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

Considerable interest exists in the HIV-1 protease for biochemical studies as a potential therapeutic target of acquired immunodeficiency syndrome. We have produced the retroviral enzyme in E. coli from a synthetic gene encoding the protease that was constructed by assembling six overlapping and complementary oligonucleotides into the vector pKK223-3. When expressed in E. coli, the recombinant protease was able to correctly process the HIV-1 core protein p24 from a beta-galactosidase-gag fusion protein and to use a heptapeptide as a substrate for proteolytic cleavage. A single base pair mutation was identified in a recombinant that resulted in the substitution of lysine for asparagine at position 88 and a significant loss of enzyme activity. Through site-directed mutagenesis, the Asn88 was changed to five other residues representative of all classes of amino acids. The correlation between enzyme activity and amino acid substitution suggests that the protease domain surrounding position 88 affects the protein's potential for forming an active homodimeric protein and hence, indicates a biochemical interaction that could be inhibited by novel antiviral compounds.
 ...
PMID:HIV-1 protease: mutagenesis of asparagine 88 indicates a domain required for dimer formation. 269 24

The mature proteins of retroviruses originate as a result of proteolytic cleavages of polyprotein precursors. Retroviruses encode proteases responsible for several of these processing events, making them potential antiviral drug targets. A 99-amino acid HIV-1 protease, produced by chemical synthesis or by expression in bacteria, is shown here to hydrolyze peptides corresponding to all of the known cleavage sites in the HIV-1 gag and pol polyproteins. It does not hydrolyze peptides corresponding to an env cleavage site or a distantly related retroviral gag cleavage site.
 ...
PMID:HIV-1 protease specificity of peptide cleavage is sufficient for processing of gag and pol polyproteins. 305 48

The genetic locus and primary structure of the human immunodeficiency virus (HIV) protease was determined by comparing the data of protein analyses with the published data of the gene analysis. The complete sequence of HIV-1 and HIV-2 protease was synthesized by solid-phase peptide synthesis. The synthetic protease was capable of accurately cleaving synthetic peptide substrates corresponding to known cleavage sites in gag polyproteins of HIV-1, HIV-2, and murine leukemia virus. The chemical synthesis of protease confirms the DNA sequence and provides a means of rapidly producing active protease in substantial quantities for biochemical and physical studies.
 ...
PMID:Genetic locus, primary structure, and chemical synthesis of human immunodeficiency virus protease. 306 43

A protein corresponding to the putative protease of the human immunodeficiency virus 1 (HIV-1) has been prepared by total chemical synthesis. This 99 residue synthetic enzyme showed specific proteolytic activity on fragments of the natural gag precursor and on synthetic peptide substrates, two of which released fragments corresponding to the N terminus and C terminus of the protease molecule itself. The observed substrate specificity was not restricted to cleavage at Phe/Tyr-Pro bonds. Inhibition studies provided direct evidence that the HIV-1 protease belongs to the family of aspartic proteases. The availability of the HIV-1 protease as a defined molecular species has important implications for the design of specific inhibitors that do not interfere with the host cell metabolism as a possible route to antiviral agents against acquired immunodeficiency syndrome (AIDS).
 ...
PMID:Enzymatic activity of a synthetic 99 residue protein corresponding to the putative HIV-1 protease. 329 1

Human plasma impairs the activity of the human immunodeficiency virus (HIV-1) proteinase to cleave the HIV-1 gag-polyprotein precursor. The inhibition is due to the entrapment of the proteinase by plasma alpha 2-macroglobulin (alpha 2M). In methylamine-treated plasma, where alpha 2M is inactivated, HIV proteinase is not blocked. The interaction of alpha 2M and HIV-1 proteinase resulting in covalent complexes of proteinase and alpha 2M was demonstrated by immunoblotting with antiserum either to alpha 2M or to the HIV proteinase. We suggest if HIV-1 proteinase would be released in vivo from infected patients' cells, alpha 2M entrapment may prevent or minimize a conceivable cleavage of extracellular matrix or plasma proteins by the HIV-1 enzyme.
 ...
PMID:Human immunodeficiency virus type 1 proteinase is rapidly and efficiently inactivated in human plasma by alpha 2-macroglobulin. 753 87

An enzyme immunoassay was developed for monitoring protease reactions of human immunodeficiency virus (HIV). The protease and its substrate, the gag precursor, were generated separately in Escherichia coli. The HIV-1 protease was generated with a glutathione-S-transferase expression system and the gag substrate, named Pin17/24, was prepared with a PinPoint expression system. Pin17/24 consists of an N-terminal peptide, which is biotinylated in E. coli, fused with a C-terminal peptide that contains a protease cleavage site flanked by p17 and p24 segments. Through its biotin in the N-terminal region, Pin17/24 bound to ELISA plates coated with avidin, whereas through its C-terminal region, the same molecule of Pin17/24 could be recognized by an anti-p24 monoclonal antibody. When the protease was added to Pin17/24, the p24 fragment was released from the biotinylated fusion protein and could no longer be retained on the avidin plates, and as a result, binding of the anti-p24 monoclonal antibody decreased. The binding was specific and the reaction was inhibited by a known HIV protease inhibitor. Due to the specific interactions between avidin and biotin, monoclonal antibody and antigen, and the HIV protease and the gag substrate, crude preparations of these reagents can be used readily in the assay. The simplicity and feasibility of this method should be useful for simultaneous monitoring of many enzyme reactions, particularly for screening possible HIV protease inhibitors.
 ...
PMID:Assay of HIV-1 protease activity by use of crude preparations of enzyme and biotinylated substrate. 763 27

Some retroviruses, including HIV-1, regulate the relative amounts of gag and pol gene products by a translational frameshift mechanism. The consequences of altering the ratios of the Gag and Pol proteins were tested using vaccinia virus expression vectors, in which the gag and pol genes were fused by placing them in the same open reading frame. Immunoblotting of cell lysates indicated that a protein of approximately 160 kDa, the expected translation product of the fused gag-pol gene, was the dominant species detected with HIV-specific antiserum during the first several hours of infection with this recombinant virus. Subsequently, the full-length polyprotein diminished in amount and a series of Gag-related intermediate size proteins appeared. Later in infection, p24 and myristoylated p17 Gag proteins predominated and larger amounts of intracellularly processed reverse transcriptase, integrase, and protease were detected compared to the amounts formed with the wild-type gag-pol gene. Large numbers of budding, immature, and mature retrovirus-like particles were visualized by electron microscopy when the wild-type gag-pol gene was expressed, whereas no particles were detected in cells that expressed the fused gag-pol gene. The block to virus assembly was partially overcome by (i) inhibition of the HIV-1 protease with a peptidomimetic inhibitor, (ii) mutagenesis of the active site of the protease, or (iii) shortening of the Gag-Pol polyprotein by deletion of most of the reverse transcriptase gene. Nevertheless, budding was inefficient and the structures appeared immature and frequently aberrant. These results indicated that overproduction of the full-length Gag-Pol polyprotein and increased intracellular protease activity were both detrimental to viral assembly. Further experiments indicated that intracellular processing of Gag and Gag-Pol polyproteins occurred in the absence of particle formation when myristoylation was prevented.
 ...
PMID:Overexpression of the HIV-1 gag-pol polyprotein results in intracellular activation of HIV-1 protease and inhibition of assembly and budding of virus-like particles. 768 10

Upon in vitro processing of the recombinant HIV-1/gag p24 protein, expressed in Escherichia coli as a fusion protein, by HIV-1 protease, a cleavage site within the staphylococcal protein A fusion partner was found. N-terminal sequencing of the protein A fragments showed that HIV-1 protease cleavage occurred between phenylalanine-235 and tyrosine-236 within the sequence Gln-Asn-Ala-Phe/Tyr-Glu-Ile-Leu (QNAF/YEIL) in the IgG-binding domain C of the protein A encoded by the pRIT2T fusion gene vector (Pharmacia). Results presented here have proven that the protease-sensitive site is viable in vitro on the protein A alone and other chimeric protein, protein A/beta-galactosidase. A possible significance of this phenomenon in biotechnology work is discussed.
 ...
PMID:Staphylococcal protein A is a novel heterologous substrate for the HIV-1 protease. 776 14

The aspartyl protease of the human immunodeficiency virus (HIV) is an important target for chemotherapeutic intervention because of its key role in cleaving the HIV gag-pol polyprotein during viral assembly and budding. Short peptides and peptidomimetics, which bind to the active site of the HIV aspartyl protease and inhibit processing of the polyprotein, have been synthesized. These compounds are active against HIV in vitro, but many face substantial development problems because of their rapid elimination from the body in bile and urine. Refinement of these agents appears to be necessary if they are to become useful clinically. Recently, we developed a novel chemical strategy for increasing plasma levels of HIV protease inhibitory peptides, which involves the attachment of a biodegradable phospholipid group to the C-terminus of a pentapeptide, iBOC-[L-Phe]-[D-beta-Nal]-Pip-[alpha-(OH)-Leu]-Val (7194). We coupled phosphatidylethanolamine to the C-terminal valine of 7194 to make a phospholipid prodrug (7196). In vitro assays in HT4-6C cells infected with HIV-1 showed that the antiviral activity of the C-terminal phospholipid prodrug, 7196, was equal to that of the free peptide, 7194. Similar results were obtained in vitro when a related pentapeptide (7140) was derivatized at the N-terminal with dipalmitoylphosphatidylethanolamine-succinic acid (7172). Tritium-labeled 7194 and 7196 were prepared and injected intravenously into rats at 3 mumol/kg; then the plasma was assayed for native compound and metabolites by HPLC radioactivity flow detection. The peak plasma level of the tritium-labeled lipid prodrug (7196) was 36 microM versus 1.6 microM for the free protease inhibitor pentapeptide (7194). The area under the curve of the phospholipid prodrug (7196) was 48-fold greater and its mean residence time was increased 43-fold versus the free peptide (7194). Phospholipid prodrugs appear to offer an alternative approach to optimizing in vivo performance of HIV protease inhibitors and other small peptides.
 ...
PMID:Phospholipid prodrug inhibitors of the HIV protease. Antiviral activity and pharmacokinetics in rats. 794 39

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.
 ...
PMID:The gag precursor contains a specific HIV-1 protease cleavage site between the NC (P7) and P1 proteins. 822 64


<< Previous 1 2 3 4 5 6 7 8 9 Next >>