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991.
Chatterji U Bobardt MD Stanfield R Ptak RG Pallansch LA Ward PA Jones MJ Stoddart CA Scalfaro P Dumont JM Besseghir K Rosenwirth B Gallay PA 《The Journal of biological chemistry》2005,280(48):40293-40300
In this study, we asked if a naturally occurring HIV-1 variant exists that circumvents CypA dependence in human cells. To address this issue, we sought viruses for CypA independence using Debio-025, a cyclosporine A (CsA) analog that disrupts CypA-capsid interaction. Surprisingly, viral variants from the Main group replicate even in the presence of the drug. Sequencing analyses revealed that these viruses encode capsid substitutions within the CypA-binding site (V86P/H87Q/I91V/M96I). When we introduced these substitutions into viruses that normally rely on CypA for replication, these mutants no longer depended on CypA, suggesting that naturally occurring capsid substitutions obviate the need for CypA. This is the first demonstration that isolates from the Main group naturally develop CypA-independent strategies to replicate in human cells. Surprisingly, we found that these capsid substitutions render HIV-1 capable of infecting Owl monkey (OMK) cells that highly restrict HIV-1. OMK cell resistance to HIV-1 is mediated via TRIM-Cyp, which arose from a retrotransposition of CypA into the TRIM5 alpha gene. Interestingly, saturation experiments suggest that the Pro86/Gln87/Val91/Ile96 capsid core is "invisible" to TRIM-Cyp. This study demonstrates that specific capsid substitutions can release HIV-1 from both CypA dependence in human cells and TRIM-Cyp restriction in monkey cells. 相似文献
992.
Evidence for a role of the exocyst in insulin-stimulated Glut4 trafficking in 3T3-L1 adipocytes 总被引:1,自引:0,他引:1
Ewart MA Clarke M Kane S Chamberlain LH Gould GW 《The Journal of biological chemistry》2005,280(5):3812-3816
Insulin stimulates glucose transport in adipocytes and muscle by inducing the redistribution of Glut4 from intracellular locations to the plasma membrane. The fusion of Glut4-containing vesicles at the plasma membrane is known to involve the target SNAREs syntaxin 4 and SNAP-23 and the vesicle SNARE VAMP2. Little is known about the initial docking of Glut4 vesicles with the plasma membrane. A recent report has implicated Exo70, a component of the mammalian exocyst complex, in the initial interaction of Glut4 vesicles with the adipocyte plasma membrane. Here, we have examined the role of two other exocyst components, rsec6 and rsec8. We show that insulin promotes a redistribution of rsec6 and rsec8 to the plasma membrane and to cytoskeletal fractions within 3T3-L1 adipocytes but does not modulate levels of these proteins co-localized with Glut4. We further show that adenoviral-mediated overexpression of either rsec6 or rsec8 increases the magnitude of insulin-stimulated glucose transport in 3T3-L1 adipocytes. By contrast, overexpression of rsec6 or rsec8 did not increase the extent of the secretion of adipsin or ACRP30 from adipocytes and had no discernible effect on transferrin receptor traffic. Collectively, our data support a role for the exocyst in insulin-stimulated glucose transport and suggest a model by which insulin-dependent relocation of the exocyst to the plasma membrane may contribute to the specificity of Glut4 vesicle docking and fusion with the adipocyte plasma membrane. 相似文献
993.
Alderwick LJ Radmacher E Seidel M Gande R Hitchen PG Morris HR Dell A Sahm H Eggeling L Besra GS 《The Journal of biological chemistry》2005,280(37):32362-32371
The cell wall of Mycobacterium tuberculosis has a complex ultrastructure that consists of mycolic acids connected to peptidoglycan via arabinogalactan (AG) and abbreviated as the mAGP complex. The mAGP complex is crucial for the survival and pathogenicity of M. tuberculosis and is the target of several anti-tubercular agents. Apart from sharing a similar mAGP and the availability of the complete genome sequence, Corynebacterium glutamicum has proven useful in the study of orthologous M. tuberculosis genes essential for viability. Here we examined the effects of particular genes involved in AG polymerization by gene deletion in C. glutamicum. The anti-tuberculosis drug ethambutol is thought to target a set of arabinofuranosyltransferases (Emb) that are involved in arabinan polymerization. Deletion of emb in C. glutamicum results in a slow growing mutant with profound morphological changes. Chemical analysis revealed a dramatic reduction of arabinose resulting in a novel truncated AG structure possessing only terminal arabinofuranoside (t-Araf) residues with a corresponding loss of cell wall bound mycolic acids. Treatment of wild-type C. glutamicum with ethambutol and subsequent cell wall analyses resulted in an identical phenotype comparable to the C. glutamicum emb deletion mutant. Additionally, disruption of ubiA in C. glutamicum, the first enzyme involved in the biosynthesis of the sugar donor decaprenol phosphoarabinose (DPA), resulted in a complete loss of cell wall arabinan. Herein, we establish for the first time, (i) that in contrast to M. tuberculosis embA and embB mutants, deletion of C. glutamicum emb leads to a highly truncated AG possessing t-Araf residues, (ii) the exact site of attachment of arabinan chains in AG, and (iii) DPA is the only Araf sugar donor in AG biosynthesis suggesting the presence of a novel enzyme responsible for "priming" the galactan domain for further elaboration by Emb, resulting in the final maturation of the native AG polysaccharide. 相似文献
994.
Harmon LJ Kolbe JJ Cheverud JM Losos JB 《Evolution; international journal of organic evolution》2005,59(2):409-421
Convergent evolution has played an important role in the development of the ecological niche concept. We investigated patterns of convergent and divergent evolution of Caribbean Anolis lizards. These lizards diversified independently on each of the islands of the Greater Antilles, producing the same set of habitat specialists on each island. Using a phylogenetic comparative framework, we examined patterns of morphological convergence in five functionally distinct sets of morphological characters: body size, body shape, head shape, lamella number, and sexual size dimorphism. We find evidence for convergence among members of the habitat specialist types for each of these five datasets. Furthermore, the patterns of convergence differ among at least four of the five datasets; habitat specialists that are similar for one set of characters are often greatly different for another. This suggests that the habitat specialist niches into which these anoles have evolved are multidimensional, involving several distinct and independent aspects of morphology. 相似文献
995.
Shiffman D Ellis SG Rowland CM Malloy MJ Luke MM Iakoubova OA Pullinger CR Cassano J Aouizerat BE Fenwick RG Reitz RE Catanese JJ Leong DU Zellner C Sninsky JJ Topol EJ Devlin JJ Kane JP 《American journal of human genetics》2005,77(4):596-605
Family history is a major risk factor for myocardial infarction (MI). However, known gene variants associated with MI cannot fully explain the genetic component of MI risk. We hypothesized that a gene-centric association study that was not limited to candidate genes could identify novel genetic associations with MI. We studied 11,053 single-nucleotide polymorphisms (SNPs) in 6,891 genes, focusing on SNPs that could influence gene function to increase the likelihood of identifying disease-causing gene variants. To minimize false-positive associations generated by multiple testing, two studies were used to identify a limited number of nominally associated SNPs; a third study tested the hypotheses that these SNPs are associated with MI. In the initial study (of 340 cases and 346 controls), 637 SNPs were associated with MI (P<.05); these were evaluated in a second study (of 445 cases and 606 controls), and 31 of the 637 SNPs were associated with MI (P<.05) and had the same risk allele as in the first study. For each of these 31 SNPs, we tested the hypothesis that it is associated with MI, using a third study (of 560 cases and 891 controls). We found that four of these gene variants were associated with MI (P<.05; false-discovery rate <10%) and had the same risk allele as in the first two studies. These gene variants encode the cytoskeletal protein palladin (KIAA0992 [odds ratio (OR) 1.40]), a tyrosine kinase (ROS1 [OR 1.75]), and two G protein-coupled receptors (TAS2R50 [OR 1.58] and OR13G1 [OR 1.40]); all ORs are for carriers of two versus zero risk alleles. These findings could lead to a better understanding of MI pathophysiology and improved patient risk assessment. 相似文献
996.
Elucidating the specificity of binding of sulfonylurea herbicides to acetohydroxyacid synthase 总被引:2,自引:0,他引:2
Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) is the target for the sulfonylurea herbicides, which act as potent inhibitors of the enzyme. Chlorsulfuron (marketed as Glean) and sulfometuron methyl (marketed as Oust) are two commercially important members of this family of herbicides. Here we report crystal structures of yeast AHAS in complex with chlorsulfuron (at a resolution of 2.19 A), sulfometuron methyl (2.34 A), and two other sulfonylureas, metsulfuron methyl (2.29 A) and tribenuron methyl (2.58 A). The structures observed suggest why these inhibitors have different potencies and provide clues about the differential effects of mutations in the active site tunnel on various inhibitors. In all of the structures, the thiamin diphosphate cofactor is fragmented, possibly as the result of inhibitor binding. In addition to thiamin diphosphate, AHAS requires FAD for activity. Recently, it has been reported that reduction of FAD can occur as a minor side reaction due to reaction with the carbanion/enamine of the hydroxyethyl-ThDP intermediate that is formed midway through the catalytic cycle. Here we report that the isoalloxazine ring has a bent conformation that would account for its ability to accept electrons from the hydroxyethyl intermediate. Most sequence and mutation data suggest that yeast AHAS is a high-quality model for the plant enzyme. 相似文献
997.
Xia Z Azurmendi HF Lairson LL Withers SG Gabelli SB Bianchet MA Amzel LM Mildvan AS 《Biochemistry》2005,44(25):8989-8997
GDP-mannose hydrolase (GDPMH) catalyzes the hydrolysis of GDP-alpha-d-sugars by nucleophilic substitution with inversion at the anomeric C1 atom of the sugar, with general base catalysis by H124. Three lines of evidence indicate a mechanism with dissociative character. First, in the 1.3 A X-ray structure of the GDPMH-Mg(2+)-GDP.Tris(+) complex [Gabelli, S. B., et al. (2004) Structure 12, 927-935], the GDP leaving group interacts with five catalytic components: R37, Y103, R52, R65, and the essential Mg(2+). As determined by the effects of site-specific mutants on k(cat), these components contribute factors of 24-, 100-, 309-, 24-, and >/=10(5)-fold, respectively, to catalysis. Both R37 and Y103 bind the beta-phosphate of GDP and are only 5.0 A apart. Accordingly, the R37Q/Y103F double mutant exhibits partially additive effects of the two single mutants on k(cat), indicating cooperativity of R37 and Y103 in promoting catalysis, and antagonistic effects on K(m). Second, the conserved residue, D22, is positioned to accept a hydrogen bond from the C2-OH group of the sugar undergoing substitution at C1, as was shown by modeling an alpha-d-mannosyl group into the sugar binding site. The D22A and D22N mutations decreased k(cat) by factors of 10(2.1) and 10(2.6), respectively, for the hydrolysis of GDP-alpha-d-mannose, and showed smaller effects on K(m), suggesting that the D22 anion stabilizes a cationic oxocarbenium transition state. Third, the fluorinated substrate, GDP-2F-alpha-d-mannose, for which a cationic oxocarbenium transition state would be destabilized by electron withdrawal, exhibited a 16-fold decrease in k(cat) and a smaller, 2.5-fold increase in K(m). The D22A and D22N mutations further decreased the k(cat) with GDP-2F-alpha-d-mannose to values similar to those found with GDP-alpha-d-mannose, and decreased the K(m) of the fluorinated substrate. The choice of histidine as the general base over glutamate, the preferred base in other Nudix enzymes, is not due to the greater basicity of histidine, since the pK(a) of E124 in the active complex (7.7) exceeded that of H124 (6.7), and the H124E mutation showed a 10(2.2)-fold decrease in k(cat) and a 4.0-fold increase in K(m) at pH 9.3. Similarly, the catalytic triad detected in the X-ray structure (H124- - -Y127- - -P120) is unnecessary for orienting H124, since the Y127F mutation had only 2-fold effects on k(cat) and K(m) with either H124 or E124 as the general base. Hence, a neutral histidine rather than an anionic glutamate may be necessary to preserve electroneutrality in the active complex. 相似文献
998.
Amyloid diseases are caused by the aberrant assembly of a protein in the extracellular space. Folded proteins are not amyloidogenic; however, the native state is generally in equilibrium with a minor population of unfolded or partially folded aggregation-competent conformers outside of the cell. Understanding how the partially unfolded conformers kinetically partition between the competing refolding and aggregation pathways provides insight into how misfolding, which occurs continuously, becomes pathogenic. Towards this end, we have previously studied the amyloidogenicity of transthyretin (TTR), a human beta-sheet-rich homotetrameric protein that must undergo rate-limiting tetramer dissociation and partial monomer unfolding to misassemble into amyloid and other aggregates. We demonstrate herein that TTR homotetramers reassemble by an unusual monomer-dimer-trimer-tetramer (MDRT) pathway. Therefore, the rate of every step in the reassembly pathway is dependent on the concentration of folded TTR monomer. Partitioning soluble TTR monomers between the reassembly pathway and the aggregation pathway should therefore depend on the relative concentrations of aggregates and assembly intermediates. Aggregate clearance is envisioned to play an important role in the partitioning of protein in vivo, where partitioning to the aggregation pathway becomes increasingly favorable under conditions where the concentration of aggregates is increased because aggregate clearance is slow relative to the rate of aggregation. This shift from efficient to inefficient aggregate clearance could occur with aging, offering an explanation for the age-associated nature of these neurodegenerative diseases. 相似文献
999.
Complex I, a key component of the mitochondrial respiratory chain, exhibits diminished activity as a result of cardiac ischemia/reperfusion. Cardiac ischemia/reperfusion is associated with increases in the levels of mitochondrial Ca(2+) and pro-oxidants. In the current in vitro study, we sought evidence for a mechanistic link between Ca(2+), pro-oxidants, and inhibition of complex I utilizing mitochondria isolated from rat heart. Our results indicate that addition of Ca(2+) to solubilized mitochondria results in loss in complex I activity. Ca(2+) induced a maximum decrease in complex I activity of approximately 35% at low micromolar concentrations over a narrow physiologically relevant pH range. Loss in activity required reducing equivalents in the form of NADH and was not reversed upon addition of EGTA. The antioxidants N-acetylcysteine and superoxide dismutase, but not catalase, prevented inhibition, indicating the involvement of superoxide anion (O2(*-)) in the inactivation process. Importantly, the sulfhydryl reducing agent DTT was capable of fully restoring complex I activity implicating the formation of sulfenic acid and/or disulfide derivatives of cysteine in the inactivation process. Finally, complex I can reactivate endogenously upon Ca(2+) removal if NADH is present and the enzyme is allowed to turnover catalytically. Thus, the present study provides a mechanistic link between three alterations known to occur during cardiac ischemia/reperfusion, mitochondrial Ca(2+) accumulation, free radical production, and complex I inhibition. The reversibility of these processes suggests redox regulation of Ca(2+) handling. 相似文献
1000.
Hanlon MH Porter DJ Furfine ES Spaltenstein A Carter HL Danger D Shu AY Kaldor IW Miller JF Samano VA 《Biochemistry》2004,43(45):14500-14507
The arylsulfonamide derivatives described herein were such potent inhibitors of human immunodeficiency virus type 1 (HIV-1) protease (enzyme, E) that values for the inhibition constants (K(i)) could not be determined by conventional steady-state kinetic techniques (i.e., the minimal enzyme concentration usable for the activity assay was much greater than the value of the dissociation constant). Consequently, two alternative methods were developed for estimation of K(i) values. The first method employed kinetic determinations of values for k(1) and k(-1), from which K(i) was determined (k(-1)/k(1)). The second method was a competitive displacement assay used to determine binding affinities of other inhibitors relative to that of GW0385. In these assays, the inhibitor of unknown affinity was used to displace [(3)H]GW0385 from E.[(3)H]GW0385. From the concentration of E.[(3)H]GW0385 at equilibrium, the concentrations of enzyme-bound and free inhibitors were calculated, and the ratio of the K(i) value of the unknown to that of GW0385 was determined (K(i,unknown)/K(i,GW0385)). The values of k(1) were calculated from data in which changes in the intrinsic protein fluorescence of the enzyme associated with inhibitor binding were directly or indirectly monitored. In the case of saquinavir, the fluorescence changes associated with complex formation were large enough to monitor directly. The value of k(1) for saquinavir was 62 +/- 2 microM(-1) s(-1). In the case of GW0385, the fluorescence changes associated with complex formation were too small to monitor directly. Consequently, the value of k(1) was estimated from a competition experiment in which the effect of GW0385 on the binding of E to saquinavir was determined. The value of k(1) for GW0385 was estimated from these experiments to be 137 +/- 4 microM(-1) s(-1). Because E.[(3)H]GW0385 was stable in the standard buffer at room temperature for greater than 33 days, the value of the first-order rate constant for dissociation of E.[(3)H]GW0385 (k(-1)) could be estimated from the time-course for exchange of E.[(3)H]GW0385 with excess unlabeled GW0385. The value of k(-1) calculated from these data was (2.1 +/- 0.1) x10(-6) s(-1) (t(1/2) = 91 h). The K(i) value of wild-type HIV-1 protease for GW0385, calculated from these values for k(1) and k(-1), was 15 +/- 1 fM. Three multidrug resistant enzymes had K(i) values for GW0385 that were less than 5 pM. 相似文献