; Interferon Type II; Length; Malignant Neoplasms; Mediating; Modeling; Molecular Chaperones; Molecular Conformation; Motion; Mutation; Nature; Peptides; Population; Positioning Attribute; Pre-Eclampsia; Predisposition; Proteins; Psoriasis; Regulation; Research; Retinal Diseases; Risk; Role; Shapes; Sodium Chloride; Solvents; Specificity; Structure; Testing; Variant; Virus; Work; Zinc; antigen processing; base; conformational alteration; conformer; endoplasmic reticulum stress; enzyme activity; genetic association; genetic variant; in vivo; inhibitor/antagonist; insight; member; paralogous gene; small molecule inhibitor; tapasin; therapeutic development; tool; tumor

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

项目概要/摘要 ERAP1、ERAP2 和 IRAP 是 M1 家族锌氨基肽酶，在修剪抗原中发挥重要作用 用于加载 MHC-I 蛋白的肽前体与 erap1 基因中常见的多态性相关。 对自身免疫性疾病的易感性增加，包括强直性脊柱炎、牛皮癣、白塞氏病 疾病和鸟热性视网膜病，对某些癌症的易感性增加，以及原发性高血压。 多态性残基位于酶活性位点的远端，及其作用机制 ERAP2 多态性对酶活性的影响不太常见，并且与酶活性的相关性更弱。 ER 氨肽酶的关键问题包括其作用机制。 作用，特别是独特的长度依赖性裂解活性的机制基础， 多态性变异与自身免疫性疾病的联系，以及对 ERAP1 机制的了解程度 可以扩展到催产素酶亚家族 ERAP2 和 IRAP 的其他成员。 该提议的假设是大规模的构象改变为 ER氨肽酶多态性对酶活性和疾病关联的影响，以及 构象平衡通过与内质网中其他蛋白质的相互作用来调节。 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 将开发和调节酶活性的 ERAP1 催化机制的详细机制模型。 该模型将肽底物 N 端和 C 端附近的 ERAP1 结合相互作用与 大规模结构域闭合运动可稳定关键活性位点残基的催化活性构型。 使用已知会改变 ERAP1 构象动力学的盐桥突变和小分子抑制剂 改变 ERAP1 构象平衡，我们将测试与疾病相关的多态性是否通过 开放和封闭构象异构体的稳定性差异，我们将确定 ERAP2 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。 确定 ERp44 介导的内质网保留的结构基础和功能后果 我们的目标是确定 ERp44 与 ERAP1 和 ERAP2 的复合物的结构。 表征 ERp44 相互作用对 ERAP1 和 ERAP2 加工的影响，并评估 ERAP1-ERp44 相互作用在 ER 应激下生成新表位第三个具体目标是评估 ER 伴侣 Tapsin 和 TAPBPR 对表位前体 ERAP1 修剪的影响 与 MHC-I 结合。 1