Mechanism of G protein Activation by Ric-8A - competitive revision of R01GM105993

Ric-8A 激活 G 蛋白的机制 - R01GM105993 的竞争性修订版

• 批准号: 8960270
• 负责人: Stephen R Sprang
• 金额: $ 12.14万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8960270
• 关键词: Active Sites; Address; Affect; Antibodies; Attenuated; Behavior; Binding; Binding Sites; Cell division; Cell membrane; Cells; Collaborations; Complex; Coupled; Crystallization; Cytoplasm; Deuterium; Disease; Electric Conductivity; Electrons; Embryonic Development; Energy Transfer; Epitopes; Eukaryota; Event; Exhibits; Family; Funding; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Genetic Transcription; Goals; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Guanosine Triphosphate; Heterogeneity; Heterotrimeric GTP-Binding Proteins; Human; Human Development; Hydrogen; Investigation; Knowledge; Laboratories; Life; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Medicine; Membrane; Metabolism; Molecular; Molecular Chaperones; Molecular Conformation; Nucleotides; Parents; Physiological Processes; Play; Process; Protein Dynamics; Proteins; Reaction; Research; Role; Site; Spectrum Analysis; Structure; Testing; Work; X-Ray Crystallography; biophysical techniques; cell motility; in vivo; insight; nanobodies; protein activation; protein function; public health relevance; research study; scaffold; single molecule

 DESCRIPTION (provided by applicant): In this competitive revision of R01GM105993, a new aim is introduced to use camelid variable heavy chain antibody domains (nanobodies) as structural scaffolds and functional probes to investigate the interaction between Ric-8A and Gai1. R01GM105993 funds research to understand how G proteins are activated in the cytoplasm of the cell by a protein factor called Ric-8A. Heterotrimeric G proteins modulate cell metabolism, secretion, electrical conductivity, gene transcription, cell division and cellular motility, and therefore are essential to life in the domain of eukaryotes to which humans belong. Misregulation of G proteins is associated with cancer and a range of other diseases of relevance to general medicine. While most processes controlled by heterotrimeric G proteins occur at cell membranes, recent research has shown that G alpha subunits (Ga) also control certain events in cell cytoplasm. Important among these is asymmetric cell division, which is essential for embryonic development. Ric-8A is critical regulator of Ga in this process. Ric-8A is a Guanine nucleotide Exchange Factor (GEF) that activates Ga by catalyzing the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) at the active site of Ga. The intermediate in this reaction is the nucleotide-free Ga:Ric-8A complex. Describing the structural changes that occur when Ric-8A binds to Ga*GDP is key to understanding how Ric-8A activates Ga. Ric-8A is also a chaperone that promotes proper folding of Ga in cells. The first three aims of R01GM105993 address the structure and the dynamic behavior of the Ric-8A:Gai1 complex, and the structural changes in Ga and Ric-8A that accompany its formation. In these aims, Double Electron-Electron Resonance (DEER) spectroscopy, Hydrogen-Deuterium eXchange coupled with Mass Spectrometry (HDX-MS) and single molecule Förster Resonance Energy Transfer (smFRET) experiments will be conducted to probe changes in Gai1 and Ric-8A structure and dynamics. The new aim is to use nanobodies to stabilize discrete structural states of the Ric-8A:Gai1 complex to render them amenable to crystallization and structure determination by X-ray crystallography. Nanobodies that alter the GEF or chaperone activity of Ric-8A will be used to identify functional sites in Ric-8A and Ga using HDX-MS. DEER and smFRET will be used to determine whether nanobodies that impair or enhance Ric-8A activity either attenuate or amplify global structural changes or protein dynamics.

 描述（由申请人提供）：在 R01GM105993 的竞争性修订中，引入了一个新目标，即使用骆驼科动物可变重链抗体结构域（纳米抗体）作为结构支架和功能探针来研究 Ric-8A 和 Gai1 之间的相互作用。 R01GM105993 资助研究以了解细胞质中的 G 蛋白如何被称为 Ric-8A 的蛋白因子激活。异三聚体 G 蛋白调节细胞代谢、分泌、电导率、基因转录、细胞分裂和细胞运动，因此对于人类所属的真核生物领域的生命至关重要。 G 蛋白的失调与癌症和一系列与普通医学相关的其他疾病有关。虽然异源三聚体 G 蛋白控制的大多数过程发生在细胞膜上，但最近的研究表明 G α 亚基 (Ga) 也控制细胞质中的某些事件。其中重要的是不对称细胞分裂，这对于胚胎发育至关重要。 Ric-8A在此过程中是Ga的关键调节剂。 Ric-8A 是一种鸟嘌呤核苷酸交换因子 (GEF)，通过在 Ga 活性位点催化二磷酸鸟苷 (GDP) 交换为三磷酸鸟苷 (GTP) 来激活 Ga。该反应的中间体是无核苷酸的 Ga:Ric-8A 复合物。描述 Ric-8A 与 Ga*GDP 结合时发生的结构变化是了解 Ric-8A 如何激活 Ga 的关键。Ric-8A 也是促进细胞中 Ga 正确折叠的伴侣。 R01GM105993 的前三个目标解决 Ric-8A:Gai1 复合物的结构和动态行为，以及伴随其形成的 Ga 和 Ric-8A 的结构变化。在这些目标中，将进行双电子共振（DEER）光谱、氢氘交换质谱（HDX-MS）和单分子福斯特共振能量转移（smFRET）实验来探测Gai1和Ric-8A结构和动力学的变化。新的目标是利用纳米抗体来稳定 Ric-8A:Gai1 复合物的离散结构状态，使其能够通过 X 射线晶体学进行结晶和结构测定。改变 Ric-8A 的 GEF 或分子伴侣活性的纳米抗体将用于通过 HDX-MS 鉴定 Ric-8A 和 Ga 中的功能位点。 DEER 和 smFRET 将用于确定损害或增强 Ric-8A 活性的纳米抗体是否会减弱或放大整体结构变化或蛋白质动态。