ArabadopsisNEET is involved in Development, Iron Management, Senescence and Stress Response.

The NEET family is a newly discovered class of human proteins involved in a diverse array of biological processes that include autophagy, apoptosis, aging, type 2 diabetes, Wolfram Syndrome 2, and the regulation of redox and reactive oxygen accumulation in mitochondria. They contain a unique homo-dimeric fold, the "NEET fold", in which two protomers intertwine to form a two domain structure, a beta cap, and a unique redox-active labile 2Fe-2S cluster- binding domain. To accelerate the functional study of NEET proteins and to identify an evolutionary conserved function for them, we identified and characterized a plant NEET protein. We found that the Arabidopsis thaliana At5g51720 protein is a NEET protein with similar biochemical and biophysical characteristics to those of the human NEET proteins. Moreover, the plant NEET protein shows a highly similar structure to that of mitoNEET and Miner1, two mammalian NEET proteins. However, in contrast to human NEET proteins, At5g51720 is the only member of this gene family in plants, and is primarily localized to chloroplasts (therefore termed AtChloroNEET). My student Andrea Conlan cloned and expressed this gene and began biological characterization of this protein in vivo with our collaborators Ron Mittler and Rachel Nechushtai in Israel.  Phenotypic characterization of At5g51720 using knockdown and RNAi lines revealed a key role for this protein in plant development, senescence and iron metabolism (Figure 7). A role in iron metabolism is further supported by biochemical and cell biology studies of AtChloroNEET in plant and animal cells, as well as mutational analysis of its cluster binding domain. Taken together, our findings support the hypothesis that NEET proteins have an ancient role in cells that is associated with iron homeostasis/metabolism.

Human mitoNEET is a Cluster Transfer Protein.

Since the exciting discovery of the novel mitochondrial target, mitoNEET, for the thiazolidinedione (TZD) insulin sensitizing drug pioglitazone, the NEET family has emerged as an important class of human proteins that determine multiple pathologies and aging. MitoNEET is a uniquely structured 2Fe-2S protein that plays a key role in regulating maximal capacity for electron transport and oxidative phosphorylation, and the accumulation of reactive oxygen species. We recently discovered that mitoNEET is a cluster transfer protein that delivers Fe into the mitochondria under oxidative conditions (please see Figure 8). Binding of the TZD pioglitazone abrogates this transfer in vitro and in cells.  We showed that Miner1 is a structural homolog that harbors the conserved two redox-active 2Fe-2S clusters. A mutation leading to loss of the cluster binding domain of Miner1 Wolfram Syndrome 2 (WFS2) results in early onset optic atrophy, diabetes, deafness and decreased lifespan in humans. We have found that the respective 2Fe-2S cluster stabilities as well as drug/natural product and protein binding affinities of mitoNEET and Miner1 are oxidation state dependent. Clearly, cluster stability and redox-dependent modulation of functional motifs is a significant point of regulation of mitoNEET and Miner1 and is an important area of investigation for our laboratory and future drug design.  Specifically we are asking the question: What are the Molecular Determinants of Differential Drug/Natural Product Binding to the NEET Proteins MitoNEET and Miner1?  Crystal structures of mitoNEET and Miner1 revealed they constitute a novel family of 2Fe-2S cluster containing proteins.  These proteins are generating high interest due to their non-canonical binding of TZDs in a fashion completely distinct from the paradigm established by the TZD target PPARγ. It is our goal to understand the molecular interactions that modulate specific mitoNEET or Miner1 binding and ask how small molecules regulate the functional properties of the protein.

The Folding and Long Range Communication within an Alpha/Beta Protein : Interdomain Communication Revealed in MitoNEET.

Energy landscape theory indicates that proteins fold in a funneled fashion with minimal frustration, with the native state and functional fluctuations occurring towards the bottom of this funnel. Because proteins are active on the same landscape that they fold on, their functional motions may introduce ruggedness in to the folding landscape. Thus identifying residues that contribute frustration in folding may be an effective way to predict and identify important sites for protein protein interactions as well as binding regions for potential drug targets.

MitoNEET is a recently identified drug target for a commonly prescribed diabetes drug, Pioglitazone. It belongs to a previously uncharacterized ancient family of proteins for which the hallmark is the presence of a unique 39 amino acid CDGSH domain. In order to characterize the folding landscape of this novel fold, we performed thermodynamic simulations on MitoNEET using a structure-based model. Additionally, we implement a method of contact map clustering to partition out alternate pathways in folding. This cluster analysis reveals a detour late in folding and enables us to carefully examine the folding mechanism of each pathway rather than the macroscopic average. We observe that tightness in a region distal to the iron–sulfur cluster creates a constraint in folding and additionally appears to mediate communication in folding between the two domains of the protein. We demonstrate that by making changes at this site we are able to alter the order of folding events in the cluster binding domain as well as decrease the barrier to folding (Figure 5).  We have demonstrated that these long-range effects are also active in folding studies in vitro.