Superoxide dismutase-1 (SOD1) mutants, including those with unaltered enzymatic activity, are known to cause amyotrophic lateral sclerosis (ALS). Several destabilizing factors contribute to pathogenicity including a reduced ability to complete the normal maturation process which comprises folding, metal cofactor acquisition, intra-subunit disulphide bond formation and dimerization. Immature SOD1 forms toxic oligomers and characteristic large insoluble aggregates within motor system cells. Here we report that the cysteine-reactive molecule ebselen efficiently confers the SOD1 intra-subunit disulphide and directs correct SOD1 folding, depopulating the globally unfolded precursor associated with aggregation and toxicity. Assisted formation of the unusual SOD1 cytosolic disulphide bond could have potential therapeutic applications. In less reducing environments, ebselen forms a selenylsulphide with Cys111 and restores the monomer–dimer equilibrium of A4V SOD1 to wild-type. Ebselen is therefore a potent bifunctional pharmacological chaperone for SOD1 that combines properties of the SOD1 chaperone hCCS and the recently licenced antioxidant drug, edaravone.

Mutations in the gene encoding Cu/Zn superoxide dismutase-1 cause amyotrophic lateral sclerosis. Superoxide dismutase-1 mutations decrease protein stability and promote aggregation. The mutant monomer is thought to be an intermediate in the pathway from the superoxide dismutase-1 dimer to aggregate. Here we find that the monomeric copper-apo, zinc-holo protein is structurally perturbed and the apo-protein aggregates without reattainment of the monomer–dimer equilibrium. Intervention to stabilize the superoxide dismutase-1 dimer and inhibit aggregation is regarded as a potential therapeutic strategy. We describe protein–ligand interactions for two compounds, Isoproterenol and 5-fluorouridine, highlighted as superoxide dismutase-1 stabilizers. We find both compounds interact with superoxide dismutase-1 at a key region identified at the core of the superoxide dismutase-1 fibrillar aggregates, β-barrel loop II–strand 3, rather than the proposed dimer interface site. This illustrates the need for direct structural observations when developing compounds for protein-targeted therapeutics.

Few proteins have come under such intense scrutiny as superoxide dismutase-1 (SOD1). For almost a century, scientists have dissected its form, function and then later its malfunction in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We now know SOD1 is a zinc and copper metalloenzyme that clears superoxide as part of our antioxidant defence and respiratory regulation systems. The possibility of reduced structural integrity was suggested by the first crystal structures of human SOD1 even before deleterious mutations in thesod1gene were linked to the ALS. This concept evolved in the intervening years as an impressive array of biophysical studies examined the characteristics of mutant SOD1 in great detail. We now recognise how ALS-related mutations perturb the SOD1 maturation processes, reduce its ability to fold and reduce its thermal stability and half-life. Mutant SOD1 is therefore predisposed to monomerisation, non-canonical self-interactions, the formation of small misfolded oligomers and ultimately accumulation in the tell-tale insoluble inclusions found within the neurons of ALS patients. We have also seen that several post-translational modifications could push wild-type SOD1 down this toxic pathway. Recently we have come to view ALS as a prion-like disease where both the symptoms, and indeed SOD1 misfolding itself, are transmitted to neighbouring cells. This raises the possibility of intervention after the initial disease presentation. Several small-molecule and biologic-based strategies have been devised which directly target the SOD1 molecule to change the behaviour thought to be responsible for ALS. Here we provide a comprehensive review of the many biophysical advances that sculpted our view of SOD1 biology and the recent work that aims to apply this knowledge for therapeutic outcomes in ALS.

Significance: Adult onset neurodegenerative diseases are viewed as protein destabilization, misfolding, and aggregation diseases. TAR DNA binding protein-43 (TDP-43) protein is strongly associated with many neurological disorders, particularly amyotrophic lateral sclerosis and frontotemporal lobar degeneration. All of the disease-associated TDP-43 mutants tested have been shown to increase TDP-43 half-life and this correlates inversely with the age at which the sufferer first becomes aware of symptoms. Here we show that disease mutations in two TDP-43 nucleic acid binding domains also increase the protein’s half-life and this is commensurate with increased structural stability and resistance to aggregation. Our results are an unusual contrast to other neurodegenerative diseases and provide a potential link between the molecular characteristics of mutant TDP-43 and the symptoms of these debilitating diseases.

LAT1 (SLC7A5) is a transporter for both the uptake of large neutral amino acids and a number of pharmaceutical drugs. It is expressed in numerous cell types including T-cells, cancer cells and brain endothelial cells. However, mechanistic knowledge of how it functions and its interactions with lipids are unknown or limited due to inability of obtaining stable purified protein in sufficient quantities. Our data show that depleting cellular cholesterol reduced the V_max but not the K_m of the LAT1 mediated uptake of a model substrate into cells (L-DOPA). A soluble cholesterol analogue was required for the stable purification of the LAT1 with its chaperon CD98 (4F2hc,SLC3A2) and that this stabilised complex retained the ability to interact with a substrate. We propose cholesterol interacts with the conserved regions in the LAT1 transporter that have been shown to bind to cholesterol/CHS in Drosophila melanogaster dopamine transporter. In conclusion, LAT1 is modulated by cholesterol impacting on its stability and transporter activity. This novel finding has implications for other SLC7 family members and additional eukaryotic transporters that contain the LeuT fold.

The symbiotic nitrogen-fixing bacterium Bradyrhizobium japonicum is critical to the agro-industrial production of soybean because it enables the production of high yields of soybeans with little use of nitrogenous fertilizers. The FixL and FixJ two-component system (TCS) of this bacterium ensures that nitrogen fixation is only stimulated under conditions of low oxygen. When it is not bound to oxygen, the histidine kinase FixL undergoes autophosphorylation and transfers phosphate from adenosine triphosphate (ATP) to the response regulator FixJ, which, in turn, stimulates the expression of genes required for nitrogen fixation. We purified full-length B. japonicum FixL and FixJ proteins and defined their structures individually and in complex using small-angle x-ray scattering, crystallographic, and in silico modeling techniques. Comparison of active and inactive forms of FixL suggests that intramolecular signal transduction is driven by local changes in the sensor domain and in the coiled-coil region connecting the sensor and histidine kinase domains. We also found that FixJ exhibits conformational plasticity not only in the monomeric state but also in tetrameric complexes with FixL during phosphotransfer. This structural characterization of a complete TCS contributes both a mechanistic and evolutionary understanding to TCS signal relay, specifically in the context of the control of nitrogen fixation in root nodules.