Iron Transport Proteins: Gateways of Cellular and Systemic Iron Homeostasis

Iron transport proteins: Gateways of cellular and systemic iron homeostasis

Mitchell D. Knutson, PhD University of Florida Essential Vocabulary

Fe Heme Membrane Transport DMT1 FLVCR Ferroportin HRG1 Mitoferrin Nramp1 ZIP14

Transferrin receptor 1

Cytosolic Transport PCBP1, PCBP2 Timeline of identification in mammalian iron transport

Year Protein Original Publications 1947 Transferrin Laurell and Ingelman, Acta Chem Scand 1959 Transferrin receptor 1 Jandl et al., J Clin Invest 1997 DMT1 Gunshin et al., Nature; Fleming et al. Nature Genet. 1999 Nramp1 Barton et al., J Leukocyt Biol 2000 Ferroportin Donovan et al., Nature; McKie et al., Cell; Abboud et al. J. Biol Chem 2004 FLVCR Quigley et al., Cell 2006 Mitoferrin Shaw et al., Nature 2006 ZIP14 Liuzzi et al., Proc Natl Acad Sci USA 2008 PCBP1, PCBP2 Shi et al., Science 2013 HRG1 White et al., Cell Metab DMT1 (SLC11A2)

• Divalent metal-ion transporter-1

• Former names: Nramp2, DCT1 Fleming et al. Nat Genet, 1997; Gunshin et al., Nature 1997

• Mediates uptake of Fe2+, Mn2+, Cd2+

• H+ coupled transporter (cotransporter, symporter)

• Main roles: • intestinal iron absorption Illing et al. JBC, 2012 • iron assimilation by erythroid cells DMT1 (SLC11A2)

Yanatori et al. BMC Cell Biology 2010

• 4 different isoforms: 557 – 590 a.a. (hDMT1) Hubert & Hentze, PNAS, 2002 • Function similarly in iron transport • Differ in tissue/subcellular distribution and regulation • Regulated by iron: transcriptionally (via HIF2α) post-transcriptionally (via IRE) IRE = Iron-Responsive Element Enterocyte

Lumen

DMT1 Fe2+ Fe2+

Portal blood Enterocyte

Lumen

DMT1 Fe2+ Fe2+ Fe2+ Fe2+ Ferroportin

Portal blood Ferroportin (SLC40A1)

• Only known mammalian iron exporter Donovan et al., Nature 2000; McKie et al., Cell 2000; Abboud et al. J. Biol Chem 2000

• Former names: IREG1, MTP1

• 572 a.a. (human FPN), 62 kDa, 12 TM domains

• Regulated by iron: transcriptionally post-transcriptionally (via IRE) systemically (via hepcidin)

Hepcidin

• 25 a.a. peptide hormone produced by the liver Krause et al. FEBS Lett, 2000; Park et al. JBC, 2001; Pigeon et al; JBC, 2001

Jordan et al. JBC, 2009

• Main regulator of systemic iron homeostasis

• Binds to ferroportin, causing its internalization

and degradation Nemeth et al. Science, 2004

Regulation of iron absorption

Lumen Hepcidin

DMT1 Fe2+ Fe2+ Fe2+ Fe2+ Ferroportin

Portal blood Iron absorption in iron deficiency

Lumen α HIF2- Hepcidin Fe2+ Fe2+

DMT1 Fe2+ Fe2+ Fe2+ Fe2+ HIF2-α Ferroportin Fe2+ Fe2+

HIF2-α Fe2+ Fe2+

Portal blood Intestinal iron absorption

Lumen Fe3+ Hephaestin ferroxidase

DMT1 Fe2+ Fe2+ Fe2+ Fe2+ Ferroportin

Dcytb ferrireductase Fe3+ Portal blood Transferrin

• Iron transport protein in blood plasma

• Binds 1 or 2 atoms of iron as Fe3+ (pH dependent)

• Synthesized by the liver

• 80-kDa glycoprotein with two lobes (N and C) Transferrin

N-lobe C-lobe

Holo-TF = TF with Fe https://pdb101.rcsb.org/motm/35 Apo-TF = TF without Fe Mathies et al. J Biol Inorg Chem. 2015 Transferrin receptor 1 (TFR1)

• Membrane glycoprotein homodimer

TF • Each TFR1 binds 2 molecules of TF

• Binds holo-TF better than apo-TF TFR1

• Ubiquitously expressed TFR1-mediated endocytosis of TF

Holo-TF Apo-TF

TFR1 Endosome STEAP3 Reductase

H+ ○○ H+ ○ ○ DMT1 Erythroid cells The most avid consumers of TF-bound iron

Muckenthaler et al. Cell, 2017 Erythroid cell

○ ○ Mitoferrin 1

○ ○ Heme Mitoferrin 1 (SLC25A37)

• Member of the SLC25 family (MC, mitochondrial carrier) Shaw et al., Nature, 2006 • Localizes to inner mitochondrial membrane

• 338 a.a. (human), 37 kDa, 6 TM domains

• Transports iron into mitochondria

• rMfrn1 can transport Fe2+, Mn2+, Cd2+, Zn2+, Cu1+

Christenson et al. JBC 2018 Mitoferrin 2 (SLC25A28)

• 364 a.a. (human), 39 kDa, 6 TM domains

• Expressed in non-erythroid cells Paradkar et al. Mol Cell Biol. 2009

• In vivo function remains to be determined Erythroid cell

○ ○ Mitoferrin 1

○ ○ Heme

FLVCR1b Hemoglobin FLVCR1 (SLC49A1)

• Feline leukemia virus subgroup C cellular receptor Quigley et al., Cell 2004 • Member of the major facilitator superfamily

• Exports heme FLVCR1 isoforms

plasma membrane

mitochondrial membrane

Chiabrando et al. JCI 2012 Erythroid cell

○ ○ Mitoferrin 1

○ ○ Heme

FLVCR1b Hemoglobin FLVCR1a Erythroid cells The most avid consumers of TF-bound iron

Muckenthaler et al. Cell, 2017 Macrophage (of spleen, liver, bone marrow)

Senescent erythrocyte

Phagosome

HRG1

FPN HMOX1 Phagolysosome HRG-1 (SLC48A1)

• Heme-responsive gene 1 Rajagopal et al., Nature. 2008

• 146 a.a. (human), 17 kDa, 4 TM

Kahn & Quigley., Mol Asp Med 2013

• Transports heme from phagolysosome into cytosol. White et al., Cell Metab. 2013

Macrophage (of spleen, liver, bone marrow)

Senescent erythrocyte

Phagosome

HRG1 Nramp1

FPN HMOX1 Phagolysosome Nramp1 (SLC11A1)

• Natural resistance-associated macrophage protein-1 Vidal et al., Cell 1993

• Homogolous to DMT1: Nramp1 = SLC11A1 Nramp2 = SLC11A2 = DMT1

• Localizes intracellularly (lysosomal membranes)

• Transports iron from phagolysosome to cytosol Soe-Lin et al., PNAS, 2009

• Often associated with immunity Essential Vocabulary

Fe Heme Membrane Transport DMT1 FLVCR Ferroportin HRG1 Mitoferrin Nramp1 ZIP14

Serum Transport Transferrin

Transferrin receptor 1

Cytosolic Transport PCBP1, PCBP2 PCBPs

• poly rC-binding proteins

• Cytosolic/nuclear iron chaperone proteins Shi et al., Science, 2008

• Deliver iron to ferritin and iron enzymes

PCBP1 erythroid cells

PCBP2 binds to DMT1 and FPN Erythroid cell

PCBP1 Ferritin

○ ○ Mitoferrin 1

○ ○ Heme

FLVCR1b Hemoglobin FLVCR1a Essential Vocabulary

Fe Heme Membrane Transport DMT1 FLVCR Ferroportin HRG1 Mitoferrin Nramp1 ZIP14

Serum Transport Transferrin

Transferrin receptor 1

Cytosolic Transport PCBP1, PCBP2 Cellular uptake of iron

Transferrin-bound iron Non-transferrin-bound iron Hepatocytes Pancreatic acinar cells NTBI Cardiomyocytes

Reductase ZIP14 (SLC39A14)

• Member of the ZIP family of metal-ion transporters

• Can transport Zn2+, Fe2+, Mn2+, Cd2+ Taylor et al., FEBS Letters, 2005; Liuzzi et al., PNAS, 2006; Pinilla-Tenas, AJP, 2011

• Transports Fe2+ optimally at pH 7.4

• Most abundantly expressed in liver, pancreas, heart Roles of SLC39A14 in iron overload

Liver Pancreas

Jenkitkasemwong et al., Cell Metab, 2015 ZIP8 (SLC39A8)

• Can transport Zn2+, Fe2+, Mn2+, Cd2+ Wang et al., JBC, 2012

• Most abundantly expressed in lung and placenta

• May play a role in erythropoiesis Gálvez-Peralta M et al. PLoS One, 2013; Tanimura et al. Dev Cell. 2018

• In vivo roles in NTBI uptake remain to be defined Essential Vocabulary

Fe Heme Membrane Transport DMT1 FLVCR Ferroportin HRG1 Mitoferrin Nramp1 ZIP14

Serum Transport Transferrin

Transferrin receptor 1

Cytosolic Transport PCBP1, PCBP2 Timeline of identification in mammalian iron transport

Year Protein # of PubMed citations 1947 Transferrin 14764 1959 Transferrin receptor 1 5156 1997 DMT1 1000

1999 Nramp1 146 2000 Ferroportin 1691

2004 FLVCR 55 2006 Mitoferrin 55 2006 ZIP14 125 2008 PCBP1, PCBP2 188 2013 HRG1 18 TF

Ferroportin

TFR1

DMT1 ZIP14 PCBP

# of #of BioIron2019 talks or posters Mitoferrin, HRG1, FLVCR

# of PubMed citations FPN membrane topology

Aschemeyer et al. Blood, 2018 FPN crystal structure (bacterial)

Taniguchi et al. Nat Comm, 2015 FPN transport mechanism

Extracellular

Intracellular

Taniguchi et al. Nat Comm, 2015 Hepcidin-FPN interaction

Ubiquitination Internalization Degradation

Block transport

Taniguchi et al. Nat Comm, 2015 Revised FPN transport mechanism

• Fe2+ binding site likely in TM7 (vs.TM1–6 as originally reported).

Deshpande et al. Nat Comm, 2018

• Ca2+ binding is required for Fe2+ transport

(NB: This slide was added in response to comment by Tom Ganz during Q & A session) Iron transport proteins: Gateways of cellular and systemic iron homeostasis

Mitchell D. Knutson, PhD University of Florida

Thank you