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

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

doi:10.1021/pr100990s

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

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

28 Citations (Scopus)

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.

Original language	English
Journal	Journal of Proteome Research
Volume	10
Issue number	3
Pages (from-to)	1216-27
Number of pages	12
ISSN	1535-3893
DOIs	https://doi.org/10.1021/pr100990s
Publication status	Published - 4 Mar 2011

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@article{b6086288249d469298d0f8d974aae6cc,

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.",

author = "Sophie Lachapelle and Jean-Philippe Gagn{\'e} and Chantal Garand and Myriam Desbiens and Yan Coulombe and Bohr, {Vilhelm A} and Hendzel, {Michael J} and Jean-Yves Masson and Poirier, {Guy G} and Michel Lebel",

year = "2011",

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doi = "10.1021/pr100990s",

language = "English",

volume = "10",

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journal = "Journal of Proteome Research",

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TY - JOUR

T1 - Proteome-wide identification of WRN-interacting proteins in untreated and nuclease-treated samples

AU - Lachapelle, Sophie

AU - Gagné, Jean-Philippe

AU - Garand, Chantal

AU - Desbiens, Myriam

AU - Coulombe, Yan

AU - Bohr, Vilhelm A

AU - Hendzel, Michael J

AU - Masson, Jean-Yves

AU - Poirier, Guy G

AU - Lebel, Michel

PY - 2011/3/4

Y1 - 2011/3/4

N2 - 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.

AB - 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.

U2 - 10.1021/pr100990s

DO - 10.1021/pr100990s

M3 - Journal article

C2 - 21210717

SN - 1535-3893

VL - 10

SP - 1216

EP - 1227

JO - Journal of Proteome Research

JF - Journal of Proteome Research

IS - 3

ER -

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

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