Dynamic properties of MHC class II allotypes

主要组织相容性复合体(MHC)的蛋白质在人类群体中以数百种不同的等位基因变体(同种异型)表达。MHC基因位点和相应蛋白变体表达的这种多态性质的结果是，不同个体的免疫肽是不同的，表现致病抗原、自身免疫相关抗原或肿瘤相关抗原的倾向也不同。因此，为了了解想要的和不想要的免疫反应，了解肽选择的关键步骤是必不可少的。对于理解MHC II类分子的抗原提呈，核心是人类白细胞抗原-DM催化的占位符片段(II类相关不变链肽)与高级仿射肽的交换。理解多肽交换率的巨大差异不能仅用静态晶体结构来解释，而需要一种捕捉MHCII-多肽复合体的基本动态特征的方法。在这项建议中，我们的目标是(I)鉴定在对HLA-DM依赖性方面有很大不同的MHC-肽复合体，(Ii)通过核磁共振波谱研究这些MHCII同种异型的动态性质，以及(Iii)确定DM催化的交换的酶参数。这将使我们能够定义决定单个MHCII-肽复合体对人类白细胞抗原-糖尿病易感性的关键氨基酸和分子中间体。在项目的第二部分，最初是独立的部分，我们将描绘(Iv)在肿瘤细胞系的第一部分和(V)部分中研究的MHCII多肽复合体的细胞多肽。最后，将分子机制和细胞学结果进行比较，以确定MHCII动力学对细胞抗原提呈的影响。

Proteins of the Major Histocompatibility Complex (MHC) are expressed as hundreds of different allelic variants (allotypes) across the human population. This polymorphic nature of the MHC gene locus and the corresponding expression of protein variants has the consequence that the immune peptidomes of individuals are distinct, and that the propensities to display pathogenic, autoimmune-related or tumor-associated antigens also differ. For the understanding of wanted and unwanted immune responses it is therefore essential to know the critical steps of peptide selection. Central for an understanding of antigen presentation by MHC class II molecules is the HLA-DM-catalyzed exchange of the placeholder CLIP (class II associated invariant chain peptide) against higher affine peptides. The understanding of the large differences in peptide exchange rates cannot be explained by static crystal structures alone, but requires an approach that captures the essential dynamic features of MHCII-peptide complexes. In this proposal we aim to (i) identify MHC-peptide complexes that strongly differ in their HLA-DM dependency, (ii) investigate the dynamic properties of these MHCII allotypes by NMR spectroscopy and to (iii) determine the enzymatic parameters of DM-catalyzed exchange. This will allow us to define the critical amino acids and molecular intermediates that determine HLA-DM susceptibility of individual MHCII-peptide complexes. In a second, initially independent part of the project we will delineate the cellular peptidomes of (iv) the MHCII peptide complexes investigated in Part 1 and (v) of tumor cell lines. Finally, the molecular mechanistic and cellular findings will be compared to define the impact of MHCII dynamics on cellular antigen presentation.