Structural basis for the resilience of efavirenz (DMP-266) to drug resistance mutations in HIV-1 reverse transcriptase.
(57/992)
BACKGROUND: Efavirenz is a second-generation non-nucleoside inhibitor of HIV-1 reverse transcriptase (RT) that has recently been approved for use against HIV-1 infection. Compared with first-generation drugs such as nevirapine, efavirenz shows greater resilience to drug resistance mutations within HIV-1 RT. In order to understand the basis for this resilience at the molecular level and to help the design of further-improved anti-AIDS drugs, we have determined crystal structures of efavirenz and nevirapine with wild-type RT and the clinically important K103N mutant. RESULTS: The relatively compact efavirenz molecule binds, as expected, within the non-nucleoside inhibitor binding pocket of RT. There are significant rearrangements of the drug binding site within the mutant RT compared with the wild-type enzyme. These changes, which lead to the repositioning of the inhibitor, are not seen in the interaction with the first-generation drug nevirapine. CONCLUSIONS: The repositioning of efavirenz within the drug binding pocket of the mutant RT, together with conformational rearrangements in the protein, could represent a general mechanism whereby certain second-generation non-nucleoside inhibitors are able to reduce the effect of drug-resistance mutations on binding potency. (+info)
A new non-radioactive method for IL-2 bioassay.
(58/992)
An oxidation-reduction (redox) indicator, alamarBlue, was used to measure the bioactivity of interleukin 2 (IL-2). This assay system has several advantages over other bioassays for measuring IL-2. It is a nonradioactive method unlike the conventional tritium-labeled thymidine ([3H]TdR) incorporation assay. The alamarBlue assay is also easier to use than other colorimetric methods, such as the MTT assay, because the alamarBlue assay does not depend on the extraction of insoluble formazan salt, which is time-consuming, error-prone, and cumbersome. Due to its solubility in culture medium and its nontoxicity to cells, alamarBlue provides an easy method to monitor cellular growth using either a fluorescence- or an absorbance-based instrument. The alamarBlue assay is not sample-destructive, unlike the thymidine incorporation and MTT methods. This adds another advantage to the alamarBlue method as the measurement of cellular growth by sample-destructive methods requires as many tubes as time points whereas the alamarBlue method requires only one tube for the entire growth period. In this study, alamarBlue was used to measure the proliferation of the IL-2-dependent cytotoxic T cell line, CTLL-2. The colorimetric change of alamarBlue at 570 nm compared to the reference wavelength, 600 nm, was proportional to the number of viable cells. The sensitivity of the IL-2 assay using alamarBlue was comparable to that of the [3H]thymidine incorporation method. These results demonstrate that the alamarBlue assay is valid for the IL-2 bioassay and that alamarBlue can replace the [3H]thymidine employed in the conventional proliferation assays. (+info)
The mechanism of substrate (aglycone) specificity in beta -glucosidases is revealed by crystal structures of mutant maize beta -glucosidase-DIMBOA, -DIMBOAGlc, and -dhurrin complexes.
(59/992)
The mechanism and the site of substrate (i.e., aglycone) recognition and specificity were investigated in maize beta-glucosidase (Glu1) by x-ray crystallography by using crystals of a catalytically inactive mutant (Glu1E191D) in complex with the natural substrate 2-O-beta-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOAGlc), the free aglycone DIMBOA, and competitive inhibitor para-hydroxy-S-mandelonitrile beta-glucoside (dhurrin). The structures of these complexes and of the free enzyme were solved at 2.1-, 2.1-, 2.0-, and 2.2-A resolution, respectively. The structural data from the complexes allowed us to visualize an intact substrate, free aglycone, or a competitive inhibitor in the slot-like active site of a beta-glucosidase. These data show that the aglycone moiety of the substrate is sandwiched between W378 on one side and F198, F205, and F466 on the other. Thus, specific conformations of these four hydrophobic amino acids and the shape of the aglycone-binding site they form determine aglycone recognition and substrate specificity in Glu1. In addition to these four residues, A467 interacts with the 7-methoxy group of DIMBOA. All residues but W378 are variable among beta-glucosidases that differ in substrate specificity, supporting the conclusion that these sites are the basis of aglycone recognition and binding (i.e., substrate specificity) in beta-glucosidases. The data also provide a plausible explanation for the competitive binding of dhurrin to maize beta-glucosidases with high affinity without being hydrolyzed. (+info)
Therapy with efavirenz plus indinavir in patients with extensive prior nucleoside reverse-transcriptase inhibitor experience: a randomized, double-blind, placebo-controlled trial.
(60/992)
A randomized, double-blind, placebo-controlled trial compared efavirenz (600 mg every 24 h) plus indinavir (1000 mg every 8 h) with placebo (every 24 h) plus indinavir (800 mg every 8 h) among 327 nucleoside analogue reverse-transcriptase inhibitor (NRTI)-experienced human immunodeficiency virus (HIV)-infected adults. Patients received 50 cells/mm(3), >10,000 plasma HIV-1 RNA copies/mL, and no prior protease inhibitor or non-NRTI therapy. Patients had a mean of 2.8 years of prior NRTI therapy. At 24 weeks, plasma HIV-1 RNA level was <400 copies/mL in 68.2% of efavirenz versus 52.4% of placebo recipients (P=.004). CD4 cell count increases were 104+/-9 cells/mm(3) and 77+/-10 cells/mm(3) in efavirenz and placebo recipients, respectively (P=.023). Responses in efavirenz recipients were sustained at 48 weeks. Thus, efavirenz plus indinavir with concomitant NRTIs is effective therapy for NRTI-experienced patients. (+info)
Engineering of a functional human NADH-dependent cytochrome P450 system.
(61/992)
A functional human NADH-dependent cytochrome P450 system has been developed by altering the cofactor preference of human NADPH cytochrome P450 reductase (CPR), the redox partner for P450s. This has been achieved by a single amino acid change of the conserved aromatic amino acid Trp-676, which covers the re-side of the FAD isoalloxazine ring in the nicotinamide-binding site. Of the mutations made, the substitution of Trp-676 with alanine (W676A) resulted in a functional NADH-dependent enzyme, which catalyzed the reduction of cytochrome c and ferricyanide as well as facilitated the metabolism of 7-ethoxyresorufin by CYP1A2. Kinetic analysis measuring cytochrome c activity revealed that the NADH-dependent k(cat) of W676A is equivalent (90%) to the NADPH-dependent k(cat) of the wild-type enzyme, with W676A having an approximately 1,000-fold higher specificity for NADH. The apparent K(M)(NADPH) and K(M)(NADH) values of W676A are 80- and 150-fold decreased, respectively. In accordance with structural data, which show a bipartite binding mode of NADPH, substitution of Trp-676 does not affect 2'-AMP binding as seen by the inhibition of both wild-type CPR and the W676A mutant. Furthermore, NADPH was a potent inhibitor of the W676A NADH-dependent cytochrome c reduction and CYP1A2 activity. Overall, the results show that Trp-676 of human CPR plays a major role in cofactor discrimination, and substitution of this conserved aromatic residue with alanine results in an efficient NADH-dependent cytochrome P450 system. (+info)
Isolation and localization of a cytosolic 10 S triacylglycerol biosynthetic multienzyme complex from oleaginous yeast.
(62/992)
Triacylglycerol is one of the major storage forms of metabolic energy in eukaryotic cells. Biosynthesis of triacylglycerol is known to occur in membranes. We report here the isolation, purification, and characterization of a catalytically active cytosolic 10 S multienzyme complex for triacylglycerol biosynthesis from Rhodotorula glutinis during exponential growth. The complex was characterized and was found to contain lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, acyl-acyl carrier protein synthetase, and acyl carrier protein. The 10 S triacylglycerol biosynthetic complex rapidly incorporates free fatty acids as well as fatty acyl-coenzyme A into triacylglycerol and its biosynthetic intermediates. Lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, and diacylglycerol acyltransferase from the complex were microsequenced. Antibodies were raised against the synthetic peptides corresponding to lysophosphatidic acid acyltransferase and phosphatidic acid phosphatase sequences. Immunoprecipitation and immunolocalization studies show the presence of a cytosolic multienzyme complex for triacylglycerol biosynthesis. Chemical cross-linking studies revealed that the 10 S multienzyme complex was held together by protein-protein interactions. These results demonstrate that the cytosol is one of the sites for triacylglycerol biosynthesis in oleaginous yeast. (+info)
A plastidic ABC protein involved in intercompartmental communication of light signaling.
(63/992)
Plants perceive light via specialized photoreceptors of which the phytochromes (phyA-E), absorbing far-red (FR) and red light (R) are best understood. Several nuclear and cytoplasmic proteins have been characterized whose deficiencies lead to changes in light-dependent morphological responses and gene expression. However, no plastid protein has yet been identified to play a role in phytochrome signal transduction. We have isolated a new Arabidopsis mutant, laf (long after FR) 6, with reduced responsiveness preferentially toward continuous FR light. The disrupted gene in laf6 encodes a novel plant ATP-binding-cassette (atABC1) protein of 557 amino acids with high homology to ABC-like proteins from lower eukaryotes. In contrast to lower eukaryotic ABCs, however, atABC1 contains an N-terminal transit peptide, which targets it to chloroplasts. atABC1 deficiency in laf6 results in an accumulation of the chlorophyll precursor protoporphyrin IX and in attenuation of FR-regulated gene expression. The long hypocotyl phenotype of laf6 and the accumulation of protoporphyrin IX in the mutant can be recapitulated by treating wild-type (WT) seedlings with flumioxazin, a protoporphyrinogen IX oxidase (PPO) inhibitor. Moreover, protoporphyrin IX accumulation in flumioxazin-treated WT seedlings can be reduced by overexpression of atABC1. Consistent with the notion that ABC proteins are involved in transport, these observations suggest that functional atABC1 is required for the transport and correct distribution of protoporphyrin IX, which may act as a light-specific signaling factor involved in coordinating intercompartmental communication between plastids and the nucleus. (+info)
Accumulation of HIF-1alpha under the influence of nitric oxide.
(64/992)
The key player for adaptation to reduced oxygen availability is the transcription factor hypoxia-inducible factor 1 (HIF-1), composed of the redox-sensitive HIF-1alpha and the constitutively expressed HIF-1beta subunits. Under normoxic conditions, HIF-1alpha is rapidly degraded, whereas hypoxia, CoCl(2), or desferroxamine promote protein stabilization, thus evoking its transcriptional activity. Because HIF-1 is regulated by reactive oxygen species, investigation of the impact of reactive nitrogen species was intended. By using different nitric oxide (NO) donors, dose- and time-dependent HIF-1alpha accumulation in close correlation with the release of NO from chemically distinct NO donors was established. Intriguingly, small NO concentrations induced a faster but transient HIF-1alpha accumulation than higher doses of the same NO donor. In contrast, NO attenuated up-regulation of HIF-1alpha evoked by CoCl(2) in a concentration- and time-dependent manner, whereas the desferroxamine-elicited HIF-1alpha signal remained unaltered. To demonstrate an autocrine or paracrine signaling function of NO, we overexpressed the inducible NO synthase and used a coculture system of activated macrophages and tubular cells. Expression of the NO synthase induced HIF-1alpha accumulation, which underscored the role of NO as an intracellular activator for HIF-1. In addition, macrophage-derived NO triggered HIF-1alpha up-regulation in LLC-PK(1) target cells, which points to intercellular signaling properties of NO in achieving HIF-1 accumulation. Our results show that NO does not only modulate the HIF-1 response under hypoxic conditions, but it also functions as a HIF-1 inducer. We conclude that accumulation of HIF-1 occurs during hypoxia but also under inflammatory conditions that are characterized by sustained NO formation. (+info)