Small molecules present in the cerebrospinal fluid metabolome influence superoxide dismutase 1 aggregation

Abstract

Superoxide dismutase 1 (SOD1) aggregation is one of the pathological markers of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. The underlying molecular grounds of SOD1 pathologic aggregation remains obscure as mutations alone are not exclusively the cause for the formation of protein inclusions. Thus, other components in the cell environment likely play a key role in triggering SOD1 toxic aggregation in ALS. Recently, it was found that ALS patients present a specific altered metabolomic profile in the cerebrospinal fluid (CSF) where SOD1 is also present and potentially interacts with metabolites. Here we have investigated how some of these small molecules affect apoSOD1 structure and aggregation propensity. Our results show that as co-solvents, the tested small molecules do not affect apoSOD1 thermal stability but do influence its tertiary interactions and dynamics, as evidenced by combined biophysical analysis and proteolytic susceptibility. Moreover, these compounds influence apoSOD1 aggregation, decreasing nucleation time and promoting the formation of larger and less soluble aggregates, and in some cases polymeric assemblies apparently composed by spherical species resembling the soluble native protein. We conclude that some components of the ALS metabolome that shape the chemical environment in the CSF may influence apoSOD1 conformers and aggregation.

Figure 1

Metabolites impact on apoSOD1 tertiary structure. (A) Tryptophan fluorescence emission spectra of 5 μM apoSOD1 (black curve) and upon incubation with 200 μM of each metabolite (colors); (B) Concentration dependence of apoSOD1 tryptophan fluorescence emission maximum at 348 nm, for the different metabolites. See experimental section for further details.

Figure 2

Metabolites increase apoSOD1 proteolytic susceptibility. (A) SDS-PAGE analysis of a time course trypsin digestion experiment (50 μM apoSOD1 and 200 μM of metabolites); (B) Percentage of digested apoSOD1 in the presence of the different metabolites after 15, 30 and 45 min of incubation with trypsin. See experimental section for further details.

Figure 3

Metabolites promote aggregation of apoSOD1. (A) Aggregation kinetics of 50 μM apoSOD1 monitored by ThT fluorescence emission, in the absence (black) and in presence (colors) of 200 μM metabolites; (B) Plot of ThT fluorescence intensity at 200 h (from traces in panel A). Both panels are representative of 3 independent measurements with 50 μM apoSOD1 at pH 7.4 under agitation at 37 °C with and without 200 μM of each metabolite. The ThT bar represents the fluorescence intensity of the probe in the absence of protein and metabolites. See experimental section for further details.

Figure 4

Metabolites boost the formation of larger apoSOD1 aggregates. Nanoparticle Tracking Analysis (NTA) of the distribution of total apoSOD1 aggregates generated from 50 μM apoSOD1, alone and in the presence of 200 μM of each metabolite after incubation at pH 7.4 under agitation at 37 °C for 200 h (metabolite:protein = 4). Blue bars correspond to the distribution of small aggregates (70–350 nm) and red bars correspond to larger aggregates (350–2000 nm). See experimental section for further details.

Figure 5

Metabolites influence apoSOD1 aggregation towards insoluble aggregates and increase the size of soluble aggregates. (A) Ratio of soluble and insoluble aggregates formed from 50 μM apoSOD1 with 200 μM of each metabolite. SDS-PAGE gel: first band correspond to soluble aggregates and second band corresponds to insoluble aggregates, after incubation of apoSOD1 under agitation at 37 °C for 200 h; (B) DLS size distribution of soluble aggregates of apoSOD1 alone and with 200 μM of each metabolite after incubation at pH 7.4 under agitation at 37 °C for 200 h. Red bar correspond to apoSOD1 particles (50–100 nm) and blue bars correspond to larger aggregates formed in the presence of metabolites (100–1000 nm). See experimental section for further details.

Figure 6

Metabolites modulate the morphology of apoSOD1 aggregates. TEM images of apoSOD1 with or without metabolites at a metabolite:protein ratio of 4, after incubation at pH 7.4 under agitation at 37 °C for 200 h. (A,B) Control, apoSOD1 with no added metabolite; (C) +lysine; (D) +erythronic acid; (E) +glyceric acid; (F) +glyceric acid, magnification of panel E; (G) +phthalate; (H) +arginine; (I) +fructose; (J) +ornithine; (K) +ornithine, magnification of panel J. See experimental section for further details.