GSK3β is a key regulator of the ROS-dependent necrotic death induced by the quinone DMNQ
GSK3β is a key regulator of the ROS-dependent necrotic death induced by the quinone DMNQ
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DOI：
10.1038/s41419-019-2202-0
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发表时间：
2020-01-02
影响因子：
9
通讯作者：
Brancolini, Claudio
中科院分区：
文献类型：
--
作者：
Ciotti, Sonia;Iuliano, Luca;Brancolini, Claudio
控制坏死的信号通路仍然是神秘和有争议的。我们应用基于shRNA的活力筛选来鉴定坏死反应的关键要素。我们利用了小分子（G5），使共价加合物与游离硫醇迈克尔加成和elevently多重应力。在抗凋亡的细胞中，G5通过诱导蛋白质解折叠、谷胱甘肽耗竭、ER应激、蛋白酶体损伤和细胞骨架应激来触发坏死。激酶GSK 3 β在筛选的最高命中中分离。使用醌DMNQ，ROS发生器，我们证明，GSK 3 β参与ROS依赖性坏死的调节。我们的结果已经使用siRNA和通过CRISPR/Cas9技术敲除GSK 3 β进行了验证。响应于DMNQ，GSK 3 β通过丝氨酸9去磷酸化而被激活，伴随着Akt失活。在醌诱导的促坏死应激期间，在线粒体膜电位崩溃之前，GSK 3 β逐渐积聚到细胞核中。由于缺乏GSK 3 β，响应DMNQ的ROS积累受损。我们提供的证据表明，强制性的双电子还原黄素酶，NQO 1（NAD（P）H醌脱氢酶1）和NQO 2的活动是必需的，以抑制DMNQ诱导的坏死。在缺乏GSK 3 β的情况下，NQO 1和NQO 2的表达显著增加，这可能是由于NRF 2的转录活性增加。总之，GSK 3 β通过钝化抗氧化反应，特别是NQO 1和NQO 2表达，有利于响应于ROS的坏死的出现，如由醌DMNQ产生的。
Signaling pathways controlling necrosis are still mysterious and debated. We applied a shRNA-based viability screen to identify critical elements of the necrotic response. We took advantage from a small molecule (G5) that makes covalent adducts with free thiols by Michael addition and elicits multiple stresses. In cells resistant to apoptosis, G5 triggers necrosis through the induction of protein unfolding, glutathione depletion, ER stress, proteasomal impairments, and cytoskeletal stress. The kinase GSK3 beta was isolated among the top hits of the screening. Using the quinone DMNQ, a ROS generator, we demonstrate that GSK3 beta is involved in the regulation of ROS-dependent necrosis. Our results have been validated using siRNA and by knocking-out GSK3 beta with the CRISPR/Cas9 technology. In response to DMNQ GSK3 beta is activated by serine 9 dephosphorylation, concomitantly to Akt inactivation. During the quinone-induced pro-necrotic stress, GSK3 beta gradually accumulates into the nucleus, before the collapse of the mitochondrial membrane potential. Accumulation of ROS in response to DMNQ is impaired by the absence of GSK3 beta. We provide evidence that the activities of the obligatory two-electrons reducing flavoenzymes, NQO1 (NAD(P)H quinone dehydrogenase 1) and NQO2 are required to suppress DMNQ-induced necrosis. In the absence of GSK3 beta the expression of NQO1 and NQO2 is dramatically increased, possibly because of an increased transcriptional activity of NRF2. In summary, GSK3 beta by blunting the anti-oxidant response and particularly NQO1 and NQO2 expression, favors the appearance of necrosis in response to ROS, as generated by the quinone DMNQ.