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Biomedical subjects

B M Dunn

Publications and source records attributed to B M Dunn.

At least 19 recordsLinked to original sources
PubMed1997

Escape mutants of HIV-1 proteinase: enzymic efficiency and susceptibility to inhibition.

Genes encoding a number of mutants of HIV-1 proteinase were sub-cloned and expressed in E. coli. The proteinases containing mutations of single residues (e.g., G48V, V82F, I84V and L90M) were purified and their catalytic efficiencies relative to that of wild-type proteinase were examined using a polyprotein (recombinant HIV-1 gag) substrate and several series of synthetic peptides based on the -Hydrophobic * Hydrophobic-, -Aromatic * Pro- and pseudo-symmetrical types of cleavage junction. The L90M proteinase showed only small changes, whereas the activity of the other mutant enzymes was compromised more severely, particularly towards substrates of the -Aromatic * Pro- and pseudo-symmetrical types. The susceptibility of the mutants and the wild-type proteinase to inhibition by eleven different compounds was compared. The L90M proteinase again showed only marginal changes in its susceptibility to all except one of the inhibitors examined. The K(i) values determined for one inhibitor (Ro31-8959) showed that its potency towards the V82F, L90M, I84V and G48V mutant proteinases respectively was 2-, 3-, 17- and 27-fold less than against the wild-type proteinase. Several of the other inhibitors examined form a systematic series with Ro31-8959. The inhibition constants derived with these and a number of other inhibitors, including ABT-538 and L-735,524, are used in conjunction with the data on enzymic efficiency to assess whether each mutation in the proteinase confers an advantage for viral replication in the presence of any given inhibitor.

Anti-HIV Agents↗
PubMed1997

Expression and characterisation of plasmepsin I from Plasmodium falciparum.

Two aspartic proteinases, plasmepsins I and II, are present in the digestive vacuole of the human malarial parasite Plasmodium falciparum and are believed to be essential for parasite degradation of haemoglobin. Here we report the expression and kinetic characterisation of functional recombinant plasmepsin I. In order to generate active plasmepsin I from its precursor, an autocatalytic cleavage site was introduced into the propart of the zymogen by mutation of Lys110P to Val (P indicates a propart residue). Appropriate refolding of the mutated zymogen then permitted pH-dependent autocatalytic processing of the zymogen to the active mature proteinase. A purification scheme was devised that removed aggregated and misfolded protein to yield pure, fully processable, proplasmepsin I. Kinetic constants for two synthetic peptide substrates and four inhibitors were determined for both recombinant plasmepsin I and recombinant plasmepsin II. Plasmepsin I had 5-10-fold lower k(cat)/Km values than plasmepsin II for the peptide substrates, while the aspartic proteinase inhibitors, selected for their ability to inhibit P. falciparum growth, were found to have up to 80-fold lower inhibition constants for plasmepsin I compared to plasmepsin II. The most active plasmepsin I inhibitors were antagonistic to the antimalarial action of chloroquine on cultured parasites. Northern blot analysis of RNA, isolated from specific stages of the erythrocytic cycle of P. falciparum, showed that the proplasmepsin I gene is expressed in the ring stages whereas the proplasmepsin II gene is not transcribed until the later trophozoite stage of parasite growth. The differences in kinetic properties and temporal expression of the two plasmepsins suggest they are not functionally redundant but play distinct roles in the parasite.

Animals↗
PubMed1996

Expression, characterisation and mutagenesis of the aspartic proteinase from equine infectious anaemia virus.

The gene encoding the proteinase from equine infectious anaemia virus (EIAV) was cloned and expressed in Escherichia coli. The recombinant EIAV proteinase was purified to homogeneity and shown to have the ability to process polyprotein and synthetic peptide substrates of human immunodeficiency virus (HIV) origin with an efficiency that can approach that exhibited by HIV proteinase. EIAV proteinase, however, was not susceptible to inhibition by a wide variety of inhibitors of HIV-1 proteinase, including those which have been licenced as anti-AIDS drugs. In this respect, EIAV proteinase behaves like an extreme case of a drug-resistant mutant of HIV-1 proteinase that has arisen under selective drug pressure. Only one potent inhibitor (HBY-793) of HIV-1 proteinase showed comparable efficiency against the EIAV enzyme; the compounds A-77003 and A-76889, which differ only in their stereochemistry and which are otherwise structurally identical to HBY-793 from residues P2 to P2' [nomenclature of Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162], were not effective inhibitors of EIAV proteinase. Mutant forms of EIAV proteinase (Thr30-->Asp and Ile54-->Gly) were generated and their ability to interact with substrates and inhibitors was characterised. HBY-793 inhibited [Gly54]proteinase as effectively as the wild-type proteinase but was tenfold less potent against [Asp30]proteinase. Data interpretations are presented, based on the structure solved for the complex between HBY-793 and EIAV [Gly54]proteinase [Gustchina A., Kervinen, J., Powell, D. J., Zdanov, A., Kay, J. & Wlodawer, A. (1996) Protein Sci. 5, 1453-1465].

Amino Acid Sequence↗
PubMed1996

Self-activation of recombinant human lysosomal procathepsin D at a newly engineered cleavage junction, "short" pseudocathepsin D.

To obtain a recombinant model of human cathepsin D with kinetic properties that are identical with native human liver enzyme, we have addressed the significant differences in structure and catalytic function between naturally occurring enzyme and bacterially derived pseudocathepsin D. Human procathepsin D was expressed in a baculovirus system to obtain correctly folded, glycosylated enzyme that upon acidification completely converts to the active intermediate, pseudocathepsin D. The oligosaccharide moieties of this recombinant enzyme contributed to about 5% of the apparent molecular mass of the enzyme, and the carbohydrate composition was quite similar to the native material. However, specificity constants (kcat/Km) of this glycosylated pseudoform for several synthetic chromogenic substrates were considerably less (33%-50%) than those for the native enzyme and were virtually identical with those observed with nonglycosylated pseudocathepsin D. A cleavable junction suitable for self-processing at the normal maturation point of human cathepsin D was engineered into procathepsin D according to known specificity requirements of this enzyme, and the construct was expressed using baculovirus. Following experiments that demonstrated that the new proenzyme failed to process to the expected point, the new cleavage junction was moved 6 residues toward the amino terminus of procathepsin D and expressed in Escherichia coli. After refolding, the protein containing the newly engineered junction self-processed, generating a shortened mutant form of pseudocathepsin D that is 6 residues longer at the amino terminus than the native material. The kinetic properties of this newly engineered pseudoform proved to be identical with those of the native enzyme, thus establishing an improved recombinant model for this important aspartic proteinase.

Amino Acid Sequence↗
PubMed1996

Natural variation in HIV-1 protease, Gag p7 and p6, and protease cleavage sites within gag/pol polyproteins: amino acid substitutions in the absence of protease inhibitors in mothers and children infected by human immunodeficiency virus type 1.

Reduced sensitivity of human immunodeficiency virus type 1 (HIV-1) to protease inhibitors is associated with multiple amino acid substitutions in the virus-encoded protease. The combination of changes that contribute to drug resistance is dependent in part upon the amino acid residues comprising protease alleles prior to drug therapy. We analyzed within peripheral blood mononuclear cells from HIV-1-infected mothers and their children viral gag/pol regions, which included p7, transframe p6/p6*, and protease coding sequences, as well as six protease cleavage sites. Sixty protease alleles from 12 individuals differed by at least 3 to as many as 10 amino acids from proteases encoded by molecular clones of HIV-1, indicating that there is no prototype or consensus wild-type HIV-1 protease sequence. Protease variants with a proline at position 63, a substitution associated with resistance to protease inhibitors, appeared in the absence of antiprotease therapy in 7 patients and were transmitted by 2 mothers to their infants. Gag p7 p6 regions were significantly more variable than protease. The p6/p6* region contained length variants and amino acid repeats in both reading frames. Five protease cleavage sites (B, D', D, E, and F) contained highly conserved amino acid sequences in individuals infected by epidemiologically distinct viruses. In contrast, C cleavage sites, localized between Gag p2 and Gag p7, displayed considerable amino acid variability, were unique among groups of infected individuals, and appeared to be related to particular protease alleles. Genetic variability in vivo in protease, in cleavage sites, and in proteins upstream of protease provides the potential to modulate enzyme activity and susceptibility to protease inhibitors.

Alleles↗
PubMed1996

Substrate specificities of pepstatin-insensitive carboxyl proteinases from gram-negative bacteria.

Pseudomonas carboxyl proteinase (PCP), isolated from Pseudomonas sp. 101, and Xanthomonas carboxyl proteinase (XCP), isolated from Xanthomonas sp. T-22, are the first and second examples of unique carboxyl proteinases [EC 3.4.23.33] which are insensitive to aspartic proteinase inhibitors, such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3(p-nitrophenoxy)propane. The substrate specificities of PCP and XCP were studied using a series of synthetic chromogenic peptide substrates with the general structure, P5-P4-P3-P2-Phe-Nph-P2'-P3' (P5, P4, P3, P2, P2', P3': a variety of amino acids, Nph is p-nitro-L-phenylalanine, and the Phe-Nph bond is cleaved). PCP and XCP were shown to hydrolyze a synthetic substrate, Lys-Pro-Ala-Leu-Phe-Nph-Arg-Leu, most effectively among 28 substrates. The kinetic parameters of this peptide for PCP were Km = 6.3 microM, Kcat = 51.4 s-1, and kcat/Km = 8.16 microM-1.s-1. The kinetic parameters for XCP were Km = 3.6 microM, kcat = 52.2 s-1, and kcat/Km = 14.5 microM-1.s-1. PCP showed a stricter substrate specificity than XCP. That is, the specificity constant (kcat/Km) of each substrate for PCP was in general < 0.5 microM-1.s-1, but was drastically improved by the replacement of Lys by Leu at the P2 position. On the other hand, XCP showed a less stringent substrate specificity, with most of the peptides exhibiting reasonable kcat/Km values (> 1.0 microM-1.s-1). Thus it was found that the substrate specificities of PCP and XCP differ considerably, in spite of the high similarity in their primary structures. In addition, tyrostatin was found to be a competitive inhibitor for XCP, with a Ki value of 2.1 nM, as well as for PCP (Ki = 2.6 nM).

Aspartic Acid Endopeptidases↗
PubMed1995

Monomeric human cathepsin E.

Cathepsin E is a homodimer, consisting of two monomers linked by an inter-molecular disulphide bond. The cysteine residue involved is located near to the N-terminus of the mature proteinase. By mutating this residue to alanine, a monomeric form of human cathepsin E was engineered and purified. The activity of the resultant enzyme was not altered significantly (in terms of its ability to hydrolyse two chromogenic peptide substrates; and its susceptibility to inhibition by pepstatin). However, the stability of the mutant enzyme to alkaline pH and to temperature was markedly reduced.

Amino Acid Sequence↗
PubMed1995

A continuous fluorescence-based assay of human cytomegalovirus protease using a peptide substrate.

The 28-kDa protease from human cytomegalovirus (hCMV) has been successfully cloned, expressed, and purified to homogeneity. An internally quenched fluorescent substrate (4-4'-dimethylaminophenazo)benzoyl-Arg-Gly-Val-Val-Asn-Ala-Ser-Ser -Arg-Leu-Ala-5-[(2'-aminoethyl)-amino]-naphthalene-1-sulfonic acid; DABCYL-CMV-EDANS) based on the maturational cleavage site (M-site) junction was synthesized in an effort to develop a fluorescence-based assay. This substrate is cleaved specifically between the Ala-Ser peptide bond thereby liberating the C-terminal peptide-EDANS fragment from the proximity quenching effect of the DABCYL group. This results in greater than a 10-fold increase in fluorescence that is observed at the EDANS emission wavelength of 495 nm. Human CMV protease efficiently cleaved this synthetic substrate permitting continuous assay at peptide concentrations lower than 10 microM. At substrate concentrations greater than 10 microM, linearity was lost due to the "inner filter effect." This represents the first fluorescence-based assay for any of the herpes virus proteases. Additionally, a peptidyl inhibitor, H-Arg-Gly-Val-Val-Asn-Ala-psi[CH2NH]-Ser-Ser-Arg-Leu-Ala-OH, was prepared. This inhibitor was also based on the same M-site cleavage junction with a nonhydrolyzable reduced peptide bond incorporated at the cleavage site. Using the fluorescence-based assay, this reduced peptide bond analog was observed to be an inhibitor of hCMV protease with an inhibition constant of > 500 microM.

Amino Acid Sequence↗
PubMed1995

Effect of point mutations on the kinetics and the inhibition of human immunodeficiency virus type 1 protease: relationship to drug resistance.

Mutations of human immunodeficiency virus type 1 (HIV-1) protease at four positions, Val82, Asp30, Gly48, and Lys45 were analyzed for the resulting effects on kinetics and inhibition. In these mutants, Val82 was substituted separately by Asn, Glu, Ala, Ser, Asp, and Gln; Asp30 was individually substituted by Phe or Trp; Gly48 by His, Asp, and Tyr, respectively; and Lys45 by Glu. By examination of the inhibition of a single inhibitor, the differences in Ki values between the native and mutant enzymes can range from very large to insignificant even for the mutants with substitutions at the same position. By examination of a single mutant enzyme, the same broad range of Ki changes was observed for a group of inhibitors: Thus, how much the inhibition changes from the wild-type enzyme to a mutant is dependent on both the mutation and the inhibitor. The examination of Ki changes of inhibitors with closely related structures binding to Val82 mutants also reveals that the change of inhibition involves subsites in which Val82 is not in direct contact, indicating a considerable flexibility of the conformation of HIV protease. For the catalytic activities of the mutants, the kcat and Km values of many Val82 mutants and a Lys45 mutant are comparable to the native enzyme. Surprisingly, Gly48 mutations produce enzymes with catalytic efficiency superior to that of the wild-type enzyme by as much as 10-fold. Modeling of the structure of the mutants suggests that the high catalytic efficiency of some substrates is related to an increase of rigidity of the flap region of the mutants. The examination of the relative changes of inhibition and catalysis of mutants suggests that some of the Val82 and Gly48 mutants are potential resistance mutants. However, the resistance is specific with respect to individual inhibitors.

Amino Acid Sequence↗
PubMed1995

Engineering the substrate specificity of rhizopuspepsin: the role of Asp 77 of fungal aspartic proteinases in facilitating the cleavage of oligopeptide substrates with lysine in P1.

Rhizopuspepsin and other fungal aspartic proteinases are distinct from the mammalian enzymes in that they are able to cleave substrates with lysine in the P1 position. Sequence and structural comparisons suggest that two aspartic acid residues, Asp 30 and Asp 77 (pig pepsin numbering), may be responsible for generating this unique specificity. Asp 30 and Asp 77 were changed to the corresponding residues in porcine pepsin, Ile 30 and Thr 77, to create single and double mutants. The zymogen forms of the wild-type and mutant enzymes were overexpressed in Escherichia coli as inclusion bodies. Following solubilization, denaturation, refolding, activation, and purification to homogeneity, structural and kinetic comparisons were made. The mutant enzymes exhibited a high degree of structural similarity to the wild-type recombinant protein and a native isozyme. The catalytic activities of the recombinant proteins were analyzed with chromogenic substrates containing lysine in the P1, P2, or P3 positions. Mutation of Asp 77 resulted in a loss of 7 kcal mol-1 of transition-state stabilization energy in the hydrolysis of the substrate containing lysine in P1. An inhibitor containing the positively charged P1-lysine side chain inhibited only the enzymes containing Asp 77. Inhibition of the Asp 77 mutants of rhizopuspepsin and several mammalian enzymes was restored upon acetylation of the lysine side chain. These results suggest that an exploitation of the specific electrostatic interaction of Asp 77 in the active site of fungal enzymes may lead to the design of compounds that preferentially inhibit a variety of related Candida proteinases in immunocompromised patients.

Amino Acid Sequence↗
PubMed1995

Exploring the binding preferences/specificity in the active site of human cathepsin E.

Aspartic proteinases are produced in the human body by a variety of cells. Some of these proteins, examples of which are pepsin, gastricsin, and renin, are secreted and exert their effects in the extracellular spaces. Cathepsin D and cathepsin E on the other hand are intracellular enzymes. The least characterized of the human aspartic proteinases is cathepsin E. Presented here are results of studies designed to characterize the binding specificities in the active site of human cathepsin E with comparison to other mechanistically similar enzymes. A peptide series based on Lys-Pro-Ala-Lys-Phe*Nph-Arg-Leu was generated to elucidate the specificity in the individual binding pockets with systematic substitutions in the P5-P2, and P2'-P3' based on charge, hydrophobicity, and hydrogen bonding. Also, to explore the S2 binding preferences, a second series of peptides based on Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu was generated with systematic replacements in the P2 position. Kinetic parameters were determined for both sets of peptides. The results were correlated to a rule-based structural model of human cathepsin E, constructed on the known three-dimensional structures of several highly homologous aspartic proteinases; porcine pepsin, bovine chymosin, yeast proteinase A, human cathepsin D, and mouse and human renin. Important specificity-determining interactions were found in the S3 (Glu-13) and S2 (Thr-222, Gln-287, Leu-289, Ile-300) subsites.

Amino Acid Sequence↗