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首页> 外文期刊>Oncogene >Nrf2 is an inhibitor of the Fas pathway as identified by Achilles' Heel Method, a new function-based approach to gene identification in human cells
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Nrf2 is an inhibitor of the Fas pathway as identified by Achilles' Heel Method, a new function-based approach to gene identification in human cells

机译：Nrf2是Fas通路的抑制剂，通过Achilles的Heel方法鉴定，这是一种基于功能的新方法来鉴定人细胞中的基因

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Here we describe the Achilles' Heel Method (AHM), a new function-based approach for identification of inhibitors of signaling pathways, optimized for human cells. The principle of AHM is the identification of 'sensitizing' cDNAs based on their decreased abundance following selection. As a proof of principle, we have employed AHM for the identification of Fas/CD95/APO-1 pathway inhibitors. HeLa cells were transfected with an antisense cDNA expression library in an episomal vector followed by selection with a suboptimal dose of the apoptotic inducer. Antisense inactivation of Fas inhibitors rendered the cells more sensitive to apoptosis resulting in their preferential death and consequent loss of their sensitizing episomes that were identified by subtraction. We show that the resulting products were enriched for sensitizing cDNAs as seven out of eight candidates tested were confirmed as inhibitors of Fas-induced killing either by transfection or by pharmacological inhibition. Furthermore, we demonstrate by multiple approaches that one candidate, NF-E2 related factor 2 (Nrf2), is an inhibitor of Fas-induced apoptosis. Inactivation of Nrf2 by antisense or by a membrane permeable dominant-negative polypeptide sensitized cells while overexpression of Nrf2 protected cells from Fas-induced apoptosis. In addition, dicumarol, an inhibitor of the phase II detoxifying enzyme NQO1, a downstream target of Nrf2, sensitized cells. Nrf2 induces the production of Glutathione (GSH) and we demonstrated that N-acetyl L-cysteine (NAC), a precursor to GSH, protected cells from Fas-mediated killing. Taken together, AHM is a powerful approach for the identification of inhibitors of a signaling pathway with a low rate of false positives that opens new avenues for function profiling of human genes and discovery of new drug targets.
机译：在这里，我们描述了阿基里斯的脚跟法（AHM），这是一种新的基于功能的方法，可用于信号传导途径的抑制剂的鉴定，针对人类细胞进行了优化。 AHM的原理是根据选择后丰度降低来鉴定“致敏” cDNA。作为原理的证明，我们已使用AHM鉴定Fas / CD95 / APO-1途径抑制剂。用反义cDNA表达文库在游离型载体中转染HeLa细胞，然后用次适量的凋亡诱导剂进行选择。 Fas抑制剂的反义失活使细胞对凋亡更加敏感，导致其优先死亡，并因此失去了通过减法鉴定的致敏附加体。我们表明，所得产物富集了用于致敏cDNA的抗体，因为通过转染或通过药理学抑制作用，证实了八分之七的候选候选物是Fas诱导的杀伤的抑制剂。此外，我们通过多种方法证明了一种候选因子NF-E2相关因子2（Nrf2）是Fas诱导的凋亡的抑制剂。 Nrf2的反义或膜可渗透的显性阴性多肽致敏细胞的失活，而Nrf2的过表达保护细胞免受Fas诱导的细胞凋亡。此外，狄古洛尔（II期排毒酶NQO1的抑制剂，Nrf2的下游靶标）的抑制剂可使细胞敏感。 Nrf2诱导谷胱甘肽（GSH）的产生，我们证明了N-乙酰基L-半胱氨酸（NAC）（GSH的前体）保护细胞免受Fas介导的杀伤。综上所述，AHM是一种有效的方法，可用于识别具有低假阳性率的信号传导途径抑制剂，从而为人类基因功能分析和发现新的药物靶标开辟了新途径。

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