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

来自父R01的项目摘要 细胞黏附和细胞信号之间的相互作用对所有器官的发育都是必不可少的。 例如大脑，以及神经系统等系统的功能。Teneurins(TEN1-4)是一种 调节细胞间通讯的鲜为人知的家族。它们在胚胎发育过程中起着至关重要的作用 发育和神经回路连接；并与许多人类疾病有关，包括神经学 疾病和癌症。TENS是具有较大C末端胞外区(ECR)的II型膜蛋白 主要表现为没有可识别的领域。ECR介导TENS的跨细胞异嗜性相互作用 与拉特罗菲林(LPHN1-3)，一个家族的G蛋白偶联受体；调节突触功能。《欧洲经济报告》 也调节TEN与自身的跨细胞同嗜性相互作用，以指导神经回路连接。 然而，十种作用背后的分子机制主要是由于 缺乏关于ECR的结构性信息。我们最近通过确定高分辨率 Ten2 ECR的低温EM结构，并揭示了与细菌TC毒素令人惊讶的同源性。我们也 显示了选择性剪接插入物作为开关来调节LPHN结合和其他十种功能 比如突触的形成。本应用程序中提出的研究的最终目标是理解 由其ECR调解的各种十种功能的机械细节。我们提出了三个具体目标， 是基于十项功能中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反式均二聚的分子决定因素。第三，我们的目标是 揭示TEN是否通过类似于细菌毒素的自我蛋白分解发挥作用。然后，我们的目标是使用 来自前三项的信息旨在研究突触形成分析中的十项功能。这项研究有一个 多学科方法，在PI的实验室中执行的结构和功能数据范围为 电子显微镜，生物物理和生化方法，细胞生物学的神经元分析和IS 并辅之以密切合作者的实验室提供或执行的专门知识。建议数 实验将建立在令人兴奋的结果上，包括非常不寻常的TEN2结构，令人惊讶的参与 10个功能的选择性剪接，在提纯所有需要的10个片段方面的关键进展，以及 蛋白分解产物的观察。我们预计，这项研究将为 十种功能的机械细节，有助于建立关于细胞间通信的新原理， 对许多细胞功能都至关重要。

Project Summary from the Parent R01 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.