Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), an acyclic nucleoside phosphonate analog, is active against several retroviruses and herpesviruses and has shown anti-human immunodeficiency virus (HIV) activity in clinical trials. Serial passage of HIV type 1 (strain IIIb, in MT2 cells in increasing concentrations of PMEA resulted in viruses with > 12-fold increases in their 50% inhibitory concentrations of PMEA compared with that for strain IIIb. Sequence analyses of these PMEA-selected viruses demonstrated the presence of a novel lysine-to-glutamic acid mutation at amino acid 70 (K70E) in HIV reverse transcriptase. A recombinant virus carrying the K70E mutation was constructed and showed a 10-fold increase in its 50% inhibitory concentrations of PMEA and 2',3'-dideoxy-3'-thiacytidine but showed wild-type susceptibility levels to 2',3'-dideoxycytosine, 2',3'-dideoxyinosine,2',3'-didehydro-2'3'-dideoxythymidine, 3'-azido-3'-deoxythymidine, foscarnet, and two additional phosphonates, 9-[(R)-2-(phosphonomethoxy)propyl]adenine and 9-[2,5-dihydro-5-(phosphonomethoxy)-2-furanyl]adenine. Additionally, the K70E recombinant showed a minor reduction in growth kinetics compared with those of the wild-type virus in vitro.
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PMID:Novel mutation (K70E) in human immunodeficiency virus type 1 reverse transcriptase confers decreased susceptibility to 9-[2-(phosphonomethoxy)ethyl]adenine in vitro. 887 11

The substitution of a glycine for glutamic acid at position 89 in human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT) (E89G) confers resistance to several nucleoside and non-nucleoside inhibitors of RT. As residue 89 contacts the template strand, it has been suggested that this mutation may modulate the conformation of the RT.template/primer complex. In addition, certain mutations in RT that confer resistance to nucleoside analogs, such as M184V, are located near the polymerase active site. To characterize further these substitutions, we performed processivity assays alongside an analysis of pausing profiles with wild-type (wt) RT and recombinant RTs containing substitutions at E89G, M184V, or both. We now show that E89G RT has higher processivity than wt enzyme as well as a different pattern of pausing sites. Similar findings were obtained with the doubly mutated RT, although enzyme containing only the M184V mutation had lower processivity than wt. Consistent with these observations, and from a mechanistic standpoint, both E89G-containing as well as doubly mutated RT had decreased dissociation constants from a complex consisting of RT and template-primer, in comparison with either wt RT or M184V-containing RT. No significant differences were observed among the various enzymes in regard to Km values for the heteropolymeric RNA template used in these studies. Viruses containing the E89G mutation synthesized longer strand DNA products than either wt viruses or viruses containing only the M184V mutation in endogenous RT assays. Thus, the E89G substitution is a dominant determinant in regard to each of the koff values from an RT.template/primer complex, RT processivity, and specific patterns of pausing during DNA polymerization.
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PMID:Dominance of the E89G substitution in HIV-1 reverse transcriptase in regard to increased polymerase processivity and patterns of pausing. 970 31

A minimal, nonamer epitope (TEMEKEGKI) from the reverse transcriptase protein of HIV-1, restricted by H-2Kk, was identified and the function of individual residues determined. Besides classical anchor residues at positions 2 and 9, methionine at position 3 was identified as an important MHC anchor and improved binding of a different (malarial) nonamer epitope to H-2Kk, albeit while also abolishing CTL recognition. Lysine at position 5 was replaceable by alanine for CTL raised against wild-type peptide but abolished recognition for CTL raised against the variant 5ALA peptide, indicating a unidirectional cross-reactivity. Interestingly, one CTL line raised against the 5ALA substituted peptide was permissive for a double substitution at positions 5 and 6, in which lysine was permissive at position 5 only if the adjacent glutamic acid was replaced by alanine. Extensive analysis revealed three distinct patterns of responses with peptides doubly substituted in this region: recognition of both single substitutions but not the double substitution, recognition of only one single substitution but also the double substitution, or recognition of both single substitutions and the double substitution. A second complementary substitution can therefore restore function lost through a first substitution. Thus, no residue acts independently of its neighbors, and pairs of substitutions may give results not predictable from the effects of each taken singly. This finding may have bearing on viral infections (such as HIV), in which the accumulation of two mutations in the epitope may lead to the reengagement of memory CTL previously silenced by the initial mutation.
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PMID:The importance of pairwise interactions between peptide residues in the delineation of TCR specificity. 979 3

A novel metallocarboxypeptidase inhibitor was isolated from the medical leech Hirudo medicinalis. Amino acid sequence analysis provided a nearly complete primary structure. which was subsequently verified and completed by cDNA cloning using reverse transcriptase-polymerase chain reaction/rapid amplification of cDNA end techniques. The inhibitor, called LCI (leech carboxypeptidase inhibitor), is a cysteine-rich polypeptide composed of 66 amino acid residues. It does not show sequence similarity to any other protein except at its C-terminal end. In this region, the inhibitor shares the amino acid sequence -Thr-Cys-X-Pro-Tyr-Val-X with Solanacea carboxypeptidase inhibitors, suggesting a similar mechanism of inhibition where the C-terminal tail of the inhibitor interacts with the active center of metallocarboxypeptidases in a substrate-like manner. This hypothesis is supported by the hydrolytic release of the C-terminal glutamic acid residue of LCI after binding to the enzyme. Heterologous overexpression of LCI in Escherichia coli, either into the medium or as an intracellular thioredoxin fusion protein, yields a protein with full inhibitory activity. Both in the natural and recombinant forms, LCI is a tightly binding, competitive inhibitor of different types of pancreatic-like carboxypeptidases, with equilibrium dissociation constants Ki of 0.2-0.4 x 10(-9) M for the complexes with the pancreatic enzymes A1, A2, and B and plasma carboxypeptidase B. Circular dichroism and nuclear magnetic resonance spectroscopy analysis indicate that recombinant LCI is a compactly folded globular protein, stable to a wide range of pH and denaturing conditions.
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PMID:A carboxypeptidase inhibitor from the medical leech Hirudo medicinalis. Isolation, sequence analysis, cDNA cloning, recombinant expression, and characterization. 983 43

Variations in glucuronidation activities among different individuals have been reported; however, genetic polymorphisms in the genes encoding phase II drug metabolizing UDP-glucuronosyltransferases have not been studied extensively. A novel UGT2B cDNA clone UGT2B4(E458) was isolated from human prostate and LNCaP cell cDNA libraries. The cDNA encoding UGT2B4(E458) is 2097 bp in length and has an open reading frame of 1584 nucleotides encoding a protein of 528 amino acids. Characterization of the UGT2B4(E458) cDNA revealed nucleotide differences with the previously published UGT2B4 and UGT2B11 cDNAs. These variations in the UGT2B4 sequence lead to an amino acid change from aspartic acid to glutamic acid at position 458. In the previous UGT2B11 cDNA (which has subsequently been renamed UGT2B4 (L109,396, D458)), leucine residues are found at positions 109 and 396, whereas phenylalanines are present at these positions in the UGT2B4(D458) and UGT2B4(E458) enzymes. Analysing the genomic DNA of 26 unrelated Caucasian individuals demonstrated the presence of variant alleles encoding UGT2B4(D458) and UGT2B4(E458). Stable expression of UGT2B4(E458) cDNA in HK293 cells demonstrates the presence of a 52 kDa protein, which is in agreement with other characterized (UGT2B proteins. UGT2B4(E458) conjugates hyodeoxycholic acid (HDCA) as well as 4-hydroxyestrone (4-OH-E1), androstane-3alpha,17beta-diol (3alpha-diol) and androsterone (ADT). Specific reverse transcriptase-polymerase chain reaction analysis revealed expression of UGT2B4(D458) and UGT2B4(E458) transcripts in a wide range of extrahepatic tissues, including the liver, kidney, testis, mammary gland, prostate, placenta, adipose, adrenal, skin and lung. Our results suggest that UGT2B4(E458) and UGT2B(E458) are two widely expressed isoenzymes, and that polymorphism in the UGT2B4 gene might be responsible for differences in UGT2B4 enzymatic properties.
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PMID:Characterization and substrate specificity of UGT2B4 (E458): a UDP-glucuronosyltransferase encoded by a polymorphic gene. 1037 68

We describe a new human immunodeficiency virus type 1 (HIV-1) mutational pattern associated with phenotypic resistance to lamivudine (3TC) in the absence of the characteristic replacement of methionine by valine at position 184 (M184V) of reverse transcriptase. Combined genotypic and phenotypic analyses of clinical isolates revealed the presence of moderate levels of phenotypic resistance (between 4- and 50-fold) to 3TC in a subset of isolates that did not harbor the M184V mutation. Mutational cluster analysis and comparison with the phenotypic data revealed a significant correlation between moderate phenotypic 3TC resistance and an increased incidence of replacement of glutamic acid by aspartic acid or alanine and of valine by isoleucine at residues 44 and 118 of reverse transcriptase, respectively. This occurred predominantly in those isolates harboring zidovudine resistance-associated mutations (41L, 215Y). The requirement of the combination of mutations 41L and 215Y with mutations 44D and 44A and/or 118I for phenotypic 3TC resistance was confirmed by site-directed mutagenesis experiments. These data support the assumption that HIV-1 may have access to several different genetic pathways to escape drug pressure or that the increase in the frequency of particular mutations may affect susceptibility to drugs that have never been part of a particular regimen.
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PMID:A novel human immunodeficiency virus type 1 reverse transcriptase mutational pattern confers phenotypic lamivudine resistance in the absence of mutation 184V. 1068 19

DNA replication and repair require a specific mechanism to join the 3'- and 5'-ends of two strands to maintain DNA continuity. In order to understand the details of this process, we studied the activity of the 5' nucleases with substrates containing an RNA template strand. By comparing the eubacterial and archaeal 5' nucleases, we show that the polymerase domain of the eubacterial enzymes is critical for the activity of the 5' nuclease domain on RNA containing substrates. Analysis of the activity of chimeric enzymes between the DNA polymerases from Thermus aquaticus (TaqPol) and Thermus thermophilus (TthPol) reveals two regions, in the "thumb" and in the "palm" subdomains, critical for RNA-dependent 5' nuclease activity. There are two critical amino acids in those regions that are responsible for the high activity of TthPol on RNA containing substrates. Mutating glycine 418 and glutamic acid 507 of TaqPol to lysine and glutamine, respectively, increases its RNA-dependent 5' nuclease activity 4-10-fold. Furthermore, the RNA-dependent DNA polymerase activity is controlled by a completely different region of TaqPol and TthPol, and mutations in this region do not affect the 5' nuclease activity. The results presented here suggest a novel substrate binding mode of the eubacterial DNA polymerase enzymes, called a 5' nuclease mode, that is distinct from the polymerizing and editing modes described previously. The application of the enzymes with improved RNA-dependent 5' nuclease activity for RNA detection using the invasive signal amplification assay is discussed.
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PMID:RNA template-dependent 5' nuclease activity of Thermus aquaticus and Thermus thermophilus DNA polymerases. 1082 84

The hepatitis C virus (HCV) encodes an RNA-dependent RNA polymerase (NS5B), which is indispensable for the viral genome replication. Although structural comparison among HCV NS5B, poliovirus 3D-pol, and human immunodeficiency virus-reverse transcriptase RNA-dependent polymerase reveals the canonical palm, fingers, and thumb domains, the crystal structure of HCV NS5B highlights the presence of a unique A1-loop, which extends from the fingers to the thumb domain (amino acids 12-46), providing many contact points for the proposed "closed" conformation of the enzyme. The polymerase also possesses a tunnel, which starts at the active site and terminates on the back surface of the enzyme. This tunnel of 19 A contains five basic amino acids, which may be engaged in NTP trafficking. In the present study, we exploited the crystal structure of the enzyme to elucidate the involvement of these two structural motifs in enzyme activity by site-directed mutagenesis. As predicted, the replacement of leucine 30 located in the Lambda 1-loop is detrimental to the NS5B activity. Heparin-Sepharose column chromatography and analytical ultracentrifugation experiments strongly suggest a local alteration in the structure of the Leu-30 mutant. An analysis of amino acid substitutions in Arg-222 and Lys-151 within the putative NTP tunnel indicates that Arg-222 was critical in delivering NTPs to the active site, whereas Lys-151 was dispensable. Interestingly, the substitution of lysine 151 for a glutamic acid resulted in an enzyme that was consistently more active in de novo synthesis as well as by "copy-back" mechanism of a self-primed substrate when compared with the wild type NS5B enzyme. Burst kinetic analyses indicate that the gain in function of K151E enzyme was primarily the result of the formation of more productive pre-initiation complexes that were used for the elongation reaction. In contrast to the recent observations, both the wild type and mutant enzymes were monomeric in solution, whereas molecules of higher order were apparent in the presence of RNA template.
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PMID:Modulation of hepatitis C virus RNA-dependent RNA polymerase activity by structure-based site-directed mutagenesis. 1214 89

We have previously described a high molecular mass, asexual stage antigen from Eimeria maxima (EmTFP250), implicated as a target of maternal antibodies produced by breeding hens infected with this protozoan parasite. Following partial purification of the protein by ion exchange chromatography, N-terminal and internal peptide sequences were generated and used in the design of degenerate PCR primers. Using a rapid amplification of cDNA ends PCR-based strategy, the cDNA encoding EmTFP250 has been cloned and sequenced. Translation predicts a mature polypeptide with a molecular mass of 246kDa and an isoelectric point of 4.2. Analysis of the amino acid sequence has revealed a novel member of the TRAP (thrombospondin-related anonymous protein) family, containing 16 thrombospondin type-1 repeats and 31 epidermal growth factor-like calcium binding domains. EmTFP250 also contains two low complex, hydrophilic regions rich in glutamic acid and glycine residues, and a transmembrane domain/cytosolic tail associated with parasite gliding motility that is highly conserved within apicomplexan microneme proteins. The protein has 61% identity (71% similarity) with EtMIC4, a 218kDa microneme protein of Eimeria tenella also rich in epidermal growth factor-like and thrombospondin type-1 domains. Using Southern blotting, the gene encoding EmTFP250 has been determined to be present as a single copy within the genome, and reverse transcriptase-PCR has shown that expression is confined to the asexual stages of development. By employing a PCR-based method, a region of the E. maxima Houghton strain EmTFP250 gene was found conserved in Australian isolates of several (at least four) Eimeria species that parasitise chickens. The characterisation of EmTFP250 adds to the expanding apicomplexan TRAP family and suggests a functional significance for the protein.
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PMID:Molecular characterisation of EmTFP250: a novel member of the TRAP protein family in Eimeria maxima. 1281 49

Pathogenic molecular pathways in cirrhotic liver diseases such as hepatitis C virus (HCV), autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) are poorly characterized. Differentially expressed genes are often important in disease pathogenesis. Suppression subtractive hybridization (SSH) is a genome-wide approach that enriches for differentially expressed mRNA transcripts. We aimed to make novel observations of differential gene expression in cirrhosis using SSH combined with quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Liver transcriptomes in HCV cirrhosis, AIH cirrhosis, PBC, and nondiseased liver tissue were examined by SSH. Resulting complementary DNA (cDNA) clones were rescreened for differential expression by dot-blot hybridization and then sequenced. Selected gene expression was quantified by real-time RT-PCR. Following SSH, 694 clones were rescreened for differential gene expression, of which 145 were sequenced and found to derive from 89 different genes. Seven clones were homologous only with expressed sequence tag (EST) sequences encoding genes having no known function. Up-regulated expression of four genes was confirmed by real-time RT-PCR: transmembrane 4 superfamily member 3 (tetraspanin CO-029) in all forms of cirrhosis, hedgehog interacting protein (HIP) in AIH cirrhosis and chitinase 3-like-1 (HC gp-39 or ykl-40) and arginine-glutamic acid repeat (RERE) in HCV cirrhosis. RERE gene polymorphisms and splice variants were observed in all tissues examined. Tetraspanin CO-029 up-regulation was primarily localized to bile ductular cells. In conclusion, novel observations of differential gene expression in human cirrhosis were made using SSH as the primary discovery tool. In particular, further studies of the RERE gene and its products in HCV associated liver disease are warranted.
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PMID:Novel differential gene expression in human cirrhosis detected by suppression subtractive hybridization. 1293 84


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