Biomedical subjects

Ying-Kai Wang

Publications and source records attributed to Ying-Kai Wang.

5 recordsLinked to original sources

PubMed2006

A homogeneous, solid-phase assay for hepatitis C virus RNA-dependent RNA polymerase.

Discovery and development of effective antiviral agents to combat hepatitis C virus (HCV) is the focus of intensive research both in academia and in pharmaceutical companies. One of the HCV nonstructural proteins, NS5B (an RNA-dependent RNA polymerase), represents an attractive target in light of the clinical success of human immunodeficiency virus reverse transcriptase inhibitors. To identify and evaluate NS5B inhibitors, we developed a homogeneous, solid-phase, high-throughput biochemical assay for detecting NS5B enzymatic activity. In this assay, a biotinylated oligo(dT(12)) primer was immobilized onto streptavidin-coated scintillation proximity assay (SPA) beads, and after addition of homopolymeric A template, NS5B enzyme, and radiolabeled uridine 5'-triphosphate, the primer-dependent RNA synthesis occurred on beads. Optimization of the on-bead reaction resulted in the use of significantly less RNA template and NS5B enzyme while producing a faster steady state reaction rate compared to the solution-phase or off-bead SPA. The newly developed solid-phase assay is functionally comparable to the solution-phase assay as similar potencies of HCV NS5B inhibitors tested were obtained with the two assays. Furthermore, the solid-phase assay offers the advantage of delaying initiation, mimicking a physical preincubation step required for evaluating time-dependent inhibitors.

Antiviral Agents↗

PubMed2006

Conserved fungal genes as potential targets for broad-spectrum antifungal drug discovery.

The discovery of novel classes of antifungal drugs depends to a certain extent on the identification of new, unexplored targets that are essential for growth of fungal pathogens. Likewise, the broad-spectrum capacity of future antifungals requires the target gene(s) to be conserved among key fungal pathogens. Using a genome comparison (or concordance) tool, we identified 240 conserved genes as candidates for potential antifungal targets in 10 fungal genomes. To facilitate the identification of essential genes in Candida albicans, we developed a repressible C. albicans MET3 (CaMET3) promoter system capable of evaluating gene essentiality on a genome-wide scale. The CaMET3 promoter was found to be highly amenable to controlled gene expression, a prerequisite for use in target-based whole-cell screening. When the expression of the known antifungal target C. albicans ERG1 was reduced via down-regulation of the CaMET3 promoter, the CaERG1 conditional mutant strain became hypersensitive, specifically to its inhibitor, terbinafine. Furthermore, parallel screening against a small compound library using the CaERG1 conditional mutant under normal and repressed conditions uncovered several hypersensitive compound hits. This work therefore demonstrates a streamlined process for proceeding from selection and validation of candidate antifungal targets to screening for specific inhibitors.

Antifungal Agents↗

PubMed2005

Loss and gain of chromosome 5 controls growth of Candida albicans on sorbose due to dispersed redundant negative regulators.

A reversible decrease or increase of Candida albicans chromosome copy number was found to be a prevalent means of survival of this opportunistic pathogen, under conditions that kill cells or inhibit their propagation. The utilization of a secondary carbon source, l-sorbose, by reversible loss of chromosome 5, serves as a model system. We have determined that an approximately 209-kbp portion of the right arm of chromosome 5 contains at least five spatially separated, functionally redundant regions that control utilization of l-sorbose. The regions bear no structural similarity among themselves, and four of them contain sequences that bear no similarity with any known sequence. We identified a regulatory gene in region A that encodes a helix-loop-helix protein. Most important, the multiple redundant regulators scattered along chromosome 5 explain, in a simple, elegant way, why the loss of the entire homologue is usually required for growth on sorbose. Thus, an entire chromosome acts as a single regulatory unit, a feature not previously considered. Our finding appears to be a paradigm for the control of other phenotypes in C. albicans that also depend on chromosome loss, thus implying that C. albicans genes are not distributed randomly among different chromosomes.

Amino Acid Sequence↗

PubMed2004

Role of the 14-3-3 protein in carbon metabolism of the pathogenic yeast Candida albicans.

We previously demonstrated that the pathogenic yeast Candida albicans effectively adapts to utilize L-sorbose (Sou+) by a novel mechanism based on the loss of one copy of chromosome 5, probably due to the reduction of copy number of a negative regulator located on this chromosome. We report here another negative regulator of L-sorbose utilization, an orthologue of the Saccharomyces cerevisiae BMH1 gene, which encodes the evolutionarily conserved protein 14-3-3. This essential gene is located on chromosome 1, does not have paralogues, and is supposedly a component of the regulatory network. Experiments involving disruption of one allele of BMH1 and overexpression of BMH1 revealed that BMH1 represses the transcription of SOU1, which is responsible for the utilization of L-sorbose. Although the exact mechanism of the interaction between BMH1 and SOU1 is not known, it is clear that the control is based on the ratio of gene copy number, and that BMH1 does not control the loss of chromosome 5, the major mechanism producing Sou+ mutants. We propose that function of BMH1 as a negative regulator of SOU1 contributes to a general cellular homeostasis. This is a first report on the role of the C. albicans essential gene BMH1 as a negative regulator of the utilization of secondary carbon source in yeast, which further substantiates the involvement of 14-3-3 proteins in diverse functions.

14-3-3 Proteins↗

PubMed2003

Nucleotide-dependent conformational changes in the sigma54-dependent activator DctD.

Activators of sigma(54)-RNA polymerase holoenzyme couple ATP hydrolysis to formation of an open promoter complex. DctD(Delta1-142), a truncated and constitutively active form of the sigma(54)-dependent activator DctD from Sinorhizobium meliloti, displayed an altered DNase I footprint at its binding site located upstream of the dctA promoter in the presence of ATP. The altered footprint was not observed for a mutant protein with a substitution at or near the putative arginine finger, a conserved arginine residue thought to contact the nucleotide. These data suggest that structural changes in DctD(Delta1-142) during ATP hydrolysis can be detected by alterations in the DNase I footprint of the protein and may be communicated by interactions between bound nucleotide and the arginine finger. In addition, kinetic data for changes in fluorescence energy transfer upon binding of 2'(3')-O-(N-methylanthraniloyl)-ATP (Mant-ATP) to DctD(Delta1-142) and DctD suggested that these proteins undergo multiple conformational changes following ATP binding.

Adenosine Triphosphate↗