Design of a C/EBP-specific, dominant-negative bZIP protein with both inhibitory and gain-of-function properties
Design of a C/EBP-specific, dominant-negative bZIP protein with both inhibitory and gain-of-function properties
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DOI：
10.1074/jbc.271.4.2040
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发表时间：
1996-01-26
影响因子：
4.8
通讯作者：
Vinson, C
中科院分区：
文献类型：
--
作者：
Olive, M;Williams, SC;Vinson, C
我们开发了一种具有显性负性（DN）和功能获得特性的bZIP蛋白GBF - F。GBF - F是一种由两部分组成的嵌合体：来自植物bZIP蛋白GBF - 1（GBF）的DNA结合（碱性）区域和一个亮氨酸拉链（F），其被设计为优先与C/EBPα亮氨酸拉链异源二聚化。生化研究表明，GBF - F优先与C/EBPα形成异源二聚体，因此能结合由C/EBP和GBF碱性区域所识别的半位点组成的嵌合DNA序列。在HepG2肝癌细胞中的瞬时转染表明，GBF - F的两个组成部分对于抑制C/EBPα的反式激活都是必需的。当C/EBPα亮氨酸拉链被GCN4或VBP的亮氨酸拉链取代时，所得蛋白质能够反式激活一个C/EBP顺式元件，但不受GBF - F抑制，这表明显性负性作用的特异性是由亮氨酸拉链决定的。C/EBP家族的所有已知成员都包含相似的亮氨酸拉链区域，并且都受GBF - F抑制。GBF - F还表现出功能获得特性，因为在C/EBP家族成员的必要协作下，它能够反式激活一个含有嵌合C/EBP\GBF位点的启动子。因此，这种蛋白质作为C/EBP功能的显性负性抑制剂以及具有新型DNA序列特异性的激活蛋白都具有潜在的用途。
We have developed a bZIP protein, GBF-F, with both dominant-negative (DN) and gain-of-function properties, GBF-F is a chimera consisting of two components: the DNA binding (basic) region from the plant bZIP protein GBF-1 (GBF) and a leucine zipper (F) designed to preferentially heterodimerize with the C/EBP alpha leucine zipper. Biochemical studies show that GBF-F preferentially forms heterodimers with C/EBP alpha and thus binds a chimeric DNA sequence composed of the half-sites recognized by the C/EBP and GBF basic regions. Transient transfections in HepG2 hepatoma cells show that both components of GBF-F are necessary for inhibition of C/EBP alpha transactivation. When the C/EBP alpha leucine zipper is replaced with that of either GCN4 or VBP, the resulting protein can transactivate a C/EBP cis-element but is not inhibited by GBF-F, indicating that the specificity of dominant-negative action is determined by the leucine zipper. All known members of the C/EBP family contain similar leucine zipper regions and are inhibited by GBF-F, GBF-F also exhibits gain-of-function properties, since, with the essential cooperation of a C/EBP family member, it can transactivate a promoter containing the chimeric C/EBP\GBF site. This protein therefore has potential utility both as a dominant-negative inhibitor of C/EBP function and as an activator protein with novel DNA sequence specificity.