Mutational analysis of the amino and carboxy termini of the HIV-2 Tat protein.

The transactivator proteins of HIV-1 and HIV-2, Tat-1 and Tat-2, are highly homologous in the center of each molecule but are divergent in the amino and carboxy termini. The structure of Tat-1 has been extensively characterized by mutagenesis studies, whereas little is as yet known specifically about the structure of Tat-2. To characterize the Tat-2 protein, we performed a mutational analysis of the amino and carboxy termini of the fully functional first exon (99 residues) of the Tat-2 protein. We found that deletion of residues 8 through 33 in the amino terminus drastically reduced transactivation activity, whereas deletion of residues 8 through 47 largely abolished transactivation activity. We also analyzed chimeric proteins in which the amino termini of the Tat-1 and Tat-2 proteins were exchanged precisely at the first cysteine in the cysteine-rich regions. Both chimeric proteins possessed very low levels of transactivation activity, indicating that the amino termini of Tat-1 and Tat-2 are not interchangeable. Truncation mutants in the carboxy terminus were analyzed and amino acid 90 at the end of the basic domain was found to be at or near the limit of carboxy residues that can be deleted without abolishing Tat-2 function. A Tat-2 mutant truncated after residue 84 within the basic domain was found to be a transdominant mutant able to inhibit wild-type Tat-1 and wild-type Tat-2 activities. Additionally, the results of immunoprecipitations suggested that deletions in the Tat-2 amino terminus can reduce protein stability.

Wild-type and mutant HIV-1 and HIV-2 Tat proteins expressed in Escherichia coli as fusions with glutathione S-transferase.

Human immunodeficiency virus type 1 (HIV-1) and HIV-2 encode related transcriptional activators known as Tat-1 and Tat-2, respectively, that are required for efficient viral replication. The Tat proteins have been studied extensively, and it appears that their mechanism of action is unique to the primate immunodeficiency viruses or a few distantly related lentiviruses. Here we describe a collection of 24 wild-type and mutant Tat-1 and Tat-2 proteins that are expressed in Escherichia coli as fusions with glutathione S-transferase (GST). The GST-Tat fusions can be used for biochemical studies after simple purification from E. coli lysates in a single step under nondenaturing conditions. The availability of these GST-Tat fusions should be useful to investigators examining biochemical properties of Tat-1 and Tat-2 proteins. E. coli cultures harboring GST-Tat fusions described here are available through the National Institute of Health AIDS Research and Reference Reagent Program.

Construction and characterization of a potent HIV-2 Tat transdominant mutant protein.

The human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) Tat proteins Tat-1 and Tat-2 stimulate transcription of the viral long terminal repeat (LTR) sequences and are required for efficient viral replication. A class of mutant Tat proteins, termed "transdominant mutants," has been described that possesses relatively low transactivation activity, yet is able to inhibit the function of wild-type Tat. These mutant proteins contain a nonfunctional TAR RNA-binding domain but apparently retain a functional activation domain. A potential limitation for therapeutic use of transdominant mutants described to date is their low but significant basal level of transactivation for the HIV-1 or HIV-2 LTRs. In order to make an improved transdominant mutant, we have constructed Tat-2 proteins that contain mutations in four contiguous arginines at residues 81 to 84 in the RNA-binding domain. Using purified proteins and in vitro RNA-binding assays, we verified that these mutant Tat-2 proteins are defective for TAR RNA binding. We also verified that these mutant Tat-2 proteins bind to a cellular protein kinase in vitro that we have previously shown to bind specifically to the Tat-1 and Tat-2 activation domain. Using plasmid cotransfection assays, we compared the phenotypes of these mutant Tat-2 proteins with the most potent Tat-1 transdominant mutant described to date. One Tat-2 mutant, named "R81-84A," was found to be equivalent to the Tat-1 mutant in ability to inhibit wild-type Tat transactivation of HIV-1 and HIV-2 LTRs. Moreover, the R81-84A mutant possessed a significantly lower basal level of transactivation than the Tat-1 mutant. The R81-84A Tat-2 mutant is therefore a promising reagent for future development as an anti-HIV agent. Additionally, our results suggest that wild-type Tat-2 transactivation of the HIV-2 LTR is especially sensitive to inhibition by transdominant mutants.

Hepatic tyrosine aminotransferase from the rainbow lizard Agama agama: purification and some properties.

Tyrosine aminotransferase, induced by dexamethasone in the liver of the rainbow lizard, Agama agama, was extracted under optimal conditions which yield the native undegraded enzyme; purified by heat treatment at 65 degrees C, ammonium sulfate precipitation, chromatography on DEAE-Sephacel and Sephadex G-150-120 and then characterized. The enzyme was purified over 2000-fold to a specific activity of 2653 units/mg of protein. It had an optimum pH of 7.6 in potassium phosphate buffer, KmTyr: 1.0 mM; K alpha-KGm: 0.32 mM; Vmax: 1.33 nmol/min and a molecular weight of about 130,000. It was inhibited by L-glutamate (competitively, Ki, 2.5 mM), and by metal ions Ca2+, Mn2+, Zn2+, Hg2+ and Ag2+, but was unaffected by chelating agents and other divalent cations. Lizard hepatic cytosolic tyrosine aminotransferase was specific for L-tyrosine and alpha-ketoglutarate as substrates sensitive to sulfhydryl inactivation and to protection from thermal lability by alpha-ketoglutarate and pyridoxal phosphate.

Extracellular lipase and proteinase of Basidiobolus haptosporus: possible role in subcutaneous mycosis.

Properties of proteinase(s) and phospholipase A in dialyzed culture filtrate of Basidiobolus haptosporus were studied. Lysolecithin, one of the hydrolytic products of phosphatidyl choline by phospholipase A was prepared and found able to hemolyse human red blood cells and 'clear' rat skin and muscle homogenates. The proteinase was able to digest human serum proteins. The pathogenic mechanism employed by this fungus may involve hydrolysis of lecithin to yield lysolecithin, which destroys cell membranes thereby liberating intracellular contents. The protein components of these contents are then digested by the proteinase(s) yielding amino acids which along with other metabolites serve as nutrients for the growth of the pathogen.

Rapid purification of monomer HIV-2 Tat protein expressed in Escherichia coli.

Human immunodeficiency viruses types 1 and 2 encode transactivator proteins, named Tat-1 and Tat-2, that stimulate transcription directed by the viral long terminal repeat sequences. The Tat-1 and Tat-2 proteins are related in protein sequence and mechanism of transactivation. We expressed Tat proteins by in vitro translation and found, as expected, that both Tat-1 and Tat-2 were monomers. Unexpectedly, we found that the Tat-1 and Tat-2 proteins displayed significantly different tertiary structures. As determined by velocity sedimentation and gel filtration, Tat-1 acted as a compact structure and Tat-2 acted as an extended, asymmetric structure. Additionally, we found that the in vitro-expressed Tat-2 protein was significantly less susceptible to aggregation than the Tat-1 protein. This observation led us to overexpress Tat-2 in Escherichia coli and develop a simple and rapid method to purify monomer Tat-2 to approximately 50% purity. These results suggest that large amounts of Tat-2 protein can be purified in suitable form for biochemical and biophysical studies of Tat protein structure.

Localization of regulatory protein binding sites in the proximal region of human myometrial connexin 43 gene.

Parturition is preceded by a large increase in gap junctions between myometrial smooth muscle cells. Connexin 43 is the major structural protein of myometrial gap junctions. To explore transcriptional regulation of the myometrial Cx43 gene, we used DNase I footprinting, electrophoretic mobility shift and transient transfection assays to examine a 312 bp promoter region (-164 to +148) of the gene, utilizing human myometrial cell cultures and nuclear extracts. The DNase I studies showed four regions of nucleoprotein interactions. Protection of region 1 (-80 to -31) encompassed an Activator Protein 1 (AP1) (-44 to -36) and two Specificity Protein 1 (Sp1) (-77 to -69 and -59 to -48) consensus sequences. Regions 2 to 4 included the transcription initiation site (-10 to +25), an Ets/NF-kB consensus sequence (+47 to +74) and a TA-rich region (+81 to +101) respectively. Gel mobility shift and supershift assays demonstrated c-Jun and Sp1 binding at the AP1 and Sp1 sites respectively. Promoter mutagenesis and transient transfection analyses combined with Sp1 and c-Jun/c-Fos over-expression studies indicate that both Sp1 and c-Jun are required for maximal promoter activity and, therefore, may positively regulate transcription of myometrial Cx43 during the initiation of labour.