Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.23.16 (HIV-1 protease)
 2,107 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human immunodeficiency virus type 1 (HIV-1) and HIV-2 proteases are dimers of identical subunits. We made a construct for the expression of recombinant one-chain HIV-2 protease dimer, which, like the previously described one-chain HIV-1 protease dimer, is fully active. The constructs for the one-chain dimers of HIV-1 and HIV-2 proteases were modified to produce hybrid one-chain dimers consisting of both HIV-1 and HIV-2 protease monomers. Although the monomers share only 47.5% sequence identity, the hybrid one-chain dimers are fully active, suggesting that the folding of both HIV-1 and HIV-2 protease monomers is functionally similar.
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PMID:Human immunodeficiency virus type 1 and type 2 protease monomers are functionally interchangeable in the dimeric enzymes. 173 Nov 2

Human immunodeficiency virus type 1 (HIV-1) protease optimally catalyzes in the pH range of 4-6 in contrast to nearly all of the other eukaryotic aspartic proteases, which catalyze best in the pH range of 2-4. A possible structural reason for the higher optimal pH of HIV-1 protease is the absence of a hydrogen bond to the carboxyl group of active-site Asp25, which is nearly universally present in others. To investigate this hypothesis, we have mutated residue 28 in HIV-1 protease from alanine to serine. Both the wild-type and the mutant A28S enzymes have been overexpressed in Escherichia coli using a chemically synthesized gene and purified for a comparative study in enzyme kinetics. The kcat and Km values were determined by a radiometric assay for the wild-type enzyme from pH 3.2 to 7.0, and for the mutant enzyme from pH 3.2 to 6.0. The low pK values of the active site of the free enzyme, pKe1, are 3.3 and 3.4 for the wild-type and mutant enzymes, respectively. The low pK values of the active site of the enzyme bound to substrate, pKes1, are 5.1 and 4.3 for the wild-type and mutant enzymes, respectively. The high pK values of the free enzyme, pKe2, are 6.8 and 5.6, and the corresponding ones for the substrate-bound enzyme, pKes2, are 6.9 and 6.0 for the wild-type and mutant enzymes, respectively. The lowering of pK values in mutant HIV-1 protease indicates that the hydroxyl group of Ser28 forms a new hydrogen bond to active-site Asp25 to increase its acidity.
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PMID:Kinetic studies of human immunodeficiency virus type 1 protease and its active-site hydrogen bond mutant A28S. 176 38

Human immunodeficiency virus type 1 (HIV-1) protease was expressed in Escherichia coli as a fusion protein with the N-terminal sequence of IGF-2. The protein accumulated in inclusion bodies as a 40:60 mixture of unprocessed fusion protein and processed protein. A simple purification procedure was developed that yielded 30-40 mg of active protease per liter of fermentation broth with a recovery of 30-40%. The purification process involved the selective extraction of HIV-1 protease from E. coli inclusion bodies with 50% acetic acid and fractional diafiltration to remove impurities and low-molecular-weight protease-related fragments. No chromatographic steps were employed, yet the HIV-1 protease produced by this procedure was greater than 95% pure by SDS-PAGE, reverse-phase HPLC, and N-terminal sequence analysis.
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PMID:Large-scale production of HIV-1 protease from Escherichia coli using selective extraction and membrane fractionation. 852 38

Human immunodeficiency virus type 1 (HIV-1) expresses its structural and functional proteins within Gag-Pol precursor polyproteins. Specific proteolytic processing of the precursors by the viral protease is critical for the maturation and infectivity of viral particles. To observe the influence of autoprocessing on the activation of recombinant HIV-1 protease, we constructed different HIV-1 protease forms, with or without the Phe-Pro bond directly upstream of the protease domain, and expressed them in Escherichia coli systems. We found that the presence of a short upstream sequence of the protease domain, which could generate the original N-terminus of the protease by autoproteolysis of the Phe-Pro bond, resulted in processing of active protease, whereas for a wild-type protease extended only with the initiator methionine, the proteolytic activity was not recovered. Our results suggested that autoprocessing of the direct upstream sequence of the protease domain is an essential step for the activation of recombinant HIV-1 protease in the E. coli expression system. Expression of HIV-1 protease as fusion proteins revealed that the existence of a fusion portion increased the accumulation of expressed protease by affecting its homotypic dimer formation.
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PMID:Autoprocessing: an essential step for the activation of HIV-1 protease. 868 2

Thymidylate synthase (TS) is an essential enzyme of DNA metabolism. We have carried out an extensive insertional mutagenesis of the Escherichia coli TS gene (thyA) using three different methods. Insertion of exogenous sequences at unique restriction sites or at random positions produced defective mutants, whereas comparison of TS sequences from different species allowed us to identify six zones permissive for insertions of exogenous sequences. The insertion of Human immunodeficiency virus type 1 (HIV-1) protease substrate sequences into the permissive sites converted TS to an HIV-1 protease substrate, and the in vivo cleavage of these insertions by the cloned HIV-1 protease conferred a thymidylate synthase-deficient phenotype in some of our E. coli mutant strains. In agreement with crystallographic data, these results show that the permissive sites are located in regions of the TS protein not essential for enzyme activity and accessible to cleavage by HIV protease. These results also show that it is possible to control a growth phenotype in E. coli through the protease-mediated destruction of an essential metabolic enzyme. Because both wild type and thymidylate synthase-deficient phenotypes are selectable on the appropriate growth medium, these thyA mutants could be used for genetic selections of protease inhibitors and analysis of protease specificities.
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PMID:Conversion of thymidylate synthase into an HIV protease substrate. 870 91

Human immunodeficiency virus type 2 (HIV-2) Nef is proteolytically cleaved by the HIV-2-encoded protease. The proteolysis is not influenced by the absence or presence of the N-terminal myristoylation. The main cleavage site is located between residues 39 and 40, suggesting a protease recognition sequence, GGEY-SQFQ. As observed previously for Nef protein from HIV-1, a large, stable core domain with an apparent molecular mass of 30 kDa is produced by the proteolytic activity. Cleavage of Nef from HIV-1 in two domains by its own protease or the protease from HIV-2 is also independent of Nef myristoylation. However, processing of HIV-1 Nef by the HIV-2 protease is less selective than that by the HIV-1 protease: the obtained core fragment is heterogeneous at its N terminus and has an additional cleavage site between amino acids 99 and 100. Preliminary experiments suggest that the full-length Nef of HIV-2 and the core domain are part of the HIV-2 particles, analogous to the situation reported recently for HIV-1.
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PMID:Specific cleavage sites of Nef proteins from human immunodeficiency virus types 1 and 2 for the viral proteases. 897 Oct 42

Human immunodeficiency virus type 1 (HIV-1) protease inhibitors are a promising class of antiretroviral agents that compromise enzymatic function through substrate mimicry. The in vitro susceptibility of a panel of HIV-1 clinical isolates demonstrating various drug resistance phenotypes to combinations of the HIV-1 protease inhibitors saquinavir and indinavir was determined. Antiviral effect was assessed by an HIV-1 p24 antigen reduction assay in phytohemagglutinin-stimulated peripheral blood mononuclear cells after harvesting of cell-free supernatant fluids at peak antigen production (days 4-7). Drug interactions were determined by median-dose-effect analysis, with the combination index (CI) calculated at several inhibitory concentrations (IC50, IC75, IC90, IC95, IC99). The interactive effects ranged from synergy at low efficacy doses to antagonism at higher doses against a pan-susceptible clinical isolate of HIV-1. Against a zidovudine-resistant isolate as well as a multidrug-resistant isolate, the combination of saquinavir and indinavir demonstrated antagonism at all doses.
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PMID:Antagonism between human immunodeficiency virus type 1 protease inhibitors indinavir and saquinavir in vitro. 920 79

Human immunodeficiency virus type 1 (HIV-1) Vif is required for productive infection of T lymphocytes and macrophages. Virions produced in the absence of Vif have abnormal core morphology and those produced in primary T cells carry immature core proteins and low levels of mature capsid (M. Simm, M. Shahabuddin, W. Chao, J. S. Allan, and D. J. Volsky, J. Virol. 69:4582-4586, 1995). To investigate whether Vif influences the activity of HIV-1 protease (PR), the viral enzyme which is responsible for processing Gag and Gag-Pol precursor polyproteins into mature virion components, we transformed bacteria to inducibly express truncated Gag-Pol fusion proteins and Vif. We examined the cleavage of polyproteins consisting of matrix to PR (Gag-PR), capsid to PR (CA-PR), and p6Pol to PR (p6Pol-PR) and evaluated HIV-1 protein processing at specific sites by Western blotting using antibodies against matrix, capsid, and PR proteins. We found that Vif modulates HIV-1 PR activity in bacteria mainly by preventing the release of mature MA and CA from Gag-PR, CA from CA-PR, and p6Pol from p6Pol-PR, with other cleavages being less affected. Using subconstructs of Vif, we mapped this activity to the N-terminal half of the molecule, thus identifying a new functional domain of Vif. Kinetic study of p6Pol-PR autocatalysis in the presence or absence of Vif revealed that Vif and N'Vif reduce the rate of PR-mediated proteolysis of this substrate. In an assay of in vitro proteolysis of a synthetic peptide substrate by purified recombinant PR we found that recombinant Vif and the N-terminal half of the molecule specifically inhibit PR activity at a molar ratio of the N-terminal half of Vif to PR of about 1. These results suggest a mechanism and site of action of Vif in HIV-1 replication and demonstrate novel regulation of a lentivirus PR by an autologous viral protein acting in trans.
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PMID:Human immunodeficiency virus type 1 (HIV-1) protein Vif inhibits the activity of HIV-1 protease in bacteria and in vitro. 922 65

Human immunodeficiency virus type-1 (HIV-1) Rev overcomes negative elements within viral RNAs to allow expression of gag, pol, and env. The effect of Rev on protein and RNA expression of HIV-1 protease (PR)-containing constructs was investigated utilizing transient transfection of COS cells. Rev, through the Rev response element (RRE), resulted in a large increase in proteolytic activity and cytoplasmic RNA accumulation. Furthermore, Rev increased the level of total RNA produced by a PR-containing construct. The increase in cytoplasmic RNA accumulation in the presence of Rev indicated the presence of cis-acting repressor sequences (CRS) within the RNA produced by this construct. Therefore, components of the construct were analyzed for CRS activity. PR sequences in both sense and antisense orientations exhibited CRS activity. RRE sequences alone conferred a small CRS effect. Additional CRS activity was present within an unspliced RNA containing only nef and LTR sequences. These results indicate a novel form of cis-acting repressor activity within HIV-1 PR; this activity is exerted regardless of the orientation of PR and appears to function at the level of cytoplasmic or nuclear RNA stability.
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PMID:Identification of cis-acting repressor activity within human immunodeficiency virus type 1 protease sequences. 926 56

Human immunodeficiency virus type 1 (HIV-1) protease hydrolysis of the Gag CA-p2 cleavage site is crucial for virion maturation and is optimal at acidic pH. To understand the processing of the CA-p2 site, we have determined the structure of HIV-1 protease complexed with an analog of the CA-p2 site, the reduced peptide inhibitor Arg-Val-Leu-r-Phe-Glu-Ala-Ahx-NH2 [r denotes the reduced peptide bond and Ahx 2-aminohexanoic acid (norleucine), respectively]. The crystal structure was refined to an R-factor of 0.17 at 0.21-nm resolution. The crystals have nearly the same lattice as related complexes in P2(1)2(1)2(1) which have twofold disordered inhibitor, but are in space group P2(1). and the asymmetric unit contains two dimers of HIV-1 protease related by 180 degrees rotation. An approximate non-crystallographic symmetry has replaced the exact crystal symmetry resulting in well-ordered inhibitor structure. Each protease dimer binds one ordered inhibitor molecule, but in opposite orientations. The interactions of the inhibitor with the two dimers are very similar for the central P2 Val to P2' Glu residues, but show more variation for the distal P3 Arg and P4' Ahx residues. Importantly, the carboxylate oxygens of Glu at P2' in the inhibitor are within hydrogen-bonding distance of a carboxylate oxygen of Asp30 of the protease suggesting that the two side chains share a proton. This interaction suggests that the enzyme-substrate complex is additionally stabilized at lower pH. The importance of this interaction is emphasized by the absence of polymorphisms of Asp30 in the protease and variants of P2' Glu in the critical CA-p2 cleavage site.
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PMID:Crystallographic analysis of human immunodeficiency virus 1 protease with an analog of the conserved CA-p2 substrate -- interactions with frequently occurring glutamic acid residue at P2' position of substrates. 937 Mar 63


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