Backbone and side chain dynamics of mutant calmodulin-peptide complexes

The mechanism of long-range coupling of allosteric sites in calcium-saturated calmodulin (CaM) has been explored by characterizing structural and dynamics effects of mutants of calmodulin in complex with a peptide corresponding to the smooth muscle myosin light chain kinase calmodulin-binding domain (smMLCKp). Four CaM mutants were examined: D95N and D58N, located in Ca2+-binding loops; and M124L and E84K, located in the target domain-binding site of CaM. Three of these mutants have altered allosteric coupling either between Ca2+-binding sites (D58N and D95N) or between the target- and Ca2+-binding sites (E84K). The structure and dynamics of the mutant calmodulins in complex with smMLCKp were characterized using solution NMR. Analysis of chemical shift perturbations was employed to detect largely structural perturbations. 15N and 2H relaxation was employed to detect perturbations of the dynamics of the backbone and methyl-bearing side chains of calmodulin. The least median squares method was found to be robust in the detection of perturbed sites. The main chain dynamics of calmodulin are found to be largely unresponsive to the mutations. Three mutants show significantly perturbed dynamics of methyl-bearing side chains. Despite the pseudosymmetric location of Ca2+-binding loop mutations D58N and D95N, the dynamic response of CaM is asymmetric, producing long-range perturbation in D58N and almost none in D95N. The mutations located at the target domain-binding site have quite different effects. For M124L, a local perturbation of the methyl dynamics is observed, while the E84K mutation produces a long-range propagation of dynamic perturbations along the target domain-binding site.     

Peptide YY and neuropeptide Y induce villin expression, reduce adhesion, and enhance migration in small intestinal cells through the regulation of CD63, matrix metalloproteinase-3, and Cdc42 activity

Peptide YY (PYY) and neuropeptide Y (NPY) are regulatory peptides synthesized in the intestine and brain, respectively, that modify physiological functions affecting nutrient assimilation and feeding behavior. Because PYY and NPY also alter the expression of intestine-specific differentiation marker proteins and the tetraspanin CD63, which is involved in cell adhesion, we investigated whether intestinal cell differentiation could be linked to mucosal cell adhesion and migration through these peptides. PYY and NPY significantly decreased cell adhesion and increased cell migration in a dose-dependent manner prior to cell confluency in our model system, non-tumorigenic small intestinal hBRIE 380i cells. Both peptides reduced CD63 expression and CD63-dependent cell adhesion. CD63 overexpression increased and antisense CD63 cDNA decreased intestinal cell adhesion. In parallel, both PYY and NPY increased expression of matrix metalloproteinase-3 (MMP-3) to a level sufficient to induce cell migration by activating the Rho GTPase Cdc42. The effects of both peptides on cell migration were blocked in cells constitutively overexpressing dominant-negative Cdc42. PYY and NPY also significantly induced the expression of the differentiation marker villin, which could be eliminated by an MMP inhibitor at a concentration that inhibits cell migration. Increased MMP-3 activity, which enhanced cell migration, also induced villin mRNA levels. Therefore, these data indicate that the alteration of adhesion and migration by PYY and NPY occurs in part by synchronous modulation of three proteins that are involved in extracellular matrix-basolateral membrane interactions, CD63, MMP-3 and Cdc42, and that PYY/NPY regulation of expression of mucosal proteins such as villin is linked to the process of cell migration and adhesion.     

Formylchromone derivatives as irreversible and selective inhibitors of human protein tyrosine phosphatase 1B. Kinetic and modeling studies

A series of formylchromone derivatives were synthesized as PTP1B inhibitors and some of them were potent against PTP1B with IC50 values as low as 1.0 microM. They exhibited remarkable selectivity for PTP1B over other human PTPases. Kinetic studies revealed that formylchromone derivatives are irreversible and active site-directed inhibitors. Molecular modeling study identified the orientation of the inhibitor bound at the active site of PTP1B.     

Structural chemoproteomics and drug discovery

Our laboratories have developed several technologies to accelerate drug discovery process on the basis of structural chemoproteomics. They include SPS technology for the efficient determination of protein structures, SCP technology for the rapid lead generation and SDF technology for the productive lead optimization. Using these technologies, we could determine many 3D structures of target proteins bound with biologically active chemicals including the structure of phosphodiesterase 5/Viagra complex and obtain highly potent compounds in animal models of obesity, diabetes, cancer and inflammation. In this paper, we will discuss concepts and applications of structural chemoproteomics for drug discovery.     

Alternatively spliced human genes by exon skipping--a database (ASHESdb)

Alternative splicing of mRNA allows many gene products with different functions to be produced from a single coding sequence. Exon skipping is the most commonly known alternative splicing mechanism. A comprehensive database of alternative splicing by exon skipping is made available for the human genome data. 1,229 human genes are identified to exhibit alternative splicing by exon skipping. Availability: http://sege.ntu.edu.sg/wester/ashes/.     

Biochemical characterization of RssA-RssB, a two-component signal transduction system regulating swarming behavior in Serratia marcescens

Our previous study had identified a pair of potential two-component signal transduction proteins, RssA-RssB, involved in the regulation of Serratia marcescens swarming. When mutated, both rssA and rssB mutants showed precocious swarming phenotypes on LB swarming agar, whereby swarming not only occurred at 37 degrees C but also initiated on a surface of higher agar concentration and more rapidly than did the parent strain at 30 degrees C. In this study, we further show that the predicted sensor kinase RssA and the response regulator RssB bear characteristics of components of the phosphorelay signaling system. In vitro phosphorylation and site-directed mutagenesis assays showed that phosphorylated RssA transfers the phosphate group to RssB and that histidine 248 and aspartate 51 are essential amino acid residues involved in the phosphotransfer reactions in RssA and RssB, respectively. Accordingly, while wild-type rssA could, the mutated rssA(H248A) in trans could not complement the precocious swarming phenotype of the rssA mutant. Although RssA-RssB regulates expressions of shlA and ygfF of S. marcescens (ygfF(Sm)), in vitro DNA-binding assays showed that the phosphorylated RssB did not bind directly to the promoter regions of these two genes but bound to its own rssB promoter. Subsequent assays located the RssB binding site within a 63-bp rssB promoter DNA region and confirmed a direct negative autoregulation of the RssA-RssB signaling pathway. These results suggest that when activated, RssA-RssB acts as a negative regulator for controlling the initiation of S. marcescens swarming.     

Denaturing gradient gel electrophoresis analysis of bacterial populations in 5-stage biological nutrient removal process with step feed system for wastewater treatment

Changes in the bacterial populations of a 5-stage biological nutrient removal (BNR) process, with a step feed system for wastewater treatment, were monitored by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA fragments. DGGE analysis indicated seasonal community changes were observed, however, community profiles of the total bacteria of each reactor showed only minor differences in the samples obtained from the same season. The number of major bands was higher in the summer samples, and decreased during the winter period, indicating that the microbial community structure became simpler at low temperatures. Since the nitrogen and phosphate removal efficiencies were highly maintained throughout the winter operation period, the bacteria which still remaining in the winter sample can be considered important, playing a key role in the present 5-stage BNR sludge. The prominent DGGE bands were excised, and sequenced to gain insight into the identities of the predominant bacterial populations present, and most were found to not be closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods for the quality control of wastewater treatment.