title: Proteome-wide identification of WRN-interacting proteins in untreated and nuclease-treated samples.
abstract: Werner syndrome (WS) is characterized by the premature onset of several age-associated pathologies. The protein defective in WS patients (WRN) is a helicase/exonuclease involved in DNA repair, replication, telomere maintenance, and transcription. Here, we present the results of a large-scale proteome analysis to determine protein partners of WRN. We expressed fluorescent tagged-WRN (eYFP-WRN) in human 293 embryonic kidney cells and detected interacting proteins by co-immunoprecipitation from cell extract. We identified by mass spectrometry 220 nuclear proteins that complexed with WRN. This number was reduced to 40 when broad-spectrum nucleases were added to the lysate. We consider these 40 proteins as directly interacting with WRN. Some of these proteins have previously been shown to interact with WRN, whereas most are new partners. Among the top 15 hits, we find the new interactors TMPO, HNRNPU, RPS3, RALY, RPS9 DDX21, and HNRNPM. These proteins are likely important components in understanding the function of WRN in preventing premature aging and deserve further investigation. We have confirmed endogenous WRN interaction with endogenous RPS3, a ribosomal protein with endonuclease activities involved in oxidative DNA damage recognition. Our results suggest that the use of nucleases during cell lysis severely restricts interacting protein partners and thus enhances specificity.
authors.affiliation: Centre de Recherche en Cancérologie de l'Université Laval, Hôpital Hôtel-Dieu de Québec, Québec City, Québec, G1R 2J6, Canada.
authors.forename: Sophie
authors.initials: S
authors.lastname: Lachapelle
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Gagné
authors.forename: Chantal
authors.initials: C
authors.lastname: Garand
authors.forename: Myriam
authors.initials: M
authors.lastname: Desbiens
authors.forename: Yan
authors.initials: Y
authors.lastname: Coulombe
authors.forename: Vilhelm A
authors.initials: VA
authors.lastname: Bohr
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Hendzel
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Masson
authors.forename: Guy G
authors.initials: GG
authors.lastname: Poirier
authors.forename: Michel
authors.initials: M
authors.lastname: Lebel
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D003851:Deoxyribonucleases / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding; D020543:Proteome / Q000032:analysis*; D053484:RecQ Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods; D014898:Werner Syndrome / Q000473:pathology / Q000503:physiopathology; D000071657:Werner Syndrome Helicase
chemical_list: D020543:Proteome; C031016:RPS3A protein, human; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D003851:Deoxyribonucleases; D005090:Exodeoxyribonucleases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase