Cell surface clusters of MHC class I molecules: origin, structure, and functions

MHC I 类分子的细胞表面簇：起源、结构和功能

• 批准号: 447012451
• 负责人: Professor Dr. Sebastian Springer
• 金额: --
• 依托单位: Department of Life Sciences and Chemistry
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/447012451?language=en
• 关键词: Cell; surface; clusters; MHC; class

MHC (major histocompatibility complex) class I proteins transport peptide fragments of the cellular proteome to the plasma membrane to present them to cytotoxic T cells, which can recognize non-self peptides and kill the presenting cells in case of virus infection or tumorigenic aberration. At the cell surface, MHC class I heavy chains can lose the ligand peptide and the light chain (beta-2 microglobulin) and form non-covalent associations, as we have shown. We have characterized these 'MHC class I free heavy chain clusters' with co-immunoprecipitation, with a novel antibody micropatterning two-hybrid assay, with fluorescence recovery after photobleaching (FRAP), and with single-molecule fluorescence microscopy. Our preliminary data point to the alpha-3 domain of the class I heavy chain as the interaction site and suggests that the clusters are transient. We hypothesize that the class I free heavy chain clusters are mostly dimers of heavy chains, and that they play a role in the highly efficient endocytic sorting of the different conformations of class I at the cell surface, the mechanism of which is so far unknown. Another potential function is the signaling to T cells and NK cells, for which there is evidence in the literature. The first objective of this work is to understand which MHC allotypes form MHC free heavy chain clusters, and how. We will test different murine and human allotypes for cluster formation and mutagenize and truncate them to find the interaction site. To detect the clusters, we will use the micropatterning assay (Springer and Lanzerstorfer groups) and FRAP (Lanzerstorfer group). The second objective is to establish the molecular structure and dynamics of the clusters. We will generate in silico models with the help of our collaborator Martin Zacharias (Technical University of Munich, Computational Biology) and produce the clusters from recombinant proteins for X-ray crystallography. We'll use the advanced spectroscopy techniques of our collaborators Jacob Piehler (Osnabrück University, for quantitative single-molecule microscopy) and Gerhard Schütz (Vienna Technical University, for artificial membrane systems) to investigate the size, dynamics, and affinity of the clusters in live cells and on membranes. The third objective is to characterize the biological function of the clusters. To investigate their role in class I endocytic sorting, we will monitor the rate of endocytosis and cell surface return of the clusters and compare them with peptide-loaded (monomeric) class I molecules. To investigate their role in signaling, we will stimulate reporter T and NK cells with cells expressing clusters, vs. control cells. We will also test whether free heavy chain clusters plays a role in the formation of covalent dimers of HLA-B*27:05, which are involved in the pathogenesis of inflammatory autoimmune diseases.

MHC（主要组织相容性复合体）I类蛋白将细胞蛋白质组的肽片段转运到质膜，呈递给细胞毒性T细胞，在病毒感染或致瘤性畸变的情况下，细胞毒性T细胞可以识别非自身肽并杀死呈递给细胞的细胞。在细胞表面，MHC I类重链可以失去配体肽和轻链（β -2微球蛋白）并形成非共价结合，正如我们所示。我们用共免疫沉淀、新型抗体微图双杂交试验、光漂白后荧光恢复（FRAP）和单分子荧光显微镜对这些“MHC I类游离重链团簇”进行了表征。我们的初步数据指向I类重链的α -3结构域作为相互作用位点，并表明簇是短暂的。我们假设I类自由重链簇主要是重链二聚体，它们在细胞表面对I类不同构象的高效内吞分选中起作用，其机制迄今尚不清楚。另一个潜在的功能是对T细胞和NK细胞的信号传导，这在文献中有证据。这项工作的第一个目标是了解哪些MHC异体形成MHC无重链簇，以及如何形成。我们将测试不同的小鼠和人类同种异体的簇形成，并对它们进行诱变和截断以找到相互作用位点。为了检测簇，我们将使用微图分析（施普林格组和Lanzerstorfer组）和FRAP （Lanzerstorfer组）。第二个目标是建立簇的分子结构和动力学。我们将在合作者Martin Zacharias（慕尼黑工业大学计算生物学专业）的帮助下生成计算机模型，并从重组蛋白中生成用于x射线晶体学的簇。我们将使用我们的合作者Jacob Piehler （osnabrck大学，用于定量单分子显微镜）和Gerhard sch<e:1> tz（维也纳技术大学，用于人工膜系统）的先进光谱技术来研究活细胞和膜上簇的大小、动力学和亲和力。第三个目标是描述集群的生物学功能。为了研究它们在I类内吞分选中的作用，我们将监测这些簇的内吞率和细胞表面返回率，并将它们与肽负载（单体）I类分子进行比较。为了研究它们在信号传导中的作用，我们将用表达簇的细胞刺激报告细胞T和NK细胞，而不是对照细胞。我们还将测试游离重链簇是否在HLA-B*27:05共价二聚体的形成中发挥作用，这参与了炎症性自身免疫性疾病的发病机制。