title: Accurately mapping the location of the binding site for the interaction between hepatitis B virus X protein and cytochrome c oxidase III.
abstract: The hepatitis B virus (HBV) X protein (HBx) plays an important pathogenetic role in hepatocarcinoma tumorigenesis. As HBx does not have the ability to bind to double-stranded DNA (dsDNA), protein-protein interaction is crucial for HBx functions. In a previous study, we screened a novel HBx-interacting protein, the cytochrome c oxidase subunit III (COXIII). In the present study, we aimed to accurately map the location of the binding site for the interaction of HBx with COXIII. Two fragments of HBx mutants (X1 aa1-72 and X2 aa1-117) were amplified by polymerase chain reaction (PCR) and separately inserted into the pAS2-1 plasmid. PCR and gene sequencing confirmed the correct insertion of the mutant fragments in the plasmid. The transcription of the mutant fragments in yeast cells was demonstrated by RT-PCR and western blot analysis confirmed that they were accurately translated into fusion proteins. Hybridization on solid medium and the detection of β-galactosidase (β-gal) activity indicated that the binding site for the interaction between HBx and COXIII was located between aa72 and aa117. Specific interactions between the HBxX2 protein and COXIII were verified by co-immunoprecipitation. To the best of our knowledge, this is the first study showing to demonstrate that aa72-117 in HBx is the key region for binding with COXIII.
authors.affiliation: Department of Gastroenterology, Union Hospital of Fujian Medical University, Fuzhou, Fujian 350001, P.R. China.
authors.forename: Dan
authors.initials: D
authors.lastname: Li
authors.affiliation: Department of Gastroenterology, First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian 350001, P.R. China.
authors.forename: Jian
authors.initials: J
authors.lastname: Ding
authors.affiliation: Department of Gastroenterology, Union Hospital of Fujian Medical University, Fuzhou, Fujian 350001, P.R. China.
authors.forename: Zhixin
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Department of Gastroenterology, Union Hospital of Fujian Medical University, Fuzhou, Fujian 350001, P.R. China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Gastroenterology, Union Hospital of Fujian Medical University, Fuzhou, Fujian 350001, P.R. China.
authors.forename: Na
authors.initials: N
authors.lastname: Lin
authors.affiliation: Department of Gastroenterology, Union Hospital of Fujian Medical University, Fuzhou, Fujian 350001, P.R. China.
authors.forename: Fenglin
authors.initials: F
authors.lastname: Chen
authors.affiliation: Department of Gastroenterology, Union Hospital of Fujian Medical University, Fuzhou, Fujian 350001, P.R. China.
authors.forename: Xiaozhong
authors.initials: X
authors.lastname: Wang
mesh_terms: D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism*; D006509:Hepatitis B / Q000235:genetics / Q000378:metabolism*; D006515:Hepatitis B virus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D012441:Saccharomyces cerevisiae; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D003576:Electron Transport Complex IV