Novel mechanisms of TCR quality control

描述(申请人提供)：T细胞受体对适应性免疫系统的主要功能是必不可少的，是最复杂的细胞表面受体之一。它由八条多肽链组成，必须在内质网中以适当的化学计量比组装，才能发挥其重要功能。因此，它的组装对ER质量控制机构提出了一项艰巨的任务。尽管有大量关于TCR的组装和质量控制的文献，但这些努力主要集中在单个链的不寻常的跨膜域上。它们含有带电残基，当没有配对时，会加速TCR链的降解，并被认为可以驱动组装。然而，这一假定的“整体膜质量控制”步骤的基础还没有被阐明。此外，很少有人注意到这种受体的管腔部分的可能作用，这些部分很可能是已知的内质网质量控制机制仔细检查的区域。为了弥补这一不足，我们建议将生物物理学和基于细胞的研究相结合，以获得与细胞中检查点相关的TCR折叠和组装的高分辨率结构和动力学数据。我们从这些方法获得的初步数据已经揭示了TCR链的两个意想不到的特征。我们发现它的恒定结构域在没有与？链结合的情况下是非结构化的，并且当它单独表达时，它的跨膜区没有整合到内质网中。这两个特征很可能在受体组装的质量控制中提供检查点。这些初步的见解将被扩展，以获得帮助和监测TCR生物合成的内质网机制的综合视图，仅允许正确组装的受体在细胞表面表达。 公共卫生相关性：尽管在确定ER质量控制机制的组成部分方面取得了很大进展，但执行这些机制的基本机制仍然知之甚少。对于控制多细胞生物体中重要功能的跨膜蛋白来说尤其如此，并且通常是药理靶标。我们建议进行研究，以破译基本T细胞抗原受体(TCR)的组装和质量控制方面的新方面，TCR是已知的最复杂的细胞表面受体之一。我们选择TCR是因为众所周知，严格的质量控制检查点可以仔细检查这种八链受体的组装，但其潜在的分子机制尚不清楚。它的管腔结构域在质量控制中的作用还没有得到充分的研究，而且假定的“跨膜质量控制”步骤仍然含糊不清。这两个层面的质量控制将在我们提议的项目中得到解决。从我们提出的体外和体内技术的结合中获得的机制洞察有可能有助于开发更直接的方法来评估细胞中的蛋白质折叠，目前仅限于间接方法，如二硫键的形成，与分子伴侣的相互作用，以及沿着分泌途径的进一步运输而不是降解。除了增加我们对ER质量控制的总体理解外，我们的研究还将为TCR的生物合成提供新的见解，这可能适用于其他免疫受体，并可能提供新的治疗方法来操纵它们。

DESCRIPTION (provided by applicant): The ¿¿T-cell receptor is essential for major functions of the adaptive immune system and is one of the most complex cell surface receptors. It is composed of eight polypeptide chains that must be assembled in the ER in the proper stoichiometry for it to perform its vital functions. As such, its assembly poses a formidable task for the ER quality control machinery. Although a large body of literature exists on the assembly and quality control of the ¿¿TCR, these efforts have focused primarily on the unusual transmembrane domains of the individual chains. These possess charged residues that, when unpaired, accelerate degradation of the ¿¿TCR chains and are believed to drive assembly. However, the basis of this presumed "integral membrane quality control" step has not been elucidated. Furthermore, very little attention has been directed to possible roles for the lumenal portions of this receptor, which are likely to be the regions scrutinized by the known quality control machinery of the ER. To remedy this deficiency, we propose to combine biophysical and cell based studies to obtain high resolution structural and kinetic data on the folding and assembly of the ¿¿TCR that can be correlated with checkpoints in the cell. Our preliminary data obtained from these approaches have already revealed two unanticipated features of the TCR ¿-chain. We find that its constant domain is unstructured in the absence of association with the ¿- chain and that its transmembrane region is not integrated into the ER membrane when expressed alone. These two features are very likely to provide checkpoints in the quality control of receptor assembly. These preliminary insights will be expanded in order to obtain an integrated view of the ER mechanisms that aid and monitor TCR biosynthesis allowing only properly assembled receptors to be expressed on the cell surface. PUBLIC HEALTH RELEVANCE: Although there has been much progress in identifying components of the ER quality control machinery, the underlying mechanisms for executing them remain poorly understood. This is particularly true for transmembrane proteins that control important functions in multicellular organisms and are often pharmacological targets. We propose studies to decipher novel aspects in the assembly and quality control of the essential T cell antigen receptor (TCR), one of the most complex cell surface receptors known. We chose the TCR because strict quality control checkpoints are known to scrutinize the assembly of this eight-chain receptor, yet the underlying molecular mechanisms are not well understood. The role of its lumenal domains in quality control has not been adequately investigated, and a putative "transmembrane quality control" step has remained vague. Both layers of quality control will be addressed within our proposed project. The mechanistic insights gained from our proposed combination of in vitro and in vivo techniques have the potential to contribute to the development of more direct methods for assessing protein folding in the cell, which is currently limited to indirect methods like disulfide bond formation, interactions with molecular chaperones, and further transport along the secretory pathway versus degradation. In addition to increasing our general understanding of ER quality control, our study will provide novel insights into the biosynthesis of TCR that will likely apply to other immunoreceptors and might provide novel ways to manipulate them therapeutically.