Abstract 1256 Development of cell permeable peptide-based PROTACs to study peroxisomal biochemistry

MitoNEET is a protein that was originally identified in 2004 as a binding partner of the anti-diabetes drug pioglitazone.Additionally, it has been assoicated with diabetes, cancer, neurological disorders, and metabolic diseases.The development of mitoNEET-targeting treatments has been limited by the lack of understanding exactly how mitoNEET integrates with pathophysiological processes.MitoNEET is a dimer that contains a [2Fe-2S] cluster per monomer and ligated by three histidine and one cysteine residues.The ligating histidine residues is in a hydrogen bonding network with the Lys55 residue of the opposite monomer, making that residues particularly reactive towards the coenzyme pyridoxal phosphate.The Konkle Lab identified mitoNEET as a transaminase enzyme in 2022.MitoNEET catalyzes the conversion of cysteine and 2-oxoglutarate to 3-mercaptopyruvate and glutamate.Interestingly, the Konkle Lab also identified that Lys55 is selectively modified by the reactive electrophile 4oxononenal, that is produced during oxidative stress.Mito-NEET is recombinantly expressed and purified and the products of the transaminase enzyme assay are analyzed by gas chromatography-mass spectrometry (GC-MS) after derivatization with TBDMS groups.The impact of modification of mitoNEET by 4-hydroxynonenal or 4-oxononenal on the transaminase activity is analyzed to better understand how the newly appreciated enzymatic activity of mitoNEET contributes to the metabolome of cells under oxidative stress.