The iron uptake and utilization pathways play a critical role in allowing human pathogens, including Cryptococcus neoformans, the causative agent of fatal meningoencephalitis, to survive within the mammalian body by competing with the host for iron. Here we show that the iron regulon is also required for diverse environmental stress responses and that in C. neoformans, it is regulated by the high-osmolarity glycerol response (HOG) pathway. Between CFO1 and CFO2, two ferroxidase genes in the iron regulon, CFO1 but not CFO2 was induced during oxidative and osmotic stress. Interestingly, we found that the HOG pathway repressed basal expression of both CFO1 and CFO2. Furthermore, when the HOG pathway was blocked, CFO2 also responded to oxidative and osmotic stress and the response of CFO1 was increased. We also established that CFO1 plays a major role in responding and adapting to diverse environmental stresses, including oxidative and genotoxic damage, osmotic fluctuations, heavy metal stress, and stress induced by cell membrane destabilizers. Therefore, our findings indicate that in C. neoformans, the iron uptake and utilization pathways are not only required for iron acquisition and survival, but also play a significant role in the environmental stress response through crosstalk with the HOG pathway.
Cell Membrane ; Ceruloplasmin* ; Cryptococcus neoformans* ; Glycerol ; Humans ; Iron ; Meningoencephalitis ; Osmotic Pressure ; Regulon

Type 2 diabetes mellitus is a complex metabolic disorder associated with multiple genetic, developmental and environmental factors. The recent advances in gene expression microarray technologies as well as network-based analysis methodologies provide groundbreaking opportunities to study type 2 diabetes mellitus. In the present study, we used previously published gene expression microarray datasets of human skeletal muscle samples collected from 20 insulin sensitive individuals before and after insulin treatment in order to construct insulin-mediated regulatory network. Based on a motif discovery method implemented by iRegulon, a Cytoscape app, we identified 25 candidate regulons, motifs of which were enriched among the promoters of 478 up-regulated genes and 82 down-regulated genes. We then looked for a hierarchical network of the candidate regulators, in such a way that the conditional combination of their expression changes may explain those of their target genes. Using Genomica, a software tool for regulatory network construction, we obtained a hierarchical network of eight regulons that were used to map insulin downstream signaling network. Taken together, the results illustrate the benefits of combining completely different methods such as motif-based regulatory factor discovery and expression level-based construction of regulatory network of their target genes in understanding insulin induced biological processes and signaling pathways.
Biological Processes ; Dataset ; Diabetes Mellitus, Type 2 ; Gene Expression* ; Humans ; Insulin ; Methods* ; Muscle, Skeletal ; Regulon ; Transcription Factors*

The Escherichia coli fadR protein product, a paradigm/prototypical FadR regulator, positively regulates fabA and fabB, the two critical genes for unsaturated fatty acid (UFA) biosynthesis. However the scenario in the other Ɣ-proteobacteria, such as Shewanella with the marine origin, is unusual in that Rodionov and coworkers predicted that only fabA (not fabB) has a binding site for FadR protein. It raised the possibility of fad regulon contraction. Here we report that this is the case. Sequence alignment of the FadR homologs revealed that the N-terminal DNA-binding domain exhibited remarkable similarity, whereas the ligand-accepting motif at C-terminus is relatively-less conserved. The FadR homologue of S. oneidensis (referred to FadR_she) was over-expressed and purified to homogeneity. Integrative evidence obtained by FPLC (fast protein liquid chromatography) and chemical cross-linking analyses elucidated that FadR_she protein can dimerize in solution, whose identity was determined by MALDI-TOF-MS. In vitro data from electrophoretic mobility shift assays suggested that FadR_she is almost functionally-exchangeable/equivalent to E. coli FadR (FadR_ec) in the ability of binding the E. coli fabA (and fabB) promoters. In an agreement with that of E. coli fabA, S. oneidensis fabA promoter bound both FadR_she and FadR_ec, and was disassociated specifically with the FadR regulatory protein upon the addition of long-chain acyl-CoA thioesters. To monitor in vivo effect exerted by FadR on Shewanella fabA expression, the native promoter of S. oneidensis fabA was fused to a LacZ reporter gene to engineer a chromosome fabA-lacZ transcriptional fusion in E. coli. As anticipated, the removal of fadR gene gave about 2-fold decrement of Shewanella fabA expression by β-gal activity, which is almost identical to the inhibitory level by the addition of oleate. Therefore, we concluded that fabA is contracted to be the only one member of fad regulon in the context of fatty acid synthesis in the marine bacteria Shewanella genus.
Amino Acid Sequence ; Bacterial Proteins ; chemistry ; metabolism ; Base Sequence ; Binding Sites ; DNA, Bacterial ; metabolism ; Escherichia coli ; genetics ; metabolism ; Fatty Acid Synthase, Type II ; genetics ; metabolism ; Fatty Acids ; biosynthesis ; Gene Expression Regulation, Bacterial ; drug effects ; Molecular Sequence Data ; Oleic Acid ; pharmacology ; Protein Binding ; drug effects ; Regulon ; genetics ; Repressor Proteins ; chemistry ; metabolism ; Shewanella ; genetics ; metabolism