DOCSLIB.ORG

Serum Nardilysin, a Surrogate Marker for Epithelial-Mesenchymal Transition, Predicts Prognosis of Intrahepatic Cholangiocarcinoma After Surgical Resection

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Serum Nardilysin, a Surrogate Marker for Epithelial-Mesenchymal Transition, Predicts Prognosis of Intrahepatic Cholangiocarcinoma After Surgical Resection Author Manuscript Published OnlineFirst on October 23, 2018; DOI: 10.1158/1078-0432.CCR-18-0124 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Title Page Serum nardilysin, a surrogate marker for epithelial-mesenchymal transition, predicts prognosis of intrahepatic cholangiocarcinoma after surgical resection 5 Authors Tomoaki Yoh1), Etsuro Hatano2), Yosuke Kasai1), Hiroaki Fuji1), Kiyoto Nishi3), Kan Toriguchi2), Hideaki Sueoka2), Mikiko Ohno4), Satoru Seo1), Keiko Iwaisako5), Kojiro Taura1), Rina Yamaguchi6), Masato Kurokawa6), Jiro Fujimoto2), Takeshi Kimura3), Shinji Uemoto1), Eiichiro 10 Nishi4) Affiliations 1) Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan 2) Department of Surgery, Hyogo College of Medicine, Nishinomiya, Japan 15 3) Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan 4) Department of Pharmacology, Shiga University of Medical Science, Otsu, Japan 5) Department of Medical Life Systems, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan 20 6) Sanyo Chemical Industries Ltd., Kyoto, Japan Corresponding author: Eiichiro Nishi, MD, PhD Department of Pharmacology, Shiga University of Medical Science, Seta-Tsukinowa-cho, Otsu, 25 520-2192, Japan Tel.: +81-77-548-2181, Fax: +81-77-548-2183, E-mail: [email protected] 1 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 23, 2018; DOI: 10.1158/1078-0432.CCR-18-0124 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Etsuro Hatano, MD, PhD Department of Surgery, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo, 663-8501, Japan Tel.: +81-798-45-6582, Fax: +81-798-45-6581, E-mail: [email protected] 5 Running title: Nardilysin is a novel prognostic biomarker for ICC Key words: 10 Intrahepatic cholangiocarcinoma, nardilysin, epithelial-mesenchymal transition, biomarker, surgery Conflicts of Interest: The authors declare no conflict of interest. 15 Abbreviations ICC: intrahepatic cholangiocarcinoma HCC: hepatocellular carcinoma OS: overall survival NRDC: nardilysin (N-arginine dibasic convertase) 20 HB-EGF: heparin-binding epidermal growth factor TNF-α: tumor necrosis factor-α ADAM: a disintegrin and metalloproteinase HDAC3: Histone deacetylase 3 PGC-1α: peroxisome proliferator-activated receptor gamma coactivator 1-alpha 25 IHC: immunohistochemistry AJCC: American Joint Committee on Cancer 2 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 23, 2018; DOI: 10.1158/1078-0432.CCR-18-0124 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ELISA: enzyme-linked immunosorbent assay CM: condition medium FBS: fetal bovine serum 5 KD: knockdown NC: negative control qRT-PCR: quantitative real-time polymerase chain reaction WST-8: tetrazolium salt-based proliferation assay CCK8: Cell Counting Kit-8 10 PBS: Phosphate-buffered saline GAPDH: glyceraldehyde-3-phosphate dehydrogenase DFS: disease-free survival ROC: receiver operating characteristic AUC: area under the curve 15 SD: standard deviation HC: healthy controls LN: lymph node CEA: Carcinoembryonic antigen CA19-9: carbohydrate antigen 19-9 20 EMT: epithelial-mesenchymal transition ZEB1: zinc finger E-box binding homeobox 1 EMT-TFs: EMT-inducing transcription factors SOX2: sex-determining region Y-box 2 CSC: cancer stem cell 25 HIF-1α: hypoxia-inducible factor-1α IGF-1: insulin-like growth factors-1 3 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 23, 2018; DOI: 10.1158/1078-0432.CCR-18-0124 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. TGF-β: transforming growth factor-β EGF: epidermal growth factor 4 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 23, 2018; DOI: 10.1158/1078-0432.CCR-18-0124 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Purpose: Few studies have investigated prognostic biomarkers in patients with intrahepatic 5 cholangiocarcinoma (ICC). Nardilysin (NRDC), a metalloendopeptidase of the M16 family, has been suggested to play important roles in inflammation and several cancer types. We herein examined the clinical significance and biological function of NRDC in ICC. Experimental Design: We measured serum NRDC levels in 98 ICC patients who underwent surgical resection in two 10 independent cohorts to assess its prognostic impact. We also analyzed NRDC mRNA levels in cancerous tissue specimens from 43 ICC patients. We investigated the roles of NRDC in cell proliferation, migration, gemcitabine sensitivity, and gene expression in ICC cell lines using gene silencing. Results: 15 High serum NRDC levels were associated with shorter overall survival and disease-free survival in the primary (n=79) and validation (n=19) cohorts. A correlation was observed between serum protein levels and cancerous tissue mRNA levels of NRDC (Spearman’s ρ=0.413, p=0.006). The gene knockdown of NRDC in ICC cell lines attenuated cell proliferation, migration, and tumor growth in xenografts, and increased sensitivity to gemcitabine. The gene knockdown of NRDC was 20 also accompanied by significant changes in the expression of several epithelial-mesenchymal transition (EMT)-related genes. Strong correlations were observed between the mRNA levels of NRDC and EMT-inducing transcription factors, ZEB1 and SNAI1, in surgical specimens from ICC patients. Conclusions: 25 Serum NRDC, a possible surrogate marker reflecting the EMT state in primary tumors, predicts the outcome of ICC after surgical resection. 5 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 23, 2018; DOI: 10.1158/1078-0432.CCR-18-0124 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Translational Relevance Few studies have investigated prognostic biomarkers in intrahepatic cholangiocarcinoma (ICC) that 5 may contribute to establishing adjuvant strategies. Nardilysin (NRDC), a metalloendopeptidase of the M16 family, has been suggested to play important roles in inflammation and several cancer types. The present results revealed 1) a correlation between serum NRDC levels and cancerous tissue mRNA levels of NRDC, 2) that preoperative serum NRDC levels were associated with survival and recurrence, and 3) strong correlations between the mRNA levels of NRDC and EMT- 10 inducing transcription factors, ZEB1 and SNAI1, in ICC cell lines and cancerous tissue. Based on the potential relationship between NRDC and EMT, the preoperative evaluation of serum NRDC has potential as a clinical tool for predicting the postoperative outcomes of ICC patients undergoing surgical resection. 6 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 23, 2018; DOI: 10.1158/1078-0432.CCR-18-0124 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer following hepatocellular carcinoma (HCC), accounting for 5-15% of all primary liver cancers [1-3]. There are marked geographic variations in the incidence of ICC, with a higher incidence in East Asia, while 5 the number of ICC patients has been reported to be increasing worldwide [3, 4]. The survival rate of ICC patients is poor because of the late presentation of the disease and limited therapies. Although surgical resection is the only curative treatment, 30-40% of ICC patients have surgical indications [3]. Moreover, the recurrence rate after surgical resection is 50–60% and the 5-year overall survival (OS) rate after surgical resection is only 25–31% [3, 5, 6], highlighting the need to optimize 10 adjuvant strategies. Recent evidence has suggested that adjuvant chemotherapy is associated with prolonged survival, particularly in some advanced cases [7, 8]. However, there are no established methods to define patient subgroups that need adjuvant strategies. The preoperative measurement of serum tumor markers may identify high-risk patients; however, few studies have investigated biomarkers in ICC patients possibly due to the difficulties associated with collecting large numbers 15 of serum samples from ICC patients [9-11]. Nardilysin (N-arginine dibasic convertase, NRDC) is a zinc peptidase of the M16 family that selectively cleaves dibasic sites [12, 13]. NRDC exhibits widespread expression throughout the body, and regulates multiple biological processes, such as myelination [14], body temperature homeostasis [15], and insulin secretion [16]. Although NRDC is a soluble cytosolic protein without 20 an obvious signal peptide or nuclear localization signal, it shuttles between the cytoplasm and nucleus and is secreted via an as yet unknown mechanism [17]. We
Load more

Recommended publications
  • Nardilysin Is Involved in Autoimmune Arthritis Via the Regulation of Tumour Necrosis Factor Alpha Secretion
    Animal models RMD Open: first published as 10.1136/rmdopen-2017-000436 on 13 July 2017. Downloaded from ORIGINAL articLE Nardilysin is involved in autoimmune arthritis via the regulation of tumour necrosis factor alpha secretion Takayuki Fujii,1 Eiichiro Nishi,2,3 Hiromu Ito,1 Hiroyuki Yoshitomi,4 Moritoshi Furu,5 Namiko Okabe,6 Mikiko Ohno,2 Kiyoto Nishi,2 Yusuke Morita,2 Yugo Morita,1 Masayuki Azukizawa,1 Akinori Okahata,1 Takuya Tomizawa,1 Takeshi Kimura,2 Shuichi Matsuda1 To cite: Fujii T, Nishi E, ABSTRACT Key messages Ito H, et al. Nardilysin is Objective Tumour necrosis factor alpha (TNF-α) plays involved in autoimmune an important role in rheumatoid arthritis (RA). TNF-α is arthritis via the regulation synthesised as a membrane-anchored precursor and is What is already known about this subject? of tumour necrosis factor fully activated by a disintegrin and metalloproteinase 17 ► Tumour necrosis factor alpha (TNF-α), which alpha secretion. RMD Open (ADAM17)-mediated ectodomain shedding. Nardilysin is activated by ectodomain shedding, plays an 2017;3:e000436. doi:10.1136/ important role in rheumatoid arthritis (RA). rmdopen-2017-000436 (NRDC) facilitates ectodomain shedding via activation of ADAM17. This study was undertaken to elucidate the role ► Nardilysin facilitates TNF-α shedding via enhancing of NRDC in RA. a disintegrin and metalloproteinase 17 activity. Additional material is –/– ► Methods NRDC-deficient Nrdc( ) mice and macrophage- published online only. To view What does this study add? specific NRDC-deficientNrdc ( delM) mice were examined please visit the journal online ► Deletion or inhibition of nardilysin prevents (http:// dx. doi. org/ 10.
    [Show full text]
  • Functional Coverage of the Human Genome by Existing Structures, Structural Genomics Targets, and Homology Models
    Functional Coverage of the Human Genome by Existing Structures, Structural Genomics Targets, and Homology Models Lei Xie, Philip E. Bourne* San Diego Supercomputer Center and Department of Pharmacology, University of California, San Diego, California, United States of America The bias in protein structure and function space resulting from experimental limitations and targeting of particular functional classes of proteins by structural biologists has long been recognized, but never continuously quantified. Using the Enzyme Commission and the Gene Ontology classifications as a reference frame, and integrating structure data from the Protein Data Bank (PDB), target sequences from the structural genomics projects, structure homology derived from the SUPERFAMILY database, and genome annotations from Ensembl and NCBI, we provide a quantified view, both at the domain and whole-protein levels, of the current and projected coverage of protein structure and function space relative to the human genome. Protein structures currently provide at least one domain that covers 37% of the functional classes identified in the genome; whole structure coverage exists for 25% of the genome. If all the structural genomics targets were solved (twice the current number of structures in the PDB), it is estimated that structures of one domain would cover 69% of the functional classes identified and complete structure coverage would be 44%. Homology models from existing experimental structures extend the 37% coverage to 56% of the genome as single domains and 25% to 31% for complete structures. Coverage from homology models is not evenly distributed by protein family, reflecting differing degrees of sequence and structure divergence within families. While these data provide coverage, conversely, they also systematically highlight functional classes of proteins for which structures should be determined.
    [Show full text]
  • The Ectodomain Shedding Database for Membrane-Bound Shed Markers Wei-Sheng Tien1,2, Jun-Hong Chen3 and Kun-Pin Wu1*
    The Author(s) BMC Bioinformatics 2017, 18(Suppl 3):42 DOI 10.1186/s12859-017-1465-7 RESEARCH Open Access SheddomeDB: the ectodomain shedding database for membrane-bound shed markers Wei-Sheng Tien1,2, Jun-Hong Chen3 and Kun-Pin Wu1* From The Fifteenth Asia Pacific Bioinformatics Conference Shenzhen, China. 16-18 January 2017 Abstract Background: A number of membrane-anchored proteins are known to be released from cell surface via ectodomain shedding. The cleavage and release of membrane proteins has been shown to modulate various cellular processes and disease pathologies. Numerous studies revealed that cell membrane molecules of diverse functional groups are subjected to proteolytic cleavage, and the released soluble form of proteins may modulate various signaling processes. Therefore, in addition to the secreted protein markers that undergo secretion through the secretory pathway, the shed membrane proteins may comprise an additional resource of noninvasive and accessible biomarkers. In this context, identifying the membrane-bound proteins that will be shed has become important in the discovery of clinically noninvasive biomarkers. Nevertheless, a data repository for biological and clinical researchers to review the shedding information, which is experimentally validated, for membrane-bound protein shed markers is still lacking. Results: In this study, the database SheddomeDB was developed to integrate publicly available data of the shed membrane proteins. A comprehensive literature survey was performed to collect the membrane proteins that were verified to be cleaved or released in the supernatant by immunological-based validation experiments. From 436 studies on shedding, 401 validated shed membrane proteins were included, among which 199 shed membrane proteins have not been annotated or validated yet by existing cleavage databases.
    [Show full text]
  • Enzyme DHRS7
    Toward the identification of a function of the “orphan” enzyme DHRS7 Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Selene Araya, aus Lugano, Tessin Basel, 2018 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Alex Odermatt (Fakultätsverantwortlicher) und Prof. Dr. Michael Arand (Korreferent) Basel, den 26.6.2018 ________________________ Dekan Prof. Dr. Martin Spiess I. List of Abbreviations 3α/βAdiol 3α/β-Androstanediol (5α-Androstane-3α/β,17β-diol) 3α/βHSD 3α/β-hydroxysteroid dehydrogenase 17β-HSD 17β-Hydroxysteroid Dehydrogenase 17αOHProg 17α-Hydroxyprogesterone 20α/βOHProg 20α/β-Hydroxyprogesterone 17α,20α/βdiOHProg 20α/βdihydroxyprogesterone ADT Androgen deprivation therapy ANOVA Analysis of variance AR Androgen Receptor AKR Aldo-Keto Reductase ATCC American Type Culture Collection CAM Cell Adhesion Molecule CYP Cytochrome P450 CBR1 Carbonyl reductase 1 CRPC Castration resistant prostate cancer Ct-value Cycle threshold-value DHRS7 (B/C) Dehydrogenase/Reductase Short Chain Dehydrogenase Family Member 7 (B/C) DHEA Dehydroepiandrosterone DHP Dehydroprogesterone DHT 5α-Dihydrotestosterone DMEM Dulbecco's Modified Eagle's Medium DMSO Dimethyl Sulfoxide DTT Dithiothreitol E1 Estrone E2 Estradiol ECM Extracellular Membrane EDTA Ethylenediaminetetraacetic acid EMT Epithelial-mesenchymal transition ER Endoplasmic Reticulum ERα/β Estrogen Receptor α/β FBS Fetal Bovine Serum 3 FDR False discovery rate FGF Fibroblast growth factor HEPES 4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic Acid HMDB Human Metabolome Database HPLC High Performance Liquid Chromatography HSD Hydroxysteroid Dehydrogenase IC50 Half-Maximal Inhibitory Concentration LNCaP Lymph node carcinoma of the prostate mRNA Messenger Ribonucleic Acid n.d.
    [Show full text]
  • Serine Proteases with Altered Sensitivity to Activity-Modulating
    (19) & (11) EP 2 045 321 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 08.04.2009 Bulletin 2009/15 C12N 9/00 (2006.01) C12N 15/00 (2006.01) C12Q 1/37 (2006.01) (21) Application number: 09150549.5 (22) Date of filing: 26.05.2006 (84) Designated Contracting States: • Haupts, Ulrich AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 51519 Odenthal (DE) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Coco, Wayne SK TR 50737 Köln (DE) •Tebbe, Jan (30) Priority: 27.05.2005 EP 05104543 50733 Köln (DE) • Votsmeier, Christian (62) Document number(s) of the earlier application(s) in 50259 Pulheim (DE) accordance with Art. 76 EPC: • Scheidig, Andreas 06763303.2 / 1 883 696 50823 Köln (DE) (71) Applicant: Direvo Biotech AG (74) Representative: von Kreisler Selting Werner 50829 Köln (DE) Patentanwälte P.O. Box 10 22 41 (72) Inventors: 50462 Köln (DE) • Koltermann, André 82057 Icking (DE) Remarks: • Kettling, Ulrich This application was filed on 14-01-2009 as a 81477 München (DE) divisional application to the application mentioned under INID code 62. (54) Serine proteases with altered sensitivity to activity-modulating substances (57) The present invention provides variants of ser- screening of the library in the presence of one or several ine proteases of the S1 class with altered sensitivity to activity-modulating substances, selection of variants with one or more activity-modulating substances. A method altered sensitivity to one or several activity-modulating for the generation of such proteases is disclosed, com- substances and isolation of those polynucleotide se- prising the provision of a protease library encoding poly- quences that encode for the selected variants.
    [Show full text]
  • Handbook of Proteolytic Enzymes Second Edition Volume 1 Aspartic and Metallo Peptidases
    Handbook of Proteolytic Enzymes Second Edition Volume 1 Aspartic and Metallo Peptidases Alan J. Barrett Neil D. Rawlings J. Fred Woessner Editor biographies xxi Contributors xxiii Preface xxxi Introduction ' Abbreviations xxxvii ASPARTIC PEPTIDASES Introduction 1 Aspartic peptidases and their clans 3 2 Catalytic pathway of aspartic peptidases 12 Clan AA Family Al 3 Pepsin A 19 4 Pepsin B 28 5 Chymosin 29 6 Cathepsin E 33 7 Gastricsin 38 8 Cathepsin D 43 9 Napsin A 52 10 Renin 54 11 Mouse submandibular renin 62 12 Memapsin 1 64 13 Memapsin 2 66 14 Plasmepsins 70 15 Plasmepsin II 73 16 Tick heme-binding aspartic proteinase 76 17 Phytepsin 77 18 Nepenthesin 85 19 Saccharopepsin 87 20 Neurosporapepsin 90 21 Acrocylindropepsin 9 1 22 Aspergillopepsin I 92 23 Penicillopepsin 99 24 Endothiapepsin 104 25 Rhizopuspepsin 108 26 Mucorpepsin 11 1 27 Polyporopepsin 113 28 Candidapepsin 115 29 Candiparapsin 120 30 Canditropsin 123 31 Syncephapepsin 125 32 Barrierpepsin 126 33 Yapsin 1 128 34 Yapsin 2 132 35 Yapsin A 133 36 Pregnancy-associated glycoproteins 135 37 Pepsin F 137 38 Rhodotorulapepsin 139 39 Cladosporopepsin 140 40 Pycnoporopepsin 141 Family A2 and others 41 Human immunodeficiency virus 1 retropepsin 144 42 Human immunodeficiency virus 2 retropepsin 154 43 Simian immunodeficiency virus retropepsin 158 44 Equine infectious anemia virus retropepsin 160 45 Rous sarcoma virus retropepsin and avian myeloblastosis virus retropepsin 163 46 Human T-cell leukemia virus type I (HTLV-I) retropepsin 166 47 Bovine leukemia virus retropepsin 169 48
    [Show full text]
  • Protein Symbol Protein Name Rank Metric Score 4F2 4F2 Cell-Surface
    Supplementary Table 2 Supplementary Table 2. Ranked list of proteins present in anti-Sema4D treated macrophage conditioned media obtained in the GSEA analysis of the proteomic data. Proteins are listed according to their rank metric score, which is the score used to position the gene in the ranked list of genes of the GSEA. Values are obtained from comparing Sema4D treated RAW conditioned media versus REST, which includes untreated, IgG treated and anti-Sema4D added RAW conditioned media. GSEA analysis was performed under standard conditions in November 2015. Protein Rank metric symbol Protein name score 4F2 4F2 cell-surface antigen heavy chain 2.5000 PLOD3 Procollagen-lysine,2-oxoglutarate 5-dioxygenase 3 1.4815 ELOB Transcription elongation factor B polypeptide 2 1.4350 ARPC5 Actin-related protein 2/3 complex subunit 5 1.2603 OSTF1 teoclast-stimulating factor 1 1.2500 RL5 60S ribomal protein L5 1.2135 SYK Lysine--tRNA ligase 1.2135 RL10A 60S ribomal protein L10a 1.2135 TXNL1 Thioredoxin-like protein 1 1.1716 LIS1 Platelet-activating factor acetylhydrolase IB subunit alpha 1.1067 A4 Amyloid beta A4 protein 1.0911 H2B1M Histone H2B type 1-M 1.0514 UB2V2 Ubiquitin-conjugating enzyme E2 variant 2 1.0381 PDCD5 Programmed cell death protein 5 1.0373 UCHL3 Ubiquitin carboxyl-terminal hydrolase isozyme L3 1.0061 PLEC Plectin 1.0061 ITPA Inine triphphate pyrophphatase 0.9524 IF5A1 Eukaryotic translation initiation factor 5A-1 0.9314 ARP2 Actin-related protein 2 0.8618 HNRPL Heterogeneous nuclear ribonucleoprotein L 0.8576 DNJA3 DnaJ homolog subfamily
    [Show full text]
  • UCLA Electronic Theses and Dissertations
    UCLA UCLA Electronic Theses and Dissertations Title A Liquid Chromatography-Mass Spectrometry Platform for Identifying Protein Targets of Small-Molecule Binding Relevant to Disease and Metabolism Permalink https://escholarship.org/uc/item/8pn170t7 Author O'Brien Johnson, Reid Publication Date 2018 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA Los Angeles A Liquid Chromatography-Mass Spectrometry Platform for Identifying Protein Targets of Small-Molecule Binding Relevant to Disease and Metabolism A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biochemistry and Molecular Biology by Reid Lee O’Brien Johnson 2018 ABSTRACT OF THE DISSERTATION A Liquid Chromatography-Mass Spectrometry Platform for Identifying Protein Targets of Small-Molecule Binding Relevant to Disease and Metabolism by Reid Lee O’Brien Johnson Doctor of Philosophy in Biochemistry and Molecular Biology University of California, Los Angeles, 2018 Professor Joseph Ambrose Loo, Chair Identifying small-molecule binders to protein targets remains a daunting task due to the huge diversity in compound structure, activity, and mechanisms of action. Affinity- based target identification techniques are limited by the necessity to modify each drug individually (without losing bioactivity), while non-affinity based approaches are dependent on the drug’s ability to induce specific biochemical/cellular readouts. To overcome these limitations, we have developed a high-throughput liquid chromatography- mass spectrometry (LC-MS) platform coupled to a universally applicable target identification approach that analyzes direct small-molecule binding to its protein target(s). DARTS (drug affinity responsive target stability) relies on a well-known phenomenon in which ligand binding causes thermodynamic stabilization of its target protein’s structure such that the protein becomes resistant to a variety of insults, including proteolysis.
    [Show full text]
  • 12) United States Patent (10
    US007635572B2 (12) UnitedO States Patent (10) Patent No.: US 7,635,572 B2 Zhou et al. (45) Date of Patent: Dec. 22, 2009 (54) METHODS FOR CONDUCTING ASSAYS FOR 5,506,121 A 4/1996 Skerra et al. ENZYME ACTIVITY ON PROTEIN 5,510,270 A 4/1996 Fodor et al. MICROARRAYS 5,512,492 A 4/1996 Herron et al. 5,516,635 A 5/1996 Ekins et al. (75) Inventors: Fang X. Zhou, New Haven, CT (US); 5,532,128 A 7/1996 Eggers Barry Schweitzer, Cheshire, CT (US) 5,538,897 A 7/1996 Yates, III et al. s s 5,541,070 A 7/1996 Kauvar (73) Assignee: Life Technologies Corporation, .. S.E. al Carlsbad, CA (US) 5,585,069 A 12/1996 Zanzucchi et al. 5,585,639 A 12/1996 Dorsel et al. (*) Notice: Subject to any disclaimer, the term of this 5,593,838 A 1/1997 Zanzucchi et al. patent is extended or adjusted under 35 5,605,662 A 2f1997 Heller et al. U.S.C. 154(b) by 0 days. 5,620,850 A 4/1997 Bamdad et al. 5,624,711 A 4/1997 Sundberg et al. (21) Appl. No.: 10/865,431 5,627,369 A 5/1997 Vestal et al. 5,629,213 A 5/1997 Kornguth et al. (22) Filed: Jun. 9, 2004 (Continued) (65) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2005/O118665 A1 Jun. 2, 2005 EP 596421 10, 1993 EP 0619321 12/1994 (51) Int. Cl. EP O664452 7, 1995 CI2O 1/50 (2006.01) EP O818467 1, 1998 (52) U.S.
    [Show full text]
  • Production of an Antigenic Peptide by Insulin-Degrading Enzyme
    ARTICLES Production of an antigenic peptide by insulin- degrading enzyme Nicolas Parmentier1,2, Vincent Stroobant1,2, Didier Colau1,2, Philippe de Diesbach3, Sandra Morel1,2,6, Jacques Chapiro1,2,6, Peter van Endert4,5 & Benoît J Van den Eynde1,2 Most antigenic peptides presented by major histocompatibility complex (MHC) class I molecules are produced by the proteasome. Here we show that a proteasome-independent peptide derived from the human tumor protein MAGE-A3 is produced directly by insulin-degrading enzyme (IDE), a cytosolic metallopeptidase. Cytotoxic T lymphocyte recognition of tumor cells was reduced after metallopeptidase inhibition or IDE silencing. Separate inhibition of the metallopeptidase and the proteasome impaired degradation of MAGE-A3 proteins, and simultaneous inhibition of both further stabilized MAGE-A3 proteins. These results suggest that MAGE-A3 proteins are degraded along two parallel pathways that involve either the proteasome or IDE and produce different sets of antigenic peptides presented by MHC class I molecules. Degradation of intracellular proteins is a constitutive physiological cancer8,9. This peptide, which corresponds to positions 168–176 process that ensures maintenance of cellular integrity. This highly reg- (EVDPIGHLY) of the MAGE-A3 protein, has been widely used to ulated process essentially occurs along two pathways: the ubiquitin- immunize people with melanoma in clinical trials of cancer immuno- proteasome pathway and the autophagy pathway1. The proteasome therapy10. To investigate the processing of this antigenic peptide, is a self-compartmentalizing protease that degrades proteins tagged we examined whether recognition of tumor cells was affected by for degradation by covalent binding of ubiquitin. Autophagy is the inhibition of the proteasome, which produces the majority of anti- degradation of cellular components in the lysosomal compartment.
    [Show full text]
  • Mass Spectrometry-Based Proteomics Reveals Distinct Mechanisms of Astrocyte Protein Secretion
    University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Summer 2009 Mass Spectrometry-Based Proteomics Reveals Distinct Mechanisms of Astrocyte Protein Secretion Todd M. Greco University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Cell Biology Commons, and the Molecular and Cellular Neuroscience Commons Recommended Citation Greco, Todd M., "Mass Spectrometry-Based Proteomics Reveals Distinct Mechanisms of Astrocyte Protein Secretion" (2009). Publicly Accessible Penn Dissertations. 22. https://repository.upenn.edu/edissertations/22 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/22 For more information, please contact [email protected]. Mass Spectrometry-Based Proteomics Reveals Distinct Mechanisms of Astrocyte Protein Secretion Abstract The ability of astrocytes to secrete proteins subserves many of its known function, such as synapse formation during development and extracellular matrix remodeling after cellular injury. Protein secretion may also play an important, but less clear, role in the propagation of inflammatory responses and neurodegenerative disease pathogenesis. While potential astrocyte-secreted proteins may number in the thousands, known astrocyte-secreted proteins are less than 100. To address this fundamental deficiency, mass spectrometry-based proteomics and bioinformatic tools were utilized for global discovery, comparison, and quantification of astrocyte-secreted proteins. A primary mouse astrocyte cell culture model was used to generate a collection of astrocyte-secreted proteins termed the astrocyte secretome. A multidimensional protein and peptide separation approach paired with mass spectrometric analysis interrogated the astrocyte secretome under control and cytokine-exposed conditions, identifying cytokine- induced secreted proteins, while extending the depth of known astrocyte-secreted proteins to 169.
    [Show full text]
  • A Meta-Analysis of the Effects of High-LET Ionizing Radiations in Human Gene Expression
    Supplementary Materials A Meta-Analysis of the Effects of High-LET Ionizing Radiations in Human Gene Expression Table S1. Statistically significant DEGs (Adj. p-value < 0.01) derived from meta-analysis for samples irradiated with high doses of HZE particles, collected 6-24 h post-IR not common with any other meta- analysis group. This meta-analysis group consists of 3 DEG lists obtained from DGEA, using a total of 11 control and 11 irradiated samples [Data Series: E-MTAB-5761 and E-MTAB-5754]. Ensembl ID Gene Symbol Gene Description Up-Regulated Genes ↑ (2425) ENSG00000000938 FGR FGR proto-oncogene, Src family tyrosine kinase ENSG00000001036 FUCA2 alpha-L-fucosidase 2 ENSG00000001084 GCLC glutamate-cysteine ligase catalytic subunit ENSG00000001631 KRIT1 KRIT1 ankyrin repeat containing ENSG00000002079 MYH16 myosin heavy chain 16 pseudogene ENSG00000002587 HS3ST1 heparan sulfate-glucosamine 3-sulfotransferase 1 ENSG00000003056 M6PR mannose-6-phosphate receptor, cation dependent ENSG00000004059 ARF5 ADP ribosylation factor 5 ENSG00000004777 ARHGAP33 Rho GTPase activating protein 33 ENSG00000004799 PDK4 pyruvate dehydrogenase kinase 4 ENSG00000004848 ARX aristaless related homeobox ENSG00000005022 SLC25A5 solute carrier family 25 member 5 ENSG00000005108 THSD7A thrombospondin type 1 domain containing 7A ENSG00000005194 CIAPIN1 cytokine induced apoptosis inhibitor 1 ENSG00000005381 MPO myeloperoxidase ENSG00000005486 RHBDD2 rhomboid domain containing 2 ENSG00000005884 ITGA3 integrin subunit alpha 3 ENSG00000006016 CRLF1 cytokine receptor like
    [Show full text]