Interrelationships between 3-hydroxy-3-methylglutaryl-CoA synthase, acetoacetyl-CoA and ketogenesis

Kinetic and physical approaches have been employed to investigate the binding of acetoacetyl-CoA to hydroxymethylglutaryl-CoA synthase. The enzyme has an apparent Km for acetoacetyl-CoA (0.35 microM) which is more than an order of magnitude lower than the Ki (6--10 microM) measured for substrate inhibition by this metabolite. Hepatic acetoacetyl-CoA concentration, as measured by a sensitive and highly specific radioactive assay appears to be in the 1--10 microM range; the concentration decreases during diabetic ketoacidosis. Total hepatic activity of hydroxymethylglutaryl-CoA synthase and levels of mitochondrial enzyme protein, determined by radioimmunoassay, are not appreciably different in livers from control or ketoacidotic animals. In contrast to the decrease in hepatic acetoacetyl-CoA concentration observed during ketoacidosis, myocardial acetoacetyl-CoA levels are increased by at least tenfold when compared to controls. Elevated acetoacetyl-CoA levels may serve to inhibit fatty acid utilization by the heart. Thus, a consideration of the multiple interactions of acetoacetyl-CoA with the enzymes involved in ketone body production and utilization may be useful in evaluating the metabolic significance of this intermediate.     

Catalytic properties of MGAT3, a putative triacylgycerol synthase

Acyl-coenzyme A:monoacylglycerol acyltransferase 3 (MGAT3) is a member of the MGAT family of enzymes that catalyze the synthesis of diacylglycerol (DAG) from monoacylglycerol (MAG), a committed step in dietary fat absorption. Although named after the initial identification of its MGAT activity, MGAT3 shares higher sequence homology with acyl-coenzyme A:diacylglycerol acyltransferase 2 (DGAT2) than with other MGAT enzymes, suggesting that MGAT3 may also possess significant DGAT activity. This study compared the catalytic properties of MGAT3 with those of MGAT1 and MGAT2 enzymes using both MAG and DAG as substrates. Our results showed that in addition to the expected MGAT activity, the recombinant MGAT3 enzyme expressed in Sf-9 insect cells displayed a strong DGAT activity relative to that of MGAT1 and MGAT2 enzymes in the order MGAT3 > MGAT1 > MGAT2. In contrast, none of the three MGAT enzymes recognized biotinylated acyl-CoA or MAG as a substrate. Although MGAT3 possesses full DGAT activity, it differs from DGAT1 in catalytic properties and subcellular localization. The MGAT3 activity was sensitive to inhibition by the presence of 1% CHAPS, whereas DGAT1 activity was stimulated by the detergent. Consistent with high sequence homology with DGAT2, the MGAT3 enzyme demonstrated a similar subcellular distribution pattern to that of DGAT2, but not DGAT1, when expressed in COS-7 cells. Our data suggest that MGAT3 functions as a novel triacylglycerol (TAG) synthase that catalyzes efficiently the two consecutive acylation steps in TAG synthesis.     

Hepoxilin A3 synthase

Hepoxilins constitute a group of 12S-hydroperoxyeicosatetraenoic acid (12S-HpETE)-derived epoxy-hydroxy fatty acids that have been detected in various cell types and tissues. Although hepoxilin A3 (HXA3) exhibits a myriad of biological activities, its biosynthetic mechanism was not investigated in detail. Here we review the isolation, cloning, and characterization of a leukocyte-type 12S-lipoxygenase (12S-LOX) from rat insulinoma cells RINm5F, which exhibits an intrinsic hepoxilin A3 synthase activity. Confirmation for this observation was achieved by coimmunoprecipitation of HXA3 synthase activity with an anti-leukocyte 12S-LOX antibody, preparation of recombinant rat 12S-LOX enzyme from RINm5F cells, and assay of HXA3 synthase activity therein. Site-directed mutagenesis studies performed on rat 12S-LOX showed that 12-lipoxygenating enzyme species exhibit a strong HXA3 synthase activity that is impaired when the positional specificity of arachidonic acid is altered in favor of 15-lipoxygenation. Inasmuch as cellular glutathione peroxidases (cGPx and PHGPx) and HXA3 synthase compete for the same substrate 12S-HpETE, it can be proposed that the overall activity of glutathione peroxidases, representing the overall peroxide tone, finely tunes the rate of HXA3 formation.     

Mechanistic studies on three 2-oxoglutarate-dependent oxygenases of flavonoid biosynthesis: anthocyanidin synthase, flavonol synthase, and flavanone 3beta-hydroxylase

Anthocyanidin synthase (ANS), flavonol synthase (FLS), and flavanone 3beta-hydroxylase (FHT) are involved in the biosynthesis of flavonoids in plants and are all members of the family of 2-oxoglutarate- and ferrous iron-dependent oxygenases. ANS, FLS, and FHT are closely related by sequence and catalyze oxidation of the flavonoid "C ring"; they have been shown to have overlapping substrate and product selectivities. In the initial steps of catalysis, 2-oxoglutarate and dioxygen are thought to react at the ferrous iron center producing succinate, carbon dioxide, and a reactive ferryl intermediate, the latter of which can then affect oxidation of the flavonoid substrate. Here we describe work on ANS, FLS, and FHT utilizing several different substrates carried out in 18O2/16OH2, 16O2/18OH2, and 18O2/18OH2 atmospheres. In the 18O2/16OH2 atmosphere close to complete incorporation of a single 18O label was observed in the dihydroflavonol products (e.g. (2R,3R)-trans-dihydrokaempferol) from incubations of flavanones (e.g. (2S)naringenin) with FHT, ANS, and FLS. This and other evidence supports the intermediacy of a reactive oxidizing species, the oxygen of which does not exchange with that of water. In the case of products formed by oxidation of flavonoid substrates with a C-3 hydroxyl group (e.g. (2R,3R)-trans-dihydroquercetin), the results imply that oxygen exchange can occur at a stage subsequent to initial oxidation of the C-ring, probably via an enzyme-bound C-3 ketone/3,3-gem-diol intermediate.     

Functional characterization of an unusual phytochelatin synthase, LjPCS3, of Lotus japonicus

In plants and many other organisms, phytochelatin synthase (PCS) catalyzes the synthesis of phytochelatins from glutathione in the presence of certain metals and metalloids. We have used budding yeast (Saccharomyces cerevisiae) as a heterologous system to characterize two PCS proteins, LjPCS1 and LjPCS3, of the model legume Lotus japonicus. Initial experiments revealed that the metal tolerance of yeast cells in vivo depends on the concentrations of divalent cations in the growth medium. Detailed in vivo (intact cells) and in vitro (broken cells) assays of PCS activity were performed with yeast expressing the plant enzymes, and values of phytochelatin production for each metal tested were normalized with respect to those of cadmium to correct for the lower expression level of LjPCS3. Our results showed that lead was the best activator of LjPCS1 in the in vitro assay, whereas, for both assays, arsenic, iron, and aluminum were better activators of LjPCS3 and mercury was similarly active with the two enzymes. Most interestingly, zinc was a powerful activator, especially of LjPCS3, when assayed in vivo, whereas copper and silver were the strongest activators in the in vitro assay. We conclude that the in vivo and in vitro assays are useful and complementary to assess the response of LjPCS1 and LjPCS3 to a wide range of metals and that the differences in the C-terminal domains of the two proteins are responsible for their distinct expression levels or stabilities in heterologous systems and patterns of metal activation.     

P24, a glycogen synthase kinase 3 (GSK 3) inhibitor.

A heat resistant glycogen synthase kinase 3 (GSK 3) binding protein, p24, that inhibits its kinase activity at a low magnesium concentration (in a way similar to that of lithium) was found in microtubules from adult rat brains. This protein associates with GSK 3 in microtubules and corresponds to one previously described in the literature as p25, although it has a relative molecular weight of 23472. p24 is a poor substrate for GSK 3 but it could be phosphorylated by other protein kinases such as cAMP dependent protein kinase and cdk 5. Since p24 could form complexes with GSK 3, it may not only regulate GSK 3 activity but also it might act as an anchoring protein for the kinase.     

The regulation of activity of main mevalonic acid pathway enzymes: farnesyl diphosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, and squalene synthase in yeast Saccharomyces cerevisiae

The co-regulation of the main mevalonic acid pathway enzymes was investigated in the yeast Saccharomyces cerevisiae. It was found that a 6-fold increase in FPPS activity compared with that of the wild-type strain FL100 did not cause significant changes in HMG-CoA reductase activity, while the amounts of synthesized dolichols and ergosterol increased by 80 and 32%, respectively. The disruption of the SQS gene in the strain grown in the presence of ergosterol repressed the activities of both FPP synthase and HMG-CoA reductase to a comparable degree, whereas in the same strain starved for ergosterol the activity of FPPS was 10-fold higher and HMG-CoA reductase activity was practically unchanged. We show that FPPS is the enzyme that regulates the flow rate of synthesized mevalonic acid pathway products independent of HMG-CoA reductase and SQS.     

Glycogen synthase kinase-3 regulates microglial migration,inflammation, and inflammation-induced neurotoxicity

Microglia play a prominent role in the brain's inflammatory response to injury or infection by migrating to affected locations, secreting inflammatory molecules, and phagocytosing damaged tissue. However, because severe or chronic neuroinflammation exacerbates many neurological conditions, controlling microglia actions may provide therapeutic benefits in a diverse array of diseases. Since glycogen synthase kinase-3 (GSK3) promotes inflammatory responses in peripheral immune cells, we investigated if inhibitors of GSK3 attenuated microglia responses to inflammatory stimuli. Treatment of BV-2 microglia with GSK3 inhibitors greatly reduced the migration of microglia in both a scratch assay and in a transwell migration assay. Treatment of BV-2 microglia with lipopolysaccharide (LPS) stimulated the production of interleukin-6 and increased the expression of inducible nitric oxide synthase (iNOS) and NO production. Each of these microglia responses to inflammatory stimulation were greatly attenuated by GSK3 inhibitors. However, GSK3 inhibitors did not cause a general impairment of microglia functions, as the LPS-induced stimulated expression of cylcooxygenase-2 was unaltered. Regulation of microglia functions were also evident in cultured mouse hippocampal slices where GSK3 inhibitors reduced cytokine production and microglial migration, and provided protection from inflammation-induced neuronal toxicity. These findings demonstrate that GSK3 promotes microglial responses to inflammation and that the utilization of GSK3 inhibitors provides a means to limit the inflammatory actions of microglia.     

Purification to homogeneity of GD3 synthase and partial purification of GM3 synthase from rat brain

A CMP-sialic acid: GM3 sialyltransferase (GD3 synthase) and a CMP-sialic acid: LacCer sialyltransferase (GM3 synthase) have been purified 10,000- and 3,000-fold, respectively, from the Triton X-100 extract of rat brain. The two enzymes were purified and resolved by affinity chromatography on two successive CDP-Sepharose columns by NaCl gradient elution. Final purification of GD3 synthase was achieved by specific elution from a 'GM3 acid'-Sepharose column with buffer containing GM3. Sodium dodecylsulfate-gel electrophoresis of GD3 synthase revealed a single major protein band with an apparent molecular weight of 55,000.     

Interaction of the 5'-phosphates of the anti-HIV agents, 3'-azido-3'-deoxythymidine and 3'-azido-2',3'-dideoxyuridine, with thymidylate synthase

A study has been made of the interaction of 3'-azido-3'-deoxythymidine 5'-phosphate (AZTMP) and 3'-azido-2',3'-dideoxy-uridine 5'-phosphate (AZdUMP) with thymidylate synthase. With the enzyme from L1210 cells and the tapeworm Hymenolepis diminuta, AZTMP was a weak inhibitor competitive with respect to dUMP (Ki = 6.3 mM and 0.5 mM); hence cytotoxicity of AZT, in cells in which accumulation of AZTMP is not high, is not due to inhibition of cellular thymidylate synthase. AZdUMP, with the L1210 enzyme, was a weak substrate (competition with dUMP described by apparent Ki = 4.7 mM), excluding conversion of AZdUMP to AZTMP as a source of toxicity of 3'-azido-2',3'-dideoxyuridine. An efficient procedure is described for enzymatic phosphorylation on a preparative scale of dideoxynucleosides.     

Enterococcus faecalis 3-hydroxy-3-methylglutaryl coenzyme A synthase,an enzyme of isopentenyl diphosphate biosynthesis

Biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) proceeds via two distinct pathways. Sequence comparisons and microbiological data suggest that multidrug-resistant strains of gram-positive cocci employ exclusively the mevalonate pathway for IPP biosynthesis. Bacterial mevalonate pathway enzymes therefore offer potential targets for development of active site-directed inhibitors for use as antibiotics. We used the PCR and Enterococcus faecalis genomic DNA to isolate the mvaS gene that encodes 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, the second enzyme of the mevalonate pathway. mvaS was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on Ni(2+)-agarose to apparent homogeneity and a specific activity of 10 micromol/min/mg. Analytical ultracentrifugation showed that the enzyme is a dimer (mass, 83.9 kDa; s(20,w), 5.3). Optimal activity occurred in 2.0 mM MgCl(2) at 37(o)C. The DeltaH(a) was 6,000 cal. The pH activity profile, optimum activity at pH 9.8, yielded a pK(a) of 8.8 for a dissociating group, presumably Glu78. The stoichiometry per monomer of acetyl-CoA binding was 1.2 +/- 0.2 and that of covalent acetylation was 0.60 +/- 0.02. The K(m) for the hydrolysis of acetyl-CoA was 10 microM. Coupled conversion of acetyl-CoA to mevalonate was demonstrated by using HMG-CoA synthase and acetoacetyl-CoA thiolase/HMG-CoA reductase from E. faecalis.     

Molecular identification, tissue distribution and subcellular localization of mST3GalV/GM3 synthase

A molecular screen for a mouse homologue of a Drosophila carbohydrate binding protein, called Gliolectin, yielded a cDNA encoding mST3GalV/GM3 synthase (CMP-NeuAc: lactosylceramide alpha2, 3-sialyltransferase). By in situ hybridization and immunohistochemistry, mST3GalV exhibits differential expression in neural and non-neural tissues. Although expressed by all neurons in the central nervous system, neuronal populations that contribute their axons to myelinated efferent projections, such as cerebellar Purkinje cells and spinal motorneurons, demonstrate the highest ST3GalV expression. When stained with anti-mST3GalV antiserum (designated CS2), subpopulations of neurons display an elaborate Golgi apparatus, frequently extending into one or more dendritic processes. The extended spatial distribution of the neuronal Golgi apparatus, particularly in spinal motorneurons, allowed the confocal immunohistochemical colocalization of mST3GalV with markers for medial/trans-Golgi but not the cis-Golgi or trans-Golgi network, consistent with previous observations suggesting that ganglioside glycosyltransferases are enriched in late Golgi compartments. Among non-neural tissues, liver and testes demonstrate cell-type specific CS2 staining. In liver, endothelial cells lining a ring of sinusoids, concentric with the central vein, express mST3GalV. Kupffer cells are also stained with CS2 antiserum but hepatocyte expression is undetectable. In the seminiferous tubules of the testes, ST3GalV is found in somatic (Leydig, Sertoli) and early germline cells (spermatogonia and primary spermatocytes); the epididymal epithelium exhibits intense ST3GalV expression. Since GM3 is a precursor for the synthesis of a- and b-series gangliosides, the range of mST3GalV/GM3 synthase expression among various cell populations indicates that certain cell types possess greater reliance on ganglioside function than others.     

Expression, purification, and characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Pyrococcus furiosus

The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P). DAH7PS from the hyperthermophile Pyrococcus furiosus has been expressed in Escherichia coli. The expressed protein was insoluble but was partially solubilized as a dimer by the inclusion of 200 mM KCl in the cell lysis buffer. An effective two step purification procedure has been developed. The first step resulted in a high degree of purification and involved lysis by sonication at approximately 40 degrees C followed by a heat treatment at 70 degrees C. A continuous assay measuring the loss of PEP at 232 nm at elevated temperatures was also developed. Temperature, pH, and divalent metal ions all had an effect on the extinction coefficient of PEP. Purified recombinant P. furiosus DAH7PS is a dimer with a subunit Mr of 29,226 (determined by ESMS), shows resistance to denaturation by SDS, has activity over a broad pH range, and has an activation energy of 88 kJmol-1. The kinetic parameters are Km (PEP) 120 microM, Km (E4P) 28 microM, and kcat 1.5s-1, at 60 degrees C and pH 6.8. DAH7PS is not inhibited by phenylalanine, tyrosine, or tryptophan. EDTA inactivates the enzyme and enzyme activity is restored by a wide range of divalent metal ions including (in order of decreasing effectiveness): Zn2+, Cd2+, Mn2+, Co2+, Ni2+, Ca2+, Hg2+, and Cu2+. This detailed characterization of the DAH7PS from P. furiosus raises the possibility that the subfamily Ibeta DAH7PS enzymes are metal ion dependent, contrary to previous predictions.     

Inhibitory effect of polycations on phosphorylation of glycogen synthase by glycogen synthase kinase 3

Several polycations were tested for their abilities to inhibit the activity of glycogen synthase kinase 3 (GSK-3). L-Polylysine was the most powerful inhibitor of GSK-3 with half-maximal inhibition of glycogen synthase phosphorylation occurring at approx. 100 nM. D-Polylysine and histone H1 were also inhibitory, but the concentration dependence was complex, and DL-polylysine was the least effective inhibitor. Spermine caused about 50% inhibition of GSK-3 at 0.7 mM and 70% inhibition at 4 mM. Inhibition of GSK-3 by L-polylysine could be blocked or reversed by heparin. A heat-stable polycation antagonist isolated from swine kidney cortex also blocked the inhibitory effect of L-polylysine on GSK-3 and blocked histone H1 stimulation of protein phosphatase 2A activity. Under the conditions tested, L-polylysine also inhibited GSK-3 catalyzed phosphorylation of type II regulatory subunit of cAMP-dependent protein kinase and a 63 kDa brain protein, but only slightly inhibited phosphorylation of inhibitor 2 or proteolytic fragments of glycogen synthase that contain site 3 (a + b + c). L-Polylysine at a concentration (200 nM) that caused nearly complete inhibition of GSK-3 stimulated casein kinase I and casein kinase II, but had virtually no effect on the catalytic subunit of cAMP-dependent protein kinase. These results suggest that polycations can be useful in controlling GSK-3 activity. Polycations have the potential to decrease the phosphorylation state of glycogen synthase at site 3, both by inhibiting GKS-3 as shown in this study and by stimulating the phosphatase reaction as shown previously (Pelech, S. and Cohen, P. (1985) Eur. J. Biochem. 148, 245-251).     

Ubiquitylation of neuronal nitric-oxide synthase by CHIP, a chaperone-dependent E3 ligase

It is established that neuronal nitric-oxide synthase (nNOS) is ubiquitylated and proteasomally degraded. The proteasomal degradation of nNOS is enhanced by suicide inactivation of nNOS or by the inhibition of hsp90, which is a chaperone found in a native complex with nNOS. In the current study, we have examined whether CHIP, a chaperone-dependent E3 ubiquitin-protein isopeptide ligase that is known to ubiquitylate other hsp90-chaperoned proteins, could act as an ubiquitin ligase for nNOS. We found with the use of HEK293T or COS-7 cells and transient transfection methods that CHIP overexpression causes a decrease in immunodetectable levels of nNOS. The extent of the loss of nNOS is dependent on the amount of CHIP cDNA used for transfection. Lactacystin (10 microM), a selective proteasome inhibitor, attenuates the loss of nNOS in part by causing the nNOS to be found in a detergent-insoluble form. Immunoprecipitation of the nNOS and subsequent Western blotting with an anti-ubiquitin IgG shows an increase in nNOS-ubiquitin conjugates because of CHIP. Moreover, incubation of nNOS with a purified system containing an E1 ubiquitin-activating enzyme, an E2 ubiquitin carrier protein conjugating enzyme (UbcH5a), CHIP, glutathione S-transferase-tagged ubiquitin, and an ATP-generating system leads to the ubiquitylation of nNOS. The addition of purified hsp70 and hsp40 to this in vitro system greatly enhances the amount of nNOS-ubiquitin conjugates, suggesting that CHIP is an E3 ligase for nNOS whose action is facilitated by (and possibly requires) its interaction with nNOS-bound hsp70.