title: 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.
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.
authors.affiliation: INSERM Unit 1073, University of Rouen, Rouen, France; Institute for Research and Innovation in Biomedicine, University of Rouen, Rouen, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Bertrand
authors.forename: Naouel
authors.initials: N
authors.lastname: Tennoune
authors.forename: Rachel
authors.initials: R
authors.lastname: Marion-Letellier
authors.forename: Alexis
authors.initials: A
authors.lastname: Goichon
authors.forename: Philippe
authors.initials: P
authors.lastname: Chan
authors.forename: Khaly
authors.initials: K
authors.lastname: Mbodji
authors.forename: David
authors.initials: D
authors.lastname: Vaudry
authors.forename: Pierre
authors.initials: P
authors.lastname: Déchelotte
authors.forename: Moïse
authors.initials: M
authors.lastname: Coëffier
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003092:Colitis / Q000139:chemically induced / Q000378:metabolism; D003106:Colon / Q000737:chemistry / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D016209:Interleukin-8 / Q000032:analysis / Q000378:metabolism; D007413:Intestinal Mucosa / Q000737:chemistry / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar; D014302:Trinitrobenzenesulfonic Acid / Q000633:toxicity; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism; D057149:Ubiquitinated Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D016209:Interleukin-8; D008565:Membrane Proteins; D025801:Ubiquitin; D057149:Ubiquitinated Proteins; C018551:glucose-regulated proteins; D014302:Trinitrobenzenesulfonic Acid; D046988:Proteasome Endopeptidase Complex
keywords: Cell biology; HSPs; Inflammation; Intestine; Proteasome; Ubiquitin