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K A Bostian

Publications and source records attributed to K A Bostian.

At least 19 recordsLinked to original sources
PubMed1998

Type A botulinum neurotoxin proteolytic activity: development of competitive inhibitors and implications for substrate specificity at the S1' binding subsite.

Type A botulinum neurotoxin (botox A) is a zinc metalloprotease that cleaves only one peptide bond in the synaptosomal protein, SNAP-25. Single-residue changes in a 17-residue substrate peptide were used to develop the first specific, competitive inhibitors of its proteolytic activity. Substrate analog peptides with P4, P3, P2' or P3' cysteine were readily hydrolyzed by the toxin, but those with P1 or P2 cysteine were not cleaved and were inhibitors. Peptides with either D- or L-cysteine as the N-terminus, followed by the last six residues of the substrate, were the most effective inhibitors, each with a Ki value of 2 microM. Elimination of the cysteine sulfhydryl group yielded much less effective inhibitors, suggesting that inhibition was primarily due to binding of the active-site zinc by the sulfhydryl group. Botox A displayed an unusual requirement for arginine as the P1' inhibitor residue, demonstrating that the S1' binding subsite of botox A is dissimilar to those of most other zinc metalloproteases. This characteristic is an important element in shaping the substrate specificity of botox A.

Amino Acid Sequence↗
PubMed1997

Endoproteinase activity of type A botulinum neurotoxin: substrate requirements and activation by serum albumin.

Type A botulinum neurotoxin, a zinc-dependent endoproteinase that selectively cleaves the neuronal protein SNAP-25, can also cleave relatively short peptides. We found that bovine and other serum albumins stimulated the type A-catalyzed hydrolysis of synthetic peptide substrates, through a direct effect on the kinetic constants of the reaction. Furthermore, with bovine serum albumin in the assays, the optimum substrate size was 16 residues (11 on the amino-terminal side of the cleavage site and 5 on the carboxy-terminal side). To further investigate the catalytic requirements of the neurotoxin, peptides were synthesized with various amino acid substitutions at the P5 through P5' substrate sites. Changes at all of these locations affected values for both kcat and K(m). Substitutions at the P2, P1', and P2' sites had more pronounced effects on hydrolysis rates than did substitutions at the P1 site. Enzyme-substrate interactions at the P3' threonine probably involved the side-chain methyl group rather than the hydroxyl group. Replacing the P2' alanine with leucine eliminated detectable hydrolysis, but not binding, since this peptide was an inhibitor. A negatively charged residue was preferred at P5, but not at P4. The data indicate that type A botulinum neurotoxin has an extended substrate recognition region and a requirement for arginine as the P1' residue.

Binding Sites↗
PubMed1995

Proteolysis of synthetic peptides by type A botulinum neurotoxin.

Type A botulinum neurotoxin catalyzed the hydrolysis of synthetic peptides based on the sequence of the 25-kD synaptosomal protein SNAP-25. In each peptide, the toxin cleaved at a single glutaminyl-arginine bond corresponding to residues 197 and 198 of SNAP-25, confirming earlier reports on the enzymatic specificity of the toxin in synaptosomal preparations. Metal chelators inhibited catalysis, consistent with a metalloprotease activity. In contrast to tetanus toxin and other botulinum toxin serotypes, type A toxin hydrolyzed relatively short, 17- to 20-residue peptides. In the substrates, SNAP-25 residue 202 and one or more of residues 187-191 were required for efficient hydrolysis, but residues 167-186 and 203-206 were not. The highest rates of hydrolysis were found when the C-terminal residues of the peptides were amidated.

Amides↗
PubMed1993

Calcineurin-dependent growth of an FK506- and CsA-hypersensitive mutant of Saccharomyces cerevisiae.

The immunosuppressants FK506 and cyclosporin A (CsA) bound to their receptors, FKBP12 or cyclophilin, inhibit the Ca2+/calmodulin-dependent protein phosphatase, calcineurin, preventing T cell activation or, in yeast, recovery from alpha-mating factor arrest. Vegetative growth of yeast does not require calcineurin, and in strains sensitive to FK506 or CsA, growth is inhibited by concentrations of drug much higher than those required to inhibit T cell activation or recovery from mating factor arrest. We now describe the isolation of a mutant of Saccharomyces cerevisiae which is 100-1000-fold more sensitive to the growth inhibitory properties of these drugs. The mutation (fks1) also confers a slow growth phenotype which is partially suppressed by exogenously added Ca2+ and exacerbated by EGTA. Simultaneous disruption of the two genes (CNA1 and CNA2) encoding the alternative forms of the catalytic A subunit of calcineurin, or of the gene (CNB1) encoding the regulatory B subunit, is lethal in an fks1 mutant. Disruption of the gene encoding FKBP12 (FKB1) or the major, cytosolic cyclophilin (CPH1) in fks1 cells results in the loss of hypersensitivity to the relevant drug. Overexpression of CNA1 or CNA2, in conjunction with CNB1, results in a significant decrease in hypersensitivity to FK506 and CsA. The results show that the hypersensitivity of the fks1 mutant is due to the inhibition of calcineurin phosphatase activity by the receptor-drug complexes. The growth dependence of the mutant on the Ca2+/calcineurin signal pathway provides an important tool for studying in yeast certain aspects of immune suppression by these drugs.

Amino Acid Isomerases↗
PubMed1993

Production of L-dihydroxyphenylalanine in Escherichia coli with the tyrosine phenol-lyase gene cloned from Erwinia herbicola.

The gene (tutA) encoding tyrosine phenol-lyase from Erwinia herbicola was cloned into Escherichia coli, and fusions to the lac and tac promoters were constructed. The enzyme was expressed at high levels in E. coli in the presence of isopropyl-beta-D-thiogalactopyranoside or lactose as an inducer. L-Dihydroxyphenylalanine was synthesized in high yield from catechol, pyruvate, and ammonia by induced cells.

Amino Acid Sequence↗
PubMed1992

Calcineurin mediates inhibition by FK506 and cyclosporin of recovery from alpha-factor arrest in yeast.

The structurally unrelated immunosuppressants FK506 and cyclosporin A (CsA) act similarly, inhibiting a Ca(2+)-dependent signal required for interleukin-2 transcription and T-cell activation. Each drug binds to its cytosolic receptor, FKBP-12 and cyclophilin, respectively, and the drug-receptor complexes inhibit the Ca2+/calmodulin-dependent protein phosphatase, calcineurin. In yeast, calcineurin has been implicated in recovery from alpha-mating factor arrest. Here we show that FK506 bound to yeast FKBP-12 appears to form a complex with yeast calcineurin. Moreover, recovery from mating factor arrest is highly sensitive to FK506 or CsA, and this sensitivity requires the presence of FKBP-12 or cyclophilin, respectively. These results define a key physiological target of an FK506- and CsA-sensitive signal pathway in yeast, suggest a high degree of mechanistic conservation with mammalian cells, and indicate that further examination of the yeast system should provide insight into the same process in T cells.

Alleles↗
PubMed1991

Schistosoma mansoni: two-dimensional gel electrophoretic analysis of antigens uniquely immunoreactive with protective rat serum.

Candidate vaccine antigens are defined by their differential immunoreactivity with antisera which are distinguishable by their capacity to confer passive resistance to infection. This "contrasting antisera" immunoassay has been successfully used in previous analyses of 4-week-old worm biosynthetically radiolabeled Schistosoma mansoni proteins to identify potentially protective antigens. Twice-infected Fischer (F-2x) and Wistar-Furth (W-2x) rat sera were the sources of protective and non-protective antibody, respectively. We have extended our original analysis by applying two-dimensional gel electrophoresis to resolve total and immunoreactive soluble proteins of the 4-week worms. Total proteins were characterized by silver staining and autoradiography. Radiolabeled protein antigens immunoprecipitated by F-2x and W-2x antisera were compared, and several were shown to be uniquely reactive with the protective immune serum. In a companion molecular approach to clone the candidate vaccine antigens, screening of a lambda gt11 adult S. mansoni cDNA expression library by the contrasting antisera assay has identified a clone (lambda 40) producing a fusion protein with epitopes uniquely reactive with F-2x. A rabbit antiserum to the lambda 40 fusion protein (anti-FP40) reacted with radiolabeled worm proteins in the 20-kDa size range. By 2D gel electrophoretic analysis, we can now demonstrate that anti-FP40 specifically immunoprecipitates most of the members of a multicomponent protein antigen subset 18-22 kDa in Mr, focusing over a pI range of 5.3-5.8, and recognized uniquely by F-2x.

Animals↗
PubMed1991

Cloning and sequence analysis of the Schistosoma mansoni membrane glycoprotein antigen gene GP22.

A family of Schistosoma mansoni proteins (18-22 kDa, pI 5.3-5.8) are biosynthesized in juvenile worms and immunoprecipitated by antibodies uniquely present in protective Fischer rat antiserum. A cDNA clone, lambda gt11-40, expressing epitopes common to this protein family was used to obtain a genomic DNA clone, by hybridization with a lambda gt11-40 oligonucleotide probe. In the 1.37 kb of genomic DNA sequenced, an open reading frame of 182 amino acids was identified on the strand corresponding to lambda gt11-40 coding sequences, and those of identical independently isolated cDNA clones defining a 25-kDa surface membrane glycoprotein. The new S. mansoni gene is termed GP22. There are two candidate promoters, confirmed by primer extension studies with worm RNA. Promoter 1 (P1) is preceded by a G + C-rich region and potential CAAT sequences, and is to the 5'-side of P2. Transcription from P1 is initiated at 2 different sites, apparently producing mRNAs with different translation start sites (ATG). Decoding these mRNAs yields protein products of 182 (P1), 175 (P1), 140 (P2) and 136 (P2) amino acids. The polypeptides share the following features: a hydrophobic segment near the carboxy terminus sufficient to span a lipid bilayer, with a consensus sequence for thio-esterification by a fatty acid; an external domain containing 2 potential N-linked glycosylation sites; and a candidate leucine-zipper motif, suggesting the protein may exist as a dimer on the worm surface. While sharing these common features in their carboxy terminal regions, the three proteins differ in the length and properties of their amino termini. The 140-amino acid protein has a short hydrophobic amino terminus, while the 175- and 182-amino acid proteins have more extensive hydrophobic sequences, each preceded by a hydrophilic amino terminal sequence. The heterogeneity observed in 2-dimensional gels of the antigen may be explained in part by the size and charge differences among the proteins deduced from the sequence and transcription pattern of this gene. The possibility of stage-specific regulated expression of this candidate vaccine antigen family is an attractive concept, potentially accounting for the phenomenon of concomitant immunity observed in the rat and perhaps other schistosome hosts.

Amino Acid Sequence↗
PubMed1991

Antifungal properties of the immunosuppressant FK-506: identification of an FK-506-responsive yeast gene distinct from FKB1.

FK-506 is a novel and potent antagonist of T-cell activation and an inhibitor of fungal growth. Its immunosuppressive activity can be antagonized by the structurally related antibiotic rapamycin, and both compounds interact with cytoplasmic FK-506-binding proteins (FKBPs) in T cells and yeast cells. In this paper, we show that FK-506 and two analogs inhibit vegetative growth of Saccharomyces cerevisiae in a fashion that parallels the immunosuppressive activity of these compounds. Yeast mutants resistant to FK-506 were isolated, and at least three complementation groups (fkr1, fkr2, and fkr3) were defined. These fkr mutants show no alteration in their levels of FK-506-binding activity. Likewise, strains carrying null alleles of FKB1 (the yeast gene coding for the FKBP) remain FK-506 sensitive, indicating that depletion of yeast FKBP is not sufficient to confer an FK-506 resistance phenotype, although fkb1 null mutants are resistant to rapamycin. FKB1 does not map to the three fkr loci defined here. These results suggest that yeast FKBP mediates the inhibitory effect of rapamycin but that at least one other protein is directly involved in mediating the activity of FK-506. Interestingly, the ability of FK-506 to rescue a temperature-sensitive growth defect of the fkr3 mutant suggests that the FKR3 gene may define such a protein.

Anti-Bacterial Agents↗
PubMed1990

Schistosoma mansoni: cloning of antigen gene sequences in Escherichia coli.

Fischer rat protective antiserum (F-2x) prepared from Schistosoma mansoni-infected rats was used to screen an adult worm cDNA library constructed in a lambda gt11 bacteriophage expression vector. This led to the isolation of several clones yielding proteins reactive with antibodies in the infection serum. Counter-screening of these clones with Wistar-Furth rat nonprotective antiserum (W-2x) enabled identification of clones either uniquely or preferentially reacting with F-2x, in addition to clones of nearly equal reactivity with both antisera. Six clones were further characterized. Five expressed beta-galactosidase/S. mansoni fusion proteins which migrated more slowly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than beta-galactosidase and all were reactive in a Western immunoblot assay. The cDNA insert sizes in the clones ranged from 150 to 900 base pairs. Rabbit antibodies prepared against fusion proteins from three of the clones recognized biosynthetically radio-labeled 4-week worm proteins of sizes 20, 38, and 70 kDa, respectively. The 20- and 38-kDa proteins were among the protein antigens uniquely recognized by the F-2x protective antiserum. These proteins are therefore candidates for protective vaccine antigens and the recombinant lambda clones are now serving as useful reagents for obtaining the corresponding nucleotide gene sequences.

Amino Acid Sequence↗
PubMed1989

Comparison of in vivo and in vitro toxic effects of microcystin-LR in fasted rats.

The toxic effects of microcystin-LR, a cyclic heptapeptide isolated from the cyanobacterium Microcystis aeruginosa, were studied in the fasted rat model and in subcellular fractions from fasted, toxin-treated and control rats. Hepatotoxic effects of a lethal dose (100 micrograms/kg) were examined 15-90 min post-injection. Elevations of serum enzymes, particularly sorbitol dehydrogenase, specific for liver mitochondria, correlated with hepatic damage. Electron micrographs showed progressive cellular disruption, including dilation of rough endoplasmic reticulum, incorporation of cellular components into cytolysosomes, hydropic mitochondria devoid of electron-opaque deposits, loss of desmosome-associated intermediate filaments, disruption of sinusoidal architecture and, ultimately, lysis of hepatocytes. The appearance of hydropic mitochondria correlated with loss of coupled electron transport. Changes in plasma membrane-associated cytoskeletal filaments correlated with loss of desmosome tonofilaments. In contrast to in vivo exposure to microcystin-LR, in vitro exposure to toxin had no effect on mitochondria or cytoskeletal filaments, suggesting that the toxic effects observed in vivo were indirect and may be dependent on bioactivation of the toxin or a cascade of events not supported in in vitro models.

Adenosine Diphosphate↗
PubMed1988

Role of protein processing, intracellular trafficking and endocytosis in production of and immunity to yeast killer toxin.

Yeast strains harboring M1-dsRNA and its packaging virus ScV-L secrete a disulfide-linked, heterodimeric toxin which kills sensitive yeast cells by disrupting plasma membrane function. The mature toxin is derived from a precursor (preprotoxin) which undergoes post-translational processing steps during export via the established yeast secretory pathway. Cleavage by both the KEX1 and KEX2 endopeptidases is required for expression of killing activity. The same 1.0 kb open reading frame on M1-dsRNA directs the expression of immunity to toxin. Differentially processed derivatives of protoxin, as well as protoxin itself, have been proposed to serve as mediators of immunity. To understand the mechanisms by which the killing and immunity phenotypes can be derived from a common precursor, we have: 1) studied cellular processes implicated in expression of the phenotypes; and 2) developed a system to produce mutants defective in immunity, killing, or both. In the first approach, the role played by both endocytosis and vesicular traffiking in expression of killing and immunity was examined. Strains defective in endocytosis (end1, end2) or vacuolar protein localization (vpl3, vpl6) were transformed with a plasmid encoding killer toxin under control of the pho5 promoter. When induced by phosphate starvation, both end mutants and all vpl mutants expressed killing activity. Immunity to exogenous toxin, however, was significantly decreased in strains carrying both vpl mutant alleles and in one of the endocytosis mutants (end1]. This suicidal phenotype (rex for resistance expression) has been described previously in M1-containing strains as a leaky phenocopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane↗
PubMed1987

Gene-protein assignments within the yeast Yarrowia lipolytica dsRNA viral genome.

Some strains of the yeast Yarrowia lipolytica possess virus-like particles (VLPs) which encapsidate a double-stranded RNA (dsRNA) genome designated Ly. We report here that these VLPs have two associated polypeptides of molecular weights 83 kd (VLy-P1) and 77 kd (VLy-P2). Denatured Ly-dsRNA was used to program a cell-free rabbit reticulocyte translation system, resulting in the appearance of four major products, viz. Ly-P1 (83 kd); Ly-P2 (77 kd); Ly-P3 (74 kd) and Ly-P4 (68 kd). The in vivo viral-associated protein VLy-P1 co-migrated on SDS-polyacrylamide gels with the in vitro product Ly-P1 and, similarly, VLy-P2 co-migrated with Ly-P2. Peptide mapping data confirm the identity of the in vivo products (VLy-P1 and VLy-P2) and their in vitro counterparts. The conclusion made is that VLy-P1 and VLy-P2 are almost identical primary translation products of the Ly genome, derived from a single or multiple species of Ly-dsRNA. RNA blot hybridizations using L1A M1 and separately, L2A M2 probes prepared from appropriate K1 and K2 Saccharomyces cerevisiae killer strains, failed to show any detectable homology to Ly-dsRNA, substantiating the uniqueness of the Ly genome with respect to the K1 and K2 S. cerevisiae dsRNA killer systems.

Animals↗
PubMed1987

Viruses in fungi: infection of yeast with the K1 and K2 killer viruses.

We demonstrate here that yeast killer viruses, previously thought to be transmitted only by cytoplasmic mixing during division, mating, or other induced forms of cell fusion, are capable of extracellular transmission. Viral particles from standard K1 and K2 killer strains were used to inoculate sensitive cells of Saccharomyces cerevisiae, rendered competent by spheroplasting, lithium acetate treatment, or by natural mating. Extracellular transmission of the killer viruses was judged by the following criteria and controls. Filter-sterilized virus inocula were shown to be free of viable yeast cells, and host cells treated in the absence of added virus did not yield killer progeny. Infected clones originating from spheroplasts or lithium acetate-treated cells were shown to possess the genotype of the host strain and the killer phenotype of the infecting virus. Infected clones derived from complementary mating pairs were found to be wild-type diploids, whose meiotic segregants exhibited 2:2 segregation for unlinked nutritional markers and 4:0 segregation for the killer phenotype. This technique is generally applicable to the study of interactions between yeast viruses and different hosts and suggests that extracellular transmission may be a natural route for the inheritance and dissemination of mycoviruses.

Crosses, Genetic↗
PubMed1986

Mapping of functional domains within the Saccharomyces cerevisiae type 1 killer preprotoxin.

Strains of Saccharomyces cerevisiae harboring M1-dsRNA, the determinant of type 1 killer and immunity phenotypes, secrete a dimeric 19-kd toxin that kills sensitive yeast cells by the production of cation-permeable pores in the cytoplasmic membrane. The preprotoxin, an intracellular precursor to toxin, has the domain sequence delta-alpha-gamma-beta where alpha and beta are the 9.5-and 9.0-kd subunits of secreted toxin. Plasmids containing a partial cDNA copy of M1, in which alpha, gamma, and beta are fused to the PH05 promoter and signal peptide, have previously been shown to express phosphate-repressible toxin production and immunity. Here the construction of a complete DNA copy of the preprotoxin gene and its mutagenesis are described. Analysis of the expression of these mutants from the PH05 promoter elucidates the functions of the preprotoxin domains. delta acts as a leader peptide and efficiently mediates the secretion, glycosylation and maturation of killer toxin. Mutations within the beta subunit indicate it to be essential for binding of toxin to and killing of whole cells but unnecessary for the killing of spheroplasts. Mutations within the putative active site of alpha prevent killing of both cells and spheroplasts. The probable role of beta is therefore recognition and binding to the cell wall receptor whereas alpha is the active ionophore. Mutations within alpha causing loss of toxicity also cause loss of immunity, while the mutants described within gamma and beta retain partial or complete immunity. Expression of gamma without alpha or beta confers no phenotype. The immunity determinant may minimally consist of the alpha domain and the N-terminal portion of gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1986

Isolation of the positive-acting regulatory gene PHO4 from Saccharomyces cerevisiae.

We have isolated a 10.2-kb fragment of yeast DNA from a genomic library of recombinant centromeric YCp50 plasmids, which complements a mutation in the PHO4 gene of Saccharomyces cerevisiae. The identity of the PHO4 gene on this plasmid was established by integration of a subfragment into the PHO4 region of the yeast chromosome. Analysis of a series of plasmid subclones covering different regions of the original yeast DNA insert localized the PHO4 gene within a 2.25-kb sequence. Southern hybridization of total genomic DNA prepared from wild-type strains and from integrative transformants show that the PHO4 gene consists of unique yeast DNA sequences and is present at a single copy in the S. cerevisiae genome. RNA blot hybridization mapping of transcripts within this genomic region identify the PHO4 transcript as a 1.7-kb, low-abundancy, constitutively expressed and polyadenylated RNA.

Acid Phosphatase↗