Glutathione and Redox Control in the Mitochondrial Intermembrane Space

描述(由申请人提供)： 细胞内的硫醇-二硫键平衡对于具有重要功能的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代谢是细胞内硫醇氧化还原动态平衡的重要组成部分，因为该细胞器是有氧代谢产生ROS的主要来源和靶点。线粒体的基本功能，如氧化磷酸化、蛋白质输入和铁-S簇生物发生，直接依赖于硫醇-二硫键的平衡。因此，线粒体硫醇-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酿酒酵母为模型系统来表征线粒体硫醇氧化还原动态平衡。线粒体被一层双膜分成两个不同的隔室：基质和膜间间隙。控制基质中硫醇-二硫键平衡的氧化还原调节系统具有很好的特性。然而，IMS中硫醇-二硫键氧化还原控制的机制以及GSH代谢在IMS硫醇氧化还原途径中的作用是我们对线粒体氧化还原系统了解的关键空白。这项建议的目标是确定在IMS中维持硫醇-二硫键平衡的机制。针对IMS和基质的基于GFP的氧化还原传感器已经被开发出来，可以通过体内荧光测量来实现局部的硫醇氧化还原监测。这些传感器与局部还原/氧化谷胱甘肽(GSH：GSSG)池保持平衡，并通过形成二硫键记录硫醇氧化还原的变化。为了确定GSH代谢如何影响IMS氧化还原环境，将使用这些传感器来表征氧化还原压力下IMS、基质和胞浆之间的GSH：GSSG交换，并将确定线粒体GSH转运体(目标1)。此外，将通过确定GSH：GSSG或IMS蛋白输入机制的变化如何相互影响来破译GSH代谢和IMS中依赖硫醇的蛋白质输入之间的联系(目标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)。通过在高度易驯化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，并对涉及线粒体功能障碍的疾病具有重要意义。

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.