Project description:NIH 3T3 fibroblasts that express little detectable levels of the MIF binding receptor CD74 but do express the signalling component CD44 were used for the identification of interacting proteins that are shared by MIF and D-DT. Endogenously expressed MIF and D-DT were tagged at the C-terminus using a short sequence that is recognized and biotinylated by the bacterial biotin ligase BirA (Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc. Natl. Acad. Sci. U. S. A. 100, 7480–5) as described in detail for MIF Filip, A. M. et al. (2009). Ribosomal Protein S19 Interacts with Macrophage Migration Inhibitory Factor and Attenuates Its Pro-inflammatory Function. J. Biol. Chem. 284, 7977–7985). After pulling down biotinylated proteins from stable NIH 3T3 clones that express tagged MIF or D-DT and BirA ligase with streptavidin agarose, bound proteins were separated by SDS-PAGE and identified after elution and tryptic digestion by mass spectrometry in order to characterize the MIF and D-DT interactomes.
Project description:We have adapted the DamID protocol for use with high throughput sequencing. We have used DamID to identify the positions within the Drosophila genome where the transcription factor DSX is bound. We sequenced DpnI-digested genomic DNA from fly tissues containing UAS-Dam (control) or UAS-Dam-DsxF or UAS-Dam-DsxM. We have performed DamID-seq on adult male and female fatbody and on ovary. We used two biological replicates for each tissue and sex.
Project description:The Bae, Cpx, Psp, Rcs and ?E pathways constitute the Escherichia coli signaling systems that detect and respond to alterations of the bacterial envelope. Contributions of these systems to stress response have previously been examined individually; however, the possible interconnections between these pathways are unknown. Here we investigate the dynamics between the five stress response pathways by i) determining the specificities of each system with respect to signal inducing conditions, and ii) monitoring global transcriptional changes in response to transient over-expression of each of the effectors.
Project description:PINA is a novel ATPase and DNA helicase highly conserved in archaea, the third domain of life. The PINA from Sulfolobus islandicus (SisPINA) forms a hexameric ring, promotes Holliday junction (HJ) migration, and physically and functionally interacts with Hjc, the HJ specific endonuclease. Here, we show that SisPINA has strong physical interaction with Hjm (Hel308a), a helicase presumably targeting replication forks. In vitro biochemical analysis revealed that Hjm, Hjc, and SisPINA are able to coordinate HJ migration and cleavage in a concerted way. Deletion of the carboxyl 13 amino acid residues abolishes the interaction between SisPINA and Hjm. Crystal structure analysis showed that the carboxyl 70 amino acid residues fold into a type II KH domain which, in other proteins, functions in binding RNA and ssDNA. The KH domain not only mediates the interactions of PINA with Hjm and Hjc but also regulates the hexameric assembly of PINA. Our results collectively suggest possible mechanisms for SisPINA, Hjm, and Hjc to work together in replication fork regression, HJ formation, and HJ cleavage.
Project description:HIF-1 is an important transcription factor for immune responses to bacterial infection. We wanted to analyze the HIF-1 dependent gene expression upon S. aureus infection and analyzed the gene expression of HepG2 nt and HepG2 HIF-1-/- cells four hours upon infection using affymetrix human gene 1.0 st. gene arrays. triplicates of HepG2 nt and HepG2 HIF-1-/- cells were infected with S. aureus 8325-4 (4 h, MOI: 20), triplicates of uninfected cells were used as control
Project description:The function of the Staphylococcus aureus eukaryotic-like serine/threonine protein kinase PknB was investigated by transcriptome analysis using DNA-microarray technology and biochemical assays. The transcriptional profile reveals a strong regulatory impact of PknB on the expression of genes encoding proteins which are involved in purine and pyrimidine biosynthesis, cell wall metabolism, autolysis, and glutamine synthesis. To investigate the functional role of PknB in S. aureus DNA microarray experiments were performed. In this experiment, the impact of the deletion of PknB was investigated. Each experiment was performed 5 times including a dye swap resulting in five chips per competitive comparison to increase reproducibility. All hybridizations were done with equal amounts of cDNA probes.
Project description:The Staphylococcus aureus ClpXP protease is an important regulator of cell homeostasis and virulence. Here we utilize a high-throughput screen against the ClpXP complex and identify a specific inhibitor of the ClpX chaperone that disrupts its oligomeric state. Synthesis of 34 derivatives revealed that the molecular scaffold is restrictive for diversification with only minor changes tolerated. Subsequent analysis of the most active compound revealed strong attenuation of S. aureus toxin production which was quantified via a novel MS-based assay platform. Transcriptome and whole proteome studies further confirmed the global reduction of virulence and unraveled characteristic signatures of protein expression in compound treated cells. Although these partially matched the pattern of ClpX knockout cells, further depletion of toxins was observed leading to the intriguing perspective that additional virulence pathways may be directly or indirectly addressed by the small molecule.
Project description:To combat methicillin-resistant Staphylococcus aureus (MRSA) infections novel drugs are desperately needed. From a screen, we found that the anti-cancer drug sorafenib effectively kills MRSA strains. By synthetic variation of key structural features, we identified a potent analog, PK150, exhibiting activities against several pathogenic strains at sub-micromolar concentrations. The antibiotic induced rapid killing of S. aureus, including challenging persisters, and eradicated established biofilms. PK150 holds promising therapeutic potential as it did not induce in vitro resistance and exhibited oral bioavailability and in vivo efficacy. Mode of action analysis by chemical proteomics revealed several targets, including interference with menaquinone biosynthesis by inhibiting demethylmenaquinone methyltransferase and stimulation of protein secretion by altering the activity of signal peptidase IB. Reduced endogenous menaquinone levels along with enhanced levels of extracellular proteins of PK150-treated bacteria support this hypothesis. The associated antibiotic effects, especially the lack of resistance development, likely stem from the compound’s polypharmacology attribute.
Project description:Heterozygous point mutations or genomic deletions of NR0B1 in Xp21.2 result in congenital adrenal hypoplasia and hypogonadotropic hypogonadism, whereas the NR0B1 locus duplications in XY individuals lead to gonadal dysgenesis and a male-to-female dosage-sensitive sex reversal. We previously reported an ~ 257 kb deletion mapping 11 kb upstream to NR0B1 in a XY female with primary amenorrhea, small immature uterus, and gonadal dysgenesis pointing to an alteration of its regulatory region. To identify the potential regulatory elements of NR0B1, we have analyzed its 2 Mb flanking regions using chromosome conformation capture-on-chip (4C) in Sertoli cells and lymphoblasts. We confirm the involvement of the previously proposed regulatory region in the control of NR0B1 expression and describe several novel potential chromatin interactions within the NR0B1 locus that may be involved in sex differentiation. Custom designed 3x720K tiling microarrays covering 4 Mb region (chrX:28,082,100-32,087,100) flanking NR0B1 gene of interest
Project description:Transcription factor SOX9 is essential for the differentiation of chondrocytes and the development of testes. Heterozygous point mutations and genomic deletions involving SOX9 lead to campomelic dysplasia (CD) often associated with sex reversal. Chromosomal rearrangements with breakpoints mapping up to 1.3 Mb up- and downstream to SOX9, and likely disrupting its distant cis-regulatory elements, have been described in patients with milder forms of CD. Performing chromosome conformation capture-on-chip (4C) analysis in Sertoli cells and lymphoblasts we identified several novel potentially cis-interacting regions both up- and downstream to SOX9, with some of them overlapping lncRNA genes preferentially expressed in testes. Custom designed 3x720K tiling microarrays covering 4 Mb region (chr17:68,117,161-72,122,560) flanking SOX9 gene of interest