Sato R, Yang J, Wang X, Evans M J, Ho Y K, Goldstein J L, Brown M S
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1994 Jun 24;269(25):17267-73.
Transcription of the low density lipoprotein receptor gene and other sterol-regulated genes is stimulated by sterol regulatory element-binding protein-1 (SREBP-1), a basic-helix-loop-helix-leucine zipper (bHLH-ZIP) transcription factor. Human SREBP-1 is synthesized as an 1147-amino acid precursor that is attached intrinsically to membranes of the nuclear envelope and endoplasmic reticulum. In sterol-depleted cells the precursor is cleaved to generate an NH2-terminal fragment that enters the nucleus and activates transcription by binding to sterol regulatory element-1 (SRE-1). Sterols prevent transcriptional activation by blocking the proteolytic cleavage. In the current studies, performed with hamster SREBP-1, we used mutational analysis to localize the transcriptional activation domain to an acidic NH2-terminal sequence. Deletion of this sequence converted SREBP-1 from an activator to an inhibitor of transcription. DNA binding was assigned to the basic region of the bHLH-ZIP domain. Binding was abolished by substitution of 3 amino acids that were previously implicated in DNA binding by Max, another bHLH-ZIP protein. The membrane attachment domain was localized to two hydrophobic regions at residues 477-497 and 536-556. Truncation of SREBP-1 prior to these regions gave rise to an NH2-terminal fragment that was soluble and entered the nucleus. This fragment was more than 30-fold more active than full-length SREBP-1 in stimulating transcription of an SRE-1 containing reporter gene in transfected cells. Deletion of the hydrophobic sequences (delta 476-556) yielded a protein that appeared cytosolic by immunofluorescence microscopy but failed to enter the nucleus readily, apparently because of inhibition by sequences in the remaining COOH-terminal domain. This study provides a picture of the domain structure of SREBP-1 and further elucidates the mechanism by which it adjusts gene transcription to maintain cholesterol homeostasis in animal cells.
低密度脂蛋白受体基因和其他固醇调节基因的转录受到固醇调节元件结合蛋白-1（SREBP-1）的刺激，SREBP-1是一种碱性螺旋-环-螺旋-亮氨酸拉链（bHLH-ZIP）转录因子。人SREBP-1作为一种1147个氨基酸的前体被合成，该前体内在地附着于核膜和内质网的膜上。在固醇缺乏的细胞中，前体被切割以产生一个NH2末端片段，该片段进入细胞核并通过与固醇调节元件-1（SRE-1）结合来激活转录。固醇通过阻断蛋白水解切割来阻止转录激活。在目前对仓鼠SREBP-1进行的研究中，我们使用突变分析将转录激活域定位到一个酸性的NH2末端序列。删除该序列将SREBP-1从激活剂转变为转录抑制剂。DNA结合被指定到bHLH-ZIP结构域的碱性区域。通过替换先前与另一种bHLH-ZIP蛋白Max的DNA结合有关的3个氨基酸，结合被消除。膜附着域被定位到残基477-497和536-556处的两个疏水区域。在这些区域之前截断SREBP-1会产生一个可溶的NH2末端片段并进入细胞核。在转染细胞中，该片段在刺激含SRE-1的报告基因转录方面比全长SREBP-1活跃30多倍。删除疏水序列（δ476-556）产生一种通过免疫荧光显微镜观察似乎位于胞质中的蛋白质，但不能轻易进入细胞核，显然是由于剩余COOH末端结构域中的序列抑制所致。这项研究提供了SREBP-1结构域结构的图景，并进一步阐明了其调节基因转录以维持动物细胞胆固醇稳态的机制。
