Proteome-wide identification of WRN-interacting proteins in untreated and nuclease-treated samples

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Sophie Lachapelle, Jean-Philippe Gagné, Chantal Garand, Myriam Desbiens, Yan Coulombe, Vilhelm A Bohr, Michael J Hendzel, Jean-Yves Masson, Guy G Poirier, Michel Lebel

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.
Original languageEnglish
JournalJournal of Proteome Research
Volume10
Issue number3
Pages (from-to)1216-27
Number of pages12
ISSN1535-3893
DOIs
Publication statusPublished - 4 Mar 2011

ID: 33492757