Understanding ERAP molecular mechanism of MHC I antigen processing

摘要 在过去的十年中，对第I类抗原提呈途径的表征方面的进展告诉我们 我们知道，抗原处理、肽转运、肽修剪、肽选择和肽负载是 主要组织相容性呈现的最佳多肽谱系开发的关键事件 复合体(MHC)I类分子对细胞毒性T淋巴细胞(CTL)的作用。这些事件影响了紧密的捆绑 产生构象稳定的MHC I/多肽复合体所需的多肽。有证据表明 假设MCH I多肽库的整体质量的变化可以对CTL产生深远的影响 回应。因此，表征MHC I成熟过程中的基本生物学事件的研究是必要的 不仅是为了抗原处理和呈递的基本原理的进步，而且也是为了更好地 了解这些成熟事件是如何调节免疫反应的。 我们研究了多肽负载和选择机制的分子和结构基础。 MHC I已经有20多年了。近年来，与抗原相关的内质网氨基肽酶 加工(ERAP1、ERAP2和ERAP1，2异二聚体；称为ERAP)已成为关键蛋白质 以影响MHC I多肽库的形成。到目前为止，我们对ERAP如何发挥作用的理解 然而，多肽编辑器却鲜为人知。同样，ERAP1和ERAP2的多态如何在 在慢性自身免疫性疾病中的作用，如与人类白细胞抗原-B*27相关的强直性脊柱炎(AS)，以及如何 ERAP1和ERAP2与疾病相关的MHC I分子相互作用仍不清楚。在此应用程序中， 我们将使用一种综合的方法来阐明ERAP的功能和分子 ERAP和MHC I分子之间的串扰。使用由ERAP组成的无细胞系统，MHC I类 分子，以及合成的和天然的N末端延伸的多肽，并结合生物化学， 分子和结晶学技术，我们将表征ERAP对FREE和MHC的作用 I结合肽(目标1)；阐明ERAP1、HLA-B*27和AS之间的功能联系(目标2)；以及 确定具有和不具有ERAP1的人类白细胞抗原-B*0801/前体复合体的X射线晶体结构(目标3)。 总体而言，我们的研究具有根本意义，因为它将提供新的知识，以更好地理解 ERAP如何发挥作用并影响MHC I肽谱的形成，对分子的新见解 AS发病机制的基础事件，并将揭示ERAP1和MHC之间的分子相互作用 I分子。我们的研究具有巨大的医学意义。

ABSTRACT Advances in characterization of the class I antigen presentation pathway over the last decade have taught us that antigen processing, peptide transport, peptide trimming, peptide selection, and peptide loading are critical events for the development of optimal peptide repertoires presented by major histocompatibility complex (MHC) class I molecules to cytotoxic T lymphocytes (CTLs). These events influence the tight binding of peptides needed to generate conformationally stable MHC I/peptide complexes. Evidence has been provided that changes in the overall quality of the MCH I peptide repertoire can have profound effects on CTL responses. Therefore, studies that characterize basic biological events in MHC I maturation are essential not only for advances in fundamental principles of antigen processing and presentation, but also to better comprehend how these maturation events modulates immune responses. We have studied the molecular and structural basis of mechanisms of peptide loading and selection by MHC I for more than 20 years. Recently, the endoplasmic reticulum-aminopeptidases associated with antigen processing (ERAP1, ERAP2, and ERAP1,2 heterodimer; referred to as ERAP) have emerged as key proteins for influencing formation of the MHC I peptide repertoire. To date, our understanding of how ERAP functions as a peptide editor is however obscure. Similarly, the question of how ERAP1 and ERAP2 polymorphisms play a role in chronic autoimmune diseases such as HLA-B*27-associated ankylosing spondylitis (AS), and how ERAP1 and ERAP2 interact with disease-associated MHC I molecules, still remain unclear. In this application, we will use a comprehensive approach to shed some light on the function of ERAPs and on the molecular crosstalk between ERAPs and MHC I molecules. Using a cell-free system composed of ERAPs, MHC class I molecules, and synthetic and natural N-terminally extended peptides, and in combination with biochemical, molecular, and crystallographic techniques, we will characterize the function of ERAPs towards free and MHC I-bound peptides (Aim #1); elucidate the functional links between ERAP1, HLA-B*27, and AS (Aim #2); and determine the x-ray crystal structure of an HLA-B*0801/precursor complex, with and without ERAP1 (Aim #3). Overall, our study is fundamentally significant because it will provide new knowledge to better understand how ERAPs function and influence formation of the MHC I peptide repertoire, novel insights into molecular events underlying the pathogenesis of AS, and will reveal the molecular interaction between ERAP1 and MHC I molecules. The medical relevance of our studies is immense.