Evaluation of ubiquitinated proteins by proteomics reveals the role of the ubiquitin proteasome system in the regulation of Grp75 and Grp78 chaperone proteins during intestinal inflammation

Authors

  • Julien Bertrand,

    1. INSERM Unit 1073, University of Rouen, Rouen, France
    2. Institute for Research and Innovation in Biomedicine, University of Rouen, Rouen, France
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  • Naouel Tennoune,

    1. INSERM Unit 1073, University of Rouen, Rouen, France
    2. Institute for Research and Innovation in Biomedicine, University of Rouen, Rouen, France
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  • Rachel Marion-Letellier,

    1. INSERM Unit 1073, University of Rouen, Rouen, France
    2. Institute for Research and Innovation in Biomedicine, University of Rouen, Rouen, France
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  • Alexis Goichon,

    1. INSERM Unit 1073, University of Rouen, Rouen, France
    2. Institute for Research and Innovation in Biomedicine, University of Rouen, Rouen, France
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  • Philippe Chan,

    1. Institute for Research and Innovation in Biomedicine, University of Rouen, Rouen, France
    2. Platform in Proteomics PISSARO, Mont-Saint-Aignan, France
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  • Khaly Mbodji,

    1. INSERM Unit 1073, University of Rouen, Rouen, France
    2. Institute for Research and Innovation in Biomedicine, University of Rouen, Rouen, France
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  • David Vaudry ,

    1. Institute for Research and Innovation in Biomedicine, University of Rouen, Rouen, France
    2. Platform in Proteomics PISSARO, Mont-Saint-Aignan, France
    3. INSERM Unit 982, University of Rouen, Mont-Saint-Aignan, France
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  • Pierre Déchelotte,

    1. INSERM Unit 1073, University of Rouen, Rouen, France
    2. Institute for Research and Innovation in Biomedicine, University of Rouen, Rouen, France
    3. Nutrition Unit, Rouen University Hospital, Rouen, France
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  • Moïse Coëffier

    Corresponding author
    1. INSERM Unit 1073, University of Rouen, Rouen, France
    2. Institute for Research and Innovation in Biomedicine, University of Rouen, Rouen, France
    3. Nutrition Unit, Rouen University Hospital, Rouen, France
    • Correspondence: Dr. Moïse Coëffier, INSERM Unit 1073, University of Rouen, 22 Boulevard Gambetta, 76183 Rouen Cedex, France

      E-mail: moise.coeffier@univ-rouen.fr

      Fax: +33-2-35-14-82-26

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Abstract

The ubiquitin proteasome system (UPS) is the major pathway of intracellular protein degradation and may be involved in the pathophysiology of inflammatory bowel diseases or irritable bowel syndrome. UPS specifically degrades proteins tagged with an ubiquitin chain. We aimed to identify polyubiquitinated proteins during inflammatory response in intestinal epithelial HCT-8 cells by a proteomic approach. HCT-8 cells were incubated with interleukin 1β, tumor necrosis factor-α, and interferon-γ for 2 h. Total cellular protein extracts were separated by 2D gel electrophoresis and analyzed by an immunodetection using antiubiquitin antibody. Differential ubiquitinated proteins were then identified by LC-ESI MS/MS. Seven proteins were differentially ubiquitinated between control and inflammatory conditions. Three of them were chaperones: Grp75 and Hsc70 were more ubiquitinated (p < 0.05) and Grp78 was less ubiquitinated (p < 0.05) under inflammatory conditions. The results for Grp75 and Grp78 were then confirmed in HCT-8 cells and in 2-4-6-trinitrobenzen sulfonic acid induced colitis in rats mimicking inflammatory bowel disease by immunoprecipitation. No difference was observed in irritable bowel syndrome like model. In conclusion, we showed that a proteomic approach is suitable to identify ubiquitinated proteins and that UPS-regulated expression of Grp75 and Grp78 may be involved in inflammatory response. Further studies should lead to the identification of ubiquitin ligases responsible for Grp75 and Grp78 ubiquitination.

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