Select Heterozygous Keap1 Mutations Have a Dominant-Negative Effect on Wild-Type Keap1 In Vivo
Select Heterozygous Keap1 Mutations Have a Dominant-Negative Effect on Wild-Type Keap1 In Vivo
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DOI：
10.1158/0008-5472.can-10-2939
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发表时间：
2011-03-01
期刊：
影响因子：
11.2
通讯作者：
Yamamoto, Masayuki
中科院分区：
文献类型：
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作者：
Suzuki, Takafumi;Maher, Jonathan;Yamamoto, Masayuki
在稳态条件下，Keap1组成性地介导蛋白酶体降解Nrf2。然而，Keap1响应于细胞环境的三级变化允许Nrf2的释放，从而转录调节下游细胞保护基因，帮助细胞存活。据估计，KEAP1/NRF2体细胞突变导致组成性NRF2激活发生在大约25%的人类肺肿瘤中，其他组织亚型中也存在类似的比例。由于Keap1和Nrf2之间的化学计量是2:1，我们假设杂合Keap1突变可以通过突变体Keap1（突变体）与Keap1（WT）的异源二聚化而作为显性负性蛋白抑制野生型Keap1（WT）的活性。当表达Keap1（G430C）或Keap1（G364C）突变体代替Keap1（WT）时，观察到幼鱼过早死亡。为了验证显性负效应的假设，Keap1缺失小鼠被改造成共同表达Keap1（WT）和Keap1（G430C）转基因，这种表型类似于先前从人类肺肿瘤中观察到的表型。这些小鼠存活，但表型上表现出下游Nrf2靶基因的过度激活和中度食管角化过度。与转基因小鼠Keap1（G430C）突变体相似，Keap1（C273&288A）的替换也降低了Keap1（WT）在体内的活性。为了进一步阐明显性-阴性异二聚体的作用，在Keap1（WT）存在的情况下，产生了Broad-complex、Tramtrack和brick -a- brac （BTB）结构域缺失或战略性突变的转基因小鼠，这些基因域不允许Keap1二聚体的存在，并且在体内未能诱导显性-阴性效应。因此，这些结果表明，将Keap1（WT）隔离到Keap1（突变体）-Keap1（WT）异源二聚体中会导致体内的显性负作用，并引起Nrf2激活。癌症Res;71 (5);1700 - 9。AACR (C) 2010。
Under homeostatic conditions, Keap1 constitutively mediates the proteasomal degradation Nrf2. However, tertiary changes in Keap1 in response to the cellular environment allow for liberation of Nrf2 to transcriptionally regulate downstream cytoprotective genes that aid in cell survival. KEAP1/NRF2 somatic mutations causing constitutive NRF2 activation have been estimated to occur in approximately 25% of human lung tumors, with similar rates believed to exist in other tissue subtypes. As the stoichiometry between Keap1 and Nrf2 is 2: 1, we hypothesized that heterozygous Keap1 mutations could suppress wild-type Keap1 (Keap1(WT)) activity by functioning as a dominant-negative protein through heterodimerization of mutant Keap1 (Keap1(mutant)) with Keap1(WT). When Keap1(G430C) or Keap1(G364C) mutants were expressed in lieu of Keap1(WT), premature juvenile mortality was observed. To test the hypothesis of a dominant-negative effect, Keap1-null mice were engineered to coexpress both Keap1(WT) and Keap1(G430C) transgenes, a phenotype analogous to that previously observed from a human lung tumor. These mice were viable, but phenotypically displayed hyperactivation of downstream Nrf2 target genes and moderate esophageal hyperkeratosis. Similar to the transgenic mouse Keap1(G430C) mutant, Keap1(C273&288A) substitutions also diminished Keap1(WT) activity in vivo. To further delineate involvement of the dominant-negative heterodimer, transgenic mice with a deletion or strategic mutation in Broad-complex, Tramtrack and Bric-a-Brac (BTB) domain that disallowed Keap1 dimerization were generated in the presence of Keap1(WT), and failed to induce the dominant-negative effect in vivo. These results thus demonstrate that sequestration of Keap1(WT) into a Keap1(mutant)-Keap1(WT) heterodimer leads to a dominant-negative effect in vivo and gives rise to Nrf2 activation. Cancer Res; 71(5); 1700-9. (C)2010 AACR.