The aspartic proteinase from equine infectious anaemia virus.
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Animals↗
Biomedical subjects
D Bur
Publications and source records attributed to D Bur.
Studies on plasmepsins I and II from the malarial parasite Plasmodium falciparum and their exploitation as drug targets.
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Animals↗
Glycosylation of Asn-632 and Asn-651 is important for functional expression of endothelin-converting enzyme-1.
It has been shown previously that N-glycosylation of Asn-144 and/or Asn-627 is important for functional expression of neutral endopeptidase-24.11 (NEP). All glycosylation sites of NEP are conserved within endothelin-converting enzyme-1 (ECE-1). In the present study we investigated the importance of proper glycosylation for the biologic function of ECE-1. We show that the double mutation of Asn-632 and Asn-651 leads to expression of an enzymatically inactive ECE-1 protein. In contrast, the single mutation of either Asn-632 or Asn-651 did not alter the enzymatic activity of ECE-1b.
Animals↗
Cathepsin E. The best laid plans of mice and men.
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Amino Acid Sequence↗
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↗
N-acyl-(alpha, gamma diaminobutyric acid)n hydrazide as an efficient gene transfer vector in mammalian cells in culture.
PURPOSE: This study investigates the structure/activity relationship of a series of N-acyl-peptides (lipopeptides) for the transfection of mammalian cells. METHODS: Lipopeptides comprising 1 to 3 basic amino-acids and a single fatty acid chain were synthesized. Transfecting complexes between lipopeptide, plasmid DNA and dioleoyl phosphatidylethanolamine were prepared and applied on cells in culture. Transfection efficiency was evaluated by measuring beta-galactosidase activity 48 h post-transfection. Lipopeptide-DNA binding was also investigated by physical means and molecular modelling. RESULTS: Besides the length of the fatty acid chain, the nature of the basic amino-acid and the C-terminal group were crucial parameters for high transfection efficiency. The N-acyl-(diaminobutyric acid)n derivatives were the most potent transfecting agents among those tested and induced a beta-galactosidase activity 2 to 20 times higher than the N-acyl-lysine, -ornithine or -diaminopropionic acid derivatives. Furthermore, a hydrazide C-terminal modification greatly enhanced transfection efficiency for all compounds tested. The reason why alpha, gamma-diaminobutyric acid hydrazide-based lipopeptides were the most potent in transfection is not fully understood but could be related to their high DNA binding. CONCLUSIONS: Poly- or oligo-diaminobutyric acid containing or not a hydrazide C-terminus could advantageously be used in peptide-based gene delivery systems.
Aminobutyrates↗
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↗
NMR studies of DNA duplexes singly cross-linked by different synthetic linkers.
Molecular modelling studies resulted in the design of a variety of non-nucleotidic covalent linkers to bridge the 3'-end of the (+)-strand and the 5'-end of the (-)-strand in DNA duplexes. Three of these linkers were synthesized and used to prepare singly cross-linked duplexes d(GTGGAATTC)-linker-d(GAATTCCAC). Linker I is an assembly of a propylene-, a phosphate- and a second propylene-group and is thought to mimic the backbone of two nucleotides. Linkers II and III consist of five and six ethyleneglycol units, respectively. The melting temperatures of the cross-linked duplexes are 65 degrees C for I and 73 degrees C for II and III, as compared with 36 degrees C for the corresponding non-linked nonadeoxynucleotide duplex. The three cross-linked duplexes were structurally characterized by nuclear magnetic resonance spectroscopy. The 1H and 31P resonance assignments in the DNA stem were obtained using standard methods. For the resonance assignment of the linker protons, two-dimensional 1H-31P heteronuclear COSY and two-quantum-experiments were used. Distance geometry calculations with NOE-derived distance constraints were performed and the resulting structures were energy-minimized. In duplex I, the nucleotides flanking the propylene-phosphate-propylene-linker do not form a Watson-Crick base pair, whereas in duplexes II and III the entire DNA stem is in a B-type double helix conformation.
Base Sequence↗
On the effect on specificity of Thr246----Gly mutation in L-lactate dehydrogenase of Bacillus sterothermophilus.
The function of the amino acid Thr246 in L-lactate dehydrogenase from Bacillus stearothermophilus has been investigated by site-directed replacement with glycine. Kinetic experiments with a number of 2-oxo acids showed strongly reduced activity for the mutated enzyme. However, the mutant enzyme shows a relative preference for the large hydrophobic sidechains of alpha-keto acids and an even higher specific activity than the wild-type lactate dehydrogenase for the polar oxaloacetate substrate. Graphic analyses indicate that the loss of one hydrogen bond, or intrusion of water into the active site, might be responsible for the reduced activity. The kinetic results suggest that the binding modes of bulky hydrophobic or polar substrates compensate to some degree for the partially disrupted active site.
Amino Acid Sequence↗
Piperidine renin inhibitors: from leads to drug candidates.
Non-peptidomimetic renin inhibitors of the piperidine type represent a novel structural class of compounds potentially free of the drawbacks seen with peptidomimetic compounds so far. Synthetic optimization in two structural series focusing on improvement of potency, as well as on physicochemical properties and metabolic stability, has led to the identification of two candidate compounds 14 and 23. Both display potent and long-lasting blood pressure lowering effects in conscious sodium-depleted marmoset monkeys and double transgenic rats harboring both the human angiotensinogen and the human renin genes. In addition, 14 normalizes albuminuria and kidney tissue damage in these rats when given over a period of 4 weeks. These data suggest that treatment of chronic renal failure patients with a renin inhibitor might result in a significant improvement of the disease status.
Animals↗