MitoNEET is an outer mitochondrial membrane protein with a [2Fe-2S] cluster, characterized as a binding site to pioglitazone, an anti-type 2 diabetic drug. However, its physiological role(s) is/are still unclear. Due to its asymmetrical ligation pattern through three cysteine-one histidine residues, the [2Fe-2S] cluster is labile and likely to release free iron into the cell. Free iron is known to promote the formation of the reactive electrophiles 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) through the oxidation of liberated poly-unsaturated fatty acids. HNE and ONE are known to react with the nucleophilic amino acids cysteine, histidine, and lysine.  MitoNEET is a homodimer in crystal structures and reacts with glutamate dehydrogenase-1 (GDH1) through the formation of a disulfide bond, indicating a high reactivity of one of the ligating cysteine residues (Cys74). MitoNEET also forms higher order oligomers through disulfide bond formation between Cys84 and either Cys72 or Cys74 showing that there is specificity dependent upon the binding partners. Additionally, the lysine of the opposite protomer (Lys55’) is in a hydrogen bonding network with the ligating histidine residue (His87). Previous literature identifies HNE modification at His90 of mitoNEET from a model of alcoholic liver disease. To fully characterize which amino acids residues in mitoNEET bind to HNE and ONE, proteomic analysis of mitoNEET-HNE and mitoNEET-ONE adducts were analyzed. SDS-PAGE revealed that both HNE and ONE disrupt the mitoNEET-mitoNEET dimer formation, increasing the relative concentration of the mitoNEET monomer under denaturing conditions. However, the mitoNEET-ONE adduct creates a redox insensitive homodimer while the mitoNEET-HNE adduct homodimer remains redox sensitive. Prior reaction of HNE and ONE with mitoNEET does not disrupt the formation of the mitoNEET-GDH1 dimer. Taken together, these results show that mitoNEET is vulnerable to reaction with reactive electrophiles and that there is chemical specificity that leads to diverse products. <br/>

Reactions of reactive electrophiles with mitoNEET

2019 Spring National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.