Immature ALS-associated mutant superoxide dismutases form variable aggregate structures through distinct oligomerization processes.

Protein misfolding and aggregation are hallmarks of many diseases, including amyotrophic lateral sclerosis (ALS). In familial ALS, aberrant self-association of mutant Cu,Zn-superoxide dismutase (SOD1) is implicated as a key contributor to disease. Mutations have the largest impacts on the stability of the most immature form of SOD1, the unmetallated, disulfide-reduced monomer (apoSH SOD1). Here we demonstrate that, despite the marginal stability of apoSH SOD1, aggregation is little correlated with the degree of protein unfolding, and multiple modes of aggregation occur, depending on the mutation and solution conditions. Light scattering and atomic force microscopy reveal two distinct mutant SOD1 behaviours: high aggregator mutants form abundant small assemblies, while low aggregator mutants form fewer, more fibre-like aggregates. Attenuated total reflectance-Fourier transform infrared spectroscopy and Thioflavin T binding show the aggregates maintain native-like anti-parallel beta structure. These results provide new evidence that ALS-associated mutations promote the aggregation of apoSH SOD1 through multiple pathways, with broad implications for understanding mechanisms of protein self-association in disease and biotechnology.

Methodological advances and strategies for high resolution structure determination of cellular protein aggregates.

Aggregation of proteins is at the nexus of molecular processes crucial to aging, disease, and employing proteins for biotechnology and medical applications. There has been much recent progress in determining the structural features of protein aggregates that form in cells; yet, owing to prevalent heterogeneity in aggregation, many aspects remain obscure and often experimentally intractable to define. Here, we review recent results of structural studies for cell-derived aggregates of normally globular proteins, with a focus on high-resolution methods for their analysis and prediction. Complementary results obtained by solid-state NMR spectroscopy, FTIR spectroscopy and microspectroscopy, cryo-EM, and amide hydrogen/deuterium exchange measured by NMR and mass spectrometry, applied to bacterial inclusion bodies and disease inclusions, are uncovering novel information on in-cell aggregation patterns as well as great diversity in the structural features of useful and aberrant protein aggregates. Using these advances as a guide, this review aims to advise the reader on which combination of approaches may be the most appropriate to apply to their unique system.

High-Resolution NMR H/D Exchange of Human Superoxide Dismutase Inclusion Bodies Reveals Significant Native Features Despite Structural Heterogeneity.

Protein aggregation is central to aging, disease and biotechnology. While there has been recent progress in defining structural features of cellular protein aggregates, many aspects remain unclear due to heterogeneity of aggregates presenting obstacles to characterization. Here we report high-resolution analysis of cellular inclusion bodies (IBs) of immature human superoxide dismutase (SOD1) mutants using NMR quenched amide hydrogen/deuterium exchange (qHDX), FTIR and Congo red binding. The extent of aggregation is correlated with mutant global stability and, notably, the free energy of native dimer dissociation, indicating contributions of native-like monomer associations to IB formation. This is further manifested by a common pattern of extensive protection against H/D exchange throughout nine mutant SOD1s despite their diverse characteristics. These results reveal multiple aggregation-prone regions in SOD1 and illuminate how aggregation may occur via an ensemble of pathways.