Glutathione and Redox Control in the Mitochondrial Intermembrane Space

描述（由申请人提供）： 细胞内巯基-二硫键平衡对于具有重要功能半胱氨酸残基的蛋白质的活性至关重要。三肽谷胱甘肽（GSH）和氧化还原酶如谷氧还蛋白（GRX）和硫氧还蛋白（TRX）通过催化二硫键交换反应和保护半胱氨酸残基免受活性氧（ROS）氧化来帮助维持硫醇-二硫键平衡。线粒体GSH代谢是细胞巯基氧化还原稳态的关键组成部分，因为该细胞器是有氧代谢产生的ROS的主要来源和靶点。重要的线粒体功能，如氧化磷酸化，蛋白质输入和Fe-S簇生物合成直接依赖于硫醇-二硫键平衡。因此，线粒体巯基-二硫键平衡的破坏与癌症、神经退行性疾病和衰老有关。这项研究计划的长期目标是以酵母酿酒酵母为模型系统，表征线粒体巯基氧化还原稳态。 细胞膜被双层膜分为两个不同的区室：基质和膜间空间（IMS）。控制基质中硫醇-二硫化物平衡的氧化还原调节系统已得到充分表征。然而，IMS中巯基-二硫化物氧化还原控制的机制和GSH代谢在IMS巯基氧化还原途径中的作用代表了我们对线粒体氧化还原系统的认识中的关键空白。本提案的目标是确定维持IMS中硫醇-二硫化物平衡的机制。已经开发了靶向IMS和基质的基于GFP的氧化还原传感器，以允许通过体内荧光测量进行局部硫醇氧化还原监测。这些传感器与局部还原型/氧化型谷胱甘肽（GSH：GSSG）池平衡，并通过二硫键形成记录硫醇氧化还原变化。为了确定GSH代谢如何影响IMS氧化还原环境，将使用这些传感器表征氧化还原应激下IMS、基质和胞质溶胶之间的GSH：GSSG交换，并鉴定线粒体GSH转运蛋白（目的1）。此外，通过确定GSH：GSSG或IMS蛋白质输入机制的改变如何相互影响，将解释GSH代谢和巯基依赖性蛋白质输入IMS之间的联系（目的2）。最后，将确定有助于维持IMS硫醇氧化还原平衡的其他硫醇氧化还原系统/途径（目标3）。通过在高度易处理的生物体中使用分子遗传学方法，这些研究将为IMS中的硫醇氧化还原控制途径提供新的线索，对涉及线粒体功能障碍的疾病具有强烈的影响。

DESCRIPTION (provided by applicant): Intracellular thiol-disulfide balance is critical for the activity of proteins with functionally important cysteine residues. The tripeptide glutathione (GSH) and oxidoreductases like glutaredoxins (GRXs) and thioredoxins (TRX) help maintain thiol-disulfide balance by catalyzing disulfide exchange reactions and protecting cysteines residues from oxidation by reactive oxygen species (ROS). Mitochondrial GSH metabolism is a key component of cellular thiol redox homeostasis since this organelle is the main source and target of ROS produced from aerobic metabolism. Essential mitochondrial functions such as oxidative phosphorylation, protein import, and Fe-S cluster biogenesis are directly dependent on thiol-disulfide balance. Consequently, disruption of mitochondrial thiol-disulfide balance has been linked to cancer, neurodegenerative diseases, and aging. The long-term objective of this research program is to characterize mitochondrial thiol redox homeostasis using the yeast Saccharomyces cerevisiae as a model system. The mitochondrion is divided by a double membrane into two distinct compartments: the matrix and the intermembrane space (IMS). Redox regulatory systems that govern thiol-disulfide balance in the matrix are well-characterized. However, the mechanisms for thiol-disulfide redox control in the IMS and the role of GSH metabolism in IMS thiol redox pathways represent key gaps in our knowledge of mitochondrial redox systems. The goal of this proposal is to determine the mechanisms for maintaining thiol-disulfide balance in the IMS. GFP-based redox sensors that are targeted to the IMS and matrix have been developed to allow localized thiol redox monitoring via in vivo fluorescence measurements. These sensors equilibrate with the local reduced/oxidized glutathione (GSH:GSSG) pool and register thiol redox changes via disulfide bond formation. To determine how GSH metabolism influences the IMS redox environment, GSH:GSSG exchange between the IMS, matrix, and cytosol under redox stress will be characterized using these sensors and mitochondrial GSH transporters will be identified (Aim 1). Furthermore, the connection between GSH metabolism and thiol- dependent protein import into the IMS will be deciphered by determining how alterations in GSH:GSSG or the IMS protein import machinery influence each other (Aim 2). Finally, additional thiol redox systems/pathways that help maintain IMS thiol redox balance will be identified (Aim 3). By using a molecular genetics approach in a highly tractable organism, these studies will shed new light on thiol redox control pathways in the IMS, with strong implications for disorders involving mitochondrial dysfunction.