Publications by authors named "Wen-Chi Cheng"

12 Publications

  • Page 1 of 1

The distribution of cultivable oral anaerobic microbiota identified by MALDI-TOF MS in healthy subjects and in patients with periodontal disease.

J Pharm Biomed Anal 2021 Jan 22;192:113647. Epub 2020 Sep 22.

Institute of Bioinformatics and Structural Biology and Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan. Electronic address:

In this study, we aimed to identify the cultivatable oral anaerobic bacterial distribution in oral cavity by MALDI-TOF Biotyper. The bacterial distribution of three groups, including subjects with/without periodontal disease, two clusters of age (60 years as the cutoff), and before/after treatment, were investigated in this study. There were 38 participants recruited in this study, involving 18 subjects with moderate to severe periodontal-infected patients and 20 healthy controls. Total number of 126 bacterial species were identified by MALDI-TOF MS. The relative abundance of Streptococcus gordonii and Streptococcus intermedius in periodontal patients is higher than healthy controls indicating potential biomarkers for periodontal disease. Participants with periodontal disease were subdivided in to two clusters of age (60 years as the cutoff), 11 and 7 participants were age <60 years and>60 years, respectively. Meanwhile, the incidence of Streptococcus pneumoniae and Streptococcus oralis infection were higher in the subjects above 60 years old than below. Moreover, the bacterial distribution between pre-treatment and post-treatment was similar indicating that basic treatment without the ability to redistribute the microbiota. In summary, the cultivable oral anaerobic bacteria were identified by MALDI-TOF MS and the bacterial distribution shifting was shown to be associated with the progress of periodontal disease to aging and basic treatment. This study provided information for diagnosis and treatment guidelines for oral healthcare.
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http://dx.doi.org/10.1016/j.jpba.2020.113647DOI Listing
January 2021

Identification of hyperglycemia-associated microbiota alterations in saliva and gingival sulcus.

Arch Biochem Biophys 2020 03 23;682:108278. Epub 2020 Jan 23.

Institute of Bioinformatics and Structural Biology and Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan. Electronic address:

Oral microbes are a contributing factor to hyperglycemia by inducing an increase in insulin resistance resulting in uncontrolled blood glucose levels. However, the relationship between the distribution of oral flora and hyperglycemia is still controversial. Combining the power of MALDI-Biotyper with anaerobic bacterial culture, this study explores the correlation between anaerobic bacteria in the oral cavity and blood glucose levels. The results demonstrated that altered blood glucose levels contributed to a varied bacterial distribution in the oral cavity. Specifically, Veillonella spp. and Prevotella spp. were identified in a higher proportion in people with elevated blood glucose levels. Six bacterial species identified in this study (Prevotella melaninogenica, Campylobacter rectus, Streptococcus gordonii, Streptococcus mitis, Streptococcus salivarius, and Veillonella parvula) not only demonstrated a positive association with higher blood glucose levels, but also likely contribute to the development of the condition. The data demonstrated MALDI-TOF MS to be a simpler, faster, and more economical clinical identification tool that provides clarity and depth to the research on blood glucose and oral microbiota.
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http://dx.doi.org/10.1016/j.abb.2020.108278DOI Listing
March 2020

The Antimicrobial Peptides P-113Du and P-113Tri Function against Candida albicans.

Antimicrob Agents Chemother 2016 10 23;60(10):6369-73. Epub 2016 Sep 23.

Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan

Two antimicrobial P-113 peptide derivatives, P-113Du and P-113Tri, were investigated in this study. Notably, P-113Du and P-113Tri contained significant fractions of α-helix conformation and were less sensitive to high salt and low pH than P-113. Moreover, compared to P-113, these peptides exhibited increased antifungal activity against planktonic cells, biofilm cells, and clinical isolates of Candida albicans and non-albicans Candida spp. These results suggest that P-113Du and P-113Tri are promising candidates for development as novel antifungal agents.
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http://dx.doi.org/10.1128/AAC.00699-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5038333PMC
October 2016

Functional characterization of ECP-heparin interaction: a novel molecular model.

PLoS One 2013 11;8(12):e82585. Epub 2013 Dec 11.

Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China ; Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.

Human eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are two ribonuclease A (RNaseA) family members secreted by activated eosinophils. They share conserved catalytic triad and similar three dimensional structures. ECP and EDN are heparin binding proteins with diverse biological functions. We predicted a novel molecular model for ECP binding of heparin hexasaccharide (Hep6), [GlcNS(6S)-IdoA(2S)]3, and residues Gln(40), His(64) and Arg(105) were indicated as major contributions for the interaction. Interestingly, Gln(40) and His(64) on ECP formed a clamp-like structure to stabilize Hep6 in our model, which was not observed in the corresponding residues on EDN. To validate our prediction, mutant ECPs including ECP Q40A, H64A, R105A, and double mutant ECP Q40A/H64A were generated, and their binding affinity for heparins were measured by isothermal titration calorimetry (ITC). Weaker binding of ECP Q40A/H64A of all heparin variants suggested that Gln(40)-His(64) clamp contributed to ECP-heparin interaction significantly. Our in silico and in vitro data together demonstrate that ECP uses not only major heparin binding region but also use other surrounding residues to interact with heparin. Such correlation in sequence, structure, and function is a unique feature of only higher primate ECP, but not EDN.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0082585PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859622PMC
October 2014

JMJD5 regulates PKM2 nuclear translocation and reprograms HIF-1α-mediated glucose metabolism.

Proc Natl Acad Sci U S A 2014 Jan 16;111(1):279-84. Epub 2013 Dec 16.

Institute of Molecular and Cellular Biology and Department of Life Sciences, National Tsing Hua University, Hsinchu 30013, Taiwan.

JMJD5, a Jumonji C domain-containing dioxygenase, is important for embryonic development and cancer growth. Here, we show that JMJD5 is up-regulated by hypoxia and is crucial for hypoxia-induced cell proliferation. JMJD5 interacts directly with pyruvate kinase muscle isozyme (PKM)2 to modulate metabolic flux in cancer cells. The JMJD5-PKM2 interaction resides at the intersubunit interface region of PKM2, which hinders PKM2 tetramerization and blocks pyruvate kinase activity. This interaction also influences translocation of PKM2 into the nucleus and promotes hypoxia-inducible factor (HIF)-1α-mediated transactivation. JMJD5 knockdown inhibits the transcription of the PKM2-HIF-1α target genes involved in glucose metabolism, resulting in a reduction of glucose uptake and lactate secretion in cancer cells. JMJD5, along with PKM2 and HIF-1α, is recruited to the hypoxia response element site in the lactate dehydrogenase A and PKM2 loci and mediates the recruitment of the latter two proteins. Our data uncover a mechanism whereby PKM2 can be regulated by factor-binding-induced homo/heterooligomeric restructuring, paving the way to cell metabolic reprogram.
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http://dx.doi.org/10.1073/pnas.1311249111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890888PMC
January 2014

Pathway-based screening strategy for multitarget inhibitors of diverse proteins in metabolic pathways.

PLoS Comput Biol 2013 4;9(7):e1003127. Epub 2013 Jul 4.

Institute of Bioinformatics and Systems Biology, National Chiao Tung University, Hsinchu, Taiwan.

Many virtual screening methods have been developed for identifying single-target inhibitors based on the strategy of "one-disease, one-target, one-drug". The hit rates of these methods are often low because they cannot capture the features that play key roles in the biological functions of the target protein. Furthermore, single-target inhibitors are often susceptible to drug resistance and are ineffective for complex diseases such as cancers. Therefore, a new strategy is required for enriching the hit rate and identifying multitarget inhibitors. To address these issues, we propose the pathway-based screening strategy (called PathSiMMap) to derive binding mechanisms for increasing the hit rate and discovering multitarget inhibitors using site-moiety maps. This strategy simultaneously screens multiple target proteins in the same pathway; these proteins bind intermediates with common substructures. These proteins possess similar conserved binding environments (pathway anchors) when the product of one protein is the substrate of the next protein in the pathway despite their low sequence identity and structure similarity. We successfully discovered two multitarget inhibitors with IC50 of <10 µM for shikimate dehydrogenase and shikimate kinase in the shikimate pathway of Helicobacter pylori. Furthermore, we found two selective inhibitors (IC50 of <10 µM) for shikimate dehydrogenase using the specific anchors derived by our method. Our experimental results reveal that this strategy can enhance the hit rates and the pathway anchors are highly conserved and important for biological functions. We believe that our strategy provides a great value for elucidating protein binding mechanisms and discovering multitarget inhibitors.
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http://dx.doi.org/10.1371/journal.pcbi.1003127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3701698PMC
February 2014

Structures of Helicobacter pylori shikimate kinase reveal a selective inhibitor-induced-fit mechanism.

PLoS One 2012 16;7(3):e33481. Epub 2012 Mar 16.

Institute of Molecular and Cellular Biology and Department of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan.

Shikimate kinase (SK), which catalyzes the specific phosphorylation of the 3-hydroxyl group of shikimic acid in the presence of ATP, is the enzyme in the fifth step of the shikimate pathway for biosynthesis of aromatic amino acids. This pathway is present in bacteria, fungi, and plants but absent in mammals and therefore represents an attractive target pathway for the development of new antimicrobial agents, herbicides, and antiparasitic agents. Here we investigated the detailed structure-activity relationship of SK from Helicobacter pylori (HpSK). Site-directed mutagenesis and isothermal titration calorimetry studies revealed critical conserved residues (D33, F48, R57, R116, and R132) that interact with shikimate and are therefore involved in catalysis. Crystal structures of HpSK·SO(4), R57A, and HpSK•shikimate-3-phosphate • ADP show a characteristic three-layer architecture and a conformationally elastic region consisting of F48, R57, R116, and R132, occupied by shikimate. The structure of the inhibitor complex, E114A • 162535, was also determined, which revealed a dramatic shift in the elastic LID region and resulted in conformational locking into a distinctive form. These results reveal considerable insight into the active-site chemistry of SKs and a selective inhibitor-induced-fit mechanism.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033481PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306394PMC
August 2012

Core site-moiety maps reveal inhibitors and binding mechanisms of orthologous proteins by screening compound libraries.

PLoS One 2012 29;7(2):e32142. Epub 2012 Feb 29.

Institute of Bioinformatics and Systems Biology, National Chiao Tung University, Hsinchu, Taiwan.

Members of protein families often share conserved structural subsites for interaction with chemically similar moieties despite low sequence identity. We propose a core site-moiety map of multiple proteins (called CoreSiMMap) to discover inhibitors and mechanisms by profiling subsite-moiety interactions of immense screening compounds. The consensus anchor, the subsite-moiety interactions with statistical significance, of a CoreSiMMap can be regarded as a "hot spot" that represents the conserved binding environments involved in biological functions. Here, we derive the CoreSiMMap with six consensus anchors and identify six inhibitors (IC(50)<8.0 µM) of shikimate kinases (SKs) of Mycobacterium tuberculosis and Helicobacter pylori from the NCI database (236,962 compounds). Studies of site-directed mutagenesis and analogues reveal that these conserved interacting residues and moieties contribute to pocket-moiety interaction spots and biological functions. These results reveal that our multi-target screening strategy and the CoreSiMMap can increase the accuracy of screening in the identification of novel inhibitors and subsite-moiety environments for elucidating the binding mechanisms of targets.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0032142PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3290551PMC
July 2012

Helicobacter pylori cholesteryl glucosides interfere with host membrane phase and affect type IV secretion system function during infection in AGS cells.

Mol Microbiol 2012 Jan 22;83(1):67-84. Epub 2011 Nov 22.

Institute of Molecular and Cellular Biology and Department of Life Sciences, National Tsing-Hua University, Hsinchu, 30013, Taiwan.

Helicobacter pylori infection is an aetiological cause of gastric disorders worldwide. H. pylori has been shown to assimilate and convert host cholesterol into cholesteryl glucosides (CGs) by cholesterol-α-glucosyltransferase encoded by capJ. Here, we show that CapJ-deficient (ΔcapJ) H. pylori resulted in greatly reduced type IV secretion system (TFSS)-associated activities, including the hummingbird phenotype of AGS cells, IL-8 production, CagA translocation/phosphorylation and CagA-mediated signalling events. Complementation of the ΔcapJ mutation with wild type cagJ or by adding CGs-containing lysates or exogenous fluorophore-tagged CGs reversed the mutant phenotypes. We also show that the wild-type but not ΔcapJ H. pylori recruited raft-associated components to sites of bacterial attachment. Fluorescence recovery after photobleaching (FRAP) analysis of AGS cells treated with fluorescence-tagged cholesterol/CGs revealed that there was a higher proportion of CGs associated with immobile fractions. CGs-associated membranes were also more resistant to a cold detergent extraction. Thus, we propose that CGs synthesized by H. pylori around host-pathogen contact sites partition in detergent-resistant membranes (DRMs), alters lateral-phase segregation in membrane and reorganizes membrane architecture. These processes together promote the formation of a functional TFSS and H. pylori infection.
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http://dx.doi.org/10.1111/j.1365-2958.2011.07910.xDOI Listing
January 2012

Structure, mechanistic action, and essential residues of a GH-64 enzyme, laminaripentaose-producing beta-1,3-glucanase.

J Biol Chem 2009 Sep 29;284(39):26708-15. Epub 2009 Jul 29.

Department of Life Science, Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 300, Taiwan.

Laminaripentaose-producing beta-1,3-glucanase (LPHase), a member of glycoside hydrolase family 64, cleaves a long-chain polysaccharide beta-1,3-glucan into specific pentasaccharide oligomers. The crystal structure of LPHase from Streptomyces matensis DIC-108 was solved to 1.62 A resolution using multiple-wavelength anomalous dispersion methods. The LPHase structure reveals a novel crescent-like fold; it consists of a barrel domain and a mixed (alpha/beta) domain, forming a wide-open groove between the two domains. The liganded crystal structure was also solved to 1.80 A, showing limited conformational changes. Within the wide groove, a laminaritetraose molecule is found to sit in an electronegatively charged central region and is proximal to several conserved residues including two carboxylates (Glu(154) and Asp(170)) and four other sugar-binding residues (Thr(156), Asn(158), Trp(163), and Thr(167)). Molecular modeling using a laminarihexaose as a substrate suggests roles for Glu(154) and Asp(170) as acid and base catalysts, respectively, whereas the side chains of Thr(156), Asn(158), and Trp(163) demarcate subsite +5. Site-directed mutagenesis of Glu(154) and Asp(170) confirms that both carboxylates are essential for catalysis. Together, our results suggest that LPHase uses a direct displacement mechanism involving Glu(154) and Asp(170) to cleave a beta-1,3-glucan into specific alpha-pentasaccharide oligomers.
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http://dx.doi.org/10.1074/jbc.M109.010983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785358PMC
September 2009

Structure-based inhibitor discovery of Helicobacter pylori dehydroquinate synthase.

Biochem Biophys Res Commun 2008 Aug 27;373(1):1-7. Epub 2008 May 27.

Institute of Molecular and Cellular Biology and Department of Life Sciences, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsinchu 300, Taiwan.

Dehydroquinate synthase (DHQS) is a nicotinamide adenine dinucleotide (NAD)-dependent enzyme that converts 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) into 3-dehydroquinate (DHQ). Since it catalyzes the second key step in the shikimate pathway, which is crucial for the aromatic amino acid metabolism in bacteria, fungi, and plants, but not in mammals, DHQS is a potential target for new antimicrobial agents, anti-parasitic agents and herbicides. The crystal structure of Helicobacter pylori DHQS (HpDHQS) complexed with NAD has been determined at 2.4-A resolution and was found to possess an N-terminal Rossmann-fold domain and a C-terminal alpha-helical domain. Structural comparison reveals that the binary complex adopts an open-state conformation and shares conserved residues in the binding pocket. Virtual docking of compounds into the active site of the HpDHQS structure using the GOLD docking program led to the identification of several inhibitors. The most active compound had an IC(50) value of 61 microM, which may serve as a lead for potent inhibitors.
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http://dx.doi.org/10.1016/j.bbrc.2008.05.070DOI Listing
August 2008

Structural basis for shikimate-binding specificity of Helicobacter pylori shikimate kinase.

J Bacteriol 2005 Dec;187(23):8156-63

Institute of Molecular and Cellular Biology and Department of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan.

Shikimate kinase (EC 2.7.1.71) catalyzes the specific phosphorylation of the 3-hydroxyl group of shikimic acid in the presence of ATP. As the fifth key step in the shikimate pathway for aromatic amino acid biosynthesis in bacteria, fungi, and plants, but not mammals, shikimate kinase represents an attractive target for the development of new antimicrobial agents, herbicides, and antiparasitic agents. Here, we report the 1.8-Angstroms crystal structure of Helicobacter pylori shikimate kinase (HpSK). The crystal structure shows a three-layer alpha/beta fold consisting of a central sheet of five parallel beta-strands flanked by seven alpha-helices. An HpSK-shikimate-PO(4) complex was also determined and refined to 2.3 Angstroms, revealing induced-fit movement from an open to a closed form on substrate binding. Shikimate is located above a short 3(10) helix formed by a strictly conserved motif (GGGXV) after beta(3). Moreover, several highly conserved charged residues including Asp33 (in a conserved DT/SD motif), Arg57, and Arg132 (interacting with shikimate) are identified, guiding the development of novel inhibitors of shikimate kinase.
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http://dx.doi.org/10.1128/JB.187.23.8156-8163.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1291267PMC
December 2005