Novel mechanisms of TCR quality control

TCR质量控制的新机制

• 批准号: 8303743
• 负责人: Linda M Hendershot
• 金额: $ 7.84万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-07 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8303743
• 关键词: Address; Anabolism; Antibodies; Attention; Biologic Characteristic; Biological Assay; Biological Models; CD3 Antigens; Cell Culture Techniques; Cell Surface Receptors; Cell surface; Cells; Characteristics; Charge; Client; Complex; Data; Development; Endoplasmic Reticulum; Ensure; Evolution; Extracellular Domain; Immune response; Immune system; In Vitro; Individual; Integral Membrane Protein; Jaw; Kinetics; Literature; Measures; Membrane; Methods; Modeling; Molecular; Molecular Chaperones; Monitor; Organism; Pathway interactions; Play; Polyproteins; Process; Property; Proteins; Proteolysis; Quality Control; Receptor Cell; Reporting; Resolution; Role; Site; T-Cell Receptor; Techniques; Transmembrane Domain; Travel; Vertebrates; Work; base; disulfide bond; extracellular; in vivo; insight; interdisciplinary approach; invariant chain; membrane assembly; novel; oxidation; polypeptide; protein folding; receptor; research study; stoichiometry

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.

描述（由申请人提供）： ¿ ¿ T 细胞受体对于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，它由八条多肽链组成，必须以正确的化学计量在 ER 中组装才能发挥其重要功能。因此，尽管有大量关于 ER 的组装和质量控制的文献，但它的组装对 ER 质量控制机器提出了艰巨的任务。 ¿ TCR，这些努力主要集中在单个链的不寻常跨膜结构域上，这些结构域具有带电残基，当未配对时，会加速 ¿ ¿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，很少有人关注该受体的腔部分的可能作用。为了弥补这一缺陷，我们建议将生物物理和细胞研究结合起来，以获得有关 ¿ 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 的两个意想不到的特征 ¿我们发现它的恒定域在没有与 ¿ 关联的情况下是非结构化的。 - 链并且其跨膜区域在单独表达时不会整合到 ER 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，以便获得对受体组装的综合看法。 ER 机制帮助和监测 TCR 生物合成，仅允许正确组装的受体在细胞表面表达。 公共健康相关性：尽管在识别 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中重要功能且通常是药理学靶点的跨膜蛋白尤其如此。我们提出研究来破译必需 T 细胞抗原受体 (TCR) 的组装和质量控制的新方面，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。仔细检查这种八链受体的组装，但其潜在的分子机制尚未得到充分研究，并且其腔域在质量控制中的作用尚未得到充分研究，并且假定的“跨膜质量控制”步骤仍然模糊。质量控制的问题将在我们提出的项目中得到解决，从我们提出的体外和体内技术组合中获得的机制见解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，而目前这种方法是有限的。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转运与降解除了增加我们对 ER 质量控制的一般理解外，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和药物。可能会提供新的方法来操纵它们进行治疗。