Thirty small molecular weight peptides related to the chemotactic peptide N-formylmethionylleucylphenylalanine (CHO-Met-Leu-Phe-OH) have been prepared by both solidphase and classical peptide synthesis. Compounds were prepared to investigate the structural requirements in the 1 position (N-formylmethionine) and the 3 position (phenylalanine). Each analogue was tested for its ability to induce lysosomal enzyme release from cytochalasin B treated rabbit polymorphonuclear leukocytes in vitro. In addition, some were also tested for their ability to stimulate neutrophil chemotaxis in vitro and for inhibition of specific binding of a 3H-labeled chemotactic peptide, CHO-Nle-Leu-Phe-OH. The results show that the formyl group of CHO-Met-Leu-Phe-OH is essential for good biological activity since N-acetylation, removal of the a-amino group (Le., desamino), or replacement by an ethyl group results in a drastic loss of chemotactic potency (approximately 5000-fold). In addition, the sulfur-

Aureobasidin A (LY295337) is a cyclic depsipeptide antifungal agent with activity against Candida spp. The mechanism of action of LY295337 remains unknown. LY295337 also shows activity against the yeast Saccharomyces cerevisiae. Generation of a mutant of S. cerevisiae resistant to LY295337 is reported. Resistance was found to reside in a dominant mutation of a single gene which has been named AUR1 (for aureobasidin resistance). This gene was cloned and sequenced. A search for homologous sequences in GenBank and by BLAST did not elucidate the function of this gene, although sequence homology to an open reading frame from the Saccharomyces genome sequencing project and several other adjacent loci was noted. Deletion of aur1 was accomplished in a diploid S. cerevisiae strain. Subsequent sporulation and dissection of the aur1/aur1⌬ diploid resulted in tetrads demonstrating 2:2 segregation of viable and nonviable spores, indicating that deletion of aur1 is lethal. As LY295337 is fungicidal and deletion of aur1 is lethal, aur1 represents a potential candidate for the target of LY295337.Aureobasidins are antifungal cyclic depsipeptides isolated from Aureobasidium pullulans R106 (15, 25). Aureobasidin A (LY295337) is the major factor isolated from the fermentation broth of A. pullulans R106 (25). This compound has potent in vitro antifungal activity against Candida albicans, other species of Candida, and Cryptococcus neoformans (9,26).The model yeast Saccharomyces cerevisiae is susceptible to LY295337. The study of mutants of S. cerevisiae resistant to the action of LY295337 could lead to an understanding of its mechanism of action. In this study, generation of a dominant mutant of S. cerevisiae which is resistant to LY295337 has allowed the cloning of a gene, ABR1, which encodes resistance to LY295337 (12, 13). Hashida-Okado and coworkers and Okado et al. have recently isolated the same gene as ABR1 through resistance to aureobasidin A (named AUR1) from S. cerevisiae (11,16). The gene encoding resistance to aureobasidin A will hereafter be referred to as AUR1. AUR1 is unique and essential for viability of S. cerevisiae. AUR1 may encode the target for LY295337. MATERIALS AND METHODSStrains, media, plasmids, and transformations. S. cerevisiae strains used in this study are presented in Table 1. Cultivation, storage, and genetic manipulation of S. cerevisiae were carried out as previously described (19). The yeast shuttle plasmid pRS416 was used for construction of the yeast library, and pRS406 was used for construction of a deletion plasmid (24). Transformation of yeast strains was carried out by the method of Reddy and Maley (18). Transformants bearing the URA3-marked plasmids were cured by the use of 5-fluoroorotic acid (5-FOA) (4).Selective medium for drug resistance was standard yeast-peptone-dextrose (YPD) agar with 5 g of LY295337 per ml and 1% ethanol (19).Library construction and propagation of plasmids were performed with Escherichia coli DH5␣, grown and selected for by standard methods (21).Determination of ant...

Syringomycin E is an antifungal cyclic lipodepsinonapeptide that inhibits the growth of Saccharomyces cerevisiae by interaction with the plasma membrane. A screen conducted to find the yeast genes necessary for its fungicidal action identified two novel syringomycin E response genes, SYR3 and SYR4. A syr3 mutant allele was complemented by ELO2 and ELO3. These genes encode enzymes that catalyze the elongation of sphingolipid very long chain fatty acids. Tetrad analysis showed that SYR3 was ELO2. Strains with deletions of SYR3/ELO2 and ELO3 were resistant to syringomycin E, and lipid analyses of both mutants revealed shortened fatty acid chains and lower levels of sphingolipids. SYR4 was identified by Tn5 inactivation of genomic library plasmids that complemented a syr4 mutant allele. SYR4 was found to be identical to IPT1, which encodes the terminal sphingolipid biosynthetic enzyme, mannosyl-diinositolphosphoryl-ceramide synthase. Deletion ⌬syr4/ ipt1 strains were viable, were resistant to syringomycin E, did not produce mannosyl-diinositolphosphorylceramide, and accumulated mannosyl-inositolphosphoryl-ceramide. Accumulation of mannosyl-inositolphosphoryl-ceramide was not responsible for resistance since a temperature-sensitive secretory pathway mutant (sec14-3 ts ) accumulated this sphingolipid and was sensitive to syringomycin E. Finally, ⌬csg1/sur1 and ⌬csg2 strains defective in the transfer of mannose to inositolphosphoryl-ceramide were resistant to syringomycin E. These findings show that syringomycin E growth inhibition of yeast is promoted by the production of sphingolipids with fully elongated fatty acid chains and the mannosyl and terminal phosphorylinositol moieties of the polar head group.Syringomycin E is a member of a family of small cyclic lipodepsinonapeptides (ca. 1,200 Da) produced by the plant bacterium Pseudomonas syringae pv. syringae (38). Other members include syringomycin A 1 and G, the syringostatins, the syringotoxins, and the pseudomycins (2, 38). All possess a characteristic tetrapeptidyl sequence (dehydroaminobutanoic acid-hydroxyaspartic acid-chlorothreonine-serine) and a ␤-hydroxy fatty acid attached to the N-terminal serine. These metabolites are fungicidal to a broad range of fungi, including yeast and human pathogens (33), and they show relatively low levels of toxicity to plants (21) and cutaneous animal tissues (33). Syringomycin E was recently shown to be partly responsible for the biological control of fungal pathogens on postharvest citrus fruits by certain P. syringae pv. syringae strains (5). Syringomycin E interacts with the fungal plasma membrane, where it causes K ϩ efflux, Ca 2ϩ influx, and changes in membrane potential by processes that are likely related to channel formation (14, 38).Molecular genetic studies with yeast were initiated to more precisely define the antifungal mechanism of action of syringomycin E. Syringomycin E-resistant mutants of Saccharomyces cerevisiae were generated to permit identification of the mutated genes by complementation (39). Two genes, ...

The IPC1 gene from Saccharomyces cerevisiae, which encodes inositolphosphorylceramide (IPC) synthase, was first identified as a novel and essential gene encoding resistance to the natural product antifungal aureobasidin A (AUR1). The formation of IPC in fungi is essential for viability, suggesting inhibitors of IPC1p function would make ideal antifungal drug candidates. Homologs of the AUR1/IPC1 gene were identified from a number of human pathogenic fungi, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Cryptococcus neoformans. Comparison of these genes with other homologous genes from Candida albicans, Aspergillus fumigatus, Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe reveals a conserved structural motif for inositolphosphoryl transferases which is similar to a motif recently described for lipid phosphatases, but with unique characteristics.

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Protein phosphorylation is a primary form of information transfer in cell signaling pathways and plays a crucial role in regulating biological responses. Aberrant phosphorylation has been implicated in a number of diseases, and kinases and phosphatases, the cellular enzymes that control dynamic phosphorylation events, present attractive therapeutic targets. However, the innate complexity of signaling networks has presented many challenges to therapeutic target selection and successful drug development. Approaches in phosphoproteomics can contribute functional, systems-level datasets across signaling networks that can provide insight into suitable drug targets, more broadly profile compound activities, and identify key biomarkers to assess clinical outcomes. Advances in MS-based phosphoproteomics efforts now provide the ability to quantitate phosphorylation with throughput and sensitivity to sample a significant portion of the phosphoproteome in clinically relevant systems. This review will discuss recent work and examples of application data that demonstrate the utility of MS, with a particular focus on the use of quantitative phosphoproteomics and phosphotyrosine-directed signaling analyses to provide robust measurement for functional biological interpretation of drug action on signaling and phenotypic outcomes.

Radioiodination of rat-derived Pneumocystis carinii obtained from an in vitro culture demonstrated the presence of a major surface glycoprotein (gpl20). The glycoprotein was of the high-mannose type. It exhibited adherence properties similar to those observed in the intact organism. Under nonreducing conditions, it existed as an aggregate with a molecular weight in excess of 2 x 106. Since its aggregating behavior and adherent quality prevented isolation of the glycoprotein by conventional methods, the glycoprotein was purified by chromatography on hydroxyapatite in the presence of sodium dodecyl sulfate under reducing conditions.

Syringomycin E is an antifungal cyclic lipodepsinonapeptide produced by Pseudomonas syringae pv. syringae. To understand the mechanism of action of syringomycin E, a novel resistant Saccharomyces cerevisiae strain, BW7, was isolated and characterized. Lipid analyses revealed that BW7 contained only the hydrophobic subspecies of sphingolipids that are normally minor components in wild type strains. This aberrant sphingolipid composition was the result of lack of K K-hydroxylation of the amide-linked very long chain fatty acids, suggesting a defective sphingolipid K K-hydroxylase encoded by the FAH1 gene. A yeast strain that lacks the FAH1 gene was resistant to syringomycin E, and failed to complement BW7. These results demonstrate that BW7 carries a mutation in the FAH1 gene, and that the lack of K K-hydroxylated very long chain fatty acids in yeast sphingolipids confers resistance to syringomycin E. ß