Apolipoprotein B gene regulatory factor-2 (BRF-2) is structurally and immunologically highly related to hepatitis B virus X associated protein-1 (XAP-1).
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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载脂蛋白 B 基因调节因子 2 (BRF-2) 在结构和免疫学上与乙型肝炎病毒 X 相关蛋白 1 (XAP-1) 高度相关。
DOI：
10.1021/bi961407c
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Das,HK
中科院分区：
文献类型：
--
作者：
Krishnamoorthy,RR;Lee,TH;Butel,JS;Das,HK
人载脂蛋白 B (apoB) 基因的肝细胞特异性表达至少由位于 -128 和 +122 之间的四顺式作用元件控制 [Chuang, S. S., & Das, H. K. (1996)Biochem.Biophys.Res.Commun.220, 553−562]。远端元件（-128 至 -85）似乎是肝脏特异性的，因为它在 HepG2 细胞中显示出阳性活性，在 HeLa 细胞中显示出阴性活性。 ApoB 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，其分子量已确定为 ∼120 kDa [Zhuang et al. 2017]。 (1992)Mol.Cell.Biol.12, 3183−3191]。在本文中，我们报告了通过使用高效液相色谱进一步纯化来分离 BRF-2 的两种亚型。通过银染检测，两种亚型在十二烷基硫酸钠聚丙烯酰胺凝胶电泳中均产生单个 ~120-kDa 条带。衍生自 HPLC 纯化的较重 BRF-2 亚型的两种胰蛋白酶肽的氨基酸序列被确定为 YLAIAPPIIK 和 ALYYLQIHPQELR。研究发现这两种肽与人乙型肝炎病毒 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。在蛋白质印迹分析中，针对 XAP-1 的两种合成肽产生的抗肽抗血清在两种 BRF-2 亚型中识别出~120-kDa 的多肽条带。通过使用含有各种内部缺失和取代突变的 apoB 启动子片段作为凝胶迁移率变化测定的模板，我们确定了 -104 和 -85 之间的区域对于高分子量形式的结合至关重要。相反，较低分子量的异构体与所有测试的 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 50%。这些结果表明BRF-2和XAP-1是apoB基因的结构和免疫学高度相关的反式激活剂。我们认为 BRF-2 在溶液中既以单体 (BRF-2M) 又以同源寡聚物（可能是同源二聚体 (BRF-2D)）的形式存在；寡聚化似乎是赋予 BRF-2 蛋白序列特异性的重要步骤，从而促进其作为 apoB 基因的 atrans 激活剂的作用。
Hepatic cell-specific expression of the human apolipoprotein B (apoB) gene is controlled by at least fourcis-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 ∼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 inhibitin vitrotranscription 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 relatedtrans-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 atrans-activator of the apoB gene.