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Liu H, Li M, Deng Y, Hou Y, Hou L, Zhang X, Zheng Z, Guo F, Sun K. The Roles of DMT1 in Inflammatory and Degenerative Diseases. Mol Neurobiol 2025; 62:6317-6332. [PMID: 39775481 DOI: 10.1007/s12035-025-04687-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2024] [Accepted: 01/02/2025] [Indexed: 01/11/2025]
Abstract
Iron homeostasis is critical for multiple physiological and pathological processes. DMT1, a core iron transporter, is expressed in almost all cells and organs and altered in response to various conditions, whereas, there is few reviews focusing on DMT1 in diseases associated with aberrant iron metabolism. Based on available knowledge, this review described a full view of DMT1 and summarized the roles of DMT1 and DMT1-mediated iron metabolism in the onset and development of inflammatory and degenerative diseases. This review also provided an overview of DMT1-related treatment in these disorders, highlighting its therapeutic potential in chronic inflammatory and degenerative diseases.
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Affiliation(s)
- Haigang Liu
- Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China
| | - Mi Li
- Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China
| | - Yi Deng
- Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China
| | - Yanjun Hou
- Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China
| | - Liangcai Hou
- Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China
| | - Xiong Zhang
- Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China
| | - Zehang Zheng
- Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China
| | - Fengjing Guo
- Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China.
| | - Kai Sun
- Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China.
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2
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Liziczai M, Fuchs A, Manatschal C, Dutzler R. Structural basis for metal ion transport by the human SLC11 proteins DMT1 and NRAMP1. Nat Commun 2025; 16:761. [PMID: 39824808 PMCID: PMC11742427 DOI: 10.1038/s41467-024-54705-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2024] [Accepted: 11/19/2024] [Indexed: 01/30/2025] Open
Abstract
Iron and manganese are essential nutrients whose transport across membranes is catalyzed by members of the SLC11 family. In humans, this protein family contains two paralogs, the ubiquitously expressed DMT1, which is involved in the uptake and distribution of Fe2+ and Mn2+, and NRAMP1, which participates in the resistance against infections and nutrient recycling. Despite previous studies contributing to our mechanistic understanding of the family, the structures of human SLC11 proteins and their relationship to functional properties have remained elusive. Here we describe the cryo-electron microscopy structures of DMT1 and NRAMP1 and relate them to their functional properties. We show that both proteins catalyze selective metal ion transport coupled to the symport of H+, but additionally also mediate uncoupled H+ flux. Their structures, while sharing general properties with known prokaryotic homologs, display distinct features that lead to stronger transition metal ion selectivity.
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Affiliation(s)
- Márton Liziczai
- Department of Biochemistry, University of Zurich, Zurich, Switzerland
| | - Ariane Fuchs
- Department of Biochemistry, University of Zurich, Zurich, Switzerland
| | | | - Raimund Dutzler
- Department of Biochemistry, University of Zurich, Zurich, Switzerland.
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3
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Loveridge KM, Sigala PA. Identification of a divalent metal transporter required for cellular iron metabolism in malaria parasites. Proc Natl Acad Sci U S A 2024; 121:e2411631121. [PMID: 39467134 PMCID: PMC11551425 DOI: 10.1073/pnas.2411631121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2024] [Accepted: 09/23/2024] [Indexed: 10/30/2024] Open
Abstract
Plasmodium falciparum malaria parasites invade and multiply inside red blood cells (RBCs), the most iron-rich compartment in humans. Like all cells, P. falciparum requires nutritional iron to support essential metabolic pathways, but the critical mechanisms of iron acquisition and trafficking during RBC infection have remained obscure. Parasites internalize and liberate massive amounts of heme during large-scale digestion of RBC hemoglobin within an acidic food vacuole (FV) but lack a heme oxygenase to release porphyrin-bound iron. Although most FV heme is sequestered into inert hemozoin crystals, prior studies indicate that trace heme escapes biomineralization and is susceptible to nonenzymatic degradation within the oxidizing FV environment to release labile iron. Parasites retain a homolog of divalent metal transporter 1 (DMT1), a known mammalian iron transporter, but its role in P. falciparum iron acquisition has not been tested. Our phylogenetic studies indicate that P. falciparum DMT1 (PfDMT1) retains conserved molecular features critical for metal transport. We localized this protein to the FV membrane and defined its orientation in an export-competent topology. Conditional knockdown of PfDMT1 expression is lethal to parasites, which display broad cellular defects in iron-dependent functions, including impaired apicoplast biogenesis and mitochondrial polarization. Parasites are selectively rescued from partial PfDMT1 knockdown by supplementation with exogenous iron, but not other metals. These results support a cellular paradigm whereby PfDMT1 is the molecular gatekeeper to essential iron acquisition by blood-stage malaria parasites and suggest that therapeutic targeting of PfDMT1 may be a potent antimalarial strategy.
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Affiliation(s)
- Kade M. Loveridge
- Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT84112
| | - Paul A. Sigala
- Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT84112
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Kwong RWM. Trace metals in the teleost fish gill: biological roles, uptake regulation, and detoxification mechanisms. J Comp Physiol B 2024; 194:749-763. [PMID: 38916671 DOI: 10.1007/s00360-024-01565-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2024] [Revised: 05/10/2024] [Accepted: 05/21/2024] [Indexed: 06/26/2024]
Abstract
In fish, the gill plays a vital role in regulating the absorption of trace metals and is also highly susceptible to metal toxicity. Trace metals such as iron (Fe), copper (Cu), zinc (Zn), and manganese (Mn) are involved in various catalytic activities and molecular binding within the gill, thereby supporting a range of physiological processes in this organ. While beneficial at normal levels, these metals can become toxic when present in excess. Conversely, nonessential metals like cadmium (Cd) and lead (Pb) can gain entry into gill cells through similar metal transport pathways, potentially interfering with various cellular processes. The transepithelial transport of these metals across the gill epithelium is governed by a variety of metal transport and metal binding proteins. These include the Cu transporter 1 (CTR1), divalent metal transporter 1 (DMT1), and members of the Zrt-/Irt-like protein (ZIP) and zinc transport (ZnT) families. Additionally, some of these metals can compete with major ions (e.g., calcium, sodium) for absorption sites in the gill. This complex crosstalk suggests an interdependent mechanism that balances metal uptake to meet physiological needs while preventing excessive accumulation. In this article, I review the roles of trace metals in proteins/enzymes that support the different functions in the gill of teleost fish. I also discuss current understanding of the pathways involved in regulating the branchial uptake of metals and their influence on ionic regulation, and the potential detoxification mechanisms in the gill. Finally, I summarize knowledge gaps and potential areas for further investigation.
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Affiliation(s)
- Raymond W M Kwong
- Department of Biology, York University, 4700 Keele Street, Toronto, ON, M3J 1P3, Canada.
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5
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Pan X, Köberle M, Ghashghaeinia M. Vitamin C-Dependent Uptake of Non-Heme Iron by Enterocytes, Its Impact on Erythropoiesis and Redox Capacity of Human Erythrocytes. Antioxidants (Basel) 2024; 13:968. [PMID: 39199214 PMCID: PMC11352176 DOI: 10.3390/antiox13080968] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2024] [Revised: 08/06/2024] [Accepted: 08/07/2024] [Indexed: 09/01/2024] Open
Abstract
In the small intestine, nutrients from ingested food are absorbed and broken down by enterocytes, which constitute over 95% of the intestinal epithelium. Enterocytes demonstrate diet- and segment-dependent metabolic flexibility, enabling them to take up large amounts of glutamine and glucose to meet their energy needs and transfer these nutrients into the bloodstream. During glycolysis, ATP, lactate, and H+ ions are produced within the enterocytes. Based on extensive but incomplete glutamine oxidation large amounts of alanine or lactate are produced. Lactate, in turn, promotes hypoxia-inducible factor-1α (Hif-1α) activation and Hif-1α-dependent transcription of various proton channels and exchangers, which extrude cytoplasmic H+-ions into the intestinal lumen. In parallel, the vitamin C-dependent and duodenal cytochrome b-mediated conversion of ferric iron into ferrous iron progresses. Finally, the generated electrochemical gradient is utilized by the divalent metal transporter 1 for H+-coupled uptake of non-heme Fe2+-ions. Iron efflux from enterocytes, subsequent binding to the plasma protein transferrin, and systemic distribution supply a wide range of cells with iron, including erythroid precursors essential for erythropoiesis. In this review, we discuss the impact of vitamin C on the redox capacity of human erythrocytes and connect enterocyte function with iron metabolism, highlighting its effects on erythropoiesis.
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Affiliation(s)
- Xia Pan
- Physiological Institute, Department of Vegetative and Clinical Physiology, Eberhard Karls University of Tübingen, 72074 Tübingen, Germany
| | - Martin Köberle
- Department of Dermatology and Allergology, School of Medicine and Health, Technical University of Munich, Biedersteinerstr. 29, 80802 München, Germany
| | - Mehrdad Ghashghaeinia
- Physiological Institute, Department of Vegetative and Clinical Physiology, Eberhard Karls University of Tübingen, 72074 Tübingen, Germany
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6
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Loveridge KM, Sigala PA. Identification of a divalent metal transporter required for cellular iron metabolism in malaria parasites. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.05.10.587216. [PMID: 38798484 PMCID: PMC11118319 DOI: 10.1101/2024.05.10.587216] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/29/2024]
Abstract
Plasmodium falciparum malaria parasites invade and multiply inside red blood cells (RBCs), the most iron-rich compartment in humans. Like all cells, P. falciparum requires nutritional iron to support essential metabolic pathways, but the critical mechanisms of iron acquisition and trafficking during RBC infection have remained obscure. Parasites internalize and liberate massive amounts of heme during large-scale digestion of RBC hemoglobin within an acidic food vacuole (FV) but lack a heme oxygenase to release porphyrin-bound iron. Although most FV heme is sequestered into inert hemozoin crystals, prior studies indicate that trace heme escapes biomineralization and is susceptible to non-enzymatic degradation within the oxidizing FV environment to release labile iron. Parasites retain a homolog of divalent metal transporter 1 (DMT1), a known mammalian iron transporter, but its role in P. falciparum iron acquisition has not been tested. Our phylogenetic studies indicate that P. falciparum DMT1 (PfDMT1) retains conserved molecular features critical for metal transport. We localized this protein to the FV membrane and defined its orientation in an export-competent topology. Conditional knockdown of PfDMT1 expression is lethal to parasites, which display broad cellular defects in iron-dependent functions, including impaired apicoplast biogenesis and mitochondrial polarization. Parasites are selectively rescued from partial PfDMT1 knockdown by supplementation with exogenous iron, but not other metals. These results support a cellular paradigm whereby PfDMT1 is the molecular gatekeeper to essential iron acquisition by blood-stage malaria parasites and suggest that therapeutic targeting of PfDMT1 may be a potent antimalarial strategy.
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Affiliation(s)
- Kade M. Loveridge
- Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT, United States
| | - Paul A. Sigala
- Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT, United States
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7
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Jormakka M. Structural insights into ferroportin mediated iron transport. Biochem Soc Trans 2023; 51:BST20230594. [PMID: 38115725 DOI: 10.1042/bst20230594] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2023] [Revised: 12/08/2023] [Accepted: 12/11/2023] [Indexed: 12/21/2023]
Abstract
Iron is a vital trace element for almost all organisms, and maintaining iron homeostasis is critical for human health. In mammals, the only known gatekeeper between intestinally absorbed iron and circulatory blood plasma is the membrane transporter ferroportin (Fpn). As such, dysfunction of Fpn or its regulation is a key driver of iron-related pathophysiology. This review focuses on discussing recent insights from high-resolution structural studies of the Fpn protein family. While these studies have unveiled crucial details of Fpn regulation and structural architecture, the associated functional studies have also at times provided conflicting data provoking more questions than answers. Here, we summarize key findings and illuminate important remaining questions and contradictions.
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Affiliation(s)
- Mika Jormakka
- Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
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Cutts A, Chowdhury S, Ratkay LG, Eyers M, Young C, Namdari R, Cadieux JA, Chahal N, Grimwood M, Zhang Z, Lin S, Tietjen I, Xie Z, Robinette L, Sojo L, Waldbrook M, Hayden M, Mansour T, Pimstone S, Goldberg YP, Webb M, Cohen CJ. Potent, Gut-Restricted Inhibitors of Divalent Metal Transporter 1: Preclinical Efficacy against Iron Overload and Safety Evaluation. J Pharmacol Exp Ther 2023; 386:4-14. [PMID: 36958846 DOI: 10.1124/jpet.122.001435] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2022] [Revised: 01/26/2023] [Accepted: 02/21/2023] [Indexed: 03/25/2023] Open
Abstract
Divalent metal transporter 1 (DMT1) cotransports ferrous iron and protons and is the primary mechanism for uptake of nonheme iron by enterocytes. Inhibitors are potentially useful as therapeutic agents to treat iron overload disorders such as hereditary hemochromatosis or β-thalassemia intermedia, provided that inhibition can be restricted to the duodenum. We used a calcein quench assay to identify human DMT1 inhibitors. Dimeric compounds were made to generate more potent compounds with low systemic exposure. Direct block of DMT1 was confirmed by voltage clamp measurements. The lead compound, XEN602, strongly inhibits dietary nonheme iron uptake in both rats and pigs yet has negligible systemic exposure. Efficacy is maintained for >2 weeks in a rat subchronic dosing assay. Doses that lowered iron content in the spleen and liver by >50% had no effect on the tissue content of other divalent cations except for cobalt. XEN602 represents a powerful pharmacological tool for understanding the physiologic function of DMT1 in the gut. SIGNIFICANCE STATEMENT: This report introduces methodology to develop potent, gut-restricted inhibitors of divalent metal transporter 1 (DMT1) and identifies XEN602 as a suitable compound for in vivo studies. We also report novel animal models to quantify the inhibition of dietary uptake of iron in both rodents and pigs. This research shows that inhibition of DMT1 is a promising means to treat iron overload disorders.
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Affiliation(s)
- Alison Cutts
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Sultan Chowdhury
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Laszlo G Ratkay
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Maryanne Eyers
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Clint Young
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Rostam Namdari
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Jay A Cadieux
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Navjot Chahal
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Michael Grimwood
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Zaihui Zhang
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Sophia Lin
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Ian Tietjen
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Zhiwei Xie
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Lee Robinette
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Luis Sojo
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Matthew Waldbrook
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Michael Hayden
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Tarek Mansour
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Simon Pimstone
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Y Paul Goldberg
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Michael Webb
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
| | - Charles J Cohen
- Xenon Pharmaceuticals Inc., Burnaby, British Columbia, Canada(A.C., S.C., L.G.R., M.E., C.Y., R.N., J.A.C., N.C., M.G., Z.Z., S.L., I.T., Z.X., L.R., L.S., M.W., M.H., T.M., S.P., Y.P.G., M.W., C.J.C.) and Division of General Internal Medicine, University of British Columbia, Vancouver, British Columbia, Canada (S.P.)
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9
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Azucenas CR, Ruwe TA, Bonamer JP, Qiao B, Ganz T, Jormakka M, Nemeth E, Mackenzie B. Comparative analysis of the functional properties of human and mouse ferroportin. Am J Physiol Cell Physiol 2023; 324:C1110-C1118. [PMID: 36939203 PMCID: PMC10191125 DOI: 10.1152/ajpcell.00063.2023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2023] [Revised: 03/16/2023] [Accepted: 03/16/2023] [Indexed: 03/21/2023]
Abstract
Ferroportin (Fpn)-expressed at the plasma membrane of macrophages, enterocytes, and hepatocytes-mediates the transfer of cellular iron into the blood plasma. Under the control of the iron-regulatory hormone hepcidin, Fpn serves a critical role in systemic iron homeostasis. Although we have previously characterized human Fpn, a great deal of research in iron homeostasis and disorders uses mouse models. By way of example, the flatiron mouse, a model of classical ferroportin disease, bears the mutation H32R in Fpn and is characterized by systemic iron deficiency and macrophage iron retention. The flatiron mouse also appears to exhibit a manganese phenotype, raising the possibility that mouse Fpn serves a role in manganese metabolism. At odds with this observation, we have found that human Fpn does not transport manganese, so we considered the possibility that a species difference could explain this discrepancy. We tested the hypothesis that mouse but not human Fpn can transport manganese and performed a comparative analysis of mouse and human Fpn. We examined the functional properties of human Fpn, mouse Fpn, and mutant mouse Fpn by using radiotracer assays in RNA-injected Xenopus oocytes. We found that neither mouse nor human Fpn transports manganese. Mouse and human Fpn share identical properties with respect to substrate profile, calcium dependence, optimal pH, and hepcidin sensitivity. We have also demonstrated that Fpn is not an ATPase pump. Our findings validate the use of mouse models of ferroportin function in iron homeostasis and disease.
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Affiliation(s)
- Corbin R Azucenas
- Department of Pharmacology & Systems Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States
- Medical Sciences Program, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States
- Systems Biology & Physiology Program, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States
| | - T Alex Ruwe
- Department of Pharmacology & Systems Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States
- Systems Biology & Physiology Program, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States
| | - John P Bonamer
- Department of Pharmacology & Systems Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States
| | - Bo Qiao
- Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, United States
| | - Tomas Ganz
- Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, United States
- Department of Pathology, David Geffen School of Medicine at UCLA, Los Angeles, California, United States
| | - Mika Jormakka
- Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Elizabeta Nemeth
- Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, United States
| | - Bryan Mackenzie
- Department of Pharmacology & Systems Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States
- Medical Sciences Program, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States
- Systems Biology & Physiology Program, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States
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10
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Cegarra L, Aguirre P, Nuñez MT, Gerdtzen ZP, Salgado JC. Calcium is a noncompetitive inhibitor of DMT1 on the intestinal iron absorption process: empirical evidence and mathematical modeling analysis. Am J Physiol Cell Physiol 2022; 323:C1791-C1806. [PMID: 36342159 DOI: 10.1152/ajpcell.00411.2022] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Iron absorption is a complex and highly controlled process where DMT1 transports nonheme iron through the brush-border membrane of enterocytes to the cytoplasm but does not transport alkaline-earth metals such as calcium. However, it has been proposed that high concentrations of calcium in the diet could reduce iron bioavailability. In this work, we investigate the effect of intracellular and extracellular calcium on iron uptake by Caco-2 cells, as determined by calcein fluorescence quenching. We found that extracellular calcium inhibits iron uptake by Caco-2 cells in a concentration-dependent manner. Chelation of intracellular calcium with BAPTA did not affect iron uptake, which indicates that the inhibitory effect of calcium is not exerted through intracellular calcium signaling. Kinetic studies performed, provided evidence that calcium acts as a reversible noncompetitive inhibitor of the iron transport activity of DMT1. Based on these experimental results, a mathematical model was developed that considers the dynamics of noncompetitive inhibition using a four-state mechanism to describe the inhibitory effect of calcium on the DMT1 iron transport process in intestinal cells. The model accurately predicts the calcein fluorescence quenching dynamics observed experimentally after an iron challenge. Therefore, the proposed model structure is capable of representing the inhibitory effect of extracellular calcium on DMT1-mediated iron entry into the cLIP of Caco-2 cells. Considering the range of calcium concentrations that can inhibit iron uptake, the possible inhibition of dietary calcium on intestinal iron uptake is discussed.
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Affiliation(s)
- Layimar Cegarra
- Laboratory of Process Modeling and Distributed Computing, Department of Chemical Engineering, Biotechnology and Materials, University of Chile, Santiago, Chile.,Mammalian Cell Culture Laboratory, Department of Chemical Engineering, Biotechnology and Materials, University of Chile, Santiago, Chile.,Centre for Biotechnology and Bioengineering, University of Chile, Santiago, Chile
| | - Pabla Aguirre
- Iron and Biology of Aging Laboratory, Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile
| | - Marco T Nuñez
- Iron and Biology of Aging Laboratory, Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile
| | - Ziomara P Gerdtzen
- Mammalian Cell Culture Laboratory, Department of Chemical Engineering, Biotechnology and Materials, University of Chile, Santiago, Chile.,Centre for Biotechnology and Bioengineering, University of Chile, Santiago, Chile.,Center of Interventional Medicine for Precision and Advanced Cellular Therapy, Santiago, Chile.,Millennium Nucleus Marine Agronomy of Seaweed Holobionts, Puerto Mont, Chile
| | - J Cristian Salgado
- Laboratory of Process Modeling and Distributed Computing, Department of Chemical Engineering, Biotechnology and Materials, University of Chile, Santiago, Chile.,Centre for Biotechnology and Bioengineering, University of Chile, Santiago, Chile
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11
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Ramanadane K, Straub MS, Dutzler R, Manatschal C. Structural and functional properties of a magnesium transporter of the SLC11/NRAMP family. eLife 2022; 11:74589. [PMID: 35001872 PMCID: PMC8806188 DOI: 10.7554/elife.74589] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2021] [Accepted: 12/17/2021] [Indexed: 11/13/2022] Open
Abstract
Members of the ubiquitous SLC11/NRAMP family catalyze the uptake of divalent transition metal ions into cells. They have evolved to efficiently select these trace elements from a large pool of Ca2+ and Mg2+, which are both orders of magnitude more abundant, and to concentrate them in the cytoplasm aided by the cotransport of H+ serving as energy source. In the present study, we have characterized a member of a distant clade of the family found in prokaryotes, termed NRMTs, that were proposed to function as transporters of Mg2+. The protein transports Mg2+ and Mn2+ but not Ca2+ by a mechanism that is not coupled to H+. Structures determined by cryo-EM and X-ray crystallography revealed a generally similar protein architecture compared to classical NRAMPs, with a restructured ion binding site whose increased volume provides suitable interactions with ions that likely have retained much of their hydration shell.
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Affiliation(s)
| | - Monique S Straub
- Department of Biochemistry, University of Zurich, Zürich, Switzerland
| | - Raimund Dutzler
- Department of Biochemistry, University of Zurich, Zürich, Switzerland
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12
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Anemia feriprivă – manifestare de debut al unei boli celiace oculte. ONCOLOG-HEMATOLOG.RO 2022. [DOI: 10.26416/onhe.60.3.2022.7153] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
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13
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Dirweesh A, Anugwom CM, Li Y, Vaughn BP, Lake J. Proton pump inhibitors reduce phlebotomy burden in patients with HFE-related hemochromatosis: a systematic review and meta-analysis. Eur J Gastroenterol Hepatol 2021; 33:1327-1331. [PMID: 32769410 DOI: 10.1097/meg.0000000000001857] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/10/2022]
Abstract
BACKGROUND AND AIMS Proton pump inhibitors (PPIs) may reduce iron absorption and serum ferritin levels in patients with homeostatic iron regulator (HFE)-related hemochromatosis, reducing the need for frequent phlebotomies. Our study aimed to perform for the first time a meta-analysis of existing observational and randomized controlled studies to ascertain the overall effect of PPI use in patients with HFE-related hemochromatosis. METHODS Studies in adults reporting the outcomes of PPIs use in hereditary hemochromatosis patients from Medline, Embase, Scopus and Google Scholar databases from inception to December 2019 were systematically searched. The study outcomes were the serum ferritin levels and annual requirement for phlebotomies. Pooled mean difference, and 95% confidence intervals (CIs) were obtained by the random-effects model. Forrest plots were constructed to show the summary pooled estimate. Heterogeneity was assessed by using I2 measure of inconsistency. RESULTS Following an initial search of 202 manuscripts, a total of three studies involving 68 patients with hemochromatosis (34 in the PPIs group and 34 in the placebo or non-PPI group) were included. A minimum duration of PPI use was 1 year. Patients who received PPIs therapy did not have a statistically significant lower serum ferritin levels (mean difference: -18.86, 95% CI: -60.44, 22.72, P = 0.37, I2 = 88%) but required significantly less sessions of phlebotomies annually (mean difference: -3.10, 95% CI: -4.46, -3.08, P < 0.00001, I2 = 93%). No publication bias was found on Egger (P = 0.94) or Begg (P = 0.98) tests. CONCLUSION PPIs can be used as an adjuvant therapy to reduce phlebotomy burden in patients with HFE-related hemochromatosis.
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Affiliation(s)
- Ahmed Dirweesh
- Division of Gastroenterology, Hepatology and Nutrition, University of Minnesota, Minneapolis, Minnesota
| | - Chimaobi M Anugwom
- Division of Gastroenterology, Hepatology and Nutrition, University of Minnesota, Minneapolis, Minnesota
| | - Yiting Li
- Department of Internal Medicine, Seton Hall University, South Orange, New Jersey, USA
| | - Byron P Vaughn
- Division of Gastroenterology, Hepatology and Nutrition, University of Minnesota, Minneapolis, Minnesota
| | - John Lake
- Division of Gastroenterology, Hepatology and Nutrition, University of Minnesota, Minneapolis, Minnesota
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14
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Kosman DJ. A holistic view of mammalian (vertebrate) cellular iron uptake. Metallomics 2021; 12:1323-1334. [PMID: 32766655 DOI: 10.1039/d0mt00065e] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Cell iron uptake in mammals is commonly distinguished by whether the iron is presented to the cell as transferrin-bound or not: TBI or NTBI. This generic perspective conflates TBI with canonical transferrin receptor, endosomal iron uptake, and NTBI with uptake supported by a plasma membrane-localized divalent metal ion transporter, most often identified as DMT1. In fact, iron uptake by mammalian cells is far more nuanced than this somewhat proscribed view suggests. This view fails to accommodate the substantial role that ZIP8 and ZIP14 play in iron uptake, while adhering to the traditional premise that a relatively high endosomal [H+] is thermodynamically required for release of iron from holo-Tf. The canonical view of iron uptake also does not encompass the fact that plasma membrane electron transport - PMET - has long been linked to cell iron uptake. In fact, the known mammalian metallo-reductases - Dcytb and the STEAP proteins - are members of this cohort of cytochrome-dependent oxido-reductases that shuttle reducing equivalents across the plasma membrane. A not commonly appreciated fact is the reduction potential of ferric iron in holo-Tf is accessible to cytoplasmic reducing equivalents - reduced pyridine and flavin mono- and di-nucleotides and dihydroascorbic acid. This allows for the reductive release of Fe2+ at the extracellular surface of the PM and subsequent transport into the cytoplasm by a neutral pH transporter - a ZIP protein. What this perspective emphasizes is that there are two TfR-dependent uptake pathways, one which does and one which does not involve clathrin-dependent, endolysosomal trafficking. This raises the question as to the selective advantage of having two Tf, TfR-dependent routes of iron accumulation. This review of canonical and non-canonical iron uptake uses cerebral iron trafficking as a point of discussion, a focus that encourages inclusion also of the importance of ferritin as a circulating 'chaperone' of ferric iron.
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Affiliation(s)
- Daniel J Kosman
- Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, The University of Buffalo, Suite 4102, 995 Main St., Buffalo, NY 14203, USA.
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15
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Molecular Mechanism of Nramp-Family Transition Metal Transport. J Mol Biol 2021; 433:166991. [PMID: 33865868 DOI: 10.1016/j.jmb.2021.166991] [Citation(s) in RCA: 52] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2021] [Revised: 04/02/2021] [Accepted: 04/05/2021] [Indexed: 02/06/2023]
Abstract
The Natural resistance-associated macrophage protein (Nramp) family of transition metal transporters enables uptake and trafficking of essential micronutrients that all organisms must acquire to survive. Two decades after Nramps were identified as proton-driven, voltage-dependent secondary transporters, multiple Nramp crystal structures have begun to illustrate the fine details of the transport process and provide a new framework for understanding a wealth of preexisting biochemical data. Here we review the relevant literature pertaining to Nramps' biological roles and especially their conserved molecular mechanism, including our updated understanding of conformational change, metal binding and transport, substrate selectivity, proton transport, proton-metal coupling, and voltage dependence. We ultimately describe how the Nramp family has adapted the LeuT fold common to many secondary transporters to provide selective transition-metal transport with a mechanism that deviates from the canonical model of symport.
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16
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Cinquetti R, Imperiali FG, Bozzaro S, Zanella D, Vacca F, Roseti C, Peracino B, Castagna M, Bossi E. Characterization of Transport Activity of SLC11 Transporters in Xenopus laevis Oocytes by Fluorophore Quenching. SLAS DISCOVERY 2021; 26:798-810. [PMID: 33825579 DOI: 10.1177/24725552211004123] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Membrane proteins are involved in different physiological functions and are the target of pharmaceutical and abuse drugs. Xenopus laevis oocytes provide a powerful heterologous expression system for functional studies of these proteins. Typical experiments investigate transport using electrophysiology and radiolabeled uptake. A two-electrode voltage clamp is suitable only for electrogenic proteins, and uptake measurements require the existence of radiolabeled substrates and adequate laboratory facilities.Recently, Dictyostelium discoideum Nramp1 and NrampB were characterized using multidisciplinary approaches. NrampB showed no measurable electrogenic activity, and it was investigated in Xenopus oocytes by acquiring confocal images of the quenching of injected fluorophore calcein.This method is adequate to measure the variation in emitted fluorescence, and thus transporter activity indirectly, but requires long experimental procedures to collect statistically consistent data. Considering that optimal expression of heterologous proteins lasts for 48-72 h, a slow acquiring process requires the use of more than one batch of oocytes to complete the experiments. Here, a novel approach to measure substrate uptake is reported. Upon injection of a fluorophore, oocytes were incubated with the substrate and the transport activity measured, evaluating fluorescence quenching in a microplate reader. The technique permits the testing of tens of oocytes in different experimental conditions simultaneously, and thus the collection of significant statistical data for each batch, saving time and animals.The method was tested with different metal transporters (SLC11), DMT1, DdNramp1, and DdNrampB, and verified with the peptide transporter PepT1 (SLC15). Comparison with traditional methods (uptake, two-electrode voltage clamp) and with quenching images acquired by fluorescence microscopy confirmed its efficacy.
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Affiliation(s)
| | | | | | - Daniele Zanella
- University of Insubria, Varese, Lombardia, Italy.,The University of Alabama, Birmingham, AL, USA
| | - Francesca Vacca
- University of Insubria, Varese, Lombardia, Italy.,Italian Institute of Technology (IIT), Genova, Italy
| | | | | | | | - Elena Bossi
- University of Insubria, Varese, Lombardia, Italy
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17
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Bae DH, Gholam Azad M, Kalinowski DS, Lane DJR, Jansson PJ, Richardson DR. Ascorbate and Tumor Cell Iron Metabolism: The Evolving Story and Its Link to Pathology. Antioxid Redox Signal 2020; 33:816-838. [PMID: 31672021 DOI: 10.1089/ars.2019.7903] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Significance: Vitamin C or ascorbate (Asc) is a water-soluble vitamin and an antioxidant that is involved in many crucial biological functions. Asc's ability to reduce metals makes it an essential enzyme cofactor. Recent Advances: The ability of Asc to act as a reductant also plays an important part in its overall role in iron metabolism, where Asc induces both nontransferrin-bound iron and transferrin-bound iron uptake at physiological concentrations (∼50 μM). Moreover, Asc has emerged to play an important role in multiple diseases and its effects at pharmacological doses could be important for their treatment. Critical Issues: Asc's role as a regulator of cellular iron metabolism, along with its cytotoxic effects and different roles at pharmacological concentrations, makes it a candidate as an anticancer agent. Ever since the controversy regarding the studies from the Mayo Clinic was finally explained, there has been a renewed interest in using Asc as a therapeutic approach toward cancer due to its minimal side effects. Numerous studies have been able to demonstrate the anticancer activity of Asc through selective oxidative stress toward cancer cells via H2O2 generation at pharmacological concentrations. Studies have demonstrated that Asc's cytotoxic mechanism at concentrations (>1 mM) has been associated with decreased cellular iron uptake. Future Directions: Recent studies have also suggested other mechanisms, such as Asc's effects on autophagy, polyamine metabolism, and the cell cycle. Clearly, more has yet to be discovered about Asc's mechanism of action to facilitate safe and effective treatment options for cancer and other diseases.
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Affiliation(s)
- Dong-Hun Bae
- Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, The University of Sydney, Sydney, Australia
| | - Mahan Gholam Azad
- Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, The University of Sydney, Sydney, Australia
| | - Danuta S Kalinowski
- Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, The University of Sydney, Sydney, Australia
| | - Darius J R Lane
- The Florey Institute of Neuroscience and Mental Health, Melbourne Dementia Research Centre, The University of Melbourne, Parkville, Australia
| | - Patric J Jansson
- Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, The University of Sydney, Sydney, Australia
| | - Des R Richardson
- Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, The University of Sydney, Sydney, Australia.,Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Showa-ku, Japan
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18
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Electrophysiology Measurements of Metal Transport by MntH2 from Enterococcus faecalis. MEMBRANES 2020; 10:membranes10100255. [PMID: 32987882 PMCID: PMC7599946 DOI: 10.3390/membranes10100255] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/29/2020] [Revised: 09/16/2020] [Accepted: 09/22/2020] [Indexed: 12/30/2022]
Abstract
Transition metals are essential trace elements and their high-affinity uptake is required for many organisms. Metal transporters are often characterised using metal-sensitive fluorescent dyes, limiting the metals and experimental conditions that can be studied. Here, we have tested whether metal transport by Enterococcus faecalis MntH2 can be measured with an electrophysiology method that is based on the solid-supported membrane technology. E. faecalis MntH2 belongs to the Natural Resistance-Associated Macrophage Protein (Nramp) family of proton-coupled transporters, which transport divalent transition metals and do not transport the earth metals. Electrophysiology confirms transport of Mn(II), Co(II), Zn(II) and Cd(II) by MntH2. However, no uptake responses for Cu(II), Fe(II) and Ni(II) were observed, while the presence of these metals abolishes the uptake signals for Mn(II). Fluorescence assays confirm that Ni(II) is transported. The data are discussed with respect to properties and structures of Nramp-type family members and the ability of electrophysiology to measure charge transport and not directly substrate transport.
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19
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Li Z, Jiang L, Toyokuni S. Role of carbonic anhydrases in ferroptosis-resistance. Arch Biochem Biophys 2020; 689:108440. [PMID: 32485154 DOI: 10.1016/j.abb.2020.108440] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2020] [Revised: 05/18/2020] [Accepted: 05/22/2020] [Indexed: 12/14/2022]
Abstract
Iron is essential for all the lives on earth but may trigger a switch toward ferroptosis, a novel form of regulated necrosis. Carbonic anhydrases (CAs) are ubiquitous enzymes from microbes to humans. The primary function of CAs is to regulate cellular pH by hydrating carbon dioxide (CO2) to protons (H+) and bicarbonate ions (HCO3-). Furthermore, CAs play roles in biosynthetic reactions, such as gluconeogenesis, lipogenesis, ureagenesis and are also associated with tumor metabolism, suggesting that CAs may be a potential target for the treatment of cancers. We have recently revealed a novel function of CA IX in ferroptosis-resistance by using human malignant mesothelioma cells. Herein, we aim to review the potential molecular association between ferroptosis and CAs, from the viewpoint of iron-metabolism, lipogenesis and signaling pathways both under physiological and pathological contexts.
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Affiliation(s)
- Zan Li
- Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan
| | - Li Jiang
- Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan
| | - Shinya Toyokuni
- Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan; Center for Low-temperature Plasma Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8603, Japan; Sydney Medical School, The University of Sydney, NSW, Australia.
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20
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Bozzi AT, McCabe AL, Barnett BC, Gaudet R. Transmembrane helix 6b links proton and metal release pathways and drives conformational change in an Nramp-family transition metal transporter. J Biol Chem 2020. [DOI: 10.1016/s0021-9258(17)49881-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
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21
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Bozzi AT, McCabe AL, Barnett BC, Gaudet R. Transmembrane helix 6b links proton and metal release pathways and drives conformational change in an Nramp-family transition metal transporter. J Biol Chem 2019; 295:1212-1224. [PMID: 31882536 DOI: 10.1074/jbc.ra119.011336] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2019] [Revised: 12/10/2019] [Indexed: 12/15/2022] Open
Abstract
The natural resistance-associated macrophage protein (Nramp) family encompasses transition metal and proton cotransporters that are present in many organisms from bacteria to humans. Recent structures of Deinococcus radiodurans Nramp (DraNramp) in multiple conformations revealed the intramolecular rearrangements required for alternating access of the metal-binding site to the external or cytosolic environment. Here, using recombinant proteins and metal transport and cysteine accessibility assays, we demonstrate that two parallel cytoplasm-accessible networks of conserved hydrophilic residues in DraNramp, one lining the wide intracellular vestibule for metal release and the other forming a narrow proton transport pathway, are essential for metal transport. We further show that mutagenic or posttranslational modifications of transmembrane helix (TM) 6b, which structurally links these two pathways, impede normal conformational cycling and metal transport. TM6b contains two highly conserved histidines, His232 and His237 We found that different mutagenic perturbations of His232, just below the metal-binding site along the proton exit route, differentially affect DraNramp's conformational state, suggesting that His232 serves as a pivot point for conformational changes. In contrast, any replacement of His237, lining the metal exit route, locked the transporter in a transport-inactive outward-closed state. We conclude that these two histidines, and TM6b more broadly, help trigger the bulk rearrangement of DraNramp to the inward-open state upon metal binding and facilitate return of the empty transporter to an outward-open state upon metal release.
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Affiliation(s)
- Aaron T Bozzi
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138
| | - Anne L McCabe
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138
| | - Benjamin C Barnett
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138
| | - Rachelle Gaudet
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138
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22
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Manatschal C, Pujol-Giménez J, Poirier M, Reymond JL, Hediger MA, Dutzler R. Mechanistic basis of the inhibition of SLC11/NRAMP-mediated metal ion transport by bis-isothiourea substituted compounds. eLife 2019; 8:51913. [PMID: 31804182 PMCID: PMC6917499 DOI: 10.7554/elife.51913] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2019] [Accepted: 11/22/2019] [Indexed: 12/23/2022] Open
Abstract
In humans, the divalent metal ion transporter-1 (DMT1) mediates the transport of ferrous iron across the apical membrane of enterocytes. Hence, its inhibition could be beneficial for the treatment of iron overload disorders. Here we characterize the interaction of aromatic bis-isothiourea-substituted compounds with human DMT1 and its prokaryotic homologue EcoDMT. Both transporters are inhibited by a common competitive mechanism with potencies in the low micromolar range. The crystal structure of EcoDMT in complex with a brominated derivative defines the binding of the inhibitor to an extracellular pocket of the transporter in direct contact with residues of the metal ion coordination site, thereby interfering with substrate loading and locking the transporter in its outward-facing state. Mutagenesis and structure-activity relationships further support the observed interaction mode and reveal species-dependent differences between pro- and eukaryotic transporters. Together, our data provide the first detailed mechanistic insight into the pharmacology of SLC11/NRAMP transporters.
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Affiliation(s)
| | - Jonai Pujol-Giménez
- Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland.,Membrane Transport Discovery Lab, Department of Nephrology and Hypertension, Inselspital, University of Bern, Bern, Switzerland.,Department of Biomedical Research, University of Bern, Bern, Switzerland
| | - Marion Poirier
- Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland
| | - Jean-Louis Reymond
- Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland
| | - Matthias A Hediger
- Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland.,Membrane Transport Discovery Lab, Department of Nephrology and Hypertension, Inselspital, University of Bern, Bern, Switzerland.,Department of Biomedical Research, University of Bern, Bern, Switzerland
| | - Raimund Dutzler
- Department of Biochemistry, University of Zurich, Zurich, Switzerland
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23
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Bozzi AT, Bane LB, Zimanyi CM, Gaudet R. Unique structural features in an Nramp metal transporter impart substrate-specific proton cotransport and a kinetic bias to favor import. J Gen Physiol 2019; 151:1413-1429. [PMID: 31619456 PMCID: PMC6888756 DOI: 10.1085/jgp.201912428] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2019] [Accepted: 09/26/2019] [Indexed: 01/01/2023] Open
Abstract
Natural resistance-associated macrophage protein (Nramp) transporters enable uptake of essential transition metal micronutrients in numerous biological contexts. These proteins are believed to function as secondary transporters that harness the electrochemical energy of proton gradients by "coupling" proton and metal transport. Here we use the Deinococcus radiodurans (Dra) Nramp homologue, for which we have determined crystal structures in multiple conformations, to investigate mechanistic details of metal and proton transport. We untangle the proton-metal coupling behavior of DraNramp into two distinct phenomena: ΔpH stimulation of metal transport rates and metal stimulation of proton transport. Surprisingly, metal type influences substrate stoichiometry, leading to manganese-proton cotransport but cadmium uniport, while proton uniport also occurs. Additionally, a physiological negative membrane potential is required for high-affinity metal uptake. To begin to understand how Nramp's structure imparts these properties, we target a conserved salt-bridge network that forms a proton-transport pathway from the metal-binding site to the cytosol. Mutations to this network diminish voltage and ΔpH dependence of metal transport rates, alter substrate selectivity, perturb or eliminate metal-stimulated proton transport, and erode the directional bias favoring outward-to-inward metal transport under physiological-like conditions. Thus, this unique salt-bridge network may help Nramp-family transporters maximize metal uptake and reduce deleterious back-transport of acquired metals. We provide a new mechanistic model for Nramp proton-metal cotransport and propose that functional advantages may arise from deviations from the traditional model of symport.
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Affiliation(s)
- Aaron T Bozzi
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA
| | - Lukas B Bane
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA
| | - Christina M Zimanyi
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA
| | - Rachelle Gaudet
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA
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24
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Steimle BL, Smith FM, Kosman DJ. The solute carriers ZIP8 and ZIP14 regulate manganese accumulation in brain microvascular endothelial cells and control brain manganese levels. J Biol Chem 2019; 294:19197-19208. [PMID: 31699897 DOI: 10.1074/jbc.ra119.009371] [Citation(s) in RCA: 32] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2019] [Revised: 10/29/2019] [Indexed: 12/29/2022] Open
Abstract
Manganese supports numerous neuronal functions but in excess is neurotoxic. Consequently, regulation of manganese flux at the blood-brain barrier (BBB) is critical to brain homeostasis. However, the molecular pathways supporting the transcellular trafficking of divalent manganese ions within the microvascular capillary endothelial cells (BMVECs) that constitute the BBB have not been examined. In this study, we have determined that ZIP8 and ZIP14 (Zrt- and Irt-like proteins 8 and 14) support Mn2+ uptake by BMVECs and that neither DMT1 nor an endocytosis-dependent pathway play any significant role in Mn2+ uptake. Specifically, siRNA-mediated knockdown of ZIP8 and ZIP14 coincided with a decrease in manganese uptake, and kinetic analyses revealed that manganese uptake depends on pH and bicarbonate and is up-regulated by lipopolysaccharide, all biochemical markers of ZIP8 or ZIP14 activity. Mn2+ uptake also was associated with cell-surface membrane presentation of ZIP8 and ZIP14, as indicated by membrane protein biotinylation. Importantly, surface ZIP8 and ZIP14 biotinylation and Mn2+-uptake experiments together revealed that these transporters support manganese uptake at both the apical, blood and basal, brain sides of BMVECs. This indicated that in the BMVECs of the BBB, these two transporters support a bidirectional Mn2+ flux. We conclude that BMVECs play a critical role in controlling manganese homeostasis in the brain.
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Affiliation(s)
- Brittany L Steimle
- Department of Biochemistry, State University of New York at Buffalo, Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York 14203
| | - Frances M Smith
- Department of Biochemistry, State University of New York at Buffalo, Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York 14203
| | - Daniel J Kosman
- Department of Biochemistry, State University of New York at Buffalo, Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York 14203
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25
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Cegarra L, Colins A, Gerdtzen ZP, Nuñez MT, Salgado JC. Mathematical modeling of the relocation of the divalent metal transporter DMT1 in the intestinal iron absorption process. PLoS One 2019; 14:e0218123. [PMID: 31181103 PMCID: PMC6557526 DOI: 10.1371/journal.pone.0218123] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2019] [Accepted: 05/27/2019] [Indexed: 11/18/2022] Open
Abstract
Iron is essential for the normal development of cellular processes. This metal has a high redox potential that can damage cells and its overload or deficiency is related to several diseases, therefore it is crucial for its absorption to be highly regulated. A fast-response regulatory mechanism has been reported known as mucosal block, which allows to regulate iron absorption after an initial iron challenge. In this mechanism, the internalization of the DMT1 transporters in enterocytes would be a key factor. Two phenomenological models are proposed for the iron absorption process: DMT1's binary switching mechanism model and DMT1's swinging-mechanism model, which represent the absorption mechanism for iron uptake in intestinal cells. The first model considers mutually excluding processes for endocytosis and exocytosis of DMT1. The second model considers a Ball's oscillator to represent the oscillatory behavior of DMT1's internalization. Both models are capable of capturing the kinetics of iron absorption and represent empirical observations, but the DMT1's swinging-mechanism model exhibits a better correlation with experimental data and is able to capture the regulatory phenomenon of mucosal block. The DMT1 swinging-mechanism model is the first phenomenological model reported to effectively represent the complexity of the iron absorption process, as it can predict the behavior of iron absorption fluxes after challenging cells with an initial dose of iron, and the reduction in iron uptake observed as a result of mucosal block after a second iron dose.
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Affiliation(s)
- Layimar Cegarra
- Laboratory of Process Modeling and Distributed Computing, Department of Chemical Engineering, Biotechnology and Materials, University of Chile, Santiago, Chile
- Centre for Biotechnology and Bioengineering, Department of Chemical Engineering, Biotechnology and Materials, University of Chile, Santiago, Chile
| | - Andrea Colins
- Laboratory of Process Modeling and Distributed Computing, Department of Chemical Engineering, Biotechnology and Materials, University of Chile, Santiago, Chile
| | - Ziomara P. Gerdtzen
- Centre for Biotechnology and Bioengineering, Department of Chemical Engineering, Biotechnology and Materials, University of Chile, Santiago, Chile
| | - Marco T. Nuñez
- Iron and Biology of Aging Laboratory, Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile
| | - J. Cristian Salgado
- Laboratory of Process Modeling and Distributed Computing, Department of Chemical Engineering, Biotechnology and Materials, University of Chile, Santiago, Chile
- Centre for Biotechnology and Bioengineering, Department of Chemical Engineering, Biotechnology and Materials, University of Chile, Santiago, Chile
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26
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Abstract
Most cells in the body acquire iron via receptor-mediated endocytosis of transferrin, the circulating iron transport protein. When cellular iron levels are sufficient, the uptake of transferrin decreases to limit further iron assimilation and prevent excessive iron accumulation. In iron overload conditions, such as hereditary hemochromatosis and thalassemia major, unregulated iron entry into the plasma overwhelms the carrying capacity of transferrin, resulting in non-transferrin-bound iron (NTBI), a redox-active, potentially toxic form of iron. Plasma NTBI is rapidly cleared from the circulation primarily by the liver and other organs (e.g., pancreas, heart, and pituitary) where it contributes significantly to tissue iron overload and related pathology. While NTBI is usually not detectable in the plasma of healthy individuals, it does appear to be a normal constituent of brain interstitial fluid and therefore likely serves as an important source of iron for most cell types in the CNS. A growing body of literature indicates that NTBI uptake is mediated by non-transferrin-bound iron transporters such as ZIP14, L-type and T-type calcium channels, DMT1, ZIP8, and TRPC6. This review provides an overview of NTBI uptake by various tissues and cells and summarizes the evidence for and against the roles of individual transporters in this process.
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Affiliation(s)
- Mitchell D Knutson
- Food Science and Human Nutrition Department, University of Florida, Gainesville, FL, USA.
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27
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Bozzi AT, Zimanyi CM, Nicoludis JM, Lee BK, Zhang CH, Gaudet R. Structures in multiple conformations reveal distinct transition metal and proton pathways in an Nramp transporter. eLife 2019; 8:41124. [PMID: 30714568 PMCID: PMC6398981 DOI: 10.7554/elife.41124] [Citation(s) in RCA: 42] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2018] [Accepted: 01/31/2019] [Indexed: 01/03/2023] Open
Abstract
Nramp family transporters—expressed in organisms from bacteria to humans—enable uptake of essential divalent transition metals via an alternating-access mechanism that also involves proton transport. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in multiple conformations to provide a thorough description of the Nramp transport cycle by identifying the key intramolecular rearrangements and changes to the metal coordination sphere. Strikingly, while metal transport requires cycling from outward- to inward-open states, efficient proton transport still occurs in outward-locked (but not inward-locked) DraNramp. We propose a model in which metal and proton enter the transporter via the same external pathway to the binding site, but follow separate routes to the cytoplasm, which could facilitate the co-transport of two cationic species. Our results illustrate the flexibility of the LeuT fold to support a broad range of substrate transport and conformational change mechanisms. Cells use transport proteins embedded in their membrane to acquire many of the nutrients they need to survive and grow. Different transport proteins transport different nutrients; for example, the Nramp transporters move transition metal ions across cell membranes. Nramps are found in a wide range of organisms. Bacteria use them to acquire the metals they need during the course of an infection, and humans rely on Nramps to absorb iron from food. Nramps can also transport hydrogen ions (known as protons). Understanding how the structure of an Nramp transporter changes as it transports metal ions and protons can help researchers to understand how it works. These structures can be studied using a technique called X-ray crystallography, which captures snapshots of the proteins at different stages of their task. Bozzi, Zimanyi et al. used X-ray crystallography to study the structures of an Nramp transporter from the bacterium Deinococcus radiodurans. The results reveal four of the shapes that the Nramp transporter takes on at different stages in its transport process, including the first structure to show an Nramp binding to a metal ion from the outside of the cell. Taken together, the structures suggest a new transport mechanism that has not been seen in previously studied transport proteins with similar structures. An unexpected feature of this mechanism is that Nramps transport metal ions and protons along different pathways. Studying the transport mechanisms used by Nramp transporters will help researchers to understand how cells maintain appropriate levels of metal ions, an important component of human health. The mechanisms of relatively few transport proteins are understood at a structural level, yet many share common origins and have shared characteristics. Understanding how Nramps work could therefore help us to understand how wider classes of transporters work as well.
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Affiliation(s)
- Aaron T Bozzi
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States
| | - Christina M Zimanyi
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States
| | - John M Nicoludis
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States
| | - Brandon K Lee
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States
| | - Casey H Zhang
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States
| | - Rachelle Gaudet
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States
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28
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Landry GM, Furrow E, Holmes HL, Hirata T, Kato A, Williams P, Strohmaier K, Gallo CJR, Chang M, Pandey MK, Jiang H, Bansal A, Franz MC, Montalbetti N, Alexander MP, Cabrero P, Dow JAT, DeGrado TR, Romero MF. Cloning, function, and localization of human, canine, and Drosophila ZIP10 (SLC39A10), a Zn 2+ transporter. Am J Physiol Renal Physiol 2018; 316:F263-F273. [PMID: 30520657 DOI: 10.1152/ajprenal.00573.2017] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Zinc (Zn2+) is the second most abundant trace element, but is considered a micronutrient, as it is a cofactor for many enzymes and transcription factors. Whereas Zn2+ deficiency can cause cognitive immune or metabolic dysfunction and infertility, excess Zn2+ is nephrotoxic. As for other ions and solutes, Zn2+ is moved into and out of cells by specific membrane transporters: ZnT, Zip, and NRAMP/DMT proteins. ZIP10 is reported to be localized at the apical membrane of renal proximal tubules in rats, where it is believed to play a role in Zn2+ import. Renal regulation of Zn2+ is of particular interest in light of growing evidence that Zn2+ may play a role in kidney stone formation. The objective of this study was to show that ZIP10 homologs transport Zn2+, as well as ZIP10, kidney localization across species. We cloned ZIP10 from dog, human, and Drosophila ( CG10006), tested clones for Zn2+ uptake in Xenopus oocytes and localized the protein in renal structures. CG10006, rather than foi (fear-of-intimacy, CG6817) is the primary ZIP10 homolog found in Drosophila Malpighian tubules. The ZIP10 antibody recognizes recombinant dog, human, and Drosophila ZIP10 proteins. Immunohistochemistry reveals that ZIP10 in higher mammals is found not only in the proximal tubule, but also in the collecting duct system. These ZIP10 proteins show Zn2+ transport. Together, these studies reveal ZIP10 kidney localization, a role in renal Zn2+ transport, and indicates that CG10006 is a Drosophila homolog of ZIP10.
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Affiliation(s)
- Greg M Landry
- Physiology and Biomedical Engineering, Mayo Clinic College of Medicine and Science , Rochester, Minnesota.,Nephrology and Hypertension, Mayo Clinic College of Medicine and Science , Rochester, Minnesota.,O'Brien Urology Research Center, Mayo Clinic College of Medicine and Science , Rochester, Minnesota
| | - Eva Furrow
- Veterinary Clinical Sciences, College of Veterinary Medicine, University of Minnesota , St. Paul, Minnesota
| | - Heather L Holmes
- Physiology and Biomedical Engineering, Mayo Clinic College of Medicine and Science , Rochester, Minnesota
| | - Taku Hirata
- Physiology and Biomedical Engineering, Mayo Clinic College of Medicine and Science , Rochester, Minnesota.,Nephrology and Hypertension, Mayo Clinic College of Medicine and Science , Rochester, Minnesota.,O'Brien Urology Research Center, Mayo Clinic College of Medicine and Science , Rochester, Minnesota
| | - Akira Kato
- Physiology and Biomedical Engineering, Mayo Clinic College of Medicine and Science , Rochester, Minnesota.,Center for Biological Resources and Informatics and Department of Biological Sciences, Tokyo Institute of Technology , Yokohama , Japan
| | - Paige Williams
- Physiology and Biomedical Engineering, Mayo Clinic College of Medicine and Science , Rochester, Minnesota.,Nephrology and Hypertension, Mayo Clinic College of Medicine and Science , Rochester, Minnesota.,O'Brien Urology Research Center, Mayo Clinic College of Medicine and Science , Rochester, Minnesota
| | - Käri Strohmaier
- Physiology and Biomedical Engineering, Mayo Clinic College of Medicine and Science , Rochester, Minnesota.,Nephrology and Hypertension, Mayo Clinic College of Medicine and Science , Rochester, Minnesota.,O'Brien Urology Research Center, Mayo Clinic College of Medicine and Science , Rochester, Minnesota
| | - Chris J R Gallo
- Physiology and Biomedical Engineering, Mayo Clinic College of Medicine and Science , Rochester, Minnesota.,O'Brien Urology Research Center, Mayo Clinic College of Medicine and Science , Rochester, Minnesota
| | - Minhwang Chang
- Physiology and Biomedical Engineering, Mayo Clinic College of Medicine and Science , Rochester, Minnesota
| | - Mukesh K Pandey
- Nuclear Medicine, Mayo Clinic College of Medicine and Science , Rochester, Minnesota
| | - Huailei Jiang
- Nuclear Medicine, Mayo Clinic College of Medicine and Science , Rochester, Minnesota
| | - Aditya Bansal
- Nuclear Medicine, Mayo Clinic College of Medicine and Science , Rochester, Minnesota
| | - Marie-Christine Franz
- Physiology and Biomedical Engineering, Mayo Clinic College of Medicine and Science , Rochester, Minnesota
| | - Nicolas Montalbetti
- Physiology and Biomedical Engineering, Mayo Clinic College of Medicine and Science , Rochester, Minnesota
| | - Mariam P Alexander
- Laboratory of Medicine and Pathology, Mayo Clinic College of Medicine and Science , Rochester, Minnesota
| | - Pablo Cabrero
- Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, University of Glasgow , Glasgow , United Kingdom
| | - Julian A T Dow
- Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, University of Glasgow , Glasgow , United Kingdom
| | - Timothy R DeGrado
- Nuclear Medicine, Mayo Clinic College of Medicine and Science , Rochester, Minnesota
| | - Michael F Romero
- Physiology and Biomedical Engineering, Mayo Clinic College of Medicine and Science , Rochester, Minnesota.,Nephrology and Hypertension, Mayo Clinic College of Medicine and Science , Rochester, Minnesota.,O'Brien Urology Research Center, Mayo Clinic College of Medicine and Science , Rochester, Minnesota
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29
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Sherman HG, Jovanovic C, Stolnik S, Baronian K, Downard AJ, Rawson FJ. New Perspectives on Iron Uptake in Eukaryotes. Front Mol Biosci 2018; 5:97. [PMID: 30510932 PMCID: PMC6254016 DOI: 10.3389/fmolb.2018.00097] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2018] [Accepted: 10/23/2018] [Indexed: 12/20/2022] Open
Abstract
All eukaryotic organisms require iron to function. Malfunctions within iron homeostasis have a range of physiological consequences, and can lead to the development of pathological conditions that can result in an excess of non-transferrin bound iron (NTBI). Despite extensive understanding of iron homeostasis, the links between the “macroscopic” transport of iron across biological barriers (cellular membranes) and the chemistry of redox changes that drive these processes still needs elucidating. This review draws conclusions from the current literature, and describes some of the underlying biophysical and biochemical processes that occur in iron homeostasis. By first taking a broad view of iron uptake within the gut and subsequent delivery to tissues, in addition to describing the transferrin and non-transferrin mediated components of these processes, we provide a base of knowledge from which we further explore NTBI uptake. We provide concise up-to-date information of the transplasma electron transport systems (tPMETSs) involved within NTBI uptake, and highlight how these systems are not only involved within NTBI uptake for detoxification but also may play a role within the reduction of metabolic stress through regeneration of intracellular NAD(P)H/NAD(P)+ levels. Furthermore, we illuminate the thermodynamics that governs iron transport, namely the redox potential cascade and electrochemical behavior of key components of the electron transport systems that facilitate the movement of electrons across the plasma membrane to the extracellular compartment. We also take account of kinetic changes that occur to transport iron into the cell, namely membrane dipole change and their consequent effects within membrane structure that act to facilitate transport of ions.
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Affiliation(s)
- Harry G Sherman
- Division of Regenerative Medicine and Cellular Therapies, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom
| | | | - Snow Stolnik
- Division of Molecular Therapeutics and Formulation, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom
| | - Kim Baronian
- School of Biological Sciences, University of Canterbury, Christchurch, New Zealand
| | - Alison J Downard
- MacDiarmid Institute for Advanced Materials and Nanotechnology, School of Physical and Chemical Sciences, University of Canterbury, Christchurch, New Zealand
| | - Frankie J Rawson
- Division of Regenerative Medicine and Cellular Therapies, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom
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30
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Pietak A, Levin M. Bioelectrical control of positional information in development and regeneration: A review of conceptual and computational advances. PROGRESS IN BIOPHYSICS AND MOLECULAR BIOLOGY 2018; 137:52-68. [PMID: 29626560 PMCID: PMC10464501 DOI: 10.1016/j.pbiomolbio.2018.03.008] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/10/2018] [Revised: 03/23/2018] [Accepted: 03/26/2018] [Indexed: 12/16/2022]
Abstract
Positional information describes pre-patterns of morphogenetic substances that alter spatio-temporal gene expression to instruct development of growth and form. A wealth of recent data indicate bioelectrical properties, such as the transmembrane potential (Vmem), are involved as instructive signals in the spatiotemporal regulation of morphogenesis. However, the mechanistic relationships between Vmem and molecular positional information are only beginning to be understood. Recent advances in computational modeling are assisting in the development of comprehensive frameworks for mechanistically understanding how endogenous bioelectricity can guide anatomy in a broad range of systems. Vmem represents an extraordinarily strong electric field (∼1.0 × 106 V/m) active over the thin expanse of the plasma membrane, with the capacity to influence a variety of downstream molecular signaling cascades. Moreover, in multicellular networks, intercellular coupling facilitated by gap junction channels may induce directed, electrodiffusive transport of charged molecules between cells of the network to generate new positional information patterning possibilities and characteristics. Given the demonstrated role of Vmem in morphogenesis, here we review current understanding of how Vmem can integrate with molecular regulatory networks to control single cell state, and the unique properties bioelectricity adds to transport phenomena in gap junction-coupled cell networks to facilitate self-assembly of morphogen gradients and other patterns. Understanding how Vmem integrates with biochemical regulatory networks at the level of a single cell, and mechanisms through which Vmem shapes molecular positional information in multicellular networks, are essential for a deep understanding of body plan control in development, regeneration and disease.
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Affiliation(s)
| | - Michael Levin
- Allen Discovery Center at Tufts, USA; Center for Regenerative and Developmental Biology, Tufts University, Medford, MA, USA
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31
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Zhang Q, Li C, Zhang T, Ge Y, Han X, Sun S, Ding J, Lu M, Hu G. Deletion of Kir6.2/SUR1 potassium channels rescues diminishing of DA neurons via decreasing iron accumulation in PD. Mol Cell Neurosci 2018; 92:164-176. [PMID: 30171894 DOI: 10.1016/j.mcn.2018.08.006] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2018] [Revised: 07/16/2018] [Accepted: 08/25/2018] [Indexed: 11/25/2022] Open
Abstract
ATP-sensitive potassium (K-ATP) channels express in the central nervous system extensively which coupling cell metabolism and cellular electrical activity. K-ATP channels in mature substantia nigra (SN) dopaminergic (DA) neurons are composed of inwardly rectifying potassium channel (Kir) subunit 6.2 and sulfonylurea receptor 1 (SUR1). Our previous study revealed that regulating K-ATP channel exerts the protective effect on DA neurons in a mouse model of Parkinson's disease (PD). However, the detailed mechanism underlying the role of Kir6.2/K-ATP remains unclear. In the present study, we found the deletion of Kir6.2 dramatically alleviated PD-like motor dysfunction of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) PD model. We further found that Kir6.2 knockout selectively restored the reduction of both DA neuronal number and dopamine transmitter level in the nigrostriatal of MPTP-treated PD mice. To gain some understanding on the molecular basis of this effect, we focused on the regulation of Kir6.2 deletion on iron metabolism which is tightly associated with DA neuron damage. We found that Kir6.2 knockout suppressed the excessive iron accumulation in MPTP-treated mouse midbrain and inhibited the upregulation of ferritin light chain (FTL), which is a main intracellular iron storage protein. We probed further and found out that the deletion of Kir6.2 inhibited the excessive production of FTL via IRP-IRE regulatory system, and thereby protecting SN DA neurons against MPTP challenge. Our findings suggest that Kir6.2 plays a crucial role in the pathogenesis of PD and regulating Kir6.2/K-ATP channel may be a promising strategy for PD treatment.
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Affiliation(s)
- Qian Zhang
- Department of Pharmacology, School of Medicine and Life Sciences, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing, Jiangsu 210023, China
| | - Chengwu Li
- Department of Pharmacology, School of Medicine and Life Sciences, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing, Jiangsu 210023, China
| | - Ting Zhang
- Department of Pharmacology, School of Medicine and Life Sciences, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing, Jiangsu 210023, China
| | - Yaping Ge
- Department of Pharmacology, School of Medicine and Life Sciences, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing, Jiangsu 210023, China
| | - Xiaojuan Han
- Department of Pharmacology, School of Medicine and Life Sciences, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing, Jiangsu 210023, China
| | - Sifan Sun
- First Clinic Medical School, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing, Jiangsu 210023, China
| | - Jianhua Ding
- Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University, 101 Longmian Avenue, Nanjing, Jiangsu 211166, China
| | - Ming Lu
- Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University, 101 Longmian Avenue, Nanjing, Jiangsu 211166, China.
| | - Gang Hu
- Department of Pharmacology, School of Medicine and Life Sciences, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing, Jiangsu 210023, China; Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University, 101 Longmian Avenue, Nanjing, Jiangsu 211166, China.
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32
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Ballesteros C, Geary JF, Mackenzie CD, Geary TG. Characterization of Divalent Metal Transporter 1 (DMT1) in Brugia malayi suggests an intestinal-associated pathway for iron absorption. Int J Parasitol Drugs Drug Resist 2018; 8:341-349. [PMID: 29957332 PMCID: PMC6038845 DOI: 10.1016/j.ijpddr.2018.06.003] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2018] [Revised: 06/06/2018] [Accepted: 06/08/2018] [Indexed: 01/12/2023]
Abstract
Lymphatic filariasis and onchocerciasis are neglected parasitic diseases which pose a threat to public health in tropical and sub-tropical regions. Strategies for control and elimination of these diseases by mass drug administration (MDA) campaigns are designed to reduce symptoms of onchocerciasis and transmission of both parasites to eventually eliminate the burden on public health. Drugs used for MDA are predominantly microfilaricidal, and prolonged rounds of treatment are required for eradication. Understanding parasite biology is crucial to unravelling the complex processes involved in host-parasite interactions, disease transmission, parasite immune evasion, and the emergence of drug resistance. In nematode biology, large gaps still exist in our understanding of iron metabolism, iron-dependent processes and their regulation. The acquisition of iron from the host is a crucial determinant of the success of a parasitic infection. Here we identify a filarial ortholog of Divalent Metal Transporter 1 (DMT1), a member of a highly conserved family of NRAMP proteins that play an essential role in the transport of ferrous iron in many species. We cloned and expressed the B. malayi NRAMP ortholog in the iron-deficient fet3fet4 strain of Saccharomyces cerevisiae, performed qPCR to estimate stage-specific expression, and localized expression of this gene by immunohistochemistry. Results from functional iron uptake assays showed that expression of this gene in the iron transport-deficient yeast strain significantly rescued growth in low-iron medium. DMT1 was highly expressed in adult female and male B. malayi and Onchocerca volvulus. Immunolocalization revealed that DMT1 is expressed in the intestinal brush border, lateral chords, and reproductive tissues of males and females, areas also inhabited by Wolbachia. We hypothesize based on our results that DMT1 in B. malayi functions as an iron transporter. The presence of this transporter in the intestine supports the hypothesis that iron acquisition by adult females requires oral ingestion and suggests that the intestine plays a functional role in at least some aspects of nutrient uptake.
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Affiliation(s)
- Cristina Ballesteros
- Institute of Parasitology, McGill University, 21111 Lakeshore Road, Sainte-Anne-de-Bellevue, Quebec, H9X 3V9, Canada
| | - James F Geary
- Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, USA
| | - Charles D Mackenzie
- Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI, 48824, USA
| | - Timothy G Geary
- Institute of Parasitology, McGill University, 21111 Lakeshore Road, Sainte-Anne-de-Bellevue, Quebec, H9X 3V9, Canada.
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33
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Franz MC, Pujol-Giménez J, Montalbetti N, Fernandez-Tenorio M, DeGrado TR, Niggli E, Romero MF, Hediger MA. Reassessment of the Transport Mechanism of the Human Zinc Transporter SLC39A2. Biochemistry 2018; 57:3976-3986. [PMID: 29791142 DOI: 10.1021/acs.biochem.8b00511] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
The human zinc transporter SLC39A2, also known as ZIP2, was shown to mediate zinc transport that could be inhibited at pH <7.0 and stimulated by HCO3-, suggesting a Zn2+/HCO3- cotransport mechanism [Gaither, L. A., and Eide, D. J. (2000) J. Biol. Chem. 275, 5560-5564]. In contrast, recent experiments in our laboratory indicated that the functional activity of ZIP2 increases at acidic pH [Franz, M. C., et al. (2014) J. Biomol. Screening 19, 909-916]. The study presented here was therefore designed to reexamine the findings about the pH dependence and to extend the functional characterization of ZIP2. Our current results show that ZIP2-mediated transport is modulated by extracellular pH but independent of the H+ driving force. Also, in our experiments, ZIP2-mediated transport is not modulated by extracellular HCO3-. Moreover, a high extracellular [K+], which induces depolarization, inhibited ZIP2-mediated transport, indicating that the transport mechanism is voltage-dependent. We also show that ZIP2 mediates the uptake of Cd2+ ( Km ∼ 1.57 μM) in a pH-dependent manner ( KH+ ∼ 66 nM). Cd2+ transport is inhibited by extracellular [Zn2+] (IC50 ∼ 0.32 μM), [Cu2+] (IC50 ∼ 1.81 μM), and to a lesser extent [Co2+], but not by [Mn2+] or [Ba2+]. Fe2+ is not transported by ZIP2. Accordingly, the substrate selectivity of ZIP2 decreases in the following order: Zn2+ > Cd2+ ≥ Cu2+ > Co2+. Altogether, we propose that ZIP2 is a facilitated divalent metal ion transporter that can be modulated by extracellular pH and membrane potential. Given that ZIP2 expression has been reported in acidic environments [Desouki, M. M., et al. (2007) Mol. Cancer 6, 37; Inoue, Y., et al. (2014) J. Biol. Chem. 289, 21451-21462; Tao, Y. T., et al. (2013) Mol. Biol. Rep. 40, 4979-4984], we suggest that the herein described H+-mediated regulatory mechanism might be important for determining the velocity and direction of the transport process.
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Affiliation(s)
- Marie C Franz
- University of Bern , Institute of Biochemistry and Molecular Medicine, and National Center of Competence in Research, NCCR TransCure , Bühlstrasse 28 , 3012 Bern , Switzerland
| | - Jonai Pujol-Giménez
- University of Bern , Institute of Biochemistry and Molecular Medicine, and National Center of Competence in Research, NCCR TransCure , Bühlstrasse 28 , 3012 Bern , Switzerland
| | - Nicolas Montalbetti
- University of Bern , Institute of Biochemistry and Molecular Medicine, and National Center of Competence in Research, NCCR TransCure , Bühlstrasse 28 , 3012 Bern , Switzerland
| | | | - Timothy R DeGrado
- Department of Physiology and Biomedical Engineering , Mayo Clinic College of Medicine and Science , Rochester , Minnesota 55905 , United States
| | - Ernst Niggli
- University of Bern , Department of Physiology , Buehlplatz 5 , 3012 Bern , Switzerland
| | - Michael F Romero
- Department of Physiology and Biomedical Engineering , Mayo Clinic College of Medicine and Science , Rochester , Minnesota 55905 , United States
| | - Matthias A Hediger
- University of Bern , Institute of Biochemistry and Molecular Medicine, and National Center of Competence in Research, NCCR TransCure , Bühlstrasse 28 , 3012 Bern , Switzerland
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A Functional Study Identifying Critical Residues Involving Metal Transport Activity and Selectivity in Natural Resistance-Associated Macrophage Protein 3 in Arabidopsis thaliana. Int J Mol Sci 2018; 19:ijms19051430. [PMID: 29748478 PMCID: PMC5983769 DOI: 10.3390/ijms19051430] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2018] [Revised: 04/24/2018] [Accepted: 04/28/2018] [Indexed: 11/16/2022] Open
Abstract
Arabidopsis thaliana natural resistance-associated macrophage protein 3 (AtNRAMP3) is involved in the transport of cadmium (Cd), iron (Fe), and manganese (Mn). Here, we present a structure-function analysis of AtNRAMP3 based on site-directed mutagenesis and metal toxicity growth assays involving yeast mutants, combined with three-dimensional (3D) structure modeling based on the crystal structure of the Eremococcus coleocola NRAMP family transporter, EcoDMT. We demonstrated that two conservative sites, D72 and N75, are essential for the transport activity. The M248A mutation resulted in a decrease in Cd sensitivity, while maintaining Mn transport. The mutation involving G61 caused a significant impairment of Fe and Mn transport, thereby indicating the importance of the conserved residue for proper protein function. The mutation involving G171 disrupted Fe transport activity but not that of Mn and Cd, suggesting that G171 is essential to metal binding and selectivity. Two residues, E194 and R262, may play an important role in stabilizing outward-facing conformation, which is essential for transport activity. Deletion assays indicated that the N-terminus is necessary for the function of AtNRAMP3. The findings of the present study revealed the structure-function relationship of AtNRAMP3 and metal transport activity and selectivity, which may possibly be applied to other plant NRAMP proteins.
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Bai X, Moraes TF, Reithmeier RAF. Structural biology of solute carrier (SLC) membrane transport proteins. Mol Membr Biol 2018; 34:1-32. [PMID: 29651895 DOI: 10.1080/09687688.2018.1448123] [Citation(s) in RCA: 102] [Impact Index Per Article: 14.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
The human solute carriers (SLCs) comprise over 400 different transporters, organized into 65 families ( http://slc.bioparadigms.org/ ) based on their sequence homology and transport function. SLCs are responsible for transporting extraordinarily diverse solutes across biological membranes, including inorganic ions, amino acids, lipids, sugars, neurotransmitters and drugs. Most of these membrane proteins function as coupled symporters (co-transporters) utilizing downhill ion (H+ or Na+) gradients as the driving force for the transport of substrate against its concentration gradient into cells. Other members work as antiporters (exchangers) that typically contain a single substrate-binding site with an alternating access mode of transport, while a few members exhibit channel-like properties. Dysfunction of SLCs is correlated with numerous human diseases and therefore they are potential therapeutic drug targets. In this review, we identified all of the SLC crystal structures that have been determined, most of which are from prokaryotic species. We further sorted all the SLC structures into four main groups with different protein folds and further discuss the well-characterized MFS (major facilitator superfamily) and LeuT (leucine transporter) folds. This review provides a systematic analysis of the structure, molecular basis of substrate recognition and mechanism of action in different SLC family members.
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Affiliation(s)
- Xiaoyun Bai
- a Department of Biochemistry , University of Toronto , Toronto , Canada
| | - Trevor F Moraes
- a Department of Biochemistry , University of Toronto , Toronto , Canada
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36
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Sherman HG, Jovanovic C, Stolnik S, Rawson FJ. Electrochemical System for the Study of Trans-Plasma Membrane Electron Transport in Whole Eukaryotic Cells. Anal Chem 2018; 90:2780-2786. [DOI: 10.1021/acs.analchem.7b04853] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Affiliation(s)
- Harry G. Sherman
- Division
of Regenerative Medicine and Cellular Therapies, School of Pharmacy, University of Nottingham, Nottingham, NG7 2RD, United Kingdom
| | | | - Snow Stolnik
- Division
of Molecular Therapeutics and Formulation, School of Pharmacy, University of Nottingham, Nottingham, NG7 2RD, United Kingdom
| | - Frankie J. Rawson
- Division
of Regenerative Medicine and Cellular Therapies, School of Pharmacy, University of Nottingham, Nottingham, NG7 2RD, United Kingdom
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37
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A role for divalent metal transporter (DMT1) in mitochondrial uptake of iron and manganese. Sci Rep 2018; 8:211. [PMID: 29317744 PMCID: PMC5760699 DOI: 10.1038/s41598-017-18584-4] [Citation(s) in RCA: 100] [Impact Index Per Article: 14.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2017] [Accepted: 12/13/2017] [Indexed: 01/01/2023] Open
Abstract
Much of iron and manganese metabolism occurs in mitochondria. Uptake of redox-active iron must be tightly controlled, but little is known about how metal ions enter mitochondria. Recently, we established that the divalent metal transporter 1 (DMT1) is present in the outer mitochondrial membrane (OMM). Therefore we asked if it mediates Fe2+ and Mn2+ influx. Mitochondria were isolated from HEK293 cells permanently transfected with inducible rat DMT1 isoform 1 A/+IRE (HEK293-rDMT1). Fe2+-induced quenching of the dye PhenGreen™SK (PGSK) occurred in two phases, one of which reflected OMM DMT1 with stronger Fe2+ uptake after DMT1 overexpression. DMT1-specific quenching showed an apparent affinity of ~1.5 µM for Fe2+and was blocked by the DMT1 inhibitor CISMBI. Fe2+ influx reflected an imposed proton gradient, a response that was also observed in purified rat kidney cortex (rKC) mitochondria. Non-heme Fe accumulation assayed by ICPOES and stable 57Fe isotope incorporation by ICPMS were increased in HEK293-rDMT1 mitochondria. HEK293-rDMT1 mitochondria displayed higher 59Fe2+ and 54Mn2+ uptake relative to controls with 54Mn2+ uptake blocked by the DMT1 inhibitor XEN602. Such transport was defective in rKC mitochondria with the Belgrade (G185R) mutation. Thus, these results support a role for DMT1 in mitochondrial Fe2+ and Mn2+ acquisition.
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38
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Pietak A, Levin M. Bioelectric gene and reaction networks: computational modelling of genetic, biochemical and bioelectrical dynamics in pattern regulation. J R Soc Interface 2017; 14:20170425. [PMID: 28954851 PMCID: PMC5636277 DOI: 10.1098/rsif.2017.0425] [Citation(s) in RCA: 56] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2017] [Accepted: 08/31/2017] [Indexed: 12/17/2022] Open
Abstract
Gene regulatory networks (GRNs) describe interactions between gene products and transcription factors that control gene expression. In combination with reaction-diffusion models, GRNs have enhanced comprehension of biological pattern formation. However, although it is well known that biological systems exploit an interplay of genetic and physical mechanisms, instructive factors such as transmembrane potential (Vmem) have not been integrated into full GRN models. Here we extend regulatory networks to include bioelectric signalling, developing a novel synthesis: the bioelectricity-integrated gene and reaction (BIGR) network. Using in silico simulations, we highlight the capacity for Vmem to alter steady-state concentrations of key signalling molecules inside and out of cells. We characterize fundamental feedbacks where Vmem both controls, and is in turn regulated by, biochemical signals and thereby demonstrate Vmem homeostatic control, Vmem memory and Vmem controlled state switching. BIGR networks demonstrating hysteresis are identified as a mechanisms through which more complex patterns of stable Vmem spots and stripes, along with correlated concentration patterns, can spontaneously emerge. As further proof of principle, we present and analyse a BIGR network model that mechanistically explains key aspects of the remarkable regenerative powers of creatures such as planarian flatworms. The functional properties of BIGR networks generate the first testable, quantitative hypotheses for biophysical mechanisms underlying the stability and adaptive regulation of anatomical bioelectric pattern.
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Affiliation(s)
- Alexis Pietak
- Allen Discovery Center, Tufts University, Medford, MA, USA
| | - Michael Levin
- Allen Discovery Center, Tufts University, Medford, MA, USA
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Bakouh N, Bellanca S, Nyboer B, Moliner Cubel S, Karim Z, Sanchez CP, Stein WD, Planelles G, Lanzer M. Iron is a substrate of the Plasmodium falciparum chloroquine resistance transporter PfCRT in Xenopus oocytes. J Biol Chem 2017; 292:16109-16121. [PMID: 28768767 DOI: 10.1074/jbc.m117.805200] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2017] [Revised: 08/01/2017] [Indexed: 01/01/2023] Open
Abstract
The chloroquine resistance transporter of the human malaria parasite Plasmodium falciparum, PfCRT, is an important determinant of resistance to several quinoline and quinoline-like antimalarial drugs. PfCRT also plays an essential role in the physiology of the parasite during development inside erythrocytes. However, the function of this transporter besides its role in drug resistance is still unclear. Using electrophysiological and flux experiments conducted on PfCRT-expressing Xenopus laevis oocytes, we show here that both wild-type PfCRT and a PfCRT variant associated with chloroquine resistance transport both ferrous and ferric iron, albeit with different kinetics. In particular, we found that the ability to transport ferrous iron is reduced by the specific polymorphisms acquired by the PfCRT variant as a result of chloroquine selection. We further show that iron and chloroquine transport via PfCRT is electrogenic. If these findings in the Xenopus model extend to P. falciparum in vivo, our data suggest that PfCRT might play a role in iron homeostasis, which is essential for the parasite's development in erythrocytes.
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Affiliation(s)
- Naziha Bakouh
- From INSERM, Centre de Recherche des Cordeliers, Unité 1138, CNRS ERL8228, Université Pierre et Marie Curie and Université Paris-Descartes, Paris 75006, France
| | - Sebastiano Bellanca
- the Center of Infectious Diseases, Parasitology, Heidelberg University Hospital, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
| | - Britta Nyboer
- the Center of Infectious Diseases, Parasitology, Heidelberg University Hospital, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
| | - Sonia Moliner Cubel
- the Center of Infectious Diseases, Parasitology, Heidelberg University Hospital, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
| | - Zoubida Karim
- INSERM, UMR1149, CNRS ERL 8252, Université Paris Diderot Paris 75890, France, and
| | - Cecilia P Sanchez
- the Center of Infectious Diseases, Parasitology, Heidelberg University Hospital, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
| | - Wilfred D Stein
- Biological Chemistry, Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel
| | - Gabrielle Planelles
- From INSERM, Centre de Recherche des Cordeliers, Unité 1138, CNRS ERL8228, Université Pierre et Marie Curie and Université Paris-Descartes, Paris 75006, France,
| | - Michael Lanzer
- the Center of Infectious Diseases, Parasitology, Heidelberg University Hospital, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany,
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40
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Pujol-Giménez J, Hediger MA, Gyimesi G. A novel proton transfer mechanism in the SLC11 family of divalent metal ion transporters. Sci Rep 2017; 7:6194. [PMID: 28754960 PMCID: PMC5533754 DOI: 10.1038/s41598-017-06446-y] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2017] [Accepted: 06/13/2017] [Indexed: 12/17/2022] Open
Abstract
In humans, the H+-coupled Fe2+ transporter DMT1 (SLC11A2) is essential for proper maintenance of iron homeostasis. While X-ray diffraction has recently unveiled the structure of the bacterial homologue ScaDMT as a LeuT-fold transporter, the exact mechanism of H+-cotransport has remained elusive. Here, we used a combination of molecular dynamics simulations, in silico pK a calculations and site-directed mutagenesis, followed by rigorous functional analysis, to discover two previously uncharacterized functionally relevant residues in hDMT1 that contribute to H+-coupling. E193 plays a central role in proton binding, thereby affecting transport properties and electrogenicity, while N472 likely coordinates the metal ion, securing an optimally "closed" state of the protein. Our molecular dynamics simulations provide insight into how H+-translocation through E193 is allosterically linked to intracellular gating, establishing a novel transport mechanism distinct from that of other H+-coupled transporters.
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Affiliation(s)
- Jonai Pujol-Giménez
- Institute of Biochemistry and Molecular Medicine and National Center of Competence in Research, NCCR TransCure, University of Bern, Bern, Switzerland
| | - Matthias A Hediger
- Institute of Biochemistry and Molecular Medicine and National Center of Competence in Research, NCCR TransCure, University of Bern, Bern, Switzerland.
| | - Gergely Gyimesi
- Institute of Biochemistry and Molecular Medicine and National Center of Competence in Research, NCCR TransCure, University of Bern, Bern, Switzerland.
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41
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Colins A, Gerdtzen ZP, Nuñez MT, Salgado JC. Mathematical Modeling of Intestinal Iron Absorption Using Genetic Programming. PLoS One 2017; 12:e0169601. [PMID: 28072870 PMCID: PMC5225013 DOI: 10.1371/journal.pone.0169601] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2016] [Accepted: 12/18/2016] [Indexed: 01/08/2023] Open
Abstract
Iron is a trace metal, key for the development of living organisms. Its absorption process is complex and highly regulated at the transcriptional, translational and systemic levels. Recently, the internalization of the DMT1 transporter has been proposed as an additional regulatory mechanism at the intestinal level, associated to the mucosal block phenomenon. The short-term effect of iron exposure in apical uptake and initial absorption rates was studied in Caco-2 cells at different apical iron concentrations, using both an experimental approach and a mathematical modeling framework. This is the first report of short-term studies for this system. A non-linear behavior in the apical uptake dynamics was observed, which does not follow the classic saturation dynamics of traditional biochemical models. We propose a method for developing mathematical models for complex systems, based on a genetic programming algorithm. The algorithm is aimed at obtaining models with a high predictive capacity, and considers an additional parameter fitting stage and an additional Jackknife stage for estimating the generalization error. We developed a model for the iron uptake system with a higher predictive capacity than classic biochemical models. This was observed both with the apical uptake dataset used for generating the model and with an independent initial rates dataset used to test the predictive capacity of the model. The model obtained is a function of time and the initial apical iron concentration, with a linear component that captures the global tendency of the system, and a non-linear component that can be associated to the movement of DMT1 transporters. The model presented in this paper allows the detailed analysis, interpretation of experimental data, and identification of key relevant components for this complex biological process. This general method holds great potential for application to the elucidation of biological mechanisms and their key components in other complex systems.
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Affiliation(s)
- Andrea Colins
- Laboratory of Process Modeling and Distributed Computing, Department of Chemical Engineering and Biotechnology, University of Chile, Santiago, Chile
| | - Ziomara P. Gerdtzen
- Centre for Biotechnology and Bioengineering, Department of Chemical Engineering and Biotechnology, University of Chile, Santiago, Chile
| | - Marco T. Nuñez
- Iron and Biology of Aging Laboratory, Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile
| | - J. Cristian Salgado
- Laboratory of Process Modeling and Distributed Computing, Department of Chemical Engineering and Biotechnology, University of Chile, Santiago, Chile
- Centre for Biotechnology and Bioengineering, Department of Chemical Engineering and Biotechnology, University of Chile, Santiago, Chile
- * E-mail:
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42
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Ehrnstorfer IA, Manatschal C, Arnold FM, Laederach J, Dutzler R. Structural and mechanistic basis of proton-coupled metal ion transport in the SLC11/NRAMP family. Nat Commun 2017; 8:14033. [PMID: 28059071 PMCID: PMC5230734 DOI: 10.1038/ncomms14033] [Citation(s) in RCA: 55] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2016] [Accepted: 11/23/2016] [Indexed: 02/01/2023] Open
Abstract
Secondary active transporters of the SLC11/NRAMP family catalyse the uptake of iron
and manganese into cells. These proteins are highly conserved across all kingdoms of
life and thus likely share a common transport mechanism. Here we describe the
structural and functional properties of the prokaryotic SLC11 transporter EcoDMT.
Its crystal structure reveals a previously unknown outward-facing state of the
protein family. In proteoliposomes EcoDMT mediates proton-coupled uptake of
manganese at low micromolar concentrations. Mutants of residues in the
transition-metal ion-binding site severely affect transport, whereas a mutation of a
conserved histidine located near this site results in metal ion transport that
appears uncoupled to proton transport. Combined with previous results, our study
defines the conformational changes underlying transition-metal ion transport in the
SLC11 family and it provides molecular insight to its coupling to protons. Cellular uptake of transition metal ions is mediated by members of the SLC11/NRAMP
family. Here the authors determine the structural and functional properties of EcoDMT, a
bacterial SLC11 transporter, gathering molecular insight into its transport mechanism
and proton coupling process.
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Affiliation(s)
- Ines A Ehrnstorfer
- Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
| | - Cristina Manatschal
- Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
| | - Fabian M Arnold
- Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
| | - Juerg Laederach
- Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
| | - Raimund Dutzler
- Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
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Thévenod F, Wolff NA. Iron transport in the kidney: implications for physiology and cadmium nephrotoxicity. Metallomics 2016; 8:17-42. [PMID: 26485516 DOI: 10.1039/c5mt00215j] [Citation(s) in RCA: 62] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The kidney has recently emerged as an organ with a significant role in systemic iron (Fe) homeostasis. Substantial amounts of Fe are filtered by the kidney, which have to be reabsorbed to prevent Fe deficiency. Accordingly Fe transporters and receptors for protein-bound Fe are expressed in the nephron that may also function as entry pathways for toxic metals, such as cadmium (Cd), by way of "ionic and molecular mimicry". Similarities, but also differences in handling of Cd by these transport routes offer rationales for the propensity of the kidney to develop Cd toxicity. This critical review provides a comprehensive update on Fe transport by the kidney and its relevance for physiology and Cd nephrotoxicity. Based on quantitative considerations, we have also estimated the in vivo relevance of the described transport pathways for physiology and toxicology. Under physiological conditions all segments of the kidney tubules are likely to utilize Fe for cellular Fe requiring processes for metabolic purposes and also to contribute to reabsorption of free and bound forms of Fe into the circulation. But Cd entering tubule cells disrupts metabolic pathways and is unable to exit. Furthermore, our quantitative analyses contest established models linking chronic Cd nephrotoxicity to proximal tubular uptake of metallothionein-bound Cd. Hence, Fe transport by the kidney may be beneficial by preventing losses from the body. But increased uptake of Fe or Cd that cannot exit tubule cells may lead to kidney injury, and Fe deficiency may facilitate renal Cd uptake.
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Affiliation(s)
- Frank Thévenod
- Institute of Physiology, Pathophysiology & Toxicology, Center for Biomedical Training and Research (ZBAF), University of Witten/Herdecke, Stockumer Str. 12, 58453 Witten, Germany.
| | - Natascha A Wolff
- Institute of Physiology, Pathophysiology & Toxicology, Center for Biomedical Training and Research (ZBAF), University of Witten/Herdecke, Stockumer Str. 12, 58453 Witten, Germany.
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44
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Du X, Xu H, Shi L, Jiang Z, Song N, Jiang H, Xie J. Activation of ATP-sensitive potassium channels enhances DMT1-mediated iron uptake in SK-N-SH cells in vitro. Sci Rep 2016; 6:33674. [PMID: 27646472 PMCID: PMC5028757 DOI: 10.1038/srep33674] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2016] [Accepted: 08/30/2016] [Indexed: 12/13/2022] Open
Abstract
Iron importer divalent metal transporter 1 (DMT1) plays a crucial role in the nigal iron accumulation in Parkinson’s disease (PD). Membrane hyperpolarization is one of the factors that could affect its iron transport function. Besides iron, selective activation of the ATP-sensitive potassium (KATP) channels also contributes to the vulnerability of dopaminergic neurons in PD. Interestingly, activation of KATP channels could induce membrane hyperpolarization. Therefore, it is of vital importance to study the effects of activation of KATP channels on DMT1-mediated iron uptake function. In the present study, activation of KATP channels by diazoxide resulted in the hyperpolarization of the membrane potential and increased DMT1-mediated iron uptake in SK-N-SH cells. This led to an increase in intracellular iron levels and a subsequent decrease in the mitochondrial membrane potential and an increase in ROS production. Delayed inactivation of the Fe2+-evoked currents by diazoxide was recorded by patch clamp in HEK293 cells, which demonstrated that diazoxide could prolonged DMT1-facilitated iron transport. While inhibition of KATP channels by glibenclamide could block ferrous iron influx and the subsequent cell damage. Overexpression of Kir6.2/SUR1 resulted in an increase in iron influx and intracellular iron levels, which was markedly increased after diazoxide treatment.
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Affiliation(s)
- Xixun Du
- Collaborative Innovation Center for Brain Science, Department of Physiology, Shandong Provincial Collaborative Innovation Center for Neurodegenerative Disorders, Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University, Qingdao, 266071, China
| | - Huamin Xu
- Collaborative Innovation Center for Brain Science, Department of Physiology, Shandong Provincial Collaborative Innovation Center for Neurodegenerative Disorders, Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University, Qingdao, 266071, China
| | - Limin Shi
- Collaborative Innovation Center for Brain Science, Department of Physiology, Shandong Provincial Collaborative Innovation Center for Neurodegenerative Disorders, Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University, Qingdao, 266071, China
| | - Zhifeng Jiang
- Collaborative Innovation Center for Brain Science, Department of Physiology, Shandong Provincial Collaborative Innovation Center for Neurodegenerative Disorders, Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University, Qingdao, 266071, China
| | - Ning Song
- Collaborative Innovation Center for Brain Science, Department of Physiology, Shandong Provincial Collaborative Innovation Center for Neurodegenerative Disorders, Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University, Qingdao, 266071, China
| | - Hong Jiang
- Collaborative Innovation Center for Brain Science, Department of Physiology, Shandong Provincial Collaborative Innovation Center for Neurodegenerative Disorders, Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University, Qingdao, 266071, China
| | - Junxia Xie
- Collaborative Innovation Center for Brain Science, Department of Physiology, Shandong Provincial Collaborative Innovation Center for Neurodegenerative Disorders, Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University, Qingdao, 266071, China
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Shawki A, Engevik MA, Kim RS, Knight PB, Baik RA, Anthony SR, Worrell RT, Shull GE, Mackenzie B. Intestinal brush-border Na+/H+ exchanger-3 drives H+-coupled iron absorption in the mouse. Am J Physiol Gastrointest Liver Physiol 2016; 311:G423-30. [PMID: 27390324 PMCID: PMC5076011 DOI: 10.1152/ajpgi.00167.2016] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/26/2016] [Accepted: 06/29/2016] [Indexed: 01/31/2023]
Abstract
Divalent metal-ion transporter-1 (DMT1), the principal mechanism by which nonheme iron is taken up at the intestinal brush border, is energized by the H(+)-electrochemical potential gradient. The provenance of the H(+) gradient in vivo is unknown, so we have explored a role for brush-border Na(+)/H(+) exchanger (NHE) isoforms by examining iron homeostasis and intestinal iron handling in mice lacking NHE2 or NHE3. We observed modestly depleted liver iron stores in NHE2-null (NHE2(-/-)) mice stressed on a low-iron diet but no change in hematological or blood iron variables or the expression of genes associated with iron metabolism compared with wild-type mice. Ablation of NHE3 strongly depleted liver iron stores, regardless of diet. We observed decreases in blood iron variables but no overt anemia in NHE3-null (NHE3(-/-)) mice on a low-iron diet. Intestinal expression of DMT1, the apical surface ferrireductase cytochrome b reductase-1, and the basolateral iron exporter ferroportin was upregulated in NHE3(-/-) mice, and expression of liver Hamp1 (hepcidin) was suppressed compared with wild-type mice. Absorption of (59)Fe from an oral dose was substantially impaired in NHE3(-/-) compared with wild-type mice. Our data point to an important role for NHE3 in generating the H(+) gradient that drives DMT1-mediated iron uptake at the intestinal brush border.
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Affiliation(s)
- Ali Shawki
- Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio; Systems Biology and Physiology Program, University of Cincinnati College of Medicine, Cincinnati, Ohio; and
| | - Melinda A Engevik
- Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio; Systems Biology and Physiology Program, University of Cincinnati College of Medicine, Cincinnati, Ohio; and
| | - Robert S Kim
- Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio
| | - Patrick B Knight
- Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio
| | - Rusty A Baik
- Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio
| | - Sarah R Anthony
- Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio
| | - Roger T Worrell
- Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio; Systems Biology and Physiology Program, University of Cincinnati College of Medicine, Cincinnati, Ohio; and
| | - Gary E Shull
- Systems Biology and Physiology Program, University of Cincinnati College of Medicine, Cincinnati, Ohio; and Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio
| | - Bryan Mackenzie
- Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio; Systems Biology and Physiology Program, University of Cincinnati College of Medicine, Cincinnati, Ohio; and
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White RS, Bhattacharya AK, Chen Y, Byrd M, McMullen MF, Siegel SJ, Carlson GC, Kim SF. Lysosomal iron modulates NMDA receptor-mediated excitation via small GTPase, Dexras1. Mol Brain 2016; 9:38. [PMID: 27080392 PMCID: PMC4832449 DOI: 10.1186/s13041-016-0220-8] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2015] [Accepted: 04/08/2016] [Indexed: 12/23/2022] Open
Abstract
Background Activation of NMDA receptors can induce iron movement into neurons by the small GTPase Dexras1 via the divalent metal transporter 1 (DMT1). This pathway under pathological conditions such as NMDA excitotoxicity contributes to metal-catalyzed reactive oxygen species (ROS) generation and neuronal cell death, and yet its physiological role is not well understood. Results We found that genetic and pharmacological ablation of this neuronal iron pathway in the mice increased glutamatergic transmission. Voltage sensitive dye imaging of hippocampal slices and whole-cell patch clamping of synaptic currents, indicated that the increase in excitability was due to synaptic modification of NMDA receptor activity via modulation of the PKC/Src/NR2A pathway. Moreover, we identified that lysosomal iron serves as a main source for intracellular iron signaling modulating glutamatergic excitability. Conclusions Our data indicates that intracellular iron is dynamically regulated in the neurons and robustly modulate synaptic excitability under physiological condition. Since NMDA receptors play a central role in synaptic neurophysiology, plasticity, neuronal homeostasis, neurodevelopment as well as in the neurobiology of many diseases, endogenous iron is therefore likely to have functional relevance to each of these areas.
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Affiliation(s)
- Rachel S White
- Department of Psychiatry, Center for Neurobiology and Behavior, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA
| | - Anup K Bhattacharya
- Department of Psychiatry, Center for Neurobiology and Behavior, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA
| | - Yong Chen
- Department of Psychiatry, Center for Neurobiology and Behavior, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA
| | - Madeleine Byrd
- Department of Psychiatry, Center for Neurobiology and Behavior, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA
| | - Mary F McMullen
- Department of Psychiatry, Center for Neurobiology and Behavior, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA
| | - Steven J Siegel
- Department of Psychiatry, Center for Neurobiology and Behavior, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA
| | - Gregory C Carlson
- Department of Psychiatry, Center for Neurobiology and Behavior, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA
| | - Sangwon F Kim
- Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine at the University of Pennsylvania, 125 S 31st, TRL RM 2207, Philadelphia, PA, 19104, USA.
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Iron overload of human colon adenocarcinoma cells studied by synchrotron-based X-ray techniques. J Biol Inorg Chem 2016; 21:241-9. [DOI: 10.1007/s00775-015-1331-x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2015] [Accepted: 12/30/2015] [Indexed: 01/05/2023]
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Shawki A, Anthony SR, Nose Y, Engevik MA, Niespodzany EJ, Barrientos T, Öhrvik H, Worrell RT, Thiele DJ, Mackenzie B. Intestinal DMT1 is critical for iron absorption in the mouse but is not required for the absorption of copper or manganese. Am J Physiol Gastrointest Liver Physiol 2015; 309:G635-47. [PMID: 26294671 PMCID: PMC4609933 DOI: 10.1152/ajpgi.00160.2015] [Citation(s) in RCA: 105] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/22/2015] [Accepted: 08/18/2015] [Indexed: 01/31/2023]
Abstract
Divalent metal-ion transporter-1 (DMT1) is a widely expressed iron-preferring membrane-transport protein that serves a critical role in erythroid iron utilization. We have investigated its role in intestinal metal absorption by studying a mouse model lacking intestinal DMT1 (i.e., DMT1(int/int)). DMT1(int/int) mice exhibited a profound hypochromic-microcytic anemia, splenomegaly, and cardiomegaly. That the anemia was due to iron deficiency was demonstrated by the following observations in DMT1(int/int) mice: 1) blood iron and tissue nonheme-iron stores were depleted; 2) mRNA expression of liver hepcidin (Hamp1) was depressed; and 3) intraperitoneal iron injection corrected the anemia, and reversed the changes in blood iron, nonheme-iron stores, and hepcidin expression levels. We observed decreased total iron content in multiple tissues from DMT1(int/int) mice compared with DMT1(+/+) mice but no meaningful change in copper, manganese, or zinc. DMT1(int/int) mice absorbed (64)Cu and (54)Mn from an intragastric dose to the same extent as did DMT1(+/+) mice but the absorption of (59)Fe was virtually abolished in DMT1(int/int) mice. This study reveals a critical function for DMT1 in intestinal nonheme-iron absorption for normal growth and development. Further, this work demonstrates that intestinal DMT1 is not required for the intestinal transport of copper, manganese, or zinc.
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Affiliation(s)
- Ali Shawki
- 1Department of Molecular & Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio; ,2Systems Biology & Physiology Program, University of Cincinnati College of Medicine, Cincinnati, Ohio;
| | - Sarah R. Anthony
- 1Department of Molecular & Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio;
| | - Yasuhiro Nose
- 3Department of Pharmacology & Cancer Biology, Duke University Medical Center, Durham, North Carolina;
| | - Melinda A. Engevik
- 1Department of Molecular & Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio; ,2Systems Biology & Physiology Program, University of Cincinnati College of Medicine, Cincinnati, Ohio;
| | - Eric J. Niespodzany
- 1Department of Molecular & Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio;
| | - Tomasa Barrientos
- 3Department of Pharmacology & Cancer Biology, Duke University Medical Center, Durham, North Carolina;
| | - Helena Öhrvik
- 3Department of Pharmacology & Cancer Biology, Duke University Medical Center, Durham, North Carolina; ,4Department of Medical Biochemistry & Microbiology, Uppsala University, Uppsala, Sweden; and
| | - Roger T. Worrell
- 1Department of Molecular & Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio; ,2Systems Biology & Physiology Program, University of Cincinnati College of Medicine, Cincinnati, Ohio;
| | - Dennis J. Thiele
- 3Department of Pharmacology & Cancer Biology, Duke University Medical Center, Durham, North Carolina; ,5Department of Biochemistry, Duke University Medical Center, Durham, North Carolina
| | - Bryan Mackenzie
- Department of Molecular & Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio; Systems Biology & Physiology Program, University of Cincinnati College of Medicine, Cincinnati, Ohio;
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Wolff NA, Garrick LM, Zhao L, Garrick MD, Thévenod F. Mitochondria represent another locale for the divalent metal transporter 1 (DMT1). Channels (Austin) 2015; 8:458-66. [PMID: 25483589 DOI: 10.4161/19336950.2014.956564] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
The divalent metal transporter (DMT1) is well known for its roles in duodenal iron absorption across the apical enterocyte membrane, in iron efflux from the endosome during transferrin-dependent cellular iron acquisition, as well as in uptake of non-transferrin bound iron in many cells. Recently, using multiple approaches, we have obtained evidence that the mitochondrial outer membrane is another subcellular locale of DMT1 expression. While iron is of vital importance for mitochondrial energy metabolism, its delivery is likely to be tightly controlled due to iron's damaging redox properties. Here we provide additional support for a role of DMT1 in mitochondrial iron acquisition by immunofluorescence colocalization with mitochondrial markers in cells and isolated mitochondria, as well as flow cytometric quantification of DMT1-positive mitochondria from an inducible expression system. Physiological consequences of mitochondrial DMT1 expression are discussed also in consideration of other DMT1 substrates, such as manganese, relevant to mitochondrial antioxidant defense.
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Key Words
- AIF, apoptosis-inducing factor
- BSA, bovine serum albumin
- CHO, Chinese hamster ovary
- COXII, cytochrome C oxidase subunit II
- DMT1, divalent metal transporter 1
- HEK293, human embryonic kidney cells
- IRE, iron responsive element
- Lamp1, lysosome-associated membrane protein 1
- MRB, Mitochondrial Resuspending Buffer
- OMM, outer mitochondrial membrane
- PBS, phosphate-buffered saline
- Tf, transferrin
- Tom6/Tom20, translocase of the outer mitochondrial membrane 6 kDa subunit homolog/20 kDa subunit, respectively
- VDAC1, voltage-dependent anion-selective channel protein 1
- divalent metal transporter 1 (DMT1)
- flow cytometry
- immunofluorescence microscopy
- iron transport
- mitochondrial outer membrane
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Affiliation(s)
- Natascha A Wolff
- a Institute of Physiology; Pathophysiology & Toxicology ; University of Witten/Herdecke ; Witten , Germany
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Buracco S, Peracino B, Cinquetti R, Signoretto E, Vollero A, Imperiali F, Castagna M, Bossi E, Bozzaro S. Dictyostelium Nramp1, which is structurally and functionally similar to mammalian DMT1 transporter, mediates phagosomal iron efflux. J Cell Sci 2015; 128:3304-16. [PMID: 26208637 PMCID: PMC4582194 DOI: 10.1242/jcs.173153] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2015] [Accepted: 07/21/2015] [Indexed: 01/01/2023] Open
Abstract
The Nramp (Slc11) protein family is widespread in bacteria and eukaryotes, and mediates transport of divalent metals across cellular membranes. The social amoeba Dictyostelium discoideum has two Nramp proteins. Nramp1, like its mammalian ortholog (SLC11A1), is recruited to phagosomal and macropinosomal membranes, and confers resistance to pathogenic bacteria. Nramp2 is located exclusively in the contractile vacuole membrane and controls, synergistically with Nramp1, iron homeostasis. It has long been debated whether mammalian Nramp1 mediates iron import or export from phagosomes. By selectively loading the iron-chelating fluorochrome calcein in macropinosomes, we show that Dictyostelium Nramp1 mediates iron efflux from macropinosomes in vivo. To gain insight in ion selectivity and the transport mechanism, the proteins were expressed in Xenopus oocytes. Using a novel assay with calcein, and electrophysiological and radiochemical assays, we show that Nramp1, similar to rat DMT1 (also known as SLC11A2), transports Fe(2+) and manganese, not Fe(3+) or copper. Metal ion transport is electrogenic and proton dependent. By contrast, Nramp2 transports only Fe(2+) in a non-electrogenic and proton-independent way. These differences reflect evolutionary divergence of the prototypical Nramp2 protein sequence compared to the archetypical Nramp1 and DMT1 proteins.
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Affiliation(s)
- Simona Buracco
- Department of Clinical and Biological Sciences, University of Torino, AOU S. Luigi, Orbassano 10043, Italy
| | - Barbara Peracino
- Department of Clinical and Biological Sciences, University of Torino, AOU S. Luigi, Orbassano 10043, Italy
| | - Raffaella Cinquetti
- Department of Biotechnology and Life Sciences, University of Insubria, Via J. H. Dunant 3, Varese 21100, Italy
| | - Elena Signoretto
- Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Via Trentacoste 2, Milano 20133, Italy
| | - Alessandra Vollero
- Department of Biotechnology and Life Sciences, University of Insubria, Via J. H. Dunant 3, Varese 21100, Italy
| | - Francesca Imperiali
- Department of Biotechnology and Life Sciences, University of Insubria, Via J. H. Dunant 3, Varese 21100, Italy
| | - Michela Castagna
- Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Via Trentacoste 2, Milano 20133, Italy
| | - Elena Bossi
- Department of Biotechnology and Life Sciences, University of Insubria, Via J. H. Dunant 3, Varese 21100, Italy
| | - Salvatore Bozzaro
- Department of Clinical and Biological Sciences, University of Torino, AOU S. Luigi, Orbassano 10043, Italy
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