Publications by authors named "Sung-Fang Chen"

33 Publications

Tenofovir Hampers the Efficacy of Sorafenib in Prolonging Overall Survival in Hepatocellular Carcinoma.

Biomedicines 2021 Oct 26;9(11). Epub 2021 Oct 26.

Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taoyuan 333, Taiwan.

Sorafenib is a first-line treatment for patients with advanced hepatocellular carcinoma (HCC). These patients may simultaneously receive anti-hepatitis B treatment if they are viremic. The () gene can serve as a biomarker to guide HCC treatments. However, the enzyme substrates of its gene product, GalNAc-T14 (a glycosyltransferase), remained uncharacterized. Here, we conducted a glycoproteome-wide search for GalNAc-T14 substrates using lectin affinity chromatography followed by tandem mass spectrometry. Seventeen novel GalNAc-T14 substrates were identified. A connective map analysis showed that an antiviral drug, tenofovir, was the leading medicinal compound to down-regulate the expression of these substrates. In vitro assays showed that HCC cells were resistant to sorafenib if pretreated by tenofovir but not entecavir. Clinical analysis showed that the concomitant use of tenofovir and sorafenib was a previously unrecognized predictive factor for unfavorable overall survival (hazard ratio = 2.060, 95% confidence interval = [1.256, 3.381], = 0.004) in a cohort of 181 hepatitis-B-related, sorafenib-treated HCC patients (concomitant tenofovir versus entecavir treatment; = 0.003). In conclusion, by conducting a glycoproteome-wide search for GalNAc-T14 substrates, we unexpectedly found that tenofovir was a major negative regulator of GalNAc-T14 substrates and an unfavorable anti-hepatitis B drug in HCC patients receiving sorafenib.
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http://dx.doi.org/10.3390/biomedicines9111539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8614833PMC
October 2021

Quantitative Proteomics of Th-MYCN Transgenic Mice Reveals Aurora Kinase Inhibitor Altered Metabolic Pathways and Enhanced ACADM To Suppress Neuroblastoma Progression.

J Proteome Res 2019 11 11;18(11):3850-3866. Epub 2019 Oct 11.

Institute of Biomedical Informatics , National Yang-Ming University , Taipei 112 , Taiwan.

Neuroblastoma is a neural crest-derived embryonal tumor and accounts for about 15% of all cancer deaths in children. MYCN amplification is associated with aggressive and advanced stage of high-risk neuroblastoma, which remains difficult to treat and exhibits poor survival under current multimodality treatment. Here, we analyzed the transcriptomic profiles of neuroblastoma patients and showed that aurora kinases lead to poor survival and had positive correlation with MYCN amplification and high-risk disease. Further, pan-aurora kinase inhibitor (tozasertib) treatment not only induces cell-cycle arrest and suppresses cell proliferation, migration, and invasion ability in MYCN-amplified (MNA) neuroblastoma cell lines, but also inhibits tumor growth and prolongs animal survival in Th-MYCN transgenic mice. Moreover, we performed quantitative proteomics and identified 150 differentially expressed proteins after tozasertib treatment in the Th-MYCN mouse model. The functional and network-based enrichment revealed that tozasertib alters metabolic processes and identified a mitochondrial flavoenzyme in fatty acid β-oxidation, ACADM, which is correlated with aurora kinases and neuroblastoma patient survival. Our findings indicate that the aurora kinase inhibitor could cause metabolic imbalance, possibly by disturbing carbohydrate and fatty acid metabolic pathways, and ACADM may be a potential target in MNA neuroblastoma.
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http://dx.doi.org/10.1021/acs.jproteome.9b00245DOI Listing
November 2019

Comparison of different fractionation strategies for in-depth phosphoproteomics by liquid chromatography tandem mass spectrometry.

Anal Bioanal Chem 2019 Jun 22;411(15):3417-3424. Epub 2019 Apr 22.

Department of Chemistry, National Taiwan Normal University, Taipei, 11677, Taiwan.

Phosphorylation, a major posttranslational modification of proteins, plays an important role in protein activity and cell signaling. However, it is difficult to detect protein phosphorylation because of its low abundance and the fact that the analysis can be hindered by the presence of highly abundant non-phosphoproteins. In order to reduce the sample complexity and improve the efficiency of identification of phosphopeptides, aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) was utilized to enrich phosphopeptides from a murine macrophage cell lysate. Strong cation chromatography (SCX), electrostatic repulsion hydrophilic interaction chromatography (ERLIC), and solution isoelectric focusing (sIEF) were investigated in detail for phosphopeptide fractionation strategies followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. A total of 5744 non-redundant phosphopeptides and 2159 phosphoproteins were identified from the cell lysates in three fractionation approaches. The SCX fractionation contained the largest number of phosphoproteins and phosphopeptides that were identified. In addition, 4336, 2064, and 2424 phosphopeptides were identified from SCX-LC-MS/MS, ERLIC-LC-MS/MS, and sIEF-LC/MS-MS, including 2430, 438, and 751 phosphopeptides that were only specifically found in SCX, ERLIC, and sIEF fractionations. In conclusion, these three fractionation strategies demonstrated great complementarity, which greatly improved the efficiency of identification of phosphopeptides and can be suitable for use in in-depth phosphoproteome research. Graphical Abstract.
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http://dx.doi.org/10.1007/s00216-019-01823-0DOI Listing
June 2019

Analysis of heterocyclic amines in meat products by liquid chromatography - Tandem mass spectrometry.

J Food Drug Anal 2019 04 27;27(2):595-602. Epub 2018 Oct 27.

National Taiwan Normal University, Department of Chemistry, Taiwan. Electronic address:

Heterocyclic amines (HCAs), a class made up of more than 25 compounds, are unintended hazardous substances that are generated by the heating or processing of proteinaceous foods at high temperatures. The International Agency for Research on Cancer (IARC) has classified four such HCAs (IQ, MeIQ, MeIQx, and PhIP) as being probable or possible human carcinogens. In this study, two sample preparation strategies, liquid-liquid extraction (LLE) with solid-phase extraction (SPE) and a rapid, easy, cheap, effective, rugged, and safe extraction (QuEChERS) method, were investigated for the determination of 11 types of HCAs in meat products by LC-MS/MS. The HCAs in the samples were first extracted with acetonitrile by LLE, and followed by SPE. In the case of QuEChERS extraction, acetonitrile is used as the LLME solvent, and PSA, C18EC and MgSO were used as the dSPE sorbent. Both methods showed good performance with respect to precision (RSD < 15.15%), accuracy (79.80-117.64%), recovery (52.39-116.88%), limit of quantitation for a spiked meat extract (0.01-10 ppb) and correlation coefficients (>0.993). The QuEChERS extraction strategy provided a better linear dynamic range and superior sensitivity in comparison with the LLE-SPE approach. HCAs were successfully quantified in real samples using the two proposed approaches by LC-MS/MS.
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http://dx.doi.org/10.1016/j.jfda.2018.10.002DOI Listing
April 2019

Differential dynamics of hepatic protein expressions with long-term cultivated hepatitis C virus infection.

J Microbiol Immunol Infect 2020 Oct 21;53(5):715-723. Epub 2019 Feb 21.

Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan; Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan. Electronic address:

Background: The liver maintains blood chemical homeostasis by active uptake and secretion through endocytosis, exocytosis, and intracellular trafficking between the plasma and intracellular membranes. Hepatitis C virus (HCV) infection affects the host membrane architecture and might thus impair the regulation of the cellular transportation machinery. Additionally, the hepatic expressions of differential protein dynamics with long-term HCV infection remain fully recover.

Methods: In this study, comparative proteomic analysis was performed in HCV-infected and mock-control Huh7 cells according to the viral dynamics of exponential, plateau, declined, and silencing phases at the acute stage, and the chronic stage. The proteins with <0.8-fold and ≥1.25-fold changes in expression were analyzed using functional pathway clustering prediction.

Results: The combined experimental repetitions identified full-spectrum cellular proteins in each of 5 sample sets from acute exponential, plateau, declined, and silencing phases, and the chronic stage. The clustering results revealed that HCV infection might differentiate regulatory pathways involving extracellular exosome, cadherin, melanosome, and RNA binding. Overall host proteins in HCV-infected cells exhibited kinetic pattern 1, in which cellular expression was downregulated from the acute exponential to plateau phases, reached a nadir, and was then elevated at the chronic stage. The proteins involved in the membrane-budding pathway exhibited kinetic pattern 2, in which their expressions were distinctly downregulated at the chronic stage.

Conclusion: The current comparative proteomics revealed the differential regulatory effects of HCV infection on host intracellular transport functional pathways, which might contribute to the pathogenic mechanisms of HCV in hepatocytes that sustain long-term infection.
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http://dx.doi.org/10.1016/j.jmii.2019.01.003DOI Listing
October 2020

Monitoring the Disulfide Bonds of Folding Isomers of Synthetic CTX A3 Polypeptide Using MS-Based Technology.

Toxins (Basel) 2019 01 17;11(1). Epub 2019 Jan 17.

National Health Research Institutes, National Institute of Infectious Diseases and Vaccinology, Miaoli 35053, Taiwan.

Native disulfide formation is crucial to the process of disulfide-rich protein folding in vitro. As such, analysis of the disulfide bonds can be used to track the process of the folding reaction; however, the diverse structural isomers interfere with characterization due to the non-native disulfide linkages. Previously, a mass spectrometry (MS) based platform coupled with peptide demethylation and an automatic disulfide bond searching engine demonstrated the potential to screen disulfide-linked peptides for the unambiguous assignment of paired cysteine residues of toxin components in cobra venom. The developed MS-based platform was evaluated to analyze the disulfide bonds of structural isomers during the folding reaction of synthetic cardiotoxin A3 polypeptide (syn-CTX A3), an important medical component in cobra venom. Through application of this work flow, a total of 13 disulfide-linked peptides were repeatedly identified across the folding reaction, and two of them were found to contain cysteine pairings, like those found in native CTX A3. Quantitative analysis of these disulfide-linked peptides showed the occurrence of a progressive disulfide rearrangement that generates a native disulfide bond pattern on syn-CTX A3 folded protein. The formation of these syn-CTX A3 folded protein reaches a steady level in the late stage of the folding reaction. Biophysical and cell-based assays showed that the collected syn-CTX A3 folded protein have a β-sheet secondary structure and cytotoxic activity similar to that of native CTX A3. In addition, the immunization of the syn-CTX A3 folded proteins could induce neutralization antibodies against the cytotoxic activity of native CTX A3. In contrast, these structure activities were poorly observed in the other folded isomers with non-native disulfide bonds. The study highlights the ability of the developed MS platform to assay isomers with heterogeneous disulfide bonds, providing insight into the folding mechanism of the bioactive protein generation.
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http://dx.doi.org/10.3390/toxins11010052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356385PMC
January 2019

Identifying Specific and Differentially Linked Glycosyl Residues in Mammalian Glycans by Targeted LC-MS Analysis.

Anal Sci 2018 Sep 24;34(9):1049-1054. Epub 2018 Aug 24.

Institute of Biological Chemistry, Academia Sinica.

Glycans, which are widespread in nature, consist of a large number of monosaccharides linked via glycosidic bonds. Due to the complex nature of glycan structures on glycoproteins, assessing the configuration and positions of the glycosidic linkages of a glycan is a subject of considerable interest. In this study, a method for accomplishing this using partially O-methylated alditols (PMAs) from glycans combined with LC-MS analysis is reported. N-Glycans were first per-methylated with methyl iodide, and the levels of methylation were further confirmed by MALDI-TOF. PMAs were then produced via complete hydrolysis and reduction. PMAs derived from Fetuin N-glycan and Lewis antigen carbohydrates were successfully detected by LC-MS analysis. This analysis can be performed without the need for an additional derivatization step for GC analysis, and should be suitable for use in conjunction with a LC-MS-based analysis platform.
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http://dx.doi.org/10.2116/analsci.18SCP01DOI Listing
September 2018

Combination of on-line desalting and HPLC-UV-ESI-MS for simultaneous detection and identification of FIP-fve and flammutoxin in Flammulina velutipes.

J Food Drug Anal 2018 07 17;26(3):1045-1053. Epub 2018 Jan 17.

Graduate Institute of Food Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan, ROC. Electronic address:

A rapid analytical approach, on-line desalting HPLC-UV-ESI-MS method, for the analysis of FIP-fve and flammutoxin (FTX), two important bioactive proteins in the fruiting bodies of Flammulina velutipes, was developed. In this study, a highly efficient desalting method is provided using molecular weight cut-off centrifugal filtration and on-line desalting. Sample preparation followed by an on-line desalting HPLC-UV-ESI-MS system was employed for simultaneous desalting and detection and identification of FIP-fve and FTX. Results indicated that using trifluoroacetic acid as a modifier on a C18 reversed-phase column renders effective separation. ESI-MS revealed that the apparent molecular masses of FIP-fve and FTX were 12,749.1 Da and 21,912.5 Da, respectively. Eleven milligrams of FIP-fve was obtained from 100 g of fresh fruiting bodies, and UV detection was performed at 280 nm using bovine serum albumin as the standard protein. The calibration curve was linear in the concentration range of 0.29-4.69 mg/mL (r = 0.9999). FTX and a series of degradation products were isolated from F. velutipes using 35% saturated ammonium sulfate on a DEAE cellulose column. The complete identification of FTX and a series of degradation products were carried out by precipitation of various ammonium sulfate concentrations (0-45%, 45-65% and 65-90%), in-gel trypsin digestion, and MS analysis with combined database search. The molecular weights of FTX and a series of degradation products were 29,957.2 Da, 27,480.2 Da, 26,512.5 Da, and 21,912.5 Da.
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http://dx.doi.org/10.1016/j.jfda.2017.12.004DOI Listing
July 2018

Application of thermal stability difference to remove flammutoxin in fungal immunomodulatory protein, FIP-fve, extract from Flammulina velutipes.

J Food Drug Anal 2018 07 17;26(3):1005-1014. Epub 2018 Jan 17.

Graduate Institute of Food Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, 10617, Taiwan, ROC. Electronic address:

Fungal immunomodulatory protein (FIP-fve) is a potential functional food ingredient. However, undesirable component flammutoxin (FTX) would occur in the extracted fraction of FIP-fve. In this paper, an application of heating processing instead of the intensive separation process was employed in fractionation of FIP-fve, meanwhile, exclusion of FTX was reached. Contents of FIP-fve and FTX were monitored by HPLC-UV-ESI-MS. Both FIP-fve and FTX had higher thermal stability in a lower concentration solution. Cold water could effectively extract FIP-fve and FTX from fresh mushroom without acetic acid and disulfide-bond breaking agent β-mercaptoethanol commonly used in biochemical studies. Heating cold water extract contained 580 μg/mL FIP-fve and 452 μg/mL FTX at 60 °C for 5 min could effectively exclude FTX and remain 75% of FIP-fve. Adding 0.1 M trehalose or 20% ethanol did not significantly alter the stability of both proteins. The method developed is an applicable procedure for preparing FIP-fve solution free of FTX.
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http://dx.doi.org/10.1016/j.jfda.2017.12.010DOI Listing
July 2018

Quantification of Trans-resveratrol in Red Wines Using QuEChERS Extraction Combined with Liquid Chromatography-Tandem Mass Spectrometry.

Anal Sci 2018 ;34(4):439-444

Department of Chemistry, National Taiwan Normal University.

Resveratrol is one of representative ingredient in red wine, but its quantification is a challenge because of a complex and abundant matrix. In this study, two sample pretreatments, direct dilution and QuEChERS extraction, coupling LCMS analysis were examined for resveratrol quantification. Similar recoveries of 106.4 to 93.7% were obtained for direct dilution and QuEChERS, respectively. With the aid of condition optimization, QuEChERS extraction could concentrate the resveratrol from red wines to improve the detection sensitivity with a LOD value of 2.5 ng/mL, which is four-times greater than the direct dilution approach. As a result, the QuEChERS method can provide a high linearity within the concentration range of 5 - 500 ng/mL, in which direct dilution produced the linear calibration curve within the concentrations of 25 - 500 ng/mL. A high consistency was obtained for both approaches in which intra-day precisions were within 0.5 to 7.2% (n = 3), and the inter-day precisions were within 7.8 to 16.0% (n = 9). Overall, the sample pretreatment of QuEChERS can effectively reduce the matrix effect, which leads LCMS to quantify the low resveratrol abundance of 8.0 ppb in each red wine sample, which is not achieved with the direct dilution approach.
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http://dx.doi.org/10.2116/analsci.17P528DOI Listing
September 2018

A Brief Review of Bioinformatics Tools for Glycosylation Analysis by Mass Spectrometry.

Mass Spectrom (Tokyo) 2017 24;6(Spec Iss):S0064. Epub 2017 Feb 24.

Department of Chemistry, National Taiwan Normal University.

The purpose of this review is to provide updated information regarding bioinformatic software for the use in the characterization of glycosylated structures since 2013. A comprehensive review by Woodin 138: 2793-2803, 2013 (ref. 1) described two main approaches that are introduced for starting researchers in this area; analysis of released glycans and the identification of glycopeptide in enzymatic digests, respectively. Complementary to that report, this review focuses on mass spectrometry related bioinformatics tools for the characterization of N-linked and O-linked glycopeptides. Specifically, it also provides information regarding automated tools that can be used for glycan profiling using mass spectrometry.
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http://dx.doi.org/10.5702/massspectrometry.S0064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358406PMC
February 2017

Identification of Cofilin-1 Induces G0/G1 Arrest and Autophagy in Angiotensin-(1-7)-treated Human Aortic Endothelial Cells from iTRAQ Quantitative Proteomics.

Sci Rep 2016 10 17;6:35372. Epub 2016 Oct 17.

Cardiovascular &Translational Medicine Laboratory, Department of Biotechnology, Hung Kuang University, No. 1018, Sec. 6, Taiwan Boulevard, Shalu District, Taichung Taiwan 43302, R.O.C.

The angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis is a pathway that acts against the detrimental effects of the renin-angiotensin system. However, the effects of angiotensin-(1-7) on endothelial protein expression and the related phenotypes are unclear. We performed a duplicate of iTRAQ quantitative proteomic analysis on human aortic endothelial cells (HAECs) treated with angiotensin-(1-7) for 6 hours. Cofilin-1 was identified as a highly abundant candidate with consistent >30% coverage and >1.2-fold overexpression in the angiotensin-(1-7)-treated group. Gene ontology analysis showed that the "regulation_of_mitosis" was significantly altered, and cell cycle analysis indicated that the 6-hour angiotensin-(1-7) treatment significantly induced G0/G1 arrest. Knockdown of the cofilin-1 (CFL1) gene suggested the G0/G1 phase arrest was mediated by the modulation of p27 and the p21/Cyclin/CDK complex by Cofilin-1. Interestingly, quiescent HAECs escaped G0/G1 arrest upon angiotensin-(1-7) treatment for 24 hours, and angiotensin-(1-7) induced autophagy by upregulating Beclin-1 and microtubule-associated protein 1 light chain 3b-II expression, which was also attenuated by A779 pre-treatment and CFL1 knockdown. After pre-treatment with 3-methyladenine (3MA), treatment with angiotensin-(1-7) for 24 h induced significant G0/G1 phase arrest and apoptosis, suggesting a pro-survival role of autophagy in this context. In conclusion, Cofilin-1 plays a dominant role in angiotensin-(1-7)-induced G0/G1 arrest and autophagy to maintain cellular homeostasis in HAECs.
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http://dx.doi.org/10.1038/srep35372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066316PMC
October 2016

Evaluation of disulfide scrambling during the enzymatic digestion of bevacizumab at various pH values using mass spectrometry.

Biochim Biophys Acta 2016 09 27;1864(9):1188-1194. Epub 2016 May 27.

National Taiwan Normal University, Department of Chemistry, Taipei, Taiwan. Electronic address:

Disulfide linkages play an important role in protein stability and activity. Thus, it is critical to characterize disulfide bonds to ensure the quality and function of protein pharmaceuticals. There are, however, problems associated with maintaining disulfide linkages in the conventional procedures that are used to digest a protein. In order to preserve enzyme activity during the digestion of a protein, it is commonly carried out at neutral to basic environment which increases the possibilities of disulfide bond scrambling. However, it is not easy to differentiate whether the scrambled disulfide linkages are initiated by the sample itself or whether they are induced during the protease digestion process. In this study, the optimum pH for minimizing disulfide bond rearrangements during the digestion process was determined. Three sets of proteases, trypsin plus Glu-C, Lys-C and thermolysin were used, followed by dimethyl labeling and mass spectrometry for a bevacizumab (Avastin) disulfide linkage analysis. No disulfide linkage scrambling was detected at pH6 when Lys-C or trypsin plus Glu-C were used as enzymes. When thermolysin was applied, some scrambled disulfide bonds were identified at pH5, 6 and 7. Nevertheless, there was less disulfide bond scrambling at a lower pH. All correct disulfide bonds on bevacizumab could be identified using this approach. The results demonstrated that by choosing the proper enzymes, using a lower pH environment for the digestion could reduce the degree of artifact disulfide scrambling.
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http://dx.doi.org/10.1016/j.bbapap.2016.05.011DOI Listing
September 2016

GALNT14 genotype effectively predicts the therapeutic response in unresectable hepatocellular carcinoma treated with transcatheter arterial chemoembolization.

Pharmacogenomics 2016 Mar 12;17(4):353-66. Epub 2016 Feb 12.

Liver Research Center, Chang Gung Memorial Hospital, No. 5, Fu-Sing Street, Guishan Township, Taoyuan, Taiwan.

Aim: Transcatheter arterial chemoembolization is currently the standard treatment in hepatocellular carcinoma patients with Barcelona Clinic Liver Cancer stage B. Genomic variants of GALNT14 were recently identified as effective predictors for chemotherapy responses in Barcelona Clinic Liver Cancer stage C patients.

Methods: We investigated the prognosis predictive value of GALNT14 genotypes in 327 hepatocelluar carcinoma patients treated by transcatheter arterial chemoembolization.

Result: Cox proportional hazards model analysis showed that the genotype 'TT' was associated with shorter time-to-response (multivariate p < 0.001), time-to-complete-response (p = 0.004) and longer time-to-tumor progression (p < 0.001), compared with the genotype 'non-TT'. In patients with albumin <3.5 g/dl, genotype 'TT' was associated with longer overall survival (p = 0.027). Finally, genotype 'TT' correlated with higher cancer-to-noncancer ratios of GALNT14 protein levels, lower cancer-to-noncancer ratios of antiapoptotic cFLIP-S, and a clustered glycosylation pattern in the extracellular domain of death receptor 5.

Conclusion: GALNT14 genotypes were significantly associated with clinical outcomes of transcatheter arterial chemoembolization. The differential status of extrinsic apoptotic signaling between cancerous and non-cancerous tissues might underlie the clinical association.
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http://dx.doi.org/10.2217/pgs.15.179DOI Listing
March 2016

Adenine supplement delays senescence in cultured human follicle dermal papilla cells.

Exp Dermatol 2016 Feb 23;25(2):162-4. Epub 2015 Nov 23.

Department of Life Science, Catholic Fu-Jen University, New Taipei City, Taiwan.

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http://dx.doi.org/10.1111/exd.12881DOI Listing
February 2016

Differential proteomics of monosodium urate crystals-induced inflammatory response in dissected murine air pouch membranes by iTRAQ technology.

Proteomics 2015 Oct 17;15(19):3338-48. Epub 2015 Aug 17.

Department of Chemistry, National Taiwan Normal University, Taipei 116, Taiwan.

The precipitation of monosodium urate crystals within joints triggers an acute inflammatory reaction that is the root cause of gout. The inflammation induced by the injection of MSU crystals into the murine air pouch for 1, 3, and 5 h was examined by iTRAQ-based proteomic profiling. The iTRAQ-labeled peptides were fractionated by SCX, basic-RP or solution-IEF, followed by LC-MS/MS analysis. A total of 951 proteins were quantified from the total combined fractions. Among them, 317 proteins exhibited a differential expression, compared to that of the controls at one time point or more. The majority of the differentially expressed proteins were found in the sample after a 5-h MSU treatment. Western blot revealed that the expression levels of cathelin-related antimicrobial peptide and S100A9 were positively correlated with the time-course treated with MSU. Further analysis of GeneGO pathway demonstrated that these differentially expressed proteins are primarily related to the immune-related complement system and the tricarboxylic acid cycle. Moreover, seven genes from the TCA cycle were found to be significantly downregulated at the transcriptional level and its correlation with gout and possible therapeutic applications are worth further investigation. Last, we found that pyruvate carboxylation could be potential targets for antigout treatment.
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http://dx.doi.org/10.1002/pmic.201400626DOI Listing
October 2015

iTRAQ quantitative proteomics-based identification of cell adhesion as a dominant phenotypic modulation in thrombin-stimulated human aortic endothelial cells.

Thromb Res 2015 May 27;135(5):944-50. Epub 2015 Feb 27.

Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan. Electronic address:

Introduction: The phenotypic changes in thrombin-stimulated endothelial cells include alterations in permeability, cell shape, vasomotor tone, leukocyte trafficking, migration, proliferation, and angiogenesis. Previous studies regarding the pleotropic effects of thrombin on the endothelium used human umbilical vein endothelial cells (HUVECs)-cells derived from fetal tissue that does not exist in adults. Only a few groups have used screening approaches such as microarrays to profile the global effects of thrombin on endothelial cells. Moreover, the proteomic changes of thrombin-stimulated human aortic endothelial cells (HAECs) have not been elucidated.

Materials And Methods: HAECs were stimulated with 2 units/mL thrombin for 5h and their proteome was investigated using isobaric tags for the relative and absolute quantification (iTRAQ) and the MetaCore(TM) software.

Results: A total of 627 (experiment A) and 622 proteins (experiment B) were quantified in the duplicated iTRAQ analyses. MetaCore(TM) pathway analysis identified cell adhesion as a dominant phenotype in thrombin-stimulated HAECs. Replicated iTRAQ data revealed that "Cell adhesion_Chemokines and adhesion," "Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity," and "Cell adhesion_Integrin-mediated cell adhesion and migration" were among the top 10 statistically significant pathways. The cell adhesion phenotype was verified by increased THP-1 adhesion to thrombin-stimulated HAECs. In addition, the expression of ICAM-1, VCAM-1, and SELE was significantly upregulated in thrombin-stimulated HAECs.

Conclusions: Several regulatory pathways are altered in thrombin-stimulated HAECs, with cell adhesion being the dominant altered phenotype. Our findings show the feasibility of the iTRAQ technique for evaluating cellular responses to acute stimulation.
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http://dx.doi.org/10.1016/j.thromres.2015.02.031DOI Listing
May 2015

Quantitative analysis of prostate specific antigen isoforms using immunoprecipitation and stable isotope labeling mass spectrometry.

Anal Chem 2015 Jan 12;87(1):545-53. Epub 2014 Dec 12.

Department of Chemistry, National Taiwan Normal University , Taipei 11677 Taiwan.

Prostate specific antigen (PSA) is a widely used serum marker for prostate cancer (PCa), but has limited specificity for distinguishing early PCa from benign prostatic hyperplasia (BPH). Recently, proPSAs, comprised of native proPSA, as well as truncated proPSA forms, [-2] proPSA, [-5] proPSA, and [-7] proPSA, have been shown to be better diagnostic targets than PSA for PCa. Stable isotope labeling-multiple reaction monitoring mass spectrometry (SIL/MRM-MS) has been frequently used to measure low-abundance biomarkers in tissues and biofluids, owing to its high sensitivity and specificity, simplicity, and multiplexing capability. In this study, we have developed and optimized a strategy using immunoprecipitation in conjunction with SIL/MRM-MS assay which is capable of sensitive and accurate quantification of proPSA in serum. Since serum and plasma are by far the most complex biological fluids, the immunoprecipitation workflow was optimized to achieve sufficient sensitivity, efficiencies of protein purification with immunoaffinity depletion were determined. The developed strategy can detect proPSA and PSA with a limit of detection (LOD) and limit of quantitation (LOQ) at nanogram per milliliter levels, corresponding to a concentration 6 orders-of-magnitude lower than the most abundant serum proteins. Furthermore, the simultaneous measurement of multiple biomarkers, including the mature and precursor forms of PSA, can be achieved in a single multiplexed analysis using LC/MRM-MS. The strategy demonstrated here provides an attractive alternative to ELISAs or RIAs for the reliably measurement of proPSA to improve the specificity of PCa diagnosis.
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http://dx.doi.org/10.1021/ac5033066DOI Listing
January 2015

Global disulfide bond profiling for crude snake venom using dimethyl labeling coupled with mass spectrometry and RADAR algorithm.

Anal Chem 2014 Sep 20;86(17):8742-50. Epub 2014 Aug 20.

Mithra Biotechnology Inc., 7F, No. 104, Sec. 1, Xintai 5th Road, Xizhi Dist., New Taipei City 221, Taiwan.

Snake venom consists of toxin proteins with multiple disulfide linkages to generate unique structures and biological functions. Determination of these cysteine connections usually requires the purification of each protein followed by structural analysis. In this study, dimethyl labeling coupled with LC-MS/MS and RADAR algorithm was developed to identify the disulfide bonds in crude snake venom. Without any protein separation, the disulfide linkages of several cytotoxins and PLA2 could be solved, including more than 20 disulfide bonds. The results show that this method is capable of analyzing protein mixture. In addition, the approach was also used to compare native cytotoxin 3 (CTX III) and its scrambled isomer, another category of protein mixture, for unknown disulfide bonds. Two disulfide-linked peptides were observed in the native CTX III, and 10 in its scrambled form, X-CTX III. This is the first study that reports a platform for the global cysteine connection analysis on a protein mixture. The proposed method is simple and automatic, offering an efficient tool for structural and functional studies of venom proteins.
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http://dx.doi.org/10.1021/ac501931tDOI Listing
September 2014

Differential proteomic analysis of cancer stem cell properties in hepatocellular carcinomas by isobaric tag labeling and mass spectrometry.

J Proteome Res 2013 Aug 17;12(8):3573-85. Epub 2013 Jul 17.

Strategic Business and Innovation Technology Development Division, Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute, 195 Sec.4 Chung Hsing Road, Chutung, 31040 Hsinchu, Taiwan.

Malignant tumors are relatively resistant to treatment due to their heterogeneous nature, drug resistance, and tendency for metastasis. Recent studies suggest that a subpopulation of cancer cells is responsible for the malignant outcomes. These cells are considered as cancer stem cells (CSC). Although a number of molecules have been identified in different cancer cells as markers for cancer stem cells, no promising markers are currently available for hepatocellular carcinoma cells. In this study, two clones of Hep3B cell lines were functionally characterized as control or CSC-like cells, based on properties including spheroid formation, drug resistance, and tumor initiation. Furthermore, their protein expression profiles were investigated by isobaric tags for relative and absolute quantitation (iTRAQ), and a total of 1,127 proteins were identified and quantified from the combined fractions; 50 proteins exhibited at least 2-fold differences between these two clones. These 50 proteins were analyzed by GeneGo and were found to be associated with liver neoplasms, hepatocellular carcinoma (HCC), and liver diseases. They were also components of metabolic pathways, immune responses, and cytoskeleton remodeling. Among these proteins, the expressions of S100P, S100A14, and vimentin were verified in several HCC cell lines, and their expressions were correlated with tumorigenicity in HCC cell lines. The functional significance of vimentin and S100A14 were also investigated and verified.
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http://dx.doi.org/10.1021/pr4004294DOI Listing
August 2013

Evaluation of peptide fractionation strategies used in proteome analysis.

J Sep Sci 2012 Dec 29;35(23):3293-301. Epub 2012 Oct 29.

Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan.

Peptide fractionation is extremely important for the comprehensive analysis of complex protein mixtures. Although a few comparisons of the relative separation efficiencies of 2-D methodologies using complex biological samples have appeared, a systematic evaluation was conducted in this study. Four different fractionation methods, namely strong-cation exchange, hydrophilic interaction chromatography, alkaline-RP and solution isoelectric focusing, which can be used prior to LC-MS/MS analysis, were compared. Strong-cation exchange × RPLC was used after desalting the sample; significantly more proteins were identified, compared with the nondesalted sample (1990 and 1375). We also found that the use of a combination of analytical methods resulted in a dramatic increase in the number of unique peptides that could be identified, compared with only a small increase in protein levels. The increased number of distinct peptides that can be identified is especially beneficial, not only for unequivocally identifying proteins but also for proteomic studies involving posttranslational modifications and peptide-based quantification approaches using stable isotope labeling. The identification and quantification of more peptides per protein provide valuable information that improves both the quantification of, and confidence of protein identification.
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http://dx.doi.org/10.1002/jssc.201200631DOI Listing
December 2012

Essential role of β-human 8-oxoguanine DNA glycosylase 1 in mitochondrial oxidative DNA repair.

Environ Mol Mutagen 2013 Jan 11;54(1):54-64. Epub 2012 Oct 11.

Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.

8-Oxoguanine (8-OG) is the major mutagenic base lesion in DNA caused by reactive oxygen species (ROS) and accumulates in both nuclear and mitochondrial DNA (mtDNA). In humans, 8-OG is primarily removed by human 8-OG DNA glycosylase 1 (hOGG1) through the base excision repair (BER) pathway. There are two major hOGG1 isoforms, designated α- and β-hOGG1, generated by alternative splicing, and they have distinct subcellular localization: cell nuclei and mitochondria, respectively. Using yeast two-hybrid screening assays, we found that β- but not α-hOGG1 directly interacts with the mitochondrial protein NADH:ubiquinone oxidoreductase 1 beta subcomplex 10 (NDUFB10), an integral factor in Complex 1 on the mitochondrial inner membrane. Using coimmunoprecipitation and immunofluorescence studies, we found that this interaction was greatly increased by hydrogen peroxide-induced oxidative stress, suggesting that β- but not α-hOGG1 is localized in the mitochondrial inner membrane. Analyses of nuclear and mtDNA damage showed that the β- but not α- hogg1 knockdown (KD) cells were severely defective in mitochondrial BER, indicating an essential requirement of β-hOGG1 for mtDNA repair. β-hogg1 KD cells were also found to be mildly deficient in Complex I activity, suggesting that β-hOGG1 is an accessory factor for the mitochondrial integral function for ATP synthesis. In summary, our findings define β-hOGG1 as an important factor for mitochondrial BER and as an accessory factor in the mitochondrial Complex I function.
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http://dx.doi.org/10.1002/em.21742DOI Listing
January 2013

miR-27b-regulated TCTP as a novel plasma biomarker for oral cancer: from quantitative proteomics to post-transcriptional study.

J Proteomics 2012 Dec 9;77:154-66. Epub 2012 Aug 9.

Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan.

We combined an iTRAQ-based quantitative proteomic analysis and the miRNA determination to profile potentially novel biomarker from oral cancer. There are 757 and 674 unique proteins identified from proteomic analysis, and 13 proteins displayed consistent underexpression (<0.67 fold) in normal tissues in comparison with the corresponding tumor tissues. After preliminary screening, EGFR, OAT, TPT1, ITGA6, G3BP1 and CB39L were the six genes validated in the 37 oral cancer patients (T1, n=10; T2, n=10; T3, n=10 and T4, n=7). The TPT1, ITGA6 and CAB39L genes were displayed the higher transcriptions level in the tumor tissues and the TPT1, ITGA6 and CAB39L proteins were also shown overexpression in the tumor tissues from the same patients. The miR-19a, 19b, 27a, 27b, 186, 203 and 377 transcripts were predicted and the miR-27b level was shown to significantly reduce in the tumor tissues and the plasma of OSCC patients. In the in vitro study, the overexpression of miR-27b only significantly decreased TCTP protein and gene levels in both HSC-3 and Cal-27 cell lines. Our results demonstrate that human miR-27b regulates the expression of the TCTP tumor protein, and circulating miR-27b may be useful as a biomarker for oral cancer research.
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http://dx.doi.org/10.1016/j.jprot.2012.07.039DOI Listing
December 2012

Automatic disulfide bond assignment using a1 ion screening by mass spectrometry for structural characterization of protein pharmaceuticals.

Anal Chem 2012 Jun 8;84(11):4900-6. Epub 2012 May 8.

Mithra Biotechnology Inc., 5F, Number 79, Section 1, Sintai Fifth Road, Sijhih District, New Taipei City 221, Taiwan.

An automatic method for disulfide bond assignment using dimethyl labeling and computational screening of a(1) ions with customized software, RADAR, is developed. By utilization of the enhanced a(1) ions generated from labeled peptides, the N-terminal amino acids from disulfide-linked peptides can be determined. In this study, we applied this method for structural characterization of recombinant monoclonal antibodies, an important group of therapeutic proteins. In addition to a(1) ion screening and molecular weight match, new RADAR is capable of confirming the matched peptide pairs by further comparing the collision-induced dissociation (CID) fragment ions. With the N-terminal amino acid identities as a threshold, the identification of disulfide-linked peptide pairs can be achieved rapidly at a higher confidence level. Unlike most current approaches, prior knowledge of disulfide linkages or a high-end mass spectrometer is not required, and tedious work or deliberate interpretation can be avoided in this study. Our approach makes it possible to analyze unknown disulfide bonds of protein pharmaceuticals as well as their degraded forms without further protein separation. It can be used as a convenient quality examination tool during biopharmaceutical development and manufacturing processes.
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http://dx.doi.org/10.1021/ac3005007DOI Listing
June 2012

Nonmuscle myosin IIA (myosin heavy polypeptide 9): a novel class of signal transducer mediating the activation of G alpha h/phospholipase C-delta 1 pathway.

Endocrinology 2010 Mar 12;151(3):876-85. Epub 2010 Jan 12.

School of Pharmacy, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.

The dimeric Gh protein is comprised of alpha (tissue transglutaminase) and beta (Calreticulin) subunits and known to be associated with FSH-, oxytocin-, or epinephrine-receptors/functions in their respective target cells. After establishing the FSH-induced activation of G alpha h/phospholipase C (PLC)-delta 1 pathway in rat Sertoli cells (SCs), we have attempted to identify a possible G alpha h-coupled novel FSH receptor (FSH-R). Remarkably, a protein with approximately 240-kDa molecular mass was coimmunoprecipitated with G alpha h in the fractionated membrane proteins of rat SCs. The protein was identified as myosin heavy polypeptide 9 (MyH9) by mass spectrometric analysis and immunoblotting. In addition, immunoprecipitation analysis reveals that MyH9 is constitutively associated with classical Gs-coupled FSH-R and inactive GDP-bound G alpha h at resting state of rat SCs, but did not interact with FSH directly as judged by Far-Western analysis. Upon the stimulation of higher levels of extracellular FSH (>1000 IU/liter), classical FSH-R induces the phosphorylation of MyH9, the dissociation of active GTP-bound G alpha h from FSH-R:MyH9 complexes, and the elicitation of G alpha h/PLC-delta 1 pathway-dependent Ca(2+)-influx in rat SCs. Furthermore, the specific inhibition of MyH9 ATPase activity with Blebbistatin dose-dependently suppressed FSH-induced G alpha h/PLC-delta 1 signaling and Ca(2+)-influx, but not intracellular cAMP accumulation in rat SCs, implying that MyH9 mediates FSH-induced activation of G alpha h/PLC-delta 1/IP(3)/Ca(2+)-influx pathway in rat SCs. This is the first to demonstrate that the filament protein MyH9 constitutively forms a ternary complex with FSH-R and inactive GDP-bound G alpha h. At higher FSH levels, this ternary complex executes an alternative signaling of classical Gs-coupled FSH-R through activating a Gs/cAMP-independent, G alpha h/PLC-delta 1 pathway in rat SCs.
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http://dx.doi.org/10.1210/en.2009-0722DOI Listing
March 2010

Protein profilings in mouse liver regeneration after partial hepatectomy using iTRAQ technology.

J Proteome Res 2009 Feb;8(2):1004-13

Biomedical Engineering Research Laboratories, Industrial Technology Research Institute, Hsinchu 310, Taiwan.

Liver is unique in its capability to regenerate after an injury. Liver regeneration after a 2/3 partial hepatectomy served as a classical model and is adopted frequently to study the mechanism of liver regeneration. In the present study, semiquantitative analysis of protein expression in mouse liver regeneration following partial hepatectomy was performed using an iTRAQ technique. Proteins from pre-PHx control livers and livers regenerating for 24, 48 and 72 h were extracted and inspected using 4-plex isotope labeling, followed by liquid chromatography fractionation, mass spectrometry and statistical differential analysis. A total of 827 proteins were identified in this study. There were 270 proteins for which quantitative information was available at all the time points in both biologically duplicate experiments. Among the 270 proteins, Car3, Mif, Adh1, Lactb2, Fabp5, Es31, Acaa1b and LOC100044783 were consistently down-regulated, and Mat1a, Dnpep, Pabpc1, Apoa4, Oat, Hpx, Hp and Mt1 were up-regulated by a factor of at least 1.5 from that of the controls at one time point or more. The regulation of each differential protein was also demonstrated by monitoring its time-dependent expression changes during the regenerating process. We believe this is the first report to profile the protein changes in liver regeneration utilizing the iTRAQ proteomic technique.
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http://dx.doi.org/10.1021/pr800696mDOI Listing
February 2009

Screening, purification, and identification of a copper-dependent FITC-binding protein in human plasma: albumin.

J Chromatogr B Analyt Technol Biomed Life Sci 2008 Feb 18;863(1):187-91. Epub 2008 Jan 18.

Graduate Institute of Pharmacy, Taipei Medical University, Taipei, Taiwan.

In this study, a protein purified by fluorescein isothiocyanate (FITC)-affinity chromatography from human plasma was identified as albumin by MALDI-TOF-MS. Albumin was found to conjugate with FITC-labeled molecules through a copper-dependent reaction. The formation of this complex was confirmed by methods including a newly developed "charcoal-based fluorescence assay" (CFA), gel-filtration, affinity chromatography, and ultrafiltration. The binding was identified as disulfide bridge formation. This is the first to demonstrate that copper induces a covalent binding of FITC-labeled molecules with albumin. In addition, the developed CFA method facilitates the screening of small fluorescent dyes binding to macromolecules.
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http://dx.doi.org/10.1016/j.jchromb.2008.01.019DOI Listing
February 2008

The possible interaction of CDA14 and protein elongation factor 1alpha.

Biochim Biophys Acta 2008 Feb 17;1784(2):312-8. Epub 2007 Oct 17.

Department of Proteomics Technology, Industrial Technology Research Institute, 195, Section 4 Chung Hsing Road, Chutung, Hsinchu 310, Taiwan.

The CDA14, a 45kD protein, is currently annotated as PTX1-like protein or ERGIC 32. Over expressing CDA14 can slow PC11 cell proliferation rate. In HepG2 cells, it had been demonstrated that CDA14 is involved in protein transportation. The knowledge about the protein is very limited and not clarified. The CDA14 and its homologous proteins form a family and are restricted to eukaryotes. In the family, there are no homologous sequences with resolved three-dimensional structure and their functions are difficult to predict. Transcriptional expression of CDA14 in three hepatoma cell lines, Hu7, HCC and HepG2, was lower than normal liver tissue and liver carcinoma tissue. In this study, functional proteomic techniques were utilized in searching the interacting counterpart of CDA14. Several proteins involved in protein translation and folding were selectively precipitated with CDA14 and identified mass spectrometry. Interaction of CDA14 and elongation factor 1alpha was confirmed by Western blotting and confocal microscopy. Elongation factor 1alpha is a multiple function protein and involved in several biological mechanisms, including protein synthesis, cell proliferation, apoptosis and tumorigensis. Over-expression of CDA14 down regulated the proliferation of HepG2 cells. These results suggest that CDA14 participated in the elongation factor 1alpha regulated mechanisms.
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http://dx.doi.org/10.1016/j.bbapap.2007.10.006DOI Listing
February 2008

Subcellular and functional proteomic analysis of the cellular responses induced by Helicobacter pylori.

Mol Cell Proteomics 2006 Apr 9;5(4):702-13. Epub 2006 Jan 9.

Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei 100, Taiwan.

Helicobacter pylori infection is a crucial factor in the pathogenesis of several digestive disorders, including peptic ulcers, chronic gastritis, and gastric cancer. Moreover H. pylori induces disease-specific protein expression in gastric epithelial cells. The aim of the present study was to characterize proteins differentially expressed in H. pylori-infected gastric epithelial AGS cells. An in vitro model was established using a multiplicity of infection of 100 and evaluating the effectiveness of H. pylori infection by functional analyses. Changes in protein patterns were identified using a proteomic approach consisting of two-dimensional fluorescence difference gel electrophoresis and mass spectrometry. The expression of many proteins was found to be altered, and 28 of these were identified and classified as protein synthesis- and folding-related proteins, cytoskeleton proteins, metabolic enzymes, transcription- and translation-related proteins, angiogenesis/metastasis-related proteins, cell communication/signal transduction-related proteins, or others (oxygen-regulated protein and oncoprotein). The expression profiles of eight of these proteins, laminin gamma-1 chain precursor, valosin-containing protein, heat shock 70-kDa protein, mitochondrial matrix protein P1, FK506-binding protein 4, T-complex protein 1, enolase alpha, and 14-3-3 beta were further examined in cancerous and paired surrounding normal tissues by immunoblot assay and immunohistochemical staining to identify molecular targets that may be involved in the pathogenesis of H. pylori-induced gastric diseases. On the basis of our results, valosin-containing protein, mitochondrial matrix protein P1, T-complex protein 1, enolase alpha, and 14-3-3 beta may play a crucial role in H. pylori-induced gastric carcinogenesis by mediating antiapoptotic and proliferative responses.
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http://dx.doi.org/10.1074/mcp.M500029-MCP200DOI Listing
April 2006

Assembly of homotrimeric type XXI minicollagen by coexpression of prolyl 4-hydroxylase in stably transfected Drosophila melanogaster S2 cells.

Biochem Biophys Res Commun 2005 Oct;336(2):375-85

Department of Applied Gene Technology, Biomedical Engineering Center, Industrial Technology Research Institute, Taiwan, ROC.

We established stably transfected insect cell lines containing cDNAs encoding the alpha and beta subunits of human prolyl 4-hydroxylase in both Trichoplusia ni and Drosophila melanogaster S2 cells. The expression level and enzymatic activity of recombinant prolyl 4-hydroxylase produced in the Drosophila expression system were significantly higher than those produced in the T. ni system. We further characterized the involvement of prolyl 4-hydroxylase in the assembly of the three alpha chains to form trimeric type XXI minicollagen, which comprises the intact C-terminal non-collagenous (NC1) and collagenous domain (COL1), in the Drosophila system. When minicollagen XXI was stably expressed in Drosophila S2 cells alone, negligible amounts of interchain disulfide-bonded trimers were detected in the culture media. However, minicollagen XXI was secreted as disulfide-bonded homotrimers by coexpression with prolyl 4-hydroxylase in the stably transfected Drosophila S2 cells. Minicollagen XXI coexpressed with prolyl 4-hydroxylase contained sufficient amounts of hydroxyproline to form thermal stable pepsin-resistant triple helices consisting of both interchain and non-interchain disulfide-bonded trimers. These results demonstrate that a sufficient amount of active prolyl 4-hydroxylase is required for the assembly of type XXI collagen triple helices in Drosophila cells and the trimeric assembly is governed by the C-terminal collagenous domain.
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http://dx.doi.org/10.1016/j.bbrc.2005.08.018DOI Listing
October 2005
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