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

分泌的蛋白质是细胞与环境相互作用所必需的。内质网（ER）中大多数分泌蛋白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至关重要。蛋白质二硫异构酶（PDI）和内质网硫醇氧化酶（ERO）共同构成内质网的氧化蛋白折叠机制。它们构成了一个二硫中继系统，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，因为内质网中氧化蛋白折叠的需求可以根据发育阶段和环境条件发生巨大变化。植物作为一种无根生物，经常暴露在特别剧烈的环境变化中，这就需要细胞水平上的快速适应反应。在酵母和哺乳动物中，折叠机制的活性由EROs上的调节巯基开关调节，允许快速的翻译后控制。虽然植物对氧化蛋白折叠的控制尚不清楚，但与酵母和哺乳动物相比，植物的ERO异构体含有几种额外的半胱氨酸。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硫醇开关或形成替代二硫化物，构成额外的，植物特异性水平的ERO氧化还原控制。为了研究植物内质网中基于硫醇的氧化蛋白折叠调控，拟南芥和小壶属植物将被用作模型系统。在拟南芥中，敲除和敲除两种ERO亚型的组合导致对DTT的严重敏感性和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。该项目的目标是鉴定和表征植物ERO蛋白中的调节硫醇开关及其与PDI作为开关操作员和PDI靶蛋白的相互作用。ERO调控的生化分析将与ERO中硫醇氧化还原平衡的体内研究相结合。由于缺乏特异性和敏感的传感器，动态测量内质膜中的谷胱甘肽氧化还原电位和H2O2仍然受到限制，因此将开发改进的基于荧光蛋白的传感器变体，用于氧化氧化还原环境。这些尝试将建立在对rogfp相互作用的机制理解和蛋白质-蛋白质相互作用建模的基础上。能够成像内质网中的氧化还原状态和H2O2水平将为定量氧化还原分析提供重要的一步，并为深入了解内质网及其调节硫醇在设置内质网氧化还原稳态中的作用提供新的视角。

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.