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.

2,5-Dimethyl-Celecoxib Inhibits Cell Cycle Progression and Induces Apoptosis in Human Leukemia Cells

“…The mechanism by which DMC decreases GRP78 protein expression was not investigated in our study. GRP78 is regulated by protein degradation through the proteasome but also cathepsins (Shi et al, 2009;Bertrand et al, 2013). In our study, we could not revert Mcl-1 downregulation and DMC-induced apoptosis with the proteasome inhibitor MG132, thus suggesting a proteasome-independent effect.…”

SPECT-computed tomography in rats with TNBS-induced colitis: A first step toward functional imaging

“…Similarly to previous studies[13-15,17-20], we observed colon inflammation demonstrated by stiffened colon, histological inflammatory score or increased inflammatory markers. We also noted SPECT signs of inflammation such as an increased maximal thickness of colon.…”

“…Colitis was induced at day 0 by administration of TNBS (Sigma-Aldrich, Saint-Quentin Fallavier, France) as previously described[13,15,16]. The rats were then briefly anesthetized with an intraperitoneal injection of ketamine and chlorpromazine following 24-h food deprivation.…”

Glutamine enema regulates colonic ubiquitinated proteins but not proteasome activities during TNBS‐induced colitis leading to increased mitochondrial activity

“…reported single nucleotide polymorphisms in several genes involved in the UPS in CD patients and that adherent‐invasive Escherichia coli modified NF‐κB activation through the regulation of UPS turnover in intestinal epithelial cells . In a previous study, we showed that UPS also regulated the expression of other proteins, that is, HSPs, during intestinal inflammatory response both in vitro and in vivo by using a proteomic approach .…”

“…After transfer, membranes were blocked with 5% w/v BSA (fraction V, Eurobio) in (TBS (10 mM Tris, pH 8; 150 mM NaCl) plus 0.05% v/v Tween 20 = TBS‐T). After washes with TBS‐T, membranes were incubated overnight at 4°C with rabbit polyclonal anti‐ubiquitin (1:500, SantaCruz Biotechnology, Le Perray en Yvelines, France), then washed again and incubated with swine anti‐rabbit IgG HRP conjugated (1:5000, Dako, Trappes, France) as previously described . Immunocomplexes were revealed by using the ECL detection system (GE Healthcare).…”