MitoNEET is a member of the CDGSH iron sulfur domain (CISD) protein family. This family is characterized by the presence of a [2Fe-2S] cluster ligated by a unique 3Cys-1His pattern. MitoNEET was discovered in a screen for proteins that bind the thiazolidinedione molecules (TZDs) used to treat type-II diabetes. With its primary physiological function(s) still unknown, a program to identify molecules (large and small) that bind to mitoNEET through both non-covalent and reversible covalent interactions has been undertaken. Several dehydrogenase enzymes including glutamate dehydrogenase 1 (GDH1), malate dehydrogenases (MDH1 and MDH2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were identified as binding partners for mitoNEET using a protein pull-down assay from both mouse and human liver cells. MitoNEET forms a redox-sensitive covalent bond with GDH1, but not with MDH or GAPDH. Considering the preponderance of dehydrogenase enzymes on the pull-down list, further efforts were focused on developing an assay that could determine if mitoNEET binds to the cofactors of dehydrogenase enzymes, NAD(P)H. We demonstrate that the soluble portion of mitoNEET has one tyrosine and tryptophan residue per monomer which undergo fluorescence emission quenching upon ligand binding. This feature allows for the development of a rapid fluorescence-based assay to characterize binding of physiologically relevant water-soluble ligands. Excitation at l = 280 nm results in fluorescent emission of both tyrosine and tryptophan residues, while excitation at l = 295 nm generates an emission signal for tryptophan exclusively. By monitoring the fluorescent emission of mitoNEET excited at l = 280 nm or l = 295 nm, we demonstrate that reduced nicotinamide adenine dinucleotide (NADH) quench fluorescence of both amino acids, while the oxidized form and adenine nucleotides such as ATP only quench fluorescence of tyrosine. Taken together, this work presents detailed picture of the importance of mitoNEET reactivity towards and binding of molecules that contribute to the redox tone of the cell. Part of this work was funded by NSF CHE-1609440.

Characterization of the binding partners of MitoNEET

255th National Meeting (2018)

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