Exploring an antifungal target in the plasma membrane H(+)-ATPase of fungi.

The plasma membrane H(+)-ATPase is a promising new antifungal target that is readily probed with the sulfhydryl-reactive reagent omeprazole. Inhibition of the H(+)-ATPase by omeprazole is closely linked to cell killing, and it has been suggested that enzyme inhibition may result from a covalent interaction within the first two transmembrane segments (M1 and M2) (Monk et al. (1995) Biochim. Biophys. Acta 1239, 81-90). In this study, the molecular nature of this interaction was examined by screening a series of 26 well-characterized pma1 mutations residing in the first two transmembrane segments of the H(+)-ATPase from Saccharomyces cerevisiae. Only two pma1 mutants, A135G and G158D,G156C, were found to significantly decrease the sensitivity of cells for omeprazole. In contrast, enhanced sensitivity was observed at a number of positions, with D140C(A) and M128C producing the most significant increases in sensitivity. The introduction of cysteine at various locations within this region only marginally affected omeprazole sensitivity, suggesting that this region was not a direct site of covalent modification. Rather, its conformation influences omeprazole binding at some other locus. In order to determine the sidedness of the omeprazole interaction, a novel in vitro assay system was exploited that utilized liposomes co-reconstituted with the H(+)-ATPase and the light-driven proton pump bacteriorhodopsin. Omeprazole was found to completely inhibit proton transport by the H(+)-ATPase at 50 microM in this system. An asymmetrically-distributed chemical trap system involving glutathione was used to demonstrate that this inhibition appears localized to the extracellular portion of the enzyme. This work indicates that omeprazole can inhibit the H(+)-ATPase from its extracellular face, and this inhibition is influenced by changes in the M1, M2 region of the protein.

Regulation and pH-dependent expression of a bilaterally truncated yeast plasma membrane H+-ATPase.

Constitutive, chromosomal expression of yeast pma1 deletion alleles in Saccharomyces cerevisiae yielded functional, truncated forms of the plasma membrane H+-ATPase which were independently capable of supporting wild type yeast growth rates. Deletion of 27 amino-terminal residues affected neither the enzyme's activity nor its responsiveness to changes in glucose metabolism. By contrast, removal of 18 carboxy-terminal amino acids produced an enzyme with a Vmax that was relatively insensitive to glucose-dependent metabolic status and with a Km that was significantly lower than that of the wild type enzyme. These effects were exaggerated when the amino- and carboxy-terminal deletions were combined in a bilaterally truncated H+-ATPase, suggesting that the amino terminus may have a subtle role in modulating ATPase activity. In pma1DeltaDelta cells cultured at pH 6, plasma membrane H+-ATPase levels were much lower than those in cells expressing a wild type ATPase. Increased expression levels could be achieved by growing the pma1DeltaDelta mutant at pH 3, a result that was at least partially due to a sustained, elevated transcription of pma1DeltaDelta mRNA. Our observations suggest that intracellular proton balance can be maintained by regulation of the activity and/or quantity of H+-ATPase in the plasma membrane.

Functional complementation between transmembrane loops of Saccharomyces cerevisiae and Candida albicans plasma membrane H(+)-ATPases.

Saccharomyces cerevisiae PMA1 sequences encoding a putative antifungal target site comprising transmembrane loops 1 + 2 and/or 3 + 4 were replaced with the homologous sequences from Candida albicans PMA1 by using PCR-mediated domain transfer. The chimeric pma1 mutants and an isogenic wild type S. cerevisiae strain had similar growth rates, growth yields, glucose-dependent proton pumping rates, acid-activated omeprazole sensitivities, salt tolerances and antifungal sensitivities. The yields and kinetic properties of H(+)-ATPases in plasma membranes of mutant and wild type strains were comparable. Single heterologous transmembrane loops caused deleterious phenotypes at low pH and elevated temperature. Inclusion of both heterologous transmembrane loops fully suppressed the temperature sensitivity caused by heterologous transmembrane loop 1 + 2, partially suppressed the pH sensitivity and gave Candida-like in vitro sensitivity to vanadate, suggesting that the loops operate as a domain. The fully functional chimeric H(+)-ATPase containing C. albicans transmembrane loops 1 + 2 and 3 + 4 demonstrates this domain's complementarity to the equivalent region of the S. cerevisiae enzyme and validates the wild type S. cerevisiae H(+)-ATPase as an antifungal screening target.

The yeast plasma membrane proton pumping ATPase is a viable antifungal target. I. Effects of the cysteine-modifying reagent omeprazole.

The yeast plasma membrane proton pumping ATPase (H(+)-ATPase) was investigated as a potential molecular target for antifungal drug therapy by examining the inhibitory effects of the sulfhydryl-reactive reagent omeprazole on cell growth, glucose-induced medium acidification and H(+)-ATPase activity. Omeprazole inhibits the growth of Saccharomyces cerevisiae and the human pathogenic yeast Candida albicans in a pH dependent manner. Omeprazole action is closely correlated with inhibition of the H(+)-ATPase and is fungicidal. Glucose-dependent medium acidification is correspondingly blocked by omeprazole and appears to require the H(+)-ATPase to proceed through its reaction cycle. A strong correlation is observed between inhibition of medium acidification and H(+)-ATPase activity in plasma membranes isolated from treated cells. The inhibitory properties of omeprazole are blocked by pre-treatment of activated drug with beta-mercaptoethanol, which is consistent with the expected formation of a sulfhydryl-reactive sulfenamide derivative. Mutagenesis of the three putative membrane sector cysteine residues (C148S, C312S, C867A) in the S. cerevisiae H(+)-ATPase suggests that covalent modification of the conserved C148 residue may be important for inhibition of ATPase activity and cell growth. Other mutations (M128C and G158D/G156C) mapping near C148 support the importance of this region by modulating omeprazole inhibition of the H(+)-ATPase. These findings suggest that the plasma membrane H(+)-ATPase may serve as an important molecular target for antifungal intervention.

Asp ligand provides the trigger for closure of transferrin molecules. Direct evidence from X-ray scattering studies of site-specific mutants of the N-terminal half-molecule of human transferrin.

Recent X-ray crystallographic and solution X-ray scattering studies have shown that transferrins (serum transferrin, lactoferrin and ovotransferrin) undergo a major conformational change when iron is incorporated into the molecule. Apo-proteins show a structure with open interdomain clefts which close when iron is bound. The closed conformation has been suggested as an important step in the receptor recognition. Here, we report X-ray solution scattering experiments of the mutated N-terminal fragment of human serum transferrin with Asp63-->Ser (Cys). The data provide the first direct experimental evidence for the existence of a trigger mechanism for the closure of the interdomain cleft and that this trigger mechanism is disrupted by mutation of Asp63, the only ligand of iron from domain I.

Preliminary crystallographic analyses of the N-terminal lobe of recombinant human serum transferrin.

The N-terminal lobe of recombinant human serum transferrin (residues 1 to 337) has been crystallized in a form suitable for high-resolution three-dimensional X-ray crystallographic analyses. Crystals are of the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions of a = 44.9 A, b = 57.0 A and c = 135.9 A, and diffract to beyond 2 A resolution. Further studies show that isomorphous crystals of specifically designed mutants of this protein can also be grown. Structural studies of both recombinant and mutant protein forms will provide a basis for understanding the mechanism by which human serum transferrin functions.

Crystal structures of two mutants (K206Q, H207E) of the N-lobe of human transferrin with increased affinity for iron.

The X-ray crystallographic structures of two mutants (K206Q and H207E) of the N-lobe of human transferrin (hTF/2N) have been determined to high resolution (1.8 and 2.0 A, respectively). Both mutant proteins bind iron with greater affinity than native hTF/2N. The structures of the K206Q and H207E mutants show interactions (both H-bonding and electrostatic) that stabilize the interaction of Lys296 in the closed conformation, thereby stabilizing the iron bound forms.

[1H,13C] NMR determination of the order of lobe loading of human transferrin with iron: comparison with other metal ions.

Human serum transferrin (hTF) is a single-chain bilobal glycoprotein (80 kDa) which transports Fe3+ and a variety of other metal ions in blood. Only diferric transferrin, not the apo-protein, binds strongly to transferrin receptors and is taken up by cells via receptor-mediated endocytosis. We show here that 2D [1H,13C] NMR studies of recombinant epsilon-[13C]Met-hTF allow the order of lobe loading with various metal ions, including Fe3+, to be determined. In particular, the resonance for Met-464, a residue in the hydrophobic patch of helix 5, is very sensitive to iron binding in the C-lobe. The selectivity of lobe loading with Fe3+ is compared to loading with Fe2+ (which binds as Fe3+), Al3+, Ga3+ and Bi3+. Similar changes in shifts of the Met residues are observed for these metal ions, suggesting that they induce similar conformational changes in the protein.

Oral Candida: clearance, colonization, or candidiasis?

Candida albicans is frequently isolated from the human mouth, yet few carriers develop clinical signs of candidiasis. Oral candidiasis presents clinically in many forms. This reflects the ability of the yeast to colonize different oral surfaces and the variety of factors which predispose the host to Candida colonization and subsequent infection. Colonization of the oral cavity appears to be facilitated by several specific adherence interactions between C. albicans and oral surfaces which enable the yeast to resist host clearance mechanisms. Thus, Candida has been shown to adhere to complement receptors, various extracellular matrix proteins, and specific sugar residues displayed on host or bacterial surfaces in the oral cavity. Oral candidiasis results from yeast overgrowth and penetration of the oral tissues when the host's physical and immunological defenses have been undermined. Tissue invasion may be assisted by secreted hydrolytic enzymes, hyphal formation, and contact sensing. While these and other phenotypic characteristics may endow certain Candida species or strains with a competitive advantage in the oral cavity, it is the host's immune competence that ultimately determines whether clearance, colonization, or candidiasis occurs.

Targeting the fungal plasma membrane proton pump.

The need for new mechanistic classes of broad spectrum antifungal agents has prompted development of the membrane sector and ectodomain of the plasma membrane proton pumping ATPase as an antifungal target. The fungal proton pump is a highly abundant, essential enzyme in Saccharomyces cerevisiae. It belongs to the family of P-type ATPases, a class of enzymes that includes the Na+,K(+)-ATPase and the gastric H+,K(+)-ATPase. These enzymes are cell surface therapeutic targets for the cardiac glycosides and several anti-ulcer drugs, respectively. The effects of acid-activated omeprazole show that extensive inhibition of the S. cerevisiae ATPase is fungicidal. Fungal proton pumps possess elements within their transmembrane loops that distinguish them from other P-type ATPases. These loops, such as the conformationally sensitive transmembrane loop 1+2, can attenuate the activity of the enzyme. Expression in S. cerevisiae of fully functional chimeric ATPases that contain a foreign target comprising transmembrane loops 1+2 and/or 3+4 from the fungal pathogen Candida albicans suggests that these loops operate as a domain. The chimera containing C. albicans transmembrane loops 1+2 and 3+4 provides a prototype for mutational analysis of the target region and the screening of inhibitors directed against opportunistic fungal pathogens. Panels of mutants with modified ATPase regulation or with altered cell surface cysteine residues are also described. Information about the ATPase membrane sector and ectodomain has been integrated into a model of this region.

Salt effects on the physical properties of the transferrins.

Salts are known to have a pronounced effect on the spectroscopic, thermodynamic and kinetic properties of human serum transferrin. The present study was undertaken to examine the effect of NaCl on the related proteins ovotransferrin and lactoferrin. EPR difference spectroscopy was used to probe changes in the metal site of these proteins. Sodium chloride was found to perturb the g' = 4.3 EPR spectra of both ovotransferrin and lactoferrin but in different ways. The spectrum of ovotransferrin is reduced in amplitude with a broad feature appearing at g' = 4.8 whereas there is a loss of resolution of the doublet feature at the peak of the EPR derivative spectrum for lactoferrin. The increase in the amplitude of the ovotransferrin EPR difference spectrum (spectrum without NaCl minus spectrum with NaCl) as a function of NaCl concentration is suggestive of saturation binding. A Hill plot binding isotherm gave n = 1.87 +/- 0.32 and log K = 1.49 +/- 0.03 for ovotransferrin, where n is the number of C1- ions binding to either one or both iron containing lobes of the protein and K is the overall association constant. Preliminary measurements with lactoferrin gave n = 1.95 +/- 0.34 and log K = 1.41 +/- 0.06. These results are similar to those previously reported for serum transferrin and suggest that Cl- binds to all the transferrins with strong pairwise cooperativity. This binding may reflect a functional role for chloride and other physiological anions in the uptake and release of iron by the transferrins.

Monoclonal antibodies to the amino- and carboxyl-terminal domains of human transferrin.

Seven high affinity antibodies to human serum transferrin which recognize at least four different epitopes are described. Apparent dissociation constants (Kd's) have been determined for the binding of the antibodies to human transferrin in the presence and absence of iron. Small differences in reactivity were found. Five of the antibodies bind to the isolated amino-terminal half-molecule of human transferrin. Two of the antibodies appear to be to the C-terminal lobe since they bind to holo-transferrin but do not recognize the N-terminal half-molecule. Immunoblotting shows that six of the antibodies recognize both reduced and nonreduced transferrin. In addition, all of the antibodies bind with sufficiently high avidity to transferrin to make them useful as probes in studies in which binding of transferrin to the specific transferrin receptor is examined.

Monoclonal antibodies to the amino- and carboxyl-terminal domains of ovotransferrin.

Monoclonal antibodies to the iron transport protein ovotransferrin were produced by immunizing mice simultaneously with ovotransferrin and with the proteolytically derived amino- and carboxyl-terminal half-molecule domains of ovotransferrin. Two isolated hybridoma clones (designated alpha OT + N1 and alpha OT + N2) produced antibodies (IgG1) to determinants located on holo-ovotransferrin and the amino-terminal domain; two hybridoma clones (designated alpha OT + C1 and alpha OT + C2) produced antibodies (IgG1) to determinants on holo-ovotransferrin and the carboxyl-terminal domain. One hybridoma clone (designated alpha OT-N1) produced an antibody (IgG1) that bound only the amino-terminal domain and did not bind holo-ovotransferrin. Both alpha OT + N1, and alpha OT-N1 bound to antigen less tightly after removal of iron; antibodies alpha OT + N2, alpha OT + Cl, and alpha OT + C2 were unaffected by removal of iron from holo-ovotransferrin or the isolated domains. Intact disulfide bonds in the antigens were required for binding by the antibodies. These antibodies should prove useful as probes for discrete regions of the ovotransferrin molecule, in particular, those regions involved in binding to the transferrin receptor.

Differential effect of iodination of ovotransferrin and its two half-molecule domains on binding to transferrin receptors on chick embryo red blood cells.

Iodination of the C-terminal half-molecule domain of ovotransferrin (OTF) causes a significant reduction in binding to transferrin receptors on chick reticulocytes when compared to the binding observed with holo-OTF or the N-terminal half-molecule domain. (In such studies binding of iodinated half-molecule is measured in the presence of equimolar unlabelled complementary half-molecule). In particular iodination of the C-terminal half-molecule domain by the solid-phase reagent Iodogen resulted in half the binding found when ICl was used. The iodinated N-terminal half-molecule domain labelled by either Iodogen or ICl showed consistently higher binding than was observed with the C-terminal half-molecule or Fe2OTF. Although the molecular basis for the reduced binding of these proteins relative to the N-terminal half-molecule has not been definitively established, the implication is that there is a Tyr in the C-terminal domain which is involved in receptor recognition and binding. Addition of one or more bulky iodine atoms to the Tyr interferes with the interaction. Tryptic peptide maps of unlabelled holo-OTF and half-molecule domains and of the half-molecule domains labelled by both ICl and Iodogen are presented. The maps indicate limited access of the tyrosine residues to iodination especially in the C-terminal half-molecule domain. Equilibrium binding experiments have been carried out to compare the Kd (the apparent dissociation constant for the interaction between OTF and the transferrin receptors on chick-embryo red blood cells) with the Bmax, (binding at infinite free-ligand concentration) for Fe2OTF labelled using ICl, Iodogen, Enzymobeads and Chloramine-T. The effect of labelling Fe2OTF by Bolton-Hunter reagent has also been assessed. These studies show that ICl appears to be the reagent of choice for labelling Fe2OTF and its half-molecule domains.

Alcohol acetyltransferases and the significance of ester synthesis in yeast.

This paper reviews our current knowledge of yeast alcohol acyltransferases. Much of this information has been gathered over the past 10 years through the application of powerful yeast molecular biology techniques. Evidence from gene disruption and expression analysis of members of the alcohol acyltransferase (ATF) gene family indicates that different ester synthases are involved in the synthesis of esters during alcoholic fermentation. The natural physiological rationale behind these enzyme activities remains unclear. However, it is believed that these enzymes may be involved in very different functions, including cellular fatty acid homeostasis and detoxification mechanisms. Insights into the regulation of yeast ester synthesis by oxygen and unsaturated fatty acids have contributed to our understanding of the general mechanisms of gene regulation. In particular, control mechanisms that underpin the oxygen-mediated regulation of ATF1 gene transcription appear to be closely linked to those involved in the regulation of fatty acid metabolism. Data pertaining to the regulation of ATF1 gene transcription have been integrated into a working model for future research.

Cardiac glycoside toxicity resulting from cough linctus abuse.

Proprietry cough medicines are often abused by drug addicts as they are freely available and, in many instances, contain narcotic substances. A variety of other compounds are present in these products, including compounds structurally related to the commonly prescribed cardiac glycosides. We report a case in which severe cardiac glycoside toxicity resulted from the abuse of such a preparation.