No abstract

The GAL genes, which encode the enzymes required for normal galactose metabolism in yeast, are transcriptionally regulated by three proteins: Gal4p, an activator; Gal80p, an inhibitor; and Gal3p, a galactose sensor. These proteins control the switch between inert and active gene expression. The transcriptional activation function of Gal4p is rendered inactive in the presence of Gal80p. Here we present the three-dimensional structure of a complex between the acidic activation domain of Gal4p and Gal80p. The transactivation domain initiates with an extended region of polypeptide chain followed by two turns of an amphipathic ␣-helix. It fits into and across a deep cleft within the Gal80p dimer with the protein-protein interface defined primarily by hydrophobic interactions. A disordered loop in the apo-Gal80p structure (Asp-309 to Ser-316) becomes well-defined upon binding of the transactivation domain. This investigation provides a new molecular scaffold for understanding previous biochemical and genetic studies.In yeast, the GAL genes encode the enzymes of the Leloir pathway, which are required for the conversion of galactose into a metabolically useful form, glucose 6-phosphate (1). The regulation of these genes in response to the organism being challenged with galactose has served as a research paradigm for eukaryotic transcriptional control for over 50 years (1-5). Three key protein components form the GAL regulatory switch: a transcriptional activator, Gal4p; a transcriptional inhibitor, Gal80p; and an inducer, Gal3p. When yeast cells are grown in the absence of galactose, the GAL genes are, for the most part, transcriptionally inert. Under these conditions, Gal4p is produced in the cell and is tethered upstream of the GAL genes (6), but its activity is inhibited by its interaction with Gal80p (7). When galactose is available as a carbon source, the GAL genes are transcribed, both rapidly and to a high level (8). Although the presence of galactose within the cell triggers the activation of Gal4p, neither Gal4p nor Gal80p function as the galactose sensor. Instead, Gal3p serves in this capacity by binding both galactose and ATP and adopting the conformation required for its interaction with Gal80p (1, 9). The net result of this interaction is that Gal4p becomes active, and transcription of the GAL genes proceeds.Gal4p is a large protein of 881 amino acids with the first ϳ100 residues functioning in DNA recognition and dimerization. The last C-terminal residues serve as an acidic transactivation domain (TAD) 3 that is required, ultimately, for the recruitment of RNA polymerase II to initiate transcription (10). The amino acid residues comprising the TAD also provide the binding platform for Gal80p (11,12). Both Gal3p and Gal80p are smaller proteins containing ϳ520 and ϳ450 amino acids, respectively.Until very recently, the only three-dimensional structural information available for any of these key regulatory components of the GAL genetic switch was that of an N-terminal 65-residue fragment of Gal4p bound to D...

We have identified five α-tubulin and six β-tubulin isotypes that are expressed in adult Fasciola hepatica. Amino acid sequence identities ranged between 72 and 95% for fluke α-tubulin and between 65 and 97% for β-tubulin isotypes. Nucleotide sequence identity ranged between 68–77% and 62–80%, respectively, for their coding sequences. Phylogenetic analysis indicated that two of the α-tubulins and two of the β-tubulins were distinctly divergent from the other trematode and nematode tubulin sequences described in this study, whereas the other isotypes segregated within the trematode clades. With regard to the proposed benzimidazole binding site on β-tubulin, three of the fluke isotypes had tyrosine at position 200 of β-tubulin, two had phenylalanine and one had leucine. All had phenylalanine at position 167 and glutamic acid at position 198. When isotype RT-PCR fragment sequences were compared between six individual flukes from the susceptible Cullompton isolate and from seven individual flukes from the two resistant isolates, Sligo and Oberon, these residues were conserved.

The comparatively simple eukaryote Saccharomyces cerevisiae is composed of some 6000 individual genes. Specific sets of these genes can be transcribed co-ordinately in response to particular metabolic signals. The resultant integrated response to nutrient challenge allows the organism to survive and flourish in a variety of environmental conditions while minimal energy is expended upon the production of unnecessary proteins. The Zn(II)2Cys6 family of transcriptional regulators is composed of some 46 members in S. cerevisiae and many of these have been implicated in mediating transcriptional responses to specific nutrients. Gal4p, the archetypical member of this family, is responsible for the expression of the GAL genes when galactose is utilized as a carbon source. The regulation of Gal4p activity has been studied for many years, but we are still uncovering both nuances and fundamental control mechanisms that impinge on its function. In the present review, we describe the latest developments in the regulation of GAL gene expression and compare the mechanisms employed here with the molecular control of other Zn(II)2Cys6 transcriptional regulators. This reveals a wide array of protein-protein, protein-DNA and protein-nutrient interactions that are employed by this family of regulators.

No abstract

Albendazole is a benzimidazole drug which can be used to treat liver fluke (Fasciola hepatica) infections. Its mode of action is believed to be the inhibition of microtubule formation through binding to β-tubulin. However, F. hepatica expresses at least six different isotypes of β-tubulin, and this has confused, rather than clarified, understanding of the molecular mechanisms of benzimidazole drugs in this organism. Recombinant F. hepatica β-tubulin proteins were expressed in, and purified from, Escherichia coli. These proteins were then used in pull-down assays in which albendazole was covalently linked to Sepharose. β-Tubulin isotype 2 was pulled down in this assay, and this interaction could be reduced by adding competing albendazole. Molecular modelling of β-tubulin isotypes suggests that changes in the side change conformations of residue 200 in the putative albendazole binding site may be important in determining whether, or not, a particular isotype will bind to the drug. These results, together with previous work demonstrating that albendazole causes disruption of microtubules in the liver fluke, strongly suggest that β-tubulin isotype 2 is one of the targets of this drug.

The Escherichia coli protease inhibitor ecotin is believed to be implicated in the evasion of host defenses during infection. The protein has also attracted attention as a scaffold for the design of novel, specific protease inhibitors. Ecotin interacts with its targets through two sites. Key hydrophobic residues in both sites (Leu-64, Trp-67, Tyr-69, Met-84, and Met-85) were mutated to alanine and the effects on the inhibition of trypsin, chymotrypsin, and elastase were assessed. Each of these mutant ecotin proteins tested in kinetic assays with these enzymes exerted less inhibitory potency compared to wild-type ecotin. However, these effects were relatively small and not additive. Keywords: Protease inhibitor; trypsin; chymotrypsin; elastase; free energy of interaction; alanine substitution