Lipid-derived electrophiles induce covalent modification and aggregation of Cu,Zn-superoxide dismutase in a hydrophobicity-dependent manner

ABSTRACTLipid peroxidation generates a huge number of reactive electrophilic aldehyde products. These reactive aldehydes can modify macromolecules such as proteins, resulting in loss of function and/or aggregation. The accumulation of Cu,Zn-superoxide dismutase (SOD1) aggregates is associated with familial cases of amyotrophic lateral sclerosis (ALS). Recent studies have shown that lipid and its oxidized derivatives may play a role in this process. Here we aimed to compare and characterize the ability of lipid-derived electrophiles with different hydrophobicities to induce SOD1 modification and aggregation in vitro. SOD1 was incubated with 4-hydroxy-2-hexenal (HHE), 4-hydroxy- 2-nonenal (HNE), 2-hexen-1-al (HEX), 2,4-nonadienal (NON), 2,4-decadienal (DEC) or secosterol aldehydes (Seco-A or Seco-B) at 37°C for 24 h. Size exclusion chromatography analysis showed that hydrophobic aldehydes smarkedly enhances apo- SOD1 aggregation. More importantly, aggregation level was positively correlated to calculated aldehyde hydrophobicities (LogP). Protein sequencing by LC-MS/MS showed that aldehydes covalently modifies SOD1 at aggregation prone regions. For instance, specific lysine residues located mainly nearby the dimer interface (K3, K9) and at the electrostatic loop (K122, K128, K136) were ubiquitously modified by all aldehydes. The α,β-unsaturated aldehydes also promoted modifications on histidine and cysteine residues, with H120 and C6 being the most commonly modified residues. Overall, our data suggest that electrophile’s hydrophobicity is a critical factor that strongly influences protein aggregation propensity.Graphical abstractHighlights- Aldehyde hydrophobicity is positively correlated to SOD1 aggregation;- Lys residues located nearby the SOD1 dimer interface and electrostatic loop are ubiquitously modified by all aldehydes;- Hydrophobic aldehydes increase the lipophilic potential surface of the region where they bind;

Structural investigation on the electrostatic loop of native and mutated SOD1 and their interaction with therapeutic compounds

The electrostatic loop of the native and mutated SOD1 protein with single point mutation in the loop is subjected to MD simulation. The structure and electrostatic properties of the native and mutated loops before/after interacting with small compounds are compared.

Flexibility of the Cu,Zn superoxide dismutase structure investigated at 0.57 GPa

The 2 Å resolution crystal structure of bovine erythrocyte Cu,Zn superoxide dismutase (CuZnSOD) has been determined by X-ray diffraction at high pressure (0.57 GPa) and room temperature. At 0.57 GPa the secondary, tertiary and quaternary structures are similar to other previously determined bovine erythrocyte CuZnSOD structures. Nevertheless, pressure has a localized impact on the atomic coordinates of Cαatoms and on side chains. The compression of the crystal and of the protein backbone is anisotropic. This anisotropy is discussed, taking into account intermolecular contacts and protein conformation. Pressure perturbation highlights the more flexible zones in the protein such as the electrostatic loop. At 0.57 GPa, a global shift of the dimetallic sites in both subunits and changes in the oxidation state of Cu were observed. The flexibility of the electrostatic loop may be useful for the interaction of different metal carriers in the copper-uptake process, whereas the flexibility of the metal sites involved in the activity of the protein could contribute to explaining the ubiquitous character of CuZnSODs, which are found in organisms living in very different conditions, including the deep-sea environment. This work illustrates the potential of combining X-ray crystallography with high pressure to promote and stabilize higher energy conformational substates.