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的主要挑战。 这是由于活性氧大大降低细胞ATP水平，因此使现有的ATP依赖性伴侣丧失能力并减缓其他ATP依赖性过程。 我们以前的研究有助于认识到，生物体使用一个非常不同的类的应激特异性ATP-独立的分子伴侣，这是posterior氧化应激激活。 我们发现Get 3（哺乳动物中的TRC 40）是尾锚定蛋白（TA-蛋白）插入机制的可溶性成员，通过巯基开关进行氧化还原调节，并且在氧化应激介导的二硫键形成后迅速变成有效的分子伴侣。 ATP非依赖性伴侣功能的激活与ATP依赖性TA蛋白靶向活性的丧失密切相关，使Get 3成为真正的双功能蛋白。 在酵母中使用功能性Get 3变体的互补研究表明，Get 3的伴侣活性而不是其TA插入功能是导致许多先前观察到的Get 3缺失表型的原因。 这些结果特别令人兴奋，因为它们意味着Get 3的伴侣功能实际上可能是Get 3在生理上更重要的作用。 我们现在将应用我们在细菌氧化还原调节的伴侣蛋白Hsp 33的工作中开发的广泛的机制和结构工具集来研究氧化还原调节的Get 3/TRC 40在体外和体内的激活。 我们将特别注意的作用，其他成员的可溶性和膜为基础的GET复合物中的伴侣功能的Get 3，工作的假设，这些蛋白质促进功能的变化Get 3，影响客户端的结合和/或释放。 我们将在酵母中验证我们的体外结果，特别是利用我们以前产生的许多经过大量操作和充分表征的酵母突变体，并使用建立的siRNA介导的TRC 40机制各种组分敲低的条件在哺乳动物细胞系中测试相应的突变体。 我们将在不同的生理条件下使用酵母中的系统定位筛选来鉴定完全依赖于Get 3的伴侣蛋白功能的客户端蛋白，并将监测这些蛋白在应激期间和应激恢复后的命运。这些结果将为Get 3/TRC 40在酵母和哺乳动物中发挥的促生存作用提供重要的新见解。