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

主要组织相容性复合体(MHC)I类蛋白将细胞蛋白质组的多肽片段转运到细胞膜上，呈递给细胞毒性T细胞，在病毒感染或肿瘤发生异常的情况下，细胞毒性T细胞可以识别非自身多肽并杀死呈递细胞。在细胞表面，MHC I类重链可以失去配位肽和轻链(β-2微球蛋白)，形成非共价缔合，如我们所示。我们用免疫共沉淀法、一种新的抗体微图案化双杂交方法、光漂白后荧光恢复(FRAP)和单分子荧光显微镜对这些‘MHC I类自由重链簇’进行了表征。我们的初步数据表明，I类重链的α-3结构域是相互作用的部位，并表明这些团簇是暂时的。我们假设I类自由重链簇主要是重链的二聚体，它们在细胞表面对I类不同构象的高效内吞分选中发挥作用，其机制迄今尚不清楚。另一个潜在的功能是向T细胞和NK细胞发出信号，这在文献中是有证据的。这项工作的第一个目标是了解哪些MHC同种异型形成MHC自由重链簇，以及如何形成。我们将测试不同的小鼠和人类同种异型的簇形成，并突变和截断它们，以找到相互作用的位置。为了检测簇，我们将使用微图案分析(Springer和Lanzerstorfer组)和FRAP(Lanzerstorfer组)。第二个目标是确定团簇的分子结构和动力学。我们将在我们的合作者Martin Zacharias(慕尼黑技术大学计算生物学)的帮助下在计算机模型中产生，并从重组蛋白质中产生用于X射线结晶学的簇。我们将使用我们的合作者Jacob Piehler(奥斯纳布吕克大学定量单分子显微镜)和Gerhard Schütz(维也纳技术大学人工膜系统)的先进光谱技术来研究活细胞和膜上团簇的大小、动力学和亲和力。第三个目标是描述这些簇的生物学功能。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作用和细胞表面返回率，并将它们与负载多肽(单体)的I类分子进行比较。为了研究它们在信号传递中的作用，我们将用表达簇的细胞刺激报告T细胞和NK细胞，而不是对照细胞。我们还将测试游离重链簇是否在人类白细胞抗原-B*27：05共价二聚体的形成中发挥作用，后者参与炎症性自身免疫性疾病的发病机制。

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.