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

项目摘要： 葡萄糖-6-磷酸酶催化亚基（G6 PC）将葡萄糖-6-磷酸（G6 P）水解为葡萄糖， 无机磷酸盐G6 PC 2主要在胰岛B细胞中表达。遗传和分子 研究已经将增加的G6 PC 2活性与升高的FBG联系起来。非糖尿病和糖尿病前期的FBG较高 范围强烈影响心血管相关死亡率（CAM）的风险， 然而，在患有T2 D的个体中，升高的FBG增加了糖尿病并发症的风险 并进一步增加CAM的风险。这些观察结果强烈表明，G6 PC 2抑制剂可能 代表一种潜在的降低FBG的新疗法。我们假设基因，代谢， 生理和生化研究将确定G6 PC 2在B细胞中的功能，证明G6 PC 2代表了一种新的表达途径， 降低FBG的可行目标，并产生促进G6 PC 2抑制剂开发的数据。我们的数据显示 一种新的范例，其中葡萄糖激酶/G6 PC 2无效底物循环，而不是单独的葡萄糖激酶，是关键 B细胞中糖酵解通量的决定因素。因此，G6 pc 2的缺失增加了葡萄糖敏感性。 刺激胰岛素分泌（GSIS）转化为葡萄糖，这导致FBG降低。我们开发了新的检测方法 用于在体外和完整细胞中测量G6 PC 2活性。在目标1中，我们建议使用这些测定来鉴定 非同义G6 PC 2单核苷酸多态性（SNP），损害G6 PC 2活性， 利用范德比尔特的BioVU生物样本库研究它们对人类健康的影响。我们还将探讨这个概念， 根据初步数据，G6 PC 2调节B细胞中的代谢通量和脉动胰岛素分泌 通过一种与已知的G6 PC 2对糖酵解的作用无关的机制， 钙振荡我们假设目标1的结果将定义G6 PC 2在B细胞中的功能，并突出G6 PC 2在细胞中的作用。 G6 PC 2抑制在人体中的正面和潜在负面影响。明显缺乏结构- 对G6 PC 2的功能研究起源于缺乏高分辨率的分子模型，低的固有频率， 酶的活性和不能纯化活性G6 PC 2。开创性的出版物， AlphaFold 2算法用于蛋白质结构预测，以及我们开发的异源表达和 用于分离稳定且具有催化活性的G6 PC 2的纯化方案已经克服了这些障碍。在 目的2，我们将使用诱变来探测各种假定的氨基酸中氨基酸的功能重要性， 然后利用生物化学/生物物理测定的工具包来确定它们对G6 PC 2中的结构域的影响， G6 PC 2折叠、稳定性和催化。我们还将进行研究，以表征人类的特异性， G6 PC 2抑制剂，并探讨其在人胰岛中调节GSIS的能力。我们假设目标2的结果将 通过为理解G6 PC 2的结构提供分子蓝图， 催化的基础以及产生将提供对已知G6 PC 2抑制剂的作用模式的洞察的数据， 这将为优化化合物的合理设计提供信息。

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.