The Mechanism Of E3 Ligase Smurf2 In Liver Diseases

The ubiquitn-proteasome pathway(UPP) is a major pathway for the targeting degradation of proteins.This process plays critical roles in a wide range of biological processes,including cell-cycle progression,signal transduction,transcriptional regulation,cell metabolism,transdifferenction,proliferation,mutation and tumor.In general,protein ubiquitination is catalyzed by a cascade of enzymes,including an ubiquitin-activating enzyme E1,an ubiquitin-conjugating enzyme E2 and an ubiquitin ligase E3.E3 ubiquitin ligases are crucial in the selective recognition of target proteins and also function in subsequent protein degradation by the 26s proteasomes. Smurf2 is one C2-WW-HECT domain E3 ligase,and Smurf2 could regulate signal transduction,apoptosis and senescence.We show that Smurf2 and its down-stream substrates changes in hepatocellular carcinoma,liver cirrhosis.Also we analyze the interactions of these molecules and the ubiquitination action.Then we illustrate the mechanism of Smurf2 in human hepatic stellate cells.At last,we preliminarily screen the signal molecules of Smurf2 pathway by immunoprecipitation following LC-MS/MS,and we find some HCC associated molecules.PartⅠ:The expression,interaction and ubiquitination of Smurf2 and its substrates in HCC,liver cirrhosis and normal liverHuman liver specimens were obtained from patients who experienced a liver surgery.Selected samples distributed into HCC,cirrhosis and normal groups according to the histological study.Real-time PCR and Western Blot showed Smurf2 decreased while its substrates Smad7,TβRI increased in HCC and cirrhosis groups. Another substrate SnoN was found no changes.Then we used immunoprecipitation and Western Blot to analyze the interactions of Smurf2,Smad7 and SnoN.We found the three had interactions each other,maybe they form a heterogeneous complex.The anti-ubiquitin antibody was used to undergo ubiquitination analysis.We investigated both Smad7 and TβRI could be ubiquitnated.PartⅡ:Impaired Smad Ubiquitination Regulatory Factor 2 mediated Transforming growth faetor-βsignaling inhibition in the process of liver cirrhosisSmurf2 could be induced by TGFβ.We examined lower Smurf2 expression in rat and human liver cirrhosis.So we speculated that impaired Smurf2 mediated TGFβsignaling inhibition promoted liver cirrhosis.We cultured activated human HSC cell line LX-2 in vitro.After TGFβ1 stimulation,LX-2 cell secretion less collagens companied with high expression of Smad7 and Smurf2.Over expression of Smad7 and Smurf2 could inhibit collagen secretion.The ubiquitin-dependent proteolytic degradation of TβRI was not the main role of Smad7 to inhibit TGFβsignal transduction and collagen secretons.Over expression of Smurf2 could inhibit TGFβsignal transduction through ubiquitin-dependent degradation of Smad7 and TβRI. Proteasome inhibitor MG132 could prevent this action.PartⅢ:Screening the new snbstrates of Smurf2 and HCC associated proteins by immunopreieipitation combined with LC-MS/MSThe given results showed the key role in liver diseases of Smurf2.But the Smurf2 pathway is not clear now.According to the specific binding characteristics of Smurf2 to its signal molecules,we used anti-Smurf2 antibody to immunoprecipitate the molecules which was associated with Smurf2.And then we identified these molecules by LC-MS/MS.The results showed more than ten molecules associated with Smurf2, several of them existed in HCC,not in normal liver;the others of them existed in normal liver,not in HCC.In brief,Smurf2 and its substrates Smad7 and TβRI express abnormally in HCC and cirrhosis liver.Smurf2 and Smad7,TβRI had reverse relationship in liver diseases. Smurf2 and Smad7,TβRI had interactions with each other,formed a heterogeneous complex.Smurf2 could inhibit TGFβsignal transduction through ubiquitin-dependent degradation of Smad7 and TβRI by proteasome.Immunoprecipitation combined with high sensitive LC-MS/MS was an effective method to separate and identify signal molecules which was associated with Smurf2.That may be a new way to finding the signal molecules of Smurf2 pathway and the biomarkers of HCC.