c-Myc represses the murine Nrampl promoter

2002 ◽  
Vol 30 (4) ◽  
pp. 774-777 ◽  
Author(s):  
H. Bowen ◽  
T. E. Biggs ◽  
S. T. Baker ◽  
E. Phillips ◽  
V. H. Perry ◽  
...  
Keyword(s):  
Binding Sites ◽  
Natural Resistance ◽  
Intracellular Pathogens ◽  
Flanking Sequence ◽  
Inhibitory Effects ◽  
Late Endosomes ◽  
E Box ◽  
Macrophage Protein ◽  
Iron Pool ◽  
Divalent Cation Transporter

The Nrampl (natural resistance-associated macrophage protein 1) gene modulates the growth of intracellular pathogens and encodes a divalent cation transporter within lysosomes/late endosomes of macrophages. Nrampl modulates the cytoplasmic iron pool. Wu, Polack and Dalla-Favera [(1999) Science 283, 676–679] showed reciprocal control of H-ferritin and IRP2 by c-Myc, and suggest that c-Myc regulates genes to increase cytoplasmic iron. A role for c-Myc in Nrampl regulation was evaluated. Co-transfection studies show that c-Myc represses Nrampl promoter function. Five non-canonical Myc-max binding sites (E-box) identified within the Nrampl 5′-flanking sequence are not responsible for the inhibitory effects of c-Myc on Nrampl expression. An initiator(s) adjacent to the transcriptioninitiation site is a candidate for the inhibition observed. Results are consistent with a role for Nrampl removing iron from the cytosol and antagonizing c-Myc function.

scholarly journals Leishmania infection triggers hepcidin-mediated proteasomal degradation of Nramp1 resulting in increased phagolysosomal iron availability

2019 ◽  
Author(s):  
Sourav Banerjee ◽  
Rupak Datta
Keyword(s):  
Leishmania Major ◽  
Peptide Hormone ◽  
Parasite Growth ◽  
Natural Resistance ◽  
Intracellular Pathogens ◽  
Iron Availability ◽  
Regulatory Peptide ◽  
Ubiquitin Proteasome ◽  
Macrophage Protein ◽  
Iron Pool

AbstractNatural resistance associated macrophage protein 1 (Nramp1) was discovered as a genetic determinant of resistance against multiple intracellular pathogens, including Leishmania. It encodes a transmembrane protein of the phago-endosomal vesicles, where it functions as an iron transporter. But how Nramp1 expression is regulated in an infected macrophage is unknown. Its role in controlling iron availability to the intracellular pathogens and in determining the final outcome of an infection also remains to be fully deciphered. Here we report that Nramp1 protein abundance undergoes temporal changes in Leishmania major infected macrophages. At 12 hours post infection, there was drastic lowering of Nramp1 level accompanied by increased phagolysosomal iron availability and enhanced parasite growth. Leishmania infection-induced downregulation of Nramp1 was found to be caused by ubiquitin-proteasome degradation pathway. In fact, blocking of Nramp1 degradation with proteasome inhibitor resulted in depletion of phagolysosomal iron pool with significant reduction in the number of intracellular parasites. Further, we uncovered that this degradation process is mediated by the iron regulatory peptide hormone hepcidin that binds to Nramp1. Interestingly, Nramp1 protein level was restored to normalcy after 30 hours of infection with a concomitant drop in the phagolysosomal iron level, which is suggestive of a host counter defense strategy to deprive the pathogen of this essential micronutrient. Taken together, our study implicates Nramp1 as a central player in the host-pathogen battle for iron. It also unravels Nramp1 as a novel partner for hepcidin. The hitherto unidentified ‘hepcidin-Nramp1 axis’ may have a broader role in regulating macrophage iron homeostasis.ImportanceLeishmania parasites are the causative agents of a group of neglected tropical diseases called leishmaniasis. They reside within the phagolysosomes of mammalian macrophages. Since iron is an essential micronutrient for survival and virulence, intracellular Leishmania must acquire it from the tightly regulated macrophage iron pool. How this challenging task is accomplished remains a fundamental question in Leishmania biology. We report here that Leishmania major infection caused ubiquitin-proteasome-mediated degradation of natural resistance associated macrophage protein 1 (Nramp1). Nramp1 being an iron exporter at the phago-endosomal membrane, its degradation resulted in increased phagolysosomal iron availability thereby stimulating parasite growth. We also uncovered that Nramp1 degradation is controlled by the iron regulatory peptide hormone hepcidin. Interestingly, at a later stage of infection, Nramp1 protein level was restored to normalcy with simultaneous depletion of phagolysosomal iron. Collectively, our study implicates Nramp1 as a central player in the host-pathogen struggle for acquiring iron.

Transcriptional control of Nramp1: a paradigm for the repressive action of c-Myc

2004 ◽  
Vol 32 (6) ◽  
pp. 1084-1086 ◽  
Author(s):  
A.S. Lapham ◽  
E.S. Phillips ◽  
C.H. Barton
Keyword(s):  
Transcriptional Control ◽  
Cell Activation ◽  
Zinc Finger Protein ◽  
Specific Binding ◽  
Divalent Cations ◽  
Natural Resistance ◽  
Finger Protein ◽  
Late Endosomes ◽  
Solute Carrier ◽  
Macrophage Protein

Slc11a1/Nramp1 (solute carrier family 11 member a1/murine natural resistance-associated macrophage protein 1 gene) encodes a divalent cation transporter that resides within lysosomes/late endosomes of macrophages. Nramp1 modulates the cellular distribution of divalent cations in response to cell activation by intracellular pathogens. Nramp1 expression is repressed and activated by the proto-oncogene c-Myc and Miz-1 (c-Myc-interacting zinc finger protein 1) respectively. Here we demonstrate, using a c-Myc mutant (V394D, Val394→Asp) that is incapable of binding Miz-1, that c-Myc repression of Nramp1 transcription is dependent on its interaction with Miz-1. An oligo pull-down assay demonstrates specific binding of recombinant Miz-1 to the Nramp1 Miz-1-binding site or initiator element(s), and Miz-1-dependent c-Myc recruitment.

scholarly journals Host Resistance to Intracellular Infection: Mutation of Natural Resistance-associated Macrophage Protein 1 (Nramp1) Impairs Phagosomal Acidification

1998 ◽  
Vol 188 (2) ◽  
pp. 351-364 ◽  
Author(s):  
David J. Hackam ◽  
Ori D. Rotstein ◽  
Wei-jian Zhang ◽  
Samantha Gruenheid ◽  
Philippe Gros ◽  
...  
Keyword(s):  
Integral Membrane Protein ◽  
Natural Resistance ◽  
Wild Type ◽  
Intracellular Parasites ◽  
Late Endosomes ◽  
Latex Beads ◽  
Buffering Power ◽  
Ratio Imaging ◽  
Macrophage Protein ◽  
Phagosomal Membrane

The mechanisms underlying the survival of intracellular parasites such as mycobacteria in host macrophages remain poorly understood. In mice, mutations at the Nramp1 gene (for natural resistance-associated macrophage protein), cause susceptibility to mycobacterial infections. Nramp1 encodes an integral membrane protein that is recruited to the phagosome membrane in infected macrophages. In this study, we used microfluorescence ratio imaging of macrophages from wild-type and Nramp1 mutant mice to analyze the effect of loss of Nramp1 function on the properties of phagosomes containing inert particles or live mycobacteria. The pH of phagosomes containing live Mycobacterium bovis was significantly more acidic in Nramp1- expressing macrophages than in mutant cells (pH 5.5 ± 0.06 versus pH 6.6 ± 0.05, respectively; P <0.005). The enhanced acidification could not be accounted for by differences in proton consumption during dismutation of superoxide, phagosomal buffering power, counterion conductance, or in the rate of proton “leak”, as these were found to be comparable in wild-type and Nramp1-deficient macrophages. Rather, after ingestion of live mycobacteria, Nramp1-expressing cells exhibited increased concanamycin-sensitive H+ pumping across the phagosomal membrane. This was associated with an enhanced ability of phagosomes to fuse with vacuolar-type ATPase–containing late endosomes and/or lysosomes. This effect was restricted to live M. bovis and was not seen in phagosomes containing dead M. bovis or latex beads. These data support the notion that Nramp1 affects intracellular mycobacterial replication by modulating phagosomal pH, suggesting that Nramp1 plays a central role in this process.

Functional analysis of bovine Nramp1 and production of transgenic cloned embryosin vitro

Zygote ◽  
2013 ◽  
Vol 23 (1) ◽  
pp. 83-92 ◽  
Author(s):  
Xiang Cheng ◽  
Xiaoli Yu ◽  
Yajun Liu ◽  
Jie Deng ◽  
Xiaoling Ma ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Formation Rate ◽  
Natural Resistance ◽  
Intracellular Bacteria ◽  
Intracellular Pathogens ◽  
Qinchuan Cattle ◽  
Polymerase Chain ◽  
Macrophage Protein ◽  
Rectal Palpation

SummaryNatural resistance-associated macrophage protein 1 (Nramp1) plays an important role in restraining the growth of intracellular pathogens within macrophages. In this study,Nramp1cDNA was cloned from Qinchuan cattle and its anti-bacterial activity was demonstrated as being able to significantly inhibit the growth ofSalmonella abortusovisandBrucella abortusin macrophages. Calf fibroblasts stably transfected with pSP–NRAMP1–HA vector were used to reconstruct bovine embryos by somatic cell nuclear transfer (SCNT). Reconstructed embryos were maturatedin vitroand the blastocyst formation rate (14.0%) was similar to that of control embryos (14.5%). Transgenic blastocysts were transplanted into 43 recipient cattle, of which 14 recipients became pregnant as evidenced by non-return estrus and by rectal palpation. One fetus was aborted after 6½ months of pregnancy and transgene integration was confirmed by semi-quantitative polymerase chain reaction. Together, this study showed that bovine Nramp1 retains biological function against the growth of intracellular bacteria and can be used to reconstruct embryos and produceNramp1transgenic cattle, which may benefit the animal and enhance their ability to prevent attack by intracellular pathogens.

scholarly journals Localisation of Nramp1 in macrophages: modulation with activation and infection

1998 ◽  
Vol 111 (19) ◽  
pp. 2855-2866 ◽  
Author(s):  
S. Searle ◽  
N.A. Bright ◽  
T.I. Roach ◽  
P.G. Atkinson ◽  
C.H. Barton ◽  
...  
Keyword(s):  
Macrophage Activation ◽  
Leishmania Major ◽  
Polyclonal Antibodies ◽  
Natural Resistance ◽  
Activated Macrophages ◽  
Wild Type ◽  
Late Endosomes ◽  
Gold Labelling ◽  
Macrophage Protein ◽  
Phagosome Fusion

The murine natural resistance-associated macrophage protein, Nramp1, has multiple pleiotropic effects on macrophage activation and regulates survival of intracellular pathogens including Leishmania, Salmonella and Mycobacterium species. Nramp1 acts as an iron transporter, but precisely how this relates to macrophage activation and/or pathogen survival remains unclear. To gain insight into function, anti-Nramp1 monoclonal and polyclonal antibodies are used here to localise Nramp1 following activation and infection. Confocal microscope analysis in uninfected macrophages demonstrates that both the mutant (infection-susceptible) and wild-type (infection-resistant) forms of the protein localise to the membranes of intracellular vesicular compartments. Gold labelling and electron microscopy defines these compartments more precisely as electron-lucent late endosomal and electron-dense lysosomal compartments, with Nramp1 colocalizing with Lamp1 and cathepsins D and L in both compartments, with macrosialin in late endosomes, and with BSA-5 nm gold in pre-loaded lysosomes. Nramp1 is upregulated with interferon-(gamma) and lipopolysaccaride treatment, coinciding with an increase in labelling in lysosomes relative to late endosomes and apparent dispersion of Nramp1-positive vesicles from a perinuclear location towards the periphery of the cytoplasm along the microtubular network. In both control and activated macrophages, expression of the protein is 3- to 4-fold higher in wild-type compared to mutant macrophages. In Leishmania major-infected macrophages, Nramp1 is observed in the membrane of the pathogen-containing phagosomes, which retain a perinuclear localization in resting macrophages. In Mycobacterium avium-infected resting and activated macrophages, Nramp1-positive vesicles migrated to converge, but not always fuse, with pathogen-containing phagosomes. The Nramp1 protein is thus located where it can have a direct influence on phagosome fusion and the microenvironment of the pathogen, as well as in the more general regulation of endosomal/lysosomal function in macrophages.

Both Nramp1 and Dmt1 Are Necessary for Efficient Macrophage Iron Recycling.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1994-1994
Author(s):  
Shan Soe-Lin ◽  
Sameer S Apte ◽  
Marc R Mikhael ◽  
Lidia Kayembe ◽  
Guangjun Nie ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Natural Resistance ◽  
Heme Oxygenase 1 ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Metal Transporter ◽  
Labile Iron Pool ◽  
Raw264.7 Macrophages ◽  
Macrophage Protein ◽  
Iron Pool

Abstract Abstract 1994 Poster Board I-1016 Divalent metal transporter 1 (DMT1) and natural resistance-associated macrophage protein 1 (Nramp1) are iron transporters that localize, respectively, to the early and late endosomal compartments. DMT1 is ubiquitously expressed, while Nramp1 is found only within macrophages and neutrophils. Our previous studies have identified a role for Nramp1 during macrophage erythrophagocytosis; however, little is known about the function of DMT1 during this process. Wild-type RAW264.7 macrophages (Nramp1-/-), and those stably transfected with Nramp1 (Nramp1+/+) were treated with either DMT1-siRNA, or with ebselen, a selective inhibitor of DMT1. While macrophages lacking either functional DMT1 or Nramp1 experienced a moderate reduction in iron recycling efficiency, the ability of macrophages lacking both functional DMT1 and Nramp1 to recycle hemoglobin-derived iron was severely compromised. Compared to macrophages singly deficient in either DMT1 or Nramp1 transport ability, macrophages where DMT1 and Nramp1 were both compromised exhibited an abrogated increase in labile iron pool content, released less iron, and experienced diminished upregulation of ferroportin and heme-oxygenase 1 levels following erythrophagocytosis. These results suggest that while the loss of either Nramp1 or DMT1 transport ability results in minor impairment following erythrophagocytosis, the simultaneous loss of both Nramp1 and DMT1 iron transport activity is detrimental to the iron recycling capacity of the macrophage. Disclosures: No relevant conflicts of interest to declare.

Genetic analysis of SLC11A1 polymorphisms in multiple sclerosis patients

2004 ◽  
Vol 10 (6) ◽  
pp. 618-620 ◽  
Author(s):  
Manuel Comabella ◽  
Laura Altet ◽  
Francesc Peris ◽  
Pablo Villoslada ◽  
Armand Sánchez ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Natural Resistance ◽  
Bivalent Cation ◽  
Slc11a1 Gene ◽  
Late Endosomes ◽  
Solute Carrier ◽  
Macrophage Protein ◽  
Multiple Sclerosis Patients ◽  
Control Study

Solute carrier 11a1 (SLC11A1; formerly NRAMP1, where NRAMP stands for natural resistance-associated macrophage protein) is a proton/bivalent cation antiporter that localizes to late endosomes/lysosomes. SLC11A1 regulates macrophage functions that are of potential importance in the induction and/or maintenance of autoimmune diseases such as rheumatoid arthritis, type 1 diabetes and Crohn’s disease. We investigated SLC11A1 gene as a candidate gene for genetic susceptibility to multiple sclerosis (MS) in our population. Four SLC11A1 gene polymorphisms (5?GT repeat, D543N, 1729 -55del4 and 1729 -271del4) were analysed in a case-control study of 195 patients with MS and 125 control subjects. We found no evidence of association between SLC11A1 polymorphisms and MS susceptibility in the Spanish population.

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