Uptake mechanism of iron-phytosiderophore from the soil based on the structure of yellow stripe transporter

植物が根から鉄を吸収する機構の解明 --不良土壌を改善する次世代肥料の開発に期待--. 京都大学プレスリリース. 2022-12-08.Calcareous soils cover one-third of all land and cause severe growth defects in plants due to the poor water solubility of iron at high pH. Poaceae species use a unique chelation strategy, whereby plants secrete a high-affinity metal chelator, known as phytosiderophores (mugineic acids), and reabsorb the iron-phytosiderophore complex by the yellow stripe 1/yellow stripe 1-like (YS1/YSL) transporter for efficient uptake of iron from the soil. Here, we present three cryo-electron microscopy structures of barley YS1 (HvYS1) in the apo state, in complex with an iron-phytosiderophore complex, Fe(III)-deoxymugineic acid (Fe(III)–DMA), and in complex with the iron-bound synthetic DMA analog (Fe(III)–PDMA). The structures reveal a homodimeric assembly mediated through an anti-parallel β-sheet interaction with cholesterol hemisuccinate. Each protomer adopts an outward open conformation, and Fe(III)–DMA is bound near the extracellular space in the central cavity. Fe(III)–PDMA occupies the same binding site as Fe(III)–DMA, demonstrating that PDMA can function as a potent fertilizer in an essentially identical manner to DMA. Our results provide a structural framework for iron-phytosiderophore recognition and transport by YS1/YSL transporters, which will enable the rational design of new, high-potency fertilizers

Extracellular SOD-Derived H2O2 Promotes VEGF Signaling in Caveolae/Lipid Rafts and Post-Ischemic Angiogenesis in Mice

Reactive oxygen species (ROS), in particular, H2O2, is essential for full activation of VEGF receptor2 (VEGFR2) signaling involved in endothelial cell (EC) proliferation and migration. Extracellular superoxide dismutase (ecSOD) is a major secreted extracellular enzyme that catalyzes the dismutation of superoxide to H2O2, and anchors to EC surface through heparin-binding domain (HBD). Mice lacking ecSOD show impaired postnatal angiogenesis. However, it is unknown whether ecSOD-derived H2O2 regulates VEGF signaling. Here we show that gene transfer of ecSOD, but not ecSOD lacking HBD (ecSOD-ΔHBD), increases H2O2 levels in adductor muscle of mice, and promotes angiogenesis after hindlimb ischemia. Mice lacking ecSOD show reduction of H2O2 in non-ischemic and ischemic limbs. In vitro, overexpression of ecSOD, but not ecSOD-ΔHBD, in cultured medium in ECs enhances VEGF-induced tyrosine phosphorylation of VEGFR2 (VEGFR2-pY), which is prevented by short-term pretreatment with catalase that scavenges extracellular H2O2. Either exogenous H2O2 (<500 µM), which is diffusible, or nitric oxide donor has no effect on VEGF-induced VEGFR2-pY. These suggest that ecSOD binding to ECs via HBD is required for localized generation of extracellular H2O2 to regulate VEGFR2-pY. Mechanistically, VEGF-induced VEGFR2-pY in caveolae/lipid rafts, but non-lipid rafts, is enhanced by ecSOD, which localizes at lipid rafts via HBD. One of the targets of ROS is protein tyrosine phosphatases (PTPs). ecSOD induces oxidation and inactivation of both PTP1B and DEP1, which negatively regulates VEGFR2-pY, in caveolae/lipid rafts, but not non-lipid rafts. Disruption of caveolae/lipid rafts, or PTPs inhibitor orthovanadate, or siRNAs for PTP1B and DEP1 enhances VEGF-induced VEGFR2-pY, which prevents ecSOD-induced effect. Functionally, ecSOD promotes VEGF-stimulated EC migration and proliferation. In summary, extracellular H2O2 generated by ecSOD localized at caveolae/lipid rafts via HBD promotes VEGFR2 signaling via oxidative inactivation of PTPs in these microdomains. Thus, ecSOD is a potential therapeutic target for angiogenesis-dependent cardiovascular diseases

Copper Transport Protein Antioxidant-1 Promotes Inflammatory Neovascularization via Chaperone and Transcription Factor Function

Supplementary information accompanies this paper at http://www.nature.com/srepCopper (Cu), an essential micronutrient, plays a fundamental role in inflammation and angiogenesis; however, its precise mechanism remains undefined. Here we uncover a novel role of Cu transport protein Antioxidant-1 (Atox1), which is originally appreciated as a Cu chaperone and recently discovered as a Cu-dependent transcription factor, in inflammatory neovascularization. Atox1 expression is upregulated in patients and mice with critical limb ischemia. Atox1-deficient mice show impaired limb perfusion recovery with reduced arteriogenesis, angiogenesis, and recruitment of inflammatory cells. In vivo intravital microscopy, bone marrow reconstitution, and Atox1 gene transfer in Atox1(-/-) mice show that Atox1 in endothelial cells (ECs) is essential for neovascularization and recruitment of inflammatory cells which release VEGF and TNFα. Mechanistically, Atox1-depleted ECs demonstrate that Cu chaperone function of Atox1 mediated through Cu transporter ATP7A is required for VEGF-induced angiogenesis via activation of Cu enzyme lysyl oxidase. Moreover, Atox1 functions as a Cu-dependent transcription factor for NADPH oxidase organizer p47phox, thereby increasing ROS-NFκB-VCAM-1/ICAM-1 expression and monocyte adhesion in ECs inflamed with TNFα in an ATP7A-independent manner. These findings demonstrate a novel linkage between Atox1 and NADPH oxidase involved in inflammatory neovascularization and suggest Atox1 as a potential therapeutic target for treatment of ischemic disease.SS is a British Heart Foundation (BHF) PhD student; GDA is BHF Chair in cardiac surgery and NIHR Senior Investigator; CE is a BHF Senior Research Fellow. Sources of Funding: This research was supported by NIH R01 HL070187 (T.F.), Department of Veterans Affairs Merit Review grant 1I01BX001232 (T.F.), R01HL116976 (T.F., M.U.-F.), NIH R01 HL077524 and HL077524-S1, R21HL112293 (to M.U.-F.), Ruth L. Kirschstein-National Service Research Award (Kirschstein-NRSA) T32 Training Grant (to G-F.C.), AHA Post-doctoral Fellowship 09POST2250151 (to N.U.), and 11POST5740006 (to V.S.).Peer-reviewedPublisher Versio

IQGAP1 Is Involved in Post-Ischemic Neovascularization by Regulating Angiogenesis and Macrophage Infiltration

Neovascularization is an important repair mechanism in response to ischemic injury and is dependent on inflammation, angiogenesis and reactive oxygen species (ROS). IQGAP1, an actin-binding scaffold protein, is a key regulator for actin cytoskeleton and motility. We previously demonstrated that IQGAP1 mediates vascular endothelial growth factor (VEGF)-induced ROS production and migration of cultured endothelial cells (ECs); however, its role in post-ischemic neovascularization is unknown.Ischemia was induced by left femoral artery ligation, which resulted in increased IQGAP1 expression in Mac3(+) macrophages and CD31(+) capillary-like ECs in ischemic legs. Mice lacking IQGAP1 exhibited a significant reduction in the post-ischemic neovascularization as evaluated by laser Doppler blood flow, capillary density and α-actin positive arterioles. Furthermore, IQGAP1(-/-) mice showed a decrease in macrophage infiltration and ROS production in ischemic muscles, leading to impaired muscle regeneration and increased necrosis and fibrosis. The numbers of bone marrow (BM)-derived cells in the peripheral blood were not affected in these knockout mice. BM transplantation revealed that IQGAP1 expressed in both BM-derived cells and tissue resident cells, such as ECs, is required for post-ischemic neovascularization. Moreover, thioglycollate-induced peritoneal macrophage recruitment and ROS production were inhibited in IQGAP1(-/-) mice. In vitro, IQGAP1(-/-) BM-derived macrophages showed inhibition of migration and adhesion capacity, which may explain the defective macrophage recruitment into the ischemic tissue in IQGAP1(-/-) mice.IQGAP1 plays a key role in post-ischemic neovascularization by regulating, not only, ECs-mediated angiogenesis but also macrophage infiltration as well as ROS production. Thus, IQGAP1 is a potential therapeutic target for inflammation- and angiogenesis-dependent ischemic cardiovascular diseases

Superoxide Dismutases: Role in Redox Signaling, Vascular Function and Diseases

Excessive reactive oxygen species Revised abstract, especially superoxide anion (O2•−), play important roles in the pathogenesis of many cardiovascular diseases, including hypertension and atherosclerosis. Superoxide dismutases (SODs) are the major antioxidant defense systems against O2•−, which consist of three isoforms of SOD in mammals: the cytoplasmic Cu/ZnSOD (SOD1), the mitochondrial MnSOD (SOD2), and the extracellular Cu/ZnSOD (SOD3), all of which require catalytic metal (Cu or Mn) for their activation. Recent evidence suggests that in each subcellular location, SODs catalyze the conversion of O2•− H2O2, which may participate in cell signaling. In addition, SODs play a critical role in inhibiting oxidative inactivation of nitric oxide, thereby preventing peroxynitrite formation and endothelial and mitochondrial dysfunction. The importance of each SOD isoform is further illustrated by studies from the use of genetically altered mice and viral-mediated gene transfer. Given the essential role of SODs in cardiovascular disease, the concept of antioxidant therapies, that is, reinforcement of endogenous antioxidant defenses to more effectively protect against oxidative stress, is of substantial interest. However, the clinical evidence remains controversial. In this review, we will update the role of each SOD in vascular biologies, physiologies, and pathophysiologies such as atherosclerosis, hypertension, and angiogenesis. Because of the importance of metal cofactors in the activity of SODs, we will also discuss how each SOD obtains catalytic metal in the active sites. Finally, we will discuss the development of future SOD-dependent therapeutic strategies. Antioxid. Redox Signal. 15, 000–000