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J M Louis

Publications and source records attributed to J M Louis.

At least 55 records · Page 3Linked to original sources
PubMed1997

Studies on the symmetry and sequence context dependence of the HIV-1 proteinase specificity.

Two major types of cleavage sites with different sequence preferences have been proposed for the human immunodeficiency virus type 1 (HIV-1) proteinase. To understand the nature of these sequence preferences better, single and multiple amino acid substitutions were introduced into a type 1 cleavage site peptide, thus changing it to a naturally occurring type 2 cleavage site sequence. Our results indicated that the previous classification of the retroviral cleavage sites may not be generally valid and that the preference for a residue at a particular position in the substrate depends strongly on the neighboring residues, including both those at the same side and at the opposite side of the peptide backbone of the substrate. Based on these results, pseudosymmetric (palindromic) substrates were designed. The retroviral proteinases are symmetrical dimers of two identical subunits; however, the residues of naturally occurring cleavage sites do not show symmetrical arrangements, and no obvious symmetrical substrate preference has been observed for the specificity of HIV proteinase. To examine the role of the asymmetry created by the peptide bonds on the specificity of the respective primed and nonprimed halves of the binding site, amino acid substitutions were introduced into a palindromic sequence. In general, the results suggested that the asymmetry does not result in substantial differences in specificity of the S3 and S3' subsites, whereas its effect is more pronounced for the S2 and S2' subsites. Although it was possible to design several good palindromic substrates, asymmetrical arrangements may be preferred by the HIV proteinase.

HIV Protease↗
PubMed1996

Influence of flanking sequences on the dimer stability of human immunodeficiency virus type 1 protease.

The maturation of the human immunodeficiency virus type 1 protease (PR) from the Gag-Pol polyprotein is dependent on the intrinsic proteolytic activity of the dimeric Gag-Pol. Herein, we report the kinetics and conformational stabilities of two unique fusion proteins of the protease. In one, X28-PR, a random sequences of 28 amino acids (X28) was linked to the N terminus of the mature protease. In the second construct, X28-delta TF*PR*delta Pol, X28 is fused to the protease which is flanked at both its termini by short sequences (delta) which correspond to the native sequences of the Gag-Pol precursor. Autoprocessing of the latter protein was prevented by inserting an Ala at the native protease cleavage sites. The measured kinetic parameters and the pH-rate profile of both enzymes are nearly identical to those of the mature protease. However, these fusion proteins are more sensitive to acid and urea denaturation than the mature protease. The decrease in the conformational stability of X28-PR and X28-delta TF*PR*delta Pol is reflected by increases in their apparent dissociation constants (Kd) from < 5 nM to approximately 180 and 25 nM, respectively. These results suggest that subunit interactions and hence the dimer stability of the protease domain in the Gag-Pol polyprotein differ from those of the mature protease. The high Kd of X28-PR further suggests that addition of non-native sequences to the N terminus of the protease destablizes the dimer.

Amino Acid Sequence↗
PubMed1996

A transient precursor of the HIV-1 protease. Isolation, characterization, and kinetics of maturation.

Recently, the mechanism of autoprocessing of the protease (PR) of the human immunodeficiency virus type 1 from the model polyprotein, MBP-DeltaTF-PR-DeltaPol, which contains the protease linked to short native flanking sequences (DeltaTF and DeltaPol) fused to the maltose binding protein (MBP) of Escherichia coli, was reported (Louis, J. M., Nashed, N. T., Parris, K. D., Kimmel, A. R., and Jerina, D. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7970-7974). According to this mechanism, intramolecular cleavage of the N-terminal strands of the dimeric MBP-DeltaTF-PR-DeltaPol protein leads to the formation of the PR-DeltaPol intermediate, which is subsequently converted to the mature protease by cleavage of the C-terminal strands. We now report the purification and characterization of the PR-DeltaPol intermediate and the kinetics of its processing to the mature protease. Unlike the MBP-DeltaTF-PR-DeltaPol precursor, PR-DeltaPol has proteolytic activity similar to that of the mature enzyme at pH 5.0. The pH rate profile for kcat/Km is similar to that of the mature protease above pH 4.0. Although the PR-DeltaPol is more sensitive than the mature protease toward denaturing reagents, both the enzymatic activity and the intrinsic fluorescence of PR-DeltaPol are linearly dependent on the protein concentration, indicating that the protein is largely in its dimeric form above 10 nM. In contrast to the first-order kinetics observed for the proteolytic reaction at the N terminus of the protease, the proteolytic reaction at the C terminus of the protease is second order in protein concentration. These results are discussed in terms of a mechanism in which the C-terminally located DeltaPol peptide chains are cleaved intermolecularly to release the mature protease.

Amino Acid Sequence↗
PubMed1994

Removal of zinc is required for processing of the mature nucleocapsid protein of human immunodeficiency virus, type 1, by the viral protease.

In human immunodeficiency virus, RNA selection and packaging during assembly involve the two retroviral-type fingers of the nucleocapsid protein that are held in a constrained configuration by coordinated zinc ions. In this report, we demonstrate that the nucleocapsid protein in a metal bound state is resistant to cleavage by the viral protease, but upon removal of zinc ions by chelating agents, it is hydrolyzed within the first zinc finger between Phe-16 and Asn-17. However, 3-nitrosobenzamide and cupric ions, which release zinc through oxidation of the cysteine residues of the finger, render the nucleocapsid protein resistant to cleavage. Since protease inhibitors and 3-nitrosobenzamide restrict processes relating to steps early in infection, the cleavage of the nucleocapsid protein may represent an essential event that can be exploited for the design of novel antiviral agents.

Amino Acid Sequence↗
PubMed1994

The cAMP receptor CAR4 regulates axial patterning and cellular differentiation during late development of Dictyostelium.

Pseudoplasmodia of developing Dictyostelium are organized with anteroposterior polarity. We have isolated CAR4, the gene for a new cell-surface, G protein-linked cAMP receptor. CAR4 mRNA is initially expressed during tip elongation and continues to accumulate into culmination. CAR4 is maximally expressed in pseudoplasmodia anteriors which are centers for extracellular cAMP signaling and for organization of cellular patterning. Although car4 null cells progress unperturbed through early development, they exhibit major patterning aberrations as the anteroposterior axis becomes established. Prestalk gene expression is significantly reduced in car4 nulls, whereas prespore-specific markers are overexpressed and detected in zones normally restricted to prestalk cells. Patterning defects are similarly apparent in terminally differentiated fruiting bodies. Our results show that cAMP signaling is required for pattern formation and cellular differentiation during late Dictyostelium development.

Amino Acid Sequence↗
PubMed1994

Kinetics and mechanism of autoprocessing of human immunodeficiency virus type 1 protease from an analog of the Gag-Pol polyprotein.

Upon renaturation, the polyprotein MBP-delta TF-Protease-delta Pol, consisting of HIV-1 protease and short native sequences from the trans-frame protein (delta TF) and the polymerase (delta Pol) fused to the maltose-binding protein (MBP) of Escherichia coli, undergoes autoprocessing to produce the mature protease in two steps. The initial step corresponds to cleavage of the N-terminal sequence to release the protein intermediate Protease-delta Pol, which has enzymatic activity comparable to that of the mature enzyme. Subsequently, the mature enzyme is formed by a slower cleavage at the C terminus. The rate of increase in enzymatic activity is identical to that of the appearance of MBP-delta TF and the disappearance of the MBP-delta TF-Protease-delta Pol. Initial rates are linearly dependent on the protein concentration, indicating that the N-terminal cleavage is first-order in protein concentration. The reaction is competitively inhibited by pepstatin A and has a pH rate profile similar to that of the mature enzyme. These results and molecular modeling studies are discussed in terms of a mechanism in which a dimeric full-length fusion protein must form prior to rate-limiting intramolecular cleavage of the N-terminal sequence that leads to an increase in enzymatic activity.

ATP-Binding Cassette Transporters↗
PubMed1993

The gag precursor contains a specific HIV-1 protease cleavage site between the NC (P7) and P1 proteins.

The predicted protease cleavage site (p7/p1; [J. Virol. 66 (1992) 1856-1865]) within the nucleocapsid precursor protein (p15) of human immunodeficiency virus, type 1, was confirmed using an in vitro assay employing recombinant HIV-1 protease and a chemically synthesized 72 amino acid polypeptide containing the p7 and p1 protein domains of the native gag polyprotein. The cleavage occurred between amino acid 55 (N) and amino acid 56 (F) of the polypeptide, as determined by N-terminal sequencing. The hydrolysis was optimal at pH 6.0 and at high salt concentration. The kinetic parameters Km, kcat and kcat/Km were 99 microM (+/- 8), 0.152 s-1 (+/- 0.002) and 1.56 mM-1.s-1 (+/- 0.11), respectively. Reconstituted as well as denatured polypeptides were cleaved at approximately the same rate, demonstrating that the conformation of the p7 protein, as a result of the Zn(2+)-binding, had no significant effect on the rate of hydrolysis of the p7/p1 cleavage.

Amino Acid Sequence↗
PubMed1993

Two transmembrane signaling mechanisms control expression of the cAMP receptor gene CAR1 during Dictyostelium development.

Dictyostelium discoideum is among the best characterized organisms for the study of receptor/guanine nucleotide binding protein-mediated control of differentiation. Dictyostelium grow unicellularly but form fully differentiated multicellular organisms through a developmental program regulated by secreted cAMP activating specific cell-surface receptors. Dictyostelium respond differentially to cAMP at different developmental stages. During early development, expression of certain genes is induced by low-level oscillations of extracellular cAMP. Later, continuous, high cAMP concentrations will promote expression of specific genes in multicellular structures. Here, we show that the cAMP receptor gene CAR1, which is essential for development, utilizes two promoters that are activated at distinct stages of development and respond to different extracellular cAMP conditions. One promoter is active with low-level oscillations of cAMP; exposure to high cAMP concentrations will repress this promoter and induce a second promoter. The CAR1 mRNAs are alternatively spliced but encode identical proteins. Thus, through differential sensitivity to its own ligand, cAMP, two promoters and alternative splicing regulate CAR1 expression during Dictyostelium development.

Alternative Splicing↗
PubMed1993

CAR2, a prestalk cAMP receptor required for normal tip formation and late development of Dictyostelium discoideum.

Extracellular cAMP serves as a primary signaling molecule to regulate the development of Dictyostelium discoideum. It is required for chemotaxis, aggregation, cytodifferentiation, and morphogenetic movement. The receptors for cAMP are members of the family of cell-surface receptors that are linked to G proteins and characterized by seven putative transmembrane domains. Previously, we have isolated the gene for the cAMP receptor subtype 1 (CAR1) from Dictyostelium and suggested that several genes related to CAR1 were present in the genome. Here, we describe a family of cAMP receptor genes of Dictyostelium and the isolation and function of the gene for the cAMP receptor subtype 2, CAR2. CAR2 is structurally similar to CAR1. Overall, their transmembrane and loop domains are approximately 75% identical in amino acid sequence; however, their carboxyl termini are quite dissimilar; CAR2 possesses homopolymeric runs of histidines and asparagines that are absent from the corresponding region in CAR1. Although CAR1 is maximally expressed during the early stages of development, CAR2 is expressed only after cells have aggregated and, then, preferentially in prestalk cells. Transgenic Dictyostelium that have had their wild-type CAR2 gene replaced by a defective copy using homologous recombination proceed through early development but are detained at the tight mound stage. CAR2 may be required for cAMP-directed sorting of prestalk cells during pattern formation within the aggregation mound. Furthermore, although prestalk genes are expressed normally in aggregates that lack CAR2, they exhibit an enhanced expression of prespore-specific mRNA. Previously, we had shown that there was a requirement for CAR1 during early development. The present results demonstrate that the multiple responses of Dictyostelium to cAMP are regulated by distinct cAMP receptors that are encoded by unique genes.

Amino Acid Sequence↗
PubMed1993

[Gold salt-induced pneumonia and CD4 alveolitis].

In a 62-year old man who consulted for dyspnoea, clinical, radiological and functional examinations led to the diagnosis of immunoallergic lung disease caused by gold salt therapy. Regression of the symptoms when gold salt therapy was withdrawn supported this diagnosis. Contrary to previously published cases concerning treatment with gold salts, the study of bronchoalveolar lavage (BAL) fluid yielded a lymphocytic alveolitis with a very high CD4/CD8 ratio, as already reported with methotrexate, cyclothiazide and nitrofurantoin. This case can be added to the list of drugs that may induce CD4 alveolitis; it also reminds the existence of gold salt pneumonia and permits to discuss the value of a lymphocyte subpopulation study in the BAL fluid in patients with drug-induced immunoallergic lung diseases.

Alveolitis, Extrinsic Allergic↗
PubMed1992

Kinetic and modeling studies of S3-S3' subsites of HIV proteinases.

Kinetic analysis and modeling studies of HIV-1 and HIV-2 proteinases were carried out using the oligopeptide substrate [formula: see text] and its analogs containing single amino acid substitutions in P3-P3' positions. The two proteinases acted similarly on the substrates except those having certain hydrophobic amino acids at P2, P1, P2', and P3' positions (Ala, Leu, Met, Phe). Various amino acids seemed to be acceptable at P3 and P3' positions, while the P2 and P2' positions seemed to be more restrictive. Polar uncharged residues resulted in relatively good binding at P3 and P2 positions, while at P2' and P3' positions they gave very high Km values, indicating substantial differences in the respective S and S' subsites of the enzyme. Lys prevented substrate hydrolysis at any of the P2-P2' positions. The large differences for subsite preference at P2 and P2' positions seem to be at least partially due to the different internal interactions of P2 residue with P1', and P2' residue with P1. As expected on the basis of amino acid frequency in the naturally occurring cleavage sites, hydrophobic residues at P1 position resulted in cleavable peptides, while polar and beta-branched amino acids prevented hydrolysis. On the other hand, changing the P1' Pro to other amino acids prevented substrate hydrolysis, even if the substituted amino acid had produced a good substrate in other oligopeptides representing naturally occurring cleavage sites. The results suggest that the subsite specificity of the HIV proteinases may strongly depend on the sequence context of the substrate.

Amino Acid Sequence↗
PubMed1991

Autoprocessing of the HIV-1 protease using purified wild-type and mutated fusion proteins expressed at high levels in Escherichia coli.

Various constructs of the human immunodeficiency virus, type 1 (HIV-1) protease containing flanking Pol region sequences were expressed as fusion proteins with the maltose-binding protein of the malE gene of Escherichia coli. The full-length fusion proteins did not exhibit self-processing in E. coli, thereby allowing rapid purification by affinity chromatography on cross-linked amylose columns. Denaturation of the fusion protein in 5 M urea, followed by renaturation, resulted in efficient site-specific autoprocessing to release the 11-kDa protease. Rapid purification involving two column steps gave an HIV-1 protease preparations of greater than 95% purity (specific activity approximately 8500 pmol.min-1.micrograms protease-1) with an overall yield of about 1 mg/l culture. Incubation of an inactive mutant protease fusion protein with the purified wild-type protease resulted in specific trans cleavage and release of the mutant protease. Analysis of products of the HIV-1 fusion proteins containing mutations at either the N- or the C-terminal protease cleavage sites indicated that blocking one of the cleavage sites influences the cleavage at the non-mutated site. Such mutated full-length and truncated protease fusion proteins possess very low levels of proteolytic activity (approximately 5 pmol.min-1.micrograms protein-1).

ATP-Binding Cassette Transporters↗
PubMed1991

The effect of salt on the Michaelis Menten constant of the HIV-1 protease correlates with the Hofmeister series.

The effect of different types of salt on the proteolytic activity of HIV-1 protease was studied. At a similar ionic strength, the enzyme activity changed according to the salting out effect of the ions used (Hofmeister series). Kinetic studies showed that a stronger salting out effect of the ions rather than the higher ionic strength per se increased the affinity to the substrate (Km) but in general did not alter the Kcat value.

Amino Acid Sequence↗
PubMed1991

Purification of HIV-1 wild-type protease and characterization of proteolytically inactive HIV-1 protease mutants by pepstatin A affinity chromatography.

Recombinant wild-type protease of human immunodeficiency virus, type 1 (HIV-1) expressed in E. coli was purified by pepstatin A affinity chromatography. An 88-fold purification was achieved giving a protease preparation with a specific enzymatic activity of approximately 3700 pmol/min/micrograms. Two proteolytically inactive HIV-1 mutant proteases (Arg-87----Lys; Asn-88----Glu) were found to bind to pepstatin A agarose, and and they were purified as the wild-type protease. A third mutant protease Arg-87----Glu) was apparently unable to bind to pepstatin A under similar conditions. Binding to pepstatin A indicates the binding ability of the substrate binding site and the ability to form dimers. These features may be used to purify and to characterize other mutated HIV-1 proteases.

Amino Acid Sequence↗
PubMed1990

A solid phase assay for the protease of human immunodeficiency virus.

A solid phase assay for human immunodeficiency virus (HIV) protease using an immobilized substrate, Affi Gel 10-Gly-Gly-Gly-Gly-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-[3H]Gly-OH has been devised. The Tyr-Pro bond of the substrate was hydrolyzed by the protease, releasing the radiolabeled cleavage product, Pro-Ile-Val-Gln-[3H]Gly-OH, into the supernatant. The pH optimum was found to be 6.0, and a high ionic strength was required for maximal activity. The solid phase assay is usable for convenient monitoring of purification procedures, and rapid screening of inhibitors of HIV protease.

Amino Acid Sequence↗
PubMed1989

Substitution mutations of the highly conserved arginine 87 of HIV-1 protease result in loss of proteolytic activity.

The 297bp gene coding for the HIV-1 protease was chemically synthesized and expressed in E. coli. Single amino acid substitutions (Arg 87 - greater than Lys; Arg 87 - greater than Glu) were introduced in the C-terminally located conserved region GlyArgAsn of the protease gene in the wild-type clone. The products of the mutant and the wild-type clones were expressed at approximately similar levels at 30 minutes of induction but the mutant protease proteins accumulated as a function of time of induction unlike the wild-type protease which declined after 60 minutes. The mutants were completely devoid of proteolytic activity as determined in assays employing as substrates a synthetic nonapeptide and a gag related recombinant polyprotein.

Arginine↗