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Nramp1: a link between intracellular iron transport and innate resistance to intracellular pathogens
C H Barton1, T E Biggs, S T Baker
1Biochemistry and Molecular Biology, University of Southampton, United Kingdom. CHB@Soton.ac.uk
Abstract:
Nramp1 (natural resistance-associated macrophage protein one) regulates intracellular pathogen proliferation and macrophage inflammatory responses. Murine Nramp1 exhibits a natural polymorphism with alleles termed resistant and susceptible. Alleles restrict or allow the proliferation of intracellular pathogens, respectively. Structural predictions suggest that Nramp1 encodes the prototypic member of a transporter family. Nramp1 exhibits sequence identity to Nramp2, which regulates intestinal and reticulocyte iron uptake. Based on this sequence identity we have initiated experiments for Nramp1 to investigate its role in macrophage iron homoeostasis and using a transfection approach in the RAW264.7 murine macrophage-like cell line, which lacks a functional Nramp1 gene. Nramp1 expression supports increased acute cytoplasmic influx of iron, detected using the fluorescent iron sensor dye calcein. Analysis of the endogenous iron sensors, iron regulatory protein 1 and 2, reveals a greater flux of iron in Nramp1-expressing cells and in its exclusion from the cytoplasm. Other work supports the prediction that Nramp1 is a phosphoprotein and the extent of phosphorylation changes in response to inflammatory cytokines. Together these data support the hypothesis that control of intracellular iron homoeostasis is a vital element used by phagocytes to control the proliferation of intracellular pathogens.
Insights
Natural resistance-associated macrophage protein one (Nramp1) regulates iron homeostasis in macrophages. Nramp1 expression enhances iron uptake, impacting pathogen control and inflammatory responses.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Natural resistance-associated macrophage protein one (Nramp1) is crucial for regulating intracellular pathogen proliferation and macrophage inflammatory responses.
- Murine Nramp1 exists in resistant and susceptible alleles, influencing pathogen proliferation.
- Structural predictions indicate Nramp1 is a transporter, sharing sequence identity with Nramp2 involved in iron uptake.
Purpose of the Study:
- To investigate the role of Nramp1 in macrophage iron homeostasis.
- To explore Nramp1's function using a transfection approach in a murine macrophage cell line lacking functional Nramp1.
Main Methods:
- Transfection of RAW264.7 murine macrophage-like cells with Nramp1.
- Detection of intracellular iron influx using the fluorescent dye calcein.
- Analysis of endogenous iron sensors (IRP1 and IRP2) to assess iron flux and cytoplasmic exclusion.
- Investigation of Nramp1 phosphorylation in response to inflammatory cytokines.
Main Results:
- Nramp1 expression led to increased acute cytoplasmic iron influx.
- Nramp1-expressing cells showed greater iron flux and exclusion from the cytoplasm.
- Nramp1 was confirmed as a phosphoprotein, with phosphorylation levels varying with inflammatory cytokines.
Conclusions:
- Nramp1 plays a significant role in regulating macrophage iron homeostasis.
- Control of intracellular iron by Nramp1 is a key mechanism for phagocytes to limit intracellular pathogen proliferation.
- Nramp1's function is modulated by phosphorylation in response to inflammatory signals.