Apolipoprotein B Gene Regulatory Factor-2 (BRF-2) Is Structurally and Immunologically Highly Related to Hepatitis B Virus X Associated Protein-1 (XAP-1)
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- 作者：Raghu R. Krishnamoorthy ; Teh-Hsiu Lee ; Janet S. Butel ; and Hriday K. Das
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：January 28, 1997
- 年：1997
- 卷：36
- 期：4
- 页码：960 - 969
- 全文大小：288K
- 年卷期：v.36,no.4(January 28, 1997)
- ISSN：1520-4995
文摘
Hepatic cell-specific expression of the humanapolipoprotein B (apoB) gene is controlled byat least four cis-acting elements located between positions-128 and +122 [Chuang, S. S., & Das, H. K.(1996) Biochem. Biophys. Res.Commun. 220, 553-562]. The distal element(-128 to -85) appears tobe liver specific because it shows positive activity in HepG2 cells andnegative activity in HeLa cells.ApoB gene regulatory factor-2 (BRF-2) interacts with the sequence(-104 to -85). BRF-2 has beenpurified from rat liver nuclear extract, and its molecular weight hasbeen determined to be ~120 kDa[Zhuang et al. (1992) Mol. Cell.Biol. 12, 3183-3191]. In this paper wereport the isolation of twoisoforms of BRF-2 by further purification using high-performance liquidchromatography. Both isoformsproduced a single ~120-kDa band in sodium dodecyl sulfatepolyacrylamide gel electrophoresis detectedby silver stain. The amino acid sequences of two tryptic peptidesderived from HPLC-purified heavierBRF-2 isoform were determined to be YLAIAPPIIK and ALYYLQIHPQELR.These two peptides werefound to share 100% sequence homology with human hepatitis B virus Xassociated protein-1 (XAP-1)and monkey UV-damaged DNA-binding protein (UV-DDB). Anti-peptideantisera raised against twosynthetic peptides of XAP-1 recognized a ~120-kDa polypeptide band inboth BRF-2 isoforms in a westernblot analysis. By using apoB promoter fragments containing variousinternal deletions and a substitutionmutation as templates for gel mobility shift assays, we identified theregion between -104 and -85 ascrucial for binding by the high-molecular weight form. Incontrast, the lower molecular weight isoformbound to all apoB mutants tested. Anti-peptide 2 antiserumdirected against XAP-1 was found to inhibitin vitro transcription of the apoB gene in rat liver nuclearextracts by 50%. These results suggest thatBRF-2 and XAP-1 are structurally and immunologically highly relatedtrans-activators of the apoB gene.We propose that BRF-2 exists both as a monomer (BRF-2M) and as ahomooligomer, probably ahomodimer (BRF-2D), in solution; oligomerization appears to be anessential step for imparting sequence-specificity to BRF-2 protein and thereby facilitating its role as atrans-activator of the apoB gene.