pnas-2007-wiley-5318-23

MitoNEET is an iron-containing outer mitochondrial membrane protein that regulates oxidative capacity Sandra E. Wiley*, Anne N. Murphy*, Stuart A. Ross †‡ , Peter van der Geer § , and Jack E. Dixon* ¶ ** Departments of *Pharmacology and ¶ Cellular and Molecular Medicine, School of Medicine, and Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093; † Department of Pediatrics, University of Kentucky School of Medicine–Kentucky Pediatrics Research Institute, Lexington, KY 40536; and § Department of Chemistry and Biochemistry, San Diego State University, San Diego, CA 92182 Contributed by Jack E. Dixon, February 6, 2007 (sent for review December 29, 2006) Members of the thiazolidinedione (TZD) class of insulin-sensitizin g drugs are extensively used in the treatment of type 2 diabetes. Pioglitazon e, a member of the TZD family, has been shown to bind specifically to a protein named mitoNEET [Colca JR, McDonald WG, Waldon DJ, Leone JW, Lull JM, Bannow CA, Lund ET, Mathews WR (2004) Am J Physiol 286:E252–E260]. Bioinformatic analysis reveals that mit oNE ET is a member of a sma ll family of pr ote ins con tai nin g a domain annotated as a CDGSH-type zinc finger. Although anno- tate d as a zinc finger prote in, mitoNEE T contains no zinc, but instead contains 1.6 mol of Fe per mole of protein. The conserved sequence C-X-C-X 2 -(S/T)-X 3 -P-X-C-D-G-(S/A/T)-H is a defining fea- ture of this unique family of proteins and is likely involved in iron bind ing. Localiza tion studies demo nstrate that mitoNEET is an integral protein present in the outer mitochondrial membrane. An amino-terminal anchor sequence tethers the protein to the outer membran e with the CDGSH domain oriented toward the cyto- plas m. Card iac mito chon dria isola ted from mito NEET-n ull mice demonstrate a reduced oxidative capacity, suggesting that mito- NEET is an important iron-con taini ng protein invo lved in the control of maximal mitochondrial respiratory rates. mitochondria oxidative phosphorylati on pioglitazone T ype 2 diabetes is a complex disease involving insulin resistance, decreased insulin secretion, dyslipidemia, and altered nutrient partitioning (1). Mitochondria play a well documented role in insulin secretio n and the control of glucose and fatty acid oxidation (2, 3). Insulin resistance is associated with decreased mitochondrial mass and compromised oxidative capacity, sug- ges ting a primary role for mitochondria l dysfunction in the pathogenesis of insulin resistance (4–8). One of the dru gs of choi ce to tr eat type 2 di abetes is pioglitazone, a member of the thiazolidinedione (TZD) class of insulin sensitizers. The pharmacology of the TZDs has tradi- tionall y been attri bute d to thei r func tion as agoni sts of the peroxisome prolifera tor-activated receptor (PPAR ), a tran- scription factor involved in adipocyte differentiation and matu- ration (9). There is a substantial amount of data indicating that theTZDs exe rt onl y a por tion of the ir act ivi ty via genomi c effe cts on PPAR and that nongenomic mechanisms are significant and pote ntia llyrelevan t to thei r cli nical effec ts (10). For example, the eff icacy of the TZDs doe s not corr el ate with the bi ndi ng affinities for PPAR , and these drugs can elicit beneficial effects in the absence of PPAR in selected tissues (10, 11). In addition, TZD effects can occur too rapidly for PPAR -mediated tran- scriptional events to be primarily responsible (11). In an effort to identify cellular targets of pioglitazone, Colca et al. (12) identified a protein that was cross-linked to a radio- labele d photo affinity deri vati ve of piog litaz one. This prot ein had a mitochondrial association , c ontained the amino acid sequence As n-Gl u-Gl u-Thr (NEET), and was name d mitoNEET (12). The important role of the TZDs in treating type 2 diabetes and the identification of mitoNEET as a target of pioglitazone binding prompted us to examine this protein in greater detail. Results and Discussion MitoNEET Family Proteins Possess Unique ‘‘CDGSH-Type Zinc Finger’’ Domains. Colca et al. (12) identified mitoNEET as a target for pioglitazone binding. Because there were no additional studies on mitoNEET, we began our efforts by analyzing the domain organization of the protein. By using the simple modular archi- tecture research tool (SMART) and the National Center for Biotechnology Information (NCBI) conserved domain search alg orit hms, our anal ysis showed that mitoNEET contain s a domain of 40 aa (amino acids 55–93) annotated as a CDGSH- type zinc fing er (Fig. 1). Two addi tional cysteine s flank the CDGSH sequence. In addition to the CDGSH domain, mito- NEET has a predicted transmembrane domain localized be- tween amino acid s 14 and 32, sugg es ting tha t it may be a membrane-anchored protein (Fig. 1). To identify other proteins with a CDGSH domain, we performed BLAST searche s by using the human mitoNEET CDGSH sequence (residues 55–93). These searches yielded t wo related human proteins, which we refer to as Miner1 and Miner2 for MitoNEET-related 1 and 2 (Fig. 1). Both mitoNEET and Miner1 have a single CDGSH-type zinc finger, whereas Miner2 contains two of these domains (Fig. 1). Multiple species align- ments of the CDGSH-type zinc finger domains from mitoNEET, Miner1, and Miner2 revealed that this family contains the consensus sequence: C-X-C-X 2 -(S/T)-X 3 -P-X-C-D-G- (S/A/T)-H. In addition to the invariant proline, aspartic acid, and glycine residues, this motif also contains three invariant cysteines and an invariant histidine. Although zinc finger proteins are abundant in the genome, this CDGSH-type zinc finger domain is unique. The Annotated CDGSH-Type Zinc Finger Protein Contains Iron. To our surprise, purified recombinant mitoNEET was strikingly red in color , as were recombi nant protei ns of Miner1 and Miner2. Because zinc finger proteins are not reported to be red in color, this suggested that mitoNEET likely had an una nticipated ‘‘cofactor.’’ To explore this further, mitoNEET 27–108 (lacking Author contributions: S.E.W., A.N.M., and J.E.D. designed experiments; S.E.W., A.N.M., S.A.R., and P.v.d.G. performed research; S.E.W. and S.A.R. provided new reagents/analytic tools; and S.E.W., A.N.M., P.v.d.G., and J.E.D. wrote the paper. The authors declare no conflict of interest. Abbreviations:TZD, thiazolidinedione ; PPAR , peroxisomeproliferator-activated receptor ; Mine r1,MitoNE ET-re late d 1;Miner2,MitoNEET- rela ted2; MBP,malto sebindingprotein ; ICP-HRMS, inductively coupled plasma–high-resolution mass spectrometry; OMM, outer mitochondrial membrane. Datadeposition:Thesequenc esreportedin thispaperhavebeen depos itedin theGenBank database [accession nos. GI:37590611 (mitoNEET), GI:21619026 (Miner1), and GI:42661145 (Miner2)]. ‡ Deceased January 8, 2007. **To whom correspondence should be addressed at: Department of Pharmacology, Uni- versity of California at San Diego School of Medicine, 9500 Gilman Drive, Leichtag Research Building, Room 284, La Jolla, CA 92093-0721. E-mail: [email protected]. This article contains suppo rting informati on onlin e at www.pnas.org/cgi/content/full/ 0701078104/DC1 . © 2007 by The National Academy of Sciences of the USA 5318–5323 PNAS March 27, 2007 vol. 104 no. 13 www.pnas.orgcgidoi10.1073pnas.0701078104

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