title: Apolipoprotein B gene regulatory factor-2 (BRF-2) is structurally and immunologically highly related to hepatitis B virus X associated protein-1 (XAP-1).
abstract: Hepatic cell-specific expression of the human apolipoprotein B (apoB) gene is controlled by at 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 to be liver specific because it shows positive activity in HepG2 cells and negative activity in HeLa cells. ApoB gene regulatory factor-2 (BRF-2) interacts with the sequence (-104 to -85). BRF-2 has been purified from rat liver nuclear extract, and its molecular weight has been determined to be approximately 120 kDa [Zhuang et al. (1992) Mol. Cell. Biol. 12, 3183-3191]. In this paper we report the isolation of two isoforms of BRF-2 by further purification using high-performance liquid chromatography. Both isoforms produced a single approximately 120-kDa band in sodium dodecyl sulfate polyacrylamide gel electrophoresis detected by silver stain. The amino acid sequences of two tryptic peptides derived from HPLC-purified heavier BRF-2 isoform were determined to be YLAIAPPIIK and ALYYLQIHPQELR. These two peptides were found to share 100% sequence homology with human hepatitis B virus X associated protein-1 (XAP-1) and monkey UV-damaged DNA-binding protein (UV-DDB). Anti-peptide antisera raised against two synthetic peptides of XAP-1 recognized a approximately 120-kDa polypeptide band in both BRF-2 isoforms in a western blot analysis. By using apoB promoter fragments containing various internal deletions and a substitution mutation as templates for gel mobility shift assays, we identified the region between -104 and -85 as crucial for binding by the high-molecular weight form. In contrast, the lower molecular weight isoform bound to all apoB mutants tested. Anti-peptide 2 antiserum directed against XAP-1 was found to inhibit in vitro transcription of the apoB gene in rat liver nuclear extracts by 50%. These results suggest that BRF-2 and XAP-1 are structurally and immunologically highly related trans-activators of the apoB gene. We propose that BRF-2 exists both as a monomer (BRF-2M) and as a homooligomer. probably a homodimer (BRF-2D), in solution; oligomerization appears to be an essential step for imparting sequence-specificity to BRF-2 protein and thereby facilitating its role as a trans-activator of the apoB gene.
authors.affiliation: Department of Pharmacology, University of North Texas Health Science Center at Fort Worth 76107, USA.
authors.forename: R R
authors.initials: RR
authors.lastname: Krishnamoorthy
authors.forename: T H
authors.initials: TH
authors.lastname: Lee
authors.forename: J S
authors.initials: JS
authors.lastname: Butel
authors.forename: H K
authors.initials: HK
authors.lastname: Das
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000276:immunology*; D005809:Genes, Regulator; D006801:Humans; D007120:Immunochemistry; D066298:In Vitro Techniques; D008099:Liver / Q000737:chemistry; D015394:Molecular Structure; D008970:Molecular Weight; D015345:Oligonucleotide Probes / Q000235:genetics; D011487:Protein Conformation; D051381:Rats; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*
chemical_list: D001055:Apolipoproteins B; C091435:DDB1 protein, human; D004268:DNA-Binding Proteins; C490414:Ddb1 protein, rat; D015345:Oligonucleotide Probes; D015534:Trans-Activators