Analysis of the steps involved in Dengue virus entry into host cells.
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The initial steps of dengue viral entry have been divided into adsorption and penetration using acid glycine treatment to inactivate extracellular virus after attachment to baby hamster kidney (BHK) cells but prior to penetration. First, we showed that virus infection was accomplished within 2 h after adsorption. Second, the assay was used to examine the properties of dengue envelope E protein-specific monoclonal antibodies (MAbs), lectins, and heparin. We found that three MAbs, 17-2, 46-9, and 51-3, may neutralize dengue 2 virus (DEN-2) through inhibition of not only viral attachment but also of penetration. However, one MAb, 56-3.1, interfered specifically with attachment. Therefore, the functional domains of E protein involved in attachment and penetration may be different. Moreover, studies with lectins indicated that carbohydrates, especially alpha-mannose residues, present on the virion glycoproteins may contribute to binding and penetration of the virus into BHK and mosquito C6/36 cells. Finally, virus infectivity was inhibited by heparin through its blocking effects at both virus attachment and penetration. This suggests that cell surface heparan sulfate functions in both viral attachment and penetration of DEN-2 virus. In conclusion, our results further elucidated some aspects of the dengue virus entry process. (+info)
Pre-steady-state reaction of 5-aminolevulinate synthase. Evidence for a rate-determining product release.
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5-Aminolevulinate synthase (ALAS) is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and the alpha-subclass of purple bacteria. The pyridoxal 5'-phosphate cofactor at the active site undergoes changes in absorptive properties during substrate binding and catalysis that have allowed us to study the kinetics of these reactions spectroscopically. Rapid scanning stopped-flow experiments of murine erythroid 5-aminolevulinate synthase demonstrate that reaction with glycine plus succinyl-CoA results in a pre-steady-state burst of quinonoid intermediate formation. Thus, a step following binding of substrates and initial quinonoid intermediate formation is rate-determining. The steady-state spectrum of the enzyme is similar to that formed in the presence of 5-aminolevulinate, suggesting that release of this product limits the overall rate. Reaction of either glycine or 5-aminolevulinate with ALAS is slow (kf = 0.15 s-1) and approximates kcat. The rate constant for reaction with glycine is increased at least 90-fold in the presence of succinyl-CoA and most likely represents a slow conformational change of the enzyme that is accelerated by succinyl-CoA. The slow rate of reaction of 5-aminolevulinate with ALAS is 5-aminolevulinate-independent, suggesting that it also represents a slow isomerization of the enzyme. Reaction of succinyl-CoA with the enzyme-glycine complex to form a quinonoid intermediate is a biphasic process and may be irreversible. Taken together, the data suggest that turnover is limited by release of 5-aminolevulinate or a conformational change associated with 5-aminolevulinate release. (+info)
A gain-of-function polymorphism in a G-protein coupling domain of the human beta1-adrenergic receptor.
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The beta1-adrenergic receptor (beta1AR) is a key cell surface signaling protein expressed in the heart and other organs that mediates the actions of catecholamines of the sympathetic nervous system. A polymorphism in the intracellular cytoplasmic tail near the seventh transmembrane-spanning segment of the human beta1AR has been identified in a cohort of normal individuals. At amino acid position 389, Gly or Arg can be found (allele frequencies 0.26 and 0. 74, respectively), the former previously considered as the human wild-type beta1AR. Using site-directed mutagenesis to mimic the two variants, CHW-1102 cells were permanently transfected to express the Gly-389 and Arg-389 receptors. In functional studies with matched expression, the Arg-389 receptors had slightly higher basal levels of adenylyl cyclase activities (10.7 +/- 1.2 versus 6.1 +/- 0.4 pmol/min/mg). However, maximal isoproterenol-stimulated levels were markedly higher for the Arg-389 as compared to the Gly-389 receptor (63.3 +/- 6.1 versus 20.9 +/- 2.0 pmol/min/mg). Agonist-promoted [35S]guanosine 5'-O-(thiotriphosphate) binding was also increased with the Arg-389 receptor consistent with enhanced coupling to Gs and increased adenylyl cyclase activation. In agonist competition studies carried out in the absence of guanosine 5'-(beta, gamma-imido)triphosphate, high affinity binding could not be resolved with the Gly-389 receptor, whereas Arg-389 displayed an accumulation of the agonist high affinity receptor complex (RH = 26%). Taken together, these data indicate that this polymorphic variation of the human beta1AR results in alterations of receptor-Gs interaction with functional signal transduction consequences, consistent with its localization in a putative G-protein binding domain. The genetic variation of beta1AR at this locus may be the basis of interindividual differences in pathophysiologic characteristics or in the response to therapeutic betaAR agonists and antagonists in cardiovascular and other diseases. (+info)
Subtype-selective antagonism of N-methyl-D-aspartate receptors by felbamate: insights into the mechanism of action.
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Felbamate is an anticonvulsant used in the treatment of seizures associated with Lennox-Gastaut syndrome and complex partial seizures that are refractory to other medications. Its unique clinical profile is thought to be due to an interaction with N-methyl-D-aspartate (NMDA) receptors, resulting in decreased excitatory amino acid neurotransmission. To further characterize the interaction between felbamate and NMDA receptors, recombinant receptors expressed in Xenopus oocytes were used to investigate the subtype specificity and mechanism of action. Felbamate reduced NMDA- and glycine-induced currents most effectively at NMDA receptors composed of NR1 and NR2B subunits (IC50 = 0.93 mM), followed by NR1-2C (2.02 mM) and NR1-2A (8.56 mM) receptors. The NR1-2B-selective interaction was noncompetitive with respect to the coagonists NMDA and glycine and was not dependent on voltage. Felbamate enhanced the affinity of the NR1-2B receptor for the agonist NMDA by 3.5-fold, suggesting a similarity in mechanism to other noncompetitive antagonists such as ifenprodil. However, a point mutation at position 201 (E201R) of the epsilon2 (mouse NR2B) subunit that affects receptor sensitivity to ifenprodil, haloperidol, and protons reduced the affinity of NR1-epsilon2 receptors for felbamate by only 2-fold. Furthermore, pH had no effect on the affinity of NR1-2B receptors for felbamate. We suggest that felbamate interacts with a unique site on the NR2B subunit (or one formed by NR1 plus NR2B) that interacts allosterically with the NMDA/glutamate binding site. These results suggest that the unique clinical profile of felbamate is due in part to an interaction with the NR1-2B subtype of NMDA receptor. (+info)
Role for the leucine-responsive regulatory protein (Lrp) as a structural protein in regulating the Escherichia coli gcvTHP operon.
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The Escherichia coli glycine-cleavage enzyme system (gcvTHP and lpd gene products) provides C1 units for cellular methylation reactions. Both the GcvA and leucine-responsive regulatory (Lrp) proteins are required for regulation of the gcv operon. One model proposed for gcv regulation is that Lrp plays a structural role, bending the DNA to allow GcvA to function as either an activator or a repressor in response to environmental signals. This hypothesis was tested by replacing all but the upstream 22 bp of the Lrp-binding region in a gcvT::lacZ fusion with the I1A site from phage lambda. Integration host factor (IHF) binds the I1A site and bends the DNA about 140 degrees. Shifting the I1A site by increments of 1 base around the DNA helix resulted in IHF-dependent activation and repression of gcvT::lacZ expression that were face-of-the-helix dependent. Activation was also dependent on the GcvA protein, and repression was dependent on both the GcvA and GcvR proteins, demonstrating that the roles for these proteins were not altered. The results are consistent with Lrp playing primarily a structural role in gcv regulation, although they do not completely rule out the possibility that Lrp also interacts with another gcv-regulatory protein or with RNA polymerase. (+info)
Elevated expression of liver gamma-cystathionase is required for the maintenance of lactation in rats.
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Liver gamma-cystathionase activity increases in rats during lactation; its inhibition due to propargylglycine is followed by a significant decrease in lactation. This is reversible by N-acetylcysteine administration. To study the role of liver gamma-cystathionase and the intertissue flux of glutathione during lactation, we used lactating and virgin rats fed liquid diets. Virgin rats were divided into two groups as follows: one group was fed daily a diet containing the same amount of protein that was consumed the previous day by lactating rats (high protein diet-fed rats); the other virgin group was fed the normal liquid diet (control). The expression and activity of liver gamma-cystathionase were significantly greater in lactating rats and in high protein diet-fed virgin rats compared with control rats. The total glutathione [reduced glutathione (GSH) + oxidized glutathione (GSSG)] released per gram of liver did not differ in lactating rats or in high protein diet-fed rats, but it was significantly higher in these two groups than in control virgin rats. Liver size and the GSH + GSSG released by total liver were significantly higher in lactating rats than in high protein diet-fed virgin rats, and this difference was similar to the amount of glutathione taken up by the mammary gland (454.2 +/- 36.0 nmol/min). The uptake of total glutathione by the lactating mammary gland was much higher than the uptakes of free L-cysteine and L-cystine, which were negligible. These data suggest that the intertissue flux of glutathione is an important mechanism of L-cysteine delivery to the lactating mammary gland, which lacks gamma-cystathionase activity. This emphasizes the physiologic importance of the increased expression and activity of liver gamma-cystathionase during lactation. (+info)
The flexibility of actin filaments as revealed by fluorescence resonance energy transfer. The influence of divalent cations.
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The temperature profile of the fluorescence resonance energy transfer efficiency normalized by the fluorescence quantum yield of the donor in the presence of acceptor, f', was measured in a way allowing the independent investigation of (i) the strength of interaction between the adjacent protomers (intermonomer flexibility) and (ii) the flexibility of the protein matrix within actin protomers (intramonomer flexibility). In both cases the relative increase as a function of temperature in f' is larger in calcium-F-actin than in magnesium-F-actin in the range of 5-40 degrees C, which indicates that both the intramonomer and the intermonomer flexibility of the actin filaments are larger in calcium-F-actin than those in magnesium-F-actin. The intermonomer flexibility was proved to be larger than the intramonomer one in both the calcium-F-actin and the magnesium-F-actin. The distance between Gln41 and Cys374 residues was found to be cation-independent and did not change during polymerization at 21 degrees C. The steady-state fluorescence anisotropy data of fluorophores attached to the Gln41 or Cys374 residues suggest that the microenvironments around these regions are more rigid in the magnesium-loaded actin filament than in the calcium-loaded form. (+info)
Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease.
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The mature form of the cathepsin B-like protease of Leishmania major (LmajcatB) is a 243 amino acid protein belonging to the papain family of cysteine proteases and is 54% identical to human-liver cathepsin B. Despite the high identity and structural similarity with cathepsin B, LmajcatB does not readily hydrolyse benzyloxycarbonyl-Arg-Arg-7-amino-4-methyl coumarin (Z-Arg-Arg-AMC), which is cleaved by cathepsin B enzymes. It does, however, hydrolyse Z-Phe-Arg-AMC, a substrate typically cleaved by cathepsin L and B enzymes. Based upon computer generated protein models of LmajcatB and mammalian cathepsin B, it was predicted that this variation in substrate specificity was attributed to Gly234 at the S2 subsite of LmajcatB, which forms a larger, more hydrophobic pocket compared with mammalian cathepsin B. To test this hypothesis, recombinant LmajcatB was expressed in the Pichia pastoris yeast expression system. The quality of the recombinant enzyme was confirmed by kinetic characterization, N-terminal sequencing, and Western blot analysis. Alteration of Gly234 to Glu, which is found at the corresponding site in mammalian cathepsin B, increased recombinant LmajcatB (rLmajcatB) activity toward Z-Arg-Arg-AMC 8-fold over the wild-type recombinant enzyme (kcat/Km=3740+/-413 M-1.s-1 versus 472+/-72.4 M-1.s-1). The results of inhibition assays of rLmajcatB with an inhibitor of cathepsin L enzymes, K11002 (morpholine urea-Phe-homoPhe-vinylsulphonylphenyl, kinact/Ki=208200+/-36000 M-1. s-1), and a cathepsin B specific inhibitor, CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l- prolin e, kinact/Ki=199200+/-32900 M-1.s-1], support the findings that this protozoan protease has the P2 specificity of cathepsin L-like enzymes while retaining structural homology to mammalian cathepsin B. (+info)