Nutrient-Deprivation Autophagy Factor-1 (NAF-1): Biochemical Properties of a Novel Cellular Target for Anti-Diabetic Drugs

PLOS ONE, Dec 2019

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.

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Nutrient-Deprivation Autophagy Factor-1 (NAF-1): Biochemical Properties of a Novel Cellular Target for Anti-Diabetic Drugs

et al. (2013) Nutrient-Deprivation Autophagy Factor-1 (NAF-1): Biochemical Properties of a Novel Cellular Target for Anti-Diabetic Drugs. PLoS ONE 8(5): e61202. doi:10.1371/journal.pone.0061202 Nutrient-Deprivation Autophagy Factor-1 (NAF-1): Biochemical Properties of a Novel Cellular Target for Anti-Diabetic Drugs Sagi Tamir 0 John A. Zuris 0 Lily Agranat 0 Colin H. Lipper 0 Andrea R. Conlan 0 Dorit Michaeli 0 Yael Harir 0 Mark L. Paddock 0 Ron Mittler 0 Zvi Ioav Cabantchik 0 Patricia A. Jennings 0 Rachel Nechushtai 0 Yaakov Koby Levy, Weizmann Institute of Science, Israel 0 1 The Alexander Silberman Institute of Life Science, Hebrew University of Jerusalem, Edmond J. Safra Campus at Givat Ram, Jerusalem, Israel, 2 Departments of Chemistry and Biochemistry and Physics, University of California San Diego, La Jolla, California, United States of America, 3 Department of Biology, University of North Texas , Denton, Texas , United States of America 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 antidiabetes thiazolidinedione drugs like pioglitazone and of the natural product resveratrol, both of which interact with the protein and stabilize its labile [2Fe-2S] cluster. - Funding: This work was supported by the following: the Israel Science Foundation (ISF) grant ISF 863/09 awarded to RN; ISF 141/06 and a grant from the Canadian Friends of the Hebrew University awarded to ZIC; ISF 214/08 and the European Union Grant FP7-Marie Curie 447 awarded to RM; the Cell and Molecular Genetics Training (CMG) grant 2T32GM007240-29 awarded to ARC; National Institutes of Health (NIH) GM54038 and NIH GM101467 to PAJ; and the Heme and Blood Proteins Training grant 5T32DK007233-34 to JAZ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. NAF-1 (Nutrient-deprivation autophagy factor-1, synonyms: ERIS, Miner1) is a member of a newly discovered family of ironsulfur (FeS) proteins coded by Cisd genes that are defined by a unique CDGSH amino acid sequence in their FeS cluster binding domain [14]. Interest in NAF-1 has recently increased because the gene is in a region of chromosome associated with neuronal development [5], and it is now known to be critical for the maintenance of skeletal muscle [6] and for promoting longevity [7],[8]. Moreover, a transcriptional splicing error leads to a rare but serious disease called Wolfram Syndrome 2 [9], which is associated with hearing deficiencies, severe blindness and diabetes and a lower life expectancy. The protein, which is localized in both ER [9] and in mitochondria [7], has been functionally implicated in cell autophagy, possibly as a mediator of Bcl-2 antagonism of Beclin-1 dependent autophagy on the surface of the ER [10]. The crystal structure of NAF-1 [1] showed that it is a homodimeric [2Fe-2S] protein and each protomer harbors one 2Fe-2S cluster bound to the protein by an usual 3-Cys-1-His coordination geometry (Fig. 1). The structure bears a similar backbone fold [1] to its paralog mitoNEET (Fig. S1) [2], a previously identified target of the thiazolidinedione (TZD) class of anti-diabetes drugs [11],[12]. Recent work has suggested that there is an additional mitochondrial target for this class of drug [13] so we sought to determine if NAF-1 was a bona fide target of TZDs. Importantly, these results have bearing on the development of alternative treatments for type II diabetes as until now, the pharmacological (both beneficial and deleterious) effects of the TZD drugs have been widely linked to the peroxisome proliferator-activat (...truncated)


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Sagi Tamir, John A. Zuris, Lily Agranat, Colin H. Lipper, Andrea R. Conlan, Dorit Michaeli, Yael Harir, Mark L. Paddock, Ron Mittler, Zvi Ioav Cabantchik, Patricia A. Jennings, Rachel Nechushtai. Nutrient-Deprivation Autophagy Factor-1 (NAF-1): Biochemical Properties of a Novel Cellular Target for Anti-Diabetic Drugs, PLOS ONE, 2013, Volume 8, Issue 5, DOI: 10.1371/journal.pone.0061202