A conserved and multifunctional redox switch in yeast Get3 and mammalian TRC40

酵母 Get3 和哺乳动物 TRC40 中保守的多功能氧化还原开关

• 批准号: 251912032
• 负责人: Professorin Dr. Blanche Schwappach-Pignataro
• 金额: --
• 依托单位: Department of Biological Chemistry
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/251912032?language=en
• 关键词: conserved; multifunctional; redox; switch; yeast

Maintaining protein homeostasis during oxidative stress is a major challenge for all organisms. This is due to the fact that reactive oxygen species substantially decrease cellular ATP levels, therefore incapacitating existing ATP-dependent chaperones and slowing down other ATP-dependent processes. Our previous studies have contributed to the realization that organisms use a very different class of stress-specific ATP-independent chaperones, which are posttranslationally activated by oxidative stress. We discovered that Get3 (TRC40 in mammals), a soluble member of the tail-anchored protein (TA-protein) insertion machinery, is redox-regulated by a thiol switch, and rapidly turns into an effective molecular chaperone upon oxidative stress-mediated disulfide bond formation. Activation of the ATP-independent chaperone function goes hand in hand with a loss in ATP-dependent TA-protein targeting activity, making Get3 a bona fide dual-function protein. Complementation studies in yeast using functional Get3 variants revealed that the chaperone activity of Get3 and not its TA-insertion function is responsible for many of the previously observed get3 deletion phenotypes. These results are particularly exciting since they imply that the chaperone function of Get3 might in fact be the physiologically more significant role of Get3. We will now apply the extensive mechanistic and structural toolset that we have developed in our work on the bacterial redox-regulated chaperone Hsp33 to investigate the redox-regulated activation of Get3/TRC40 both in vitro and in vivo. We will pay particular attention to the role of the other members of the soluble and membrane-based GET complexes in the chaperone function of Get3, working on the assumption that these proteins promote functional changes in Get3, affect client binding and/or release. We will validate our in vitro results in yeast, making particular use of the many heavily manipulated and well-characterized yeast mutants that we have previously generated, and test corresponding mutants in mammalian cell lines using established conditions for the siRNA-mediated knock-down of various components of the TRC40 machinery. We will use a systematic localization screen in yeast under different physiological conditions to identify client proteins that rely exclusively on the chaperone function of Get3, and will monitor the fate of these proteins during stress and upon stress recovery. These results will provide important new insights into the pro-survival role that Get3/TRC40 plays in yeast and mammals.

在氧化应激期间维持蛋白质稳态是所有生物体面临的主要挑战。这是因为活性氧大大降低了细胞ATP水平，从而使现有的ATP依赖性伴侣丧失能力，并减缓了其他ATP依赖性过程。我们之前的研究有助于认识到生物体使用一种非常不同的应激特异性atp独立伴侣，它们在翻译后被氧化应激激活。我们发现Get3（哺乳动物中的TRC40）是尾锚蛋白（ta蛋白）插入机制的可溶性成员，受硫醇开关的氧化还原调节，并在氧化应激介导的二硫键形成中迅速转化为有效的分子伴侣。atp独立伴侣功能的激活与atp依赖的ta蛋白靶向活性的丧失密切相关，使Get3成为真正的双功能蛋白。利用功能性Get3变异对酵母进行的互补研究表明，许多先前观察到的Get3缺失表型是由Get3的伴侣活性而不是其ta插入功能造成的。这些结果特别令人兴奋，因为它们暗示了Get3的伴侣功能实际上可能是Get3在生理上更重要的作用。现在，我们将应用我们在细菌氧化还原调节伴侣Hsp33的研究中开发的广泛的机制和结构工具集来研究体外和体内氧化还原调节的Get3/TRC40激活。我们将特别关注可溶性和膜基GET复合物的其他成员在Get3的伴侣功能中的作用，假设这些蛋白质促进Get3的功能变化，影响客户端结合和/或释放。我们将在酵母中验证我们的体外结果，特别是使用我们之前生成的许多经过大量操作和特征良好的酵母突变体，并使用既定条件在哺乳动物细胞系中测试相应的突变体，以sirna介导的TRC40机制的各种成分的敲除。我们将在不同生理条件下的酵母中使用系统的定位筛选来鉴定完全依赖于Get3伴侣功能的客户蛋白，并将监测这些蛋白在应激和应激恢复过程中的命运。这些结果将为了解Get3/TRC40在酵母和哺乳动物中的促生存作用提供重要的新见解。