Apolipoprotein B gene regulatory factor-2 (BRF-2) is structurally and immunologically highly related to hepatitis B virus X associated protein-i (XAP-1)

Raghu Krishnamoorthy, Teh Hsiu Lee, Janet S. Butel, Hriday Das

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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., and 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 ~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 ~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 ~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.

Original languageEnglish
Pages (from-to)960-969
Number of pages10
JournalBiochemistry
Volume36
Issue number4
DOIs
StatePublished - 28 Jan 1997

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Apolipoproteins B
Regulator Genes
Viruses
Genes
Protein Isoforms
Peptides
Proteins
Liver Extracts
Trans-Activators
Molecular Weight
Liver
Immune Sera
High Pressure Liquid Chromatography
Molecular weight
hepatitis B virus X protein
Hep G2 Cells
DNA-Binding Proteins
Electrophoretic Mobility Shift Assay
Rats
Sequence Homology

Cite this

@article{fc0a08e71328468daf4a7bd8ddb6bdaa,
title = "Apolipoprotein B gene regulatory factor-2 (BRF-2) is structurally and immunologically highly related to hepatitis B virus X associated protein-i (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., and 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 ~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 ~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 ~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.",
author = "Raghu Krishnamoorthy and Lee, {Teh Hsiu} and Butel, {Janet S.} and Hriday Das",
year = "1997",
month = "1",
day = "28",
doi = "10.1021/bi961407c",
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volume = "36",
pages = "960--969",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "4",

}

Apolipoprotein B gene regulatory factor-2 (BRF-2) is structurally and immunologically highly related to hepatitis B virus X associated protein-i (XAP-1). / Krishnamoorthy, Raghu; Lee, Teh Hsiu; Butel, Janet S.; Das, Hriday.

In: Biochemistry, Vol. 36, No. 4, 28.01.1997, p. 960-969.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Apolipoprotein B gene regulatory factor-2 (BRF-2) is structurally and immunologically highly related to hepatitis B virus X associated protein-i (XAP-1)

AU - Krishnamoorthy, Raghu

AU - Lee, Teh Hsiu

AU - Butel, Janet S.

AU - Das, Hriday

PY - 1997/1/28

Y1 - 1997/1/28

N2 - 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., and 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 ~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 ~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 ~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.

AB - 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., and 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 ~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 ~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 ~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.

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DO - 10.1021/bi961407c

M3 - Article

VL - 36

SP - 960

EP - 969

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

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