Thiol switches controlled by the glutathione-S-transferase GDAP1

由谷胱甘肽-S-转移酶 GDAP1 控制的硫醇开关

• 批准号: 386417025
• 负责人: Professor Dr. Axel Methner
• 金额: --
• 依托单位: Institut für Immunologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/386417025?language=en
• 关键词: Thiol; switches; controlled; glutathione; transferase

Ganglioside-induced differentiation associated protein 1 (GDAP1) is a neuronal protein involved in the control of mitochondrial shape and function. Its over-expression attenuates mitochondrial respiration and causes mitochondrial fragmentation while its knockdown has opposite effects. GDAP1 is anchored in the outer mitochondrial membrane facing the cytosol and its mutation causes a peripheral neuropathy, Charcot-Marie-Tooth disease. Interestingly, mutations in mitofusin-2 (MFN2), a GTPase with a similar topology and opposite effects on mitochondrial shape and function, cause a clinically undistinguishable disease making a link of the two proteins conceivable. GDAP1 is a glutathione-S-transferase (GST) and our unpublished data demonstrate that mutation of the active site completely abrogates all functions of GDAP1 signifying that the GST function is involved in its mechanism of action. In the presence of oxidized glutathione (GSSG) - the core cellular stress indicator - MFN2 forms trans oligomers mediated by disulphide bridges involving the thiol switch cysteine 684. This then causes mitochondrial hyperfusion, an adaptive stress response. Intriguingly, in the presence of GSSG, GDAP1 migrates in the same complex with MFN2 and its over-expression inhibits stress-induced mitochondrial hyperfusion. Based on these results, we hypothesize that the GST GDAP1 mediates adaptive changes in mitochondrial shape and function upon alterations of the cellular redox homeostasis. GDAP1 can reside in a complex with MFN2 and modulate MFN2 oligomerization either by transferring GSH to the thiol switch C684 itself or to other proteins in the complex, which then modulate the thiol switch. GDAP1 thereby regulates mitochondrial hyperfusion and this is compromised in CMT disease. In Aim A, we will 1) study changes in mitochondrial shape and function mediated by the thiol switch MFN2 C684 under normal and stressed conditions using CRISPR/Cas9-altered human motoneurons derived from neural precursor cells. We will also 2) study the effect of wildtype and mutated GDAP1 on mitochondrial shape and function in MFN2 thiol switch cells. We assume that GDAP1 will have an altered function in these cells. These experiments will therefore directly connect the mechanism of action of two proteins involved in CMT disease and thus enable a better understanding of this disease.As the complex containing GDAP1 and MFN2 is much larger than the two proteins together, we also hypothesize that GDAP1 transfers GSH to other, yet uncharacterized thiol switches in other proteins involved in the adaptation of mitochondrial shape and function to stress. In Aim B we will thus identify target proteins of GDAP1 and proteins contained in the GSSG-induced protein complex by 1) using quantitative, site-specific chemical proteomics to compare patient-derived neurons and flies with perturbed GDAP1 expression. We will also 2) identify the proteins contained in the GSSG-induced complex.

神经节苷脂诱导分化相关蛋白1(GDAP1)是一种参与调控线粒体形态和功能的神经元蛋白。它的过度表达减弱了线粒体的呼吸，导致线粒体碎裂，而它的敲除则起到了相反的作用。GDAP1被锚定在面对胞浆的线粒体膜外膜上，它的突变会导致一种周围神经病变，夏科-玛丽-图斯病。有趣的是，丝裂原蛋白-2(Mfn2)的突变导致了一种临床上无法区分的疾病，使这两种蛋白之间的联系成为可能。Mfn2是一种具有类似拓扑结构而对线粒体形状和功能有相反影响的GTP酶。GDAP1是一种谷胱甘肽-S-转移酶(GST)，我们未发表的数据表明，GDAP1活性位点的突变完全丧失了GDAP1的所有功能，这意味着GST功能参与了其作用机制。在氧化谷胱甘肽(GSSG)的存在下-核心细胞应激指示剂-Mfn2形成反式低聚物，由二硫键介导，涉及硫醇开关半胱氨酸684。这会导致线粒体过度融合，这是一种适应性应激反应。有趣的是，在GSSG的存在下，GDAP1与Mfn2在同一复合体中迁移，其过度表达抑制了应激诱导的线粒体过度融合。基于这些结果，我们假设GST GDAP1介导了线粒体形状和功能在细胞氧化还原稳态改变时的适应性变化。GDAP1可以存在于与Mfn2的复合体中，通过将GSH转移到硫醇开关C684本身或复合体中的其他蛋白质来调节Mfn2的寡聚，然后这些蛋白质调节硫醇开关。GDAP1因此调节线粒体过度融合，这在CMT病中受到影响。在目标A中，我们将1)利用CRISPR/Cas9突变的人神经前体细胞来源的运动神经元，研究在正常和应激条件下，硫醇开关Mfn2 C684介导的线粒体形态和功能的变化。我们还将研究野生型和突变型GDAP1对Mfn2硫醇开关细胞线粒体形态和功能的影响。我们假设GDAP1将在这些细胞中具有改变的功能。因此，这些实验将直接连接参与CMT病的两种蛋白质的作用机制，从而使人们能够更好地了解这种疾病。由于GDAP1和Mfn2的复合体比这两种蛋白质的总和大得多，我们还假设GDAP1将GSH转移到其他尚未确定的硫醇开关，参与线粒体对应激的形状和功能的适应。因此，在目标B中，我们将通过以下方式识别GDAP1的目标蛋白质和GSSG诱导的蛋白质复合体中包含的蛋白质：1)使用定量、位点特异性的化学蛋白质组学来比较患者来源的神经元和果蝇与受干扰的GDAP1表达。我们还将鉴定GSSG诱导的复合体中包含的蛋白质。