Structure-function analysis of NEET proteins uncovers their role as key regulators of iron and ROS homeostasis in health and disease.

A novel family of 2Fe-2S proteins, the NEET family, was discovered during the last decade in numerous organisms, including archea, bacteria, algae, plant and human; suggesting an evolutionary-conserved function, potentially mediated by their CDGSH Iron-Sulfur Domain. In human, three NEET members encoded by the CISD1-3 genes were identified. The structures of CISD1 (mitoNEET, mNT), CISD2 (NAF-1), and the plant At-NEET uncovered a homodimer with a unique "NEET fold", as well as two distinct domains: a beta-cap and a 2Fe-2S cluster-binding domain. The 2Fe-2S clusters of NEET proteins were found to be coordinated by a novel 3Cys:1His structure that is relatively labile compared to other 2Fe-2S proteins and is the reason of the NEETs' clusters could be transferred to apo-acceptor protein(s) or mitochondria. Positioned at the protein surface, the NEET's 2Fe-2S's coordinating His is exposed to protonation upon changes in its environment, potentially suggesting a sensing function for this residue. Studies in different model systems demonstrated a role for NAF-1 and mNT in the regulation of cellular iron, calcium and ROS homeostasis, and uncovered a key role for NEET proteins in critical processes, such as cancer cell proliferation and tumor growth, lipid and glucose homeostasis in obesity and diabetes, control of autophagy, longevity in mice, and senescence in plants. Abnormal regulation of NEET proteins was consequently found to result in multiple health conditions, and aberrant splicing of NAF-1 was found to be a causative of the neurological genetic disorder Wolfram Syndrome 2. Here we review the discovery of NEET proteins, their structural, biochemical and biophysical characterization, and their most recent structure-function analyses. We additionally highlight future avenues of research focused on NEET proteins and propose an essential role for NEETs in health and disease. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

Nutrient-deprivation autophagy factor-1 (NAF-1): biochemical properties of a novel cellular target for anti-diabetic drugs.

Nutrient-deprivation autophagy factor-1 (NAF-1) (synonyms: Cisd2, Eris, Miner1, and Noxp70) is a [2Fe-2S] cluster protein immune-detected both in endoplasmic reticulum (ER) and mitochondrial outer membrane. It was implicated in human pathology (Wolfram Syndrome 2) and in BCL-2 mediated antagonization of Beclin 1-dependent autophagy and depression of ER calcium stores. To gain insights about NAF-1 functions, we investigated the biochemical properties of its 2Fe-2S cluster and sensitivity of those properties to small molecules. The structure of the soluble domain of NAF-1 shows that it forms a homodimer with each protomer containing a [2Fe-2S] cluster bound by 3 Cys and one His. NAF-1 has shown the unusual abilities to transfer its 2Fe-2S cluster to an apo-acceptor protein (followed in vitro by spectrophotometry and by native PAGE electrophoresis) and to transfer iron to intact mitochondria in cell models (monitored by fluorescence imaging with iron fluorescent sensors targeted to mitochondria). Importantly, the drug pioglitazone abrogates NAF-1's ability to transfer the cluster to acceptor proteins and iron to mitochondria. Similar effects were found for the anti-diabetes and longevity-promoting antioxidant resveratrol. These results reveal NAF-1 as a previously unidentified cell target of anti-diabetes thiazolidinedione drugs like pioglitazone and of the natural product resveratrol, both of which interact with the protein and stabilize its labile [2Fe-2S] cluster.

A novel tissue-specific alternatively spliced form of the A-to-I RNA editing enzyme ADAR2.

ADAR2, a member of the adenosine deaminase family of proteins, is the enzyme that edits the Q/R site in the GluR-B transcript, an important physiological A-to-I editing event. ADAR2 pre-mRNA undergoes a number of known alternative splicing events, affecting its function. Here we describe a novel alternatively spliced exon, located within intron 7 of the human gene, which we term "exon 7a". This alternatively spliced exon is highly conserved in the mammalian ADAR2 gene. It has stop codons in all three frames and is down regulated by NMD. We show that the level of exon 7a inclusion differs between different human tissues, with the highest levels of inclusion in skeletal muscle, heart and testis. In the brain, where the level of editing is known to be high, the level of exon 7a inclusion is low. The new alternative form was also found in supraspliceosomes, which constitute the nuclear pre-mRNA processing machine. The high conservation of the novel ADAR2 alternative exon in mammals indicates a physiological importance for this exon.

The editing enzyme ADAR1 and the mRNA surveillance protein hUpf1 interact in the cell nucleus.

Posttranscriptional regulation is an important step in the regulation of gene expression. In this article, we show an unexpected connection between two proteins that participate in different processes of posttranscriptional regulation that ensures the production of functional mRNA molecules. Specifically, we show that the A-to-I RNA editing protein adenosine deaminase that acts on RNA 1 (ADAR1) and the human Upf1 (hUpf1) protein involved in RNA surveillance are found associated within nuclear RNA-splicing complexes. A potential functional role for this association was revealed by RNAi-mediated down-regulation of ADAR1, which was accompanied by up-regulation of a number of genes previously shown to undergo A-to-I editing in Alu repeats and to be down-regulated by hUpf1. This study suggests a regulatory pathway by a combination of ADAR1 A-to-I editing enzyme and RNA degradation presumably with the aid of hUpf1.