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

单体谷胱甘肽过氧化物酶 8 (Gpx8) 作为哺乳动物内质网氧化还原传感器的功能评估

• 批准号: 251897775
• 负责人: Dr. Marcus Conrad
• 金额: --
• 依托单位: Institut für Metabolismus und Zelltod (MCD)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/251897775?language=en
• 关键词: Functional; evaluation; monomeric; glutathione; peroxidase

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.

巯基-二硫化物介导的酶催化不仅对内质网（ER）中的蛋白质正确折叠至关重要，而且正在出现参与额外的ER相关过程，如ER应激传感。内质网应激传感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ER驻留硫醇依赖性系统中，过氧化物氧还蛋白-4和谷胱甘肽过氧化物酶蛋白家族的两个单体成员，即谷胱甘肽过氧化物酶7（Gpx 7）和谷胱甘肽过氧化物酶8（Gpx 8），被认为有助于通过硫醇-二硫化物介导的交换反应进行ER应激传感。从机制上讲，Gpx 7通过独特的巯基-二硫键交换反应增加GRP 78/BiP（HSPA 5，热休克蛋白家族A）的伴侣活性，因此Gpx 7敲除小鼠和细胞积累错误折叠的蛋白，并显示增加的ER和氧化应激。然而，Gpx 7显示出非常低和有限的表达;因此，它不太可能赋予更普遍的作用，在传感正确的蛋白质折叠。与此形成鲜明对比的是，我们发现其相关家族成员Gpx 8在大多数组织和细胞中普遍表达，其中在肾脏、心脏、睾丸、肾上腺和胰岛中表达水平最高。此外，我们发现Gpx 8在响应ER和氧化应激时受到动态调节，尽管潜在的分子机制仍有待进一步探索。此外，我们发现Gpx 8的强制表达使细胞对脂肪酸诱导的细胞死亡具有抗性，这表明脂肪酸代谢、Gpx 8和ER应激之间存在联系。为了阐明ER中巯基依赖性氧化还原网络的这一几乎未被研究的成员，我们提出（i）鉴定和功能验证新的巯基-二硫化物介导的Gpx 8氧化还原配偶体，（ii）询问ER应激中Gpx 8损失和相关细胞死亡的细胞机制以及Gpx 8响应于ER应激的动态调节的影响，和（iii）作为最终的概念验证，以验证Gpx 8熟练或缺乏的新型小鼠模型中的一些关键发现，沿着高脂肪饮食诱导的代谢综合征和2型糖尿病的应激模型。因此，我们的研究旨在揭示尚未认识到的硫醇介导的ER应激传感机制，这将最终导致具有ER应激特征的病理条件的新概念。