Ero1α翻译后修饰介导的内质网氧化应激及抗氧化应答

The endoplasmic reticulum (ER) is the main compartment for the folding and maturation of newly synthesized secretory and membrane proteins in eukaryotic cells. Proteins that traverse the secretory pathway generally possess disulfide bonds for their stabilities and functions. The highly conserved sulfhydryl oxidase Ero1 in the ER catalyses de novo disulfide bond formation by using molecular oxygen and generating hydrogen peroxide as a byproduct. The overload of peroxide is potentially toxic to trigger ER oxidative stress. The fine-tuning regulation of Ero1 oxidase activity is critical for the maintenance of the ER redox homeostasis. This project aims to study the effects of two post-translational modifications of human Ero1α recently characterized in our lab, phosphorylation and S-nitrosylation, on the oxidative protein folding, ER redox status and ER oxidative stress, and to reveal the molecular mechanisms underlying the regulation of Ero1α activity by these post-translational modifications. This project will also study how the antioxidant response is launched by the over-oxidation signal in the ER and further transferred into the nucleus via activating the Nrf2/ARE pathway to up-regulate ER-located peroxidases. The outcomes of this study will illustrate novel cellular mechanisms of maintaining the ER redox homeostasis on the post-translational level, and shed light on a novel cellular antioxidant signaling pathway from the ER lumen to the nucleus.

内质网是真核细胞中分泌蛋白及膜蛋白折叠和加工的主要场所，二硫键形成是这些蛋白成熟的重要步骤。内质网中高度保守的巯基氧化酶Ero1利用分子氧催化二硫键形成的同时产生过氧化氢, 其过度累积会造成内质网氧化应激。Ero1氧化酶活力的精细调控对维持内质网氧化还原稳态十分重要。本项目拟研究（一）人细胞组成型表达的Ero1α的两种我们最近新鉴定的翻译后修饰——磷酸化和亚硝基化，对于蛋白质氧化折叠、内质网氧化还原状态和内质网氧化应激的影响，揭示这些修饰调节Ero1α活力的分子机制。（二）由Ero1α翻译后修饰引起的内质网氧化应激信号如何启动抗氧化应答，通过Nrf2/ARE通路上调定位于内质网的过氧化物酶的表达。本研究将阐明细胞在翻译后水平调控内质网氧化还原稳态的新机制，并力图揭示一条新的从内质网到核的抗氧化应答信号转导通路。

内质网是真核细胞中分泌蛋白及膜蛋白折叠和加工的主要场所，二硫键形成是这些蛋白成熟的重要步骤。内质网中高度保守的巯基氧化酶Ero1利用分子氧催化二硫键形成的同时产生过氧化氢, 其过度累积会造成内质网氧化应激。Ero1活力的精细调控对维持内质网氧化还原稳态十分重要。本项目研究发现：（一）Ero1α的Ser145可以被分泌途径激酶Fam20C磷酸化，磷酸化的Ero1α表现出更高的氧化酶活力，对于维持还原应激等条件下的内质网氧化还原稳态十分重要。（二）Ero1α的Cys166会发生亚硝基化，导致其失活加快，氧化酶活力降低。衰老的细胞中Ero1α亚硝基化水平增高可能是内质网氧化还原稳态失衡的重要原因。（三）定位于内质网的过氧化物酶GPx7是调控细胞衰老的关键分子，其表达受到抗氧化转录因子Nrf2的调节。进一步研究揭示了“二甲双胍-Nrf2-GPx7”这一从线虫到人保守的延缓衰老的信号通路。本研究阐明了翻译后修饰调控内质网氧化还原稳态和细胞应激的新机制，揭示了内质网相关抗氧化应答信号通路在衰老等生命过程中的重要作用。