Genistein and metformin regulate glycerol kinase and the enzymes of glycerol 3-phosphate shuttle in a differential manner in myocytes, hepatocytes and adipocytes.

Glycerol kinase (GK) and glycerol 3-phosphate dehydrogenase (GPDH) are critical in glucose homeostasis. The role of genistein and metformin on these enzymes and glucose production was investigated in C2C12, HepG2, and 3T3-L1 cells. Enzyme kinetics, Real-Time PCR and western blots were performed to determine enzyme activities and expressions of mRNAs and proteins. Glucose production and uptake were also measured in these cells. siRNAs were used to assess their impact on the enzymes and glucose production. Ki values for the compounds were determined using purified GK and GPDH. Genistein decreased GK activity by ∼45 %, while metformin reduced cGPDH and mGPDH activities by ∼32 % and âˆ¼43 %, respectively. Insignificant changes in expressions (mRNAs and proteins) of the enzymes were observed. The compounds showed dose-dependent alterations in glucose production and uptake in these cells. Genistein non-competitively inhibited His-GK activity (Ki 19.12 µM), while metformin non-competitively inhibited His-cGPDH (Ki 75.52 µM) and mGPDH (Ki 54.70 µM) activities. siRNAs transfection showed ∼50 % and âˆ¼35 % decrease in activities of GK and mGPDH and a decrease in glucose production (0.38-fold and 0.42-fold) in 3T3-L1 cells. Considering the differential effects of the compounds, this study may provide insights into the potential therapeutic strategies for type II diabetes mellitus.

Synthetic Vesicles for Sustainable Energy Recycling and Delivery of Building Blocks for Lipid Biosynthesis†.

ATP is a universal energy currency that is essential for life. l-Arginine degradation via deamination is an elegant way to generate ATP in synthetic cells, which is currently limited by a slow l-arginine/l-ornithine exchange. We are now implementing a new antiporter with better kinetics to obtain faster ATP recycling. We use l-arginine-dependent ATP formation for the continuous synthesis and export of glycerol 3-phosphate by including glycerol kinase and the glycerol 3-phosphate/Pi antiporter. Exported glycerol 3-phosphate serves as a precursor for the biosynthesis of phospholipids in a second set of vesicles, which forms the basis for the expansion of the cell membrane. We have therefore developed an out-of-equilibrium metabolic network for ATP recycling, which has been coupled to lipid synthesis. This feeder-utilizer system serves as a proof-of-principle for the systematic buildup of synthetic cells, but the vesicles can also be used to study the individual reaction networks in confinement.

Delayed diagnosis of complex glycerol kinase deficiency in a Chinese male infant: a case report.

BACKGROUND: Xp21 contiguous gene deletion syndrome is a rare genetic metabolic disorder with poor prognosis in infants, involving deletions of one or more genes in Xp21. When deletions of adrenal hypoplasia (AHC), Duchenne muscular dystrophy (DMD), and chronic granulomatosis (CGD) loci are included, complex glycerol kinase deficiency (CGKD) can be diagnosed. We present a case of CGKD that was initially misdiagnosed and died during treatment in our hospital in terms of improving our understanding of the clinical features and diagnosis of this disease, as well as highlighting the need for more precise dosing of corticosteroid replacement therapy. CASE PRESENTATION: A 48-day-old full-term male infant was transferred to our medical center with global growth delay and persistent vomiting. Routine laboratory tests revealed hyperkalemia, hyponatremia, and a high level of creatine kinase. The initial diagnosis was adrenal cortical hyperplasia (ACH), then revised to adrenocortical insufficiency with a normal level of ACTH detected. After supplementing the routine lipid test and urinary glycerol test, CGKD was diagnosed clinically due to positive triglyceridemia and urinary glycerol, and the follow-up gene screening further confirmed the diagnosis. The boy kept thriving after corticosteroid replacement and salt supplementation. While levels of serum ACTH and cortisol decreased and remained low after corticosteroid replacement was administered. The patient died of acute type 2 respiratory failure and hypoglycemia after an acute upper respiratory tract infection, which may be the result of adrenal crisis after infection. Infants with CGKD have a poor prognosis, so physicians should administer regular follow-ups, and parents counseling during treatment to improve the survival of patients. CONCLUSIONS: Overall, CGKD, although rare, cannot be easily excluded in children with persistent vomiting. Extensive blood tests can help to detect abnormal indicators. Adrenal crisis needs to be avoided as much as possible during corticosteroid replacement therapy.

Designing next generation recombinant protein expression platforms by modulating the cellular stress response in Escherichia coli.

BACKGROUND: A cellular stress response (CSR) is triggered upon recombinant protein synthesis which acts as a global feedback regulator of protein expression. To remove this key regulatory bottleneck, we had previously proposed that genes that are up-regulated post induction could be part of the signaling pathways which activate the CSR. Knocking out some of these genes which were non-essential and belonged to the bottom of the E. coli regulatory network had provided higher expression of GFP and L-asparaginase. RESULTS: We chose the best performing double knockout E. coli BW25113ΔelaAΔcysW and demonstrated its ability to enhance the expression of the toxic Rubella E1 glycoprotein by 2.5-fold by tagging it with sfGFP at the C-terminal end to better quantify expression levels. Transcriptomic analysis of this hyper-expressing mutant showed that a significantly lower proportion of genes got down-regulated post induction, which included genes for transcription, translation, protein folding and sorting, ribosome biogenesis, carbon metabolism, amino acid and ATP synthesis. This down-regulation which is a typical feature of the CSR was clearly blocked in the double knockout strain leading to its enhanced expression capability. Finally, we supplemented the expression of substrate uptake genes glpK and glpD whose down-regulation was not prevented in the double knockout, thus ameliorating almost all the negative effects of the CSR and obtained a further doubling in recombinant protein yields. CONCLUSION: The study validated the hypothesis that these up-regulated genes act as signaling messengers which activate the CSR and thus, despite having no casual connection with recombinant protein synthesis, can improve cellular health and protein expression capabilities. Combining gene knockouts with supplementing the expression of key down-regulated genes can counter the harmful effects of CSR and help in the design of a truly superior host platform for recombinant protein expression.

Glycerol kinase stimulates uncoupling protein 1 expression by regulating fatty acid metabolism in beige adipocytes.

Browning of adipose tissue is induced by specific stimuli such as cold exposure and consists of up-regulation of thermogenesis in white adipose tissue. Recently, it has emerged as an attractive target for managing obesity in humans. Here, we performed a comprehensive analysis to identify genes associated with browning in murine adipose tissue. We focused on glycerol kinase (GYK) because its mRNA expression pattern is highly correlated with that of uncoupling protein 1 (UCP1), which regulates the thermogenic capacity of adipocytes. Cold exposure-induced Ucp1 up-regulation in inguinal white adipose tissue (iWAT) was partially abolished by Gyk knockdown (KD) in vivo Consistently, the Gyk KD inhibited Ucp1 expression induced by treatment with the ß-adrenergic receptors (ßAR) agonist isoproterenol (Iso) in vitro and resulted in impaired uncoupled respiration. Gyk KD also suppressed Iso- and adenylate cyclase activator-induced transcriptional activation and phosphorylation of the cAMP response element-binding protein (CREB). However, we did not observe these effects with a cAMP analog. Therefore Gyk KD related to Iso-induced cAMP products. In Iso-treated Gyk KD adipocytes, stearoyl-CoA desaturase 1 (SCD1) was up-regulated, and monounsaturated fatty acids such as palmitoleic acid (POA) accumulated. Moreover, a SCD1 inhibitor treatment recovered the Gyk KD-induced Ucp1 down-regulation and POA treatment down-regulated Iso-activated Ucp1 Our findings suggest that Gyk stimulates Ucp1 expression via a mechanism that partially depends on the ßAR-cAMP-CREB pathway and Gyk-mediated regulation of fatty acid metabolism.

Glycerol kinase interacts with nuclear receptor NR4A1 and regulates glucose metabolism in the liver.

Glycerol kinase (Gyk), consisting of 4 isoforms, plays a critical role in metabolism by converting glycerol to glycerol 3-phosphate in an ATP-dependent reaction. Only Gyk isoform b is present in whole cells, but its function in the nucleus remains elusive. Previous studies have shown that nuclear orphan receptor subfamily 4 group A member (NR4A)-1 is an important regulator of hepatic glucose homeostasis and lipid metabolism in adipose tissue. We aimed to elucidate the functional interaction between nuclear Gyk and NR4A1 during hepatic gluconeogenesis in the unfed state and diabetes. We identified nuclear Gyk as a novel corepressor of NR4A1 in the liver; moreover, this recruitment was dependent on the C-terminal ligand-binding domain instead of the N-terminal activation function 1 domain, which interacts with other NR4A1 coregulators. NR4A1 transcriptional activity was inhibited by Gyk via protein-protein interaction but not enzymatic activity. Moreover, Gyk overexpression suppressed NR4A1 ability to regulate the expression of target genes involved in hepatic gluconeogenesis in vitro and in vivo as well as blood glucose regulation, which was observed in both unfed and diabetic mice. These results highlight the moonlighting function of nuclear Gyk, which was found to act as a coregulator of NR4A1, participating in the regulation of hepatic glucose homeostasis in the unfed state and diabetes.-Miao, L., Yang, Y., Liu, Y., Lai, L., Wang, L., Zhan, Y., Yin, R., Yu, M., Li, C., Yang, X., Ge, C. Glycerol kinase interacts with nuclear receptor NR4A1 and regulates glucose metabolism in the liver.

Glycerol kinase 5 confers gefitinib resistance through SREBP1/SCD1 signaling pathway.

BACKGROUND: Drug resistance is common in cancer chemotherapy. This study investigates the role of Glycerol kinase 5 (GK5) in mediating gefitinib resistance in NSCLC. METHODS: The exosomal mRNA of GK5 was detected using a tethered cationic lipoplex nanoparticle (TCLN) biochip. Real-time PCR and Western blot were used to examine the expression of GK5 mRNA and protein in gefitinib-sensitive and -resistant human lung adenocarcinoma cells. The cell counting kit-8, EdU assay, flow cytometry, and JC-1 dye were used to measure cell proliferation, cell cycle, and the mitochondrial membrane potential. RESULTS: We found that the exosomal mRNA of GK5 in the plasma of patients with gefitinib-resistant adenocarcinoma was significantly higher compared with that of gefitinib-sensitive patients. The mRNA and protein levels of GK5 were significantly upregulated in gefitinib-resistant human lung adenocarcinoma PC9R and H1975 cells compared with gefitinib-sensitive PC9 cells. Silencing GK5 in PC9R cells induced mitochondrial damage, caspase activation, cell cycle arrest, and apoptosis via SREBP1/SCD1 signaling pathway. CONCLUSIONS: We demonstrated that GK5 confers gefitinib resistance in lung cancer by inhibiting apoptosis and cell cycle arrest. GK5 could be a novel therapeutic target for treatment of NSCLC with resistance to EGFR tyrosine kinase inhibitors.

MicroRNA-645 promotes cell metastasis and proliferation of renal clear cell carcinoma by targeting GK5.

OBJECTIVE: To dissect the functioning mode of miR-645 on renal clear cell carcinoma cell metastasis and growth, and provide therapeutic targets for renal clear cell carcinoma. PATIENTS AND METHODS: Quantitative Real-time PCR (qRT-PCR) assay was employed to detect miR-645 expression level. Wound healing assay and transwell assay were performed to investigate metastasis capacity of renal clear cell carcinoma cells. Cell Counting Kit 8 (CCK8) assay was incorporated to assess cell proliferation capacity. Flow cytometry was used to identify cell apoptosis and cell cycle distribution. Protein levels were assessed by Western blotting assay. The target gene was predicted and verified by bioinformatics analysis and luciferase assay. RESULTS: MiR-645 was upregulated in renal clear cell carcinoma tissues when compared with para-carcinoma tissues (n=32). Downregulated miR-645 could attenuate cell migration and invasion capacities, as well as inhibited cell proliferation capacity, promoted cell apoptosis and cell cycle arrest at G0/G1 phase. GK5 was chosen as the target gene of miR-645 by bioinformatics analysis and luciferase reporter assay. Moreover, silence of GK5 could rescue tumor suppression role of downregulated miR-645 on renal clear cell carcinoma metastasis. CONCLUSIONS: Knockdown of miR-645 exerted tumor-suppressive effects on renal clear cell carcinoma metastasis and growth via targeting GK5 in vitro, which provided an innovative and candidate target for diagnose and treatment of renal clear cell carcinoma.

Glycerol kinase of African trypanosomes possesses an intrinsic phosphatase activity.

BACKGROUND: In general, glycerol kinases (GKs) are transferases that catalyze phospho group transfer from ATP to glycerol, and the mechanism was suggested to be random bi-bi. The reverse reaction i.e. phospho transfer from glycerol 3-phosphate (G3P) to ADP is only physiologically feasible by the African trypanosome GK. In contrast to other GKs the mechanism of Trypanosoma brucei gambiense glycerol kinase (TbgGK) was shown to be in an ordered fashion, and proceeding via autophosphorylation. From the unique reaction mechanism of TbgGK, we envisaged its potential to possess phosphatase activity in addition to being a kinase. METHODS: Our hypothesis was tested by spectrophotometric and LC-MS/MS analyses using paranitrophenyl phosphate (pNPP) and TbgGK's natural substrate, G3P respectively. Furthermore, protein X-ray crystallography and site-directed mutagenesis were performed to examine pNPP binding, catalytic residues, and the possible reaction mechanism. RESULTS: In addition to its widely known and expected phosphotransferase (class II) activity, TbgGK can efficiently facilitate the hydrolytic cleavage of phosphoric anhydride bonds (a class III property). This phosphatase activity followed the classical Michaelis-Menten pattern and was competitively inhibited by ADP and G3P, suggesting a common catalytic site for both activities (phosphatase and kinase). The structure of the TGK-pNPP complex, and structure-guided mutagenesis implicated T276 to be important for the catalysis. Remarkably, we captured a crystallographic molecular snapshot of the phosphorylated T276 reaction intermediate. CONCLUSION: We conclude that TbgGK has both kinase and phosphatase activities. GENERAL SIGNIFICANCE: This is the first report on a bifunctional kinase/phosphatase enzyme among members of the sugar kinase family.

Tissue-specific effects of estrogen on glycerol channel aquaporin 7 expression in an ovariectomized mouse model of menopause.

OBJECTIVE: Elevated fat mass and redistribution of body fat are commonly observed in postmenopausal women. Aquaporin 7 (AQP7), a unique glycerol permeable integral membrane protein, has been associated with the onset of obesity. We hypothesized that estrogen supplementation could counteract this fat accumulation and redistribution through tissue-specific modulation of AQP7. METHODS: We measured fat depot weight, adipocyte size, and the expression of AQP7 and glycerol kinase (GK) in visceral and subcutaneous fat tissues of ovariectomized mice supplemented with or without 17ß-estradiol. RESULTS: Removal of the ovaries resulted in a significant decrease in AQP7 expression and an increase in GK expression in visceral adipocyte tissue; expression of AQP7 and GK in subcutaneous adipose tissue remained unaltered. Supplementation with estrogen significantly restored the visceral, but not subcutaneous, fat depot mass and adipocyte size to those of sham-operated mice. A marked increase in the expression of AQP7 and a reduction of GK were observed selectively in the visceral fat depots in estrogen-treated mice. CONCLUSIONS: Our results suggest that estrogen has tissue-specific effects on AQP7 expression, and modulation of AQP7 by estrogen alters the balance of adipocyte metabolism between adipose tissue depots.

MicroRNA-451 Negatively Regulates Hepatic Glucose Production and Glucose Homeostasis by Targeting Glycerol Kinase-Mediated Gluconeogenesis.

MicroRNAs (miRNAs) are a new class of regulatory molecules implicated in type 2 diabetes, which is characterized by insulin resistance and hepatic glucose overproduction. We show that miRNA-451 (miR-451) is elevated in the liver tissues of dietary and genetic mouse models of diabetes. Through an adenovirus-mediated gain- and loss-of-function study, we found that miR-451 negatively regulates hepatic gluconeogenesis and blood glucose levels in normal mice and identified glycerol kinase (Gyk) as a direct target of miR-451. We demonstrate that miR-451 and Gyk regulate hepatic glucose production, the glycerol gluconeogenesis axis, and the AKT-FOXO1-PEPCK/G6Pase pathway in an opposite manner; Gyk could reverse the effect of miR-451 on hepatic gluconeogenesis and AKT-FOXO1-PEPCK/G6Pase pathway. Moreover, overexpression of miR-451 or knockdown of Gyk in diabetic mice significantly inhibited hepatic gluconeogenesis, alleviated hyperglycemia, and improved glucose tolerance. Further studies showed that miR-451 is upregulated by glucose and insulin in hepatocytes; the elevation of hepatic miR-451 in diabetic mice may contribute to inhibiting Gyk expression. This study provides the first evidence that miR-451 and Gyk regulate the AKT-FOXO1-PEPCK/G6Pase pathway and play critical roles in hepatic gluconeogenesis and glucose homeostasis and identifies miR-451 and Gyk as potential therapeutic targets against hyperglycemia in diabetes.

MicroRNA-135b exerts oncogenic activity in glioblastoma via the inhibition of glycerol kinase 5 expression.

Glioblastoma is the most common primary malignancy of the adult central nervous system and is associated with a markedly poor prognosis. Elucidating the pathogenesis and molecular changes will assist in further understanding the pathogenesis and progression of the disease and offer novel targets for therapies. The present study demonstrated that the expression level of GK5 was lower in high-grade glioblastoma tissues compared with low-grade ones and it can promote proliferation in glioblastoma cells. The regulatory mechanism of GK5 in glioblastoma were also investigated. It was revealed that GK5 is a target of miR-135b in U87MG glioblastoma cells. Controry to GK5, the expression of miR-135b is upregulated in glioblastoma and its expression is positively associated with the grade of the disease. Finally, it was demonstrated that miR-135b promoted the proliferation of U87MG cells. Therefore, miR-135b may function as an oncogene by inhibiting GK5 in glioblastoma.

Chimeric rabies SADB19-VSVg-pseudotyped lentiviral vectors mediate long-range retrograde transduction from the mouse spinal cord.

Lentiviral vectors have proved an effective method to deliver transgenes into the brain; however, they are often hampered by a lack of spread from the site of injection. Modifying the viral envelope with a portion of a rabies envelope glycoprotein can enhance spread in the brain by using long-range axon projections to facilitate retrograde transport. In this study, we generated two chimeric envelopes containing the extra-virion and transmembrane domain of rabies SADB19 or CVS-N2c with the intra-virion domain of vesicular stomatitis virus. Viral particles were packaged containing a green fluorescent protein reporter construct under the control of the phosphoglycerokinase promoter. Both vectors produced high-titer particles with successful integration of the glycoproteins into the particle envelope and significant transduction of neurons in vitro. Injection of the SADB19 chimeric viral vector into the lumbar spinal cord of adult mice mediated a strong preference for gene transfer to local neurons and axonal terminals, with retrograde transport to neurons in the brainstem, hypothalamus and cerebral cortex. Development of this vector provides a useful means to reliably target select populations of neurons by retrograde targeting.

Molecular basis for the reverse reaction of African human trypanosomes glycerol kinase.

The glycerol kinase (GK) of African human trypanosomes is compartmentalized in their glycosomes. Unlike the host GK, which under physiological conditions catalyzes only the forward reaction (ATP-dependent glycerol phosphorylation), trypanosome GK can additionally catalyze the reverse reaction. In fact, owing to this unique reverse catalysis, GK is potentially essential for the parasites survival in the human host, hence a promising drug target. The mechanism of its reverse catalysis was unknown; therefore, it was not clear if this ability was purely due to its localization in the organelles or whether structure-based catalytic differences also contribute. To investigate this lack of information, the X-ray crystal structure of this protein was determined up to 1.90 Å resolution, in its unligated form and in complex with three natural ligands. These data, in conjunction with results from structure-guided mutagenesis suggests that the trypanosome GK is possibly a transiently autophosphorylating threonine kinase, with the catalytic site formed by non-conserved residues. Our results provide a series of structural peculiarities of this enzyme, and gives unexpected insight into the reverse catalysis mechanism. Together, they provide an encouraging molecular framework for the development of trypanosome GK-specific inhibitors, which may lead to the design of new and safer trypanocidal drug(s).

A novel role for the non-catalytic intracellular domain of Neprilysins in muscle physiology.

BACKGROUND INFORMATION: Neprilysins (Neps) are membrane-bound M13 endopeptidases responsible for the activation and/or inactivation of peptide signalling events on cell surfaces. By hydrolysing their respective substrates, mammalian Neps are crucial to the metabolism of numerous bioactive peptides, especially in the nervous, immune, cardiovascular and inflammatory systems. On the basis of their involvement in essential physiological processes, proteins of the Nep family constitute putative therapeutic agents as well as targets in different diseases, including Alzheimer's disease. RESULTS: We here demonstrate that overexpression of Neprilysin 4 (Nep4) in Drosophila melanogaster leads to a severe muscle degeneration phenotype. This phenotype is observed for overexpression of full-length Nep4 in somatic muscles and is accompanied by severely impaired movement of larvae and lethality in late larval development. On the contrary, down-regulation of expression caused only the latter two effects. By expressing several mutated and truncated forms of Nep4 in transgenic animals, we show that the intracellular domain is responsible for the observed phenotypes while catalytic activity of the enzyme was apparently dispensable. A yeast two-hybrid screen identified a yet uncharacterised carbohydrate kinase as a first interaction partner of the intracellular domain of Nep4. CONCLUSIONS: These data demonstrate that the physiological significance of Nep4 is not limited to its function as an active peptidase but that the enzyme's intracellular N-terminus is affecting muscle integrity, independent of the protein's enzymatic activity. To our knowledge, this is the first report of an intracellular Nep domain being involved in muscle integrity.

Construction of membrane-anchoring fusion protein of Thermococcus kodakaraensis glycerol kinase and its application to repetitive batchwise reactions.

We previously demonstrated the stoichiometric conversion of glycerol to glycerol-3-phosphate (G3P) using Escherichia coli recombinants producing the ATP-dependent glycerol kinase of the hyperthermophile Thermococcus kodakaraensis (TkGK) and the polyphosphate kinase of Thermus thermophilus HB27 (TtPPK). TtPPK was associated with the membrane fraction of E. coli recombinants, whereas TkGK was released from the cells during the reaction at 70°C. In this study, TkGK was fused with either TtPPK or an E. coli membrane-intrinsic protein, YedZ, to minimize the heat-induced leakage of TkGK. When the E. coli recombinants having these fusion proteins were incubated at 70°C for 2h, more than 80% of TkGK activity was retained in the heated E. coli cells. However, the yields of G3P production by E. coli having the fusion proteins of TtPPK and TkGK were only less than 35%. Polyphosphate is a strong chelator for metal ions and has an inhibitory effect on TkGK which requires magnesium. Insufficient space between TtPPK and TkGK might enhance the inhibitory effect of polyphosphate on TkGK activity of the fusion protein. The mixture of E. coli cells having TtPPK and those having TkGK fused with YedZ converted 80% of glycerol into G3P. These recombinant cells could be easily recovered from the reaction mixture by centrifugation and repeatedly used without a significant loss of enzyme activities.

A neonate with contiguous deletion syndrome in XP21.

INTRODUCTION: We report a case of a male infant with the association of pseudohypertriglyceridemia, hypoadrenalism (hyponatremia, hyperpotasemia, dehydration), high creatine phosphokinase level (possible Duchenne's muscular dystrophy, DMD) and diagnosed contiguous gene deletion syndrome in Xp21. CASE REPORT: A 1-month-old male term infant was referred due to no weight gain. The examination revealed dehydration (decreased skin turgor), scrotal hyperpigmentation and hypotonia. Laboratory findings showed hyponatremia of 124 mmol/L, hyperpotasemia of 6.9 mg/dL, high creatine phosphokinase level of 7019 IU/L, and high blood triglyceride level of 1244 mg/dL. There was no deletion detected in the dystrophin gene with the study investigating selected exons, no dystrophin staining and nonspecific atrophic findings in the muscle biopsy. Further laboratory findings defined high glycerol concentrations both in blood and in urine that were compatible with a glycerol kinase deficiency (GKD). Array CGH study confirmed the existence of a deletion in Xp21 of the genes responsible for DMD, GKD and the congenital adrenal hypoplasia (gene DAX1 or NROB1 gene: Xp21.3-21.2). The infant showed good response to mineralocorticoid therapy, hyponatremia resolved and gained weight. CONCLUSION: Physicians should consider contiguous gene deletion syndrome in Xp21 in the infants with myopathic compromise, increased levels of creatine phosphokinase and pseudohypertriglyceridaemia to be able to prevent and treat the metabolic complications. Furthermore, geneticists should take into account to design routine deletion studies including the promoter region in this disorder.

Expression, purification, and characterization of a His6-tagged glycerokinase from Pichia farinosa for enzymatic cycling assays in mammalian cells.

The GUT1 gene of the halotolerant yeast Pichia farinosa, encoding glycerokinase (EC 2.7.1.30), was expressed in Pichia pastoris. A purification factor of approximately 61-fold was achieved by a combination of nickel affinity and anion exchange chromatography. The specific activity of the final preparation was 201.6 units per mg protein with a yield of about 21%. A nearly homogeneous enzyme preparation was confirmed by SDS-polyacrylamide gels and mass spectrometry analysis. Glycerol stabilized the purified enzyme for long-term storage at -80°C. The pH and temperature optima were in the range of 6.5-7.0 and 45-50°C, respectively. ATP was the most effective phosphoryl group donor tested. Additionally, the enzyme phosphorylated glycerol also with ITP, UTP, GTP and CTP. The K(m) values of the enzyme for ATP and ITP were 0.428 and 0.845 mM, respectively. The kinetic properties of the enzyme with respect to UTP, GTP, and CTP suggested that glycerokinase exhibited negative cooperativity as double reciprocal plots showed a biphasic response to increasing nucleoside triphosphate concentrations. The application as a coupling enzyme in the determination of pyruvate kinase activity in cell extracts of Madin-Darby canine kidney cells showed good reproducibility when compared with a commercially available preparation of bacterial glycerokinase.

Transcriptomic and network component analysis of glycerol kinase in skeletal muscle using a mouse model of glycerol kinase deficiency.

Glycerol kinase (GK) is at the interface of fat and carbohydrate metabolism and has been linked to obesity and type 2 diabetes mellitus (T2DM). The purpose of this study was to investigate the role of GK in fat metabolism and insulin signaling in skeletal muscle (an important end organ tissue in T2DM). Microarray analysis determined that there were 525 genes that were differentially expressed (1.2-fold, p value<0.05) between knockout (KO) and wild-type (WT) mice. Quantitative PCR (qPCR) confirmed the differential expression of genes including glycerol kinase (Gyk), phosphatidylinositol 3-kinase regulatory subunit, polypeptide 1 (p85 alpha) (Pik3r1), insulin-like growth factor 1 (Igf1), and growth factor receptor bound protein 2-associated protein 1 (Gab1). Network component analysis demonstrated that transcription factor activities of myogenic differentiation 1 (MYOD), myogenic regulatory factor 5 (MYF5), myogenin (MYOG), nuclear receptor subfamily 4, group A, member 1 (NUR77) are decreased in the Gyk KO whereas the activity of paired box 3 (PAX3) is increased. The activity of MYOD was confirmed using a DNA binding assay. In addition, myoblasts from Gyk KO had less ability to differentiate into myotubes compared to WT myoblasts. These findings support our previous studies in brown adipose tissue and demonstrate that the role of Gyk in muscle is due in part to its non-metabolic (moonlighting) activities.

Amino acid substitutions in the sugar kinase/hsp70/actin superfamily conserved ATPase core of E. coli glycerol kinase modulate allosteric ligand affinity but do not alter allosteric coupling.

IIA(Glc), the glucose-specific phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system, is an allosteric inhibitor of Escherichia coli glycerol kinase. A linked-functions initial-velocity enzyme kinetics approach is used to define the MgATP-IIA(Glc) heterotropic allosteric interaction. The interaction is measured by the allosteric coupling constants Q and W, which describe the mutual effect of the ligands on binding affinity and the effect of the allosteric ligand on V(max), respectively. Allosteric interactions between these ligands display K-type activation and V-type inhibition. The allosteric coupling constant Q is about 3, showing cooperative coupling such that each ligand increases the affinity for binding of the other. The allosteric coupling constant W is about 0.1, showing that the allosteric inhibition is partial such that binding of IIA(Glc) at saturation does not reduce V(max) to zero. E. coli glycerol kinase is a member of the sugar kinase/heat shock protein 70/actin superfamily, and an element of the superfamily conserved ATPase catalytic core was identified as part of the IIA(Glc) inhibition network because it is required to transplant IIA(Glc) allosteric control into a non-allosteric glycerol kinase [A.C. Pawlyk, D.W. Pettigrew, Proc. Natl. Acad. Sci. USA 99 (2002) 11115-11120]. Two of the amino acids at this locus of E. coli glycerol kinase are replaced with those from the non-allosteric enzyme to enable determination of its contributions to MgATP-IIA(Glc) allosteric coupling. The substitutions reduce the affinity for IIA(Glc) by about 5-fold without changing significantly the allosteric coupling constants Q and W. The insensitivity of the allosteric coupling constants to the substitutions may indicate that the allosteric network is robust or the locus is not an element of that network. These possibilities may arise from differences of E. coli glycerol kinase relative to other superfamily members with respect to oligomeric structure and location of the allosteric site in a single domain far from the catalytic site.