Recurrent G-to-A substitution in a single codon of SREBP cleavage-activating protein causes sterol resistance in three mutant Chinese hamster ovary cell lines.
Recurrent G-to-A substitution in a single codon of SREBP cleavage-activating protein causes sterol resistance in three mutant Chinese hamster ovary cell lines.
复制标题
SREBP 裂解激活蛋白的单个密码子中反复发生的 G 到 A 替换会导致三种突变的中国仓鼠卵巢细胞系产生甾醇抗性。
DOI：
10.1073/pnas.93.24.13709
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发表时间：
1996
影响因子：
11.1
通讯作者：
Goldstein,JL
中科院分区：
文献类型：
--
作者：
Nohturfft,A;Hua,X;Brown,MS;Goldstein,JL
含氧固醇（如25-羟基胆固醇）可杀死中国仓鼠卵巢细胞，因为它们抑制固醇调节元件结合蛋白（SREBP）的蛋白水解加工，这是一对膜结合转录因子，可激活控制胆固醇合成和从脂蛋白摄取的基因。未加工的SREBP保持膜结合，它们不能激活胆固醇生物合成途径，细胞死于胆固醇剥夺。 先前已经通过化学诱变和选择对25-羟基胆固醇杀伤的抗性分离了几种胆固醇抗性仓鼠细胞系。我们最近确定了一个这样的细胞系（25-RA细胞）中的缺陷作为一个点突变，在一个新发现的膜蛋白的1276个氨基酸，指定SREBP裂解激活蛋白（SCAP）。25-RA细胞中的突变是由SCAP 443密码子的G-到-A的转换引起的，将天冬氨酸变为天冬酰胺。 野生型SCAP，当通过转染过表达时，刺激两种SREBP的蛋白水解加工。D443 N取代是一种激活突变，可增加SCAP的活性并使其对25-羟基胆固醇的抑制具有抗性。我们在这里报告了相同的G-到-A的转变，在另外两个线的中国仓鼠卵巢细胞，诱变和分离的类似协议。 这三个突变独立发生，如使用两个基因内序列多态性的突变基因的单倍型分析所示。所有三种细胞系均用有利于G-至-A转变的烷化剂（亚硝基乙基脲或乙基甲烷磺酸盐）诱变。然而，在所有三种细胞系中相同位置处相同核苷酸取代的发现表明SCAP可能在其刺激SREBP切割的能力方面是独特的，并且残基443是蛋白质被25-羟基胆固醇抑制的能力的关键决定因素。
Oxygenated sterols such as 25-hydroxycholesterol kill Chinese hamster ovary cells because they inhibit the proteolytic processing of sterol regulatory element binding proteins (SREBPs), a pair of membrane-bound transcription factors that activate genes controlling cholesterol synthesis and uptake from lipoproteins. The unprocessed SREBPs remain membrane-bound, they cannot activate the cholesterol biosynthetic pathway, and the cells die of cholesterol deprivation. Several sterol-resistant hamster cell lines have been isolated previously by chemical mutagenesis and selection for resistance to killing by 25-hydroxycholesterol. We recently identified the defect in one such cell line (25-RA cells) as a point mutation in a newly discovered membrane protein of 1276 amino acids, designated SREBP cleavage-activating protein (SCAP). The mutation in the 25-RA cells resulted from a G-to-A transition in codon 443 of theSCAPgene, changing aspartic acid to asparagine. Wild-type SCAP, when overexpressed by transfection, stimulates the proteolytic processing of both SREBPs. The D443N substitution is an activating mutation that increases the activity of SCAP and renders it resistant to inhibition by 25-hydroxycholesterol. We here report the identical G-to-A transition in two additional lines of Chinese hamster ovary cells that were mutagenized and isolated by a similar protocol. The three mutations occurred independently as indicated by haplotype analysis of the mutant genes using two intragenic sequence polymorphisms. All three cell lines were mutagenized with alkylating agents (nitrosoethylurea or ethylmethane sulfonate) that favor G-to-A transitions. Nevertheless, the finding of the same nucleotide substitution at the same location in all three cell lines indicates that SCAP may be unique in its ability to stimulate SREBP cleavage, and residue 443 is a crucial determinant of the protein’s ability to be inhibited by 25-hydroxycholesterol.