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

Abstract

Lipid 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{degrees}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 ,{beta}-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 electrophiles hydrophobicity is a critical factor that strongly influences protein aggregation propensity.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC=\"FIGDIR/small/740688v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (26K):\norg.highwire.dtl.DTLVardef@1860bc3org.highwire.dtl.DTLVardef@16e4cb6org.highwire.dtl.DTLVardef@6e48a4org.highwire.dtl.DTLVardef@631070_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Aldehyde hydrophobicity is positively correlated to SOD1 aggregation;\n\n- Lys residues located nearby the SOD1 dimer interface and electrostatic loop are ubiquitously modified by all aldehydes;\n\n- Hydrophobic aldehydes increase the lipophilic potential surface of the region where they bind;