Vesicular transport to and from the lysosome and late endosome is defective in patients with Chediak-Higashi syndrome (CHS) and in mutant beige (bg) mice. CHS and bg cells have giant, perinuclear vesicles with characteristics of late endosomes and lysosomes that arise from dysregulated homotypic fusion. CHS and bg lysosomes also exhibit compartmental missorting of proteins, such as elastase, glucuronidase and cathepsin G. Lyst, a candidate gene for bg, was identified by direct complementary DNA selection from a yeast artificial chromosome (YAC) clone containing a 650-kilobase segment of the bg-critical region on mouse chromosome 13. Lyst is disrupted by a 5-kilobase deletion in bg mice, and Lyst messenger RNA is markedly reduced in bg homozygotes. The homologous human gene, LYST, is highly conserved with mouse Lyst, and contains a frame-shift mutation at nucleotides 117-118 of the coding domain in a CHS patient. Thus bg mice and human CHS patients have homologous disorders associated with Lyst mutations. Lyst encodes a protein with a carboxy-terminal prenylation motif and multiple potential phosphorylation sites. Lyst protein is predicted to form extended helical domains, and has a region of sequence similar to stathmin, a coiled-coil phosphoprotein thought to act as a relay integrating cellular signal response coupling.
As the global threat of drug- and antibiotic-resistant bacteria continues to rise, new strategies are required to advance the drug discovery process. This work describes the construction of an array of Escherichia coli strains for use in whole-cell screens to identify new antimicrobial compounds. We used the recombination systems from bacteriophages lambda and P1 to engineer each strain in the array for low-level expression of a single, essential gene product, thus making each strain hypersusceptible to specific inhibitors of that gene target. Screening of nine strains from the array in parallel against a large chemical library permitted identification of new inhibitors of bacterial growth. As an example of the target specificity of the approach, compounds identified in the whole-cell screen for MurA inhibitors were also found to block the biochemical function of the target when tested in vitro.
Chediak-Higashi syndrome is an autosomal recessive, immune deficiency disorder of human (CHS) and mouse (beige, bg) that is characterized by abnormal intracellular protein transport to, and from, the lysosome. Recent reports have described the identification of homologous genes that are mutated in human CHS and bg mice. Here we report the sequences of two major mRNA isoforms of the CHS gene in human and mouse. These isoforms differ both in size and in sequence at the 3' end of their coding domains, with the smaller isoform (approximately 5.8 kb) arising from incomplete splicing and reading through an intron. These mRNAs also differ in tissue distribution of transcription and in predicted biological properties. Novel mutations were identified within the region of the coding domain common to both isoforms in three CHS patients: C-->T transitions that generated stop codons (R50X and Q1029X) were found in two patients, and a novel frameshift mutation (deletion of nucleotides 3073 and 3074 of the coding domain) was found in a third. Northern blots of lymphoblastoid mRNA from CHS patients revealed loss of the largest transcript (approximately 13.5 kb) in two of seven CHS patients, while the small mRNA was undiminished in abundance. These results suggest that the small isoform alone cannot complement Chediak-Higashi syndrome.
Osmotically stabilized Escherichia coli cells subjected to freezing and thawing were utilized as the source of enzymes for a peptidoglycan pathway assay that can be used to simultaneously test all targets of the committed steps of cell wall biosynthesis. The use of 14 C-labeled UDP-N-acetylglucosamine (UDP-GlcNAc) as a substrate allows the direct detection of cross-linked peptidoglycan formed. The assay was validated with known antibiotics. Fosfomycin was the strongest inhibitor of the pathway assay, with a 50% inhibitory concentration of 1 M. Flavomycin, bacitracin, vancomycin, D-cycloserine, penicillin G, and ampicillin also inhibited formation of radiolabeled peptidoglycan by the E. coli cells. Screening of compounds identified two inhibitors of the pathway, Cpd1 and Cpd2. Subsequent tests with a biochemical assay utilizing purified enzyme implicated UDP-GlcNAc enolpyruvyl transferase (MurA) as the target of Cpd1. This compound inhibits the first enzyme of the pathway in a time-dependent manner. Moreover, enzyme inactivation is dependent on preincubation in the presence of UDP-GlcNAc, which forms a complex with MurA, exposing its active site. Cpd1 also displayed antimicrobial activity against a panel of microorganisms. The pathway assay used in conjunction with assays for individual enzymes provides an efficient means of detecting and characterizing novel antimicrobial agents.
Signal peptidases of prokaryotic organisms reside in the outer leaflet of the cytoplasmic membrane and catalyze the hydrolytic cleavage of a specific peptide bond of membrane-imbedded preproteins to liberate mature proteins for secretion. In this manuscript, we report new and efficient peptide substrates for SPase and their use to explore features of this enzyme's reaction mechanism. The enzyme used in this study was recombinant SPase I of Escherichia coli that had been solubilized with Triton X-100 and purified to near homogeneity. Our new substrates are based on the fluorogenic peptide reported by Zhong and Benkovic [(1998) Anal. Biochem. 255, 66], Y(NO2)FSASALA approximately KIK(Abz)-NH(2) (Y(NO2), 3-nitro-L-tyrosine; K(Abz), epsilon-(2-aminobenzoyl)-L-Lys; hydrolysis at A approximately K). We found that when a signal peptide-like sequence is appended onto the N-terminus of this peptide to produce K(5)-L(10)-Y(NO2)FSASALA approximately KIK(Abz)-NH(2), k(c)/K(m) increases from 85 to 2.5 x 10(6) M(-)(1) s(-)(1). k(c)/K(m) decreases with increasing concentration of Triton X-100 micelles under the condition [Triton X-100](micelle) > [S](0) > [E](0). We explain this apparent inhibition with a model of surface dilution kinetics in which "empty" micelles compete with substrate-containing micelles for micelle-bound enzyme. Fusion of micelle-bound enzyme with a substrate-containing micelle leads to formation of productive E:S substrate complexes while fusion of micelle-bound enzyme with an "empty" micelle is nonproductive and inhibitory. The dependence of steady-state kinetic parameters for the SPase-catalyzed hydrolysis of K(5)-L(10)-Y(NO2)FSASALA approximately KIK(Abz)-NH(2) on [Triton X-100](micelle) supports this model. Product inhibition and solvent isotope effects were also investigated and could be interpreted in the context of this model.
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