Probing the structure and function of the intracellular domain of cys-loop receptors

探讨cys环受体胞内结构域的结构和功能

基本信息

批准号：
10363881
负责人：
Michaela Jansen
金额：
$ 38.25万
依托单位：
TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-12-15 至 2025-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10363881
关键词：
Address Alzheimer&apos s Disease Amino Acids Anesthetics Anti-Anxiety Agents Antiemetics Antiepileptic Agents Anxiety Anxiety Disorders Architecture Atherosclerosis Attention Binding Biochemical Biological Assay Cations Cause of Death Cell Line Cell membrane Characteristics Chimera organism Choline Cholinergic Receptors Clinical Coupled Cryoelectron Microscopy Dementia Diabetes Mellitus Disease Drug Design Drug Targeting Electrophysiology (science)Environment Epilepsy Eukaryota Extracellular Domain Family Family member Foundations Funding Future Glycine Heart Diseases Homo Human Immobilization Inflammatory Inflammatory Bowel Diseases Investigation Ion Channel Ion Channel Gating Knowledge Length Ligands Link Mediating Membrane Transport Proteins Mental Depression Methods Modification Molecular Molecular Chaperones Molecular Conformation Muscle relaxants Myasthenia Gravis Nerve Degeneration Neurologic Neurotransmitter Receptor Nicotine Dependence Pain Parkinson Disease Peptides Pharmaceutical Preparations Pharmacology Plant Resins Proteins Publishing Resistance Role Schizophrenia Sepsis Serotonin Shapes Sodium Chloride Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Structure Surface Tertiary Protein Structure Testing Transmembrane Domain Western Blotting base brain tissue burden of illness clinical effect conformational conversion design disability esterase experimental study gamma-Aminobutyric Acid inhibitor innovation insight maltose-binding protein nervous system disorder neuropsychiatric disorder neuropsychiatry novel overexpression prevent protein protein interaction receptor smoking cessation targeted treatment therapeutic development therapy design

项目摘要

Project Summary Pentameric ligand-gated ion channels (pLGICs), also called Cys-loop receptors in eukaryotes, include the receptors for acetylcholine, serotonin, GABA and glycine. They are involved in numerous neuropsychiatric, neurologic, and inflammatory diseases. All Cys-loop receptor family members in metazoans contain three structural domains: an extracellular domain (ECD), a transmembrane domain (TMD), and an intracellular domain (ICD). The ECD and TMD are architecturally conserved between receptor families. The ICD is poorly conserved in both length and amino-acid composition and, for many subunits, contains large regions of predicted structural disorder. Due to the challenge of working with the ICDs of pentameric receptors, they have been essentially overlooked in terms of rigorous mechanistic characterization and direct exploration as pharmacological targets. The ICD is involved in finetuning plasma membrane expression levels, targeting, and function, in part mediated by protein-protein interactions (PPI) for example with chaperones. We envision that mechanistic knowledge of chaperone-mediated modulation will uncover new PPI drug targets. Through our published studies of the ICD we defined a linker that allowed for structure determination of the first in class structures of homo- and heteropentameric GABAA receptors. We also established that the ICD alone assembles into pentamers, establishing a novel role for the ICD in oligomeric assembly. Here, we propose a multi-layered approach leveraging both soluble ICD chimeras and full-length receptors together with results obtained during the previous funding period in careful consideration of recently-published pLGIC structures. We discovered that the resistance to inhibitors of choline esterase (Ric-3) chaperone binds to a 24-amino acid L1-MX segment of 5-HT3A subunits. With the new proposed specific aims (SA) we will: (SA1) determine the role of the L1-MX segment in RIC-3 modulation of cationic pLGIC assembly, (SA2) identify novel protein-protein interactions mediated via cationic ICDs, and (SA3) characterize the mechanism by which opening of cation-conducting pLGICs involves translocation of the L1-MX segment through the MA-helix framed portals. In each aim, we will use biochemical and electrophysiological methods, coupled with overexpressed and purified proteins or proteins in their cellular environment. We anticipate that our studies will provide the basis for a novel class of targets for therapeutic development, PPI modulators for pentameric channel intracellular domains.

项目概要五聚体配体门控离子通道 (pLGIC)，在真核生物中也称为 Cys 环受体，包括乙酰胆碱、血清素、GABA 和甘氨酸受体。他们参与了许多神经精神学、神经系统疾病和炎症疾病。后生动物中的所有 Cys 环受体家族成员都含有三个结构域：胞外域（ECD）、跨膜域（TMD）和胞内域（ICD）。 ECD 和 TMD 在受体家族之间在结构上是保守的。 ICD 保存较差在长度和氨基酸组成上，对于许多亚基来说，包含大面积的预测结构区域紊乱。由于使用五聚体受体 ICD 的挑战，它们本质上是在严格的机制表征和作为药理学目标的直接探索方面被忽视。 ICD 参与微调质膜表达水平、靶向和功能，部分介导通过蛋白质-蛋白质相互作用（PPI），例如与伴侣的相互作用。我们设想机械知识伴侣介导的调节将发现新的 PPI 药物靶点。通过我们发表的 ICD 研究我们定义了一个连接器，可以确定同源和同源类结构中的第一个结构异五聚体 GABAA 受体。我们还确定 ICD 单独组装成五聚体，确立了 ICD 在寡聚体组装中的新作用。在这里，我们提出了一种多层方法利用可溶性 ICD 嵌合体和全长受体以及在之前的资助期仔细考虑了最近发布的 pLGIC 结构。我们发现对胆碱酯酶 (Ric-3) 抑制剂的抗性分子伴侣与 5-HT3A 的 24 个氨基酸 L1-MX 片段结合亚单位。根据新提出的具体目标 (SA)，我们将： (SA1) 确定 L1-MX 部分在 RIC-3 调节阳离子 pLGIC 组装，(SA2) 鉴定通过介导的新型蛋白质-蛋白质相互作用阳离子ICD，和（SA3）表征了阳离子传导pLGIC的打开涉及的机制 L1-MX 段通过 MA 螺旋框架门户易位。在每个目标中，我们都会使用生化和电生理学方法，加上过表达和纯化的蛋白质或细胞中的蛋白质环境。我们预计我们的研究将为一类新型治疗靶点提供基础开发五聚体通道胞内结构域的 PPI 调节剂。