Identification of halohydrin dehalogenase mutants that resist COBE inhibition

The biocatalytic cascade conversion of ethyl 4-chloroacetoacetate (COBE) to ethyl (R)-4-cyano-3-hydroxybutyrate ((R)-HN) for the preparation of atorvastatin represents significant economic and environmental benefits, and is catalyzed by alcohol dehydrogenase and halohydrin dehalogenase (HHDH). However, as the activity of HHDH is inhibited by COBE, the cascade reaction is an inefficient one-pot reaction. In this study, substrate inhibition kinetics analysis was performed and the inhibition by COBE was found to be competitive reversible inhibition. Molecular simulation analysis was used to determine the inhibition mechanism by COBE. The results showed that COBE bound to the active center of HHDH via the formation of hydrogen bonds with the OH groups of S132 and Y145. Site saturation mutagenesis of residues around the active site and at the entrance of the access tunnel was performed, and two target mutant residues were identified, F136 and W249. Small focused mutagenesis on these two residues was performed and the F136V/W249F mutant was successfully found to relieve the activity inhibition of HHDH by COBE. The half inhibiting concentration of mutant F136V/W249F was found to be 20-fold higher than wild-type HHDH. The efficiency of the multi-enzymatic one-pot system for the synthesis of (R)-HN from COBE using mutant F136V/W249F was improved significantly.     

Proteomic Analysis of Cellular Response Induced by Multi-Walled Carbon Nanotubes Exposure in A549 Cells

The wide application of multi-walled carbon nanotubes (MWCNT) has raised serious concerns about their safety on human health and the environment. However, the potential harmful effects of MWCNT remain unclear and contradictory. To clarify the potentially toxic effects of MWCNT and to elucidate the associated underlying mechanisms, the effects of MWCNT on human lung adenocarcinoma A549 cells were examined at both the cellular and the protein level. Cytotoxicity and genotoxicity were examined, followed by a proteomic analysis (2-DE coupled with LC-MS/MS) of the cellular response to MWCNT. Our results demonstrate that MWCNT induces cytotoxicity in A549 cells only at relatively high concentrations and longer exposure time. Within a relatively low dosage range (30 µg/ml) and short time period (24 h), MWCNT treatment does not induce significant cytotoxicity, cell cycle changes, apoptosis, or DNA damage. However, at these low doses and times, MWCNT treatment causes significant changes in protein expression. A total of 106 proteins show altered expression at various time points and dosages, and of these, 52 proteins were further identified by MS. Identified proteins are involved in several cellular processes including proliferation, stress, and cellular skeleton organization. In particular, MWCNT treatment causes increases in actin expression. This increase has the potential to contribute to increased migration capacity and may be mediated by reactive oxygen species (ROS).     

Mass spectrometry-based N-glycoproteomics for cancer biomarker discovery

Glycosylation is estimated to be found in over 50% of human proteins. Aberrant protein glycosylation and alteration of glycans are closely related to many diseases. More than half of the cancer biomarkers are glycosylated-proteins, and specific glycoforms of glycosylated-proteins may serve as biomarkers for either the early detection of disease or the evaluation of therapeutic efficacy for treatment of diseases. Glycoproteomics, therefore, becomes an emerging field that can make unique contributions to the discovery of biomarkers of cancers. The recent advances in mass spectrometry (MS)-based glycoproteomics, which can analyze thousands of glycosylated-proteins in a single experiment, have shown great promise for this purpose. Herein, we described the MS-based strategies that are available for glycoproteomics, and discussed the sensitivity and high throughput in both qualitative and quantitative manners. The discovery of glycosylated-proteins as biomarkers in some representative diseases by employing glycoproteomics was also summarized.     

Estimating the Prevalence of Potential Enteropathogenic Escherichia coli and Intimin Gene Diversity in a Human Community by Monitoring Sanitary Sewage

Presently, the understanding of bacterial enteric diseases in the community and their virulence factors relies almost exclusively on clinical disease reporting and examination of clinical pathogen isolates. This study aimed to investigate the feasibility of an alternative approach that monitors potential enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) prevalence and intimin gene (eae) diversity in a community by directly quantifying and characterizing target virulence genes in the sanitary sewage. The quantitative PCR (qPCR) quantification of the eae, stx₁, and stx₂ genes in sanitary sewage samples collected over a 13-month period detected eae in all 13 monthly sewage samples at significantly higher abundance (93 to 7,240 calibrator cell equivalents [CCE]/100 ml) than stx₁ and stx₂, which were detected sporadically. The prevalence level of potential EPEC in the sanitary sewage was estimated by calculating the ratio of eae to uidA, which averaged 1.0% (σ = 0.4%) over the 13-month period. Cloning and sequencing of the eae gene directly from the sewage samples covered the majority of the eae diversity in the sewage and detected 17 unique eae alleles belonging to 14 subtypes. Among them, eae-β2 was identified to be the most prevalent subtype in the sewage, with the highest detection frequency in the clone libraries (41.2%) and within the different sampling months (85.7%). Additionally, sewage and environmental E. coli isolates were also obtained and used to determine the detection frequencies of the virulence genes as well as eae genetic diversity for comparison.     

Mitochondria: signaling with phosphatidic acid

Mitochondria, once viewed as functioning relatively autonomously in the cell, have increasingly been recognized to be involved in numerous signaling networks that impact on a wide range of cell biological processes. In addition to the many types of proteins that mediate these pathways, the importance of signaling functions regulated via lipids and lipid second messengers generated on the mitochondrial surface is also becoming well appreciated. We focus here on phosphatidic acid, a lipid second messenger produced via several different pathways that can in turn stimulate the formation of multiple other bioactive lipids. Taken together, fascinating roles for phosphatidic acid and the connected lipids in mitochondrial function and interaction with other organelles are being uncovered. These pathways present new opportunities for the development of therapeutic approaches relevant to reproduction, metabolism, and neurodegenerative disease.     

A novel and efficient assay for identification and quantification of Acidithiobacillus ferrooxidans in bioleaching samples

Acidithiobacillus ferrooxidans is a Gram-negative, acidophilic, and chemolithotrophic bacterium that is active in bioleaching. The leaching efficacy is directly influenced by the biomass changes of this specie in bioleaching microbial community. In order to perform a simple and sensitive assay on A. ferrooxidans from mixed strains in this process, a novel assay was developed based on sandwich hybridization assay with the aid of S1 nuclease treatment and fluorescent labeling. In the work, a designed DNA probe complementary to the conservative region of its 16S rRNA was synthesized, which showed high accuracy for distinguishing homologous species with the exclusion of even-only two base pairs difference. The specificity of this assay was verified in different systems with mixed strains, and the quantitative result was proved by comparison of microscopic cell counting. The detection sensitivity was about 8 × 10(2) cells/ml and the inter-assay coefficient of variation of three independent assays was from 3.8 to 7.7 %, respectively. In addition, the cycle of assay was about 3-4 h when the cost estimated was less than $0.5 per sample. This assay method might be applied for identifying and monitoring any kind of bacterial strain from a mixed microbial flora in bioleaching or other areas.     

Development of α1,6-fucosyltransferase inhibitors through the diversity-oriented syntheses of GDP-fucose mimics using the coupling between alkyne and sulfonyl azide

We developed α1,6-fucosyltransferase (FUT8) inhibitors through a diversity-oriented synthesis. The coupling reaction between the fucose unit containing alkyne and the guanine unit containing sulfonyl azide under various conditions afforded a series of Guanosine 5′-diphospho-β-l-fucose (GDP-fucose) analogs. The synthesized compounds displayed FUT8 inhibition activity. A docking study revealed that the binding mode of the inhibitor synthesized with FUT8 was similar to that of GDP-fucose.     

Improving the physical realism and structural accuracy of protein models by a two-step atomic-level energy minimization

Most protein structural prediction algorithms assemble structures as reduced models that represent amino acids by a reduced number of atoms to speed up the conformational search. Building accurate full-atom models from these reduced models is a necessary step toward a detailed function analysis. However, it is difficult to ensure that the atomic models retain the desired global topology while maintaining a sound local atomic geometry because the reduced models often have unphysical local distortions. To address this issue, we developed a new program, called ModRefiner, to construct and refine protein structures from Cα traces based on a two-step, atomic-level energy minimization. The main-chain structures are first constructed from initial Cα traces and the side-chain rotamers are then refined together with the backbone atoms with the use of a composite physics- and knowledge-based force field. We tested the method by performing an atomic structure refinement of 261 proteins with the initial models constructed from both ab initio and template-based structure assemblies. Compared with other state-of-art programs, ModRefiner shows improvements in both global and local structures, which have more accurate side-chain positions, better hydrogen-bonding networks, and fewer atomic overlaps. ModRefiner is freely available at http://zhanglab.ccmb.med.umich.edu/ModRefiner.