Tryptophan\xa032-mediated SOD1 aggregation is attenuated by pyrimidine-like compounds in living cells

Edward Pokrishevsky,Neil R Cashman,Mine Sher,Natalie E Farrawell,Luke Mcalary,Justin J Yerbury,Beibei Zhao

doi:10.1038/s41598-018-32835-y

Abstract

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.

Highlights

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by the selective degeneration of upper and lower motor neurons leading to patient death typically within 2 to 4 years of onset
The lack of induced aggregation of mouse superoxide dismutase 1 (SOD1) by a human wild-type SOD1 (hSOD1) seed has been confirmed in cell culture[19], even though human and mouse SOD1 have 83% sequence identity, with divergence mainly in β-strand III and loop II19,20. β-strand III contains the only tryptophan residue found within SOD1 at sequence position 32 (W32), which is solvent exposed in the native structure and is conserved across SOD1-familial amyotrophic lateral sclerosis (fALS)
The main kinetic parameters determined were the lag-phase of fibrillation, which is associated with the formation of detectable levels of β-sheet, and the elongation rate, which is associated with the rate at which β-sheet is formed

Summary

Introduction

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by the selective degeneration of upper and lower motor neurons leading to patient death typically within 2 to 4 years of onset. The SOD1 sequence contains over 160 mutations (http://alsod.iop.kcl.ac.uk), all of which are associated with the ALS syndrome and pathological neuronal inclusion bodies. These mutations can have disparate effects upon the SOD1 structure by disrupting metal ion coordination, disulphide bond formation or reduction, and protein dimerization, they all engender SOD1 misfolding and subsequent aggregation[13,14,15,16]. We have previously established that transmission of SOD1 misfolding can be significantly reduced by substituting W32 in hSOD1 by serine[19] and other work determined that W32 potentiates mutant SOD1 aggregation and toxicity in a neuronal cell system[21], further indicating the importance of this residue to pathology. We determined that the small molecules 5-Fluorouridine, and the related FDA-approved, 5-Fluorouracil, were effective at attenuating the formation of SOD1 inclusions in a cell culture model

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Discussion

Conclusion