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C Debouck

Publications and source records attributed to C Debouck.

At least 55 records · Page 3Linked to original sources
PubMed1992

Proteolysis of an active site peptide of lactate dehydrogenase by human immunodeficiency virus type 1 protease.

The muscle and heart lactate dehydrogenase (LDHs) of rabbit and pig are specifically cleaved at a single position by HIV-1 protease, resulting in the conversion of 36-kDa subunits of the oligomeric enzymes into 21- and 15-kDa protein bands as analyzed by SDS-PAGE. While the proteolysis was observed at neutral pH, it became more pronounced at pH 6.0 and 5.0. The time courses of the cleavage of the 36-kDa subunits were commensurate with the time-dependent loss of both quaternary structure and enzymatic activity. These results demonstrated that deoligomerization of rabbit muscle LDH at acidic pH rendered its subunits more susceptible to proteolysis, suggesting that a partially denatured form of the enzyme was the actual substrate. Proteolytic cleavage of the rabbit muscle enzyme occurred at a decapeptide sequence, His-Gly-Trp-Ile-Leu*Gly-Glu-His-Gly-Asp (scissile bond denoted throughout by an asterisk), which constitutes a "strand-loop" element in the muscle and heart LDH structures and contains the active site histidyl residue His-193. The kinetic parameters Km, Vmax/KmEt, and Vmax/Et for rabbit muscle LDH and the synthetic decapeptide Ac-His-Gly-Trp-Ile-Leu*Gly-Glu-His-Gly-Asp-NH2 were nearly identical, suggesting that the decapeptide within the protein substrate is conformationally mobile, as would be expected for the peptide substrate in solution. Insertion of part of this decapeptide sequence into bacterial galactokinase likewise rendered this protein susceptible to proteolysis by HIV-1 protease, and site-directed mutagenesis of this peptide in galactokinase revealed that the Glu residue at the P2' was important to binding to HIV-1 protease. Crystallographic analysis of HIV-1 protease complexed with a tight-binding peptide analogue inhibitor derived from this decapeptide sequence revealed that the "strand-loop" structure of the protein substrate must adopt a beta-sheet structure upon binding to the protease. The Glu residue in the P2' position of the inhibitor likely forms hydrogen-bonding interactions with both the alpha-amide and gamma-carboxylic groups of Asp-30 in the substrate binding site.

Amino Acid Sequence↗
PubMed1992

Use of protein unfolding studies to determine the conformational and dimeric stabilities of HIV-1 and SIV proteases.

The free energies of dimer dissociation of the retroviral proteases (PRs) of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) were determined by measuring the effects of denaturants on the protein fluorescence upon the unfolding of the enzymes. HIV-1 PR was more stable to denaturation by chaotropes and extremes of pH and temperature than SIV PR, indicating that the former enzyme has greater conformational stability. The urea unfolding curves of both proteases were sigmoidal and single phase. The midpoints of the transition curves increased with increasing protein concentrations. These data were best described by and fitted to a two-state model in which folded dimers were in equilibrium with unfolded monomers. This denaturation model conforms to cases in which protein unfolding and dimer dissociation are concomitant processes in which folded monomers do not exist [Bowie, J. U., & Sauer, R. T. (1989) Biochemistry 28, 7140-7143]. Accordingly, the free energies of unfolding reflect the stabilities of the protease dimers, which for HIV-1 PR and SIV PR were, respectively, delta GuH2O = 14 +/- 1 kcal/mol (Ku = 39 pM) and 13 +/- 1 kcal/mol (Ku = 180 pM). The binding of a tight-binding, competitive inhibitor greatly stabilized HIV-1 PR toward urea-induced unfolding (delta GuH2O = 19.3 +/- 0.7 kcal/mol, Ku = 7.0 fM). There were also profound effects caused by adverse pH on the protein conformation for both HIV-1 PR and SIV PR, resulting in unfolding at pH values above and below the respective optimal ranges of 4.0-8.0 and 4.0-7.0

Aspartic Acid Endopeptidases↗
PubMed1992

Characterization of HIV-1 p24 self-association using analytical affinity chromatography.

Analytical affinity chromatography (AAC) was used to detect and quantitate the self-association of p24gag, the major structural capsid protein of human immunodeficiency virus (HIV-1). p24gag was immobilized on a hydrophilic polymer (methacrylate) chromatographic support. The resulting affinity column was able to interact with soluble p24, as judged by the chromatographic retardation of the soluble protein upon isocratic elution under nonchaotropic binding conditions. The variation of elution volume with soluble protein concentration fit to a monomer-dimer model for self-association. The soluble p24-immobilized p24 association process was observed using both frontal and zonal elution AAC at varying pH values; the dissociation constant was 3-4 x 10(-5) M at pH 7. That p24 monomer associates to dimers was determined in solution using analytical ultracentrifugation. The solution Kd was 1.3 x 10(-5) M at pH 7. AAC in the zonal elution mode provides a simple and rapid means to screen for other HIV-1 macromolecules that may interact with p24 as well as for modulators, including antagonists, of HIV p24 protein assembly.

Antibodies, Monoclonal↗
PubMed1992

The HIV-1 protease as a therapeutic target for AIDS.

HIV produces a small , dimeric aspartyl protease which specifically cleaves the polyprotein precursors encoding the structural proteins and enzymes of the virus. This proteolytic activity is absolutely required for the production of mature, infectious virions and is therefore an attractive target for therapeutic intervention. This review summarizes the strategies and multidisciplinary efforts that have been applied to date to the identification of specific inhibitors of this critical viral enzyme. These inhibitors include rationally designed peptide substrate analogs, compounds conceived from tertiary structure information on the enzyme and natural products. Future directions in the discovery and development of HIV-1 protease inhibitors are also discussed.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Identification and characterization of novel low-temperature-inducible promoters of Escherichia coli.

Escherichia coli promoters that are more active at low temperature (15 to 20 degrees C) than at 37 degrees C were identified by using the transposon Tn5-lac to generate promoter fusions expressing beta-galactosidase (beta-Gal). Tn5-lac insertions that resulted in low-temperature-regulated beta-Gal expression were isolated by selecting kanamycin-resistant mutants capable of growth on lactose minimal medium at 15 degrees C but which grew poorly at 37 degrees C on this medium. Seven independent mutants were selected for further studies. In one such strain, designated WQ11, a temperature shift from 37 degrees C to either 20 or 15 degrees C resulted in a 15- to 24-fold induction of beta-Gal expression. Extended growth at 20 or 15 degrees C resulted in 36- to 42-fold-higher beta-Gal expression over that of cells grown at 37 degrees C. Treatment of WQ11 with streptomycin, reported to induce a response similar to heat shock, failed to induce beta-Gal expression. In contrast, treatment with either chloramphenicol or tetracycline, which mimics a cold shock response, resulted in a fourfold induction of beta-Gal expression in strain WQ11. Hfr genetic mapping studies complemented by physical mapping indicated that in at least three mutants (WQ3, WQ6, and WQ11), Tn5-lac insertions mapped at unique sites where no known cold shock genes have been reported. The Tn5-lac insertions of these mutants mapped to 81, 12, and 34 min on the E. coli chromosome, respectively. The cold-inducible promoters from two of the mutants (WQ3 and WQ11) were cloned and sequenced, and their temperature regulation was examined. Comparison of the nucleotide sequences of these two promoters with the regulatory elements of other known cold shock genes identified the sequence CCAAT as a putative conserved motif.

Anti-Bacterial Agents↗
PubMed1992

Distinction of human immunodeficiency virus type 1 neutralization and infection enhancement by human monoclonal antibodies to glycoprotein 120.

There is increasing evidence that sera from HIV-1-infected individuals contain antibodies that enhance infection by HIV-1 in vitro. Previous work has demonstrated that complement receptors on T lymphoid cells and Fc receptors for IgG (Fc gamma R) on monocytic cells are required for enhanced infection by antibody-complexed HIV-1. Characterization of such infection-enhancing antibodies is essential because immunogenic epitopes which induce enhancing antibodies should be excluded from HIV-1 vaccines. This study was conducted to identify enhancing antibodies involved in Fc R-mediated enhancement of HIV-1 infection employing IgG human monoclonal antibodies (HMAbs) reactive against gp120 of HIV-1, which were produced by B cell lines derived from an HIV-1-infected individual. A potent neutralizing HMAb N70-1.5e did not enhance infection by HIV-1 (IIIB and MN strains), whereas HMAb N70-2.3a mediated enhancement of HIV-1 infection, but had little neutralizing activity. A competition radio immunoassay demonstrated that the two antibodies bind to distinct epitopes. These results indicated that enhancing and neutralizing antibodies can be induced by different epitopes on gp120, suggesting the potential for development of safe vaccines against HIV-1 by exclusion of immunogenic epitopes for enhancing antibodies. We made attempts to identify the epitope on gp120 that is recognized by the enhancing antibody N70-2.3a by using recombinant HIV-1 proteins and found that the antibody binds to a conformational site of nonvariable sequences in the carboxyl half (aa 272-509) of gp120.

Animals↗
PubMed1991

Purification and biochemical characterization of recombinant simian immunodeficiency virus protease and comparison to human immunodeficiency virus type 1 protease.

Simian immunodeficiency virus protease (SIV-PR) was produced in Escherichia coli with a recombinant expression system in which the mature enzyme autoprocessed from a precursor form. Recombinant SIV and HIV-1 (human immunodeficiency virus, type 1) proteases were purified from bacterial cell lysates by use of sequential steps of ammonium sulfate precipitation and size-exclusion and ion-exchange chromatography. The amino acid composition, amino-terminal sequence, and molecular weight (monomer) of the recombinant SIV-PR were in accord with that of the 99 amino acid polypeptide predicted from the SIVMac-PR nucleotide sequence. The active form of SIV-PR was shown to be dimeric by gel filtration chromatography. Inhibition by pepstatin A, time-dependent inactivation by 1,2-epoxy-3-(4-nitrophenoxy)propane, and pH rate profiles using oligopeptide substrates demonstrated that SIV-PR behaves as an aspartic protease. Recombinant HIV-1 Pr55gag precursor was processed in vitro by SIV-PR and HIV-1 PR with indistinguishable proteolytic patterns upon NaDodSO4-polyacrylamide gel electrophoresis. Oligopeptide substrates for HIV-1 PR were found to be suitable substrates for recombinant SIV-PR with the exception of a peptide containing the site identified for p66/p51 cleavage (Phe*Tyr) within HIV-1 reverse transcriptase (RT). Several synthetic peptide analogue inhibitors of HIV-1 PR were also potent inhibitors of SIV-PR, indicating that SIV infection in macaques and rhesus monkeys should be useful models for the preclinical evaluation of acquired immunodeficiency syndrome (AIDS) therapeutics targeted towards the virally encoded HIV-1 protease.

Amino Acid Sequence↗
PubMed1991

Low number of functionally active B lymphocytes in the peripheral blood of HIV-1-seropositive individuals with low p24-specific serum antibody titers.

The in vitro synthesis of HIV-1, p24-, reverse transcriptase (RT)- and gp120-specific immunoglobulin (Ig) G by unstimulated peripheral blood mononuclear cells (PBMC) from 38 asymptomatic and 10 symptomatic HIV-1-seropositive individuals was analysed. In the majority of these individuals, spontaneous production of HIV-1- and gp120-specific IgG from PBMC cultures was demonstrated. In addition, in the majority of the PBMC cultures from individuals with high serum antibody titers to p24, spontaneous production of p24-specific IgG was shown. In contrast, no p24-specific IgG production was detected in PBMC cultures from seropositive individuals with low or no serum antibody titers to p24. A similar relationship between low or absent RT-specific serum antibody titers and the absence of in vitro RT-specific IgG synthesis was not demonstrated. Furthermore, it was shown that the number of p24-specific B lymphocytes in circulation, as calculated by a spot enzyme-linked immunosorbent assay, were significantly lower in individuals with low serum antibody titers to p24. These results suggest that the decline in p24-specific serum antibodies observed during progression towards AIDS is not merely a reflection of the clearance via immune complexes, but may also be attributable, at least in part, to a reduction of p24-specific antibody-producing active B lymphocytes.

Antibody-Producing Cells↗
PubMed1991

An HLA-C-restricted CD8+ cytotoxic T-lymphocyte clone recognizes a highly conserved epitope on human immunodeficiency virus type 1 gag.

A unique epitope on the gag protein of human immunodeficiency virus type 1 (HIV-1), located at amino acid 145 to 150, has been mapped by using a CD8+ cytotoxic T-lymphocyte (CTL) clone. This epitope is highly conserved among 18 HIV-1 strains. The HIV-1 gag-specific human leukocyte antigen (HLA) class I-restricted CD8+ CTL clone was generated from fresh peripheral blood mononuclear cells of an HIV-seropositive donor by stimulation with gamma-irradiated allogeneic peripheral blood mononuclear cells in the presence of an anti-CD3 monoclonal antibody and recombinant interleukin-2. This gag-specific CTL clone killed autologous target cells infected with a recombinant vaccinia virus containing the gag gene of HIV-1 and target cells pulsed with an authentic p24gag construct expressed in Escherichia coli. Fine specificity was determined by using a panel of overlapping 30-amino-acid-long synthetic peptides and subsequently using smaller peptides to precisely map the CTL domain on p24. The epitope is on a highly conserved region, and it overlaps with a major B-cell epitope of gag. This CD8+ T-cell epitope is restricted by HLA-Cw3, which has not been previously identified as a restricting element for human CTL responses.

Amino Acid Sequence↗
PubMed1991

Platelets stimulate expression of endothelin mRNA and endothelin biosynthesis in cultured endothelial cells.

Modulation of the biosynthesis of the vasoconstrictor peptide endothelin was studied in cultured endothelial cells. Immunoreactive endothelin (irET) levels were significantly elevated in conditioned medium from bovine pulmonary artery endothelial (BPAE) or human umbilical vein endothelial cells when coincubated with washed human platelets. Platelets (approximately 200,000 cells/microliters) enhanced irET levels approximately 250% over basal levels. Stimulation of irET levels in BPAE cell-conditioned medium by platelets was time and platelet number dependent. Platelets, as well as thrombin and transforming growth factor-beta 1, stimulated the expression of preproendothelin-1 mRNA in a time-dependent manner. Coincubation of low doses of thrombin (0.1 unit/ml) and subthreshold concentrations of platelets with BPAE cells resulted in a further enhancement of irET levels in conditioned medium. Platelet-mediated stimulation of irET production was not significantly affected by indomethacin (1 microM) or the platelet-activating factor receptor antagonist WEB 2086 (1 microM); however, coincubation of endotoxin (100 ng/ml) with platelets and BPAE cells resulted in significantly higher levels of irET. Whether direct contact or adhesion between platelets and endothelial cells is necessary for stimulating irET release was studied by separating platelets from BPAE cells with a 0.4 microns permeable membrane. Under these conditions, platelets still produced significant elevations (approximately 190% over basal levels) in irET levels in BPAE cell-conditioned medium. In addition, platelet-free buffer from agonist-induced platelet aggregation also significantly enhanced irET production (200% over basal values). These data indicate that a platelet-derived regulatory factor can induce the biosynthesis of endothelin from cultured endothelial cells and also suggest that platelets might play a role in vasomotor regulation via a novel intercellular interaction with the endothelium.

Animals↗
PubMed1991

Contribution of antibody response to recombinant HIV-1 gene-encoded products nef, rev, tat, and protease in predicting development of AIDS in HIV-1-infected individuals.

The relation between antibody-response profiles to Escherichia coli-produced HIV-1 nef, rev, tat, and protease proteins and the risk of developing AIDS was studied using stored serum samples taken sequentially from a cohort of 195 initially symptom-free men who were seropositive for antibodies to HIV-1 structural proteins and 72 men who seroconverted for such antibodies. The AIDS attack rates at 39 months follow-up were significantly higher in the men with negative versus positive antibody profiles to nef, tat, and protease, respectively. [Difference (D) between attack rates = 11.279, 5.884, and 8.322, respectively]. No significant difference was found between men with negative versus positive antibody profiles to rev. The above differences between AIDS attack rates were clearly lower than those reported from the same cohort for men who were serum HIV-1 antigen positive versus negative, and for men with low versus normal CD4+ lymphocyte counts, but with respect to nef antibody-response profiles, resembled the difference reported between anti-HIV-1 core antibody-negative versus antibody-positive men. In the subgroup of men without any of the markers previously found to be predictive of progression to AIDS in the cohort (persistent HIV-1 p24 antigenemia, low anti-HIV-1 anti-core antibody reactivity, and low CD4+ cell counts), antibody profiles to nef, rev, tat, and protease did not contribute to the prediction of outcome of infection. When used in combination with persistent HIV-1 p24 antigenemia and low CD4+ cell counts, negative antibody profiles to nef and protease, respectively, were equally sensitive and specific in predicting progression to AIDS, as was low anti-HIV-1 anti-core antibody reactivity.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Trans-dominant Tat mutants with alterations in the basic domain inhibit HIV-1 gene expression.

The Tat protein of the human immunodeficiency virus type 1 (HIV-1) is required for efficient viral gene expression. By means of mutational analyses, several domains of the Tat protein that are required for complete activation of HIV-1 gene expression have been defined. These include an amino-terminal activating domain, a cysteine-rich dimerization domain, and a basic domain important in the binding of Tat to the trans-activation response element (TAR) and in Tat nuclear localization. Recently, we described a mutation, known as delta tat, which resulted in a protein with a truncated basic domain. This protein had a "trans-dominant" phenotype in that it inhibited wild-type Tat activation of the HIV-1 LTR. To further characterize the requirements for generating a Tat trans-dominant phenotype, we constructed a variety of Tat proteins with truncations or substitutions in the basic domain. A number of these proteins showed a trans-dominant phenotype. These Tat mutants also inhibited activation of the HIV-1 LTR by a protein composed of Tat fused to the prokaryotic R17 (phage MS2) RNA-binding protein in which the R17 recognition element was inserted in the HIV-1 LTR in place of TAR. Thus, an intact TAR element was not required for this inhibition. We also studied the cellular localization of Tat and a trans-dominant Tat mutant by means of immunofluorescence staining with the use of antibodies reactive to different domains of the Tat protein. The results indicated that Tat becomes localized predominantly in the nucleus both in the presence and absence of the trans-dominant Tat construct, suggesting that the trans-dominant mutant does not inhibit Tat nuclear localization. These studies further define the requirements for the creation of trans-dominant Tat mutants, and suggest that the mechanism of trans-dominant Tat inhibition may be either the formation of an inactive complex between wild-type and mutant Tat or sequestration of cellular factors involved in regulating HIV-1 gene expression.

Amino Acid Sequence↗
PubMed1990

Inhibition of HIV-1 protease in infected T-lymphocytes by synthetic peptide analogues.

The gag and pol genes of the human immunodeficiency virus type 1 (HIV-1) (ref. 1) are translated as two polyproteins, Pr55gag and Pr160gag-pol (refs 2-6), which are subsequently cleaved by the action of a virus-encoded protease into the four structural gag proteins of the virion core (p17, p24, p7 and p6) and the pol-encoded enzymes essential for retrovirus replication (protease, reverse transcriptase, ribonuclease H, and endonuclease). Mutational inactivation of the proteases of HIV-1 and other retroviruses results in immature, non-infectious virions, indicating that exogenous inhibition of the protease may represent an attractive approach to anti-AIDS therapy. Here we demonstrate that synthetic peptide analogues, which are potent inhibitors of purified HIV-1 protease, inhibit the processing of the viral polyproteins in cultures of HIV-1-infected T lymphocytes and attenuate viral infectivity.

Amino Acid Sequence↗
PubMed1990

Speed of progression to AIDS and degree of antibody response to accessory gene products of HIV-1.

Antibodies to E. coli-produced HIV-1 nef, rev, tat, vpu, and vpr proteins were measured by enzyme immunoassay in serial sets of sera from 72 men seroconverting for antibodies to HIV-1 structural proteins, and from 190 initially symptom-free men who were seropositive for these antibodies at entry into the study. In the men seroconverting for antibodies to structural proteins the levels of nef-, rev-, and tat-specific antibodies, but not of vpu-, and vpr-specific antibodies, within 3 months of seroconversion, appeared to be lower in the five men progressing to AIDS, compared with the men remaining symptom-free during follow-up. Analysis of the prevalence of previously described antibody profiles to these accessory gene products was carried out. In all HIV-1 antibody seroconverters and in those HIV-1 antibody seropositive men with 15 or more months of follow-up who progressed to AIDS, there was a shift from predominantly nef- and vpu-specific antibody negative profiles in the men developing AIDS in the early years of the study to predominantly nef- and vpu-specific antibody positive profiles in men who developed AIDS later. Rev- and tat-specific antibody negative profiles were dominant in men progressing to AIDS throughout follow-up. No vpr-specific antibody profile occurred preferentially in the men progressing to AIDS throughout follow-up. Low antibody reactivity to accessory gene products nef, rev, and tat appears, like low anti-core antibody reactivity, to be associated with progression to AIDS relatively rapidly after infection with HIV-1.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Identification of an Arg-Gly-Asp (RGD) cell adhesion site in human immunodeficiency virus type 1 transactivation protein, tat.

Tat, the transactivation factor of human immunodeficiency virus type 1 (HIV-1), contains the highly conserved tripeptide sequence Arg-Gly-Asp (RGD) that characterizes sites for integrin-mediated cell adhesion. The tat protein was assayed for cell attachment activity by measuring the adhesion of monocytic, T lymphocytic, and skeletal muscle-derived cell lines to tat-coated substratum. All cell lines tested bound to tat in a dose-dependent manner and the tat cell adhesion required the RGD sequence because tat mutants constructed to contain an RGE or KGE tripeptide sequence did not mediate efficient cell adhesion. The tat-mediated cell attachment also required divalent cations and an intact cytoskeleton. In addition, cell adhesion to tat was inhibited in the presence of an RGD-containing peptide GRGDSPK or an anti-tat mAb that recognizes the RGD epitope. These results strongly suggest that cells are bound to tat through an integrin. Interestingly, myoblast cells bound to tat remained round, whereas the same cells attached through an integrin for a matrix protein typically flatten and spread. The role of this RGD-dependent cellular adhesion of tat in HIV-1 infection remains to be determined.

Amino Acid Sequence↗
PubMed1990

Characterization of murine monoclonal antibodies to the tat protein from human immunodeficiency virus type 1.

A panel of murine monoclonal antibodies (MAbs) to the human immunodeficiency virus type 1 trans-activator tat protein were characterized. The anti-tat MAbs were mapped to the different domains of the tat protein by Western blot (immunoblot) and Pepscan analyses. One-half of the MAbs tested mapped to the amino-terminal proline-rich region, and one-third of the MAbs tested mapped to the lysine-arginine-rich region of tat. The individual MAbs were tested for inhibition of tat-mediated trans activation, using a cell-based in vitro assay system. MAbs which mapped to the amino-terminal region of the tat protein demonstrated the highest degree of inhibition, whereas MAbs reactive to other portions of the molecule exhibited a less pronounced effect on tat function.

Amino Acid Sequence↗
PubMed1990

Antibody response to viral proteins U (vpu) and R (vpr) in HIV-1-infected individuals.

Antibodies to E. coli-produced HIV-1 vpr and vpu were determined by enzyme immunoassay in serial sets of sera from 72 men seroconverting for antibodies to HIV-1 structural proteins, and from 196 initially symptom-free men who were positive for such antibodies at study entry. First detection of vpr- and vpu-specific antibodies always was within 12 months of seroconversion for antibodies to structural proteins. In the combined cohort of 268 men, vpr- and vpu-specific antibodies were found persistently in 26 and 43% of men, respectively. Vpr- and vpu-specific antibodies were transiently detected in 3 and 7%, respectively, and intermittently detected in 18 and 13% of men, respectively. No association was found between the patterns of vpr- or vpu-specific antibody response and clinical outcome. In subjects with different patterns of vpr- and vpu-specific antibody response, no clear temporal relationship existed between the appearance or disappearance of antibodies and the onset of HIV-1-related disease.

Cross-Sectional Studies↗