@article{miyazawa_bogdan_hashimoto_tsuji_2019, title={Iron-induced transferrin receptor-1 mRNA destabilization: A response to "Neither miR-7-5p nor miR-141-3p is a major mediator of iron-responsive transferrin receptor-1 mRNA degradation"}, volume={25}, ISSN={["1469-9001"]}, DOI={10.1261/rna.073270.119}, abstractNote={We read with great interest the Divergent Views article by Connell and colleagues disputing our recent publication describing a role for two microRNAs in the iron-mediated regulation of transferrin receptor 1 (TfR1) mRNA stability. Our publication sought to shed light on a long-standing question in the field of cellular iron metabolism, and we welcome commentary and critique. However, there are several critical issues contained in the article by Connell and colleagues that require further consideration. We appreciate the opportunity to reply here.}, number={11}, journal={RNA}, author={Miyazawa, Masaki and Bogdan, Alexander R. and Hashimoto, Kazunori and Tsuji, Yoshiaki}, year={2019}, month={Nov}, pages={1416–1420} }
 @article{miyazawa_bogdan_tsuji_2019, title={Perturbation of Iron Metabolism by Cisplatin through Inhibition of Iron Regulatory Protein 2}, volume={26}, ISSN={["2451-9448"]}, DOI={10.1016/j.chembiol.2018.10.009}, abstractNote={Cisplatin is classically known to exhibit anticancer activity through DNA damage in the nucleus. Here we found a mechanism by which cisplatin affects iron metabolism, leading to toxicity and cell death. Cisplatin causes intracellular iron deficiency through direct inhibition of the master regulator of iron metabolism, iron regulatory protein 2 (IRP2) with marginal effects on IRP1. Cisplatin, but not carboplatin or transplatin, binds human IRP2 at Cys512 and Cys516 and impairs IRP2 binding to iron-responsive elements of ferritin and transferrin receptor-1 (TfR1) mRNAs. IRP2 inhibition by cisplatin caused ferritin upregulation and TfR1 downregulation leading to sustained intracellular iron deficiency. Cys512/516Ala mutant IRP2 made cells more resistant to cisplatin. Furthermore, combination of cisplatin and the iron chelator desferrioxamine enhanced cytotoxicity through augmented iron depletion in culture and xenograft mouse model. Collectively, cisplatin is an inhibitor of IRP2 that induces intracellular iron deficiency.}, number={1}, journal={CELL CHEMICAL BIOLOGY}, author={Miyazawa, Masaki and Bogdan, Alexander R. and Tsuji, Yoshiaki}, year={2019}, month={Jan}, pages={85-+} }
 @article{martin_gray_kilb_minchew_2016, title={Analyzing consortial "big deals" via a cost-per-cited-reference (CPCR) metric}, volume={42}, number={4}, journal={Serials Review}, author={Martin, V. and Gray, T. and Kilb, M. and Minchew, T.}, year={2016}, pages={293–305} }
 @article{ishii_yasuda_miyazawa_mitsushita_johnson_hartman_ishii_2016, title={Infertility and recurrent miscarriage with complex II deficiency-dependent mitochondrial oxidative stress in animal models}, volume={155}, journal={Mechanisms of Ageing and Development}, author={Ishii, T. and Yasuda, K. and Miyazawa, M. and Mitsushita, J. and Johnson, T. E. and Hartman, P. S. and Ishii, N.}, year={2016}, pages={22–35} }
 @misc{bogdan_miyazawa_hashimoto_tsuji_2016, title={Regulators of Iron Homeostasis: New Players in Metabolism, Cell Death, and Disease}, volume={41}, ISSN={["1362-4326"]}, DOI={10.1016/j.tibs.2015.11.012}, abstractNote={Iron is necessary for life, but can also cause cell death. Accordingly, cells evolved a robust, tightly regulated suite of genes for maintaining iron homeostasis. Previous mechanistic studies on iron homeostasis have granted insight into the role of iron in human health and disease. We highlight new regulators of iron metabolism, including iron-trafficking proteins [solute carrier family 39, SLC39, also known as ZRT/IRT-like protein, ZIP; and poly-(rC)-binding protein, PCBP] and a cargo receptor (NCOA4) that is crucial for release of ferritin-bound iron. We also discuss emerging roles of iron in apoptosis and a novel iron-dependent cell death pathway termed ‘ferroptosis’, the dysregulation of iron metabolism in human pathologies, and the use of iron chelators in cancer therapy. Iron is necessary for life, but can also cause cell death. Accordingly, cells evolved a robust, tightly regulated suite of genes for maintaining iron homeostasis. Previous mechanistic studies on iron homeostasis have granted insight into the role of iron in human health and disease. We highlight new regulators of iron metabolism, including iron-trafficking proteins [solute carrier family 39, SLC39, also known as ZRT/IRT-like protein, ZIP; and poly-(rC)-binding protein, PCBP] and a cargo receptor (NCOA4) that is crucial for release of ferritin-bound iron. We also discuss emerging roles of iron in apoptosis and a novel iron-dependent cell death pathway termed ‘ferroptosis’, the dysregulation of iron metabolism in human pathologies, and the use of iron chelators in cancer therapy. Dysregulation of iron metabolism contributes to various human pathologies, including iron overload diseases and cancer.Several new proteins have been identified as crucial iron traffickers and chaperones with important connections to human health.Ferroptosis is a unique cell death pathway that is iron-dependent, non-apoptotic, non-necroptotic, and non-autophagic.Dysregulation of the ferroportin/hepcidin regulatory axis contributes to tumor progression and is predictive of patient outcomes.New iron chelators utilize more specific mechanisms to elicit anticancer activity. Dysregulation of iron metabolism contributes to various human pathologies, including iron overload diseases and cancer. Several new proteins have been identified as crucial iron traffickers and chaperones with important connections to human health. Ferroptosis is a unique cell death pathway that is iron-dependent, non-apoptotic, non-necroptotic, and non-autophagic. Dysregulation of the ferroportin/hepcidin regulatory axis contributes to tumor progression and is predictive of patient outcomes. New iron chelators utilize more specific mechanisms to elicit anticancer activity. Iron is required in a variety of important biological processes including oxygen transport (as heme in hemoglobin), DNA biosynthesis (as a cofactor of ribonucleotide reductase), and ATP generation (as a cofactor for many proteins in the citric acid cycle and electron transport chain); therefore, cells must maintain a sufficient amount of iron. However, iron is redox-active and can generate reactive oxygen species (ROS), leading to oxidative stress and initiation of signaling pathways crucial for cell survival and cell death [1Ray P.D. et al.Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling.Cell. Signal. 2012; 24: 981-990Crossref PubMed Scopus (2799) Google Scholar]. To maintain adequate and safe amounts of iron, cells require the coordination of a wide variety of genes, which tightly control both intracellular (reviewed in [2MacKenzie E.L. et al.Intracellular iron transport and storage: from molecular mechanisms to health implications.Antioxid. Redox Signal. 2008; 10: 997-1030Crossref PubMed Scopus (343) Google Scholar, 3Hentze M.W. et al.Two to tango: regulation of mammalian iron metabolism.Cell. 2010; 142: 24-38Abstract Full Text Full Text PDF PubMed Scopus (1407) Google Scholar]) and systemic (reviewed in [4Ganz T. Nemeth E. Hepcidin and iron homeostasis.Biochim. Biophys. Acta. 2012; 1823: 1434-1443Crossref PubMed Scopus (856) Google Scholar]) iron metabolism. Extensive research by many groups has revealed key mechanisms in iron homeostasis (Box 1), as well as links between aberrations in iron homeostasis and human disease. The study of iron metabolism continues to be a dynamic field, with many breakthroughs and novel insights in the past several years. In this review we discuss recent advances in the function and regulation of key iron metabolism genes, including: ferritin (FTH1 and FTL), a protein complex that safely concentrates intracellular iron in a mineralized, redox-inactive form for later use; transferrin (TF), an iron-binding serum protein; transferrin receptor 1 (TfR1, TFRC), a plasma membrane protein that allows cellular uptake of iron-loaded transferrin; divalent metal transporter 1 (DMT1, SLC11A2), a metal transporter that is important for TfR1-mediated iron uptake and dietary iron absorption; ferroportin (Fpn, SLC40A1), the only known cellular iron efflux pump; and hepcidin (HAMP), a circulating peptide hormone that regulates serum iron levels by causing ferroportin degradation. We also examine newly identified regulators in iron metabolism, including new membrane iron transporters and cytosolic iron trafficking proteins. In addition, we review new roles for iron in cell death pathways, as well as the importance of aberrant iron metabolism in human diseases such as cancer.Box 1Control of Mammalian Iron Metabolism Occurs through Two Distinct, but Connected, Regulatory SystemsIntracellular iron metabolism is primarily controlled through coordinated post-transcriptional regulation of various iron metabolism genes (reviewed in [2MacKenzie E.L. et al.Intracellular iron transport and storage: from molecular mechanisms to health implications.Antioxid. Redox Signal. 2008; 10: 997-1030Crossref PubMed Scopus (343) Google Scholar, 3Hentze M.W. et al.Two to tango: regulation of mammalian iron metabolism.Cell. 2010; 142: 24-38Abstract Full Text Full Text PDF PubMed Scopus (1407) Google Scholar]). Many mRNAs involved in iron metabolism contain iron-responsive elements (IREs): stem-loop structures located in 5′- or 3′-untranslated regions (UTRs) flanking the coding sequence (CDS) [76Kuhn L.C. Iron regulatory proteins and their role in controlling iron metabolism.Metallomics: Int. Bio. Sci. 2015; 7: 232-243Crossref PubMed Google Scholar]. IREs bind two functionally similar iron regulatory proteins, IRP1 and IRP2. Depending upon whether the IRE is located within the 5′-UTR or in the 3′-UTR, the IRE–IRP interaction has opposite effects on target gene expression. In low iron conditions, 5′-UTR IREs are translationally repressed as a result of IRP blocking ribosome recruitment, whereas 3′-UTR IREs mediate enhanced mRNA stability, ultimately increasing protein levels (Figure IA, Low iron). High iron removes the IRPs, facilitating translational activation of 5′-UTR IRE mRNAs or degradation of 3′-UTR IRE mRNAs via endonuclease attack (Figure IA, High iron). 5′-UTR IREs are usually found in genes that lower the amount of cellular labile iron (i.e., iron that is unbound and redox active) such as ferritin and ferroportin, whereas 3′-UTR IREs are found in genes that facilitate iron uptake such as transferrin receptor 1 and divalent metal transporter 1 (Figure IA) Classically, it was believed that iron affected the IRE–IRP interaction entirely through effects on the IRPs. However, the ability of Fe2+ (and other metal ions) to directly bind to the IRE stem-loop was recently observed [77Khan M.A. et al.Direct Fe2+ sensing by iron-responsive messenger RNA:repressor complexes weakens binding.J. Biol. Chem. 2009; 284: 30122-30128Crossref PubMed Scopus (34) Google Scholar]. Fe2+ binding to 5′-UTR IREs induces a conformational change in the stem-loop, which decreases its affinity for IRPs [78Ma J. et al.Fe2+ binds iron responsive element-RNA, selectively changing protein-binding affinities and regulating mRNA repression and activation.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 8417-8422Crossref PubMed Scopus (50) Google Scholar] and increases its affinity for a ribosome recruitment factor eIF4F [79Khan M.A. et al.Rapid kinetics of iron responsive element (IRE) RNA/iron regulatory protein 1 and IRE-RNA/eIF4F complexes respond differently to metal ions.Nucleic Acids Res. 2014; 42: 6567-6577Crossref PubMed Scopus (18) Google Scholar], showing that the IRE structure itself contributes to post-transcriptional control of gene expression.In mammals, systemic iron homeostasis is controlled by the hepatocyte-secreted hormone hepcidin (reviewed in [4Ganz T. Nemeth E. Hepcidin and iron homeostasis.Biochim. Biophys. Acta. 2012; 1823: 1434-1443Crossref PubMed Scopus (856) Google Scholar]). Hepcidin circulates in the serum and binds to the cellular iron exporter ferroportin (Fpn), stimulating Fpn degradation and leading to cellular retention of iron (Figure IB). Hepcidin expression is directly correlated with both cellular and serum iron statuses and is controlled through a complex iron-sensing signaling pathway (reviewed in [4Ganz T. Nemeth E. Hepcidin and iron homeostasis.Biochim. Biophys. Acta. 2012; 1823: 1434-1443Crossref PubMed Scopus (856) Google Scholar]). Elevated serum hepcidin downregulates Fpn in duodenal enterocytes (which are responsible for dietary iron absorption), macrophages (which contain large amounts of iron from erythrocyte recycling), and hepatocytes (which act as an iron reservoir and export iron as needed). This leads to an overall reduction in serum iron (Figure IB). Some overlap exists between the IRE–IRP system and the hepcidin–Fpn axis (reviewed in [3Hentze M.W. et al.Two to tango: regulation of mammalian iron metabolism.Cell. 2010; 142: 24-38Abstract Full Text Full Text PDF PubMed Scopus (1407) Google Scholar]) because Fpn is regulated by IRPs though its 5′ -UTR IRE. Intracellular iron metabolism is primarily controlled through coordinated post-transcriptional regulation of various iron metabolism genes (reviewed in [2MacKenzie E.L. et al.Intracellular iron transport and storage: from molecular mechanisms to health implications.Antioxid. Redox Signal. 2008; 10: 997-1030Crossref PubMed Scopus (343) Google Scholar, 3Hentze M.W. et al.Two to tango: regulation of mammalian iron metabolism.Cell. 2010; 142: 24-38Abstract Full Text Full Text PDF PubMed Scopus (1407) Google Scholar]). Many mRNAs involved in iron metabolism contain iron-responsive elements (IREs): stem-loop structures located in 5′- or 3′-untranslated regions (UTRs) flanking the coding sequence (CDS) [76Kuhn L.C. Iron regulatory proteins and their role in controlling iron metabolism.Metallomics: Int. Bio. Sci. 2015; 7: 232-243Crossref PubMed Google Scholar]. IREs bind two functionally similar iron regulatory proteins, IRP1 and IRP2. Depending upon whether the IRE is located within the 5′-UTR or in the 3′-UTR, the IRE–IRP interaction has opposite effects on target gene expression. In low iron conditions, 5′-UTR IREs are translationally repressed as a result of IRP blocking ribosome recruitment, whereas 3′-UTR IREs mediate enhanced mRNA stability, ultimately increasing protein levels (Figure IA, Low iron). High iron removes the IRPs, facilitating translational activation of 5′-UTR IRE mRNAs or degradation of 3′-UTR IRE mRNAs via endonuclease attack (Figure IA, High iron). 5′-UTR IREs are usually found in genes that lower the amount of cellular labile iron (i.e., iron that is unbound and redox active) such as ferritin and ferroportin, whereas 3′-UTR IREs are found in genes that facilitate iron uptake such as transferrin receptor 1 and divalent metal transporter 1 (Figure IA) Classically, it was believed that iron affected the IRE–IRP interaction entirely through effects on the IRPs. However, the ability of Fe2+ (and other metal ions) to directly bind to the IRE stem-loop was recently observed [77Khan M.A. et al.Direct Fe2+ sensing by iron-responsive messenger RNA:repressor complexes weakens binding.J. Biol. Chem. 2009; 284: 30122-30128Crossref PubMed Scopus (34) Google Scholar]. Fe2+ binding to 5′-UTR IREs induces a conformational change in the stem-loop, which decreases its affinity for IRPs [78Ma J. et al.Fe2+ binds iron responsive element-RNA, selectively changing protein-binding affinities and regulating mRNA repression and activation.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 8417-8422Crossref PubMed Scopus (50) Google Scholar] and increases its affinity for a ribosome recruitment factor eIF4F [79Khan M.A. et al.Rapid kinetics of iron responsive element (IRE) RNA/iron regulatory protein 1 and IRE-RNA/eIF4F complexes respond differently to metal ions.Nucleic Acids Res. 2014; 42: 6567-6577Crossref PubMed Scopus (18) Google Scholar], showing that the IRE structure itself contributes to post-transcriptional control of gene expression. In mammals, systemic iron homeostasis is controlled by the hepatocyte-secreted hormone hepcidin (reviewed in [4Ganz T. Nemeth E. Hepcidin and iron homeostasis.Biochim. Biophys. Acta. 2012; 1823: 1434-1443Crossref PubMed Scopus (856) Google Scholar]). Hepcidin circulates in the serum and binds to the cellular iron exporter ferroportin (Fpn), stimulating Fpn degradation and leading to cellular retention of iron (Figure IB). Hepcidin expression is directly correlated with both cellular and serum iron statuses and is controlled through a complex iron-sensing signaling pathway (reviewed in [4Ganz T. Nemeth E. Hepcidin and iron homeostasis.Biochim. Biophys. Acta. 2012; 1823: 1434-1443Crossref PubMed Scopus (856) Google Scholar]). Elevated serum hepcidin downregulates Fpn in duodenal enterocytes (which are responsible for dietary iron absorption), macrophages (which contain large amounts of iron from erythrocyte recycling), and hepatocytes (which act as an iron reservoir and export iron as needed). This leads to an overall reduction in serum iron (Figure IB). Some overlap exists between the IRE–IRP system and the hepcidin–Fpn axis (reviewed in [3Hentze M.W. et al.Two to tango: regulation of mammalian iron metabolism.Cell. 2010; 142: 24-38Abstract Full Text Full Text PDF PubMed Scopus (1407) Google Scholar]) because Fpn is regulated by IRPs though its 5′ -UTR IRE. In recent years several new proteins have been identified as key players in intracellular iron trafficking and utilization. This section discusses novel iron transporters, iron chaperones, and ferritin-shuttling proteins. The solute carrier family 39 (SLC39), also known as the ZRT/IRT-like protein (ZIP) family, contains 14 members and has classically been understood to comprise transmembrane zinc transporters that pump extracellular Zn2+ into the cell [5Jeong J. Eide D.J. The SLC39 family of zinc transporters.Mol. Aspects Med. 2013; 34: 612-619Crossref PubMed Scopus (279) Google Scholar]. However, recent studies have uncovered a role for two ZIP family proteins in iron transport [6Jenkitkasemwong S. et al.Physiologic implications of metal-ion transport by ZIP14 and ZIP8.Biometals. 2012; 25: 643-655Crossref PubMed Scopus (169) Google Scholar]. ZIP14 was found to mediate the uptake of non-transferrin-bound iron (NTBI) [7Liuzzi J.P. et al.Zip14 (Slc39a14) mediates non-transferrin-bound iron uptake into cells.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 13612-13617Crossref PubMed Scopus (411) Google Scholar] by directly transporting NTBI across the cell membrane [8Pinilla-Tenas J.J. et al.Zip14 is a complex broad-scope metal-ion transporter whose functional properties support roles in the cellular uptake of zinc and nontransferrin-bound iron.Am J. Physiol. Cell Physiol. 2011; 301: C862-C871Crossref PubMed Scopus (156) Google Scholar] (Figure 1, left, ‘Uptake’). ZIP14-mediated NTBI transport is enhanced by the Fe3+ to Fe2+ ferrireductase activity of the prion protein PRNP [9Tripathi A.K. et al.Prion protein functions as a ferrireductase partner for ZIP14 and DMT1.Free Radic. Biol. Med. 2015; 84: 322-330Crossref PubMed Scopus (48) Google Scholar]. ZIP14 is also capable of transporting transferrin-bound iron from the endosome to the cytoplasm, similarly to DMT1 [10Zhao N. et al.ZRT/IRT-like protein 14 (ZIP14) promotes the cellular assimilation of iron from transferrin.J. Biol. Chem. 2010; 285: 32141-32150Crossref PubMed Scopus (126) Google Scholar], although subsequent studies have revealed that this is likely not the major function of ZIP14 [11Ji C. Kosman D.J. Molecular mechanisms of non-transferrin-bound and transferring-bound iron uptake in primary hippocampal neurons.J. Neurochem. 2015; 133: 668-683Crossref PubMed Scopus (57) Google Scholar]. ZIP8, the closest relative to ZIP14 [6Jenkitkasemwong S. et al.Physiologic implications of metal-ion transport by ZIP14 and ZIP8.Biometals. 2012; 25: 643-655Crossref PubMed Scopus (169) Google Scholar], was also found to transport NTBI [12Wang C.Y. et al.ZIP8 is an iron and zinc transporter whose cell-surface expression is up-regulated by cellular iron loading.J. Biol. Chem. 2012; 287: 34032-34043Crossref PubMed Scopus (254) Google Scholar] (Figure 1, left, ‘Uptake’). Interestingly, both ZIP8 and ZIP14 show maximal iron transport above pH 7, near physiological pH [8Pinilla-Tenas J.J. et al.Zip14 is a complex broad-scope metal-ion transporter whose functional properties support roles in the cellular uptake of zinc and nontransferrin-bound iron.Am J. Physiol. Cell Physiol. 2011; 301: C862-C871Crossref PubMed Scopus (156) Google Scholar, 12Wang C.Y. et al.ZIP8 is an iron and zinc transporter whose cell-surface expression is up-regulated by cellular iron loading.J. Biol. Chem. 2012; 287: 34032-34043Crossref PubMed Scopus (254) Google Scholar], whereas DMT1 is most efficient at pH 5.5 [13Gunshin H. et al.Cloning and characterization of a mammalian proton-coupled metal-ion transporter.Nature. 1997; 388: 482-488Crossref PubMed Scopus (2652) Google Scholar], which corresponds to the pH of acidified endosome. The widely disparate pH activities suggest that ZIP8/14 and DMT1 may have different biological roles. Indeed, a recent report utilizing primary rat hippocampal neurons showed that ZIP8 is the major NTBI transporter whereas DMT1 was principally responsible for transferrin-bound iron transport from the endosome [11Ji C. Kosman D.J. Molecular mechanisms of non-transferrin-bound and transferring-bound iron uptake in primary hippocampal neurons.J. Neurochem. 2015; 133: 668-683Crossref PubMed Scopus (57) Google Scholar]. ZIP8 and ZIP14 appear to have nonredundant functions because they have distinct tissue expression profiles and knockout mouse models show different phenotypes. Knockout of ZIP8 in mice causes failures in hematopoiesis and organogenesis of the spleen, liver, kidney, and lung, with associated perinatal lethality [14Galvez-Peralta M. et al.ZIP8 zinc transporter: indispensable role for both multiple-organ organogenesis and hematopoiesis in utero.PLoS ONE. 2012; 7: e36055Crossref PubMed Scopus (81) Google Scholar]. Zip8−/− mice exhibited profound anemia and have significantly lowered amounts of iron and zinc in various tissues, with the hematopoietic defect seemingly being caused by the combination of iron and zinc deficiency. By contrast, Zip14−/− mice are viable and have adequate iron stores, but exhibit dwarfism owing to aberrant bone growth. The dwarfism was found to primarily be the result of altered zinc, rather than iron, metabolism, indicating that zinc transport is more important than iron transport by ZIP14 for normal growth [15Hojyo S. et al.The zinc transporter SLC39A14/ZIP14 controls G-protein coupled receptor-mediated signaling required for systemic growth.PLoS ONE. 2011; 6: e18059Crossref PubMed Scopus (130) Google Scholar]. However, a more recent study uncovered a crucial role for ZIP14 in iron transport by knocking out ZIP14 in two mouse models for hereditary hemochromatosis, a family of iron overload diseases [16Jenkitkasemwong S. et al.SLC39A14 is required for the development of hepatocellular iron overload in murine models of hereditary hemochromatosis.Cell Metab. 2015; 22: 138-150Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar]. Hemochromatosis results in a progressive accumulation of iron primarily in the liver, resulting in hepatic fibrosis and potentially hepatocellular carcinoma. Knocking out ZIP14 in Hfe−/− (hereditary hemochromatosis protein) or Hfe2−/− (hemojuvelin) mice [modeling type 1 and type 2 (juvenile) hemochromatosis, respectively] prevented iron accumulation in the liver. ZIP14 knockout also prevented iron overload in Hfe2−/− mouse pancreas, but not heart [16Jenkitkasemwong S. et al.SLC39A14 is required for the development of hepatocellular iron overload in murine models of hereditary hemochromatosis.Cell Metab. 2015; 22: 138-150Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar], which could be attributable to the high pancreatic but low cardiac expression of ZIP14 [17Nam H. et al.ZIP14 and DMT1 in the liver, pancreas, and heart are differentially regulated by iron deficiency and overload: implications for tissue iron uptake in iron-related disorders.Haematologica. 2013; 98: 1049-1057Crossref PubMed Scopus (121) Google Scholar]. Uncovering the importance of ZIP14 in iron loading provides a new target in the treatment of iron overload diseases. Namely, inhibition of ZIP14 could potentially block hepatic and pancreatic iron overloading, and prevent subsequent organ damage. The expression of ZIP8 and ZIP14 is regulated differently than other iron transporter genes such as TfR1 and DMT1, both of which are controlled post-transcriptionally by changes of mRNA stability through the 3′ untranslated region (3′ -UTR) iron-responsive element (IRE)–iron regulatory protein (IRP) interaction (Box 1) [2MacKenzie E.L. et al.Intracellular iron transport and storage: from molecular mechanisms to health implications.Antioxid. Redox Signal. 2008; 10: 997-1030Crossref PubMed Scopus (343) Google Scholar, 3Hentze M.W. et al.Two to tango: regulation of mammalian iron metabolism.Cell. 2010; 142: 24-38Abstract Full Text Full Text PDF PubMed Scopus (1407) Google Scholar]. By contrast, ZIP8 and ZIP14 mRNA stability is unaffected by iron, and ZIP protein levels are positively correlated with iron status, in that iron loading increases ZIP8/14 protein amount whereas iron deficiency decreases protein amount [12Wang C.Y. et al.ZIP8 is an iron and zinc transporter whose cell-surface expression is up-regulated by cellular iron loading.J. Biol. Chem. 2012; 287: 34032-34043Crossref PubMed Scopus (254) Google Scholar, 18Zhao N. et al.An iron-regulated and glycosylation-dependent proteasomal degradation pathway for the plasma membrane metal transporter ZIP14.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 9175-9180Crossref PubMed Scopus (47) Google Scholar]. In summary, ZIP8 and ZIP14 are newly recognized iron transporters with important roles in the uptake of NTBI and in iron overload diseases. Further investigation will be necessary to determine the importance of ZIP iron transport in other contexts, such as disease states with disordered iron homeostasis (anemia, cancer, etc.). PCBPs are a family of four proteins that were originally identified for their high affinity binding to tracts of polycytosine and have been studied as regulators of gene expression (reviewed in [19Chaudhury A. et al.Heterogeneous nuclear ribonucleoproteins (hnRNPs) in cellular processes: focus on hnRNP E1's multifunctional regulatory roles.RNA. 2010; 16: 1449-1462Crossref PubMed Scopus (197) Google Scholar]). Each member of the PCBP family contains three highly conserved K homology (KH) domains and can form complexes with other family members. Recently, PCBP1 and PCBP2 were identified as cytosolic iron chaperones that are responsible for delivering iron to ferritin, the protein that is primarily responsible for storing intracellular iron in a non-toxic, mineralized state [20Shi H. et al.A cytosolic iron chaperone that delivers iron to ferritin.Science. 2008; 320: 1207-1210Crossref PubMed Scopus (365) Google Scholar, 21Leidgens S. et al.Each member of the poly-r(C)-binding protein 1 (PCBP) family exhibits iron chaperone activity toward ferritin.J. Biol. Chem. 2013; 288: 17791-17802Crossref PubMed Scopus (128) Google Scholar] (Figure 1, center, ‘Distribution’). PCBP3 and PCBP4 are expressed at much lower levels, with a limited tissue distribution and an unclear biological role, but there is some evidence that they can also act as iron chaperones and that PCBP3 can bind to ferritin [21Leidgens S. et al.Each member of the poly-r(C)-binding protein 1 (PCBP) family exhibits iron chaperone activity toward ferritin.J. Biol. Chem. 2013; 288: 17791-17802Crossref PubMed Scopus (128) Google Scholar]. To date, the majority of the research on the iron chaperone function of the PCBP family has focused on PCBP1 and PCBP2. Both PCBP1 and PCBP2 are necessary to form a stable ternary complex with ferritin, and ferritin–PCBP interactions were dependent upon iron-loaded PCBP1/2 [21Leidgens S. et al.Each member of the poly-r(C)-binding protein 1 (PCBP) family exhibits iron chaperone activity toward ferritin.J. Biol. Chem. 2013; 288: 17791-17802Crossref PubMed Scopus (128) Google Scholar]. In addition to ferritin, PCBP1 and PCBP2 can also deliver iron to proteins that require non-heme iron as a cofactor (‘metallation’, Figure 1, center, ‘Distribution’), such as hypoxia-inducible factor (HIF) prolyl and asparginyl hydoxylases [22Nandal A. et al.Activation of the HIF prolyl hydroxylase by the iron chaperones PCBP1 and PCBP2.Cell Metab. 2011; 14: 647-657Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar], and deoxyhypusine hydroxylase [23Frey A.G. et al.Iron chaperones PCBP1 and PCBP2 mediate the metallation of the dinuclear iron enzyme deoxyhypusine hydroxylase.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 8031-8036Crossref PubMed Scopus (86) Google Scholar]. PCBP2 is loaded with iron through binding to the iron transporter DMT1. Iron transfer to PCBP2 is mediated through interaction of the second KH domain of PCBP2 with the N-terminal cytoplasmic domain of DMT1 [24Yanatori I. et al.Chaperone protein involved in transmembrane transport of iron.Biochem. J. 2014; 462: 25-37Crossref PubMed Scopus (88) Google Scholar]. After transfer of Fe2+, PCBP2 subsequently dissociates from DMT1 (Figure 1, center, ‘Distribution’). By contrast, PCPB1 does not interact with DMT1, and the mechanism of apo-PCBP1 iron acquisition is still undetermined. A comprehensive screen of both iron-loaded and apo-PCBP1 and -2 binding to other iron metabolism genes (ZIP transporters, etc.) is crucial for understanding overall cytoplasmic iron shuttling and may shed light on PCBP1 iron loading. Many questions about the roles and mechanisms of the PCBP proteins in iron homeostasis remain unanswered. For instance, it was noted that PCBP2 can also bind to the iron exporter ferroportin (Fpn) [24Yanatori I. et al.Chaperone protein involved in transmembrane transport of iron.Biochem. J. 2014; 462: 25-37Crossref PubMed Scopus (88) Google Scholar] (Figure 1, center, ‘Distribution’), but that association remains to be characterized in depth, including what effects, if any, PCBP2 has on Fpn iron export. In addition, the question of what roles, if any, PCBP3 and PCBP4 have in intracellular iron homeostasis remains to be answered. Ferritin stores ferric iron in a mineralized, non-toxic state. However, ferritin-bound iron cannot be utilized by the cell. Consequently, iron must be released from ferritin to be biologically useful, usually through lysosomal degradation of ferritin [25Kidane T.Z. et al.Release of iron from ferritin requires lysosomal activity.Am J. Physiol. Cell Physiol. 2006; 291: C445-C455Crossref PubMed Scopus (187) Google Scholar, 26Asano T. et al.Distinct mechanisms of ferritin delivery to lysosomes in iron-depleted and iron-replete cells.Mol. Cell. Biol. 2011; 31: 2040-2052Crossref PubMed Scopus (164) Google Scholar, 27Kishi-Itakura C. et al.Ultrastructural analysis of autophagosome organization using mammalian autophagy-deficient cells.J. Cell Sci. 2014; 127: 4089-4102Crossref PubMed Scopus (148) Google Scholar, 28Zhang Y. et al.Lysosomal proteolysis is the primary degradation pathway for cytosolic ferritin and cytosolic ferritin degradation is necessary for iron exit.Antioxid. Redox Signal. 2010; 13: 999-1009Crossref PubMed Scopus (78) Google Scholar]. Ferritin protein turnover is a constant process in cells. While the rate of ferritin degradation appears to be the same in iron-deficient and iron-replete conditions, the delivery mechanism of ferritin to lysosomes seems to be different: autophagy is responsible for delivering ferritin to the lysosome during iron deficiency, whereas a non-autophagic pathway dominates during iron sufficiency [26Asano T. et al.Distinct mechanisms of ferritin delivery to lysosomes in iron-depleted and iron-replete cells.Mol. Cell. Biol. 2011; 31: 2040-2052Crossref PubMed Scopus (164) Google Scholar]. Recently, nuclear receptor coactivator 4 (NCOA4) was identified as the protein responsible for mediating ferritin autophagy [29Dowdle W.E. et al.Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo.Nat. Cell Biol. 2014; 16: 1069-1079Crossref PubMed Scopus (415) Google Scholar, 30Mancias J.D. et al.Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy.Nature. 2014; 509: 105-109Crossref PubMed Scopus (809) Google Scholar]. NCOA4 was originally identified as a coactivator of several nuclear receptors but was later found to primarily be localized in the cytoplasm and have roles in various biological processes independent of its coactivator function (reviewed in [31Kollara A. Brown T.J. Expression and function of nuclear receptor co-activato}, number={3}, journal={TRENDS IN BIOCHEMICAL SCIENCES}, author={Bogdan, Alexander R. and Miyazawa, Masaki and Hashimoto, Kazunori and Tsuji, Yoshiaki}, year={2016}, month={Mar}, pages={274–286} }
 @misc{wilson_bogdan_miyazawa_hashimoto_tsuji_2016, title={Siderophores in Iron Metabolism: From Mechanism to Therapy Potential}, volume={22}, ISSN={["1471-499X"]}, DOI={10.1016/j.molmed.2016.10.005}, abstractNote={A ‘tug-of-war’ for survival over available iron supplies can develop between a host and invading pathogens; bacteria attempt to acquire iron through ferric-siderophores, iron-bound transferrin, lactoferrin, and heme, while the host can block pathogens to access ferric-siderophores, for instance by forming a siderophore complex with lipocalin 2. Ferric-siderophore import into bacteria is mediated through specific receptors localized in the outer membrane. Secretion of siderophores from pathogens is mediated through efflux pumps including ABC transporters. Iron chelation by siderophores causes robust responses in host cells through gene expression changes involved in apoptosis, mitophagy, hypoxia, and the production of inflammatory cytokines. Siderophore-conjugated compounds that limit the availability of iron to multidrug-resistant bacteria have recently emerged as new therapeutic approaches to contain nosocomial infections. Inhibitors of bacterial siderophore biosynthesis are also promising new antimicrobial agents. Iron is an essential nutrient for life. During infection, a fierce battle of iron acquisition occurs between the host and bacterial pathogens. Bacteria acquire iron by secreting siderophores, small ferric iron-binding molecules. In response, host immune cells secrete lipocalin 2 (also known as siderocalin), a siderophore-binding protein, to prevent bacterial reuptake of iron-loaded siderophores. To counter this threat, some bacteria can produce lipocalin 2-resistant siderophores. This review discusses the recently described molecular mechanisms of siderophore iron trafficking between host and bacteria, highlighting the therapeutic potential of exploiting pathogen siderophore machinery for the treatment of antibiotic-resistant bacterial infections. Because the latter reflect a persistent problem in hospital settings, siderophore-targeting or siderophore-based compounds represent a promising avenue to combat such infections. Iron is an essential nutrient for life. During infection, a fierce battle of iron acquisition occurs between the host and bacterial pathogens. Bacteria acquire iron by secreting siderophores, small ferric iron-binding molecules. In response, host immune cells secrete lipocalin 2 (also known as siderocalin), a siderophore-binding protein, to prevent bacterial reuptake of iron-loaded siderophores. To counter this threat, some bacteria can produce lipocalin 2-resistant siderophores. This review discusses the recently described molecular mechanisms of siderophore iron trafficking between host and bacteria, highlighting the therapeutic potential of exploiting pathogen siderophore machinery for the treatment of antibiotic-resistant bacterial infections. Because the latter reflect a persistent problem in hospital settings, siderophore-targeting or siderophore-based compounds represent a promising avenue to combat such infections. Nosocomial infections (see Glossary), also known as hospital-acquired infections (HAI), are a rising health concern worldwide. In the USA alone, in 2011, at least 721 800 HAIs caused by various pathogens were estimated [1Magill S.S. et al.Multistate point-prevalence survey of health care-associated infections.N. Engl. J. Med. 2014; 370: 1198-1208Crossref PubMed Scopus (2457) Google Scholar]. Gram-positive Staphylococcus aureus (S. aureus) is of particular concern, and is estimated to be responsible for about 10% of all HAIs. The development of hyper-virulent and antibiotic-resistant strains makes treating these infections more difficult. For instance, carbapenemase-producing pathogens, such as Klebsiella pneumoniae and Escherichia coli, are resistant to the carbapenem class of antibiotics widely used to treat multidrug-resistant bacteria. Many HAIs can infect a variety of organs, including the gastrointestinal system and urinary tract, and may also infect surgical sites. These pathogens are becoming more widespread and constitute an immediate public health problem [2Temkin E. et al.Carbapenem-resistant Enterobacteriaceae: biology, epidemiology, and management.Ann. N. Y. Acad. Sci. 2014; 1323: 22-42Crossref PubMed Scopus (161) Google Scholar]. Virulence factors may aid pathogens in colonizing the host as well as enhance disease. These factors comprise a wide variety of substances including bacterial toxins, adherence factors, protective capsules, and, relevant to this review, siderophores. Iron is a necessary element in virtually all living organisms and is utilized to catalyze a wide variety of indispensable enzymatic reactions [3Soares M.P. Weiss G. The Iron Age of host–microbe interactions.EMBO Rep. 2015; 16: 1482-1500Crossref PubMed Scopus (145) Google Scholar]. Early microorganisms were able to utilize soluble ferrous iron (Fe2+), that was abundant owing to an oxygen-poor atmosphere; however, as oxygen-rich conditions arose, ferrous iron was oxidized to insoluble ferric iron (Fe3+), removing an easily bioavailable source of iron. Responding to this challenge, microorganisms evolved siderophores – small ferric iron (Fe3+)-chelating molecules [4Holden V.I. Bachman M.A. Diverging roles of bacterial siderophores during infection.Metallomics. 2015; 7: 986-995Crossref PubMed Google Scholar]. In a pathogenic context, microbes secrete siderophores to acquire and solubilize ferric iron from the host. In fact, comparisons of different Acinetobacter genomes have shown that the presence of genes involving siderophore biosynthesis is predictive of high or low virulence [5Peleg A.Y. et al.The success of acinetobacter species; genetic, metabolic and virulence attributes.PLoS One. 2012; 7: e46984Crossref PubMed Scopus (136) Google Scholar]. Consistently, in bacteria including Acinetobacter baumannii, siderophores were shown to be necessary components for the development of surface attachment and extracellular polysaccharide synthesis (termed biofilm formation) [6Harrison F. Buckling A. Siderophore production and biofilm formation as linked social traits.ISME J. 2009; 3: 632-634Crossref PubMed Scopus (58) Google Scholar, 7Gaddy J.A. et al.Role of acinetobactin-mediated iron acquisition functions in the interaction of Acinetobacter baumannii strain ATCC 19606T with human lung epithelial cells, Galleria mellonella caterpillars, and mice.Infect. Immun. 2012; 80: 1015-1024Crossref PubMed Scopus (180) Google Scholar, 8Marti S. et al.Growth of Acinetobacter baumannii in pellicle enhanced the expression of potential virulence factors.PLoS One. 2011; 6: e26030Crossref PubMed Scopus (60) Google Scholar] and the establishment of mutually-beneficial, iron-sufficient microbial communities [9Morris J.J. Black Queen evolution: the role of leakiness in structuring microbial communities.Trends Genet. 2015; 31: 475-482Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar]. Given that siderophores are involved in biofilm formation which promotes antibiotic resistance [10Su H.C. et al.The development of ciprofloxacin resistance in Pseudomonas aeruginosa involves multiple response stages and multiple proteins.Antimicrob. Agents Chemother. 2010; 54: 4626-4635Crossref PubMed Scopus (39) Google Scholar, 11Tseng B.S. et al.The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin.Environ. Microbiol. 2013; 15: 2865-2878PubMed Google Scholar], targeting siderophores by blocking siderophore synthesis or function provides a promising alternative antimicrobial approach. Iron is also essential to host cells, and is tightly regulated under various physiological conditions [12Bogdan A.R. et al.Regulators of iron homeostasis: new players in metabolism, cell death, and disease.Trends Biochem. Sci. 2016; 41: 274-286Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar]. Hosts utilize various major iron-transport systems such as iron-loaded transferrin, lactoferrin, and heme [3Soares M.P. Weiss G. The Iron Age of host–microbe interactions.EMBO Rep. 2015; 16: 1482-1500Crossref PubMed Scopus (145) Google Scholar]. However, the affinities of bacterial siderophores to iron are generally much higher than those of host proteins/molecules [4Holden V.I. Bachman M.A. Diverging roles of bacterial siderophores during infection.Metallomics. 2015; 7: 986-995Crossref PubMed Google Scholar], allowing pathogens to outcompete the host in iron acquisition. In response to the siderophore threat, mammalian immune cells (e.g., macrophages, neutrophils) can secrete a siderophore-binding protein, lipocalin 2 [Lcn2, a 24 kDa glycoprotein also known as siderocalin, 24p3, and NGAL (neutrophil gelatinase-associated lipocalin)], to intercept bacterial uptake of iron-loaded siderophores (ferric-siderophores) [13Chakraborty S. et al.The multifaceted roles of neutrophil gelatinase associated lipocalin (NGAL) in inflammation and cancer.Biochim. Biophys. Acta. 2012; 1826: 129-169PubMed Google Scholar]. This battle over limited iron is an important host–pathogen interaction for survival during infections (Box 1).Box 1Siderophore–Lipocalin 2 Interface during InfectionOne of the major challenges for pathogenic bacteria is in acquiring sufficient iron during infection and post-infection inside the host. Competition between the host and pathogen for this coveted transition metal occurs at the host–pathogen interface, a conceptual framework describing the exchange of signals and battle for common resources between host and pathogen. The fight for iron represents a robust ‘tug-of-war’, which begins with the host secreting iron-binding proteins such as lactoferrin to sequester any readily available iron (Figure I, Stage 1).When deprived of a readily available iron source, pathogens upregulate siderophore biosynthesis and iron-trafficking pathways (Figure I, Stage 2). Siderophores have much higher affinity for iron [4Holden V.I. Bachman M.A. Diverging roles of bacterial siderophores during infection.Metallomics. 2015; 7: 986-995Crossref PubMed Google Scholar] and can strip iron from lactoferrin and other host iron-binding proteins, restoring access to iron for pathogens. In response, host cells secrete lipocalin 2 (important in innate immunity). In neutrophils, Lcn2 is stored within granules and is rapidly secreted as part of a ‘first-response’ to infection. Infection also drives massive increases in de novo Lcn2 production through Toll-like receptor (TLR) and cytokine signaling [65Li C. Chan Y.R. Lipocalin 2 regulation and its complex role in inflammation and cancer.Cytokine. 2011; 56: 435-441Crossref PubMed Scopus (57) Google Scholar]. Lcn2 complexes with ferric-siderophores and prevents reuptake by pathogens, once again denying bacterial iron acquisition (Figure I, Stage 3). The Lcn2 threat is so severe that some pathogens have evolved chemically modified, Lcn2-resistant siderophores, expressing them in response to Lcn2 secretion [66Steigedal M. et al.Lipocalin 2 imparts selective pressure on bacterial growth in the bladder and is elevated in women with urinary tract infection.J. Immunol. 2014; 193: 6081-6089Crossref PubMed Scopus (48) Google Scholar]. The siderophore enterobactin produced in E. coli is readily bound by Lcn2, while glycosylated enterobactin (salmochelin) is not (Table 1 and Figure 1B). These ‘stealth siderophores’ (i.e., chemically modified and unable to bind Lcn2) are important tools for preventing host Lcn2 function (Figure I, Stage 4).Beyond simply denying iron to pathogens, Lcn2 can maintain host immune protein function. Recent data show that enterobactin can inactivate host myeloperoxidase (e.g., in neutrophils), a crucial iron-containing enzyme that generates bactericidal hypochlorous acid from H2O2 and Cl− [67Singh V. et al.Interplay between enterobactin, myeloperoxidase and lipocalin 2 regulates E. coli survival in the inflamed gut.Nat. Commun. 2015; 6: 7113Crossref PubMed Scopus (69) Google Scholar]. Enterobactin attacks the heme prosthetic group and inactivates myeloperoxidase; Lcn2 can preserve myeloperoxidase function by binding to enterobactin [67Singh V. et al.Interplay between enterobactin, myeloperoxidase and lipocalin 2 regulates E. coli survival in the inflamed gut.Nat. Commun. 2015; 6: 7113Crossref PubMed Scopus (69) Google Scholar].Another bacterial strategy is to produce siderophores that natively cannot be recognized by Lcn2, as in the case of K. pneumonia, which can cause major respiratory infections and produces yersiniabactin (Table 1 and Figure 1B) [68Bachman M.A. et al.Klebsiella pneumoniae yersiniabactin promotes respiratory tract infection through evasion of lipocalin 2.Infect. Immun. 2011; 79: 3309-3316Crossref PubMed Scopus (182) Google Scholar]. It is becoming clear that the host environment plays an important role in Lcn2-mediated immunity. Alterations in urine pH and composition can have a significant impact on the ability of Lcn2 to combat E. coli in a urinary tract infection context. Alkalinization of urine (pH above 6.45) increases the ability of Lcn2 to restrict iron and, subsequently, to inhibit the growth of E. coli [69Shields-Cutler R.R. et al.Human urinary composition controls antibacterial activity of siderocalin.J. Biol. Chem. 2015; 290: 15949-15960Crossref PubMed Scopus (39) Google Scholar]. Given that inducing urinary alkalinization in a clinical setting is trivial, modulating urinary chemistry may be an attractive option in combating uropathogenic E. coli infections and warrants further investigation. One of the major challenges for pathogenic bacteria is in acquiring sufficient iron during infection and post-infection inside the host. Competition between the host and pathogen for this coveted transition metal occurs at the host–pathogen interface, a conceptual framework describing the exchange of signals and battle for common resources between host and pathogen. The fight for iron represents a robust ‘tug-of-war’, which begins with the host secreting iron-binding proteins such as lactoferrin to sequester any readily available iron (Figure I, Stage 1). When deprived of a readily available iron source, pathogens upregulate siderophore biosynthesis and iron-trafficking pathways (Figure I, Stage 2). Siderophores have much higher affinity for iron [4Holden V.I. Bachman M.A. Diverging roles of bacterial siderophores during infection.Metallomics. 2015; 7: 986-995Crossref PubMed Google Scholar] and can strip iron from lactoferrin and other host iron-binding proteins, restoring access to iron for pathogens. In response, host cells secrete lipocalin 2 (important in innate immunity). In neutrophils, Lcn2 is stored within granules and is rapidly secreted as part of a ‘first-response’ to infection. Infection also drives massive increases in de novo Lcn2 production through Toll-like receptor (TLR) and cytokine signaling [65Li C. Chan Y.R. Lipocalin 2 regulation and its complex role in inflammation and cancer.Cytokine. 2011; 56: 435-441Crossref PubMed Scopus (57) Google Scholar]. Lcn2 complexes with ferric-siderophores and prevents reuptake by pathogens, once again denying bacterial iron acquisition (Figure I, Stage 3). The Lcn2 threat is so severe that some pathogens have evolved chemically modified, Lcn2-resistant siderophores, expressing them in response to Lcn2 secretion [66Steigedal M. et al.Lipocalin 2 imparts selective pressure on bacterial growth in the bladder and is elevated in women with urinary tract infection.J. Immunol. 2014; 193: 6081-6089Crossref PubMed Scopus (48) Google Scholar]. The siderophore enterobactin produced in E. coli is readily bound by Lcn2, while glycosylated enterobactin (salmochelin) is not (Table 1 and Figure 1B). These ‘stealth siderophores’ (i.e., chemically modified and unable to bind Lcn2) are important tools for preventing host Lcn2 function (Figure I, Stage 4). Beyond simply denying iron to pathogens, Lcn2 can maintain host immune protein function. Recent data show that enterobactin can inactivate host myeloperoxidase (e.g., in neutrophils), a crucial iron-containing enzyme that generates bactericidal hypochlorous acid from H2O2 and Cl− [67Singh V. et al.Interplay between enterobactin, myeloperoxidase and lipocalin 2 regulates E. coli survival in the inflamed gut.Nat. Commun. 2015; 6: 7113Crossref PubMed Scopus (69) Google Scholar]. Enterobactin attacks the heme prosthetic group and inactivates myeloperoxidase; Lcn2 can preserve myeloperoxidase function by binding to enterobactin [67Singh V. et al.Interplay between enterobactin, myeloperoxidase and lipocalin 2 regulates E. coli survival in the inflamed gut.Nat. Commun. 2015; 6: 7113Crossref PubMed Scopus (69) Google Scholar]. Another bacterial strategy is to produce siderophores that natively cannot be recognized by Lcn2, as in the case of K. pneumonia, which can cause major respiratory infections and produces yersiniabactin (Table 1 and Figure 1B) [68Bachman M.A. et al.Klebsiella pneumoniae yersiniabactin promotes respiratory tract infection through evasion of lipocalin 2.Infect. Immun. 2011; 79: 3309-3316Crossref PubMed Scopus (182) Google Scholar]. It is becoming clear that the host environment plays an important role in Lcn2-mediated immunity. Alterations in urine pH and composition can have a significant impact on the ability of Lcn2 to combat E. coli in a urinary tract infection context. Alkalinization of urine (pH above 6.45) increases the ability of Lcn2 to restrict iron and, subsequently, to inhibit the growth of E. coli [69Shields-Cutler R.R. et al.Human urinary composition controls antibacterial activity of siderocalin.J. Biol. Chem. 2015; 290: 15949-15960Crossref PubMed Scopus (39) Google Scholar]. Given that inducing urinary alkalinization in a clinical setting is trivial, modulating urinary chemistry may be an attractive option in combating uropathogenic E. coli infections and warrants further investigation. Siderophores are becoming better appreciated for their role in virulence beyond simple iron chelation, acting as signals leading to a robust host defense by inducing, for instance, mitophagy, hypoxic responses, and cytokine production. This review discusses recent findings in siderophore biology, with highlights on the therapeutic potential of siderophore pharmacological targeting in microbial infections (e.g., S. aureus and A. baumannii) through the use of siderophore–antibiotic drug conjugates, gallium-containing compounds, and siderophore biosynthesis inhibitors. We begin with a brief overview of nosocomial siderophore biology and import/export, followed by recent findings in host–pathogen interactions. Finally, we summarize recent advances in siderophore biology in an infection context, examining inhibitors of siderophore biosynthesis as novel antimicrobial therapeutics. Indeed, over the past few years, studies have revealed several components in siderophore production and trafficking, exposing potential vulnerabilities that can be exploited clinically. Targeting such vulnerabilities could potentially allow clinicians to circumvent the problem of traditional antibiotic-resistance in their patients, ideally reducing the burden of HAIs and improving health outcomes. Major pathogens that cause nosocomial infections and acquire multidrug resistance are summarized in Table 1. Gram-negative bacteria (e.g., Pseudomonas aeruginosa, Klebsiella pneumonia, A. baumannii, E. coli), Gram-positive bacteria (e.g., Staphylococcus aureus), and acid-fast bacilli (e.g., Mycobacterium tuberculosis) [14Sydnor E.R. Perl T.M. Hospital epidemiology and infection control in acute-care settings.Clin. Microbiol. Rev. 2011; 24: 141-173Crossref PubMed Scopus (392) Google Scholar] all utilize siderophores as important augmenters of infection and commit an abundance of resources to their production. Utilizing metabolites such as proteogenic and nonproteogenic amino acids, chorismate, and citrate, bacteria create a diverse repertoire of molecules through a ribosome-independent process [15Hider R.C. Kong X. Chemistry and biology of siderophores.Nat. Prod. Rep. 2010; 27: 637-657Crossref PubMed Scopus (1040) Google Scholar]. Siderophores may be classified according to their iron-binding moieties: catecholate, hydroxamate, phenolate, carboxylate, and mixed-type (containing more than one of the aforementioned moieties) (Figure 1A). For example, staphyloferrin A, produced by S. aureus, is a carboxylate-type with four carboxylate (R-COO−) moieties for iron binding (Figure 1B). More complicated structures also exist: pyoverdine, a mixed-type siderophore produced by P. aeruginosa, is composed of eight amino acids including: D-Ser, L-Arg, L-hf-Orn (N5-formyl-N5-hydroxyornithine), L-Lys, and L-Thr. Pyoverdine binds iron via two hydroxamate (O=CH–NROH) and one catecholate moiety (Figure 1B). Following iron capture, ferric-siderophores are imported into bacteria through siderophore-specific receptors (Table 1 and Box 2) for utilization of iron in growth and colonization during infection.Table 1Nosocomial Pathogens and their SiderophoresNosocomial pathogensTypes of diseaseAntibiotic resistanceSiderophoresSiderophore receptor (OMR)Gram-negativePseudomonas aeruginosaPneumoniaSepticemiaUrinary tract infectionCarbapenemPyoverdineFpvAPyochelinFptAKlebsiella pneumoniaeWound infectionsPneumoniaSepticemiaUrinary tract infectionsCarbapenemAerobactinIutAYersiniabactinFyuAEnterobactinFepASalmochelinIroNAcinetobacter baumanniiPneumoniaWound infectionsBone infectionsCarbapenemAcinetobactinBauAFimsbactinN.d.aN.d., not determined.BaumannoferrinsN.d.Escherichia coliUrinary tract infectionsWound infectionsSepticemiaFluoroquinoloneCarbapenemCephalosporinEnterobactinFepASalmochelinIroNAerobactinIutAYersiniabactinFyuAGram-positiveStaphylococcus aureusSepticemiaGastrointestinal infectionsUrinary tract infectionsWound infectionsPneumoniaOsteomyelitisEndocarditisMethicillinVancomycinStaphyloferrin AHtsAStaphyloferrin BSirAStaphylopineCntAMycobacteriumMycobacterium tuberculosisTuberculosisIsoniazidRifampicinEthambutolPyrazinamideStreptomycinOfloxacinKanamycinMycobactinN.d.CarboxymycobactinN.d.a N.d., not determined. Open table in a new tab Box 2Siderophore Import and Secretion MechanismsIn Gram-negative bacteria, each ferric-siderophore complex is recognized by a specific outer-membrane receptor(s) (OMR) (Figure I top left; specific OMRs are listed in Table 1). In E. coli, enterobactin is recognized by the OMR FepA. While OMRs are extremely diverse, with different bacterial species and different siderophore classes having specific receptors (Table 1), in general OMRs interact with the inner-membrane protein TonB to facilitate uptake of the ferric-siderophore complex. The current model (termed ‘rotational surveillance and energy transfer’, ROSET) suggests that TonB, driven by the inner-membrane proteins ExbB and ExbD as well as by the electrochemical proton motive force generated in the periplasm during normal cellular respiration, physically rotates within the inner membrane, causing a conformational shift in the OMR. The conformational shift of TonB in the OMR promotes internalization of the ferric-siderophore complex [70Klebba P.E. ROSET model of TonB action in Gram-negative bacterial iron acquisition.J. Bacteriol. 2016; 198: 1013-1021Crossref PubMed Scopus (32) Google Scholar]. Once enterobactin is transported across the outer membrane, the periplasmic binding protein (PBP) FepB shuttles it to the inner membrane, where a complex consisting of FepC, FepD, and FepG transports enterobactin into the cytoplasm (Figure I, top left). Other siderophores may have slightly different uptake mechanisms. Following uptake by the OMR, the ferric-pyoverdine complex in P. aeruginosa is split into its constituent components within the periplasm by the action of the PBPs FpvC and FpvF. The iron is pumped into the cytoplasm by the ABC transporter complex FpvDE, and iron-free pyoverdine is secreted back into the environment to gather more iron (reviewed in [16Schalk I.J. Guillon L. Fate of ferrisiderophores after import across bacterial outer membranes: different iron release strategies are observed in the cytoplasm or periplasm depending on the siderophore pathways.Amino Acids. 2013; 44: 1267-1277Crossref PubMed Scopus (105) Google Scholar]).Figure ISiderophore Import and Secretion Mechanisms. (Top left) Siderophore import in Gram-negative bacteria is a multistep process, involving recognition of the ferric-siderophore by a specific OMR, followed by TonB-dependent uptake into the periplasm. The ferric-siderophore is then trafficked by a PBP to the inner membrane, where an ABC transporter pumps it into the cytoplasm. (Top right) Siderophore import in Gram-positive bacteria involves recognition by a siderophore binding protein (SBP) located on the cell membrane. An associated permease is responsible for ferric-siderophore transport across the membrane. (Bottom) Enterobactin secretion in Gram-negative bacteria involves transport from the cytoplasm to the periplasmic space through major facilitator subtype (MFS) proteins. Transport across the outer membrane involves a TolC complex and an associated resistance/nodulated/cell division (RND) efflux pump.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Gram-positive bacteria, such as S. aureus, have only a single membrane and therefore possess a simpler siderophore-uptake system. In general, Gram-positive bacteria express siderophore-binding protein (SBP), associated with a permease. SPB binding to an extracellular ferric-siderophore causes a conformational change in the SBP–permease complex, allowing transport of the ferric-siderophore across the membrane into the cytoplasm. In S. aureus, carboxylate-type siderophores staphyloferrin A and staphyloferrin B are recognized by membrane-bound lipoproteins HtsA and SirA, respectively. Upon staphyloferrin binding, these proteins undergo a conformational change that activates the permeases HtsBC or SirBC, allowing staphyloferrin to cross the membrane (Figure I, top right).Siderophore secretion is a necessary step in bacterial iron acquisition. Perhaps the best-characterized siderophore secretion pathway is that of enterobactin in E. coli, which is a multistep process. First, enterobactin is transported from the cytoplasm to the periplasm via EntS (MFS-class efflux pump). Next, the concerted action of one inner-membrane RND-class efflux pump (AcrB, AcrD, or MdtABC) and the outer-membrane protein channel TolC transport enterobactin across the outer membrane [71Horiyama T. Nishino K. AcrB, AcrD, and MdtABC multidrug efflux systems are involved in enterobactin export in Escherichia coli.PLoS One. 2014; 9: e108642Crossref PubMed Scopus (74) Google Scholar] (Figure I bottom). In Gram-negative bacteria, each ferric-siderophore complex is recognized by a specific outer-membrane receptor(s) (OMR) (Figure I top left; specific OMRs are listed in Table 1). In E. coli, enterobactin is recognized by the OMR FepA. While OMRs are extremely diverse, with different bacterial species and different siderophore classes having specific receptors (Table 1), in general OMRs interact with the inner-membrane protein TonB to facilitate uptake of the ferric-siderophore complex. The current model (termed ‘rotational surveillance and energy transfer’, ROSET) suggests that TonB, driven by the inner-membrane proteins ExbB and ExbD as well as by the electrochemical proton motive force generated in the periplasm during normal cellular respiration, physically rotates within the inner membrane, causing a conformational shift in the OMR. The conformational shift of TonB in the OMR promotes internalization of the ferric-siderophore complex [70Klebba P.E. ROSET model of TonB action in Gram-negative bacterial iron acquisition.J. Bacteriol. 2016; 198: 1013-1021Crossref PubMed Scopus (32) Google Scholar]. Once enterobactin is transported across the outer membrane, the periplasmic binding protein (PBP) FepB shuttles it to the inner membrane, where a complex consisting of FepC, FepD, and FepG transports enterobactin into the cytoplasm (Figure I, top left). Other siderophores may have slightly different uptake mechanisms. Following uptake by the OMR, the ferric-pyoverdine complex in P. aeruginosa is split into its constituent components within the periplasm by the action of the PBPs FpvC and FpvF. The iron is pumped into the cytoplasm by the ABC transporter complex FpvDE, and iron-free pyoverdine is secreted back into the environment to gather more iron (reviewed in [16Schalk I.J. Guillon L. Fate of ferrisiderophores after import across bacterial outer membranes: different iron release strategies are observed in the cytoplasm or periplasm depending on the siderophore pathways.Amino Acids. 2013; 44: 1267-1277Crossref PubMed Scopus (105) Google Scholar]). Gram-positive bacteria, such as S. aureus, have only a single membrane and therefore possess a simpler siderophore-uptake system. In general, Gram-positive bacteria express siderophore-binding protein (SBP), associated with a permease. SPB binding to an extracellular ferric-siderophore causes a conformational change in the SBP–permease complex, allowing transport of the ferric-siderophore across the membrane into the cytoplasm. In S. aureus, carboxylate-type siderophores staphyloferrin A and staphyloferrin B are recognized by membrane-bound lipoproteins HtsA and SirA, respectively. Upon staphyloferrin binding, these proteins undergo a conformational change that activates the permeases HtsBC or SirBC, allowing staphyloferrin to cross the membrane (Figure I, top right). Siderophore secretion is a necessary step in bacterial iron acquisition. Perhaps the best-characterized siderophore secretion pathway is that of enterobactin in E. coli, which is a multistep process. First, enterobact}, number={12}, journal={TRENDS IN MOLECULAR MEDICINE}, author={Wilson, Briana R. and Bogdan, Alexander R. and Miyazawa, Masaki and Hashimoto, Kazunori and Tsuji, Yoshiaki}, year={2016}, month={Dec}, pages={1077–1090} }
 @article{huang_miyazawa_tsuji_2014, title={Distinct regulatory mechanisms of the human ferritin gene by hypoxia and hypoxia mimetic cobalt chloride at the transcriptional and post-transcriptional levels}, volume={26}, ISSN={["1873-3913"]}, DOI={10.1016/j.cellsig.2014.08.018}, abstractNote={Cobalt chloride has been used as a hypoxia mimetic because it stabilizes hypoxia inducible factor-1α (HIF1-α) and activates gene transcription through a hypoxia responsive element (HRE). However, differences between hypoxia and hypoxia mimetic cobalt chloride in gene regulation remain elusive. Expression of ferritin, the major iron storage protein, is regulated at the transcriptional and posttranscriptional levels through DNA and RNA regulatory elements. Here we demonstrate that hypoxia and cobalt chloride regulate ferritin heavy chain (ferritin H) expression by two distinct mechanisms. Both hypoxia and cobalt chloride increased HIF1-α but a putative HRE in the human ferritin H gene was not activated. Instead, cobalt chloride but not hypoxia activated ferritin H transcription through an antioxidant responsive element (ARE), to which Nrf2 was recruited. Intriguingly, cobalt chloride downregulated ferritin H protein expression while it upregulated other ARE-regulated antioxidant genes in K562 cells. Further characterization demonstrated that cobalt chloride increased interaction between iron regulatory proteins (IRP1 and IRP2) and iron responsive element (IRE) in the 5'UTR of ferritin H mRNA, resulting in translational block of the accumulated ferritin H mRNA. In contrast, hypoxia had marginal effect on ferritin H transcription but increased its translation through decreased IRP1-IRE interaction. These results suggest that hypoxia and hypoxia mimetic cobalt chloride employ distinct regulatory mechanisms through the interplay between DNA and mRNA elements at the transcriptional and post-transcriptional levels.}, number={12}, journal={CELLULAR SIGNALLING}, author={Huang, Bo-Wen and Miyazawa, Masaki and Tsuji, Yoshiaki}, year={2014}, month={Dec}, pages={2702–2709} }
 @article{miyazawa_tsuji_2014, title={Evidence for a novel antioxidant function and isoform-specific regulation of the human p66Shc gene}, volume={25}, ISSN={["1939-4586"]}, DOI={10.1091/mbc.e13-11-0666}, abstractNote={The mammalian Shc family, composed of p46, p52, and p66 isoforms, serves as an adaptor protein in cell growth and stress response. p66Shc was shown to be a negative lifespan regulator by acting as a prooxidant protein in mitochondria; however, the regulatory mechanisms of p66Shc expression and function are incompletely understood. This study provides evidence for new features of p66Shc serving as an antioxidant and critical protein in cell differentiation. Unique among the Shc family, transcription of p66Shc is activated through the antioxidant response element (ARE)–nuclear factor erythroid 2–related factor 2 (Nrf2) pathway in K562 human erythroleukemia and other cell types after treatment with hemin, an iron-containing porphyrin. Phosphorylated p66Shc at Ser-36, previously reported to be prone to mitochondrial localization, is increased by hemin treatment, but p66Shc remains exclusively in the cytoplasm. p66Shc knockdown inhibits hemin-induced erythroid differentiation, in which reactive oxygen species production and apoptosis are significantly enhanced in conjunction with suppression of other ARE-dependent antioxidant genes. Conversely, p66Shc overexpression is sufficient for inducing erythroid differentiation. Collectively these results demonstrate the isoform-specific regulation of the Shc gene by the Nrf2-ARE pathway and a new antioxidant role of p66Shc in the cytoplasm. Thus p66Shc is a bifunctional protein involved in cellular oxidative stress response and differentiation.}, number={13}, journal={MOLECULAR BIOLOGY OF THE CELL}, author={Miyazawa, Masaki and Tsuji, Yoshiaki}, year={2014}, month={Jul}, pages={2116–2127} }