macbecin-i and geldanamycin

[reaction: see text] A general and convergent route to the C(5)-C(15) subunits of the benzoquinone ansamycin antibiotics macbecin I, geldanamycin, and herbimycin A is described. Each subunit is prepared by the stepwise coupling of differentially functionalized aldehydes with a pentenyl dianion equivalent derived from diastereoselective pentynylation and regioselective reductive coupling.

Topics: Anti-Bacterial Agents; Benzoquinones; Lactams, Macrocyclic; Molecular Structure; Rifabutin; Stereoisomerism

In eukaryotes, production of the diverse repertoire of molecular chaperones during normal growth and in response to stress is governed by the heat shock transcription factor HSF. The HSC82 and HSP82 genes, encoding isoforms of the yeast Hsp90 molecular chaperone, were recently identified as targets of the HSF carboxyl-terminal activation domain (CTA), whose expression is required for cell cycle progression during prolonged heat stress conditions. In the present study, we have identified additional target genes of the HSF CTA, which include nearly all of the heat shock-inducible members of the Hsp90 chaperone complex, demonstrating coordinate regulation of these components by HSF. Heat shock induction of SSE1, encoding a member of the Hsp110 family of heat shock proteins, was also dependent on the HSF CTA. Disruption of SSE1 along with STI1, encoding an established subunit of the Hsp90 chaperone complex, resulted in a severe synthetic growth phenotype. Sse1 associated with partially purified Hsp90 complexes and deletion of the SSE1 gene rendered cells susceptible to the Hsp90 inhibitors macbecin and geldanamycin, suggesting functional interaction between Sse1 and Hsp90. Sse1 is required for function of the glucocorticoid receptor, a model substrate of the Hsp90 chaperone machinery, and Hsp90-based repression of HSF under nonstress conditions. Taken together, these data establish Sse1 as an integral new component of the Hsp90 chaperone complex in yeast.

Topics: Antibiotics, Antineoplastic; Benzoquinones; HSP110 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Lactams, Macrocyclic; Quinones; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

The protein kinase Gcn2 stimulates translation of the yeast transcription factor Gcn4 upon amino acid starvation. Using genetic and biochemical approaches, we show that Gcn2 is regulated by the molecular chaperone Hsp90 in budding yeast Saccharomyces cerevisiae. Specifically, we found that (i) several Hsp90 mutant strains exhibit constitutive expression of a GCN4-lacZ reporter plasmid; (ii) Gcn2 and Hsp90 form a complex in vitro as well as in vivo; (iii) the specific inhibitors of Hsp90, geldanamycin and macbecin I, enhance the association of Gcn2 with Hsp90 and inhibit its kinase activity in vitro; (iv) in vivo, macbecin I strongly reduces the levels of Gcn2; (v) in a strain expressing the temperature-sensitive Hsp90 mutant G170D, both the accumulation and activity of Gcn2 are abolished at the restrictive temperature; and (vi) the Hsp90 cochaperones Cdc37, Sti1, and Sba1 are required for the response to amino acid starvation. Taken together, these data identify Gcn2 as a novel target for Hsp90, which plays a crucial role for the maturation and regulation of Gcn2.

Topics: 5' Untranslated Regions; Benzoquinones; Cell Cycle Proteins; Chaperonins; DNA-Binding Proteins; Drosophila Proteins; Fungal Proteins; Gene Expression; Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Lac Operon; Lactams, Macrocyclic; Ligands; Molecular Chaperones; Mutagenesis; Peptide Initiation Factors; Protein Biosynthesis; Protein Kinases; Quinones; RNA, Messenger; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

The ubiquitous molecular chaperone Hsp90 acts in concert with a cohort of associated proteins to facilitate the functional maturation of a number of cellular signaling proteins, such as steroid hormone receptors and oncogene tyrosine kinases. The Hsp90-associated protein p23 is required for the assembly of functional steroid aporeceptor complexes in cell lysates, and Hsp90-binding ansamycin antibiotics disrupt the activity of Hsp90-dependent signaling proteins in cultured mammalian cells and prevent the association of p23 with Hsp90-receptor heterocomplexes; these observations have led to the hypotheses that p23 is required for the maturation of Hsp90 target proteins and that ansamycin antibiotics abrogate the activity of such proteins by disrupting the interaction of p23 with Hsp90. In this study, I demonstrate that ansamycin antibiotics disrupt the function of Hsp90 target proteins expressed in yeast cells; prevent the assembly of Sba1, a yeast p23-like protein, into steroid receptor-Hsp90 complexes; and result in the assembly of receptor-Hsp90 complexes that are defective for ligand binding. To assess the role of p23 in Hsp90 target protein function, I show that the activity of Hsp90 target proteins is unaffected by deletion of SBA1. Interestingly, steroid receptor activity in cells lacking Sba1 displays increased sensitivity to ansamycin antibiotics, and this phenotype is rescued by the expression of human p23 in yeast cells. These findings indicate that Hsp90-dependent signaling proteins can achieve a functional conformation in vivo in the absence of p23. Furthermore, while the presence of p23 decreases the sensitivity of Hsp90-dependent processes to ansamycin treatment, ansamycin antibiotics disrupt signaling through some mechanism other than altering the Hsp90-p23 interaction.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antibiotics, Antineoplastic; Benzoquinones; DNA-Binding Proteins; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Molecular Sequence Data; Oncogene Protein pp60(v-src); Phenotype; Protein-Tyrosine Kinases; Quinones; Receptors, Steroid; Saccharomyces cerevisiae; Sequence Alignment; Signal Transduction