The FadR protein of Escherichia coli has been shown to play a dual role in transcription of the genes of bacterial fatty acid metabolism. The protein acts as a repressor of ␤-oxidation and an activator of unsaturated fatty acid synthesis. FadR DNA binding is antagonized by long chain acyl-CoAs, and thus FadR acts as a sensor of fatty acid availability in the environment. When viewed from a genomic viewpoint, FadR proteins are unusual in that the DNA binding domain is very highly conserved among FadR-containing bacteria, whereas the C-terminal acyl-CoA binding domain shows only weak conservation. To further our understanding of the role of FadR in bacterial lipid metabolism we have examined the in vivo and in vitro properties of a diverse set of FadR proteins expressed in E. coli. In addition to E. coli FadR the proteins examined were those of Salmonella enterica, Vibrio cholerae, Pasteurella multocida, and Haemophilus influenzae. These FadR proteins were found to differ markedly in their effects on repression and induction of ␤-oxidation in E. coli and in their acyl-CoA binding abilities as measured by isothermal titration calorimetry. The E. coli and S. enterica proteins were the most similar, although they differed in their effects on utilization of oleic acid and acyl-CoA binding affinities, whereas the P. multocida and H. influenzae proteins showed only weak repression and poor acyl-CoA binding affinities. The V. cholerae FadR was strikingly superior to the other proteins in the amplitude of its regulatory response, and it bound long chain acyl-CoAs appreciably more strongly than the E. coli and S. enterica proteins. The significance of these findings is discussed in view of the protein sequences and the physiological niches occupied by these organisms.

Based on its genome sequence, the pathway of ␤-oxidative fatty acid degradation in Salmonella enterica serovar Typhimurium LT2 has been thought to be identical to the well-characterized Escherichia coli K-12 system. We report that wild-type strains of S. enterica grow on decanoic acid, whereas wild-type E. coli strains cannot. Mutant strains (carrying fadR) of both organisms in which the genes of fatty acid degradation (fad) are expressed constitutively are readily isolated. The S. enterica fadR strains grow more rapidly than the wild-type strains on decanoic acid and also grow well on octanoic and hexanoic acids (which do not support growth of wild-type strains). By contrast, E. coli fadR strains grow well on decanoic acid but grow only exceedingly slowly on octanoic acid and fail to grow at all on hexanoic acid. The two wild-type organisms also differed in the ability to grow on oleic acid when FadR was overexpressed. Under these superrepression conditions, E. coli failed to grow, whereas S. enterica grew well. Exchange of the wild-type fadR genes between the two organisms showed this to be a property of S. enterica rather than of the FadR proteins per se. This difference in growth was attributed to S. enterica having higher cytosolic levels of the inducing ligands, long-chain acyl coenzyme As (acyl-CoAs). The most striking results were the differences in the compositions of CoA metabolites of strains grown with octanoic acid or oleic acid. S. enterica cleanly converted all of the acid to acetyl-CoA, whereas E. coli accumulated high levels of intermediate-chain-length products. Exchange of homologous genes between the two organisms showed that the S. enterica FadE and FadBA enzymes were responsible for the greater efficiency of ␤-oxidation relative to that of E. coli.

The SNF1/AMP-activated protein kinase (AMPK) family is required for adaptation to metabolic stress and energy homeostasis. The ␥ subunit of AMPK binds AMP and ATP, and mutations that affect binding cause human disease. We have here addressed the role of the Snf4 (␥) subunit in regulating SNF1 protein kinase in response to glucose availability in Saccharomyces cerevisiae. Previous studies of mutant cells lacking Snf4 suggested that Snf4 counteracts autoinhibition by the C-terminal sequence of the Snf1 catalytic subunit but is dispensable for glucose regulation, and AMP does not activate SNF1 in vitro. We first introduced substitutions at sites that, in AMPK, contribute to nucleotide binding and regulation. Mutations at several sites relieved glucose inhibition of SNF1, as judged by catalytic activity, phosphorylation of the activation-loop Thr-210, and growth assays, although analogs of the severe human mutations R531G/Q had little effect. We further showed that alterations of Snf4 residues that interact with the glycogen-binding domain (GBD) of the ␤ subunit strongly relieved glucose inhibition. Finally, substitutions in the GBD of the Gal83 ␤ subunit that are predicted to disrupt interactions with Snf4 and also complete deletion of the GBD similarly relieved glucose inhibition of SNF1. Analysis of mutant cells lacking glycogen synthase showed that regulation of SNF1 is normal in the absence of glycogen. These findings reveal novel roles for Snf4 and the GBD in regulation of SNF1.

, and Gly 511 had no effect on MRP1 levels. Except for K503A, however, transport by these mutants was reduced by 50 to 75%, an effect largely attributable to reduced substrate binding and affinity. Studies with 32 P-labeled azido-ATP also indicated that whereas ATP binding by the G511I mutant was unchanged, vanadate-induced trapping of azido-ADP was reduced, indicating changes in the catalytic activity of MRP1. Together, these data demonstrate the multiple roles for CL5 in the membrane expression and function of MRP1.

Multidrug resistance protein 1 (MRP1/ABCC1), an integral transmembrane efflux transporter, belongs to the ATP-binding cassette (ABC) protein superfamily. MRP1 governs the absorption and disposition of a wide variety of endogenous and xenobiotic substrates including various drugs across organs and physiological barriers. Additionally, its overexpression has been implicated in multidrug resistance in chemotherapy of multiple cancers. Here, we describe the development of a high content imaging-based screening assay for MRP1 activity. This live cell-based automated microscopy assay is very robust and allows simultaneous detection of cell permeable, non-toxic and potent inhibitors. The validity of the assay was demonstrated by profiling a library of 386 anti-cancer compounds, which are under clinical trials, for interactions with MRP1. The assay identified 12 potent inhibitors including two known MRP1 inhibitors, cyclosporine A and rapamycin. On the other hand, MRP1-inhibitory activity of tipifarnib, AZD1208, deforolimus, everolimus, temsirolimus, HS-173, YM201636, ESI-09, TAK-733, and CX-6258 has not been previously reported. Inhibition of MRP1 activity was further validated using flow cytometry and confocal microscopy for the respective detection of calcein and doxorubicin in MRP1-overexpressing cells. Among the identified compounds, tipifarnib, AZD1208, rapamycin, deforolimus, everolimus, TAK-733, and temsirolimus resensitized MRP1-overexpressing H69AR cells towards vincristine, a cytotoxic chemotherapeutic agent, by 2-6-fold. Using purified HEK293 membrane vesicles overexpressing MRP1, MRP2, MRP3, and MRP4, we also demonstrated that the identified compounds exert differential and selective response on the uptake of estradiol glucuronide, an endogenous MRP substrate. In summary, we demonstrated the effectiveness of the high content imaging-based high-throughput assay for profiling compound interaction with MRP1.