Specificity of glycerol kinase.
Ontology highlight
ABSTRACT:
SUBMITTER: Eisenthal R
PROVIDER: S-EPMC1168078 | biostudies-other | 1974 Jul
REPOSITORIES: biostudies-other
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Project description:Although glycerol is the primary carbon source available to halophilic heterotrophic communities, little is known regarding haloarchaeal glycerol metabolism. In this study, a gene encoding a glycerol kinase homolog (glpK; HVO_1541) was deleted from the genome of the haloarchaeon Haloferax volcanii by a markerless knockout strategy. The glpK mutant, KS4, readily grew on yeast extract-peptone complex medium and glucose minimal medium but was incapable of growth on glycerol. Glycerol kinase activity was dependent on the glpK gene and readily detected in cells grown on glucose and/or glycerol, with the activity level higher in medium supplemented with glycerol (with or without glucose) than in medium with glucose alone. An analysis of carbon utilization revealed that glycerol suppressed the metabolism of glucose in both the parent H26 and glpK mutant strains, with catabolite repression more pronounced in the glycerol kinase mutant. Transcripts specific for glpK and an upstream gene, gpdA, encoding a homolog of glycerol-3-phosphate dehydrogenase subunit A, were upregulated (8- and 74-fold, respectively) in the presence of glycerol and glucose compared to those in the presence of glucose alone. Furthermore, glpK was transcriptionally linked to the gpdC gene of the putative glycerol-3-phosphate dehydrogenase operon (gpdABC), based on the findings of reverse transcriptase PCR analysis. The results presented here provide genetic and biochemical evidence that glycerol metabolism proceeds through a glycerol kinase encoded by glpK and suggest that a glycerol-3-phosphate dehydrogenase encoded by the upstream gpdABC operon is also involved in this pathway. Furthermore, our findings reveal a unique example of glycerol-induced repression of glucose metabolism in H. volcanii.
| S-EPMC2698487 | biostudies-literature
Project description:Glycerol kinase (GK) is an enzyme that catalyzes the formation of glycerol 3-phosphate from ATP and glycerol, the rate-limiting step in glycerol utilization. We analyzed the genome of the model organism Drosophila melanogaster and identified five GK orthologs, including two loci with sequence homology to the mammalian Xp21 GK protein. Using a combination of sequence analysis and evolutionary comparisons of orthologs between species, we characterized functional domains in the protein required for GK activity. Our findings include additional conserved domains that suggest novel nuclear and mitochondrial functions for glycerol kinase in apoptosis and transcriptional regulation. Investigation of GK function in Drosophila will inform us about the role of this enzyme in development and will provide us with a tool to examine genetic modifiers of human metabolic disorders.
| S-EPMC2807631 | biostudies-literature
Project description:Amebiasis is caused by the protozoan parasite Entamoeba histolytica. Treatment options other than metronidazole and its derivatives are few, and their low efficacy against asymptomatic cyst carriers, and experimental evidence of resistance in vitro justify the discovery/repurposing campaign for new drugs against amebiasis. To better understand the role of glycerol metabolism in this parasite, we focused on characterizing two important enzymes, glycerol kinase (GK) and glycerol 3-phosphate dehydrogenase (G3PDH). Recombinant GK was biochemically characterized in detail, while G3PDH was not due to failure of protein expression and purification. GK revealed novel characteristics and unprecedented kinetic properties in reverse reaction. Gene silencing revealed that GK is essential for optimum growth, whereas G3PDH is not. Gene silencing of G3PDH caused upregulated GK expression, while that of GK resulted in upregulation of antioxidant enzymes as shown by RNA-seq analysis. Although the precise molecular link between GK and the upregulation of antioxidant enzymes was not demonstrated, the observed increase in antioxidant enzyme expression upon GK gene silencing suggests a potential connection between GK and the cellular response to oxidative stress. Together, these results provide the first direct evidence of the biological importance and coordinated regulation of the glycerol metabolic pathways for proliferation and antioxidative defense in E. histolytica, justifying the exploitation of these enzymes as future drug targets.
2023-08-14 | GSE240436 | GEO
Project description:Glycerol kinase plays a critical role in metabolism by converting glycerol to glycerol 3-phosphate in an ATP dependent reaction. In humans, glycerol kinase deficiency results in a wide range of phenotypic variability; patients can have severe metabolic and CNS abnormalities, while others possess hyperglycerolemia and glyceroluria with no other apparent phenotype. In an effort to help understand the pathogenic mechanisms underlying the phenotypic variation, we have created a Drosophila model for glycerol kinase deficiency by RNAi targeting of dGyk (CG18374) and dGK (CG7995). As expected, RNAi flies have reduced glycerol kinase RNA expression, reduced phosphorylation activity and elevated glycerol levels. Further investigation revealed these flies to be hypersensitive to fly food supplemented with glycerol. Due to the hygroscopic nature of glycerol, we predict glycerol hypersensitivity is a result of greater susceptibility to desiccation, suggesting glycerol kinase to play an important role in desiccation resistance in insects. To evaluate a role for genetic modifier loci in determining severity of the glycerol hypersensitivity observed in knockdown flies, we performed a preliminary screen of lethal transposon insertion mutant flies using a glycerol hypersensitive survivorship assay. We demonstrate that this type of screen can identify both enhancer and suppressor genetic loci of glycerol hypersensitivity. Furthermore, we found that the glycerol hypersensitivity phenotype can be enhanced or suppressed by null mutations in eye pigmentation genes. Taken together, our data suggest proteins encoded by eye pigmentation genes play an important role in desiccation resistance and that eye pigmentation genes are strong modifiers of the glycerol hypersensitive phenotype identified in our Drosophila model for glycerol kinase deficiency.
| S-EPMC3302884 | biostudies-literature
Project description:G-protein-coupled-receptor (GPCR) signaling is exquisitely controlled to achieve spatial and temporal specificity. The endogenous protein kinase inhibitor peptide (PKI) confines the spatial and temporal spread of the activity of protein kinase A (PKA), which integrates inputs from three major types of GPCRs. Despite its wide usage as a pharmaceutical inhibitor of PKA, it was unclear whether PKI only inhibits PKA activity. Here, the effects of PKI on 55 mouse kinases were tested in in vitro assays. We found that in addition to inhibiting PKA activity, both PKI (6-22) amide and full-length PKIα facilitated the activation of multiple isoforms of protein kinase C (PKC), albeit at much higher concentrations than necessary to inhibit PKA. Thus, our results call for appropriate interpretation of experimental results using PKI as a pharmaceutical agent. Furthermore, our study lays the foundation to explore the potential functions of PKI in regulating PKC activity and in coordinating PKC and PKA activities.
| S-EPMC7878667 | biostudies-literature
Project description:Glycerol and dihydroxyacetone are metabolized by rabbit kidney-cortex tubules, isolated by collagenase treatment. Half-maximal concentrations of both substrates were determined with regard to uptake rates and product formations. Maximal uptake rates were 643 and 329 mumol/h per g of protein for dihydroxyacetone and glycerol respectively. Glucose and lactate were found as major metabolic products. Glycerol kinase, the enzyme catalysing the first step in renal glycerol and dihydroxyacetone metabolism, was measured radiochemically as described by Newsholme, Robinson & Taylor [(1967) Biochim, Biophys. Acta 132, 338-346] and adapted for studies of the localization of this enzyme along the different structures of rabbit nephron. The results show that glycerol kinase is located exclusively in the proximal segments, i.e. the proximal convoluted tubules and the pars recta, but is negligible in the other structures studied. The activities were close to the maximal dihydroxyacetone uptake rates measured in tubule suspensions.
| S-EPMC1163300 | biostudies-other
Project description:Glycerol metabolism provides a central link between sugar and fatty acid catabolism. In most bacteria, glycerol kinase plays a crucial role in regulating channel/facilitator-dependent uptake of glycerol into the cell. In the firmicute Enterococcus casseliflavus, this enzyme's activity is enhanced by phosphorylation of the histidine residue (His232) located in its activation loop, approximately 25 A from its catalytic cleft. We reported earlier that some mutations of His232 altered enzyme activities; we present here the crystal structures of these mutant GlpK enzymes. The structure of a mutant enzyme with enhanced enzymatic activity, His232Arg, reveals that residues at the catalytic cleft are more optimally aligned to bind ATP and mediate phosphoryl transfer. Specifically, the position of Arg18 in His232Arg shifts by approximately 1 A when compared to its position in wild-type (WT), His232Ala, and His232Glu enzymes. This new conformation of Arg18 is more optimally positioned at the presumed gamma-phosphate location of ATP, close to the glycerol substrate. In addition to structural changes exhibited at the active site, the conformational stability of the activation loop is decreased, as reflected by an approximately 35% increase in B factors ("thermal factors") in a mutant enzyme displaying diminished activity, His232Glu. Correlating conformational changes to alteration of enzymatic activities in the mutant enzymes identifies distinct localized regions that can have profound effects on intramolecular signal transduction. Alterations in pairwise interactions across the dimer interface can communicate phosphorylation states over 25 A from the activation loop to the catalytic cleft, positioning Arg18 to form favorable interactions at the beta,gamma-bridging position with ATP. This would offset loss of the hydrogen bonds at the gamma-phosphate of ATP during phosphoryl transfer to glycerol, suggesting that appropriate alignment of the second substrate of glycerol kinase, the ATP molecule, may largely determine the rate of glycerol 3-phosphate production.
| S-EPMC3158585 | biostudies-literature
Project description: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.
| S-EPMC6385389 | biostudies-literature
Project description:Abstract Background: Hypertriglyceridemia (HTG) is common; however, pseudo-HTG due to high glycerol in glycerol kinase deficiency (GKD, MIM: 307030) is a rare cause of HTG that need to be delineated for appropriate management. GKD is an X-linked recessive disorder characterized by hyperglycerolemia and glyceroluria. Two of three GKD subtypes are known as “isolate” GKD due to a mutation in GK gene alone: (1) symptomatic juvenile form, and (2) benign adult form, associated with an incidental finding of HTG. Since most commercial laboratories determine triglyceride (TG) levels by a glycerol measurement, TG-backbone, patients with GKD are mistakenly labelled as having HTG. Glycerol-blanking is required to reveal the actual TG, but it is costly. Since usual TG-lowering medications are ineffective or even harmful, novel methods to screen for individuals with GKD or pseudo-HTG are necessary. Objective: Through identification of a clinical case of GKD that was diagnosed by glycerol-blanking, we are proposing two potential methods to screen for pseudo-HTG, and presenting their reliability. Methods: The patient was recruited into an IRB-approved study investigating etiologies of dyslipidemia at the University of Pennsylvania. Patient provided consent for medical record review. Results: A 49-year-old man was referred for HTG management. His reported TG levels ranged between 490 and 559 mg/dL without any other adverse lipid levels for several years without a history of pancreatitis or diabetes mellitus. Intriguingly, he reported a family history of HTG. Since TG-lowering medications (fibrates and fish oil) had not reduced his TG levels, specialized lipid analyses were obtained: a non-blanked TG level of 521 mg/dL and a glycerol-blanked TG of 66 mg/dL, consistent with pseudo-HTG or hyperglycerolemia. Repeat glycerol blanked TG levels were 68 and 69 mg/dL, confirming the previous result, and the likely diagnosis of GKD. With two methods, estimated TG levels were calculated, using some of his laboratory values: (1) modified Friedewald equation to solve for TG with a direct LDL (dLDL) value, and (2) the application of a newly developed formula derived from a collection of 17,545 patient samples, to calculate the absolute TG-gap, using apolipoprotein A and B, estimating TG levels (% deltaTG), and determining whether a TG mesurement might be falsely deviated from the “plausible” TG value. Although neither methods showed perfect concordance, the calculated TG-valued derived by the two methods were significantly lower than the non-glycerol blanked TG values. The difference was statistically significant (p<0.05). Conclusion: The patient was clinically diagnosed with GKD, and was taken off of fibrate and the recently added niacin. These two methods can be used quickly to screen for pseudo-HTG or patients with GKD. Currently, it is unknown whether high glycerol levels are associated with high cardiovascular risks.
| S-EPMC7207438 | biostudies-literature
Project description:Glycerol kinase has several diverse activities in mammalian cells. Glycerol kinase deficiency is a complex, single-gene, inborn error of metabolism wherein no genotype-phenotype correlation has been established. Since glycerol kinase has been suggested to exhibit additional activities than glycerol phosphorylation, expression level perturbation in this enzyme may affect cellular physiology globally. To investigate this possibility, we conducted metabolic investigations of wild-type and two glycerol kinase-overexpressing H4IIE rat hepatoma cell lines constructed in this study. The glycerol kinase-overexpressing cell lines exhibited a significantly higher consumption of carbon sources per cell, suggesting excess carbon expenditure. Furthermore, we quantified intracellular metabolic fluxes by employing stable isotope 13C labeling with a mathematically designed substrate mixture, gas chromatography-mass spectrometry, and comprehensive isotopomer balancing. This flux analysis revealed that the pentose phosphate pathway flux in the glycerol kinase-overexpressing cell lines was 2-fold higher than that in the wild-type, in addition to subtler flux changes in other pathways of carbohydrate metabolism. Furthermore, the activity and transcript level of the lipogenic enzyme glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the pentose phosphate pathway, were also about 2-fold higher than that of the wild-type; these data corroborate the flux analysis results. This study shows that glycerol kinase affects carbon metabolism globally, possibly through its additional functions, and highlights glycerol kinase's multifaceted role in cellular physiology.
| S-EPMC2702542 | biostudies-literature