G6PC Enzymology, Structure, Function and Role in the Regulation of Fasting Blood Glucose

G6PC 酶学、结构、功能及其在空腹血糖调节中的作用

• 批准号: 10584866
• 负责人: Richard M O'Brien
• 金额: $ 43万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10584866
• 关键词: Address; Affect; Algorithms; Amino Acids; Atherosclerosis; Automobile Driving; B-Lymphocytes; Beta Cell; Biochemical; Biological Assay; Biology; Biophysics; Blood Glucose; Calcium Oscillations; Cardiovascular system; Catalysis; Catalytic Domain; Cell physiology; Cells; Complications of Diabetes Mellitus; Data; Development; Diabetes Mellitus; Endoplasmic Reticulum; Enzymatic Biochemistry; Enzymes; Failure; Fasting; Fostering; Functional disorder; Futile Cycling; G6PC2 gene; G6PC3 gene; Genetic; Genotype; Glucokinase; Glucose; Glucose-6-Phosphate; Glucosephosphates; Glycolysis; Goals; Health; Heart Diseases; Human; Hydrolysis; Hyperglycemia; Impairment; In Vitro; Incidence; Individual; Intestines; Investigation; Islets of Langerhans; Kidney; Link; Liver; Measurement; Measures; Medical; Membrane; Metabolic; Metabolic Pathway; Methodology; Molecular; Mutagenesis; Non-Insulin-Dependent Diabetes Mellitus; Patients; Phenotype; Physiological; Prediabetes syndrome; Prevention; Protein Isoforms; Protocols documentation; Publications; Publishing; Regulation; Resolution; Risk; Role; Single Nucleotide Polymorphism; Specificity; Structure; Therapeutic; Whole Organism; adverse pregnancy outcome; biobank; cardiovascular risk factor; cerebral atrophy; diabetic; enzyme activity; fasting blood glucose level; glucose-6-phosphatase; in vitro activity; inhibitor; inorganic phosphate; insight; insulin secretion; islet; molecular modeling; molecular scale; mortality; non-diabetic; novel; novel therapeutic intervention; novel therapeutics; prediction algorithm; prophylactic; protein structure prediction; rational design; structural biology

Project Summary: Glucose-6-phosphatase catalytic subunits (G6PCs) hydrolyze glucose-6-phosphate (G6P) to glucose and inorganic phosphate. G6PC2 is predominantly expressed in pancreatic islet b cells. Genetic and molecular studies have linked increased G6PC2 activity to elevated FBG. Higher FBG in the non-diabetic and pre-diabetic ranges strongly influence the risk of cardiovascular-associated mortality (CAM) and developing type 2 diabetes (T2D) whereas, in individuals with T2D, elevated FBG increases the risk for diabetic complications and further increases the risk of CAM. These observations strongly suggest that G6PC2 inhibitors may represent a potential novel therapy to reduce FBG. We hypothesize that the proposed genetic, metabolic, physiological and biochemical studies will define the functions of G6PC2 in b cells, demonstrate that G6PC2 represents a viable target for lowering FBG and generate data that fosters the development of G6PC2 inhibitors. Our data suggest a new paradigm in which a glucokinase/G6PC2 futile substrate cycle, rather than glucokinase alone, is the key determinant of glycolytic flux in b cells. Consequently, deletion of G6pc2 increases the sensitivity of glucose- stimulated insulin secretion (GSIS) to glucose, which results in reduced FBG. We have developed novel assays for measuring G6PC2 activity in vitro and in intact cells. In Aim 1 we propose using these assays to identify non-synonymous G6PC2 single nucleotide polymorphisms (SNPs) that impair G6PC2 activity and then explore their effects on human health using Vanderbilt’s BioVU biobank. We will also explore the concept, arising from preliminary data, that G6PC2 regulates metabolic fluxes and pulsatile insulin secretion in b cells through a mechanism independent of the known action of G6PC2 on glycolysis, that involves altered ER calcium oscillations. We hypothesize the results of Aim 1 will define the functions of G6PC2 in b cells and highlight the positive as well as potential negative effects of G6PC2 inhibition in humans. The conspicuous absence of structure- function studies on G6PC2 originated from the absence of a high-resolution molecular model, the low inherent activity of the enzyme and an inability to purify active G6PC2. The groundbreaking publication of the AlphaFold2 algorithm for protein structure prediction, and our development of heterologous expression and purification protocols for isolation of stable and catalytically active G6PC2, have overcome these hurdles. In Aim 2, we will use mutagenesis to probe the functional importance of amino acids within various putative domains in G6PC2 and then leverage a toolkit of biochemical/biophysical assays to define their effects on G6PC2 folding, stability and catalysis. We will also perform studies to characterize the specificity of a human G6PC2 inhibitor and explore its ability to regulate GSIS in human islets. We hypothesize the results of Aim 2 will establish a new paradigm for investigation of G6PC2 by providing a molecular blueprint for understanding the structural basis of catalysis as well as generating data that will provide insight into the mode of action of a known G6PC2 inhibitor, which will inform the rational design of optimized compounds.

项目摘要： 葡萄糖-6-磷酸酶催化亚基（G6PCS）水解6-磷酸葡萄糖（G6P）至葡萄糖和葡萄糖和 无机磷酸盐。 G6PC2主要在胰岛B细胞中表达。遗传和分子 研究已将增加的G6PC2活性与FBG升高联系起来。非糖尿病和糖尿病前的FBG较高 范围强烈影响心血管相关死亡率（CAM）的风险，并发展2型 糖尿病（T2D），而在T2D的个体中，FBG升高会增加糖尿病并发症的风险 并进一步增加了CAM的风险。这些观察结果强烈表明G6PC2抑制剂可能 代表了减少FBG的潜在新型疗法。我们假设拟议的遗传，代谢， 物理和生化研究将定义G6PC2在B细胞中的功能，证明G6PC2代表A 降低FBG并生成促进G6PC2抑制剂发展的数据的可行目标。我们的数据表明 葡萄糖酶/G6PC2徒劳的底物周期而不是单独葡萄糖酶的新范式是关键 B细胞中糖酵解通量的决定因素。因此，G6PC2的缺失增加了葡萄糖的灵敏度 刺激的胰岛素分泌（GSIS）对葡萄糖，导致FBG降低。我们已经开发了新颖的测定 用于在体外和完整细胞中测量G6PC2活性。在AIM 1中，我们建议使用这些测定法来识别 非同义G6PC2单核苷酸多态性（SNP）损害G6PC2活性，然后 使用范德比尔特的Biovu生物库来探索他们对人类健康的影响。我们还将探索这个概念， 由初步数据引起的，G6PC2调节B细胞中代谢通量和脉动胰岛素分泌 通过独立于G6PC2对糖酵解的已知作用的机制，涉及改变ER 钙振荡。我们假设AIM 1的结果将定义G6PC2在B细胞中的功能，并突出显示 G6PC2抑制在人类中的阳性和潜在负面影响。明显没有结构 - G6PC2的功能研究起源于缺乏高分辨率分子模型，低继承 酶的活性和无法纯化活性G6PC2的活性。开创性的出版物 用于蛋白质结构预测的alphafold2算法，以及我们的异源表达和 分离稳定和催化活性的G6PC2的纯化方案已经克服了这些障碍。在 AIM 2，我们将使用诱变来探测各种推定的氨基酸的功能重要性 G6PC2中的域，然后利用生化/生物物理测定的工具包来定义其对 G6PC2折叠，稳定性和催化。我们还将进行研究以表征人的特异性 G6PC2抑制剂并探索其在人类胰岛中调节GSIS的能力。我们假设AIM 2的结果将 通过提供一个分子蓝图来理解结构性，建立用于G6PC2投资的新范式 催化的基础以及生成数据，这些数据将洞悉已知G6PC2抑制剂的作用方式， 这将告知优化化合物的合理设计。