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Acute Loss of Iron-Sulfur Clusters Results in Metabolic Reprogramming and Generation of Lipid Droplets in Mammalian Cells Daniel R

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Acute Loss of Iron-Sulfur Clusters Results in Metabolic Reprogramming and Generation of Lipid Droplets in Mammalian Cells Daniel R University of Kentucky UKnowledge Center for Environmental and Systems Center for Environmental and Systems Biochemistry Faculty Publications Biochemistry 3-9-2018 Acute Loss of Iron-Sulfur Clusters Results in Metabolic Reprogramming and Generation of Lipid Droplets in Mammalian Cells Daniel R. Crooks National Institutes of Health Nunziata Maio National Institutes of Health Andrew N. Lane University of Kentucky, [email protected] Michal Jarnik National Institutes of Health Richard M. Higashi University of Kentucky, [email protected] See next page for additional authors CFolicllokw h ethire st oa ndlet ausdd knitionow alho wwork accse asts: thott thips://uknos documewnlet bedgnee.ukfitsy. eoydu/cu. esb_facpub Part of the Biochemical Phenomena, Metabolism, and Nutrition Commons, Cancer Biology Commons, and the Oncology Commons Repository Citation Crooks, Daniel R.; Maio, Nunziata; Lane, Andrew N.; Jarnik, Michal; Higashi, Richard M.; Haller, Ronald G.; Yang, Ye; Fan, Teresa Whei-Mei; Linehan, W. Marston; and Rouault, Tracey A., "Acute Loss of Iron-Sulfur Clusters Results in Metabolic Reprogramming and Generation of Lipid Droplets in Mammalian Cells" (2018). Center for Environmental and Systems Biochemistry Faculty Publications. 4. https://uknowledge.uky.edu/cesb_facpub/4 This Article is brought to you for free and open access by the Center for Environmental and Systems Biochemistry at UKnowledge. It has been accepted for inclusion in Center for Environmental and Systems Biochemistry Faculty Publications by an authorized administrator of UKnowledge. For more information, please contact [email protected]. Authors Daniel R. Crooks, Nunziata Maio, Andrew N. Lane, Michal Jarnik, Richard M. Higashi, Ronald G. Haller, Ye Yang, Teresa Whei-Mei Fan, W. Marston Linehan, and Tracey A. Rouault Acute Loss of Iron-Sulfur Clusters Results in Metabolic Reprogramming and Generation of Lipid Droplets in Mammalian Cells Notes/Citation Information Published in The Journal of Biological Chemistry, v. 293, no. 21, p. 8297-8311. This research was originally published in The Journal of Biological Chemistry. Daniel R. Crooks, Nunziata Maio, Andrew N. Lane, Michal Jarnik, Richard M. Higashi, Ronald G. Haller, Ye Yang, Teresa W-M. Fan, W. Marston Linehan, and Tracey A. Rouault. Acute Loss of Iron-Sulfur Clusters Results in Metabolic Reprogramming and Generation of Lipid Droplets in Mammalian Cells. J. Biol. Chem. 2018; 293:8297-8311. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc. The opc yright holder has granted the permission for posting the article here. Digital Object Identifier (DOI) https://doi.org/10.1074/jbc.RA118.001885 This article is available at UKnowledge: https://uknowledge.uky.edu/cesb_facpub/4 EDITORS’ PICK cro Acute loss of iron–sulfur clusters results in metabolic reprogramming and generation of lipid droplets in mammalian cells Received for publication, January 12, 2018, and in revised form, March 6, 2018 Published, Papers in Press, March 9, 2018, DOI 10.1074/jbc.RA118.001885 Daniel R. Crooks‡, Nunziata Maio§, Andrew N. Lane¶, Michal Jarnikʈ, Richard M. Higashi¶, Ronald G. Haller**‡‡§§, Ye Yang‡, Teresa W-M. Fan¶, W. Marston Linehan‡, and Tracey A. Rouault§1 From the ‡Urologic Oncology Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, the §Section on Human Iron Metabolism and ʈSection on Cell Biology and Metabolism, Eunice Kennedy Shriver NICHD, National Institutes of Health, Bethesda, Maryland 20892, the ¶Center for Environmental and Systems Biochemistry, Department of Toxicology and Cancer Biology, and Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536, the **Department of Neurology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, the ‡‡Veterans Affairs North Texas Medical Center, Dallas, Texas 75216, and the §§Neuromuscular Center, Institute for Exercise and Environmental Medicine, Dallas, Texas 75231 Edited by F. Peter Guengerich Iron–sulfur (Fe-S) clusters are ancient cofactors in cells and Iron–sulfur (Fe-S) clusters are ancient components of the participate in diverse biochemical functions, including electron cellular machinery that provide essential cofactors for numer- transfer and enzymatic catalysis. Although cell lines derived ous proteins. In addition to serving as a conduit for electron from individuals carrying mutations in the Fe-S cluster biogen- transport in respiratory chain complexes, Fe-S clusters catalyze esis pathway or siRNA-mediated knockdown of the Fe-S assem- chemical oxidation–reduction reactions and participate in cel- bly components provide excellent models for investigating Fe-S lular regulatory signaling (1). In the nucleus, Fe-S clusters are cluster formation in mammalian cells, these experimental strat- cofactors in several crucial DNA repair enzymes (2, 3), and Fe-S egies focus on the consequences of prolonged impairment of clusters are essential for the function of the eukaryotic cytosolic Fe-S assembly. Here, we constructed and expressed dominant– RNA polymerases (4). Thus, Fe-S proteins are essential for cell negative variants of the primary Fe-S biogenesis scaffold protein survival and proliferation. iron–sulfur cluster assembly enzyme 2 (ISCU2) in human Fe-S assembly scaffolds have been a focus of attention since HEK293 cells. This approach enabled us to study the early met- their discovery in nitrogen-fixing bacteria (5, 6), and mutations abolic reprogramming associated with loss of Fe-S–containing in the human Fe-S scaffold ISCU cause hereditary diseases in proteins in several major cellular compartments. Using multiple humans (7, 8). In human cells, Fe-S clusters are assembled on a metabolomics platforms, we observed a ϳ12-fold increase in multimeric complex containing the ISCU scaffold, cysteine intracellular citrate content in Fe-S–deficient cells, a surge that desulfurase NFS1, and accessory proteins ISD11 and frataxin was due to loss of aconitase activity. The excess citrate was gen- (9). Binding of the HSPA9/HSC20 chaperone/co-chaperone erated from glucose-derived acetyl-CoA, and global analysis of system facilitates ATP-dependent transfer of the nascent Fe-S cellular lipids revealed that fatty acid biosynthesis increased clusters to recipient Fe-S proteins (10, 11), which is mediated by markedly relative to cellular proliferation rates in Fe-S–defi- LYR tripeptide motifs in recipient proteins that bind to the cient cells. We also observed intracellular lipid droplet accumu- HSC20 co-chaperone (12). lation in both acutely Fe-S–deficient cells and iron-starved Fe-S clusters assembled on the ISCU scaffold are labile and cells. We conclude that deficient Fe-S biogenesis and acute iron transient, and clusters are often lost during protein purifica- deficiency rapidly increase cellular citrate concentrations, lead- tion, during exposure to oxygen, or by treatment with mild ing to fatty acid synthesis and cytosolic lipid droplet formation. reducing agents (13–15). The Fe-S cluster lability of ISCU has Our findings uncover a potential cause of cellular steatosis in been attributed to a conserved aspartate residue adjacent to one nonadipose tissues. of the Fe-S cluster–ligating cysteines (Asp-37 in Azotobacter vinelandii NifU; Asp-71 in human ISCU) (13) and has been proposed to confer the ability of the scaffold to release nascent This work was supported by the NICHD, National Institutes of Health (NIH), Intramural Research Program and the NCI/NIH Center for Cancer Research, Fe-S clusters for transfer to recipient proteins (15, 16). A D37A NIAMS/NIH Grant R01 AR050597, NIDDK/NIH Grant 1U24DK097215-01A1, mutant of IscU (NifU ) that more stably bound [2Fe-2S] and NIEHS/NIH Grant 3R01ES022191-04S1. The authors declare that they clusters (13) was later shown to trap the cysteine desulfurase have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent IscS (14) and to inhibit transfer of Fe-S clusters to purified apo- the official views of the National Institutes of Health. aconitase in vitro (16), resulting in a dominant–negative growth This article was selected as one of our Editors’ Picks. phenotype in A. vinelandii (14). Molecular modeling based on This article contains Table S1 and Figs. S1–S6. 1 To whom correspondence should be addressed. E-mail: [email protected]. the IscU-IscS crystal structure suggested that the conserved gov. aspartate in IscU served as a fourth ligand for the nascent [2Fe- J. Biol. Chem. (2018) 293(21) 8297–8311 8297 Published in the U.S.A. Metabolic reprogramming in acute Fe-S cluster deficiency 2S] cluster, allowing dissociation of a cluster-ligating cysteine in ners NFS1, ISD11, and frataxin (Fig. 1E). We observed abun- IscS and associated conformational changes (15). dant amounts of immunoprecipitated ISCU and found that Although complete loss of ISCU is lethal in all organisms NFS1 and ISD11 remained stably bound to ISCU during the tested to date (14, 17, 18), assessment of ISCU deficiency on immunoprecipitation procedure (Fig. 1E, middle panels); how- cellular metabolism in mammalian cells has been limited to ever, no frataxin protein was detected (Fig. 1E, bottom), proba- knockdown experiments (19) and/or study of patient tissues bly due to the transient nature of its interaction with the NFS1- that are naturally deficient in ISCU (7, 18, 20, 21). In this study, ISD11-ISCU initial complex. Together, these data suggest that we expressed dominant–negative
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