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Fumio Sakane - One of the best experts on this subject based on the ideXlab platform.
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Myristic Acid Enhances Diacylglycerol Kinase δ-Dependent Glucose Uptake in Myotubes
Lipids, 2016Co-Authors: Yuko Wada, Shizuka Sakiyama, Hiromichi Sakai, Fumio SakaneAbstract:Decreased expression of Diacylglycerol Kinase (DGK) δ in skeletal muscles attenuates glucose uptake and is closely related to the pathogenesis of type 2 diabetes. Therefore, up-regulation of DGKδ expression is thought to protect and improve glucose homoeostasis in type 2 diabetes. We recently determined that myristic acid (14:0), but not palmitic (16:0) or stearic (18:0) acid, significantly increased DGKδ2 protein expression in mouse C2C12 myotubes. In the current study, we analyzed whether myristic acid indeed enhances glucose uptake in C2C12 myotubes. We observed that myristic acid caused ~1.4-fold increase in insulin-independent glucose uptake. However, palmitic and stearic acids failed to enhance glucose uptake. DGKδ-specific siRNA decreased myristic acid-dependent increase of glucose uptake. Moreover, overexpression of DGKδ2 enhanced glucose uptake in C2C12 cells in the absence of myristic acid treatment. Taken together, these results strongly suggest that myristic acid enhances basal glucose uptake in myotubes in a DGKδ2 expression-dependent manner.
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Distinct 1-monoacylglycerol and 2-monoacylglycerol Kinase activities of Diacylglycerol Kinase isozymes
Biochimica et Biophysica Acta, 2016Co-Authors: Yuriko Sato, Hiromichi Sakai, Chiaki Murakami, Atsumi Yamaki, Satoru Mizuno, Fumio SakaneAbstract:Abstract Diacylglycerol Kinase (DGK) consists of ten isozymes and is involved in a wide variety of patho-physiological events. However, the enzymological properties of DGKs have not been fully understood. In this study, we performed a comprehensive analysis on the 1-monoacylglycerol Kinase (MGK) and 2-MGK activities of ten DGK isozymes. We revealed that type I (α, β and γ), type II (δ, η and κ) and type III (e) DGKs have 7.9–19.2% 2-MGK activity compared to their DGK activities, whereas their 1-MGK activities were
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Diacylglycerol Kinase η1 is a high affinity isozyme for Diacylglycerol.
FEBS Letters, 2015Co-Authors: Suguru Komenoi, Fumika Takemura, Hiromichi Sakai, Fumio SakaneAbstract:Diacylglycerol Kinase (DGK) η plays important roles in various patho-physiological events such as oncogenesis. In this study, we performed an enzymological characterization of DGKη splice variant 1 (DGKη1). The Km value for Diacylglycerol was 0.14 mol%. Intriguingly, the Km value of DGKη1 for Diacylglycerol was at least 9-fold lower than those of other DGK isozymes including DGKα, indicating that DGKη1 is a high affinity isozyme for Diacylglycerol. Therefore, DGKη1 is a unique DGK isozyme, which may function at particular membrane sites where only low concentrations of Diacylglycerol are supplied.
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evaluations of the selectivities of the Diacylglycerol Kinase inhibitors r59022 and r59949 among Diacylglycerol Kinase isozymes using a new non radioactive assay method
Pharmacology, 2013Co-Authors: Mayu Sato, Hiromichi Sakai, Ke Liu, Saori Sasaki, Naoko Kunii, Hirotaka Mizuno, Hiroshi Saga, Fumio SakaneAbstract:Ten mammalian Diacylglycerol Kinase (DGK) isozymes (α-κ) have been identified. Recent studies have revealed that DGK isozymes play pivotal roles in a wide variety of pathophysiological functions. Thus, it is important to be able to easily check DGK activity in each pathophysiological event. Moreover, the conventional DGK assay is quite laborious because it requires the use of a radioisotope and thin-layer chromatography including multiple extraction steps. In order to minimize the laborious procedures, we established a non-radioactive, single well, two-step DGK assay system. We demonstrated that, compared to the conventional method, the new assay system has comparable sensitivity and much higher efficiency, and is effective in detecting potential agents with high reliability (Z'-factor = 0.69 ± 0.12; n = 3). Using the newly developed assay, we comprehensively evaluated the DGK isozyme selectivities of commercially available DGK inhibitors, R59022 and R59949, in vitro. We found that among 10 isozymes, R59022 strongly inhibited type I DGKα and moderately attenuated type III DGKe and type V DGKθ, and that R59949 strongly inhibited type I DGK α and γ, and moderately attenuated type II DGK δ and κ.
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downregulation of Diacylglycerol Kinase delta contributes to hyperglycemia induced insulin resistance
Cell, 2008Co-Authors: Alexander V Chibalin, Fumio Sakane, Ying Leng, Elaine Vieira, Anna Krook, Marie Bjornholm, Yun Chau Long, Olga Kotova, Zhihui Zhong, Tatiana L SteilerAbstract:Type 2 (non-insulin-dependent) diabetes mellitus is a progressive metabolic disorder arising from genetic and environmental factors that impair beta cell function and insulin action in peripheral tissues. We identified reduced Diacylglycerol Kinase δ (DGKδ) expression and DGK activity in skeletal muscle from type 2 diabetic patients. In diabetic animals, reduced DGKδ protein and DGK Kinase activity were restored upon correction of glycemia. DGKδ haploinsufficiency increased Diacylglycerol content, reduced peripheral insulin sensitivity, insulin signaling, and glucose transport, and led to age-dependent obesity. Metabolic flexibility, evident by the transition between lipid and carbohydrate utilization during fasted and fed conditions, was impaired in DGKδ haploinsufficient mice. We reveal a previously unrecognized role for DGKδ in contributing to hyperglycemia-induced peripheral insulin resistance and thereby exacerbating the severity of type 2 diabetes. DGKδ deficiency causes peripheral insulin resistance and metabolic inflexibility. These defects in glucose and energy homeostasis contribute to mild obesity later in life.
Charles R Sanders - One of the best experts on this subject based on the ideXlab platform.
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Prokaryotic Diacylglycerol Kinase and Undecaprenol Kinase
Annual review of biophysics, 2011Co-Authors: Wade D. Van Horn, Charles R SandersAbstract:Prokaryotic Diacylglycerol Kinase (DAGK) and undecaprenol Kinase (UDPK) are the lone members of a family of multispan membrane enzymes that are very small, lack relationships to any other family of proteins—including water soluble Kinases—and exhibit an unusual structure and active site architecture. Escherichia coli DAGK plays an important role in recycling Diacylglycerol produced as a by-product of biosynthesis of molecules located in the periplasmic space. UDPK seems to play an analogous role in gram-positive bacteria, where its importance is evident because UDPK is essential for biofilm formation by the oral pathogen Streptococcus mutans. DAGK has also long served as a model system for studies of membrane protein biocatalysis, folding, stability, and structure. This review explores our current understanding of the microbial physiology, enzymology, structural biology, and folding of the prokaryotic DAGK family, which is based on over 40 years of studies.
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Solution nuclear magnetic resonance structure of membrane-integral Diacylglycerol Kinase
Science, 2009Co-Authors: Wade D. Van Horn, Hak Jun Kim, Charles D. Ellis, Endah S. Sulistijo, Arina Hadziselimovic, Changlin Tian, Murthy D. Karra, Frank D Sonnichsen, Charles R SandersAbstract:Escherichia coli Diacylglycerol Kinase (DAGK) represents a family of integral membrane enzymes that is unrelated to all other phosphotransferases. We have determined the three-dimensional structure of the DAGK homotrimer using solution NMR. The third transmembrane helix from each subunit is domain-swapped with the first and second transmembrane segments from an adjacent subunit. Each of DAGK’s three active sites resembles a portico. The cornice of the portico appears to be the determinant of DAGK’s lipid substrate specificity and overhangs the site of phosphoryl transfer near the water-membrane interface. Mutations to cysteine that caused severe misfolding were located in or near the active site, indicating a high degree of overlap between sites responsible for folding and for catalysis.
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solution nuclear magnetic resonance structure of membrane integral Diacylglycerol Kinase
Science, 2009Co-Authors: Wade D. Van Horn, Hak Jun Kim, Charles D. Ellis, Endah S. Sulistijo, Arina Hadziselimovic, Changlin Tian, Murthy D. Karra, Frank D Sonnichsen, Charles R SandersAbstract:Escherichia coli Diacylglycerol Kinase (DAGK) represents a family of integral membrane enzymes that is unrelated to all other phosphotransferases. We have determined the three-dimensional structure of the DAGK homotrimer with the use of solution nuclear magnetic resonance. The third transmembrane helix from each subunit is domain-swapped with the first and second transmembrane segments from an adjacent subunit. Each of DAGK's three active sites resembles a portico. The cornice of the portico appears to be the determinant of DAGK's lipid substrate specificity and overhangs the site of phosphoryl transfer near the water-membrane interface. Mutations to cysteine that caused severe misfolding were located in or near the active site, indicating a high degree of overlap between sites responsible for folding and for catalysis.
George M. Carman - One of the best experts on this subject based on the ideXlab platform.
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Transcription Factor Reb1p Regulates DGK1-encoded Diacylglycerol Kinase and Lipid Metabolism in Saccharomyces cerevisiae
The Journal of biological chemistry, 2013Co-Authors: Yixuan Qiu, Stylianos Fakas, Gil-soo Han, Symeon Siniossoglou, Antonio Daniel Barbosa, George M. CarmanAbstract:In the yeast Saccharomyces cerevisiae, the DGK1-encoded Diacylglycerol Kinase catalyzes the CTP-dependent phosphorylation of Diacylglycerol to form phosphatidate. This enzyme, in conjunction with PAH1-encoded phosphatidate phosphatase, controls the levels of phosphatidate and Diacylglycerol for phospholipid synthesis, membrane growth, and lipid droplet formation. In this work, we showed that a functional level of Diacylglycerol Kinase is regulated by the Reb1p transcription factor. In the electrophoretic mobility shift assay, purified recombinant Reb1p was shown to specifically bind its consensus recognition sequence (CGGGTAA, −166 to −160) in the DGK1 promoter. Analysis of cells expressing the PDGK1-lacZ reporter gene showed that mutations (GT→TG) in the Reb1p-binding sequence caused an 8.6-fold reduction in β-galactosidase activity. The expression of DGK1(reb1), a DGK1 allele containing the Reb1p-binding site mutation, was greatly lower than that of the wild type allele, as indicated by analyses of DGK1 mRNA, Dgk1p, and Diacylglycerol Kinase activity. In the presence of cerulenin, an inhibitor of de novo fatty acid synthesis, the dgk1Δ mutant expressing DGK1(reb1) exhibited a significant defect in growth as well as in the synthesis of phospholipids from triacylglycerol mobilization. Unlike DGK1, the DGK1(reb1) expressed in the dgk1Δ pah1Δ mutant did not result in the nuclear/endoplasmic reticulum membrane expansion, which occurs in cells lacking phosphatidate phosphatase activity. Taken together, these results indicate that the Reb1p-mediated regulation of Diacylglycerol Kinase plays a major role in its in vivo functions in lipid metabolism.
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dgk1 encoded Diacylglycerol Kinase activity is required for phospholipid synthesis during growth resumption from stationary phase in saccharomyces cerevisiae
Journal of Biological Chemistry, 2011Co-Authors: Stylianos Fakas, Chrysanthos Konstantinou, George M. CarmanAbstract:In the yeast Saccharomyces cerevisiae, triacylglycerol mobilization for phospholipid synthesis occurs during growth resumption from stationary phase, and this metabolism is essential in the absence of de novo fatty acid synthesis. In this work, we provide evidence that DGK1-encoded Diacylglycerol Kinase activity is required to convert triacylglycerol-derived Diacylglycerol to phosphatidate for phospholipid synthesis. Cells lacking Diacylglycerol Kinase activity (e.g. dgk1Δ mutation) failed to resume growth in the presence of the fatty acid synthesis inhibitor cerulenin. Lipid analysis data showed that dgk1Δ mutant cells did not mobilize triacylglycerol for membrane phospholipid synthesis and accumulated Diacylglycerol. The dgk1Δ phenotypes were partially complemented by preventing the formation of Diacylglycerol by the PAH1-encoded phosphatidate phosphatase and by channeling Diacylglycerol to phosphatidylcholine via the Kennedy pathway. These observations, coupled to an inhibitory effect of dioctanoyl-Diacylglycerol on the growth of wild type cells, indicated that Diacylglycerol Kinase also functions to alleviate Diacylglycerol toxicity.
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Characterization of the yeast DGK1-encoded CTP-dependent Diacylglycerol Kinase.
The Journal of biological chemistry, 2008Co-Authors: Gil-soo Han, Symeon Siniossoglou, Laura O'hara, George M. CarmanAbstract:The Saccharomyces cerevisiae DGK1 gene encodes a Diacylglycerol Kinase enzyme that catalyzes the formation of phosphatidate from Diacylglycerol. Unlike the Diacylglycerol Kinases from bacteria, plants, and animals, the yeast enzyme utilizes CTP, instead of ATP, as the phosphate donor in the reaction. Dgk1p contains a CTP transferase domain that is present in the SEC59-encoded dolichol Kinase and CDS1-encoded CDP-Diacylglycerol synthase enzymes. Deletion analysis showed that the CTP transferase domain was sufficient for Diacylglycerol Kinase activity. Point mutations (R76A, K77A, D177A, and G184A) of conserved residues within the CTP transferase domain caused a loss of Diacylglycerol Kinase activity. Analysis of DGK1 alleles showed that the in vivo functions of Dgk1p were specifically due to its Diacylglycerol Kinase activity. The DGK1-encoded enzyme had a pH optimum at 7.0-7.5, required Ca(2+) or Mg(2+) ions for activity, was potently inhibited by N-ethylmaleimide, and was labile at temperatures above 40 degrees C. The enzyme exhibited positive cooperative (Hill number = 2.5) kinetics with respect to Diacylglycerol (apparent K(m) = 6.5 mol %) and saturation kinetics with respect to CTP (apparent K(m) = 0.3 mm). dCTP was both a substrate (apparent K(m) = 0.4 mm) and competitive inhibitor (apparent K(i) = 0.4 mm) of the enzyme. Diacylglycerol Kinase activity was stimulated by major membrane phospholipids and was inhibited by CDP-Diacylglycerol and sphingoid bases.
David Aragao - One of the best experts on this subject based on the ideXlab platform.
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ternary structure reveals mechanism of a membrane Diacylglycerol Kinase
Nature Communications, 2015Co-Authors: Phillip J. Stansfeld, Joseph A Lyons, L Vogeley, David Aragao, Mark S. P. Sansom, Nicole Howe, Aaron P Keogh, Petra FrommeAbstract:Diacylglycerol Kinase catalyses the ATP-dependent conversion of Diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the Kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.
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Ternary structure reveals mechanism of a membrane Diacylglycerol Kinase
Nature Communications, 2015Co-Authors: Dianfan Li, Joseph A Lyons, L Vogeley, David Aragao, Phillip J. Stansfeld, Mark S. P. Sansom, Aaron Keogh, Nicole Howe, Petra Fromme, Raimund FrommeAbstract:Diacylglycerol Kinase is a small bacterial membrane-bound trimer that catalyses Diacylglycerol conversion to phosphatidic acid. Here, the authors solve the crystal structure of the Kinase bound to a lipid substrate and an ATP analogue, and show that the active site arose through convergent evolution. Diacylglycerol Kinase catalyses the ATP-dependent conversion of Diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli . The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the Kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.
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crystal structure of the integral membrane Diacylglycerol Kinase
Nature, 2013Co-Authors: Dianfan Li, Joseph A Lyons, L Vogeley, David Aragao, Colin Peter Kenyon, Syed T A Shah, Christine Doherty, Margaret AherneAbstract:This study presents the crystal structures of three functional forms of Diacylglycerol Kinase, an integral membrane protein that catalyses a crucial step in oligosaccharide and lipopolysaccharide synthesis and assembly; these X-ray structures are markedly different from the only other structure available for this unique Kinase that was solved using solution NMR. The integral membrane enzyme Diacylglycerol Kinase is involved in the synthesis of periplasmic-membrane-derived oligosaccharide and outer-membrane lipopolysaccharide in Gram-negative bacteria. Containing only 121 amino acid residues, this is the smallest known Kinase and it has become a model for the study membrane protein behaviour and enzymology. Martin Caffrey and colleagues report here the X-ray crystal structure of native Diacylglycerol Kinase and of two thermostable, functional mutants at high resolution. Of interest is the fact that the crystal structures are at variance with the only other published structure of Diacylglycerol Kinase, that solved using solution NMR. In an accompanying News & Views, Jimin Zheng and Zongchao Jia discuss the factors that may have contributed to the discrepancy between the NMR and crystal structures. Diacylglycerol Kinase catalyses the ATP-dependent phosphorylation of Diacylglycerol to phosphatidic acid for use in shuttling water-soluble components to membrane-derived oligosaccharide and lipopolysaccharide in the cell envelope of Gram-negative bacteria1. For half a century, this 121-residue Kinase has served as a model for investigating membrane protein enzymology1,2,3,4,5,6, folding7,8, assembly9,10,11,12 and stability1,13. Here we present crystal structures for three functional forms of this unique and paradigmatic Kinase, one of which is wild type. These reveal a homo-trimeric enzyme with three transmembrane helices and an amino-terminal amphiphilic helix per monomer. Bound lipid substrate and docked ATP identify the putative active site that is of the composite, shared site type. The crystal structures rationalize extensive biochemical and biophysical data on the enzyme. They are, however, at variance with a published solution NMR model14 in that domain swapping, a key feature of the solution form, is not observed in the crystal structures.
Timothy A Cross - One of the best experts on this subject based on the ideXlab platform.
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membrane protein structural validation by oriented sample solid state nmr Diacylglycerol Kinase
Biophysical Journal, 2014Co-Authors: Dylan T Murray, Philip F Gao, Huajun Qin, Timothy A CrossAbstract:The validation of protein structures through functional assays has been the norm for many years. Functional assays perform this validation for water-soluble proteins very well, but they need to be performed in the same environment as that used for the structural analysis. This is difficult for membrane proteins that are often structurally characterized in detergent environments, although functional assays for these proteins are most frequently performed in lipid bilayers. Because the structure of membrane proteins is known to be sensitive to the membrane mimetic environment, such functional assays are appropriate for validating the protein construct, but not the membrane protein structure. Here, we compare oriented sample solid-state NMR spectral data of Diacylglycerol Kinase previously published with predictions of such data from recent structures of this protein. A solution NMR structure of Diacylglycerol Kinase has been obtained in detergent micelles and three crystal structures have been obtained in a monoolein cubic phase. All of the structures are trimeric with each monomer having three transmembrane and one amphipathic helices. However, the solution NMR structure shows typical perturbations induced by a micelle environment that is reflected in the predicted solid-state NMR resonances from the structural coordinates. The crystal structures show few such perturbations, especially for the wild-type structure and especially for the monomers that do not have significant crystal contacts. For these monomers the predicted and observed data are nearly identical. The thermostabilized constructs do show more perturbations, especially the A41C mutation that introduces a hydrophilic residue into what would be the middle of the lipid bilayer inducing additional hydrogen bonding between trimers. These results demonstrate a general technique for validating membrane protein structures with minimal data obtained from membrane proteins in liquid crystalline lipid bilayers by oriented sample solid-state NMR.