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1.
Nature ; 487(7408): 482-5, 2012 Jul 25.
Article in English | MEDLINE | ID: mdl-22837004

ABSTRACT

Despite antiretroviral therapy, proviral latency of human immunodeficiency virus type 1 (HIV-1) remains a principal obstacle to curing the infection. Inducing the expression of latent genomes within resting CD4(+) T cells is the primary strategy to clear this reservoir. Although histone deacetylase inhibitors such as suberoylanilide hydroxamic acid (also known as vorinostat, VOR) can disrupt HIV-1 latency in vitro, the utility of this approach has never been directly proven in a translational clinical study of HIV-infected patients. Here we isolated the circulating resting CD4(+) T cells of patients in whom viraemia was fully suppressed by antiretroviral therapy, and directly studied the effect of VOR on this latent reservoir. In each of eight patients, a single dose of VOR increased both biomarkers of cellular acetylation, and simultaneously induced an increase in HIV RNA expression in resting CD4(+) cells (mean increase, 4.8-fold). This demonstrates that a molecular mechanism known to enforce HIV latency can be therapeutically targeted in humans, provides proof-of-concept for histone deacetylase inhibitors as a therapeutic class, and defines a precise approach to test novel strategies to attack and eradicate latent HIV infection directly.


Subject(s)
Anti-HIV Agents/therapeutic use , HIV Infections/drug therapy , HIV Infections/virology , HIV-1/drug effects , HIV-1/growth & development , Hydroxamic Acids/pharmacology , Virus Latency/drug effects , Acetylation/drug effects , Biomarkers/metabolism , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , Gene Expression Regulation, Viral/drug effects , HIV Infections/blood , HIV-1/genetics , Histone Deacetylase Inhibitors/administration & dosage , Histone Deacetylase Inhibitors/adverse effects , Histone Deacetylase Inhibitors/pharmacology , Histones/drug effects , Histones/metabolism , Humans , Hydroxamic Acids/administration & dosage , Hydroxamic Acids/adverse effects , Proviruses/drug effects , Proviruses/genetics , Proviruses/growth & development , RNA, Viral/biosynthesis , RNA, Viral/blood , Risk Assessment , Up-Regulation/drug effects , Viremia/drug therapy , Viremia/virology , Vorinostat
2.
Curr Opin Microbiol ; 4(5): 535-9, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11587929

ABSTRACT

The clinical utility of intervention in HIV-1 disease has been proven by inhibitors targeting reverse transcriptase and protease. However, novel approaches including inhibition of viral entry, integration and assembly would provide additional options to maintain long-term suppression. The identification of specific inhibitors for each of these processes has recently validated these approaches as viable alternatives for the development of new agents to treat HIV-1 infection. The most recent preclinical advances in novel antiretroviral agents are reviewed and promising new approaches that target viral processes are highlighted.


Subject(s)
Anti-HIV Agents/pharmacology , Drug Design , HIV-1/drug effects , Virus Assembly/drug effects , Virus Integration/drug effects , Anti-HIV Agents/therapeutic use , HIV Infections/drug therapy , HIV Infections/virology , HIV Protease Inhibitors/pharmacology , HIV-1/pathogenicity , HIV-1/physiology , Humans , Reverse Transcriptase Inhibitors/pharmacology
3.
Proc Natl Acad Sci U S A ; 97(21): 11244-9, 2000 Oct 10.
Article in English | MEDLINE | ID: mdl-11016953

ABSTRACT

Diketo acids such as L-731,988 are potent inhibitors of HIV-1 integrase that inhibit integration and viral replication in cells. These compounds exhibit the unique ability to inhibit the strand transfer activity of integrase in the absence of an effect on 3' end processing. To understand the reasons for this distinct inhibitory profile, we developed a scintillation proximity assay that permits analysis of radiolabeled inhibitor binding and integrase function. High-affinity binding of L-731,988 is shown to require the assembly of a specific complex on the HIV-1 long terminal repeat. The interaction of L-731,988 with the complex and the efficacy of L-731, 988 in strand transfer can be abrogated by the interaction with target substrates, suggesting competition between the inhibitor and the target DNA. The L-731,988 binding site and that of the target substrate are thus distinct from that of the donor substrate and are defined by a conformation of integrase that is only adopted after assembly with the viral end. These results elucidate the basis for diketo acid inhibition of strand transfer and have implications for integrase-directed HIV-1 drug discovery efforts.


Subject(s)
Acetoacetates/pharmacology , DNA, Viral/metabolism , HIV Integrase Inhibitors/pharmacology , HIV Integrase/chemistry , HIV-1/enzymology , Pyrroles/pharmacology , Base Sequence , Catalysis , DNA Primers , Epitopes/metabolism , HIV Integrase/metabolism , HIV-1/genetics , Substrate Specificity
4.
Bioorg Med Chem Lett ; 10(3): 235-8, 2000 Feb 07.
Article in English | MEDLINE | ID: mdl-10698443

ABSTRACT

Integric acid (1), an acyl eremophilane sesquiterpenoid, was identified as an inhibitor of HIV-1 integrase, the enzyme responsible for provirus entry into the host cell nucleus and integration in to the host genome. Chemical and enzymatic modification of integric acid led to the preparation of several selective chemical derivatives of integric acid. Preparation, HIV-1 inhibitory activity, and the structure-activity relationship against coupled and strand transfer assays are described. It appears that most of the groups present in the natural product are required for inhibition of HIV-1 integrase strand transfer activity. In contrast, inhibition of 3' processing activity is less stringent suggesting distinct SAR for the two integrase reactions.


Subject(s)
Carboxylic Acids/chemistry , Carboxylic Acids/pharmacology , HIV Integrase Inhibitors/chemistry , HIV Integrase Inhibitors/pharmacology , HIV-1/enzymology , Naphthalenes/chemistry , Naphthalenes/pharmacology , Amides/chemistry , Hydrolysis , Structure-Activity Relationship
5.
Science ; 287(5453): 646-50, 2000 Jan 28.
Article in English | MEDLINE | ID: mdl-10649997

ABSTRACT

Integrase is essential for human immunodeficiency virus-type 1 (HIV-1) replication; however, potent inhibition of the isolated enzyme in biochemical assays has not readily translated into antiviral activity in a manner consistent with inhibition of integration. In this report, we describe diketo acid inhibitors of HIV-1 integrase that manifest antiviral activity as a consequence of their effect on integration. The antiviral activity of these compounds is due exclusively to inhibition of one of the two catalytic functions of integrase, strand transfer.


Subject(s)
Acetoacetates/pharmacology , Anti-HIV Agents/pharmacology , HIV Integrase Inhibitors/pharmacology , HIV Integrase/metabolism , HIV-1/drug effects , Pyrroles/pharmacology , Virus Integration/drug effects , Acetoacetates/chemistry , Acetoacetates/metabolism , Anti-HIV Agents/chemistry , Anti-HIV Agents/metabolism , Catalysis/drug effects , Coculture Techniques , DNA, Circular/biosynthesis , DNA, Circular/metabolism , DNA, Viral/biosynthesis , DNA, Viral/metabolism , Drug Resistance, Microbial , HIV Integrase/genetics , HIV Integrase Inhibitors/chemistry , HIV Integrase Inhibitors/metabolism , HIV Long Terminal Repeat/drug effects , HIV-1/enzymology , HIV-1/genetics , HIV-1/physiology , Humans , Mutation , Pyrroles/chemistry , Pyrroles/metabolism , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , T-Lymphocytes/virology , Transcription, Genetic , Tumor Cells, Cultured , Virus Replication/drug effects
7.
J Virol ; 71(9): 7005-11, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9261430

ABSTRACT

Previous in vitro analyses have shown that the human immunodeficiency virus type 1 (HIV-1) integrase uses either manganese or magnesium to assemble as a stable complex on the donor substrate and to catalyze strand transfer. We now demonstrate that subsequent to assembly, catalysis of both 3' end processing and strand transfer requires a divalent cation cofactor and that the divalent cation requirements for assembly and catalysis can be functionally distinguished based on the ability to utilize calcium and cobalt, respectively. The different divalent cation requirements manifest by these processes are exploited to uncouple assembly and catalysis, thus staging the reaction. Staged 3' end processing and strand transfer assays are then used in conjunction with exonuclease III protection analysis to investigate the effects of integrase inhibitors on each step in the reaction. Analysis of a series of related inhibitors demonstrates that these types of compounds affect assembly and not either catalytic process, therefore reconciling the apparent disparate results obtained for such inhibitors in assays using isolated preintegration complexes. These studies provide evidence for a distinct role of the divalent cation cofactor in assembly and catalysis and have implications for both the identification and characterization of integrase inhibitors.


Subject(s)
Cations, Divalent/metabolism , HIV Integrase/metabolism , HIV-1/enzymology , Virus Assembly , Calcium/metabolism , Catalysis , Cobalt/metabolism , HIV Integrase Inhibitors , HIV-1/physiology , Humans , Magnesium/metabolism , Manganese/metabolism , Substrate Specificity
8.
J Virol ; 70(7): 4819-24, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8676515

ABSTRACT

The human cytomegalovirus (HCMV) protease is a potential target for antiviral chemotherapeutics; however, autoprocessing at internal sites, particularly at positions 143 and 209, hinders the production of large quantities of stable enzyme for either screening or structural studies. Using peptides encompassing the sequence of the natural M-site substrate (P5-P5', GVVNA/SCRLA), we previously demonstrated that substitution of glycine for valine at the P3 position in the substrate abrogates processing by the recombinant protease in vitro. We now demonstrate that introduction of the V-to-G substitution in the P3 positions of the two major internal processing sites, positions 143 and 209, in the mature HCMV protease renders the enzyme stable to autoprocessing. When expressed in Escherichia coli, the doubly substituted protease was produced almost exclusively as the 30-kDa full-length protein. The full-length V141G, V207G (V-to-G changes at positions 141 and 207) protease was purified as a soluble protein by a simple two-step procedure, ammonium sulfate precipitation followed by DEAE ion-exchange chromatography, resulting in 10 to 15 mg of greater than 95% pure enzyme per liter. The stabilized enzyme was characterized kinetically and was indistinguishable from the wild-type recombinant protease, exhibiting Km and catalytic constant values of 0.578 mM and 13.18/min, respectively, for the maturation site (M-site) peptide substrate, GVVNASCRLARR (underlined residues indicate additions to or substitutions from peptides derived from the wild-type substrate). This enzyme was also used to perform inhibition studies with a series of truncated and/or substituted maturation site peptides. Short nonsubstrate M-site-derived peptides were demonstrated to be competitive inhibitors of cleavage in vitro, and these analyses defined amino acids VVNA, P4 through P1 in the substrate, as the minimal substrate binding and recognition sequence for the HCMV protease.


Subject(s)
Cytomegalovirus/enzymology , Endopeptidases/metabolism , Protease Inhibitors/pharmacology , Viral Proteins/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , Endopeptidases/drug effects , Endopeptidases/genetics , Escherichia coli , Humans , Molecular Mimicry , Molecular Sequence Data , Oligodeoxyribonucleotides , Peptides/pharmacology , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Solubility , Viral Proteins/drug effects , Viral Proteins/genetics
9.
J Virol ; 70(3): 1424-32, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8627659

ABSTRACT

The integration of a DNA copy of the viral genome into the genome of the host cell is an essential step in the replication of all retroviruses. Integration requires two discrete biochemical reactions; specific processing of each viral long terminal repeat terminus or donor substrate, and a DNA strand transfer step wherein the processed donor substrate is joined to a nonspecific target DNA. Both reactions are catalyzed by a virally encoded enzyme, integrase. A microtiter assay for the strand transfer activity of human immunodeficiency virus type 1 integrase which uses an immobilized oligonucleotide as the donor substrate was previously published (D. J. Hazuda, J. C. Hastings, A. L. Wolfe, and E. A. Emini, Nucleic Acids Res. 22;1121-1122, 1994). We now describe a series of modifications to the method which facilitate study of both the nature and the dynamics of the interaction between integrase and the donor DNA. The enzyme which binds to the immobilized donor is shown to be sufficient to catalyze strand transfer with target DNA substrates added subsequent to assembly; in the absence of the target substrate, the complex was retained on the donor in an enzymatically competent state. Assembly required high concentrations of divalent cation, with optimal activity achieved at 25 mM MnCl2. In contrast, preassembled complexes catalyzed strand transfer equally efficiently in either 1 or 25 mM MnCl2, indicating mechanistically distinct functions for the divalent cation in assembly and catalysis, respectively. Prior incubation of the enzyme in 25 mM MnCl2 was shown to promote the multimerization of integrase in the absence of a DNA substrate and alleviate the requirement for high concentrations of divalent cation during assembly. The superphysiological requirement for MnCl2 may, therefore, reflect an insufficiency for functional self-assembly in vitro. Subunits were observed to exchange during the assembly reaction, suggesting that multimerization can occur either before or coincident with but not after donor binding. These studies both validate and illustrate the utility of this novel methodology and suggest that the approach may be generally useful in characterizing other details of this biochemical reaction.


Subject(s)
Chlorides/metabolism , DNA Nucleotidyltransferases/metabolism , HIV Long Terminal Repeat/physiology , HIV-1/enzymology , Manganese Compounds/metabolism , Base Sequence , Catalysis , Cations, Divalent , DNA/metabolism , DNA, Viral , Humans , Integrases , Molecular Sequence Data , Substrate Specificity , Time Factors , Virus Integration/physiology
10.
Antimicrob Agents Chemother ; 39(2): 320-4, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7726489

ABSTRACT

The human immunodeficiency virus type 1 (HIV-1) integrase protein is required for the productive infection of T-lymphoid cells in culture (R. L. LaFemina, C. L. Schneider, H. L. Robbins, P. L. Callahan, K. LeGrow, E. Roth, W. A. Schleif, and E. A. Emini, J. Virol. 66:7414-7419, 1992). This observation suggests that chemical inhibitors of integrase may prevent the spread of HIV in infected individuals. In our search for such potential chemotherapeutic agents, we observed that beta-conidendrol inhibits both the sequence-dependent and sequence-independent endonucleolytic activities of integrase with comparable potencies in vitro (50% inhibitory concentration, 500 nM). Structurally related compounds tested for their abilities to inhibit integrase generated a limited structure-activity analysis which demonstrated that potency is associated with the bis-catechol structure: two pairs of adjacent hydroxyls on separate benzene rings. beta-Conidendrol did not inhibit several other endonucleases and/or phosphoryltransferases. Although beta-conidendrol was not effective in preventing HIV-1 infection in cell culture, the in vitro data demonstrate that it is possible to identify selective agents targeted against this essential HIV-1 function.


Subject(s)
Catechols/pharmacology , DNA Nucleotidyltransferases/antagonists & inhibitors , HIV-1/enzymology , Base Sequence , Integrases , Molecular Sequence Data , Structure-Activity Relationship
12.
J Biol Chem ; 269(6): 3999-4004, 1994 Feb 11.
Article in English | MEDLINE | ID: mdl-8307956

ABSTRACT

A DNA binding assay was developed for the human immunodeficiency virus type 1 (HIV-1) integrase. The assay was capable of defining discrete complexes between the enzyme and the viral long terminal repeat (LTR) substrate. DNA binding reflected the sequence requirements previously demonstrated for the enzyme's 3'-end processing activity. Binding exhibited a nonlinear dependence on integrase concentration, suggesting that the enzyme functions as a multimer. The oligomeric state was investigated by UV-photo-cross-linking of integrase-LTR oligonucleotide complexes using DNA substrates substituted with 5-bromo-2'-deoxycytidine within the integrase recognition sequence. In the absence of divalent cation, integrase cross-linked to the LTR oligonucleotide as a single species whose mobility by SDS-polyacrylamide gel electrophoresis was consistent with the formation of tetramers. Using these techniques, analysis of the binding properties of integrase mutants demonstrated that the catalytic and sequence-specific DNA binding activities of the enzyme are distinct, involving residues within the conserved "DD(35)E" and zinc finger motifs, respectively.


Subject(s)
DNA Nucleotidyltransferases/metabolism , HIV Long Terminal Repeat , HIV-1/enzymology , Base Sequence , Cations, Divalent , DNA-Binding Proteins/metabolism , Integrases , Macromolecular Substances , Molecular Sequence Data , Recombinant Proteins , Structure-Activity Relationship , Substrate Specificity , Zinc Fingers
13.
Virology ; 193(1): 73-9, 1993 Mar.
Article in English | MEDLINE | ID: mdl-8382413

ABSTRACT

The origin binding protein (OBP) of herpes simplex virus (HSV) type 1 specifically interacts with two high-affinity sites in each HSV DNA replication origin. The sequence-specific DNA binding activity of OBP maps to the carboxy-terminal one-third of the protein. For a single binding site, recombinantly expressed forms of this DNA binding domain have the same sequence specificity and binding affinity as the full-length OBP. However, unlike the full-length protein, truncated OBP does not bind HSV replication origins in a cooperative manner. To determine if cooperative interactions between DNA-bound OBP molecules are essential for viral DNA replication, the 317-amino-acid carboxy-terminal DNA binding domain of OBP was expressed in chick embryo fibroblasts. Cells were infected with HSV type 1, and viral DNA synthesis and virus production were monitored. We found that cells expressing truncated OBP were severely restricted for virus replication and that HSV DNA synthesis was undetectable. The results demonstrate that the amino-terminal two-thirds of OBP is essential for HSV DNA replication and that the OBP DNA binding domain acts as a transdominant inhibitor of viral DNA replication. The results also suggest that this experimental approach could be used to generate a refined map of essential OBP functions and that the approach may be generally applicable to the analysis of the multifunction HSV DNA replication complex.


Subject(s)
DNA, Viral/metabolism , DNA-Binding Proteins/physiology , Simplexvirus/physiology , Viral Proteins/physiology , Virus Replication/physiology , Animals , Base Sequence , Cell Line , Chick Embryo , DNA Replication/genetics , DNA Replication/physiology , DNA-Binding Proteins/genetics , Fibroblasts , Molecular Sequence Data , Simplexvirus/genetics , Viral Proteins/genetics , Virus Replication/genetics
14.
J Biol Chem ; 267(20): 14309-15, 1992 Jul 15.
Article in English | MEDLINE | ID: mdl-1321142

ABSTRACT

The virally encoded origin binding protein (OBP) of herpes simplex virus (HSV) is required for viral DNA synthesis. OBP binds at the replication origin to initimultienzyme replication complex (Challberg, M. D., and Kelly, T. J. (1989) Annu Rev. Biochem. 58, 671-717), OBP binds to two sites at the replication origin. The sequence-specific interaction of OBP with each binding site is localized to the major groove, and in both HSV origins the two interaction surfaces are in phase, aligned on the same face of the helix (Hazuda, D. J., Perry, H. C., Naylor, A. M., and McClements, W. L. (1991) J. Biol. Chem. 261, 24621-24625). Using native gel electrophoresis, we now demonstrate that OBP binding to the origin is highly cooperative and that cooperativity requires the putative NH2-terminal leucine zipper. Neither the phase nor orientation of the binding sites affect cooperativity, suggesting that the interaction promotes wrapping of origin DNA around the OBP multimer. A comparison of OBP DNase I footprints with the DNase I footprints of a truncated protein defective in cooperativity demonstrates that the interaction between OBPs bound at sites I and II affects the conformation of the intervening DNA, particularly when the phase or orientation of the two sites is different from wild type. OBP may elicit a unique nucleoprotein structure which facilitates unwinding of the origin and/or assembly of the replication complex. We also demonstrate that OBP can exchange binding sites, forming interduplex complexes. This property may be important for reinitiation of DNA replication.


Subject(s)
DNA Replication , DNA, Viral/genetics , DNA-Binding Proteins/metabolism , Simplexvirus/metabolism , Viral Proteins/metabolism , Base Sequence , Binding Sites , Binding, Competitive , Chromatography, Gel , DNA, Viral/biosynthesis , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , Deoxyribonuclease I , Electrophoresis, Polyacrylamide Gel , Exonucleases/metabolism , Leucine Zippers/genetics , Leucine Zippers/physiology , Molecular Sequence Data , Oligodeoxyribonucleotides , Protein Conformation , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Restriction Mapping , Simplexvirus/genetics , Viral Proteins/genetics , Viral Proteins/isolation & purification
15.
J Biol Chem ; 266(36): 24621-6, 1991 Dec 25.
Article in English | MEDLINE | ID: mdl-1662213

ABSTRACT

The origin binding protein (OBP) of herpes simplex virus (HSV), which is essential for viral DNA replication, binds specifically to sequences within the viral replication origin(s) (for a review, see Challberg, M.D., and Kelly, T. J. (1989) Annu. Rev. Biochem. 58, 671-717). Using either a COOH-terminal OBP protein A fusion or the full-length protein, each expressed in Escherichia coli, we investigated the interaction of OBP with one HSV origin, OriS. Binding of OBP to a set of binding site variant sequences demonstrates that the 10-base pair sequence, 5' CGTTCGCACT 3', comprises the OBP-binding site. This sequence must be presented in the context of at least 15 total base pairs for high affinity binding, Ka = approximately 0.3 nM. Single base pair mutations in the central CGC sequence lower the affinity by several orders of magnitude, whereas a substitution at any of the other seven positions reduces the affinity by 10-fold or less. OBP binds with high affinity to duplex DNA containing mismatched base pairs. This property is exploited to analyze OBP binding to DNA heteroduplexes containing singly substituted mutant and wild-type DNA strands. For positions 2, 3, 5, 6, 7, 8, and 9, substitutions are tolerated on one or the other DNA strand, indicating that base-mediated interactions are limited to one base of each pair. For both Boxes I and II, these interactions are localized to one face of the DNA helix, forming a recognition surface in the major groove. In OriS, the 31 base pairs which separate Boxes I and II orient the two interaction surfaces to the same side of the DNA.


Subject(s)
DNA-Binding Proteins/metabolism , Simplexvirus/metabolism , Viral Proteins/metabolism , Base Sequence , Binding, Competitive , Cloning, Molecular , DNA Replication , DNA, Viral/metabolism , DNA-Binding Proteins/genetics , Molecular Sequence Data , Mutation , Nucleic Acid Heteroduplexes , Oligonucleotides/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Viral Proteins/genetics
16.
J Biol Chem ; 266(11): 7081-6, 1991 Apr 15.
Article in English | MEDLINE | ID: mdl-2016316

ABSTRACT

The two interleukin 1 (IL-1) genes (IL-1 alpha and beta) encode 31-kDa precursor molecules, which are cleaved upon secretion to generate the mature, active, carboxyl-terminal 17-kDa proteins. The IL-1 beta precursor is inactive, whereas the IL-1 alpha precursor is as active as the mature IL-1 alpha. In this report, we demonstrate that when either of the recombinant precursors is processed to the mature form, the mature region undergoes a conformational change from a proteinase K-sensitive structure to one that is proteinase K-insensitive. In addition, cysteine residues that are exposed to solvent in the IL-1 beta precursor become buried in the mature protein. Limited structure-activity mapping of the IL-1 beta precursor indicates that the amino-terminal 76 residues are responsible for the conformational change, whereas the most dramatic change in biological activity occurs after further removal of residues 77-94. These findings suggest that the altered structure of the mature region in precursor IL-1s has been conserved for some function. Denaturation/renaturation experiments implicate the precursor domain in protein folding, and by analogy with signal-directed secretory proteins, the unique conformation of the precursors may play a role in IL-1 secretion.


Subject(s)
Genes , Interleukin-1/genetics , Protein Precursors/genetics , Amino Acid Sequence , Animals , Cell Line , Cloning, Molecular , Endopeptidases , Escherichia coli/genetics , Humans , Interleukin-1/chemistry , Interleukin-1/isolation & purification , Interleukin-1/pharmacology , Peptide Fragments/pharmacology , Protein Conformation , Protein Denaturation , Protein Precursors/chemistry , Protein Precursors/isolation & purification , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Structure-Activity Relationship
17.
J Biol Chem ; 265(11): 6318-22, 1990 Apr 15.
Article in English | MEDLINE | ID: mdl-2156847

ABSTRACT

The processing of precursor interleukin 1 beta (IL1 beta) by elastase, cathepsin G, and collagenase, the major proteases released at sites of inflammation, was investigated using recombinant pro-IL1 beta. Each of these proteases cleaved the 31-kDa inactive precursor to a form similar in size and specific activity (greater than 10(8) units/mg) to the 17-kDa mature protein isolated from activated monocytes. Elastase, collagenase, and cathepsin G cleaved the IL1 beta precursor at distinct sites which are amino-terminal to the monocyte-processing site, Ala-117 (Cameron, P., Lumjuco, G., Rodkey, J., Bennett, C., and Schmidt, J. A. (1985) J. Exp. Med. 162, 790-801). Amino-terminal sequencing of the products of digestion by elastase and cathepsin G determined that resultant active IL1 beta proteins contained an additional 13 or 3 amino acids relative to mature IL1 beta. Synovial fluid collected from patients with inflammatory polyarthritis and bronchoalveolar lavage fluid from patients with sarcoidosis supplied similar processing activity(s). Control fluids from patients who had no symptoms of inflammatory disease did not exhibit processing activity. Lavage fluids that processed precursor IL1 beta were demonstrated to contain cathepsin G and/or elastase activity, whereas controls were negative. Because a significant fraction of IL1 beta may be secreted from monocytes as the inactive 31-kDa precursor (Hazuda, D. J., Lee, J. C., and Young, P. R. (1988) J. Biol. Chem. 263, 8473-8479, Bomford, R., Absull, E., Hughes-Jenkins, C., Simpkin, D., and Schmidt, J. (1987) Immunology 62, 543-549, and Mizel, S. B. (1988) in Cellular and Molecular Aspects of Inflammation Poste, G., and Crooke, S., eds) pp. 75-93, Plenum Publishing Corp., New York), these results suggest that in vivo the IL1 beta precursor can be processed after secretion by any of several proteases released at inflammatory sites.


Subject(s)
Inflammation/immunology , Interleukin-1/genetics , Microbial Collagenase/metabolism , Protein Precursors/genetics , Protein Processing, Post-Translational , Amino Acid Sequence , Cathepsin G , Cathepsins/metabolism , Humans , Immunoblotting , Leukocytes/enzymology , Lung/immunology , Lung/physiopathology , Molecular Sequence Data , Pancreatic Elastase/metabolism , Serine Endopeptidases , Substrate Specificity , Therapeutic Irrigation
18.
J Cell Biol ; 107(2): 447-56, 1988 Aug.
Article in English | MEDLINE | ID: mdl-3047139

ABSTRACT

To understand the secretion and processing of interleukin-1 (IL-1), a Chinese hamster fibroblast cell line (R1610) was transfected with a human IL-1 beta cDNA under the control of the SV40 early promoter and linked to the gene for neomycin resistance. After selecting for transfected cells resistant to G418, two clones were found to constitutively express the IL-1 beta 31-kD precursor which was almost exclusively located in the cytosol. Pulse-chase experiments failed to show any secretion of IL-1 and very little IL-1 activity was detectable in cell supernatants. Furthermore, surface membrane IL-1 activity could not be detected, although low levels of activity could be released upon brief trypsin treatment. Therefore, unlike monocytes, these fibroblast cells lack the mechanism for secreting and processing of IL-1 beta.


Subject(s)
DNA/genetics , Gene Expression Regulation , Interleukin-1/biosynthesis , Peptide Fragments/biosynthesis , Protein Precursors/biosynthesis , Transfection , Animals , Cell Line , Cloning, Molecular , Cricetinae , Cytoplasm/analysis , Electrophoresis, Polyacrylamide Gel , Fibroblasts , Fluorescent Antibody Technique , Genetic Vectors , Immunoassay , Interleukin-1/analysis , Interleukin-1/genetics , Interleukin-1beta , Peptide Fragments/analysis , Peptide Fragments/genetics , Plasmids , Precipitin Tests , Promoter Regions, Genetic , Protein Precursors/analysis , Protein Precursors/genetics , RNA, Messenger/analysis , Transcription, Genetic
19.
J Biol Chem ; 263(17): 8473-9, 1988 Jun 15.
Article in English | MEDLINE | ID: mdl-3259579

ABSTRACT

We have performed pulse-chase experiments to investigate the secretion and processing of interleukin 1 (IL-1) by human peripheral blood monocytes. Polyclonal antisera generated against either recombinant IL-1 alpha (p15) or IL-1 beta (p17) could distinguish the two isoelectric forms in lysates and supernatants of lipopolysaccharide-activated monocytes. In agreement with previous results, no processed IL-1 (alpha or beta) is detected in cell lysates. Both the 31-kDa precursor and 17-kDa mature forms of IL-1 were present, however, in the culture media indicating that processing is not required for secretion. The relative amounts of the secreted 31- and 17-kDa forms of IL-1 remain constant with time throughout each experiment; in addition, 31-kDa IL-1 added to monocyte cultures is not processed to the mature 17-kDa form. Precursor IL-1 beta is however, processed to 17 kDa by monocyte extracts. Therefore, the maturation and secretion of IL-1 are intimately coordinated processes. The kinetics of IL-1 secretion are unique in comparison with other secreted proteins; release of both IL-1 alpha and IL-1 beta is delayed following synthesis, and large pools of precursor IL-1 accumulate intracellularly. The intracellular half-lives of IL-1 alpha and IL-1 beta are 15 and 2.5 h, respectively. This discrepancy in half-lives is a reflection of the different kinetics with which IL-1 alpha and IL-1 beta are secreted. IL-1 beta is released continuously beginning 2 h after synthesis, whereas the secretion of IL-1 alpha is delayed for an additional 10 h. The distinct kinetics of secretion demonstrated for IL-1 alpha and IL-1 beta suggest that the release of each pI species of IL-1 is controlled by a selective mechanism(s).


Subject(s)
Interleukin-1/metabolism , Monocytes/metabolism , Animals , Immune Sera , Kinetics , Molecular Weight , Rabbits , Tumor Necrosis Factor-alpha/metabolism
20.
J Biol Chem ; 261(26): 12202-8, 1986 Sep 15.
Article in English | MEDLINE | ID: mdl-2943735

ABSTRACT

Highly purified preparations of Xenopus transcription factor A exhibit DNA-activated ATPase (ATP phosphorylase, EC 3.6.1.3) activity, which is inhibited by affinity-purified anti-factor A antibodies but not by nonspecific gamma-globulins. This enzymatic activity copurifies with both factor A and the 7 S particle, a ribonucleoprotein complex composed of factor A and 5 S RNA in a one-to-one stoichiometric ratio. At equal concentrations of protein, factor A and the 7 S particle catalyze the hydrolysis of ATP to ADP and Pi at similar rates. Kinetic analysis demonstrates that factor A is a fairly typical ATPase with a Vmax of 1.7 nmol/min/mg of protein and a KM of 5.0 X 10(-5) M, whereas the corresponding values for the 7 S particle are 2.7 nmol/min/mg of protein and 1.4 X 10(-4) M, respectively. Besides ATP, dATP is also an effective substrate for the enzyme with a Vmax of 0.7 nmol/min/mg of protein and a KM of 3.3 X 10(-5) M in reactions catalyzed by the 7 S particle. The ATPase activity of free factor A, but not the 7 S particle, can be stimulated approximately 3-fold by the addition of pBR322 plasmid DNA. Proteolytic fragments of factor A generated by treatment of the 7 S particle with papain and trypsin retain a portion of their catalytic activity, 50 and 10%, respectively, in concert with their relative size. Radioactive ATP and dATP can be photocross-linked to factor A by UV irradiation. These radioactive substrates are also cross-linked to the papain- and trypsin-generated fragments with markedly decreased efficiencies. UV photocross-linking of non-substrate nucleotides to factor A was not detectable. These results provide evidence that the ATPase activity is intrinsic to the factor A protein which is essential for the specific initiation of 5 S RNA gene transcription.


Subject(s)
Adenosine Triphosphatases/metabolism , DNA/metabolism , Trans-Activators , Transcription Factors/metabolism , Xenopus Proteins , Adenosine Triphosphate/radiation effects , Animals , Enzyme Activation , Kinetics , Molecular Weight , Photochemistry , Protein Conformation , Ultraviolet Rays , Xenopus laevis
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