The COP9 signalosome (CSN) is a conserved protein complex with homologies to the lid subcomplex of the 26S proteasome. It promotes cleavage of the Nedd8 conjugate (deneddylation) from the cullin component of SCF ubiquitin ligases. We provide evidence that cullin neddylation and deneddylation is highly dynamic, that its equilibrium can be effectively modulated by CSN, and that neddylation allows Cul1 to form larger protein complexes. CSN2 integrates into the CSN complex via its C-terminal region and its N-terminal half region is necessary for direct interaction with Cul1. The polyclonal antibodies against CSN2 but not other CSN subunits cause accumulation of neddylated Cul1/Cul2 in HeLa cell extract, indicating that CSN2 is essential in cullin deneddylation. Further, CSN inhibits ubiquitination and degradation of the cyclin-dependent kinase inhibitor p27(kip1) in vitro. Microinjection of the CSN complex impeded the G1 cells from entering the S phase. Moreover, anti-CSN2 antibodies negate the CSN-dependent p27 stabilization and the G1/S blockage, suggesting that these functions require the deneddylation activity. We conclude that CSN inhibits SCF ubiquitin ligase activity in targeting p27 proteolysis and negatively regulates cell cycle at the G1 phase by promoting deneddylation of Cul1.
Although Candida albicans and Saccharomyces cerevisiae express very similar systems of iron uptake, these species differ in their capacity to use heme as a nutritional iron source. Whereas C. albicans efficiently takes up heme, S. cerevisiae grows poorly on media containing heme as the sole source of iron. We identified a gene from C. albicans that would enhance heme uptake when expressed in S. cerevisiae. Overexpression of CaFLC1 (for flavin carrier 1) stimulated the growth of S. cerevisiae on media containing heme iron. In C. albicans, deletion of both alleles of CaFLC1 resulted in a decrease in heme uptake activity, whereas overexpression of CaFLC1 resulted in an increase in heme uptake. The S. cerevisiae genome contains three genes with homology to CaFLC1, and two of these, termed FLC1 and FLC2, also stimulated growth on heme when overexpressed in S. cerevisiae. The S. cerevisiae Flc proteins were detected in the endoplasmic reticulum and the FLC genes encoded an essential function, as strains deleted for either FLC1 or FLC2 were viable, but deletion of both FLC1 and FLC2 was synthetically lethal. FLC gene deletion resulted in pleiotropic phenotypes related to defects in cell wall integrity. High copy suppressors of this synthetic lethality included three mannosyltransferases, VAN1, KTR4, and HOC1. FLC deletion strains exhibited loss of cell wall mannose phosphates, defects in cell wall assembly, and delayed maturation of carboxypeptidase Y. Permeabilized cells lacking FLC proteins exhibited dramatic loss of FAD import activity. We propose that the FLC genes are required for import of FAD into the lumen of the endoplasmic reticulum, where it is required for disulfide bond formation.
advertising feature an8 | December 2008 | nature methods application notes cell Biology biotinylated antibody bound to streptavidin-coated donor beads and a second antibody conjugated to AlphaLISA acceptor beads. The binding of the two antibodies to the analyte brings donor and acceptor beads into proximity. Laser irradiation of donor beads at 680 nm generates a flow of singlet oxygen, triggering a cascade of chemical events in nearby acceptor beads, which results in a chemiluminescent emission at 615 nm. In competitive AlphaLISA immunoassays, a biotinylated analyte bound to streptavidin donor beads is used with an antibody conjugated to AlphaLISA acceptor beads. Rapid and simple quantification of analytesAlphaLISA assays are performed following simple 'mix-and-measure' protocols with reduced hands-on and total assay times compared to ELISAs (Fig. 2). Homogeneous AlphaLISA assays eliminate the need for multiple washes to separate bound from unbound assay components. Miniaturization and automationMiniaturization is a key consideration for reducing screening cost and increasing throughput during the drug-discovery process.AlphaLISA assays are truly miniaturizable and automatable, with ELISA is the most widely used detection platform for the quantification of analytes in biological samples. Because they require multiple washes, ELISAs are difficult to adapt to high throughput and automation. Their relatively narrow dynamic range often requires testing more than one sample dilution. There is clearly a need for simple and more robust alternatives for the quantification of biomarkers in a high-throughput screening format. The new AlphaLISA platform has been specifically designed for that purpose for both the research and drug-discovery fields.The AlphaLISA bead-based technology relies on PerkinElmer's exclusive amplified luminescent proximity homogeneous assay (AlphaScreen ® ) and uses a luminescent oxygen-channeling chemistry 1 . AlphaLISA protocols can be set up as sandwich or competition immunoassays. In a sandwich assay (Fig. 1), an analyte is captured by a AlphaLISA immunoassays: the no-wash alternative to ELISAs for research and drug discovery PerkinElmer's bead-based AlphaLISA® immunoassays are designed for the detection of analytes in biological samples. These chemiluminescent, no-wash assays are ideally suited for miniaturization and automation. They exhibit remarkable sensitivity, wide dynamic range and robust performance that compares advantageously with conventional enzyme-linked immunosorbent assay (ELISA).
Mechanism-of-action (MOA) studies of bioactive compounds are fundamental to drug discovery. However, in vitro studies alone may not recapitulate a compound's MOA in whole cells. Here, we apply a chemogenomics approach in Candida albicans to evaluate compounds affecting purine metabolism. They include the IMP dehydrogenase inhibitors mycophenolic acid and mizoribine and the previously reported GMP synthase inhibitors acivicin and 6-diazo-5-oxo-L-norleucine (DON). We report important aspects of their whole-cell activity, including their primary target, off-target activity, and drug metabolism. Further, we describe ECC1385, an inhibitor of GMP synthase, and provide biochemical and genetic evidence supporting its MOA to be distinct from acivicin or DON. Importantly, GMP synthase activity is conditionally essential in C. albicans and Aspergillus fumigatus and is required for virulence of both pathogens, thus constituting an unexpected antifungal target.
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