UC San Diego

Only recently identified, the CDGSH family of proteins is highly conserved across species. It is represented in humans by three homologs, the first of which was discovered upon binding to the anti-diabetes drug pioglitazone (ActosTM). This protein, mitoNEET, is a 2Fe- 2S binding protein with a novel fold and a unique ligand arrangement. In order to understand this family of proteins more extensively, we employed a variety of biochemical and structural techniques to probe the characteristics that are shared and differ across human homlogs and across species. Because the 2Fe-2S mitoNEET coordination is so rare, we investigate the biophysical and possible functional properties that may be impacted upon mutation of the sole histidine ligand. Results indicate this histidine ligand is important in retaining properties of this protein, but does not stabilize it, which may argue this residue is evolutionarily conserved for its specific stability. We venture beyond mitoNEET and explored the second human homolog, Miner1, the lack of which causes Wolfram's Syndrome 2. This orphan disease results in diabetes insipidus, diabetes mellitus, optic atrophy, and deafness followed by early death. We show this ER-localized protein retains many of the CDGSH characteristics, including optical and redox properties, but differs in its cluster stability and structural packing in its Beta-Cap domain, properties that could influence protein/ligand binding as well as function. Finally, to investigate an ortholog outside of humans, and to take advantage of a system with only one CISD gene, we characterize the properties of the AtNEET, an Arabidopsis thaliana protein, in vitro as well as in vivo. We show AtNEET is the first plant CDGSH protein to retain this family's optical and structural characteristics. We show that this protein is localized to the chloroplast, and we also conclude that the importance of this family in cellular health spans species, as plants with inhibited expression experience late-bolting and early senescence. Taken together, evidence shows this family maintains its unique 2Fe-2S binding across multiple homologs and species. In addition, problems arising from the lack of this protein indicate its fundamental importance in normal physiological health and the stress response