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

巯基二硫介导的酶催化不仅对内质网（ER）中适当的蛋白质折叠至关重要，而且还参与了其他内质网相关的过程，如内质网应激感应。内质网应激感知是控制未折叠蛋白反应和相关细胞死亡的关键细胞事件。在内质网巯基依赖系统中，过氧化物还蛋白-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)，被认为通过巯基二硫基介导的交换反应参与内质网应激感知。机制上，Gpx7通过独特的硫醇-二硫交换反应增加GRP78/BiP （HSPA5，热休克蛋白家族A）的伴侣活性，从而导致Gpx7敲除小鼠和细胞积累错误折叠的蛋白质，并表现出ER和氧化应激增加。而Gpx7表达极低且有限；因此，它不太可能在感知适当的蛋白质折叠中赋予更广泛的作用。与之形成鲜明对比的是，我们发现其家族成员Gpx8在大多数组织和细胞中普遍表达，在肾脏、心脏、睾丸、肾上腺和胰岛中表达量最高。此外，我们发现Gpx8在内质网和氧化应激下受到动态调节，尽管其潜在的分子机制仍有待进一步探索。此外，我们发现强迫表达Gpx8使细胞抵抗脂肪酸诱导的细胞死亡，这表明脂肪酸代谢、Gpx8和内质网应激之间存在联系。为了阐明内质网中这个几乎没有被研究过的硫醇依赖的氧化还原网络成员，我们提出(i)鉴定和功能验证新的硫醇二硫介导的Gpx8氧化还原伙伴，（ii）询问Gpx8在内质网应激和相关细胞死亡中丢失的细胞机制，以及Gpx8响应内质网应激的动态调节的影响。（iii）在Gpx8精通或缺乏的新型小鼠模型以及高脂饮食诱导代谢综合征和2型糖尿病的应激模型中，作为验证一些关键发现的最终概念证明。因此，我们的研究旨在揭示尚未被认识的硫醇介导的内质网应激感知机制，这将最终导致具有内质网应激特征的病理条件下的新概念。

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.