Characterization of the Stability and Reactivity of MitoNEET: A Novel Diabetes Drug Target

Abstract

MitoNl:ET is a mitochondrial membrane protein discovered in 2004 as a binding partner of the antidiabetic drng pioglitazone. It is sold under the trade name Actos® and used to treat type-2 diabetes. The cellular function(s) of mitoNEET are unknown. Interestingly, the non-covalent homodimer contains two [2Fe-2SJ clusters that support possible functions as an electron-transport protein, an iron-sulfur transfer protein, a mediator in cellular respiration, and a redox-sensor. MitoNEl:T uniquely ligates the [2Fc- 2SJ cluster through a 3Cys-l His ligation. In this thesis, the optimization of the expression and purification of human mitoNEET is described. Possible binding partners were identified using a pull-down assay with either a mouse liver lysatc or the human liver cancer cell line, Hep-G2, lysate. The possible binding partners were classified by their subcellular localization, presence of a cofactor, and specific mitochondrial localization. We dctem1incd that mitoNEET forms disulfide bonds through cysteine residues ligating the [2Fe-2SJ cluster. Proteomic analysis discovered that mitoNEET can form a covalent homodimer though Cys83 and either Cys72 or Cys74. Glutamate dehydrogenase I (GDH I), a mitochondrial matrix enzyme, was identified as a binding partner of mitoNEET in the pull-down assays. The interactions between mitoNEET and G DH I were further investigated by SDS-P J\ GE analysis 111 the presence or absence of a reductant resulting in a covalent complex though a mixed-disulfide bond. Proteomic analysis identified that Cys74 of mitoNEET and Cys3 I 9 of GDH l are the cysteine residues involved in the mixed-disulfide bond. The formation of the mitoNEET-GDHl covalent complex increases the enzymatic activity of GDH I and introduces a new proposed cellular function of mitoNEET as a redox sensor and a reversible covalent allosteric activator of GDH I.

Date of Award	2013
Original language	American English
Awarding Institution	Eastern Illinois University
Supervisor	Mary Konkle (Supervisor)