Thiol switches controlled by the glutathione-S-transferase GDAP1

神经节苷脂诱导分化相关蛋白1 （GDAP1）是一种参与控制线粒体形状和功能的神经元蛋白。它的过表达会减弱线粒体呼吸作用并导致线粒体断裂，而其敲低则会产生相反的效果。GDAP1固定在面对细胞质的外线粒体膜上，其突变可引起周围神经病变，即腓骨肌萎缩症。有趣的是，有丝分裂酶-2 （MFN2）的突变，一种具有相似拓扑结构和对线粒体形状和功能相反影响的GTPase，导致临床无法区分的疾病，使两种蛋白质之间的联系成为可能。GDAP1是一种谷胱甘肽- s -转移酶（GST），我们未发表的数据表明，活性位点的突变完全取消了GDAP1的所有功能，这表明GST功能参与了其作用机制。在核心细胞应激指标氧化谷胱甘肽（GSSG）存在的情况下，MFN2形成由涉及巯基开关半胱氨酸684的二硫桥介导的反式低聚物。这会导致线粒体灌注不足，这是一种适应性应激反应。有趣的是，在GSSG存在的情况下，GDAP1与MFN2在同一个复合体中迁移，其过表达抑制应激诱导的线粒体低灌注。基于这些结果，我们假设GST GDAP1在细胞氧化还原稳态改变时介导线粒体形状和功能的适应性变化。GDAP1可以存在于与MFN2的复合物中，并通过将GSH转移到硫醇开关C684本身或复合物中的其他蛋白质上，从而调节MFN2的寡聚化，然后这些蛋白质调节硫醇开关。因此，GDAP1调节线粒体灌注不足，这在CMT疾病中受到损害。在Aim A中，我们将1)利用来自神经前体细胞的CRISPR/ cas9改变的人类运动神经元，研究正常和应激条件下巯基开关MFN2 C684介导的线粒体形状和功能的变化。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状和功能的影响。我们假设GDAP1在这些细胞中具有改变的功能。因此，这些实验将直接连接CMT疾病中涉及的两种蛋白质的作用机制，从而使人们更好地了解这种疾病。由于含有GDAP1和MFN2的复合物比这两个蛋白加起来要大得多，我们也假设GDAP1将GSH转移到其他蛋白质中尚未表征的硫醇开关，这些蛋白质参与线粒体形状和功能对应激的适应。因此，在Aim B中，我们将通过以下方法鉴定GDAP1的靶蛋白和gssg诱导的蛋白复合体中含有的蛋白：1)使用定量的、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GDAP1表达紊乱的果蝇。我们还将2)鉴定gssg诱导复合物中包含的蛋白质。

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 GDAP1 is a glutathione-S-transferase (GST) and our unpublished data demonstrate that mutation of the active site completely abrogates all functions of GDAP1 signifying that the GST function is involved in its mechanism of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.