Mechanism and in vitro reconstitution of tapasin-mediated peptide exchange

Tapasin介导的肽交换的机制和体外重建

• 批准号: 528514500
• 负责人: Professor Dr. Christian Freund
• 金额: --
• 依托单位: Arbeitsgruppe Freund - Protein Biochemistry
• 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/528514500?language=en
• 关键词: Mechanism; vitro; reconstitution; tapasin; mediated

Presentation of antigens to T cells is provided by proteins encoded by the Major Histocompatibility Complex (MHC). The alleles of this highly polymorphic gene locus lead to the expression of thousands of different MHC allotypes across the population. Each allotype is capable of binding to a manifold of different antigenic peptides. When displayed on the cell surface, they can be surveilled by T cells, potentially invoking the cellular arm of adaptive immunity. Of the two major classes of canonical MHC molecules, MHC class I (MHC-I), which are present on all nucleated cells, typically derive their antigens from the proteasome. Peptides of 9-15 amino acid in length after proteasomal cleavage enter the endoplasmic reticulum through the transporter associated with antigen processing, are further trimmed by the aminopeptidase ERAP, and are loaded onto the MHC-I molecules in the peptide loading complex (PLC). At the heart of the PLC, the exchange catalyst tapasin stabilizes the MHC-I molecules and acts to replace lower affine by higher affine peptides, thus influencing the MHC-I displayed immunopeptidomes and ultimately T cell reactivity. The binding and peptide exchange activity of tapasin strongly differs for the individual MHC-I allotypes and raises the question of the mechanism behind allotypic distinction. Here, this central question is first addressed by structural biology and biochemical methods. Several human and rodent MHC-I molecules will be investigated for their binding and exchange susceptibility to tapasin. Furthermore, an in vitro reconstituted system will be developed that comprises essential components of the MHC-I loading complex. Establishing such a minimalistic experimental system allows for the identification of MHC-I bound peptidomes derived from viral proteins or from tumor proteins featuring neo-antigens. The tapasin dependency of MHC-I-peptide display will be measured and combined with the mechanistic insights to derive allotype-specific features. This knowledge can either be used to make individualized predictions of MHC-I presentation of proteins from novel viruses or patient-specific tumor neo-antigens or it will allow to develop tools that can be used to visualize, enrich, or expand antigen-specific T cell clones.

向 T 细胞呈递抗原是由主要组织相容性复合物 (MHC) 编码的蛋白质提供的。这个高度多态性基因位点的等位基因导致人群中数千种不同 MHC 同种异型的表达。每种同种异型都能够结合多种不同的抗原肽。当它们展示在细胞表面时，它们可以受到 T 细胞的监视，从而可能调用适应性免疫的细胞臂。在两类主要的典型 MHC 分子中，I 类 MHC (MHC-I) 存在于所有有核细胞上，通常从蛋白酶体中获得抗原。蛋白酶体裂解后长度为9-15个氨基酸的肽通过与抗原加工相关的转运蛋白进入内质网，被氨肽酶ERAP进一步修剪，并加载到肽加载复合物（PLC）中的MHC-I分子上。在 PLC 的核心，交换催化剂塔帕辛可稳定 MHC-I 分子，并用较高亲和力的肽取代较低亲和力的肽，从而影响 MHC-I 展示的免疫肽组并最终影响 T 细胞反应性。 Tapasin 的结合和肽交换活性对于各个 MHC-I 同种异型有很大差异，并提出了同种异型区别背后的机制问题。在这里，这个核心问题首先通过结构生物学和生化方法得到解决。将研究几种人类和啮齿动物 MHC-I 分子对 tapasin 的结合和交换敏感性。此外，还将开发一种体外重构系统，其中包含 MHC-I 加载复合物的基本成分。建立这样一个简约的实验系统可以识别来自病毒蛋白或具有新抗原特征的肿瘤蛋白的 MHC-I 结合肽组。将测量 MHC-I 肽展示的塔帕辛依赖性，并将其与机制见解相结合，以获得同种异型特异性特征。这些知识可以用于对来自新型病毒或患者特异性肿瘤新抗原的蛋白质的 MHC-I 呈递进行个性化预测，也可以开发用于可视化、富集或扩展抗原特异性 T 细胞克隆的工具。