Structural and Functional Studies of Teneurins: A bacterial toxin homolog in human

Teneurins 的结构和功能研究：人类细菌毒素同系物

• 批准号: 10675259
• 负责人: Demet Arac-Ozkan
• 金额: $ 8.41万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10675259
• 关键词: Adhesions; Affinity; Alternative Splicing; Amino Acids; Architecture; Bacterial Toxins; Binding; Biochemical; Biological; Biological Assay; Biophysics; Brain; C-terminal; Cell Adhesion; Cell Adhesion Molecules; Cell Aggregation; Cell physiology; Cell surface; Cells; Cellular biology; Communication; Complement; Complex; Coupling; Cryoelectron Microscopy; Cues; Cyclic AMP; Data; Dependence; Development; Dimerization; Disease; Electron Microscopy; Embryonic Development; Encapsulated; Excitatory Synapse; Exhibits; Family; G-Protein-Coupled Receptors; Goals; Homodimerization; Homologous Gene; Human; Immunoglobulin Domain; Inhibitory Synapse; Integral Membrane Protein; Knowledge; Laboratories; Lasers; Ligands; Link; Mechanics; Mediating; Membrane Proteins; Methods; Molecular; Monitor; Mutagenesis; Mutate; Negative Staining; Nervous system structure; Neurons; Organ; Organism; Physiological; Play; Protein Family; Protein Isoforms; Proteins; RNA Splicing; Reporting; Research; Resolution; Role; Rosaniline Dyes; Sequence Analysis; Signal Transduction; Site; Structure; Structure-Activity Relationship; Surface; Synapses; System; Testing; Tissues; Toxin; Variant; Visualization; alpha Toxin; alpha-latrotoxin receptor; analytical ultracentrifugation; anticancer research; axon guidance; base; cardiogenesis; crosslink; experimental study; extracellular; follow-up; human disease; in vivo; insight; intercellular communication; interdisciplinary approach; light scattering; malignant neurologic neoplasms; member; mutant; nervous system disorder; neural circuit; novel; novel strategies; protein function; relating to nervous system; success; synaptic function; synaptogenesis

Project Summary The interplay between cellular adhesion and cellular signaling is essential for the development of all organs such as the brain, and for the functioning of systems such as the nervous systems. Teneurins (TEN1-4) are a poorly understood family that mediates intercellular communication. They have essential roles in embryonic development and neural circuit-wiring; and are linked to numerous human diseases including neurological disorders and cancers. TENs are type-II membrane proteins with large C-terminal extracellular regions (ECR) that majorly exhibit no identifiable domains. The ECR mediates trans-cellular heterophilic interaction of TENs with Latrophilins(LPHN1-3), a family of G-Protein Coupled Receptors; to regulate synapse function. The ECR also mediates trans-cellular homophilic interaction of TENs with themselves to instruct neural circuit-wiring. However, the molecular mechanisms underlying TEN action remains poorly understood majorly due to the lack of structural information on the ECR. We recently laid the groundwork by determining the high-resolution cryo-EM structure of the TEN2 ECR and revealed a surprising homology to bacterial Tc-toxins. We also showed that an alternatively spliced insert acts as a switch to regulate LPHN binding and other TEN functions such as synapse formation. The ultimate goal of the research proposed in this application is to understand the mechanical details of various TEN functions that are mediated by its ECR. We propose three Specific Aims that are based on the major unknowns in TEN function and a Follow-up Aim to perform structure/function relationship studies: First, we aim to understand the molecular details of the TEN/LPHN interaction. Second, we aim to understand the molecular determinants for the trans-homodimerization of TEN. Third, we aim to reveal whether TEN functions via autoproteolysis similar to bacterial toxins. Then, we aim to use the information from the first three aims to study TEN function in synapse formation assays. This research has a multi-disciplinary approach where the structural and functional data performed in the PI's lab range from electron microscopy, biophysical and biochemical methods, neuronal assays to cell-biology and is complemented by the expertise provided or performed by the laboratories of close collaborators. The proposed experiments will build on exciting results, including the very unusual TEN2 structure, surprising involvement of alternative splicing in TEN function, key advances in the purification of all needed TEN fragments, and the observation of proteolytic products. We expect that this research will provide critical insights into the mechanistic details of TEN function, helping to establish novel principles on intercellular communication that are vital for numerous cellular functions.

项目概要 细胞粘附和细胞信号传导之间的相互作用对于所有器官的发育至关重要 例如大脑，以及神经系统等系统的功能。 Teneurins (TEN1-4) 是一种 人们对介导细胞间通讯的家族知之甚少。它们在胚胎发育过程中发挥着重要作用 发育和神经回路布线；并与许多人类疾病有关，包括神经系统疾病 疾病和癌症。 TEN 是具有大 C 端胞外区域 (ECR) 的 II 型膜蛋白 主要表现出没有可识别的域。 ECR 介导 TEN 的跨细胞异嗜性相互作用 与 Latrophilins (LPHN1-3) 一起，G 蛋白偶联受体家族；调节突触功能。欧洲研究报告 还介导 TEN 与其自身的跨细胞同质相互作用，以指导神经回路布线。 然而，TEN 作用的分子机制仍然知之甚少，主要是由于 缺乏 ECR 的结构信息。我们最近通过确定高分辨率奠定了基础 TEN2 ECR 的冷冻电镜结构揭示了与细菌 Tc 毒素的惊人同源性。我们也 表明选择性剪接插入片段充当调节 LPHN 结合和其他 TEN 功能的开关 例如突触的形成。本申请中提出的研究的最终目标是了解 由其 ECR 介导的各种 10 个功能的机械细节。我们提出三个具体目标 基于 TEN 功能的主要未知数和执行结构/功能的后续目标 关系研究：首先，我们的目标是了解 TEN/LPHN 相互作用的分子细节。第二， 我们的目标是了解 TEN 反式同二聚化的分子决定因素。第三，我们的目标是 揭示 TEN 是否通过类似于细菌毒素的自蛋白水解作用发挥作用。然后，我们的目标是使用 前三个信息旨在研究突触形成测定中的十个功能。这项研究有一个 多学科方法，其中 PI 实验室执行的结构和功能数据范围从 电子显微镜、生物物理和生化方法、细胞生物学的神经元测定 并由密切合作者的实验室提供或执行的专业知识进行补充。拟议的 实验将建立在令人兴奋的结果之上，包括非常不寻常的 TEN2 结构、令人惊讶的参与 TEN 功能中选择性剪接的重要性、纯化所有所需 TEN 片段的关键进展以及 观察蛋白水解产物。我们期望这项研究将为以下问题提供重要见解： 十个功能的机制细节，有助于建立细胞间通讯的新原理 对于许多细胞功能至关重要。