Oxidation of an Exposed Methionine Instigates the Aggregation of Glyceraldehyde-3-Phosphate Dehydrogenase [Molecular Biophysics]
August 1st, 2014 by Samson, A. L., Knaupp, A. S., Kass, I., Kleifeld, O., Marijanovic, E. M., Hughes, V. A., Lupton, C. J., Buckle, A. M., Bottomley, S. P., Medcalf, R. L.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous and abundant protein that participates in cellular energy production. GAPDH normally exists in a soluble form, however, following necrosis, GAPDH and numerous other intracellular proteins convert into an insoluble disulfide-crosslinked state via the process of 'Nucleocytoplasmic Coagulation'. Here, free radical-induced aggregation of GAPDH was studied as an in vitro model of Nucleocytoplasmic Coagulation. Despite the fact that disulfide-crosslinking is a prominent feature of GAPDH aggregation, our data shows that it is not a primary rate-determining step. To identify the true instigating event of GAPDH misfolding, we mapped the post-translational modifications that arise during its aggregation. Solvent accessibility and energy calculations of the mapped modifications, within the context of the high-resolution native GAPDH structure, suggested that oxidation of methionine-46 may instigate aggregation. We confirmed this by mutating methionine-46 to leucine, which rendered GAPDH highly-resistant to free radical-induced aggregation. Molecular Dynamics simulations suggest that oxidation of methionine-46 triggers a local increase in the conformational plasticity of GAPDH, which likely promotes further oxidation and eventual aggregation. Hence, methionine-46 represents a "linchpin", whereby its oxidation is a primary event permissive for the subsequent misfolding, aggregation and disulfide-crosslinking of GAPDH. A critical role for linchpin residues in Nucleocytoplasmic Coagulation and other forms of free radical-induced protein misfolding should now be investigated. Furthermore, since disulfide-crosslinked aggregates of GAPDH arise in many disorders, and because methionine-46 is irrelevant to native GAPDH function, mutation of methionine-46 in models of disease should allow the unequivocally assessment of whether GAPDH aggregation influences disease progression.