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Screening of Proteins Interact with Hepatocyte Nuclear Factor3β by IP-MS and Primary Study of Function

Author: SunZuoZuo
Tutor: WangSiYing;YangXiaoMing
School: Anhui Medical University,
Course: Pathology and Pathophysiology
Keywords: HNF3β IP MS protein-protein interaction
CLC: R341
Type: Master's thesis
Year: 2011
Downloads: 26
Quote: 0
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Abstract


Hepatocyte Nuclear Factor HNF3βis a class of liver-enriched transcription factor,engaged in the process of hepatic phenotype and functions. The expression of HNF3βis detected in the anterior primitive streak and the node at earlier embryonic day in the mouse. HNF3βvariation may be associated with type 2 diabetes. HNF3βis involved in glucose metabolism, fat metabolism, regulation of bile acid and growth-related gene expression and regulation.It regulates many types of tissue-specific expressions of hepatic genes in which many kinds of coactivors of HNF3βare required. Screeing preteins interact with HNF3βhelp us make a futher look at the functions and mechanism of HNF3β.It has been known that there are 8 moleculars(OTX2,SRC1,TLE1,HOXA5,HNF6A,GSC,EN2,LHX1) that interacted with HNF3β. The most frequently used method for protein-protein interaction is Yeast two-hybrid technology which need removed transcription activation domain is not applicable for transcription factor .Immunoprecipitation technique often be used for studying the physiological protein-protein interactions.The mass spectrometry technology developmentd quickly these years with the advantages of high accuracy and speed.For these reason thecoimmunoprecipitation combined mass spectrometry technology has been widely used to search for new protein interactions physiologically.In this study, protein complex of HNF3βwere purified and identified. Primary research has also focused on the biological functions of interaction of LMNA/HNF3β.The HNF3βcomplex was purified from HepG2 cells via the method of coimmunoprecipitation with specific antibody.Stained by Coomassie brilliant blue after SDS-PAGE electrophoresis.Then cut off the band different from the control and identified the complex by Mass Spectrometry.53 interaction candidate proteins were obtained from 4 independent experiments and 25 of them obtained for more than twice.Screening the results by Bioinformatics method we find that among the 24 interaction data, 9 have the common compart with HNF3β,5 have the direct interacting domain with HNF3β,10 are involved inthe process of transcription, and 9 have the indirect interaction with HNF3β. CO-IP, Luciferase reporter assay and indirect immunofluorescence validated the interaction between HNF3βand LMNA.RNAi decreases the level of LMNA in vivo, changing the expression of a few HNF3βtarget genes.In conclusion variety of proteins interacted with HNF3βand coinvolved in transcriptional regulation. Studies on HNF3β/LMNA demonstrates the interaction between LMNA and HNF3βis of great significance for glucose and fat metabolism and the occurrence of related diseases.

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