Mechanism of G protein Activation by Ric-8A

Ric-8A激活G蛋白的机制

• 批准号: 8482004
• 负责人: Stephen R Sprang
• 金额: $ 26.75万
• 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 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8482004
• 关键词: Active Sites; Address; Afferent Neurons; Amino Acids; Armadillo Repeat; Behavior; Binding; Cell division; Cell membrane; Cells; Complex; Coupled; Cytoplasm; Deletion Mutagenesis; Deuterium; Diffuse; Disease; Electric Conductivity; Electron Spin Resonance Spectroscopy; Electrons; Elements; Embryo; Embryonic Development; Energy Transfer; Eukaryota; Event; Exhibits; Family; Freezing; Frequencies; G Protein-Coupled Receptor Genes; 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; Homologous Gene; Human; Human Development; Hydrogen; Individual; Kinetics; Knock-out; Knowledge; Laboratories; Life; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measures; Mediating; Medicine; Metabolism; Methods; Molecular; Monitor; Motion; Nucleotides; Peptides; Physiological Processes; Play; Principal Investigator; Process; Protein Dynamics; Proteins; Proteolysis; Reaction; Reporting; Research; Resolution; Role; Scanning; Signal Transduction; Site; Solid; Solutions; Solvents; Spectrum Analysis; Spin Labels; Structure; Techniques; Testing; Time; Work; cell motility; conformational conversion; flexibility; fluorophore; inhibitor/antagonist; melting; membrane biogenesis; mutant; protein activation; public health relevance; research study; single molecule

DESCRIPTION (provided by applicant): Research will be conducted 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 doman 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 (G?) 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 G? in this process. Ric-8A is a Guanine nucleotide Exchange Factor (GEF) that catalyzes the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) at the active site of G?. This reaction transitions G? to the so-called "activated" state. In preliminary studies, the Principal Investigator's laboratory demonstrated that Ric-8A induces and stabilizes G?i1 (a Gi-class G? subunit) in a structurally heterogeneous or molten globule-like state. The nucleotide-free Ric-8A:G?i1 intermediate is stable in the absence of GTP. The hypothesis to be tested is that Ric-8A catalyzes nucleotide exchange by altering the global and local structure of G?i1, and by increasing flexibility and inducing dynamic behavior. The overall goal of the project is to characterize the trajectory of the exchange reaction from binding of Ric-8A to G?i1.GDP, to the formation of the nucleotide-free Ric-8A:G?i1 complex. The first two aims of the proposal address, respectively, the structure and the dynamic behavior of the Ric- 8A:G?i1 complex. The first aim is to use Double Electron-Electron Resonance (DEER) spectroscopy to observe large-scale changes in the structures of G?i1 and Ric-8A upon formation of the G?i1:Ric-8A complex. Hydrogen-Deuterium eXchange, followed by proteolysis and Mass Spectrometry (HDXMS) will be employed to observe structural changes at a level of detail that approaches single amino acid residues. In the second aim, Forster Resonance Energy Transfer (smFRET) studies of individual molecules, either freely diffusing or immobilized on a solid matrix, will be conducted to determine whether formation of the G?i1:Ric-8A complex is accompanied by changes in the dynamic behavior of either protein. The third aim of the proposal is to understand the mechanism by which Ric-8A alters the structure of G?i1 in the transition from the GDP- bound state to the nucleotide-free state. A combination of DEER and HDXMS methods will be used to study this transition. Site Directed Spin Labeling and Electron Paramagnetic Resonance Spectroscopy, together with mutational scanning experiments, will be used to identify Ric-8A residues that are critical to G?i1-binding and GEF activity, and the sites at which Ric-8A makes contact with G?i1.

描述（由申请人提供）：将进行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 (G?) 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 是 G 的关键调节因子？在这个过程中。 Ric-8A是鸟嘌呤核苷酸交换因子(GEF)，其在Gα的活性位点催化二磷酸鸟苷(GDP)交换为三磷酸鸟苷(GTP)。该反应转变为G?到所谓的“激活”状态。在初步研究中，首席研究员的实验室证明，Ric-8A 诱导并稳定 G?i1（Gi 类 G? 亚基）处于结构异质或熔球状状态。无核苷酸的 Ric-8A:G?i1 中间体在没有 GTP 的情况下是稳定的。要测试的假设是 Ric-8A 通过改变 G?i1 的全局和局部结构、增加灵活性和诱导动态行为来催化核苷酸交换。该项目的总体目标是表征从 Ric-8A 与 G?i1.GDP 结合到形成无核苷酸 Ric-8A:G?i1 复合物的交换反应轨迹。该提案的前两个目标分别涉及 Ric-8A:G?i1 复合体的结构和动态行为。第一个目标是使用双电子-电子共振（DEER）光谱来观察 G?i1:Ric-8A 复合物形成后 G?i1 和 Ric-8A 结构的大规模变化。将采用氢-氘交换，然后进行蛋白水解和质谱 (HDXMS)，以接近单个氨基酸残基的详细程度观察结构变化。在第二个目标中，将对自由扩散或固定在固体基质上的单个分子进行福斯特共振能量转移（smFRET）研究，以确定 G?i1:Ric-8A 复合物的形成是否伴随着任一蛋白质的动态行为。该提案的第三个目的是了解 Ric-8A 在从 GDP 结合状态向无核苷酸状态转变过程中改变 G?i1 结构的机制。将结合使用 DEER 和 HDXMS 方法来研究这一转变。定点自旋标记和电子顺磁共振波谱以及突变扫描实验将用于识别对 G?i1 结合和 GEF 活性至关重要的 Ric-8A 残基，以及 Ric-8A 与 G 接触的位点？i1。