Project Details
Projekt
Thiol switches controlled by the glutathione-S-transferase GDAP1
Subject Area
Biochemistry
Cell Biology
Cell Biology
Term
from 2017 to 2022
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 386417025
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 GDAP1 is a glutathione-S-transferase (GST) and our unpublished data demonstrate that mutation of the active site completely abrogates all functions of GDAP1 signifying that the GST function is involved in its mechanism of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.
DFG Programme
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