title: A comprehensive method for detecting ubiquitinated substrates using TR-TUBE.
abstract: The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-ε-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrates ubiquitinated by an exogenously overexpressed ubiquitin ligase, allowing detection of the specific activity of the ubiquitin ligase and isolation of its substrates. Although the diGly antibody enabled effective identification of ubiquitinated proteins in cells, overexpression of an ubiquitin ligase and treatment with a proteasome inhibitor did not increase the level of diGly peptides specific for the ligase relative to the background level of diGly peptides, probably due to deubiquitination. By contrast, in TR-TUBE-expressing cells, the level of substrate-derived diGly peptides produced by the overexpressed ubiquitin ligase was significantly elevated. We developed a method for identifying the substrates of specific ubiquitin ligases using two enrichment strategies, TR-TUBE and diGly remnant antibodies, coupled with MS. Using this method, we identified target substrates of FBXO21, an uncharacterized F-box protein.
authors.affiliation: Protein Metabolism Project, yoshida-yk@igakuken.or.jp tanaka-kj@igakuken.or.jp.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Yoshida
authors.affiliation: Laboratory of Protein Metabolism, and.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Saeki
authors.affiliation: Protein Metabolism Project, Laboratory of Protein Metabolism, and.
authors.forename: Arisa
authors.initials: A
authors.lastname: Murakami
authors.affiliation: Protein Metabolism Project.
authors.forename: Junko
authors.initials: J
authors.lastname: Kawawaki
authors.affiliation: Laboratory of Protein Metabolism, and.
authors.forename: Hikaru
authors.initials: H
authors.lastname: Tsuchiya
authors.affiliation: Laboratory of Protein Metabolism, and.
authors.forename: Hidehito
authors.initials: H
authors.lastname: Yoshihara
authors.affiliation: Center for Basic Technology Research, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Shindo
authors.affiliation: Laboratory of Protein Metabolism, and yoshida-yk@igakuken.or.jp tanaka-kj@igakuken.or.jp.
authors.forename: Keiji
authors.initials: K
authors.lastname: Tanaka
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D044783:F-Box Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D020543:Proteome / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D040901:Proteomics; D015203:Reproducibility of Results; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods; D014357:Trypsin / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D057149:Ubiquitinated Proteins / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D000906:Antibodies; D044783:F-Box Proteins; D009842:Oligopeptides; D020543:Proteome; D025801:Ubiquitin; D057149:Ubiquitinated Proteins; D044767:Ubiquitin-Protein Ligases; D014357:Trypsin; D008239:Lysine; D005998:Glycine
keywords: ubiquitin ligase; ubiquitin-binding protein; ubiquitination
references.citation: Nat Cell Biol. 1999 Aug;1(4):193-9
references.pmid: 10559916
references.citation: Nat Biotechnol. 2010 Aug;28(8):868-73
references.pmid: 20639865
references.citation: Nat Cell Biol. 2001 Mar;3(3):321-4
references.pmid: 11231585
references.citation: EMBO Rep. 2002 May;3(5):478-84
references.pmid: 11964378
references.citation: J Biol Chem. 2003 Aug 15;278(33):30854-8
references.pmid: 12840033
references.citation: Science. 2003 Dec 12;302(5652):1972-5
references.pmid: 14671306
references.citation: Nature. 2004 Mar 11;428(6979):190-3
references.pmid: 15014502
references.citation: EMBO J. 2004 May 19;23(10):2116-25
references.pmid: 15103331
references.citation: Mol Cell. 1999 Apr;3(4):527-33
references.pmid: 10230406
references.citation: J Biol Chem. 1999 May 14;274(20):13886-93
references.pmid: 10318797
references.citation: Genes Dev. 2004 Nov 1;18(21):2573-80
references.pmid: 15520277
references.citation: EMBO J. 2005 Jan 12;24(1):160-9
references.pmid: 15577944
references.citation: Biochem Biophys Res Commun. 2005 Oct 21;336(2):530-5
references.pmid: 16140271
references.citation: Mol Cell. 2005 Dec 9;20(5):699-708
references.pmid: 16337594
references.citation: Mol Cell. 2006 Sep 1;23(5):709-21
references.pmid: 16949367
references.citation: Nature. 2006 Sep 7;443(7107):50-5
references.pmid: 16906134
references.citation: Science. 2006 Oct 20;314(5798):467-71
references.pmid: 17053147
references.citation: Science. 2007 May 11;316(5826):900-4
references.pmid: 17463251
references.citation: Mol Cell. 2007 Nov 9;28(3):371-85
references.pmid: 17996702
references.citation: Nat Rev Mol Cell Biol. 2011 May;12(5):295-307
references.pmid: 21448225
references.citation: Biochem Biophys Res Commun. 2011 Jun 24;410(1):24-8
references.pmid: 21640084
references.citation: Mol Cell Biol. 2011 Sep;31(18):3687-99
references.pmid: 21768309
references.citation: Cell. 2011 Oct 14;147(2):459-74
references.pmid: 21963094
references.citation: Mol Cell. 2011 Oct 21;44(2):325-40
references.pmid: 21906983
references.citation: Annu Rev Biochem. 2012;81:203-29
references.pmid: 22524316
references.citation: Genes Dev. 2013 Jan 15;27(2):151-6
references.pmid: 23322298
references.citation: Nature. 2013 Apr 18;496(7445):372-6
references.pmid: 23503661
references.citation: Biochim Biophys Acta. 2014 Jan;1843(1):137-49
references.pmid: 23742843
references.citation: Mol Cell. 2014 Jan 9;53(1):148-61
references.pmid: 24389104
references.citation: Stem Cells. 2014 Mar;32(3):623-35
references.pmid: 24167073
references.citation: Nat Methods. 2014 Mar;11(3):319-24
references.pmid: 24487582
references.citation: Annu Rev Biochem. 2009;78:399-434
references.pmid: 19489725
references.citation: EMBO Rep. 2009 Nov;10(11):1250-8
references.pmid: 19798103
references.citation: Proc Natl Acad Sci U S A. 2010 Mar 2;107(9):4028-33
references.pmid: 20160114
references.citation: Mol Cell Biol. 2000 Dec;20(23):8889-902
references.pmid: 11073989