Over 160 mutations in superoxide dismutase 1 (SOD1) are associated with familial amyotrophic lateral sclerosis (fALS), where the main pathological feature is deposition of SOD1 into proteinaceous cytoplasmic inclusions. We previously showed that the tryptophan residue at position 32 (W32) mediates the prion-like propagation of SOD1 misfolding in cells, and that a W32S substitution blocks this phenomenon. Here, we used in vitro protein assays to demonstrate that a W32S substitution in SOD1-fALS mutants significantly diminishes their propensity to aggregate whilst paradoxically decreasing protein stability. We also show SOD1-W32S to be resistant to seeded aggregation, despite its high abundance of unfolded protein. A cell-based aggregation assay demonstrates that W32S substitution significantly mitigates inclusion formation. Furthermore, this assay reveals that W32 in SOD1 is necessary for the formation of a competent seed for aggregation under these experimental conditions. Following the observed importance of W32 for aggregation, we established that treatment of living cells with the W32-interacting 5-Fluorouridine (5-FUrd), and its FDA approved analogue 5-Fluorouracil (5-FU), substantially attenuate inclusion formation similarly to W32S substitution. Altogether, we highlight W32 as a significant contributor to SOD1 aggregation, and propose that 5-FUrd and 5-FU present promising lead drug candidates for the treatment of SOD1-associated ALS.
The underlying physical causes of SOD1---related ALS are still not well---understood. We address this problem here by computationally designing two de novo mutants, A89R and K128N, which were predicted theoretically to be either significantly destabilizing or stabilizing respectively. We subjected these in silico designed mutants to a series of experimental tests, including in vitro measures of thermodynamic stability, cell---based aggregation and toxicity assays, and an in vivo developmental model of zebrafish motor neuron axonopathy. The experimental tests validated the theoretical predictions: A89R is an unstable, highly---deleterious mutant, and K128N is a stable, non--toxic mutant. Moreover, K128N is predicted computationally to form an unusually stable heterodimer with the familial ALS mutant A4V. Consistent with this prediction, co---injection of K128N and A4V into zebrafish shows profound rescue of motor neuron pathology. The demonstrated success of these first principles calculations to predict the physical properties of SOD1 mutants holds promise for rationally designed therapies to counter the progression of ALS. SignificanceMutations in the protein superoxide dismutase cause ALS, and many of these mutants have decreased folding stability. We sought to pursue this thread using a synthetic biology approach, where we designed two de novo mutations, one stabilizing and one destabilizing, as predicted using computational molecular dynamics simulations. We then tested these mutants using in vitro, cell--based, and in vivo zebrafish models. We found that the unstable mutant was toxic, and induced a severe ALS phenotype in zebrafish; the predicted stable mutant, on the other hand, behaved even better than WT. In fact, it was able to rescue the ALS phenotype caused by mutant SOD1. We propose a mechanism for this rescue, which may provide an avenue for therapeutic intervention.\bodyMain Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons in the motor cortex, brainstem and spinal cord, with a lifetime risk of about 1:400(1). Most cases of ALS cases are sporadic with no consistently identified genetic mutation, however, a significant fraction of familial ALS (fALS) cases are due mutations in Cu,Zn superoxide dismutase (SOD1)(2---4). The ubiquity of disease---associated mutations (currently over 160 (5)) throughout the primary sequence of SOD1 suggests a physico---chemical origin for SOD1--related fALS.When natively folded, SOD1 is a 32 kDa homodimer that catalyzes the dismutation of oxygen radicals to less harmful molecular oxygen and hydrogen peroxide(4). A properly folded monomer binds one zinc and one copper atom, and contains an intramolecular disulfide bond, all of which are important for the stability of the protein(4). Loss of any of these factors increases the likelihood of SOD1 misfolding into a toxic disease state due to a decrease in stability and invariable formation of toxic aggregates(6). Indeed, SOD1---related fALS mutations ar...
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