Tyrosine Phosphorylation Of Trihydrophobin 1 And The Role Of Nuclear Receptor FXR In Alcoholic Fatty Liver

Amino acid sequence is not the only factor in determination of protein function after gene transcription and translation, whereas post-translational modifications play essential roles in the process of proterin muturation. Upon extracellular stimulations, a large number of proteins normally functions depending on dynamic regulation of post-translational modifications. More than100post-translational modifications have been discovered, and studies on post-translational modifications promote understanding the complex roles of proteins in cellular homestasis and diseases prevention. In the current study, we explored the role of tyrosine phosphorylation in the regulation of Trihydrophobin1and the role of lysine acetylation of farnesoid X receptor in murine alcoholic liver disease. We tried to explore the dynamic and comprehensive regulation of post-translational modification in physiological condition and applied translational reseach in disease model.Part I The biological functions and mechanisms of Trihydriphobin1tyrosine phosphorylated by c-src. c-Src is a non-receptor tyrosine kinase and prototype of the Src family kinases (SFKs), which play key roles in cell proliferation, survival, adhesion, morphology and motility. Expression of constitutive active mutant of c-Src promotes cell proliferation and migration. F-Src, the viral homologue of the cellular proto-oncogene c-Src encoded by the avian Rous Sarcoma Virus (RSV), is constitutively activated and causes tumor. The human trihydrophobin1(THl) gene is the homolog of Drosophila TH1, which was highly conserved from Drosophila to human. We reported that TH1served as a negative regulator of A-Raf and PAK1, inhibiting MEK/ERK signaling pathway. TH1inhibits MAPK/ERK-driven cell migration through impacting actin and adhesion dynamics. Moreover, the expression of TH1negatively correlates with the aggressiveness of human breast cancer cells and breast tumor tissues.Although the functional understading of TH1has been extended, the regulation of TH1at translational or post-translation level has been rarely studied. In this study, we identified TH1as an interaction partner of c-Src by immunoprecipitation and GST-pull down assay in HEK293T cells. The interaction depends on open-conformation of c-Src. Endogenous c-Src interacts with and colocalizes with TH1in HeLa cells.Further investigation revealed that c-Src directly phosphorylated TH1in vivo and in vitro, which also depends on c-Src activity. Selective inhibitor of c-Src, PP2almost abolished tyrosine phosphorylation on TH1by c-Src. We determined the tyrosine phosphorylation sites on TH1using fragments and point mutants of TH1. And the results show c-src phoshporylates TH1at Tyr-6directly in vivo and in vitro. Tyr-6phosphorylation of TH1decreases its inhibition on MAPK/ERK signaling becaused of reduced interaction with A-Raf and PAK1. Dephosphorylated form of TH1(TH1-Y6F) inhibited c-Src mediated MAPK/ERK pathway activation, whereas phosphorylated form of TH1did not inhibit. Moreover, c-Src driven cell migration was attenuated by TH1-Y6F over-expression. Induced tyrosine phosphorylation of TH1was found by EGF or estrogen treatment. In conclusion, we proposed a mechanism for comprehensive regulation of Ras/Raf/MEK/ERK signaling and cell migration involving tyrosine phosphorylation of TH1by c-Src.Part Ⅱ Role of Farnesoid X receptor and its acetylation in alcoholic fatty liver disease. The farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily, which plays an essential role in the regulation of bile acid, cholesterol and glucose homeostasis. FXR deficient mice display elevated serum cholesterol and triglyceride levels, while activation of FXR improves hyperglycemia and dyslipidemia in mice.In current study, wild-type and FXR deficient C57BL/6mice were fed with Lieber-DeCarli ethanol liquid diet or isocaloric control diet. We also used FXR specific agonist WAY-362450to study the role of FXR in alcoholic fatty liver diseases. Mice fed alcohol developed liver injury and intrahepatic cholestasis (hepatic bile acid:0.337+0.041μmoles/g liver in Ethanol fed vs.0.146±0.01μmoles/g liver in pair fed, p<0.001). The expressions of genes involed in bile acid metabolism were also altered in this model, with CYP7A1upregulation and BSEP downregulation. However, FXR mRNA level was unchanged. The interaction of FXR with RXRa evidently decreased. Since FXR functions as a transcriptor in a FXR/RXRa heterodimer dependent manner, we proposed FXR activity was impaired in murine alcoholic fatty liver.Upon ethanol ingestion for4weeks, FXR deficient mice developed severe liver injury and steatosis compared with the wild-type counterpart. Hepatic bile acid level was also higher in FXR deficient mice. In contrast, wild type mice fed ethanol diet received WAY-362450for5weeks shown lower serum ALT level and hepatic triglyceride level. Histological examination confirmed the therapy effect of WAY-362450. WAY-362450treatment also increased hepatic gene expression of SHP and BSEP, and reduced bile acid accumulation in livers. Moreover, WAY-362450administration decreased expression of inflammatory gene, TNF-a, MCP-1and oxidative stress gene, CYP2E1. Evidently, induced CYP2E1protein levels and oxidative makers, TBARS and4-HNE adducts, by ethanol ingestion were also attenuated by WAY-362450treatment.In conclusion, we found FXR activity was impaired in alcoholic fatty liver model. WAY-362450could activate FXR and attenuates alcoholic fatty liver disease progression by repressing steatosis and inflammation. These findings demonstrate that FXR agonists may be useful for the treatment of alcoholic fatty liver diseases.