Binding studies of mitoNEET with products of lipid peroxidation

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Arnett, Dayna Ann Maria
Konkle, Mary E. (Mary Elizabeth)
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Thesis (M.S.)
Department of Chemistry
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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. When in solution, mitoNEET folds to form a homodimer, and when reacted with Glutamate dehydrogenase-1 (GDH), mitoNEET forms a dimeric structure with GDH. Due to its asymmetrical ligation pattern of three Cystine one Histidine, the [2Fe-2S] cluster is labile and likely to release free iron into a biological system. Cystine and Histidine are also potential binding sites for products of lipid peroxidation, such as 4-hydroxy-2-nonenal (HNE), 4-oxo-2-nonenal (ONE), and malondialdehyde (MDA). To establish which amino acids residues in mitoNEET bind to HNE and ONE, Proteomics of mitoNEET-HNE and mitoNEET-ONE adducts were measured. Because mitoNEET demonstrates higher order oligomerization via denaturing and native gel electrophoresis, SDS PAGE and Native gels were ran to identify disruption of the mitoNEET-mitoNEET dimer as well as the mitoNEET-GDH dimer by HNE, ONE, and MDA. UV-visible spectroscopy was utilized to identify changes in absorbance after mitoNEET being treated with HNE, ONE, and MDA. Cell exposure was used to predict the production of apoptotic bodies after exposure to HNE and ONE, and western blotting was utilized to confirm the changes to mitoNEET’s structure with HNE and ONE. Proteomics identified binding of ONE to Lysine55 in high yields, which is hydrogen-bonded to Histidine87, a ligand of the [2Fe-2S] cluster. Previous literature also identifies residues of HNE binding at Histidine90. SDS-PAGE reveals that both HNE and ONE disrupt the mitoNEET-mitoNEET dimer formation, increasing formation of the mitoNEET monomer. However, the mitoNEET-ONE adduct creates a ß-mercaptoethanol (BME) insensitive homodimer while the mitoNEET-HNE adduct homodimer reduces to the monomer state in the presence of BME. This adduct also cannot form without the presence of the disulfide bond, which was made evident by pretreatment with BME and treatment with oxidized and reduced glutathione. HNE and ONE does not disrupt the formation of the mitoNEET-GDH dimer. UV visible spectroscopy highlights key changes in absorbance at λ = 236 nm, λ = 256 nm, and λ = 340 nm. UV-visible spectroscopy also revealed that HNE and PLP are competing for the formation of mitoNEET adducts. Overall, despite MDA having literature to support its reactivity with proteins, MDA showed no reactivity with mitoNEET. Additionally, while western blotting confirmed changes found in SDS PAGE, no apoptotic bodies formed after one-hour treatment with HNE and ONE. Ultimately, it is reasonable to suggest that mitoNEET does exhibit high modification with products of lipid peroxidation that could signal a role in oxidative stress for mitoNEET as well as a potential role as an enzyme with Lys55 as an active site