Targeting UBC9-mediated protein hyper-SUMOylation in cystic cholangiocytes halts polycystic liver disease in experimental models

Abstract

Background & Aims: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple fluid-filled biliary cysts. Most PLD-causative genes participate in protein biogenesis and/or transport. Post-translational modifications (PTMs) are implicated in protein stability, localization and activity, contributing to human pathobiology; however, their role in PLD is unknown. Herein, we aimed to unveil the role of protein SUMOylation in PLD and its potential therapeutic targeting. Methods: Levels and functional effects of SUMOylation, along with response to S-adenosylmethionine (SAMe, inhibitor of the SUMOylation enzyme UBC9) and/or short-hairpin RNAs (shRNAs) against UBE2I (UBC9), were evaluated in vitro, in vivo and/or in patients with PLD. SUMOylated proteins were determined by immunoprecipitation and proteomic analyses by mass spectrometry. Results: Most SUMOylation-related genes were found overexpressed (mRNA) in polycystic human and rat liver tissue, as well as in cystic cholangiocytes in culture compared to controls. Increased SUMOylated protein levels were also observed in cystic human cholangiocytes in culture, which decreased after SAMe administration. Chronic treatment of polycystic (PCK: Pkdhl -mut) rats with SAMe halted hepatic cystogenesis and fibrosis, and reduced liver/body weight ratio and liver volume. In vitro, both SAMe and shRNA-mediated UBE2I knockdown increased apoptosis and reduced cell proliferation of cystic cholangiocytes. High-throughput proteomic analysis of SUM01-immunoprecipitated proteins in cystic cholangiocytes identified candidates involved in protein biogenesis, ciliogenesis and proteasome degradation. Accordingly, SAMe hampered proteasome hyperactivity in cystic cholangiocytes, leading to activation of the unfolded protein response and stress-related apoptosis. Conclusions: Cystic cholangiocytes exhibit increased SUMOylation of proteins involved in cell survival and proliferation, thus promoting hepatic cystogenesis. Inhibition of protein SUMOylation with SAMe halts PLD, representing a novel therapeutic strategy.

Inhibition of NAE‐dependent protein hyper‐NEDDylation in cystic cholangiocytes halts cystogenesis in experimental models of polycystic liver disease

“… 13 , 14 All cells were cultured in fully supplemented DMEM/F‐12 medium as previously described. 2 , 3 , 11 , 15 , 16 …”

“…13,14 All cells were cultured in fully supplemented DMEM/F-12 medium as previously described. 2,3,11,15,16 RNA isolation, reverse transcription, and quantitative polymerase chain reaction RNA was isolated from human cystic wall and rat liver tissues as well as from cell cultures with TRI Reagent (Sigma-Aldrich). RNA was reverse transcribed into cDNA and quantitative real-time polymerase chain reactions (qPCRs) were performed as described in the Supplementary data.…”

“…There is elevated protein SUMOylation in cystic cholangiocytes, which drives hepatic cystogenesis in experimental models. Administration of SAMe, an inhibitor of the SUMO‐conjugating enzyme UBC9, decreased proliferation and increased apoptotic cell death of cystic cholangiocytes, thus reducing hepatic cystogenesis in vitro and in vivo 3 …”

“… 2 Furthermore, perturbations in posttranslational modifications (PTMs), particularly in SUMOylation, result in aberrant protein dynamics contributing to the pathology. 3 There is elevated protein SUMOylation in cystic cholangiocytes, which drives hepatic cystogenesis in experimental models. Administration of SAMe, an inhibitor of the SUMO‐conjugating enzyme UBC9, decreased proliferation and increased apoptotic cell death of cystic cholangiocytes, thus reducing hepatic cystogenesis in vitro and in vivo .…”

“…Administration of SAMe, an inhibitor of the SUMO‐conjugating enzyme UBC9, decreased proliferation and increased apoptotic cell death of cystic cholangiocytes, thus reducing hepatic cystogenesis in vitro and in vivo . 3 …”