Publications by authors named "Ping-Chiang Lyu"

119 Publications

Imperatorin Interferes with LPS Binding to the TLR4 Co-Receptor and Activates the Nrf2 Antioxidative Pathway in RAW264.7 Murine Macrophage Cells.

Antioxidants (Basel) 2021 Feb 27;10(3). Epub 2021 Feb 27.

Department of Pharmacology, China Medical University, Taichung 404333, Taiwan.

Imperatorin (IMP) could downregulate several inflammatory transcription factor signaling pathways. Some studies have pointed out that IMP could interfere with toll-like receptor 4 (TLR4) signaling. This study evaluates how IMP interferes with the TLR4 co-receptors signaling through the protein-ligand docking model, Western blotting, immunofluorescence (IF), and atomic force microscopy (AFM) assays in lipopolysaccharide (LPS) stimulated macrophage-like RAW264.7 cells in vitro. The results of the protein-ligand docking demonstrate that IMP interferes with LPS binding to the LPS-binding protein (LBP), the cluster of differentiation 14 (CD14), and the toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD-2) co-receptors in LPS-stimulated RAW264.7 cells. Compared with TLR4 antagonist CLI-095 or dexamethasone, IMP could suppress the protein expressions of LBP, CD14, and TLR4/MD-2 in LPS-stimulated cells. Furthermore, the three-dimensional (3D) image assay of the AFM showed IMP could prevent the LPS-induced morphological change in RAW264.7 cells. Additionally, IMP could activate the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, and it increased the antioxidative protein expression of heme oxygenase-1 (HO-1), superoxidase dismutase (SOD), and catalase (CAT). Our results are the first to reveal that the anti-inflammatory effect of IMP interferes with LPS binding to TLR4 co-receptor signaling and activates the antioxidative Nrf2 signaling pathway.
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http://dx.doi.org/10.3390/antiox10030362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997471PMC
February 2021

Cafestol Inhibits High-Glucose-Induced Cardiac Fibrosis in Cardiac Fibroblasts and Type 1-Like Diabetic Rats.

Evid Based Complement Alternat Med 2020 31;2020:4503747. Epub 2020 Dec 31.

School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan.

Diabetes is associated with the development of myocardial fibrosis, which is related to various cardiac diseases. Cafestol, one of the active ingredients in coffee, has been reported to exert biological effects. However, whether cafestol can ameliorate diabetes-induced cardiac fibrosis remains unknown. The aim of this study was to evaluate the effects of cafestol on cardiac fibrosis in high-glucose-treated cardiac fibroblasts and streptozocin- (STZ-) induced diabetic rats. Rat cardiac fibroblasts were cultured in high-glucose (25 mM) media in the absence or presence of cafestol, and the changes in collagen synthesis, transforming growth factor-1 (TGF-1) production, and related signaling molecules were assessed on the basis of H-proline incorporation, enzyme-linked immunosorbent assay, and western blotting. Cardiac fibroblasts exposed to high-glucose conditions exhibited increased collagen synthesis, TGF-1 production, and Smad2/3 phosphorylation, and these effects were mitigated by cafestol treatment. Furthermore, cafestol increased the translocation of nuclear factor erythroid 2-related factor 2 and increased the expression of heme oxygenase-1. The results of molecular docking analysis suggested a selective interaction of cafestol with Kelch-like ECH-associated protein 1. The rats with untreated STZ-induced diabetes exhibited considerable collagen accumulation, which was ameliorated by cafestol. Moreover, activities of catalase, superoxide dismutase, general matrix metalloproteinase, and reduced glutathione concentration were upregulated, whereas malondialdehyde level was downregulated by treatment with cafestol in rats with cardiac fibrosis. These findings highlight the effects of cafestol, which may be useful in treating diabetes-related cardiac fibrosis.
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http://dx.doi.org/10.1155/2020/4503747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790572PMC
December 2020

Structural Insights into the Active Site Formation of DUSP22 in N-loop-containing Protein Tyrosine Phosphatases.

Int J Mol Sci 2020 Oct 12;21(20). Epub 2020 Oct 12.

Institute of Bioinformatics and Structural Biology, Department of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan.

Cysteine-based protein tyrosine phosphatases (Cys-based PTPs) perform dephosphorylation to regulate signaling pathways in cellular responses. The hydrogen bonding network in their active site plays an important conformational role and supports the phosphatase activity. Nearly half of dual-specificity phosphatases (DUSPs) use three conserved residues, including aspartate in the D-loop, serine in the P-loop, and asparagine in the N-loop, to form the hydrogen bonding network, the D-, P-, N-triloop interaction (DPN-triloop interaction). In this study, DUSP22 is used to investigate the importance of the DPN-triloop interaction in active site formation. Alanine mutations and somatic mutations of the conserved residues, D57, S93, and N128 substantially decrease catalytic efficiency (/) by more than 10-fold. Structural studies by NMR and crystallography reveal that each residue can perturb the three loops and induce conformational changes, indicating that the hydrogen bonding network aligns the residues in the correct positions for substrate interaction and catalysis. Studying the DPN-triloop interaction reveals the mechanism maintaining phosphatase activity in N-loop-containing PTPs and provides a foundation for further investigation of active site formation in different members of this protein class.
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http://dx.doi.org/10.3390/ijms21207515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589817PMC
October 2020

An essential role of acetyl coenzyme A in the catalytic cycle of insect arylalkylamine N-acetyltransferase.

Commun Biol 2020 08 14;3(1):441. Epub 2020 Aug 14.

Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, 30013, Taiwan.

Acetyl coenzyme A (Ac-CoA)-dependent N-acetylation is performed by arylalkylamine N-acetyltransferase (AANAT) and is important in many biofunctions. AANAT catalyzes N-acetylation through an ordered sequential mechanism in which cofactor (Ac-CoA) binds first, with substrate binding afterward. No ternary structure containing AANAT, cofactor, and substrate was determined, meaning the details of substrate binding and product release remain unclear. Here, two ternary complexes of dopamine N-acetyltransferase (Dat) before and after N-acetylation were solved at 1.28 Å and 1.36 Å resolution, respectively. Combined with the structures of Dat in apo form and Ac-CoA bound form, we addressed each stage in the catalytic cycle. Isothermal titration calorimetry (ITC), crystallography, and nuclear magnetic resonance spectroscopy (NMR) were utilized to analyze the product release. Our data revealed that Ac-CoA regulates the conformational properties of Dat to form the catalytic site and substrate binding pocket, while the release of products is facilitated by the binding of new Ac-CoA.
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http://dx.doi.org/10.1038/s42003-020-01177-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7427786PMC
August 2020

Antimicrobial Peptide TP4 Targets Mitochondrial Adenine Nucleotide Translocator 2.

Mar Drugs 2020 Aug 9;18(8). Epub 2020 Aug 9.

Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, 23-10 Dahuen Rd., Jiaushi, Ilan 262, Taiwan.

Tilapia piscidin (TP) 4 is an antimicrobial peptide derived from Nile tilapia (), which shows broad-spectrum antibacterial activity and excellent cancer-killing ability in vitro and in vivo. Like many other antimicrobial peptides, TP4 treatment causes mitochondrial toxicity in cancer cells. However, the molecular mechanisms underlying TP4 targeting of mitochondria remain unclear. In this study, we used a pull-down assay on A549 cell lysates combined with LC-MS/MS to discover that TP4 targets adenine nucleotide translocator (ANT) 2, a protein essential for adenine nucleotide exchange across the inner membrane. We further showed that TP4 accumulates in mitochondria and colocalizes with ANT2. Moreover, molecular docking studies showed that the interaction requires Phe1, Ile2, His3, His4, Ser11, Lys14, His17, Arg21, Arg24 and Arg25 residues in TP4 and key residues within the cavity of ANT2. These findings suggest a mechanism by which TP4 may induce mitochondrial dysfunction to disrupt cellular energy metabolism.
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http://dx.doi.org/10.3390/md18080417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459631PMC
August 2020

MAP4K3/GLK Promotes Lung Cancer Metastasis by Phosphorylating and Activating IQGAP1.

Cancer Res 2019 10 20;79(19):4978-4993. Epub 2019 Aug 20.

Immunology Research Center, National Health Research Institutes, Zhunan, Taiwan.

Overexpression of the serine/threonine kinase GLK/MAP4K3 in human lung cancer is associated with poor prognosis and recurrence, however, the role of GLK in cancer recurrence remains unclear. Here, we report that transgenic GLK promotes tumor metastasis and cell migration through the scaffold protein IQ motif-containing GTPase-activating protein 1(IQGAP1). GLK transgenic mice displayed enhanced distant metastasis. IQGAP1 was identified as a GLK-interacting protein; two proline-rich regions of GLK and the WW domain of IQGAP1 mediated this interaction. GLK and IQGAP1 colocalized at the leading edge including filopodia and lamellipodia of migrating cells. GLK directly phosphorylated IQGAP1 at Ser-480 enhancing Cdc42 activation and subsequent cell migration. GLK-induced cell migration and lung cancer metastasis were abolished by IQGAP1 depletion. Consistently, human NSCLC patient tissues displayed increased phospho-IQGAP1, which correlated with poor survival. Collectively, GLK promotes lung cancer metastasis by binding to, phosphorylating, and activating IQGAP1. SIGNIFICANCE: These findings show the critical role of the GLK-IQGAP cascade in cell migration and tumor metastasis, suggesting it as a potential biomarker and therapeutic target for lung cancer recurrence.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-1402DOI Listing
October 2019

The ligand-mediated affinity of brain-type fatty acid-binding protein for membranes determines the directionality of lipophilic cargo transport.

Biochim Biophys Acta Mol Cell Biol Lipids 2019 12 9;1864(12):158506. Epub 2019 Aug 9.

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

The intracellular transport of lipophilic cargoes is a highly dynamic process. In eukaryotic cells, the uptake and release of long-chain fatty acids (LCFAs) are executed by fatty-acid binding proteins. However, how these carriers control the directionality of cargo trafficking remains unclear. Here, we revealed that the unliganded archetypal Drosophila brain-type fatty acid-binding protein (dFABP) possesses a stronger binding affinity than its liganded counterpart for empty nanodiscs (ND). Titrating unliganded dFABP and nanodiscs with LCFAs rescued the broadening of FABP cross-peak intensities in HSQC spectra from a weakened protein-membrane interaction. Two out of the 3 strongest LCFA contacting residues in dFABP identified by NMR HSQC chemical shift perturbation (CSP) are also part of the 30 ND-contacting residues (out of the total 130 residues in dFABP), revealed by attenuated TROSY signal in the presence of lipid ND to apo-like dFABP. Our crystallographic temperature factor data suggest enhanced αII helix dynamics upon LCFA binding, compensating for the entropic loss in the βC-D/βE-F loops. The aliphatic tail of bound LCFA impedes the charge-charge interaction between dFABP and the head groups of the membrane, and dFABP is prone to dissociate from the membrane upon ligand binding. We therefore conclude that lipophilic ligands participate directly in the control of the functionally required membrane association and dissociation of FABPs.
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http://dx.doi.org/10.1016/j.bbalip.2019.08.002DOI Listing
December 2019

CoMutPlotter: a web tool for visual summary of mutations in cancer cohorts.

BMC Med Genomics 2019 07 11;12(Suppl 5):99. Epub 2019 Jul 11.

Department of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan.

Background: CoMut plot is widely used in cancer research publications as a visual summary of mutational landscapes in cancer cohorts. This summary plot can inspect gene mutation rate and sample mutation burden with their relevant clinical details, which is a common first step for analyzing the recurrence and co-occurrence of gene mutations across samples. The cBioPortal and iCoMut are two web-based tools that allow users to create intricate visualizations from pre-loaded TCGA and ICGC data. For custom data analysis, only limited command-line packages are available now, making the production of CoMut plots difficult to achieve, especially for researchers without advanced bioinformatics skills. To address the needs for custom data and TCGA/ICGC data comparison, we have created CoMutPlotter, a web-based tool for the production of publication-quality graphs in an easy-of-use and automatic manner.

Results: We introduce a web-based tool named CoMutPlotter to lower the barriers between complex cancer genomic data and researchers, providing intuitive access to mutational profiles from TCGA/ICGC projects as well as custom cohort studies. A wide variety of file formats are supported by CoMutPlotter to translate cancer mutation profiles into biological insights and clinical applications, which include Mutation Annotation Format (MAF), Tab-separated values (TSV) and Variant Call Format (VCF) files.

Conclusions: In summary, CoMutPlotter is the first tool of its kind that supports VCF file, the most widely used file format, as its input material. CoMutPlotter also provides the most-wanted function for comparing mutation patterns between custom cohort and TCGA/ICGC project. Contributions of COSMIC mutational signatures in individual samples are also included in the summary plot, which is a unique feature of our tool. CoMutPlotter is freely available at http://tardis.cgu.edu.tw/comutplotter .
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http://dx.doi.org/10.1186/s12920-019-0510-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6624176PMC
July 2019

Introduction to the Taiwan Biophysical Society.

Authors:
Ping-Chiang Lyu

Biophys Rev 2019 Jun 22;11(3):263. Epub 2019 May 22.

Department of Medical Sciences, National Tsing Hua University, Hsinchu, Taiwan.

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http://dx.doi.org/10.1007/s12551-019-00530-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557956PMC
June 2019

Untying a Knotted SPOUT RNA Methyltransferase by Circular Permutation Results in a Domain-Swapped Dimer.

Structure 2019 08 16;27(8):1224-1233.e4. Epub 2019 May 16.

Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan; Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan. Electronic address:

YbeA from E. coli is a trefoil-knotted SpoU-TrmD (SPOUT) RNA methyltransferase. While its knotted motif plays a key functional role, it is unclear how the knotted topology emerged from evolution. Here, we reverse-engineered an unknotted circular permutant (CP) of YbeA by introducing a new opening at the knotting loop. The resulting CP folded into an unexpected domain-swapped dimer. Untying the knotted loop abrogated its function, perturbed its folding stability and kinetics, and induced allosteric dynamic changes. We speculated that the knotted loop of YbeA is under tension to keep the cofactor in a high-energy configuration while keeping the threading C-terminal helix being knotted. Circular permutation released the mechanical strain thereby allowing the spring-loaded threading helix to flip, to relax, and to form a domain-swapped dimer. Being knotted may be the consequence of selection pressure for the unique structure-function relationship of the SPOUT superfamily that exists in all kingdoms of life.
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http://dx.doi.org/10.1016/j.str.2019.04.004DOI Listing
August 2019

TNF-α-induced miR-450a mediates TMEM182 expression to promote oral squamous cell carcinoma motility.

PLoS One 2019 20;14(3):e0213463. Epub 2019 Mar 20.

National Institute of Cancer Research, National Health Research Institutes, Miaoli, Taiwan.

Distant metastasis leads oral cancer patients into a poor survival rate and a high recurrence stage. During tumor progression, dysregulated microRNAs (miRNAs) have been reported to involve tumor initiation and modulate oral cancer malignancy. MiR-450a was significantly upregulated in oral squamous cell carcinoma (OSCC) patients without functional reports. This study was attempted to uncover the molecular mechanism of novel miR-450a in OSCC. Mir-450a expression was examined by quantitative RT-PCR, both in OSCC cell lines and patients. Specific target of miR-450a was determined by software prediction, luciferase reporter assay, and correlation with target protein expression. The functions of miR-450a and TMEM182 were accessed by adhesion and transwell invasion analyses. Determination of the expression and cellular localization of TMEM182 was examined by RT-PCR and by immunofluorescence staining. The signaling pathways involved in regulation of miR-450a were investigated using the kinase inhibitors. Overexpression of miR-450a in OSCC cells impaired cell adhesion ability and induced invasiveness, which demonstrated the functional role of miR-450a as an onco-miRNA. Interestingly, tumor necrosis factor alpha (TNF-α)-mediated expression of TMEM182 was regulated by miR-450a induction. MiR-450a-reduced cellular adhesion was abolished by TMEM182 restoration. Furthermore, the oncogenic activity of TNF-α/miR-450a/TMEM182 axis was primarily through activating extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. ERK1/2 inhibitor prevented the TNF-α-induced miR-450a expression and enhanced adhesion ability. Our data suggested that TNF-α-induced ERK1/2-dependent miR-450a against TMEM182 expression exerted a great influence on increasing OSCC motility. Overall, our results provide novel molecular insights into how TNF-α contributes to oral carcinogenesis through miR-450a that targets TMEM182.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0213463PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426234PMC
December 2019

Untying a Protein Knot by Circular Permutation.

J Mol Biol 2019 02 9;431(4):857-863. Epub 2019 Jan 9.

Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan; Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan. Electronic address:

Topologically knotted proteins are tantalizing examples of how polypeptide chains can explore complex free energy landscapes to efficiently attain defined knotted conformations. The evolution trails of protein knots, however, remain elusive. We used circular permutation to change an evolutionally conserved topologically knotted SPOUT RNA methyltransferase into an unknotted form. The unknotted variant adopted the same three-dimensional structure and oligomeric state as its knotted parent, but its folding stability was markedly reduced with accelerated folding kinetics and its ligand binding was abrogated. Our findings support the hypothesis that the universally conserved knotted topology of the SPOUT superfamily evolved from unknotted forms through circular permutation under selection pressure for folding robustness and, more importantly, for functional requirements associated with the knotted structural element.
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http://dx.doi.org/10.1016/j.jmb.2019.01.005DOI Listing
February 2019

Nile Tilapia Derived Antimicrobial Peptide TP4 Exerts Antineoplastic Activity Through Microtubule Disruption.

Mar Drugs 2018 Nov 22;16(12). Epub 2018 Nov 22.

Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Ilan 262, Taiwan.

Some antimicrobial peptides (AMPs) exhibit anti-cancer activity, acting on cancer cells either by causing membrane lysis or via intracellular effects. While intracellular penetration of AMPs has been shown to cause cancer cell death, the mechanisms of toxicity remain largely unknown. Here we show that a tilapia-derived AMP, Tilapia piscidin (TP) 4, penetrates intracellularly and targets the microtubule network. A pull-down assay identified α-Tubulin as a major interaction partner for TP4, and molecular docking analysis suggested that Phe1, Ile16, and Arg23 on TP4 are required for the interaction. TP4 treatment in A549 cells was found to disrupt the microtubule network in cells, and mutation of the essential TP4 residues prevented microtubule depolymerization in vitro. Importantly, the TP4 mutants also showed decreased cytotoxicity in A549 cells, suggesting that microtubule disruption is a major mechanistic component of TP4-mediated death in lung carcinoma cells.
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http://dx.doi.org/10.3390/md16120462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315541PMC
November 2018

Cdc7-Dbf4-mediated phosphorylation of HSP90-S164 stabilizes HSP90-HCLK2-MRN complex to enhance ATR/ATM signaling that overcomes replication stress in cancer.

Sci Rep 2017 12 5;7(1):17024. Epub 2017 Dec 5.

National Institute of Cancer Research, National Health Research Institutes, Zhunan, Miaoli, Taiwan.

Cdc7-Dbf4 kinase plays a key role in the initiation of DNA replication and contributes to the replication stress in cancer. The activity of human Cdc7-Dbf4 kinase remains active and acts as an effector of checkpoint under replication stress. However, the downstream targets of Cdc7-Dbf4 contributed to checkpoint regulation and replication stress-support function in cancer are not fully identified. In this work, we showed that aberrant Cdc7-Dbf4 induces DNA lesions that activate ATM/ATR-mediated checkpoint and homologous recombination (HR) DNA repair. Using a phosphoproteome approach, we identified HSP90-S164 as a target of Cdc7-Dbf4 in vitro and in vivo. The phosphorylation of HSP90-S164 by Cdc7-Dbf4 is required for the stability of HSP90-HCLK2-MRN complex and the function of ATM/ATR signaling cascade and HR DNA repair. In clinically, the phosphorylation of HSP90-S164 indeed is increased in oral cancer patients. Our results indicate that aberrant Cdc7-Dbf4 enhances replication stress tolerance by rewiring ATR/ATM mediated HR repair through HSP90-S164 phosphorylation and by promoting recovery from replication stress. We provide a new solution to a subtyping of cancer patients with dominant ATR/HSP90 expression by combining inhibitors of ATR-Chk1, HSP90, or Cdc7 in cancer combination therapy.
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http://dx.doi.org/10.1038/s41598-017-17126-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717001PMC
December 2017

De novo protein sequencing, humanization and in vitro effects of an antihuman CD34 mouse monoclonal antibody.

Biochem Biophys Rep 2017 Mar 18;9:51-60. Epub 2016 Nov 18.

Institute of Bioinformatics and Structural Biology & Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan.

QBEND/10 is a mouse immunoglobulin lambda-chain monoclonal antibody with strict specificity against human hematopoietic progenitor cell antigen CD34. Our in vitro study showed that QBEND/10 impairs the tube formation of human umbilical vein endothelial cells (HUVECs), suggesting that the antibody may be of potential benefit in blocking tumor angiogenesis. We provided a de novo protein sequencing method through tandem mass spectrometry to identify the amino acid sequences in the variable heavy and light chains of QBEND/10. To reduce immunogenicity for clinical applications, QBEND/10 was further humanized using the resurfacing approach. We demonstrate that the de novo sequenced and humanized QBEND/10 retains the biological functions of the parental mouse counterpart, including the binding kinetics to CD34 and blockage of the tube formation of the HUVECs.
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http://dx.doi.org/10.1016/j.bbrep.2016.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614552PMC
March 2017

A clamp-like orientation of basic residues set in a parallelogram is essential for heparin binding.

FEBS Lett 2016 09 31;590(18):3089-97. Epub 2016 Aug 31.

Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.

While the majority of studies have focused on the biological roles of heparin-binding proteins, relatively little is known about their key residues and structural elements responsible for heparin interaction. In this study, we employed the IgG-binding domain B1 of Streptococcal protein G as a miniature scaffold to investigate how certain positively charged residues within the β-sheet conformation become favorable for heparin binding. By performing a series of arginine substitution mutations followed by gain-of-heparin-binding analysis, we deduced that a clamp-like orientation with discontinuous basic residues separated by ~ 5 Å with ~ 100° interior angle is advantageous for high heparin affinity.
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http://dx.doi.org/10.1002/1873-3468.12361DOI Listing
September 2016

Role of the RAD51-SWI5-SFR1 Ensemble in homologous recombination.

Nucleic Acids Res 2016 07 30;44(13):6242-51. Epub 2016 Apr 30.

Institute of Biochemical Sciences, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan Institute of Biological Chemistry, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei 11529, Taiwan

During DNA double-strand break and replication fork repair by homologous recombination, the RAD51 recombinase catalyzes the DNA strand exchange reaction via a helical polymer assembled on single-stranded DNA, termed the presynaptic filament. Our published work has demonstrated a dual function of the SWI5-SFR1 complex in RAD51-mediated DNA strand exchange, namely, by stabilizing the presynaptic filament and maintaining the catalytically active ATP-bound state of the filament via enhancement of ADP release. In this study, we have strived to determine the basis for physical and functional interactions between Mus musculus SWI5-SFR1 and RAD51. We found that SWI5-SFR1 preferentially associates with the oligomeric form of RAD51. Specifically, a C-terminal domain within SWI5 contributes to RAD51 interaction. With specific RAD51 interaction defective mutants of SWI5-SFR1 that we have isolated, we show that the physical interaction is indispensable for the stimulation of the recombinase activity of RAD51. Our results thus help establish the functional relevance of the trimeric RAD51-SWI5-SFR1 complex and provide insights into the mechanistic underpinnings of homology-directed DNA repair in mammalian cells.
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http://dx.doi.org/10.1093/nar/gkw375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291256PMC
July 2016

Pyrazolylamine Derivatives Reveal the Conformational Switching between Type I and Type II Binding Modes of Anaplastic Lymphoma Kinase (ALK).

J Med Chem 2016 04 12;59(8):3906-19. Epub 2016 Apr 12.

Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, 35 Keyan Road, Zhunan Town, Miaoli County 350, Taiwan, ROC.

Most anaplastic lymphoma kinase (ALK) inhibitors adopt a type I binding mode, but only limited type II ALK structural studies are available. Herein, we present the structure of ALK in complex with N1-(3-4-[([5-(tert-butyl)-3-isoxazolyl]aminocarbonyl)amino]-3-methylphenyl-1H-5-pyrazolyl)-4-[(4-methylpiperazino)methyl]benzamide (5a), a novel ALK inhibitor adopting a type II binding mode. It revealed binding of 5a resulted in the conformational change and reposition of the activation loop, αC-helix, and juxtamembrane domain, which are all important domains for the autoinhibition mechanism and downstream signal pathway regulation of ALK. A structure-activity relationship study revealed that modifications to the structure of 5a led to significant differences in the ALK potency and altered the protein structure of ALK. To the best of our knowledge, this is the first structural biology study to directly observe how changes in the structure of a small molecule can regulate the switch between the type I and type II binding modes and induce dramatic conformational changes.
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http://dx.doi.org/10.1021/acs.jmedchem.6b00106DOI Listing
April 2016

MMP-13 is involved in oral cancer cell metastasis.

Oncotarget 2016 03;7(13):17144-61

Department of Medical Sciences and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.

The oral cancer cell line OC3-I5 with a highly invasive ability was selected and derived from an established OSCC line OC3. In this study, we demonstrated that matrix metalloproteinases protein MMP-13 was up-regulated in OC3-I5 than in OC3 cells. We also observed that expression of epithelial-mesenchymal transition (EMT) markers including Twist, p-Src, Snail1, SIP1, JAM-A, and vinculin were increased in OC3-I5 compared to OC3 cells, whereas E-cadherin expression was decreased in the OC3-I5 cells. Using siMMP-13 knockdown techniques, we showed that siMMP-13 not only reduced the invasion and migration, but also the adhesion abilities of oral cancer cells. In support of the role of MMP-13 in metastasis, we used MMP-13 expressing plasmid-transfected 293T cells to enhance MMP-13 expression in the OC3 cells, transplanting the MMP-13 over expressing OC3 cells into nude mice led to enhanced lung metastasis. In summary, our findings show that MMP-13 promotes invasion and metastasis in oral cancer cells, suggesting altered expression of MMP-13 may be utilized to impede the process of metastasis.
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http://dx.doi.org/10.18632/oncotarget.7942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4941377PMC
March 2016

Complete Genome Sequence of Xanthomonas campestris pv. campestris Strain 17 from Taiwan.

Genome Announc 2015 Dec 17;3(6). Epub 2015 Dec 17.

Institute of Biochemistry and NCHU Biotechnological Center, National Chung Hsing University, Taichung, Taiwan

Xanthomonas campestris pv. campestris 17 is a Gram-negative bacterium that is phytopathogenic to cruciferous plants in Taiwan. The 4,994,426-bp-long genome consists of 24 contigs with 4,050 protein-coding genes, 1 noncoding RNA (ncRNA) gene, 6 rRNA genes, and 55 tRNA genes.
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http://dx.doi.org/10.1128/genomeA.01466-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683227PMC
December 2015

1H, 15N and 13C resonance assignments of light organ-associated fatty acid-binding protein of Taiwanese fireflies.

Biomol NMR Assign 2016 Apr 15;10(1):71-4. Epub 2015 Sep 15.

Institute of Bioinformatics and Structural Biology, National Tsing Hua University, No. 101, Sec. 2, Kuang Fu Rd, Hsinchu, 30013, Taiwan, ROC.

Fatty acid-binding proteins (FABPs) are a family of proteins that modulate the transfer of various fatty acids in the cytosol and constitute a significant portion in many energy-consuming cells. The ligand binding properties and specific functions of a particular type of FABP seem to be diverse and depend on the respective binding cavity as well as the cell type from which this protein is derived. Previously, a novel FABP (lcFABP; lc: Luciola cerata) was identified in the light organ of Taiwanese fireflies. The lcFABP was proved to possess fatty acids binding capabilities, especially for fatty acids of length C14-C18. However, the structural details are unknown, and the structure-function relationship has remained to be further investigated. In this study, we finished the (1)H, (15)N and (13)C chemical shift assignments of (15)N/(13)C-enriched lcFABP by solution NMR spectroscopy. In addition, the secondary structure distribution was revealed based on the backbone N, H, Cα, Hα, C and side chain Cβ assignments. These results can provide the basis for further structural exploration of lcFABP.
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http://dx.doi.org/10.1007/s12104-015-9640-0DOI Listing
April 2016

Comparative analysis of the folding dynamics and kinetics of an engineered knotted protein and its variants derived from HP0242 of Helicobacter pylori.

J Phys Condens Matter 2015 Sep 20;27(35):354106. Epub 2015 Aug 20.

Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan. Institute of Biochemical Science, National Taiwan University, Taipei 116, Taiwan. Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan.

Understanding the mechanism by which a polypeptide chain thread itself spontaneously to attain a knotted conformation has been a major challenge in the field of protein folding. HP0242 is a homodimeric protein from Helicobacter pylori with intertwined helices to form a unique pseudo-knotted folding topology. A tandem HP0242 repeat has been constructed to become the first engineered trefoil-knotted protein. Its small size renders it a model system for computational analyses to examine its folding and knotting pathways. Here we report a multi-parametric study on the folding stability and kinetics of a library of HP0242 variants, including the trefoil-knotted tandem HP0242 repeat, using far-UV circular dichroism and fluorescence spectroscopy. Equilibrium chemical denaturation of HP0242 variants shows the presence of highly populated dimeric and structurally heterogeneous folding intermediates. Such equilibrium folding intermediates retain significant amount of helical structures except those at the N- and C-terminal regions in the native structure. Stopped-flow fluorescence measurements of HP0242 variants show that spontaneous refolding into knotted structures can be achieved within seconds, which is several orders of magnitude faster than previously observed for other knotted proteins. Nevertheless, the complex chevron plots indicate that HP0242 variants are prone to misfold into kinetic traps, leading to severely rolled-over refolding arms. The experimental observations are in general agreement with the previously reported molecular dynamics simulations. Based on our results, kinetic folding pathways are proposed to qualitatively describe the complex folding processes of HP0242 variants.
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http://dx.doi.org/10.1088/0953-8984/27/35/354106DOI Listing
September 2015

Biomarker discovery for neuroendocrine cervical cancer.

Electrophoresis 2014 Jul 5;35(14):2039-45. Epub 2014 Jun 5.

Department of Medical Sciences, Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.

Neuroendocrine cervical cancer is an aggressive but rare form of cervical cancer. The majority of neuroendocrine cervical cancer patients present with advanced-stage diseases. However, the limited numbers of neuroendocrine tumor markers are insufficient for clinical purposes. Thus, we used a proteomic approach combining lysine labeling 2D-DIGE and MALDI-TOF MS to investigate the biomarkers for neuroendocrine cervical cancer. By analyzing the global proteome alteration between the neuroendocrine cervical cancer line (HM-1) and non-neuroendocrine cervical cancer lines (CaSki cells, ME-180 cells, and Hela cells), we identified 82 proteins exhibiting marked changes between HM-1 and CaSki cells, and between ME-180 and Hela cells. Several proteins involved in protein folding, cytoskeleton, transcription control, signal transduction, glycolysis, and redox regulation exhibited significant changes in abundance. Proteomic and immunoblot analyses indicated respective 49.88-fold and 25-fold increased levels of transgelin in HM-1 cells compared with that in other non-neuroendocrine cervical cancer cell lines, implying that transgelin is a biomarker for neuroendocrine cervical cancer. In summary, we used a comprehensive neuroendocrine/non-neuroendocrine cervical cancer model based proteomic approach for identifying neuroendocrine cervical cancer markers, which might contribute to the prognosis and diagnosis of neuroendocrine cervical cancer.
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http://dx.doi.org/10.1002/elps.201400014DOI Listing
July 2014

NMR assignments of a hypothetical pseudo-knotted protein HP0242 from Helicobacter pylori.

Biomol NMR Assign 2014 Oct 4;8(2):287-9. Epub 2013 Jul 4.

Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.

Many knotted proteins have been discovered recently, but the folding process of which remains elusive. HP0242 is a hypothetical protein from Helicobacter pylori, which is a model system for studying the folding pathway of a knotted protein. In this study, we report the (1)H, (13)C, and (15)N chemical shift assignments of HP0242. The results will enable us to further investigate HP0242 by NMR experiments.
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http://dx.doi.org/10.1007/s12104-013-9502-6DOI Listing
October 2014

Protein kinase inhibitor design by targeting the Asp-Phe-Gly (DFG) motif: the role of the DFG motif in the design of epidermal growth factor receptor inhibitors.

J Med Chem 2013 May 10;56(10):3889-903. Epub 2013 May 10.

Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, 35 Keyan Road, Zhunan Town, Miaoli County 350, Taiwan, ROC.

The Asp-Phe-Gly (DFG) motif plays an important role in the regulation of kinase activity. Structure-based drug design was performed to design compounds able to interact with the DFG motif; epidermal growth factor receptor (EGFR) was selected as an example. Structural insights obtained from the EGFR/2a complex suggested that an extension from the meta-position on the phenyl group (ring-5) would improve interactions with the DFG motif. Indeed, introduction of an N,N-dimethylamino tail resulted in 4b, which showed almost 50-fold improvement in inhibition compared to 2a. Structural studies confirmed this N,N-dimethylamino tail moved toward the DFG motif to form a salt bridge with the side chain of Asp831. That the interactions with the DFG motif greatly contribute to the potency of 4b is strongly evidenced by synthesizing and testing compounds 2a, 3g, and 4f: when the charge interactions are absent, the inhibitory activity decreased significantly.
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http://dx.doi.org/10.1021/jm400072pDOI Listing
May 2013

Redox-proteomic analysis of doxorubicin resistance-induced altered thiol activity in uterine carcinoma.

J Pharm Biomed Anal 2013 May 31;78-79:1-8. Epub 2013 Jan 31.

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

Doxorubicin is an anticancer drug used in a wide range of cancer therapies; however, doxorubicin-induced drug resistance is one of the most serious obstacles of cancer chemotherapy. Recent studies have indicated that reduced oxidative stress levels in cancer cells induce drug resistance. However, the redox-modifications of resistance - associated cellular targets are largely unknown. Thus, the current study employed cysteine-labeling based two-dimensional differential gel electrophoresis (2D-DIGE) combined with MALDI-TOF mass spectrometry (MALDI-TOF MS) to analyze the effect of doxorubicin resistance on redox regulation in uterine cancer and showed 33 spots that were significantly changed in thiol reactivity. These proteins involve cytoskeleton regulation, signal transduction, redox-regulation, glycolysis, and cell-cycle regulation. The current work shows that the redox 2D-DIGE-based proteomic strategy provides a rapid method to study the molecular mechanisms of doxorubicin-induced drug resistance in uterine cancer. The identified targets may be used to further evaluate their roles in drug-resistance formation and for possible diagnostic or therapeutic applications.
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http://dx.doi.org/10.1016/j.jpba.2013.01.028DOI Listing
May 2013

Residue-specific annotation of disorder-to-order transition and cathepsin inhibition of a propeptide-like crammer from D. melanogaster.

PLoS One 2013 21;8(1):e54187. Epub 2013 Jan 21.

Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.

Drosophila melanogaster crammer is a novel cathepsin inhibitor involved in long-term memory formation. A molten globule-to-ordered structure transition is required for cathepsin inhibition. This study reports the use of alanine scanning to probe the critical residues in the two hydrophobic cores and the salt bridges of crammer in the context of disorder-to-order transition and cathepsin inhibition. Alanine substitution of the aromatic residues W9, Y12, F16, Y20, Y32, and W53 within the hydrophobic cores, and charged residues E8, R28, R29, and E67 in the salt bridges considerably decrease the ability of crammer to inhibit Drosophila cathepsin B (CTSB). Far-UV circular dichroism (CD), intrinsic fluorescence, and nuclear magnetic resonance (NMR) spectroscopies show that removing most of the aromatic and charged side-chains substantially reduces thermostability, alters pH-dependent helix formation, and disrupts the molten globule-to-ordered structure transition. Molecular modeling indicates that W53 in the hydrophobic Core 2 is essential for the interaction between crammer and the prosegment binding loop (PBL) of CTSB; the salt bridge between R28 and E67 is critical for the appropriate alignment of the α-helix 4 toward the CTSB active cleft. The results of this study show detailed residue-specific dissection of folding transition and functional contributions of the hydrophobic cores and salt bridges in crammer, which have hitherto not been characterized for cathepsin inhibition by propeptide-like cysteine protease inhibitors. Because of the involvements of cathepsin inhibitors in neurodegenerative diseases, these structural insights can serve as a template for further development of therapeutic inhibitors against human cathepsins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0054187PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3551606PMC
July 2013

GI-POP: a combinational annotation and genomic island prediction pipeline for ongoing microbial genome projects.

Gene 2013 Apr 12;518(1):114-23. Epub 2013 Jan 12.

Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.

Sequencing of microbial genomes is important because of microbial-carrying antibiotic and pathogenetic activities. However, even with the help of new assembling software, finishing a whole genome is a time-consuming task. In most bacteria, pathogenetic or antibiotic genes are carried in genomic islands. Therefore, a quick genomic island (GI) prediction method is useful for ongoing sequencing genomes. In this work, we built a Web server called GI-POP (http://gipop.life.nthu.edu.tw) which integrates a sequence assembling tool, a functional annotation pipeline, and a high-performance GI predicting module, in a support vector machine (SVM)-based method called genomic island genomic profile scanning (GI-GPS). The draft genomes of the ongoing genome projects in contigs or scaffolds can be submitted to our Web server, and it provides the functional annotation and highly probable GI-predicting results. GI-POP is a comprehensive annotation Web server designed for ongoing genome project analysis. Researchers can perform annotation and obtain pre-analytic information include possible GIs, coding/non-coding sequences and functional analysis from their draft genomes. This pre-analytic system can provide useful information for finishing a genome sequencing project.
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http://dx.doi.org/10.1016/j.gene.2012.11.063DOI Listing
April 2013

Crystal structure of circular permuted RoCBM21 (CP90): dimerisation and proximity of binding sites.

PLoS One 2012 30;7(11):e50488. Epub 2012 Nov 30.

Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.

Glucoamylases, containing starch-binding domains (SBD), have a wide range of scientific and industrial applications. Random mutagenesis and DNA shuffling of the gene encoding a starch-binding domain have resulted in only minor improvements in the affinities of the corresponding protein to their ligands, whereas circular permutation of the RoCBM21 substantially improved its binding affinity and selectivity towards longer-chain carbohydrates. For the study reported herein, we used a standard soluble ligand (amylose EX-I) to characterize the functional and structural aspects of circularly permuted RoCBM21 (CP90). Site-directed mutagenesis and the analysis of crystal structure reveal the dimerisation and an altered binding path, which may be responsible for improved affinity and altered selectivity of this newly created starch-binding domain. The functional and structural characterization of CP90 suggests that it has significant potential in industrial applications.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0050488PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511584PMC
May 2013