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The authors screen for compounds that show synergistic antifungal activity when combined with the widely-used fungistatic drug fluconazole. Chemogenomic profiling explains the mode of action of synergistic drugs and allows the prediction of additional drug synergies.

The barrier imposed by lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria presents a significant challenge in treatment of these organisms with otherwise effective hydrophobic antibiotics. The absence of L-glycero-D-manno-heptose in the LPS molecule is associated with a dramatically increased bacterial susceptibility to hydrophobic antibiotics and thus enzymes in the ADP-heptose biosynthesis pathway are of significant interest. GmhA catalyzes the isomerization of D-sedoheptulose 7-phosphate into D-glycero-Dmanno-heptose 7-phosphate, the first committed step in the formation of ADP-heptose. Here we report structures of GmhA from Escherichia coli and Pseudomonas aeruginosa in apo, substrate, and product-bound forms, which together suggest that GmhA adopts two distinct conformations during isomerization through reorganization of quaternary structure. Biochemical characterization of GmhA mutants, combined with in vivo analysis of LPS biosynthesis and novobiocin susceptibility, identifies key catalytic residues. We postulate GmhA acts through an enediol-intermediate isomerase mechanism. Lipopolysaccharide (LPS)4 is an essential component of the outer membrane in Gram-negative bacteria (1). LPS not only functions as a protective barrier preventing cell entry of hydrophobic molecules, including bile salts, detergents, and lipophilic antibiotics, but also helps maintain the structural integrity of the outer membrane. Thus, LPS is vital for bacterial virulence and antibiotic sensitivity in pathogenic Gram-negative bacteria.Gram-negative pathogens are increasingly becoming a serious clinical threat. Multidrug-resistant hospital-acquired infections caused by enteric bacteria such as Escherichia coli and Klebsiella pneumoniae, and by emerging pathogens of environmental origin such as Acinetobacter baumannii and Pseudomonas aeruginosa, are the next big problem facing the infectious disease community. Furthermore, Gram-negative pathogens of animal origin such as E. coli O157-H7 are ongoing threats to agriculture and water quality. New chemotherapeutic strategies against Gram-negative bacteria are therefore required. LPS biosynthesis represents a unique Gram-negative target for new antimicrobial intervention.LPS comprises lipid A, a core oligosaccharide, and in some bacteria, an O-specific polysaccharide chain. The core oligosaccharide has an inner core region consisting of 3-deoxy-Dmanno-oct-2-ulosonic acid (Kdo) and one or more heptose units, and an outer core, consisting of additional sugar residues (Fig. 1A) (reviewed in Refs. 1-4).Lipid A and Kdo are highly conserved in Gram-negative bacteria and essential for cell viability. The biosynthesis of these molecules is therefore a target for traditional antibiotic discovery efforts. Indeed, small molecule inhibitors of lipid A biosynthesis have been reported to have anti-Gram-negative activity (5).Most Gram-negatives also contain one or more L-glycero-Dmanno-heptose molecules attached to the Kdo. Mutants in Tables S1 and S2, and a movie. The atomic coord...

Protein complexes and protein-protein interactions are essential for almost all cellular processes. Here, we establish a mammalian affinity purification and lentiviral expression (MAPLE) system for characterizing the subunit compositions of protein complexes. The system is flexible (i.e. multiple N-and C-terminal tags and multiple promoters), is compatible with Gateway TM cloning, and incorporates a reference peptide. Its major advantage is that it permits efficient and stable delivery of affinity-tagged open reading frames into most mammalian cell types. We benchmarked MAPLE with a number of human protein complexes involved in transcription, including the RNA polymerase II-associated factor, negative elongation factor, positive transcription elongation factor b, SWI/SNF, and mixed lineage leukemia complexes. In addition, MAPLE was used to identify an interaction between the reprogramming factor Klf4 and the Swi/Snf chromatin remodeling complex in mouse embryonic stem cells. We show that the SWI/SNF catalytic subunit Smarca2/Brm is up-regulated during the process of induced pluripotency and demonstrate a role for the catalytic subunits of the SWI/SNF complex during somatic cell reprogramming. Our data suggest that the transcription factor Klf4 facilitates chromatin remodeling during reprogramming. Molecular & Cellular Proteomics 9:811-823, 2010.The analysis of protein-protein interactions (PPIs) 1 and protein complexes is of central importance to biological research and facilitates our understanding of how molecular events drive phenotypic outcomes. Moreover, large scale protein interaction data can be used to generate protein interaction networks, which can then be used to predict disease genes and model biology in any living organism.A number of methods (e.g. yeast two-hybrid) have been developed to examine binary protein interactions in a systematic format and applied to model systems (1-8). However, affinity purification (AP) coupled with tandem MS has become the method of choice for the identification of protein complexes (9, 10). Large scale PPI studies using a high throughput and systematic AP-MS approach have been performed for Escherichia coli (11,12) and Saccharomyces cerevisiae (13-15). In fact, large scale efforts using AP-MS have connected an estimated 60% of the yeast proteome, demonstrating the power of coupling systematic biochemical purifications with mass spectrometry (13-16).AP-MS has also been used extensively for purification of mammalian protein complexes (17), but this has been mostly restricted to small scale studies and the use of either cell lines that are easy to transfect or highly validated antibodies against specific targets. For example, Glatter et al. (18) recently developed an integrated workflow where a high density interactome was developed for the protein phosphatase 2A complex. This workflow relies on "flip-in" technology to introduce transgenes into a common genomic site in HEK293 cells and, similar to work by other groups (19,20), utilizes an From the ‡Banting and Best

Background: Cell surface recognition of the AC133 epitope on CD133 marks many stem cell and cancer stem cell types. Results: A large scale RNA interference screen identifies genes involved in N-glycosylation that regulate cell surface AC133 recognition. Conclusion: CD133 N-glycosylation and its processing contribute to cell surface AC133 recognition. Significance: Glycobiological differences between primitive and differentiated cells may be responsible for regulating cell surface AC133.