Engineering the Redox Potential over a Wide Range within a New Class of FeS Proteins

Abstract: MitoNEET is a newly discovered mitochondrial protein and a target of the TZD class of antidiabetes drugs. MitoNEET is homodimeric with each protomer binding a [2Fe-2S] center through a rare 3-Cys and 1-His coordination geometry. Both the fold and the coordination of the [2Fe-2S] centers suggest that it could have novel properties compared to other known [2Fe-2S] proteins. We tested the robustness of mitoNEET to mutation and the range over which the redox potential (EM) could be tuned. We found that the protein could tolerate an array of mutations that modified the EM of the [2Fe-2S] center over a range of ∼700 mV, which is the largest EM range engineered in an FeS protein and, importantly, spans the cellular redox range (+200 to-300 mV). These properties make mitoNEET potentially useful for both physiological studies and industrial applications as a stable, water-soluble, redox agent

NAF-1 and mitoNEET are central to human breast cancer proliferation by maintaining mitochondrial homeostasis and promoting tumor growth

Mitochondria are emerging as important players in the transformation process of cells, maintaining the biosynthetic and energetic capacities of cancer cells and serving as one of the primary sites of apoptosis and autophagy regulation. Although several avenues of cancer therapy have focused on mitochondria, progress in developing mitochondria-targeting anticancer drugs nonetheless has been slow, owing to the limited number of known mitochondrial target proteins that link metabolism with autophagy or cell death. Recent studies have demonstrated that two members of the newly discovered family of NEET proteins, NAF-1 (CISD2) and mitoNEET (mNT; CISD1), could play such a role in cancer cells. NAF-1 was shown to be a key player in regulating autophagy, and mNT was proposed to mediate iron and reactive oxygen homeostasis in mitochondria. Here we show that the protein levels of NAF-1 and mNT are elevated in human epithelial breast cancer cells, and that suppressing the level of these proteins using shRNA results in significantly reduced cell proliferation and tumor growth, decreased mitochondrial performance, uncontrolled accumulation of iron and reactive oxygen in mitochondria, and activation of autophagy. Our findings highlight NEET proteins as promising mitochondrial targets for cancer therapy

Selenium nanoparticles trigger alterations in ovarian cancer cell biomechanics

High dose selenium acts as a cytotoxic agent, with potential applications in cancer treatment. However, clinical trials have failed to show any chemotherapeutic value of selenium at safe and tolerated doses (<90 μg/day). To enable the successful exploitation of selenium for cancer treatment, we evaluated inorganic selenium nanoparticles (SeNP), and found them effective in inhibiting ovarian cancer cell growth. In both SKOV-3 and OVCAR-3 ovarian cancer cell types SeNP treatment resulted in significant cytotoxicity. The two cell types displayed contrasting nanomechanical responses to SeNPs, with decreased surface roughness and membrane stiffness, characteristics of OVCAR-3 cell death. In SKOV-3, cell membrane surface roughness and stiffness increased, both properties associated with decreased metastatic potential. The beneficial effects of SeNPs on ovarian cancer cell death appear cell type dependent, and due to their low in vivo toxicity offer an exciting opportunity for future cancer treatment

Characterization of CDGSH family members : a new and unique class of 2Fe-2S proteins

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 respons

Characterization of CDGSH family members : a new and unique class of 2Fe-2S proteins

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 respons