Novel mechanisms of TCR quality control

描述（由申请人提供）：t细胞受体对适应性免疫系统的主要功能至关重要，是最复杂的细胞表面受体之一。它由8个多肽链组成，必须在内质网中以适当的化学计量量进行组装，以发挥其重要功能。因此，它的组装对ER质量控制机械提出了一个艰巨的任务。尽管存在大量关于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结构域上。它们具有带电残基，当不配对时，加速TCR链的降解，并被认为是驱动组装。然而，这种假定的“整体膜质量控制”步骤的基础尚未阐明。此外，很少有人关注这种受体的管腔部分的可能作用，这可能是由已知的内质网质量控制机制仔细检查的区域。为了弥补这一缺陷，我们建议结合生物物理学和基于细胞的研究来获得关于TCR折叠和组装的高分辨率结构和动力学数据，这些数据可以与细胞中的检查点相关。我们从这些方法中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，在没有与¿-链结合的情况下，其恒定结构域是非结构化的，并且当单独表达时，其跨膜区域不整合到内质网膜中。这两个特征很可能为受体组装的质量控制提供检查点。这些初步的见解将被扩展，以获得内质网机制的综合观点，该机制有助于和监测TCR生物合成，仅允许适当组装的受体在细胞表面表达。

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.