Identification Of A Novel Binding Protein Of Fat10: Eukaryotic Translation Elongation Factor1A1

Objective: To identified eukaryotic elongation factor1A1(eEF1A1) as aFAT10binding protein by screening a cDNA library from a human hepatocellularcarcinoma(HCC) cell line, Hep3B, using a yeast two-hybrid system, then toinvestigate the function of FAT10in tumorigenesis.Methods:1. To construct a yeast two-hybrid cDNA library of FAT10-overexpress Human Hepatic Carcinoma3B Cells.2. GAL4yeast two-hybrid assaywas performed to screen the Human Hep3B hepatoma cells cDNA library to obtainhost cell protein molecules which interact with FAT10by yeast two-hybrid.3.Theimmunofluoresence co-localization and co-immunoprecipitation assay was applied toconfirm the protein interactions identified.4.Then FAT10was knockdown bysiRNA to investigate the expression of eEF1A1.Results:1. The primary constructed cDNA library contained1.03±0.18×10⁶independent clones. The titer of the cDNA library was estimated as2.50±0.59×10⁶cfu/ml and that of the amplified library was3.60±0.54×10⁹cfu/ml. The inserts variedfrom0.5to3.5kb with average size was about2.0kb. The FAT10cDNA was clonedinto the pGBK7plasmid, and this served as the bait plasmid against the Hep3B cDNAlibrary.2. We first confirmed that FAT10did not demonstrate self-activation. By ayeast two-hybrid system,the positive clones was screened and sequenced。Thelongest cDNA clone for eEF1A1was1750bp, and its DNA sequence shared a99.9%identity with that of eEF1A1(GenBank accession No. NM₀01402.5), confirmingthat eEF1A1could bind strongly to FAT10in this system.3. As shown eEF1A1andFAT10were detected in the immunoprecipitant, confirming the interaction betweenFAT10and eEF1A1. Confocal laser scanning microscopy indicated identicallocalization of both eEF1A1and FAT10, which provided further evidence of theinteraction between these two proteins.4. Knockdown of FAT10via siRNA wasperformed in the Hep3B and MHCC97H cell lines. We found FAT10siRNA coulddownregulate eEF1A1mRNA expression and The FAT10siRNA coulddownregulate eEF1A1protein expression. Conclusions: We firstly confirm eEF1A1serves as a substrate of FAT10toaccomplish, in part, its functions in regulating the biological behavior of tumorcells.Furthermore, the expression of FAT10was reduced by siRNA knockdown, andthis resulted in downregulation of eEF1A1expression at both the mRNA and proteinlevels in HCC cells.