Functional evaluation of monomeric glutathione peroxidase 8 (Gpx8) as a mammalian redox sensor in the ER

硫醇-二硫化物介导的酶催化不仅对内质网(ER)中蛋白质的正确折叠是必不可少的，而且还参与了内质网相关的额外过程，如内质网应激感知。内质网应激感应是控制未折叠蛋白反应和相关细胞死亡的关键细胞事件。在内质网依赖的硫醇依赖系统中，过氧化氢氧化还蛋白-4和谷胱甘肽过氧化物酶家族的两个单体成员，即谷胱甘肽过氧化物酶7(Gpx7)和谷胱甘肽过氧化物酶8(Gpx8)，被认为通过硫醇-二硫键介导的交换反应参与内质网应激反应。在机制上，Gpx7通过不同的硫醇-二硫键交换反应增加GRP78/Bip(HSPA5，热休克蛋白家族A)的伴侣活性，从而导致Gpx7基因敲除的小鼠和细胞积累错误折叠的蛋白质，并表现出内质网和氧化应激增加。然而，Gpx7的表达非常低和有限；因此，它不太可能在检测正确的蛋白质折叠方面发挥更广泛的作用。与之形成鲜明对比的是，我们发现其相关家族成员Gpx8在大多数组织和细胞中普遍表达，其中肾脏、心脏、睾丸、肾上腺和胰岛的表达水平最高。此外，我们还发现Gpx8在内质网和氧化应激的作用下是动态调节的，尽管其潜在的分子机制仍有待进一步研究。此外，我们发现强制表达Gpx8使细胞对脂肪酸诱导的细胞死亡具有抵抗力，这表明脂肪酸代谢、Gpx8和内质网应激之间存在联系。为了揭示内质网中这个鲜有研究的硫醇依赖的氧化还原网络成员，我们建议(I)鉴定并从功能上验证Gpx8介导的新的硫醇-二硫化物介导的氧化还原伙伴；(Ii)询问Gpx8在内质网应激中丢失和相关细胞死亡的细胞机制以及Gpx8在内质网应激反应中的动态调节的影响；以及(Iii)作为最终概念验证，以验证在精通或缺乏Gpx8的新小鼠模型中的一些关键发现，以及高脂饮食诱导的代谢综合征和2型糖尿病的应激模型。因此，我们的研究旨在揭开尚未被认识的硫醇介导的内质网应激传感机制，这最终将在具有内质网应激特征的病理条件下产生新的概念。

Thiol-disulfide mediated enzyme catalysis is not only essential for proper protein folding in the endoplasmic reticulum (ER), but is emerging to be involved in additional ER-related processes, such as ER stress sensing. ER stress sensing is a critical cellular event that controls unfolded protein response and associated cell death. Among the ER-resident thiol-dependent systems, peroxiredoxin-4 and two monomeric members of the glutathione peroxidase family of proteins, i.e. glutathione peroxidase 7 (Gpx7) and glutathione peroxidase 8 (Gpx8), are being considered to contribute to ER stress sensing via thiol-disulfide mediated exchange reactions. Mechanistically, Gpx7 increases the chaperone activity of GRP78/BiP (HSPA5, heat shock protein family A) by distinctive thiol-disulfide exchange reactions, and consequently Gpx7 knockout mice and cells accumulate misfolded proteins and show increased ER and oxidative stress. Nevertheless, Gpx7 shows very low and limited expression; therefore it is unlikely that it confers a more generalized role in sensing proper protein folding. By stark contrast, we found its related family member Gpx8 to be ubiquitously expressed in most tissues and cells with highest expression levels in kidney, heart, testis, adrenal gland and islet of pancreas. Additionally, we discovered that Gpx8 is dynamically regulated in response to ER and oxidative stress, although the underlying molecular mechanisms remain to be further explored. Moreover, we found that forced expression of Gpx8 renders cells resistant to fatty acid induced cell death indicating a link between fatty acid metabolism, Gpx8 and ER stress. To shed light into this barely studied member of the thiol-dependent redox network in the ER, we propose (i) to identify and functionally validate novel thiol-disulfide mediated redox partners of Gpx8, (ii) to interrogate the cellular mechanisms of Gpx8 loss in ER stress and associated cell death as well as the impact of the dynamic regulation of Gpx8 in response to ER stress, and (iii) as ultimate proof-of-concept to validate some of the key findings in novel mouse models proficient or deficient in Gpx8, along with a stress model of high fat diet induced metabolic syndrome and Type 2 diabetes mellitus. Hence, our studies aim at unravelling yet-unrecognized thiol-mediated ER stress sensing mechanisms, which will ultimately lead to new concepts in pathological conditions with an ER stress signature.