Understanding the Moonlighting Functions of Glycerol Kinase

Glycerol kinase (GK in humans, Gyk in mice) is an enzyme that catalyzes the conversion of glycerol to glycerol 3-phosphate, which is an intermediate useful for both glycolysis and lipid synthesis. Like many other enzymes in the same metabolic pathways, GK is a moonlighting protein with abilities to perform many functions unrelated to its phosphorylative properties. Of particular interest, GK is the ATP-stimulated translocation promoter (ASTP) that interacts with glucocorticoid receptor (GR) by assisting the translocation of activated GR complex into the nucleus and promoting the transactivation of GR responsive genes. In this thesis, I investigated the protein-protein interactions of GK/GR and its contribution to the pathogenesis of isolated glycerol kinase deficiency (GKD), a X-linked inborn error of metabolism due to mutations or deletions of the GK gene.First of all, we characterized our newly developed Gyk liver-specific transgenic mouse strains. Transgenic strains exhibited statistically significant weight gain compared to wild type controls on a high fat diet. Our overall results showed that Gyk transgenic mice became obese and were at risk of developing type II diabetes mellitus (T2DM). This data is consistent with the GKD human data, showing alterations in GK expression may cause changes in the overall lipid and glucose metabolism in mice.We sought to develop more study models to facilitate our analysis of GKD pathogenesis. A mathematical model of the insulin signal transduction pathway was engineered for predicting insulin sensitivity in the Gyk knock out mice. The model covered all components in the insulin signal transduction pathway and calculated glucose uptake results similar to an actual glucose tolerance test. The model simulations suggested that the knock outs had reduced glucose uptake in response to an insulin stimulation, therefore decreased insulin sensitivity than normal mice.A new gene targeting technology, called CRISPR/Cas9 recombinase system, was incorporated into our cell culture studies to develop a human GK knock out cell line. Preliminary results showed that the knock out cells no longer produced functional GK proteins. Using lentiviral gene transfer, several GK mutants, found in individuals with GKD, were made for analysis in cell culture. Initial GKD mutations analyses in cell culture revealed that GKD mutants had reduced mRNA levels of GR direct target genes, also defined as ASTP activity, which suggests that the protein-protein interaction of GK and GR interaction is important for the pathogenesis of iGKD. We also applied the same strategy to develop site-directed GK mutants with variation in its LXXLL motif and demonstrated that the LXXLL motif in GK is critical for the interaction of GK and GR. Similar to the GKD mutants, mutations were generated in the LXXLL functional motif of GK in the knock out cells. While the enzymatic property of GK is unaffected in the LXXLL mutants, the ASTP activities were significantly reduced compared to normal cells. Overall, these experiments demonstrated GK’s moonlighting roles are just as critical to the pathogenesis of iGKD as its enzymatic role.In summary, this thesis examines the role of GK’s moonlighting functions in obesity, T2DM, and other phenotypes observed in individuals with GKD. We extensively studied the ASTP activity of GK, specifically the protein-protein interaction of GK and GR. It has become clear that moonlighting functions of GK is just as important as its enzymatic functions in regulating the lipid and carbohydrate metabolism