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A role for immunoproteasome in the regulation of intestinal permeability has been previously suggested both in mice during water avoidance stress (WAS) and in patients with irritable bowel syndrome (IBS). Here, we provide evidence that the ubiquitin-proteasome system (UPS) contributes to the pathophysiology of IBS. Indeed, we report that colonic proteome is altered in WAS mice and that β2i subunit deficiency modifies the proteome response that is associated with a limitation of colonic hyperpermeability. Interestingly, we show specific alterations of proteins involved in UPS, mitochondrial, and energy metabolism. We also report changes in the pattern of colonic ubiquitome in diarrhea-predominant IBS (IBS-D) patients and particularly a reduced expression of ubiquitinated proteins involved in the nuclear factor-kappa B (NF-κB) inflammatory signaling pathway. All these data suggest that immunoproteasome targeting may represent a new therapeutic strategy for the treatment of IBS patients with increased intestinal permeability.

The ubiquitin-proteasome system (UPS), a highly regulated mechanism including the active marking of proteins by ubiquitin to be degraded, is critical in regulating proteostasis. Dysfunctioning of the UPS has been implicated in diseases such as cancer and neurodegenerative disorders. Here we investigate the effects of proteasome malfunctioning on global proteome and ubiquitinome dynamics using SILAC proteomics in Drosophila S2 cells. dsRNA-mediated knockdown of specific proteasome target subunits is used to inactivate the proteasome. Upon this perturbation, both the global proteome and the ubiquitinome become modified to a great extent, with the overall impact on the ubiquitinome being the most dramatic. The abundances of ∼10% of all proteins are increased, while the abundances of the far majority of over 14 000 detected diGly peptides are increased, suggesting that the pool of ubiquitinated proteins is highly dynamic. Remarkably, several proteins show heterogeneous ubiquitination dynamics, with different lysine residues on the same protein showing either increased or decreased ubiquitination. This suggests the occurrence of simultaneous and functionally different ubiquitination events. This strategy offers a powerful tool to study the response of the ubiquitinome upon interruption of normal UPS activity by targeted interference and opens up new avenues for the dissection of the mode of action of individual components of the proteasome. Because this is to our knowledge the first comprehensive ubiquitinome screen upon proteasome malfunctioning in a fruit fly cell system, this data set will serve as a valuable repository for the Drosophila community.

Hepatitis C virus (HCV) causes chronic liver disease worldwide. HCV Core protein (Core) forms the viral capsid and is crucial for HCV pathogenesis and HCV-induced hepatocellular carcinoma, through its interaction with the host factor proteasome activator PA28γ. Here, using BD-PowerBlot high-throughput Western array, we attempt to further investigate HCV pathogenesis by comparing the protein levels in liver samples from Core-transgenic mice with or without the knockout of PA28γ expression (abbreviated PA28γ(-/-)CoreTG and CoreTG, respectively) against the wild-type (WT). The differentially expressed proteins integrated into the human interactome were shown to participate in compact and well-connected cellular networks. Functional analysis of the interaction networks using a newly developed data warehouse system highlighted cellular pathways associated with vesicular transport, immune system, cellular adhesion, and cell growth and death among others that were prominently influenced by Core and PA28γ in HCV infection. Follow-up assays with in vitro HCV cell culture systems validated VTI1A, a vesicular transport associated factor, which was upregulated in CoreTG but not in PA28γ(-/-)CoreTG, as a novel regulator of HCV release but not replication. Our analysis provided novel insights into the Core-PA28γ interplay in HCV pathogenesis and identified potential targets for better anti-HCV therapy and potentially novel biomarkers of HCV infection.