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A Engelman

Publications and source records attributed to A Engelman.

36 records · Page 2Linked to original sources
PubMed1995

Efficient magnesium-dependent human immunodeficiency virus type 1 integrase activity.

The integrase protein from human immunodeficiency virus type 1 (HIV-1) has generally been reported to require Mn2+ for efficient in vitro activity. We have reexamined the divalent metal ion requirements of HIV-1 integrase and find that the protein is capable of promoting efficient 3' processing and DNA strand transfer with either Mn2+ or Mg2+. The metal ion preference depended upon the reaction conditions. HIV-1 integrase displayed significantly less nonspecific nuclease activity in reaction mixtures containing Mg2+ than it did under the previously described reaction conditions with mixtures containing Mn2+.

Base Sequence↗
PubMed1994

Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases.

HIV integrase is the enzyme responsible for inserting the viral DNA into the host chromosome; it is essential for HIV replication. The crystal structure of the catalytically active core domain (residues 50 to 212) of HIV-1 integrase was determined at 2.5 A resolution. The central feature of the structure is a five-stranded beta sheet flanked by helical regions. The overall topology reveals that this domain of integrase belongs to a superfamily of polynucleotidyl transferases that includes ribonuclease H and the Holliday junction resolvase RuvC. The active site region is identified by the position of two of the conserved carboxylate residues essential for catalysis, which are located at similar positions in ribonuclease H. In the crystal, two molecules form a dimer with a extensive solvent-inaccessible interface of 1300 A2 per monomer.

Amino Acid Sequence↗
PubMed1994

Biophysical and enzymatic properties of the catalytic domain of HIV-1 integrase.

A deletion derivative of the integrase protein from human immunodeficiency virus type-1 (HIV-1) consisting of the central core domain (amino acids 50-212) has been characterized biophysically and biochemically. This deletion mutant is of particular interest for structural studies as it can carry out the disintegration reaction suggesting the presence of an active site and, under certain conditions, is more soluble than full-length integrase. The circular dichroism and fluorescence of the deletion mutant and the 288-residue full-length integrase were similar, indicating that the core residues maintain similar overall conformations in both proteins. The deletion mutant is approximately 10% more alpha-helical than the full-length protein. Analytical centrifugation demonstrated that both proteins undergo monomer-dimer association although the truncated protein showed slightly less tendency to dimerize; the dissociation constants were 2.5 x 10(-5) M for the full-length protein and 8.0 x 10(-5) M for the truncated protein. The disintegration activity of both proteins was also compared. Although a higher concentration of the truncation mutant was required for optimal activity, the mutant did not have altered pH or Mn2+ requirements relative to the full-length protein. The combined biophysical and enzymatic studies suggest that this truncated form of HIV-1 integrase is likely to be useful for structural studies.

Amino Acid Sequence↗
PubMed1994

Intermolecular disintegration and intramolecular strand transfer activities of wild-type and mutant HIV-1 integrase.

We report the activities of HIV integrase protein on a novel DNA substrate, consisting of a pair of gapped duplex molecules. Integrase catalyzed an intermolecular disintegration reaction that requires positioning of a pair of the gapped duplexes in a configuration that resembles the intgration intermediate. However, the major reaction resulted from an intramolecular reaction involving a single gapped duplex, giving rise to a hairpin. Surprisingly, a deletion mutant of integrase that lacks both the amino and carboxyl terminal regions still catalyzed the intermolecular disintegration reaction, but supported only a very low level of the intramolecular reaction. The central core region of integrase is therefore sufficient to both bind the gapped duplex DNA and juxtapose a pair of such molecules through protein-protein interactions. We suggest that the branched DNA structures of the previously reported disintegration substrate, and the intermolecular disintegration substrate described here, assist in stabilizing protein-protein interactions that otherwise require the amino and carboxy terminal regions of integrase.

Base Sequence↗
PubMed1994

Most of the avian genome appears available for retroviral DNA integration.

Although retroviral integration requires specific viral DNA sequences, factors which govern the choice of a chromosomal target site within an infected cell are less clear. For example, certain chromosomal regions may be inaccessible to the viral integration machinery, while others may favor integration. A recent paper by Withers-Ward et al.(1) addresses this issue using a polymerase chain reaction-based assay capable of identifying single integration events within a large population of infected cells. Their results show that integration can occur into many different chromosomal regions, and that local DNA structure can influence the site of integration within a given region.

Animals↗
PubMed1994

An integration-defective U5 deletion mutant of human immunodeficiency virus type 1 reverts by eliminating additional long terminal repeat sequences.

Nonoverlapping deletions that eliminated the 5' (HIV-1US/603del), middle (HIV-1U5/206del), and 3' (HIV-1U5/604del) thirds of the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) were studied for their effects on virus replication (transient transfection of HeLa cells) and infectivity (T-cell lines and peripheral blood mononuclear cells). All three mutants exhibited a wild-type phenotype in directing the production and release of virus particles from transfected HeLa cells. In infectivity assays, HIV-1U5/206del was usually indistinguishable from wild-type virus whereas HIV-1U%/603del was unable to infect human peripheral blood mononuclear cells or MT4 and CEM cells. Investigations of HIV-1U5/603del particles revealed a packaging defect resulting in a 10-fold reduction of encapsidated genomic RNA. The HIV-1U5/604del mutant either was noninfectious or exhibited delayed infection kinetics, depending on the cell type and multiplicity of infection. Quantitative competitive PCR indicated that HIV-1U5/604del synthesized normal amounts of viral DNA in newly infected cells. During the course of a long-term infectivity assay, a revertant of the HIV-1U5/604del mutant that displayed rapid infection kinetics emerged. Nucleotide sequence analysis indicated that the original 26-nucleotide deletion present in HIV-1U5/604del had been extended an additional 19 nucleotides in the revertant virus. Characterization of the HIV-1U5/604del mutant LTR in in vitro integration reactions revealed defective 3' processing and strand transfer activities that were partially restored when the revertant LTR substrate was used, suggesting that the reversion corrected a similar defect in the mutant virus.

Base Sequence↗
PubMed1994

The core and carboxyl-terminal domains of the integrase protein of human immunodeficiency virus type 1 each contribute to nonspecific DNA binding.

The integrase protein of human immunodeficiency virus type 1 removes two nucleotides from the 3' ends of reverse-transcribed human immunodeficiency virus type 1 DNA (3' processing) and covalently inserts the processed ends into a target DNA (DNA strand transfer). Mutant integrase proteins that lack the amino-and/or carboxyl-terminal domains are incapable of catalyzing 3' processing and DNA strand transfer but are competent for an apparent reversal of the DNA strand transfer reaction (disintegration) in vitro. Here, we investigate the binding of integrase to DNA by UV cross-linking. Cross-linked complexes form with a variety of DNA substrates independent of the presence of divalent metal ion. Analysis with amino- and carboxyl-terminal deletion mutant proteins shows that residues 213 to 266 of the 288-residue protein are required for efficient cross-linking in the absence of divalent metal ion. Carboxyl-terminal deletion mutants that lack this region efficiently cross-link only to the branched disintegration DNA substrate, and this reaction is dependent on the presence of metal ion. Both the core and C-terminal domains of integrase therefore contribute to nonspecific DNA binding.

Base Sequence↗
PubMed1993

Domains of the integrase protein of human immunodeficiency virus type 1 responsible for polynucleotidyl transfer and zinc binding.

The integrase protein of human immunodeficiency virus type 1 carries out a set of polynucleotidyl transfer reactions that result in the covalent attachment of the retroviral cDNA to host DNA. We have analyzed the activities of a set of deletion derivatives of the integrase protein. The analysis reveals that a central domain of only 137 amino acids is sufficient in vitro to catalyze a subset of the reactions carried out by the complete protein. This polypeptide contains an amino acid sequence motif, Asp-Xaa39-58-Asp-Xaa35-Glu (DX39-58DX35E, where X and the subscript indicate the intervening amino acids between the invariant acidic residues), that is found in the integrases of retroviruses and retrotransposons and also the transposase proteins of some bacterial transposable elements. We also find that the integrase protein can bind Zn2+, and the histidine and cysteine residues of another conserved motif (HX3-7HX23-32CX2C) are required for efficient Zn2+ binding. The activities displayed by deletion mutants suggest to us possible functions for the various parts of integrase.

Amino Acid Sequence↗
PubMed1993

Identification of discrete functional domains of HIV-1 integrase and their organization within an active multimeric complex.

HIV-1 integrase protein possesses the 3' processing and DNA strand transfer activities that are required to integrate HIV DNA into a host chromosome. The N-, C-terminal and core domains of integrase are necessary for both activities in vitro. We find that certain pairs of mutant integrase proteins, which are inactive when each protein is assayed alone, can support near wild type levels of activity when both proteins are present together in the reaction mixture. This complementation implies that HIV-1 integrase functions as a multimer and has enabled us to probe the organization of the functional domains within active mixed multimers. We have identified a minimal set of functional integrase domains that are sufficient for 3' processing and DNA strand transfer and find that some domains are contributed in trans by separate monomers within the functional complex.

Base Sequence↗
PubMed1992

Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro.

We have probed the structural organization of the human immunodeficiency virus type 1 integrase protein by limited proteolysis and the functional organization by site-directed mutagenesis of selected amino acid residues. A central region of the protein was relatively resistant to proteolysis. Proteins with altered amino acids in this region, or in the N-terminal part of the protein that includes a putative zinc-binding motif, were purified and assayed for 3' processing, DNA strand transfer, and disintegration activities in vitro. In general, these mutations had parallel effects on 3' processing and DNA strand transfer, suggesting that integrase may utilize a single active site for both reactions. The only proteins that were completely inactive in all three assays contained mutations at conserved amino acids in the central region, suggesting that this part of the protein may be involved in catalysis. In contrast, none of the mutations in the N-terminal region resulted in a protein that was inactive in all three assays, suggesting that this part of integrase may not be essential for catalysis. The disintegration reaction was particularly insensitive to these amino acid substitutions, indicating that some function that is important for 3' processing and DNA strand transfer may be dispensable for disintegration.

Amino Acid Sequence↗
PubMed1991

HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer.

Retroviral DNA integration involves a coordinated set of DNA cutting and joining reactions. Linear viral DNA is cleaved at each 3' end to generate the precursor ends for integration. The resulting recessed 3' ends are inserted into target DNA by a subsequent DNA strand transfer reaction. Purified HIV-1 integration protein carries out both of these steps in vitro. Two novel forms of the dinucleotide cleaved from HIV-1 DNA were identified and one, a cyclic dinucleotide, was used to analyze the stereochemical course of viral DNA cleavage. Both viral DNA cleavage and DNA strand transfer display inversion at chiral phosphorothioates during the course of the reaction. These results suggest that both reactions occur by a one-step mechanism without involvement of a covalent protein-DNA intermediate.

Alkaline Phosphatase↗
PubMed1991

A rapid in vitro assay for HIV DNA integration.

Retroviruses synthesize a double stranded DNA copy of their RNA genome after infection of a permissive cell and subsequent integration of this DNA copy into the host genome is necessary for normal viral replication. Integration occurs by a specialized DNA recombination reaction, mediated by the viral IN protein. Because this reaction has no known cellular counterpart, it is a particularly attractive target in the search for specific inhibitors with low toxicity that may serve as therapeutic antiviral agents. We present a simple assay system that is suitable for screening potential inhibitors of HIV DNA integration. Only short oligonucleotides matching one end of HIV DNA and purified HIV IN protein are required as substrates. Furthermore, since each step of the assay can be carried out in the wells of microtiter plates, large numbers of reactions can be processed simultaneously.

Animals↗
PubMed1990

Temperature-sensitive mutants of Abelson murine leukemia virus deficient in protein tyrosine kinase activity.

The effect of two missense mutations in abl on transformation by Abelson murine leukemia virus was evaluated. These mutations led to the substitution of a histidine for Tyr-590 and a glycine for Lys-536. Both changes gave rise to strains that were temperature dependent for transformation of both NIH 3T3 cells and lymphoid cells when expressed in the context of a truncated Abelson protein. In the context of the prototype P120 v-abl protein, the Gly-536 substitution generated a host range mutant that induced conditional transformation in lymphoid cells but had only a subtle effect on NIH 3T3 cells. The combination of both substitutions gave rise to a P120 strain that was temperature sensitive for both NIH 3T3 and lymphoid cell transformation. The Abelson proteins encoded by the temperature-sensitive strain displayed in vitro kinase activities that were reduced when compared with those of wild-type proteins. In vivo, levels of phosphotyrosine were reduced only at the restrictive temperature. Analysis of cells expressing either the wild-type P160 v-abl protein or the P210 bcr/abl protein and an Abelson protein encoded by a temperature-sensitive strain failed to correct this defect, suggesting either that tyrosine phosphorylation in vivo is an intramolecular reaction or that the protein encoded by the temperature-sensitive strain is a poor substrate for tyrosine phosphorylation in vivo. These results raise the possibility that tyrosine phosphorylation of Abelson protein plays a role in transformation.

Abelson murine leukemia virus↗
PubMed1990

bcr/abl and src but not myc and ras replace v-abl in lymphoid transformation.

Lymphoid cells transformed by temperature-sensitive Abelson virus die at the nonpermissive temperature. This property was exploited to show that bcr/abl and v-src but not myc and ras can replace the transforming signal of v-abl, a result suggesting that the former but not the latter oncogenes transform lymphoid cells via a similar pathway.

Abelson murine leukemia virus↗
PubMed1987

Isolation of temperature-sensitive Abelson virus mutants by site-directed mutagenesis.

Mutants of Abelson virus encoding temperature-sensitive protein-tyrosine kinase (EC 2.7.1.112) were created by site-directed mutagenesis using sequence information from temperature-sensitive mutants of the related v-src oncogene. Expression of these two independent mutations in Escherichia coli resulted in reduced phosphorylation of the mutant proteins at high temperature. Viruses containing one of the mutations induced conditional transformation of both NIH 3T3 and lymphoid cells when expressed in the context of a truncated transforming protein. These results underscore the functional homology between protein-tyrosine kinases and suggest that transfer of mutations within a related gene family may provide a rapid method to create mutants.

Abelson murine leukemia virus↗
PubMed1982

Enhancement of individual and community competence: the older adult as community worker.

This paper presents an evaluation of a primary prevention program designed to enhance individual and community competence in older adult community workers and in community residents with whom they worked. A total of 22 community workers and 97 community residents participated in the study; 30 residents constituted as posttest-only control group. Pre-post changes included increased knowledge of community services among all participants, as well as increased number of community information channels and increased life satisfaction for the workers. Residents, particularly black residents, became more internal, and their increased sense of personal control was related to their increased knowledge of services. Thus, the helper-therapy principle was supported for these older adult, mostly female, community workers, and their helping role had a net empowering effect.

Aged↗
PubMedDate not supplied

[The endometrium under the effects of hormone replacement therapy in menopause with percutaneous estradiol and low-dose micronized progesterone].

AIM: The aim of our study is to evaluate the effects of a new combined association of percutaneous estradiol with oral micronized progesterone during 25 days/month and to confirm that a low dose of progesterone (100 mg/day) can adequately counteract endometrial proliferation induced by estradiol. METHODS: 78 endometrial tissue samples were obtained in a multicenter study on the effects of hormonal replacement therapy of the menopause. Endometrial biopsies were performed on average at the 6.6 month of the hormonal substitution (range: 5-13 months) after 12 days minimum exposure to progesterone. The morphological evaluation was performed blindly. RESULTS: All endometria are only slightly developed, without hyperplasia. Four groups were individualized: subatrophic endometrium (3), quiescent endometrium (48), slightly active endometrium (18), and endometrium with marginal secretion (6). Rare mitosis images have only been found in slightly active endometria. Their number is always very much below the normal proliferative phase with only 3 cases between less than 3 and 6 mitoses per 1000 glandular epithelial cells. CONCLUSION: A low micronized progesterone dose (100 mg/day) over a long period (25 days per month) allows to efficiently control the estrogen-induced endometrial proliferation. This adequate endometrial response is responsible for a high incidence of amenorrhea, often asked by patients, and for rare spottings.

Administration, Cutaneous↗