Biophysical Analysis of G6PC1, a Key Factor Driving Elevated Hepatic Glucose Production in Diabetes

Glucose-6-phosphatase is a multi-component enzyme system located in the endoplasmic reticulu that plays a critical role in maintaining interprandial blood glucose homeostasis. Mediating the terminal ste of gluconeogenesis and glycogenolysis in the liver, the membrane-bound catalytic subunit, G6PC1, work in concert with accessory transport proteins to catalyze the hydrolysis of glucose-6-phosphate to glucos and inorganic phosphate. Previous studies have suggested that dysregulation of G6PC1 gene expressio contributes to elevated hepatic glucose production (HGP) associated with diabetes. Furthermore, mutations in G6PC1 that compromise activity lead to severe hypoglycemia, forming the clinical basis of glycogen storage disease (GSD). Importantly, the lack of high-resolution structural models precludes a complet understanding of G6PC1 function and impairment. The major bottleneck for a detailed structural analysis of G6PC1 is the absence of efficient heterologous expression and purification strategies that isolate the enzyme in a functional form. This proposal describes logical, time-tested approaches to express and purif G6PC1 for functional and biophysical experiments, which sets the stage for detailed structural analysis by crystallography and/or cryo-electron microscopy. The methodology employs a small-scale screenin approach utilizing the highly sensitive technique of fluorescence detection size exclusion chromatograph to rapidly identify optimal gene constructs and expression conditions, which will inform large-scal expression and purification protocols from insect cells to obtain milligram quantities of wild type and mutan G6PC1. Global structural properties of purified G6PC1 determined from a suite of biophysical tools will be correlated with in vitro hydrolase activity. The results of this work will support progress toward a detailed description of the structural basis of G6PC1 activity and the relationship to disease-associated mutations.