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，这些多肽链必须以适当的化学计量组装在ER中以执行其重要功能。因此，它的组装对ER质量控制机构来说是一项艰巨的任务。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。这些具有带电残基，当未配对时，加速TCR链的降解，并被认为驱动组装。然而，这一假定的“整体膜质量控制”步骤的基础尚未阐明。此外，很少有人注意到这种受体的内腔部分的可能作用，这可能是由ER的已知质量控制机制仔细检查的区域。为了弥补这一不足，我们建议结合联合收割机生物物理和细胞为基础的研究，以获得高分辨率的结构和动力学数据的折叠和组装的TCR，可以与检查点在细胞中。我们从这些方法中获得的初步数据已经揭示了TCR γ链的两个意想不到的特征。我们发现它的恒定区在不与μ链结合的情况下是非结构化的，并且当单独表达时，它的跨膜区不会整合到ER膜中。这两个特征很可能在受体组装的质量控制中提供检查点。这些初步的见解将扩大，以获得一个综合的观点ER机制，帮助和监测TCR的生物合成，只允许正确组装的受体在细胞表面上表达。 公共卫生相关性：虽然在确定应急反应质量控制机制的组成部分方面取得了很大进展，但对执行这些组成部分的基本机制仍然知之甚少。这对于跨膜蛋白尤其如此，跨膜蛋白控制多细胞生物体中的重要功能并且通常是药理学靶点。我们提出的研究，破译新的方面的装配和质量控制的必要的T细胞抗原受体（TCR），已知的最复杂的细胞表面受体之一。我们选择TCR是因为已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还不清楚。其内腔结构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。这两个层次的质量控制将在我们的拟议项目中得到解决。从我们提出的体外和体内技术相结合的机制的见解有可能有助于开发更直接的方法来评估蛋白质折叠在细胞中，这是目前仅限于间接的方法，如二硫键形成，与分子伴侣的相互作用，并进一步运输沿着分泌途径与降解。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，这些生物合成可能适用于其他免疫受体，并可能提供治疗上操纵它们的新方法。