Thiol-based regulation of oxidative protein folding in the ER of plants

分泌的蛋白质对于细胞与其环境相互作用是必不可少的。大多数分泌蛋白在内质网（ER）中的折叠严格要求半胱氨酸之间催化形成分子内二硫键。二硫化物对蛋白质的结构和功能至关重要，这使得它们的形成对生存必不可少。蛋白质二硫键异构酶（PDI）和ER硫醇氧化酶（ERO）共同组成ER的氧化蛋白质折叠机制。它们构成二硫键中继系统，用于将电子从新生蛋白质底物的半胱氨酸转移到分子氧。这种机制需要动态调节，因为ER中氧化蛋白质折叠的需求可能会因发育阶段和环境条件而显着变化。作为固着生物，植物经常暴露于特别严重的环境变化中，这需要细胞水平上的快速驯化反应。在酵母和哺乳动物中，折叠机制的活性由ERO上的调节性巯基开关调节，从而允许快速的翻译后控制。虽然在植物中氧化蛋白质折叠的控制尚不清楚，但与它们的酵母和哺乳动物对应物相比，植物的ERO同种型含有几个额外的半胱氨酸。基于它们在ERO蛋白中的位置，这些半胱氨酸可能通过逐步激活多个巯基开关或形成替代性二硫化物来构成额外的植物特异性ERO氧化还原控制水平。为了解决基于巯基的调节氧化蛋白质折叠在ER的植物拟南芥和小立碗藓将被用作模型系统。 拟南芥中两种ERO同种型的敲除和敲低的组合导致对DTT的严重敏感性，并且在非胁迫条件下对乙烯的显著不敏感性，这将乙烯受体ETR 1鉴定为ERO活性的敏感靶标。本项目的目标是确定和表征调节巯基开关在植物ERO蛋白和它们的相互作用与PDI开关运营商以及PDI靶蛋白。ERO调节的生化分析将与ER中巯基氧化还原平衡的体内研究相结合。由于谷胱甘肽氧化还原电位和H2 O2在内质网中的动态测量仍然受到缺乏特异性和敏感性传感器的限制，将开发用于氧化还原环境的改进的基于荧光蛋白的传感器变体。这些尝试将建立在对roGFP相互作用和蛋白质-蛋白质相互作用建模的改进的机械理解的基础上。能够在ER中成像氧化还原状态和H2 O2水平将为定量氧化还原分析向前迈出重要一步，并对ERO及其调节硫醇在设置ER氧化还原稳态中的作用产生新的深入了解。

Secreted proteins are essential for a cell to interact with its environment. The folding of most secretory proteins in the endoplasmic reticulum (ER) strictly requires the catalyzed formation of intramolecular disulfide bonds between cysteines. The disulfides are critical for protein structure and function, which makes their formation indispensable for survival. Protein disulfide isomerase (PDI) and ER thiol oxidases (ERO) together make up the oxidative protein folding machinery of the ER. They constitute a disulfide relay system for the transfer of electrons from cysteines of nascent protein substrates to molecular oxygen. This machinery needs to be dynamically regulated, as the demand for oxidative protein folding in the ER can vary dramatically, depending on developmental stage and environmental conditions. As sessile organisms plants are frequently exposed to particularly severe environmental changes, which necessitate rapid acclimation responses on cellular level. In yeast and mammals, the activity of the folding machinery is modulated by regulatory thiol switches on the EROs, allowing rapid posttranslational control. While the control of oxidative protein folding is not understood in plants, the ERO isoforms of plants contain several additional cysteines as compared to their yeast and mammalian counterparts. Based on their position in the ERO protein these cysteines are likely to constitute an additional, plant-specific level of ERO redox control by stepwise activation of multiple thiol switches or the formation of alternative disulfides. To address the thiol-based regulation of oxidative protein folding in the ER of plants Arabidopsis thaliana and Physcomitrella patens will be employed as model systems. A combination of knockout and knockdown for the two ERO isoforms in Arabidopsis results in a severe sensitivity towards DTT and under non-stress conditions a pronounced insensitivity towards ethylene which identifies the ethylene receptor ETR1 as a sensitive target of ERO activity. The goal of this project is to identify and to characterize regulatory thiol switches in plant ERO proteins and their interaction with PDIs as switch operators as well as PDI target proteins. Biochemical analysis of ERO regulation will be combined with in vivo studies of the thiol redox poise in the ER. As dynamic measurements of the glutathione redox potential and H2O2 in the ER are still limited by a lack of specific and sensitive sensors, improved fluorescent protein-based sensor variants will be developed for oxidizing redox environments. These attempts will build on improved mechanistic understanding of roGFP-interactions and modelling of protein-protein interactions. Being able to image redox state and H2O2 levels in the ER will provide a major step forward for quantitative redox analysis and generate novel depth of insight into the role of ERO and its regulatory thiols in setting ER redox homeostasis.