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

分泌的蛋白质对于细胞与环境的相互作用是必不可少的。内质网(ER)中大多数分泌性蛋白质的折叠严格要求半胱氨酸之间形成分子内二硫键。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于生存是不可或缺的。蛋白质二硫键异构酶(PDI)和内质网硫醇氧化酶(ERO)共同构成内质网的氧化蛋白质折叠机制。它们构成了一个二硫键中继系统，用于将电子从新生蛋白质底物的半胱氨酸转移到分子氧。这一机制需要动态调节，因为内质网对氧化蛋白质折叠的需求可能会根据发育阶段和环境条件而发生巨大变化。作为固着生物，植物经常暴露在特别严重的环境变化中，这就需要在细胞水平上做出快速的驯化反应。在酵母和哺乳动物中，折叠机械的活动受到性激素上调节的硫醇开关的调节，从而允许快速的翻译后控制。虽然植物中氧化蛋白质折叠的控制尚不清楚，但与酵母和哺乳动物相比，植物的ERO亚型含有几个额外的半胱氨酸。根据它们在ERO蛋白中的位置，这些半胱氨酸很可能通过逐步激活多个硫醇开关或形成替代的二硫化物，构成额外的、植物特有的ERO氧化还原控制水平。为了解决植物内质网中基于硫醇的氧化蛋白折叠的调控问题，拟南芥和小球藻将被用作模型系统。在拟南芥中，两种ERO亚型的敲除和敲除的组合导致了对DTT的严重敏感性，以及在非胁迫条件下对乙烯的明显不敏感，这表明乙烯受体ETR1是ERO活性的敏感靶标。该项目的目标是确定和表征植物ERO蛋白中的调节性硫醇开关，以及它们作为开关操纵子和PDI靶蛋白与PDI的相互作用。ERO调节的生化分析将与内质网中硫醇氧化还原平衡的体内研究相结合。由于内质网中谷胱甘肽氧化还原电位和过氧化氢的动态测量仍然受到缺乏特定和灵敏传感器的限制，因此将开发用于氧化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-蛋白质相互作用模型的改进的机制理解的基础上。能够成像内质网中的氧化还原状态和过氧化氢水平将为定量氧化还原分析提供重要的一步，并为ERO及其调节硫醇在设定内质网氧化还原动态平衡中的作用产生新的深度洞察。

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.