The mechanism of ferritin iron release has been poorly known so far. It has been suggested that iron may exit via the 8 hydrophilic channels on the 3-fold axes of the ferritin shell. However, it is thought that ferritin turnover involves its degradation by lysosomal autophagy or by the proteasome. Recently, the nuclear receptor coactivator 4 (NCOA4) has been described as selective cargo-receptor mediating ferritin degradation by autophagy (ferritinophagy). Mancias J.D. and colleagues (2014) identified NCOA4 as highly enriched protein in autophagosomes using quantitative proteomics and ferritin was recognized as its target. It was first reported a possible mechanism of ferritin iron release involving nuclear receptor coactivator 4. Further studies (Dowdle et al., 2014 ,Mancias et al., 2015; Bellelli et al., 2016) revealed more details about the NCOA4-FTH interaction, including identification of binding sites, NCOA4 as an iron-binding protein and mechanism of its iron-dependent turnover (Fig.1), as well as demonstrated the importance of NCOA4-mediated ferritinophagy in systemic and cellular iron homeostasis.

1. A STUDY OF NCOA4 EXPRESSION AND BINDING TO FERRITINS
Magdalena Gryzik, Fernando Carmona, Maura Poli and Paolo Arosio
Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia, Italy
e-mail: gryzik.m@gmail.com
INTRODUCTION
Fig.1. Proposed model of NCOA4 involvement in ferritinophagy (Mancias et al., 2015).
The mechanism of ferritin iron release has been poorly known so far. It has been suggested that iron may exit via the 8 hydrophilic channels on the 3-fold axes of the ferritin shell. However, it is thought that ferritin turnover involves its degradation by
lysosomal autophagy or by the proteasome. Recently, the nuclear receptor coactivator 4 (NCOA4) has been described as selective cargo-receptor mediating ferritin degradation by autophagy (ferritinophagy).
Mancias J.D. and colleagues (2014) identified NCOA4 as highly enriched protein in autophagosomes using quantitative proteomics and ferritin was recognized as its target. It was first reported a possible mechanism of ferritin iron release involving nuclear
receptor coactivator 4. Further studies (Dowdle et al., 2014 ,Mancias et al., 2015; Bellelli et al., 2016) revealed more details about the NCOA4-FTH interaction, including identification of binding sites, NCOA4 as an iron-binding protein and mechanism of its
iron-dependent turnover (Fig.1), as well as demonstrated the importance of NCOA4-mediated ferritinophagy in systemic and cellular iron homeostasis.
AIMS OF THE WORK
We aim to analyze:
expression level of NCOA4 mRNA in HeLa cells under different cellular conditions
NCOA4-ferritin interaction by in vitro techniques
impact of iron and other factors on NCOA4-ferritin binding
MATERIALS AND METHODS
We used human cervical cancer HeLa cell line treated with:
• ferric ammonium sulfate (FAC, 100µM) or desferoxamine (DFO, 100µM) for 24h,
• hydrogen peroxide (H2O2, 50-100µM) for 24h,
• tamoxifen (TAM, 5µM) or ammonium chloride (NH4Cl, 10mM) for 24h.
The expression of NCOA4 mRNA was analyzed by qRT-PCR.
The huNCOA4 domain (383-522aa) was cloned into pET-12a vector, expressed in E. coli and
purified by metal affinity chromatography followed by ion exchange chromatography.
The NCOA4-ferritin binding was analyzed in electrophoretic mobility shift assay (EMSA) and
enzyme-linked immunosorbent assay (ELISA).
The nuclear receptor coactivator 4 (NCOA4) is a protein of 614 amino acids and about 70 kDa, predominantly localized in the cytoplasm. NCOA4 contains four crucial domains: coiled-coil domain
(17-125aa) located at N-terminus responsible for protein oligomerization, two conserved motifs characteristic for nuclear receptor interaction (LXXLL and FXXLF) and FTH-binding site.
The ferritin binding domain has been localized in the C-terminal region of NCOA4 and this interaction involves the 383-522aa fragment predicted to contain four α-helices and constitute a sub-domain
(Mancias et al., 2015). This domain was found to bind also E3 ubiquitin ligase HERC2 and iron. A more detailed analysis restricted the ferritin binding domain to the region 488-499aa (Fig.2) and it
was shown that it binds ferritin heavy chain, but not ferritin light chain (Mancias et al., 2015).
The crystallographic structures of the ferritins are well-characterized and this facilitated the analysis of the NCOA4 binding site (Fig.3). In the approach used by Mancias J.D. and colleagues (2015) the
outer surface residues conserved in FTH orthologs, but not FTL, were mutated. Among 16 analyzed residues, arginine-23 was found to be crucial for NCOA4-FTH binding (Mancias et al., 2015).
Fig.2. NCOA4 protein with highlighted FTH1 binding site (in red).
Fig.3. H-ferritin with highlighted arginine-23 (in green), a key residue for NCOA4 binding.
Characterization of NCOA4 (383-522aa) and H-Ferritin interactionNCOA4 mRNA expression in HeLa cells Factors affecting NCOA4 (383-522aa) – Ferritin binding
CONCLUSIONS
REFERENCES
1. Arosio and Levi. 2010. Cytosolic and mitochondrial ferritins in the regulation of cellular iron homeostasis and oxidative damage. Biochim Biophys Acta 1800: 783-792
2. Bellelli et al., 2014. NCOA4 Transcriptional Coactivator Inhibits Activation of DNA Replication Origins. Mol Cell 55(1): 123-137
3. Bellelli et al., 2016. NCOA4 Deficiency Impairs Systemic Iron Homeostasis. Cell Rep 14(3): 411-421
4. Dowdle et al., 2014. Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo. Nat Cell Biol 16(11): 1069-1079
5. Kollara and Brown. 2012. Expression and function of nuclear receptor co-activator 4: evidence of a potential role independent of co-activator activity. Cell Mol Life Sci 69: 3895-3909
6. Kollara et al., 2011. Dynamic Distribution of Nuclear Coactivator 4 during Mitosis: Association with Mitotic Apparatus and Midbodies. Plos One 6: e22257
7. Mancias et al., 2014 Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy. Nature 509(7498): 105-109
8. Mancias et al., 2015. Ferritinophagy via NCOA4 is required for erythropoiesis and is regulated by iron dependent HERC2-mediated proteolysis. eLIFE 4:e10308
0
0,2
0,4
0,6
0,8
1
1,2
hNCOA4 mRNA in HeLa cells
foldincreasehNCOA4/hHPRT1
MOCK DFO 100µM FAC 100µM
**
*
0
0,2
0,4
0,6
0,8
1
1,2
hNCOA4 mRNA in HeLa cells
MOCK H2O2 100µMH2O2 50µM
*
foldincreasehNCOA4/hHPRT1
ACTIN
MOCK
H2O250
H2O2100
FTL
TFR1 95kDa
43kDa
20kDa
FTL
TFR1
Actin 43kDa
LC3B-I
LC3B-II
MOCK
TAM
NH4Cl
95kDa
16kDa
20kDa
18kDa
0
0,2
0,4
0,6
0,8
1
1,2
hNCOA4 mRNA in HeLa cells
foldincreasehNCOA4/hHPRT1
MOCK TAM 5µM NH4Cl 10mM
Ferritin level was diminished by iron deprivation conditions and induced by iron
supplementation, whereas the opposite occured with TFR1 level.
Both FAC and DFO treatments modified NCOA4 mRNA expression. The 20%
decrease of NCOA4 transcript level after both treatments was statistically
significant.
Oxidative stress induced by treatment with 50µM and 100µM of hydrogen
peroxide slightly induced FTL level.
In contrast, the level of NCOA4 mRNA decreased after 100µM H2O2 treatment
with a residual expression of about 75% of the control.
Autophagy induction (tamoxifen, TAM) or inhibition of lysosomal degradation
(NH4Cl) increased level of ferritin. The increase of LC3B-II/I level confirmed that
treatments modified autophagy.
Both treatments did not cause significant changes of NCOA4 transcript level.
FTH
FTH+NCOA4
R23A-FTH
R23A-FTH+NCOA4
FTL
FTL+NCOA4
FtMt
FtMt+NCOA4
molar ratio FTH:NCOA4
1:0 1:1 1:2 1:4 1:8 1:12 1:16 1:24 1:32 0:1
-ME - 0.5% 1% 2%
FTH
FTH + NCOA4
FTH
FTH+NCOA4
Urea - 1M 2M 4M 6M
FTH
NaCl - 0.5M 1M 1.5M
FTH+NCOA4
The recombinant NCOA4 domain (383-522aa) was
incubated with recombinant human ferritins: H-chain
(FTH), R23A H-mutant (R23A-FTH), L-chain (FTL) and
mitochondrial ferritin (FtMt) at a molar ratio 24:1.
We found that FTH was retarded by the binding of NCOA4,
while FTL was not affected. Also the mobility of FTH mutant
R23A was not modified by incubation with NCOA4.
The recombinant human H-ferritin was
incubated with increasing concentration of
purified NCOA4 domain.
We observed that FTH mobility changed when
the molar ratio of FTH to NCOA4 was 1:4, and
then it decreased from the molar ratio 1:8 until
no FTH band was observed at 1:24.
The recombinant NCOA4 domain was incubated with recombinant human H-ferritin at a molar ratio 24:1
in the presence of urea (1M, 2M, 4M, 6M), sodium chloride (0.5M, 1M, 1.5M) and -mercaptoethanol
(0.5%, 1%, 2%).
The unaltered mobility of a complex in the presence of urea, NaCl and -mercaptoethanol indicates
a high stability of the NCOA4-FTH interaction.
0
20
40
60
80
100
120
0 50 100 150 200 250 300
%ofbinding
NCOA4 concentration (nM)
FTH
R23A-FTH
FTL
FtMt
The his-tagged NCOA4 domain at various concentration was incubated with solid phase
human ferritins: H-chain (FTH), R23A H-mutant (R23A-FTH), L-chain (FTL) and
mitochondrial ferritin (FtMt) at concentration 20nM.
We observed that NCOA4 domain (383-522) binds H-ferritin producing dose-dependent
calibration plot, whereas the R23A-FTH and FTL showed low binding to NCOA4 domain.
0
20
40
60
80
100
0 40 80 120 160
%ofbinding
Fe (II) concentration (µM)
Iron (II)
0
20
40
60
80
100
0 80 160
%ofbinding
Metal concentration (µM)
Iron (II), Iron (III) and other metals
Fe (II)
Fe (III)
metals
0
20
40
60
80
100
%binding
Iron (II) before and after binding
+NCOA4 +NCOA4
+Fe (II)
+NCOA4 (30min)
+Fe (II) (30min)
To analyze NCOA4-ferritin interaction we used two different approaches: EMSA and ELISA.
EMSA (electrophoretic mobility shift assay) - Ferritin and NCOA4 domain were incubated for 30 minutes at
room temperature followed by electrophoresis on 6%/15% discontinuous non-dentauring acrylamid gel.
ELISA (enzyme-linked immunosorbent assay) - Ferritin was adsorbed onto microplates and incubated with
various concentration of his-tagged NCOA4 domain followed by incubation with anti-polyhis antibody and
secondary HRP-conjugated antibody, results were detected using TMB substrate.
The his-tagged NCOA4 domain was incubated with solid phase human H-ferritin in the
presence of ferrous ammonium sulfate (20, 40, 80, 160µM).
The addition of iron (II) resulted in inhibition of NCOA4-FTH interaction in dose-
dependent manner decreasing binding at 20µM to 77%, at 40µM to 62%, at 80µM to 45%
and at 160µM to 26%.
The Fe(II)-induced inhibition of NCOA4-ferritin binding was supressed when it was added
together with iron (II) chelator (data not shown).
The NCOA4 domain was incubated with solid phase human H-ferritin in the presence of
Fe(III) as ferric ammonium citrate and of divalent metals: Cu(II), Mn(II), Zn(II), Mg(II),
Ca(II) at concentration of 80 and 160µM.
We found that iron (III) and other divalent metals had only minor effect on the NCOA4-
FTH interaction.
After 30min incubation of NCOA4 with solid-phase FTH, we added 160µM of Fe(II) for
another 30min and after washing we evaluated binding.
Iron (II) added after the binding did not seem to modify the NCOA4-FTH complex.
PROPOSED MODEL OF FERRITINOPHAGY REGULATION BY IRON LEVEL
NCOA4 mRNA expression is regulated by iron status (DFO, FAC) and oxidative stress (H2O2)
We showed that NCOA4 mRNA expression was decreased by iron and oxidative stress, whereas manipulation of autophagy or lysosomal degradation had no effect.
The NCOA4 domain (383-522aa) binds specifically to H-ferritin, whereas does not bind to L-ferritin
We found that after incubation with the NCOA4 domain the electrophoretic mobility of ferritin was strongly retarded. The appareance of the low-mobility NCOA4(383-522)-FTH complex was
associated with the disappearance of the H-ferritin band. A complex with L-ferritin was not formed. A complex with mitochondrial ferritin was also evident, although less retarded, in keeping with
the high level of identity to the H-ferritin and the conserved Arg23.
The results were confirmed by ELISA assay showing that NCOA4(383-522aa) binds FTH with dose-dependent plot reaching the saturation at NCOA4 concentration of 150nM and the half saturation
at 18nM. It binds mitochondrial ferritin with lower affinity and does not bind FTL.
Arginine-23 of FTH is crucial for the interaction
We confirmed that Arg23 of FTH is a key amino acid for NCOA4 interaction, since the Arg23Ala mutant did not bind the NCOA4 domain.
The NCOA4-FTH complex resists to urea, sodium chloride and -mercaptoethanol
The addition of urea up to 6M, NaCl to 1.5M and -mercaptoethanol to 2% did not modify the mobility of the of NCOA4(383-522)-FTH complex showing that the complex is highly stable and the
interaction is not simply electrostatic in nature.
Interaction of NCOA4-FTH is iron(II)-dependent
Iron (II) decreased NCOA4-FTH binding in dose-dependent manner, whereas other divalent metals and iron (III) did not affect the interaction. Fe(II) addition inhibits NCOA4-FTH interaction before,
but not after complex formation.
View publication statsView publication stats