Increased metabolism is a requirement for tumor cell proliferation. To understand the dependence of tumor cells on fatty acid metabolism, we evaluated various nodes of the fatty acid synthesis pathway. Using RNAi we have demonstrated that depletion of fatty-acid synthesis pathway enzymes SCD1, FASN, or ACC1 in HCT116 colon cancer cells results in cytotoxicity that is reversible by addition of exogenous fatty acids. This conditional phenotype is most pronounced when SCD1 is depleted. We used this fatty-acid rescue strategy to characterize several small-molecule inhibitors of fatty acid synthesis, including identification of TOFA as a potent SCD1 inhibitor, representing a previously undescribed activity for this compound. Reference FASN and ACC inhibitors show cytotoxicity that is less pronounced than that of TOFA, and fatty-acid rescue profiles consistent with their proposed enzyme targets. Two reference SCD1 inhibitors show low-nanomolar cytotoxicity that is offset by at least two orders of magnitude by exogenous oleate. One of these inhibitors slows growth of HCT116 xenograft tumors. Our data outline an effective strategy for interrogation of on-mechanism potency and pathway-node-specificity of fatty acid synthesis inhibitors, establish an unambiguous link between fatty acid synthesis and cancer cell survival, and point toward SCD1 as a key target in this pathway.
The isolated ligand binding domain of the chemotaxis aspartate receptor is the focus of the present study, which both (a) identifies structural regions involved in the attractant-induced conformational change and (b) investigates the kinetic parameters of attractant binding. To analyze the attractant-induced conformational change within the homodimeric domain, 19F NMR is used to monitor six para-fluorophenylalanine (4-F-Phe) positions within each identical subunit of the homodimer. The binding one molecule of aspartate to the homodimer perturbs three of the 4-F-Phe resonances significantly: 4-F-Phe150 in the attractant binding site, 4-F-Phe107 located 26 A from the site, and 4-F-Phe180 at a distance of 40 A from the site. Comparison of the frequency shifts triggered by aspartate and glutamate reveals that these attractants generate different conformations in the vicinity of the attractant site but trigger indistinguishable long-range conformational effects at distant positions. This long-range conformational change is specific for attractant binding, since formation of the Cys36-Cys36' disulfide bond or the nonphysiological binding of 1,10-phenanthroline to an aromatic pocket distal to the attractant site each yield conformational changes which are significantly more localized. The attractant-triggered perturbations detected at 4-F-Phe107 and 4-F-Phe180 indicate that the structural change includes an intrasubunit component communicated through the domain to its C-terminal region, which, in the full-length receptor, continues through the membrane as the second membrane-spanning helix. It would thus appear that the transmembrane signal is transmitted through this helix.(ABSTRACT TRUNCATED AT 250 WORDS)
The effects of ergosterol, yeast's natural sterol, on cell cycling and a protein kinase antigenically related to pp6O' were examined in a sterol auxotroph ofSaccharomyces cerevisiae. Sterol-depleted cells accumulate in an unbudded, G1 state. Cell budding and proliferation are reinitiated upon addition of nonlimiting ergosterol or cholesterol with trace ergosterol, whereas cholesterol or trace ergosterol alone is less effective. Stimulation of a protein kinase associated with immune complexes of yeast protein and anti-pp60v1 shows a positive correlation with exit from the G1 phase following ergosterol addition. Ergosterol-stimulated cells also demonstrate an increase in phosphatidylinositol kinase activity. The data suggest that hormonal levels of ergosterol (effective concentration, 1 nM) participate in a signaling process associated with a protein kinase possibly involved in yeast cell cycle control.In the budding yeast Saccharomyces cerevisiae nutritional signals normally control cell growth and division. When yeast cells stop growing in response to nutritional deprivation they arrest in the unbudded, G1 phase of the cell cycle (1). The isolation of many temperature-sensitive cell division cycle (cdc) mutants has led to a better understanding of the regulation of yeast cell proliferation and has resulted in the description of a G1-phase "start" event that is controlled by a number of genes (2). One of these genes, CDC28, has been cloned and sequenced (3). The CDC28 gene product shares homology with a number of mammalian protein kinases, among these a vertebrate cAMP-dependent protein kinase, as well as viral oncogenes such as v-mos and v-src (3). The CDC28-encoded protein has now been shown to possess a protein kinase activity, as has a related cdc2+-encoded protein in the fission yeast (4, 5). Moreover, a decrease in the activity ofthe cdc2-encoded protein correlates with cell cycle arrest upon nitrogen starvation in Schizosaccharomyces pombe (5). cAMP-dependent protein kinase and the RAS gene product have also been implicated in growth control in yeast (6). Interestingly, an association has been demonstrated between the activities of the pp60vsrc tyrosine kinase and phosphatidylinositol (PtdIns) kinase in Rous sarcoma virustransformed chicken embryo fibroblasts (7). Although the role of the polyphosphatidylinositol phosphates in yeast physiology has yet to be determined (8-10), we have observed a marked ergosterol-mediated stimulation of the turnover of these lipids that correlates with an enhanced rate of cell proliferation (11).Studies Gl-phase cell cycle arrest (sterol starvation) or G1 release and cell proliferation (sterol addition). This analysis has led to the identification of a protein kinase having antigenic homology with the transforming protein of the Rous sarcoma virus and whose activity is positively regulated by a trace amount of ergosterol. To our knowledge, evidence for the existence of a protein kinase possibly involved in yeast cell cycle control, whose activity is exquisitely sens...
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