Krishnamoorthy R R, Lee T H, Butel J S, Das H K
Department of Pharmacology, University of North Texas Health Science Center at Fort Worth 76107, USA.
Biochemistry. 1997 Jan 28;36(4):960-9. doi: 10.1021/bi961407c.
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.
人载脂蛋白B（apoB）基因的肝细胞特异性表达受位于-128至+122位之间的至少四个顺式作用元件控制[Chuang, S. S., & Das, H. K. (1996) Biochem. Biophys. Res. Commun. 220, 553 - 562]。远端元件（-128至-85）似乎具有肝脏特异性，因为它在HepG2细胞中显示出阳性活性，而在HeLa细胞中显示出阴性活性。载脂蛋白B基因调节因子-2（BRF-2）与序列（-104至-85）相互作用。BRF-2已从大鼠肝核提取物中纯化出来，其分子量已确定约为120 kDa[Zhuang等人(1992) Mol. Cell. Biol. 12, 3183 - 3191]。在本文中，我们报告了通过高效液相色谱进一步纯化分离出两种BRF-2同工型。两种同工型在经银染检测的十二烷基硫酸钠聚丙烯酰胺凝胶电泳中均产生一条约120 kDa的单带。测定了从高效液相色谱纯化的较重的BRF-2同工型衍生的两个胰蛋白酶肽的氨基酸序列为YLAIAPPIIK和ALYYLQIHPQELR。发现这两个肽与人类乙型肝炎病毒X相关蛋白-1（XAP-1）和猴紫外线损伤DNA结合蛋白（UV-DDB）具有100%的序列同源性。针对XAP-1的两种合成肽产生的抗肽抗血清在蛋白质印迹分析中识别两种BRF-2同工型中的一条约120 kDa的多肽带。通过使用含有各种内部缺失和一个替代突变的apoB启动子片段作为凝胶迁移率变动分析的模板，我们确定-104至-85之间的区域对于高分子量形式的结合至关重要。相反，较低分子量的同工型与所有测试的apoB突变体结合。发现针对XAP-1的抗肽2抗血清可抑制大鼠肝核提取物中apoB基因的体外转录达50%。这些结果表明BRF-2和XAP-1是apoB基因在结构和免疫方面高度相关的反式激活因子。我们提出BRF-2在溶液中既以单体（BRF-2M）形式存在，也以同寡聚体形式存在，可能是同二聚体（BRF-2D）；寡聚化似乎是赋予BRF-2蛋白序列特异性并从而促进其作为apoB基因反式激活因子作用的一个必要步骤。
