Overexpression of the large subunit of the protein Ku suppresses metallothionein-I induction by heavy metals.
Overexpression of the large subunit of the protein Ku suppresses metallothionein-I induction by heavy metals.
复制标题
Ku 蛋白大亚基的过度表达可抑制重金属对金属硫蛋白-I 的诱导。
DOI：
10.1073/pnas.95.18.10390
复制
发表时间：
1998
影响因子：
11.1
通讯作者：
Jacob,ST
中科院分区：
文献类型：
--
作者：
Ghoshal,K;Li,Z;Jacob,ST
金属硫蛋白（MT）参与清除有毒重金属和保护细胞免受活性氧中间体的影响。为了研究蛋白Ku在MT表达中的潜在作用，我们测量了亲代大鼠成纤维细胞系（Rat 1）和稳定和组成型过表达Ku的小亚基、大亚基和异二聚体的细胞系中MT-1 mRNA的水平。用CdS 04或ZnS 04处理使亲本细胞和过量产生小亚基的细胞（Ku-70）或过量表达异源二聚体的细胞（Ku-7080）中的MT-I mRNA水平升高20- 30倍。而过表达Ku大亚基的细胞（Ku-80）则不能诱导MT-Ⅰ的表达。体外转录实验表明，MT-Ⅰ启动子活性在Ku-80细胞核提取物中被选择性抑制。通过在Ku-80细胞中诱导另一个Cd诱导基因hsp 70，显示了阻遏物功能的特异性。在转录反应开始时加入Ku-80细胞的核提取物消除了大鼠1细胞提取物中的MT-1启动子活性。在大鼠1细胞核提取物中形成的转录本在与Ku-80细胞提取物进一步孵育后未降解。阻遏物对热敏感。在Ku-80细胞中，至少有四种控制MT-Ⅰ启动子活性的转录因子的DNA结合活性不受影响。这些观察结果为进一步探索Ku亚基介导MT诱导抑制的机制奠定了基础。
Metallothioneins (MT) are involved in the scavenging of the toxic heavy metals and protection of cells from reactive oxygen intermediates. To investigate the potential role of the protein Ku in the expression of MT, we measured the level of MT-I mRNA in the parental rat fibroblast cell line (Rat 1) and the cell lines that stably and constitutively overexpress the small subunit, the large subunit, and the heterodimer of Ku. Treatment with CdS04or ZnS04elevated the MT-I mRNA level 20- to 30-fold in the parental cells and the cells (Ku-70) that overproduce the small subunit or those (Ku-7080) overexpressing the heterodimer. By contrast, the cells (Ku-80) overexpressing the large subunit of Ku failed to induce MT-I.In vitrotranscription assay showed that the MT-I promoter activity was suppressed selectively in the nuclear extracts from Ku-80 cells. The specificity of the repressor function was shown by the induction of hsp 70, another Cd-inducible gene, in Ku-80 cells. Addition of the nuclear extract from Ku-80 cells at the start of the transcription reaction abolished the MT-l promoter activity in the Rat 1 cell extract. The transcript once formed in Rat 1 nuclear extract was not degraded by further incubation with the extract from Ku-80 cells. The repressor was sensitive to heat. The DNA-binding activities of at least four transcription factors that control the MT-I promoter activity were not affected in Ku-80 cells. These observations have set the stage for further exploration of the mechanisms by which the Ku subunit mediates suppression of MT induction.