Graduate Program

Chemistry

Degree Name

Master of Science (MS)

Semester of Degree Completion

2013

Thesis Director

Mary Konkle

Thesis Committee Member

Michael Menze

Thesis Committee Member

Gopal Periyannan

Thesis Committee Member

Edward Treadwell

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.

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