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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Iron-mediated Aggregation and a Localized Structural Change Characterize
Ferritin from a Mutant Light Chain Polypeptide That Causes
Neurodegeneration<xref ref-type="fn" rid="fn1">*</xref><xref ref-type="fn" rid="fn2"></xref></title><author><name sortKey="Baraibar, Martin A" sort="Baraibar, Martin A" uniqKey="Baraibar M" first="Martin A." last="Baraibar">Martin A. Baraibar</name><affiliation><nlm:aff id="N0x1cd40b0N0x1f57ec0">Department of Pathology and Laboratory Medicine, Indiana University School of Medicine and the</nlm:aff></affiliation></author><author><name sortKey="Barbeito, Ana G" sort="Barbeito, Ana G" uniqKey="Barbeito A" first="Ana G." last="Barbeito">Ana G. Barbeito</name><affiliation><nlm:aff id="N0x1cd40b0N0x1f57ec0">Department of Pathology and Laboratory Medicine, Indiana University School of Medicine and the</nlm:aff></affiliation></author><author><name sortKey="Muhoberac, Barry B" sort="Muhoberac, Barry B" uniqKey="Muhoberac B" first="Barry B." last="Muhoberac">Barry B. Muhoberac</name><affiliation><nlm:aff id="N0x1cd40b0N0x1f57ec0">Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana 46202</nlm:aff></affiliation></author><author><name sortKey="Vidal, Ruben" sort="Vidal, Ruben" uniqKey="Vidal R" first="Ruben" last="Vidal">Ruben Vidal</name><affiliation><nlm:aff id="N0x1cd40b0N0x1f57ec0">Department of Pathology and Laboratory Medicine, Indiana University School of Medicine and the</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">18755684</idno><idno type="pmc">2581579</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2581579</idno><idno type="RBID">PMC:2581579</idno><idno type="doi">10.1074/jbc.M805532200</idno><date when="2008">2008</date><idno type="wicri:Area/Pmc/Corpus">000561</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000561</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Iron-mediated Aggregation and a Localized Structural Change Characterize
Ferritin from a Mutant Light Chain Polypeptide That Causes
Neurodegeneration<xref ref-type="fn" rid="fn1">*</xref><xref ref-type="fn" rid="fn2"></xref></title><author><name sortKey="Baraibar, Martin A" sort="Baraibar, Martin A" uniqKey="Baraibar M" first="Martin A." last="Baraibar">Martin A. Baraibar</name><affiliation><nlm:aff id="N0x1cd40b0N0x1f57ec0">Department of Pathology and Laboratory Medicine, Indiana University School of Medicine and the</nlm:aff></affiliation></author><author><name sortKey="Barbeito, Ana G" sort="Barbeito, Ana G" uniqKey="Barbeito A" first="Ana G." last="Barbeito">Ana G. Barbeito</name><affiliation><nlm:aff id="N0x1cd40b0N0x1f57ec0">Department of Pathology and Laboratory Medicine, Indiana University School of Medicine and the</nlm:aff></affiliation></author><author><name sortKey="Muhoberac, Barry B" sort="Muhoberac, Barry B" uniqKey="Muhoberac B" first="Barry B." last="Muhoberac">Barry B. Muhoberac</name><affiliation><nlm:aff id="N0x1cd40b0N0x1f57ec0">Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana 46202</nlm:aff></affiliation></author><author><name sortKey="Vidal, Ruben" sort="Vidal, Ruben" uniqKey="Vidal R" first="Ruben" last="Vidal">Ruben Vidal</name><affiliation><nlm:aff id="N0x1cd40b0N0x1f57ec0">Department of Pathology and Laboratory Medicine, Indiana University School of Medicine and the</nlm:aff></affiliation></author></analytic><series><title level="j">The Journal of Biological Chemistry</title><idno type="ISSN">0021-9258</idno><idno type="eISSN">1083-351X</idno><imprint><date when="2008">2008</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Nucleotide insertions in the ferritin light chain (FTL) polypeptide gene
cause hereditary ferritinopathy, a neurodegenerative disease characterized by
abnormal accumulation of ferritin and iron in the central nervous system. Here
we describe for the first time the protein structure and iron storage function
of the FTL mutant <italic>p.Phe167SerfsX26</italic> (MT-FTL), which has a C terminus
altered in sequence and extended in length. MT-FTL polypeptides assembled
spontaneously into soluble, spherical 24-mers that were ultrastructurally
indistinguishable from those of the wild type. Far-UV CD showed a decrease in
α-helical content, and 8-anilino-1-naphthalenesulfonate fluorescence
revealed the appearance of hydrophobic binding sites. Near-UV CD and
proteolysis studies suggested little or no structural alteration outside of
the C-terminal region. In contrast to wild type, MT-FTL homopolymers
precipitated at much lower iron loading, had a diminished capacity to
incorporate iron, and were less thermostable. However, precipitation was
significantly reversed by addition of iron chelators both <italic>in vitro</italic>and <italic>in vivo</italic>. Our results reveal substantial protein conformational
changes localized at the 4-fold pore of MT-FTL homopolymers and imply that the
C terminus of the MT-FTL polypeptide plays an important role in ferritin
solubility, stability, and iron management. We propose that the protrusion of
some portion of the C terminus above the spherical shell allows it to
cross-link with other mutant polypeptides through iron bridging, leading to
enhanced mutant precipitation by iron. Our data suggest that hereditary
ferritinopathy pathogenesis is likely to result from a combination of
reduction in iron storage function and enhanced toxicity associated with
iron-induced ferritin aggregates.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Biol Chem</journal-id><journal-id journal-id-type="publisher-id">jbc</journal-id><journal-title>The Journal of Biological Chemistry</journal-title><issn pub-type="ppub">0021-9258</issn><issn pub-type="epub">1083-351X</issn><publisher><publisher-name>American Society for Biochemistry and Molecular Biology</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">18755684</article-id><article-id pub-id-type="pmc">2581579</article-id><article-id pub-id-type="publisher-id">31679</article-id><article-id pub-id-type="doi">10.1074/jbc.M805532200</article-id><article-categories><subj-group subj-group-type="heading"><subject>Protein Structure and Folding</subject></subj-group></article-categories><title-group><article-title>Iron-mediated Aggregation and a Localized Structural Change Characterize
Ferritin from a Mutant Light Chain Polypeptide That Causes
Neurodegeneration<xref ref-type="fn" rid="fn1">*</xref><xref ref-type="fn" rid="fn2"></xref></article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Baraibar</surname><given-names>Martin A.</given-names></name><xref ref-type="aff" rid="N0x1cd40b0N0x1f57ec0">‡</xref></contrib><contrib contrib-type="author"><name><surname>Barbeito</surname><given-names>Ana G.</given-names></name><xref ref-type="aff" rid="N0x1cd40b0N0x1f57ec0">‡</xref></contrib><contrib contrib-type="author"><name><surname>Muhoberac</surname><given-names>Barry B.</given-names></name><xref ref-type="aff" rid="N0x1cd40b0N0x1f57ec0">§</xref></contrib><contrib contrib-type="author"><name><surname>Vidal</surname><given-names>Ruben</given-names></name><xref ref-type="aff" rid="N0x1cd40b0N0x1f57ec0">‡</xref><xref ref-type="corresp" rid="cor1">1</xref></contrib></contrib-group><aff id="N0x1cd40b0N0x1f57ec0"><label>‡</label>Department of Pathology and Laboratory Medicine, Indiana University School of Medicine and the<label>§</label>Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana 46202</aff><author-notes><fn id="cor1"><label>1</label><p>To whom correspondence should be addressed: Dept. of Pathology and Laboratory
Medicine, Indiana University School of Medicine, 635 Barnhill Dr., MSB A136,
Indianapolis, IN 46202. Tel.: 317-274-1729; Fax: 317-278-6613; E-mail:
<email>rvidal@iupui.edu</email>.
</p></fn></author-notes><pub-date pub-type="ppub"><day>14</day><month>11</month><year>2008</year></pub-date><pub-date pub-type="pmc-release"><day>14</day><month>11</month><year>2008</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on the copyright element. </pmc-comment>
<volume>283</volume><issue>46</issue><fpage>31679</fpage><lpage>31689</lpage><history><date date-type="received"><day>21</day><month>7</month><year>2008</year></date><date date-type="rev-recd"><day>26</day><month>8</month><year>2008</year></date></history><permissions><copyright-statement>Copyright © 2008, The American Society for Biochemistry
and Molecular Biology, Inc.</copyright-statement><license license-type="open-access"><p><italic>Author's Choice</italic></p><p><ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc/3.0/">Creative Commons Attribution Non-Commercial
License</ext-link> applies to Author Choice Articles</p></license></permissions><self-uri xlink:title="pdf" xlink:href="zbc04608031679.pdf"></self-uri><abstract><p>Nucleotide insertions in the ferritin light chain (FTL) polypeptide gene
cause hereditary ferritinopathy, a neurodegenerative disease characterized by
abnormal accumulation of ferritin and iron in the central nervous system. Here
we describe for the first time the protein structure and iron storage function
of the FTL mutant <italic>p.Phe167SerfsX26</italic> (MT-FTL), which has a C terminus
altered in sequence and extended in length. MT-FTL polypeptides assembled
spontaneously into soluble, spherical 24-mers that were ultrastructurally
indistinguishable from those of the wild type. Far-UV CD showed a decrease in
α-helical content, and 8-anilino-1-naphthalenesulfonate fluorescence
revealed the appearance of hydrophobic binding sites. Near-UV CD and
proteolysis studies suggested little or no structural alteration outside of
the C-terminal region. In contrast to wild type, MT-FTL homopolymers
precipitated at much lower iron loading, had a diminished capacity to
incorporate iron, and were less thermostable. However, precipitation was
significantly reversed by addition of iron chelators both <italic>in vitro</italic>and <italic>in vivo</italic>. Our results reveal substantial protein conformational
changes localized at the 4-fold pore of MT-FTL homopolymers and imply that the
C terminus of the MT-FTL polypeptide plays an important role in ferritin
solubility, stability, and iron management. We propose that the protrusion of
some portion of the C terminus above the spherical shell allows it to
cross-link with other mutant polypeptides through iron bridging, leading to
enhanced mutant precipitation by iron. Our data suggest that hereditary
ferritinopathy pathogenesis is likely to result from a combination of
reduction in iron storage function and enhanced toxicity associated with
iron-induced ferritin aggregates.</p></abstract></article-meta><notes><fn-group><fn id="fn1"><label>*</label><p>This work was supported, in whole or in part, by <grant-sponsor>National
Institutes of Health</grant-sponsor> Grant
<grant-num>NS050227</grant-num> (to R. V.). The costs of publication of this
article were defrayed in part by the payment of page charges. This article
must therefore be hereby marked “<italic>advertisement</italic>” in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.</p></fn><fn id="fn2"><label><inline-graphic xlink:href="flower.gif"></inline-graphic></label><p><italic>Author's Choice</italic>—Final version full access.</p></fn></fn-group></notes></front><body><p>Iron is an essential element needed for vital processes such as neuronal
development, myelination, synthesis, and catabolism of neurotransmitters and
electron transport, as well as heme and iron-sulfur cluster synthesis
(<xref ref-type="bibr" rid="ref1">1</xref>). Iron that is not utilized
immediately in the cell is stored in ferritin. However, when iron is
improperly regulated, it is potentially toxic leading to cell death. Mammalian
ferritin is a large, iron-storage heteropolymer composed of two
conformationally equivalent subunit types, light
(FTL)<xref ref-type="fn" rid="fn3">2</xref> and heavy
(FTH1) polypeptides, which are expressed in most kinds of cells
(<xref ref-type="bibr" rid="ref2">2</xref>–<xref ref-type="bibr" rid="ref5">5</xref>).
A single ferritin protein is composed of 24 self-assembled polypeptide
subunits related by 4-, 3-, and 2-fold symmetry axes with one polypeptide per
asymmetric unit. Each polypeptide subunit consists of a bundle of four
parallelα-helices (A–D), a long extended loop (connecting helices
B and C), and a C terminus with a shortα-helix (E), which is involved in
important stabilizing interactions around the 4-fold symmetry axes
(<xref ref-type="bibr" rid="ref2">2</xref>,
<xref ref-type="bibr" rid="ref6">6</xref>,
<xref ref-type="bibr" rid="ref7">7</xref>). Although both types of
polypeptide subunits share a high degree of conformational similarity, they
have diverse functional roles. The FTH1 subunit has a potent ferroxidase
activity that catalyzes the oxidation of ferrous iron, whereas the FTL subunit
plays important roles in iron nucleation and protein stability, giving
ferritin the dual functions of iron detoxification and iron storage
(<xref ref-type="bibr" rid="ref2">2</xref>). Interestingly, the FTL
homopolymer has iron incorporation ability, although at a substantially
reduced rate from heteropolymers containing both FTL and FTH1 subunits
(<xref ref-type="bibr" rid="ref2">2</xref>).</p><p>Recently, nucleotide insertions in the coding sequence of the <italic>FTL</italic>gene
(<xref ref-type="bibr" rid="ref8">8</xref>–<xref ref-type="bibr" rid="ref11">11</xref>)
have been found associated with an autosomal dominant neurodegenerative
disease named neuroferritinopathy
(<xref ref-type="bibr" rid="ref8">8</xref>) or hereditary
ferritinopathy (HF) (<xref ref-type="bibr" rid="ref9">9</xref>).
Clinically, HF is characterized by an abnormal involuntary movement disorder
and cognitive decline, which may appear sequentially between the 3rd and 6th
decades of life. Neuropathologically, the disease is characterized by the
presence of intracellular ferritin inclusion bodies and iron accumulation in
glia and neurons throughout the central nervous system and other organ systems
(<xref ref-type="bibr" rid="ref12">12</xref>). Three different
mutations have been found in individuals with neuropathologically confirmed
HF, an adenosine insertion at position 460–461
(<xref ref-type="bibr" rid="ref8">8</xref>), a thymidine and cytidine
insertion at position 498–499,
(<xref ref-type="bibr" rid="ref9">9</xref>), and a cytidine insertion
at position 442–443
(<xref ref-type="bibr" rid="ref10">10</xref>). As a consequence of
these mutations, the FTL polypeptides have C termini that are each changed in
length and primary amino acid sequence. Two additional mutations in the
<italic>FTL</italic> gene, a point mutation at codon 96
(<xref ref-type="bibr" rid="ref13">13</xref>) and a 16 nucleotide
insertion (<xref ref-type="bibr" rid="ref11">11</xref>), have been
described in clinically diagnosed individuals.</p><p>Here we characterize the protein structure and the iron storage function of
ferritin formed by the polypeptide <italic>p.Phe167SerfsX26</italic>, generated by the
<italic>FTL498–499InsTC</italic> mutation (MT-FTL)
(<xref ref-type="bibr" rid="ref9">9</xref>). The mutant polypeptide
assembled spontaneously into ferritin spherical shells in a soluble manner.
Spectroscopic and proteolytic analysis of these mutant ferritin homopolymers
showed a large protein conformational difference with the wild type FTL
(WT-FTL) homopolymers in the C terminus. Compared with WT-FTL homopolymers,
those of the MT-FTL were less stable, had a diminished capacity to incorporate
iron, and precipitated at iron:ferritin ratios of over 1,000 iron atoms per
ferritin 24-mer. However, precipitation was greatly reversed by the addition
of iron chelators both <italic>in vitro</italic> and <italic>in vivo</italic>. The results
shown here suggest that the C terminus of the MT-FTL polypeptide plays an
important role not only in the solubility and stability of ferritin, but also
in iron management, and provide insights into the molecular mechanisms
involved in the generation of ferritin inclusion bodies and the accumulation
of iron observed in patients with HF.</p><sec sec-type="methods"><title>EXPERIMENTAL PROCEDURES</title><p><italic>Cloning and Expression of Ferritin Polypeptides</italic>—cDNAs
containing the sequence of human WT-FTL and human mutant
<italic>FTL498–499InsTC</italic> were introduced into the pET-28a(+) expression
vector (Novagen, EMD Chemicals Inc.). The cDNAs were cloned between the BamHI
and XhoI sites, downstream from and in-frame with the sequence encoding an
N-terminal His<sub>6</sub> tag. To eliminate the His<sub>6</sub> tag (included
in the expression vector), the sequence of the vector was modified by
introducing the recognition sequence for cleavage by factor Xa before the
coding sequence of the ferritin genes. PCR amplification of the ferritin cDNAs
was performed using the upstream primer F1 5′-TGG ATC C<underline>AT CGA AGG
TCG T</underline>AT GAG CTC CCA GAT T-3′ and the downstream primer R1
5′-TTA TGC CTC GAG CCC TAT TAC TTT GCA AGG-3′. F1 contains the
factor Xa sequence (underlined). pET-28a(+) carrying WT-FTL and MT-FTL cDNAs
was transformed into BL21 (DE3) <italic>Escherichia coli</italic> (Invitrogen).
Transformed cells were grown in Luria broth medium (LB) containing 30 μg/ml
kanamycin (Invitrogen) at 37 °C up to an absorbance of 0.9–1.0 at
600 nm. Bacteria were induced to overexpress recombinant proteins by adding 1
m<sc>m</sc> isopropyl thio-β-<sc>d</sc>-galactopyranoside (ICN
Biotechnologies) for 12 h at 25 °C.</p><p><italic>Purification of Recombinant WT- and MT-FTL
Homopolymers</italic>—Cells were harvested by centrifugation and frozen at
-80 °C. The cell pellets were suspended in 50 m<sc>m</sc> sodium
phosphate, 500 m<sc>m</sc> NaCl (pH 7.4), 1 mg/ml lysozyme, and a protease
inhibitor mixture (Complete, Roche Applied Science) for 30 min. Bacteria were
disrupted by sonication, and the insoluble material was removed by
centrifugation at 21,000 × <italic>g</italic> for 30 min. The soluble fraction
was purified by nickel iminodiacetic acid affinity chromatography using an
AKTA purifier system (GE Healthcare). Purified protein was eluted with 250
m<sc>m</sc> imidazole in 50 m<sc>m</sc> sodium phosphate (pH 7.4), 0.5
<sc>m</sc> NaCl. Recombinant proteins were diluted with 50 m<sc>m</sc>Tris and 10% glycerol (v/v) down to an absorbance of 0.5 at 280 nm, and
ferritins were cleaved from the His tag by digestion with factor Xa protease
(GE Healthcare) (5 units/mg of protein). After being dialyzed against 50
m<sc>m</sc> Tris, pH 8.0, for 18 h, proteins were further purified by anion
exchange chromatography (Mono Q) using a linear NaCl elution gradient in 50
m<sc>m</sc> Tris (pH 8). Peak fractions were ∼95% pure based on SDS-12%
PAGE (Pierce) and Coomassie Blue staining. The efficiency of tag removal was
confirmed by N-terminal protein sequencing analysis, and the molecular weight
of the recombinant proteins was determined by matrix-assisted laser
desorption/ionization-time of flight mass spectrometry. Protein concentration
was determined using the BCA reagent (Pierce) with bovine serum albumin as
standard.</p><p><italic>Gel Filtration Chromatography</italic>—Size exclusion chromatography
was performed on a Superose 6 10/300 GL column (GE Healthcare) equilibrated
with 50 m<sc>m</sc> Tris, 150 m<sc>m</sc> NaCl (pH 7.4) using an AKTA
purifier. The column was calibrated with gel filtration standards (GE
Healthcare). Fractions were detected photometrically, and peak areas and
<italic>k</italic><sub>av</sub> values were evaluated using the UNICORN 5.1 software
(GE Healthcare). All gel filtration experiments were run at room
temperature.</p><p><italic>Transmission Electron Microscopy (TEM)</italic>—Ferritins were fixed
using the “single droplet” parafilm protocol. The specimens were
dropped onto a 400-mesh carbon/Formvar-coated grid (Nanoprobes) and allowed to
absorb to the Formvar for a minimum of 1 min. Excess fluid was removed using
filter paper, and the unbound protein was washed, and the grids were placed on
a 50-μl drop of Nanovan (Nanoprobes) with the section side downwards.
Finally, the grids were dried, placed in the grid chamber, and stored in
desiccators before the samples were observed with a Tecnai G2 12 Bio Twin
(FEI) transmission electron microscope.</p><p><italic>Preparation of Apoferritins</italic>—Recombinant FTL homopolymers
were treated for iron removal as described previously
(<xref ref-type="bibr" rid="ref14">14</xref>). Briefly, recombinant
ferritins were incubated with 1% thioglycolic acid (pH 5.5) and
2,2′-bipyridine, followed by dialysis against 0.1 <sc>m</sc> phosphate
buffer (pH 7.4). We consistently achieve less than five atoms of iron per
ferritin 24-mer, as determined by the colorimetric ferrozine-based assay for
the quantitation of iron
(<xref ref-type="bibr" rid="ref15">15</xref>).</p><p><italic>Iron Loading of Apoferritins</italic>—Freshly prepared ferrous
ammonium sulfate (0.5–4.5 m<sc>m</sc>) in 10 m<sc>m</sc> HCl was
added to MT- and WT-FTL apoferritin homopolymers (1 μ<sc>m</sc>) in 0.1
<sc>m</sc> Hepes buffer (pH 7.4) at room temperature
(<xref ref-type="bibr" rid="ref16">16</xref>). After 2 h, the samples
were centrifuged at 14,000 × <italic>g</italic> for 15 min. Iron incorporation
was initially monitored by measuring absorbance of the supernatants at 310 nm
(<xref ref-type="bibr" rid="ref14">14</xref>,
<xref ref-type="bibr" rid="ref17">17</xref>). Iron incorporation into
ferritin was more precisely determined by densitometric analysis of Prussian
blue staining of supernatants run on nondenaturing gel electrophoresis.
Pellets were analyzed by SDS-12% PAGE. Apoferritins were also incubated in a
molar ratio 1:3500 with ferrous ammonium sulfate and centrifuged at 14,000
× <italic>g</italic> for 15 min. Pellets were resuspended in a solution
containing 6 m<sc>m</sc> deferroxamine (DFX), 0.1 <sc>m</sc> Hepes (pH
7.4) and incubated for 2 h at 24 °C. After centrifugation, supernatants
were analyzed by nondenaturing gel electrophoresis.</p><p><italic>Circular Dichroism Spectroscopy</italic>—CD spectra of recombinant
apoferritin homopolymers were obtained in 50 m<sc>m</sc> phosphate buffer
(pH 7.4) at 25 °C in a Jasco 810 spectropolarimeter (Jasco Corp.), using a
protein concentration of 0.12 and 1.5 μ<sc>m</sc> for far-UV and near-UV,
respectively. Far-UV CD spectra were recorded in a 1.0-mm path length cell
from 250 to 190 nm with a step size of 0.1 nm and a bandwidth of 1.0 nm. Each
spectrum represents the mean of 15 scans. CD spectra of the buffer/cuvette
were recorded and subtracted from the protein spectra before averaging.
Secondary structure analyses were performed using DICHROWEB
(<xref ref-type="bibr" rid="ref18">18</xref>,
<xref ref-type="bibr" rid="ref19">19</xref>), which allows secondary
structure analyses via the software package CDPro
(<xref ref-type="bibr" rid="ref20">20</xref>). SELCON3
(<xref ref-type="bibr" rid="ref21">21</xref>), CONTINLL
(<xref ref-type="bibr" rid="ref22">22</xref>), and CDSSTR
(<xref ref-type="bibr" rid="ref23">23</xref>) programs were used for
comparing variations in the amount of secondary structure between MT- and
WT-FTL homopolymers. Normalized root mean square deviation values of < 0.1
for the three methods meant that the experimental and simulated spectra were
in close agreement. Near-UV CD spectra were recorded in a 1.0-cm path length
cell from 400 to 250 nm with a step size of 1.0 nm and a bandwidth of 1.5 nm.
For all spectra, an average of five scans was obtained.</p><p><italic>Intrinsic Protein Fluorescence and Thermal Stability Studies of
Homopolymers</italic>—Fluorescence spectra were recorded using a
spectrofluorimeter (PerkinElmer Life Sciences) equipped with a Selecta
Ultraterm water bath for temperature control. Apoferritin spectra were
obtained with excitation at 280 and 295 nm with 1.5 μ<sc>m</sc> protein
in 1-cm path length cells and with 0.1 <sc>m</sc> phosphate (pH 7.4). Blanks
without protein were subtracted from the spectra. Thermal denaturation was
induced by increasing the temperature from 20 to 100 °C at a rate of 1
°C/min. To overcome the inherent difficulty in denaturing ferritin, these
experiments were performed in 0.1 <sc>m</sc> phosphate buffer (pH 7.4)
containing 4.0 <sc>m</sc> guanidine hydrochloride (GdnHCl). Homopolymer
stability was monitored using the ratio of intrinsic fluorescence emission of
355 over 330 nm with excitation at 295 nm
(<xref ref-type="bibr" rid="ref24">24</xref>,
<xref ref-type="bibr" rid="ref25">25</xref>) with a maximum at 330 nm
signifying native ferritin (mt and WT) and 355 nm, denatured ferritin.</p><p><italic>ANS Fluorescence and Binding Studies</italic>—Extrinsic fluorescence
spectra were recorded using a spectrofluorimeter (PerkinElmer Life Sciences)
in 1.0-cm cuvettes at 25 °C. ANS binding to apoferritin homopolymers was
monitored through fluorescence enhancement with ANS excitation at 360 nm and
emission recorded from 600 to 400 nm. MT-FTL apoferritins were prepared by
diluting stock solutions to 1.5 μ<sc>m</sc> in 0.05 <sc>m</sc>phosphate buffer (pH 7.4). Stock solutions of ANS (Invitrogen) were prepared
in water, and the concentration was determined optically at 350 nm using an
extinction coefficient of 4950 <sc>m</sc><sup>-1</sup> cm<sup>-1</sup>. ANS
was added to the diluted ferritin samples and equilibrated for 30 min prior to
the measurements, and spectra were background corrected. Binding of ANS to
ferritin was quantitated by Scatchard analysis
(<xref ref-type="bibr" rid="ref26">26</xref>).</p><p><italic>Thermolysin Treatment of WT- and MT-FTL Apoferritin
Homopolymers</italic>—Proteolysis of recombinant MT- and WT-FTL homopolymers
was initiated by adding to 10 μg of ferritin a 10-fold concentrated stock
solution (36.5 units/mg) of thermolysin (Fluka) in Hepes (0.1 <sc>m</sc>)
(pH 7.0), 10 m<sc>m</sc> CaCl<sub>2</sub> to a final concentration of 0.2
mg/ml. The reaction was stopped by the addition of EDTA (50 m<sc>m</sc>) and
Laemmli sample buffer. Samples treated with thermolysin and controls without
thermolysin were boiled and loaded onto SDS-polyacrylamide gels (4–20%)
(Pierce). Gels were stained with Coomassie Blue (Total protein) or blotted
against the C-terminal antibodies (MT-1283 or WT-1278)
(<xref ref-type="bibr" rid="ref9">9</xref>) or against the N-terminal
antibody D18 (Santa Cruz Biotechnology, Inc), which recognized both
polypeptides.</p><p><italic>Astrocyte Cell Cultures and Iron/Chelator Treatment</italic>—Primary
cortical astrocyte cultures were prepared from 1-day-old mouse pups according
to the procedures of Saneto and De Vellis
(<xref ref-type="bibr" rid="ref27">27</xref>) and Cassina <italic>et
al.</italic> (<xref ref-type="bibr" rid="ref28">28</xref>), with minor
modifications. Pups were obtained from transgenic dams homozygous for the
<italic>FTL498–499InsTC</italic> mutation in C57BL/6J genetic background
(<xref ref-type="bibr" rid="ref29">29</xref>). Briefly, cerebral
cortices were removed, and the tissue was minced and dissociated in 0.25%
trypsin (Invitrogen) for 15 min at 37 °C. Cells were collected by
centrifugation and plated at a density of 2.0 × 10<sup>6</sup> cells in
25-cm<sup>2</sup> flasks (Corning Glass) in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, Hepes (25 m<sc>m</sc>), penicillin
(100 IU/ml), and streptomycin (100 μg/ml) (Invitrogen). When confluent,
cultures were shaken for 48 h at 250 rpm at 37 °C, incubated for another
48 h with 10 μ<sc>m</sc> cytosine arabinoside, and then amplified to 2.5
× 10<sup>4</sup> cells/cm<sup>2</sup> in 75-cm<sup>2</sup> flasks
(Corning Glass). The astrocyte monolayers were >98% pure as determined by
GFAP immunoreactivity. Confluent astrocyte monolayers were changed to
Dulbecco's modified Eagle's media devoid of serum prior to treatment. Stock
solutions (20 m<sc>m</sc>) of ferric ammonium citrate (FAC) (Sigma), and
1,10-phenanthroline (Phen) (Sigma) were prepared in distilled water and
directly applied to the monolayer at the indicated final concentrations. Each
flask was treated with either of the following: (<italic>a</italic>) vehicle (water)
as control group; (<italic>b</italic>) Phen at 100 μ<sc>m</sc> during 48 h
followed by 24 h at 50 μ<sc>m</sc>;(<italic>c</italic>) FAC 50 μ<sc>m</sc>during 4 days; (<italic>d</italic>) FAC treatment as in <italic>c</italic> followed by Phen
treatment as in <italic>b</italic> in the absence of iron.</p><p><italic>Characterization of Detergent-insoluble MT-FTL Ferritin from Astrocyte
Cultures</italic>—Cerebral cortical astrocytes cultures were homogenized in
lysis buffer (3 ml of 50 m<sc>m</sc> Tris-HCl (pH 7.4), 1% SDS, 30 units/ml
benzonase, 2 m<sc>m</sc> MgCl<sub>2</sub>) containing Complete protease
inhibitor mixture (Roche Applied Science) and incubated for 15 min at room
temperature. Lysates containing equal amounts of protein were ultracentrifuged
at 46,000 rpm (TLA 110, Beckman) for 25 min at 4 °C. The supernatant
(SDS-soluble) was removed, and the SDS-insoluble pellet was resuspended in
lysis buffer and then subjected to another step of centrifugation in the same
conditions. The final pellet was resuspended in 5× Laemmli sample buffer
and heated for 10 min at 95 °C. The SDS-soluble, -insoluble, and total
cell lysates (before SDS extraction) were resolved on 4–20% gradient
SDS-PAGE (Pierce) and transferred to nitrocellulose membranes (Amersham
Biosciences). Membranes were blocked for1 h in 70 m<sc>m</sc> Tris-buffered
saline, 0.1% Tween 20, and 5% nonfat dry milk, followed by an overnight
incubation with polyclonal antibodies (1283) against the MT-FTL polypeptide,
as described previously (<xref ref-type="bibr" rid="ref9">9</xref>,
<xref ref-type="bibr" rid="ref29">29</xref>) at 1:10,000. After
washing, membranes were incubated with peroxidase-conjugated secondary
antibody (GE Healthcare) for 1 h, washed, and developed using the ECL
chemiluminescent detection system (GE Healthcare). MT-FTL recombinant
polypeptides were loaded and used as positive control.</p><p><fig position="float" id="fig1"><label>FIGURE 1.</label><caption><p><bold>Sequence comparison between WT- and MT-FTL polypeptide.</bold> The wild
type FTL polypeptide (WT-FTL) consists of 175 amino acids. The
<italic>p.Phe167SerfsX26</italic> mutant polypeptide (MT-FTL) has 191 amino acids and
a different C-terminal sequence (<italic>underlined</italic>). The <italic>boxes</italic>indicate the five α-helical domains in the WT-FTL polypeptide according
to Protein Data Bank accession number 2FG4. The mutant C-terminal sequence
contains both metal-binding and hydrophobic groups.</p></caption><graphic xlink:href="zbc0460854930001"></graphic></fig></p><p><fig position="float" id="fig2"><label>FIGURE 2.</label><caption><p><bold>MT-FTL polypeptides assemble into 24-mer homopolymers.</bold> <italic>A,</italic>elution profiles of purified WT- and MT-FTL apoferritin homopolymers from a
Superose 6 column at pH 7.4 in 0.05 <sc>m</sc> Tris, 0.15 <sc>m</sc> NaCl.
Retention times for both proteins are shown. <italic>Arrows</italic> indicate the
elution time for the molecular weight markers. <italic>B,</italic> ultrastructural
characterization of WT- and MT-FTL homopolymers by TEM. The dark cores most
likely represent Nanovan that has penetrated in some cases the interior of the
24-mers. <italic>Bars,</italic> 10 nm. <italic>C,</italic> native PAGE (3–8% (pH7.4)) of
0.5 μ<sc>m</sc> WT- and MT-FTL proteins loaded before the removal of iron
and stained with Coomassie Blue (protein staining) and with Prussian blue
(iron staining).</p></caption><graphic xlink:href="zbc0460854930002"></graphic></fig></p><p><italic>Immunofluorescence of Cultured Cells</italic>—Astrocyte cultures in
Lab-Tek chambered coverglass slides (Nunc) were fixed for 15 min with 4%
paraformaldehyde in PBS at 4 °C. Briefly, the slides were washed
successively with PBS, permeabilized with 0.1% Triton X-100 for 15 min, and
incubated for 1 h at room temperature in blocking solution (0.1% Triton X-100,
2% bovine serum albumin in PBS). The cultures were incubated overnight at 4
°C with the primary antibodies diluted in blocking solution, washed with
PBS, and further incubated for 1 h at room temperature with the secondary
antibodies diluted in blocking solution. The slides were then washed with PBS,
rinsed with distilled water, and mounted with the Prolong Gold antifade
mounting reagent (Molecular Probes). Primary antibodies used were monoclonal
antibody to GFAP (1:400; Sigma) and polyclonal antibody against MT-FTL (1283).
Secondary antibodies used were Alexa 488 Fluor-conjugated goat anti-rabbit and
Alexa Fluor 594-conjugated goat anti-mouse (4 μg/ml; Molecular Probes).
Images were captured with a Zeiss LSM-510 confocal scanner attached to a Zeiss
Axiovert 100 M inverted microscope.</p></sec><sec><title>RESULTS</title><p><italic>Recombinant MT-FTL Polypeptides Assemble into 24-mer
Homopolymers</italic>—Recombinant WT- and MT-FTL polypeptides
(<xref rid="fig1" ref-type="fig">Fig. 1</xref>) were expressed in
<italic>E. coli</italic> in a soluble manner and with similar yields. Purified WT- and
MT-FTL polypeptides were analyzed by gel filtration chromatography to
determine their states of assembly at physiological pH. Both polypeptides
eluted almost exclusively as 24-mer homopolymers, but at slightly different
times (<xref rid="fig2" ref-type="fig">Fig. 2<italic>A</italic></xref>) in a
manner consistent with their difference in molecular mass. Ultrastructural
analysis by TEM showed that both recombinant ferritins had spherical shape and
a size (diameter ∼110 Å) similar to that of human ferritin
(<xref rid="fig2" ref-type="fig">Fig. 2<italic>B</italic></xref>).
Nondenaturing PAGE showed that both WT- and MT-FTL homopolymers, examined
before iron removal, were able to assemble and incorporate iron <italic>in
vivo</italic> during expression in <italic>E. coli</italic>(<xref rid="fig2" ref-type="fig">Fig. 2<italic>C</italic></xref>). Compared
with WT-FTL homopolymers, MT-FTL homopolymers showed a slower electrophoretic
mobility, which may be attributed to their larger size and different charge
(the MT-FTL polypeptide has a +1 net charge difference per subunit)
(<xref rid="fig2" ref-type="fig">Fig. 2<italic>C</italic></xref>).</p><p><italic>Enhanced Precipitation of MT-FTL Homopolymers</italic>—Iron loading
of apoferritin homopolymers was examined by an often used and well described
procedure (<xref ref-type="bibr" rid="ref14">14</xref>,
<xref ref-type="bibr" rid="ref16">16</xref>,
<xref ref-type="bibr" rid="ref17">17</xref>). In brief, WT- and MT-FTL
apoferritin homopolymers were incubated aerobically with increasing amounts of
iron (ferrous ammonium sulfate) up to 4500 iron atoms per 24-mer. After 2 h,
proteins were separated by centrifugation into soluble and insoluble (pellet)
fractions for analysis. Monitoring the 310 nm absorbance
(<xref rid="fig3" ref-type="fig">Fig. 3<italic>A</italic></xref>) suggested
that at moderate iron loading (up to 1,000 iron atoms per ferritin 24-mer),
the WT- and MT-apoferritin homopolymers incorporated similar amounts of iron
indicating that both are functional ferritins. At higher iron:ferritin ratios,
WT-FTL homopolymers continued incorporating iron, whereas MT-FTL homopolymers
began to show macroscopically visible yellow precipitates, which were stained
with Prussian blue. No precipitates were observed for WT-FTL homopolymers,
which remained in the soluble fraction during the iron loading experiment up
to a ratio of 4500:1 of iron: ferritin. The reduction in the signal observed
in native PAGE for soluble MT-FTL homopolymers correlated with the appearance
of MT-FTL in the insoluble fraction on SDS-PAGE
(<xref rid="fig3" ref-type="fig">Fig. 3<italic>B</italic></xref>). Because the
310 nm absorbance represents both iron incorporation and hydrolysis, the
soluble ferritin fractions (supernatants) were run on nondenaturing gels and
stained with Prussian blue to unambiguously quantitate iron incorporation into
the protein. There was only a modest (∼10%) decrease in iron incorporation
in MT-FTL homopolymers <italic>versus</italic> wild type at 1000:1 iron:ferritin
loading (<xref rid="fig3" ref-type="fig">Fig. 3<italic>C</italic></xref>),
which emphasizes iron mishandling through mutant ferritin precipitation. By
TEM, MT-FTL precipitates had the shape of apoferritin homopolymers (data not
shown).</p><p><fig position="float" id="fig3"><label>FIGURE 3.</label><caption><p><bold>Precipitation of MT-FTL apoferritin homopolymers mediated by iron
loading.</bold> Ferrous ammonium sulfate (0.5–4.5 m<sc>m</sc>) was added
to 1 μ<sc>m</sc> of MT- and WT-FTL homopolymers in 0.1 <sc>m</sc> Hepes
(pH 7.0) for 2 h at 24 °C. Samples were centrifuged for 15 min at 10,000
× <italic>g</italic> to separate into soluble and insoluble fractions.
<italic>A,</italic> iron uptake/hydrolysis was monitored in the soluble fractions by
measuring absorbance at 310 nm. <italic>Errors bars</italic> represent the standard
deviation of three independent experiments. <italic>B,</italic> soluble and insoluble
fractions were loaded into native gels SDS-PAGE, respectively, and stained
with Coomassie Blue. <italic>C,</italic> iron mineralization in soluble MT- and WT-FTL
homopolymers was monitored as the density of Prussian blue formed in protein
bands after separating unmineralized iron from the protein by electrophoresis
in native gels (3–8%). This experiment is representative of several with
similar results.</p></caption><graphic xlink:href="zbc0460854930003"></graphic></fig></p><p><fig position="float" id="fig4"><label>FIGURE 4.</label><caption><p><bold>Circular dichroism spectra of WT- and MT-FTL apoferritin
homopolymers.</bold> Far-UV (<italic>A</italic>) and near-UV (<italic>B</italic>) spectra were
recorded at homopolymer concentrations of 0.12 and 1.5 μ<sc>m</sc>,
respectively, at pH 7.4 and 25 °C in 50 m<sc>m</sc> potassium phosphate
buffer.</p></caption><graphic xlink:href="zbc0460854930004"></graphic></fig></p><p><italic>Spectroscopic Comparison of MT-FTL Apoprotein Homopolymers Versus
WT</italic>—Protein spectra provide molecular level information concerning
protein structure and especially structural differences between similarly
composed proteins. The far-UV CD spectrum of MT-FTL apoferritin showed minima
at 222 and 208 nm and a maximum at 191–193 nm
(<xref rid="fig4" ref-type="fig">Fig. 4<italic>A</italic></xref>). The profile
obtained was typical of a protein containing predominantly α-helical
motifs. Compared with the WT-FTL homopolymers, we observed a change in the
secondary structure of MT-FTL, with a decrease of ∼15% in the total
α-helical content and an increase in turns and unordered structures
(<xref ref-type="table" rid="tbl1">Table 1</xref>). Near-UV CD was
performed to provide a “fingerprint” profile of MT-FTL
apoferritin. The profile obtained for MT-FTL homopolymers was very similar to
that obtained for the WT-FTL, with the WT predominant peak at 286 nm and two
other peaks at 293 and 280 nm (<xref rid="fig4" ref-type="fig">Fig.
4<italic>B</italic></xref>). The intrinsic fluorescence spectra exhibited an
emission maximum at approximately the same wavelength (330 nm) for both WT-
and MT-ferritins when both proteins were excited at either 280 or 295 nm (data
not shown).</p><p><table-wrap position="float" id="tbl1"><label>TABLE 1</label><caption><p><bold>Deconvolution of far-UV CD spectra for wt- and mt-FTL apoferritin
homopolymers into percent secondary structural contributions</bold> The analysis
was performed using fitting programs ContnnLL, SELCON3, and CDSSTR available
at the website DICHROWEB as described under “Experimental
Procedures.”</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="center" valign="top"></th><th colspan="1" rowspan="1" align="center" valign="top"><bold>α-Helix</bold><xref ref-type="table-fn" rid="tblfn1"><bold><italic><sup>a</sup></italic></bold></xref></th><th colspan="1" rowspan="1" align="center" valign="top"><bold>β-Sheet</bold><xref ref-type="table-fn" rid="tblfn1"><bold><italic><sup>a</sup></italic></bold></xref></th><th colspan="1" rowspan="1" align="center" valign="top"><bold>Turns</bold></th><th colspan="1" rowspan="1" align="center" valign="top"><bold>Other</bold></th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top"><bold>MT-FTL</bold></td><td colspan="1" rowspan="1" align="center" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> ContinnLL
</td><td colspan="1" rowspan="1" align="center" valign="top"> 0.55
</td><td colspan="1" rowspan="1" align="center" valign="top"> 0
</td><td colspan="1" rowspan="1" align="center" valign="top"> 0.1
</td><td colspan="1" rowspan="1" align="left" valign="top"> 0.36
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> SELCON3
</td><td colspan="1" rowspan="1" align="center" valign="top"> 0.58
</td><td colspan="1" rowspan="1" align="center" valign="top"> 0
</td><td colspan="1" rowspan="1" align="center" valign="top"> 0.1
</td><td colspan="1" rowspan="1" align="left" valign="top"> 0.32
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> CDSSTR
</td><td colspan="1" rowspan="1" align="center" valign="top">0.57
</td><td colspan="1" rowspan="1" align="center" valign="top">0
</td><td colspan="1" rowspan="1" align="center" valign="top">0.1
</td><td colspan="1" rowspan="1" align="left" valign="top">0.34
</td></tr></tbody><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top"><bold>WT-FTL</bold></td><td colspan="1" rowspan="1" align="center" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> ContinnLL
</td><td colspan="1" rowspan="1" align="center" valign="top"> 0.72
</td><td colspan="1" rowspan="1" align="center" valign="top"> 0
</td><td colspan="1" rowspan="1" align="center" valign="top"> 0
</td><td colspan="1" rowspan="1" align="left" valign="top"> 0.28
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> SELCON3
</td><td colspan="1" rowspan="1" align="center" valign="top"> 0.69
</td><td colspan="1" rowspan="1" align="center" valign="top"> 0
</td><td colspan="1" rowspan="1" align="center" valign="top"> 0.1
</td><td colspan="1" rowspan="1" align="left" valign="top"> 0.3
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> CDSSTR
</td><td colspan="1" rowspan="1" align="center" valign="top">0.72
</td><td colspan="1" rowspan="1" align="center" valign="top">0
</td><td colspan="1" rowspan="1" align="center" valign="top">0
</td><td colspan="1" rowspan="1" align="left" valign="top">0.28
</td></tr></tbody></table><table-wrap-foot><fn id="tblfn1"><label>a</label><p>Regular fraction is indicated</p></fn></table-wrap-foot></table-wrap></p><p><italic>ANS Binding to MT-FTL Homopolymers</italic>—ANS fluorescence
intensity enhancement occurs upon its binding to hydrophobic sites on
proteins. ANS binding assays were performed to study the occurrence of exposed
hydrophobic surfaces in MT-FTL apoprotein homopolymers. In aqueous medium, ANS
shows an emission maximum at 515 nm, but when this reagent binds to a
hydrophobic moiety, its fluorescence intensity increases severalfold, and the
emission maximum is blue-shifted to 470 nm. At pH 7.4, native ferritin does
not bind ANS nor does it fully denatured ferritin
(<xref ref-type="bibr" rid="ref24">24</xref>,
<xref ref-type="bibr" rid="ref30">30</xref>). However, a large increase
in ANS fluorescence at 470 nm was observed when ANS was added to the MT-FTL
homopolymers, indicating exposed hydrophobic sites
(<xref rid="fig5" ref-type="fig">Fig. 5<italic>A</italic></xref>). A
saturation curve exhibiting noncooperative binding was observed following
titration of ANS into MT-FTL homopolymers
(<xref rid="fig5" ref-type="fig">Fig. 5<italic>B</italic></xref>). Scatchard
analyses resulted in a linear plot with an apparent dissociation constant in
the micromolar range.</p><p><italic>Decreased Thermal Stability of MT-Versus WT-FTL Apoferritin
Homopolymers</italic>—The thermal stability of the apoferritin homopolymers
was monitored by measuring the change in intrinsic fluorescence of the protein
as the structure is perturbed. The unusual stability of ferritins necessitated
the addition of a perturbing agent to partially destabilize the protein making
it accessible to denaturation by heating
(<xref ref-type="bibr" rid="ref31">31</xref>,
<xref ref-type="bibr" rid="ref32">32</xref>). Using established
procedures (<xref ref-type="bibr" rid="ref24">24</xref>,
<xref ref-type="bibr" rid="ref25">25</xref>) the protein was excited at
295 nm in the presence of 4.0 <sc>m</sc> GndHCl, and the extent of
denaturation was quantitated as a ratio of emission intensities. With
increasing temperature, the fluorescence emission maxima shifted from 330 nm
(native) to 355 nm (denatured) for both WT- and MT-FTL homopolymers
(<xref rid="fig6" ref-type="fig">Fig. 6<italic>A</italic></xref>). Both
transitions were two-state, and transition midpoint temperatures
(<italic>T<sub>m</sub></italic>) were calculated from the curves
(<xref rid="fig6" ref-type="fig">Fig. 6<italic>B</italic></xref>). WT-FTL
homopolymers had the highest <italic>T<sub>m</sub></italic> (∼90 °C), which
decreased markedly with MT-FTL (∼45 °C). Without addition of
denaturant, MT-FTL exhibited a <italic>T<sub>m</sub></italic> near 75 °C.</p><p><italic>Differential Proteolysis of WT-Versus MT-FTL Homopolymers</italic>—In
general, limited proteolysis does not usually occur with α-helices, but
largely at loops and disordered protein sequences
(<xref ref-type="bibr" rid="ref33">33</xref>,
<xref ref-type="bibr" rid="ref34">34</xref>). To investigate the
possibility of exposed disordered motifs in MT-FTL homopolymers, we conducted
limited proteolysis using thermolysin, which displays broad substrate
specificity (<xref ref-type="bibr" rid="ref35">35</xref>). Thermolysin
selectively cleaved the mutant and generated a predominant fragment of ∼17
kDa. The fragment showed immunoreactivity with an antibody that recognizes the
protein N terminus, suggesting that the mutant homopolymer is cleaved at the C
terminus. In contrast WT-FTL homopolymers were resistant to thermolysin
proteolytic digestion when reacted under identical experimental conditions
(<xref rid="fig7" ref-type="fig">Fig. 7</xref>).</p><p><italic>Chelation of Iron Reverses Iron-induced Aggregation in
Vitro</italic>—Precipitation of MT-FTL homopolymers was observed when they
were loaded with iron (ferrous ammonium sulfate) at greater than 1,000 iron
atoms per ferritin 24-mer, whereas WT-FTL homopolymers remained in the soluble
fraction at least until loading with 4,500 iron atoms. However, the
precipitation of MT-FTL homopolymers was found to be reversible. More
specifically, greater than 50% of mutant homopolymers that were precipitated
by treatment with 3500:1 iron:ferritin was resolubilized by incubation with
the chelator DFX (<xref rid="fig8" ref-type="fig">Fig. 8</xref>).</p><p><italic>Chelation of Iron Reverses Iron-induced Aggregation in
Vivo</italic>—To investigate iron-mediated aggregation <italic>in vivo</italic>, we
used primary cultures of astrocytes from cerebral cortex of transgenic mice
expressing the MT-FTL polypeptide. In this mouse model, the human MT-FTL
polypeptide forms heteropolymers with the endogenous murine wild type Ftl and
Fth1 polypeptides, which are seen to aggregate in neurons and glia throughout
the life span of the mice
(<xref ref-type="bibr" rid="ref29">29</xref>). After treatment of the
cells with FAC to increase their intracellular iron stores, we observed a
switch of ferritin from the detergent-soluble fraction to the
detergent-insoluble fraction, suggesting a change in the solubility
(<xref rid="fig9" ref-type="fig">Fig. 9<italic>A</italic></xref>). Addition of
the lipophilic and freely cell-permeant iron chelator 1,10-Phen to the
FAC-treated cells (FAC/Phen) led to a large reduction in the signal for
detergent-insoluble ferritin and the reappearance of ferritin in the
detergent-soluble fraction (<xref rid="fig9" ref-type="fig">Fig.
9<italic>A</italic></xref>). For these <italic>in vivo</italic> experiments, Phen was
preferred over the weakly cell-permeant DFX that was used for the <italic>in
vitro</italic> experiments. Addition of Phen alone did not seem to have a
significant effect on the amount of ferritin present in the
detergent-insoluble or -soluble fractions isolated from the cultured
astrocytes. Double immunolabeling experiments showed intranuclear accumulation
of ferritin as well as the presence of small, punctate ferritin deposits in
the cellular cytoplasm (<xref rid="fig9" ref-type="fig">Fig. 9</xref>,
<italic>CT</italic>). Importantly, addition of FAC to astrocytes expressing MT-FTL led
to an increase in the signal for MT-FTL
(<xref rid="fig9" ref-type="fig">Fig. 9<italic>B</italic></xref><italic>,
FAC</italic>), which correlated with an increase in the amount of
detergent-insoluble ferritin observed by Western blot analysis. We also
observed that FAC treatment led to astrocyte reactivity, characterized by a
redistribution of the intermediate filament GFAP and the formation of long
cytoplasmic processes (<xref rid="fig9" ref-type="fig">Fig.
9<italic>B</italic></xref><italic>, FAC</italic>) as described previously
(<xref ref-type="bibr" rid="ref28">28</xref>). Addition of Phen after
incubation with FAC partially reversed the astrocytic reactive phenotype and
decreased the cellular immunolabeling for ferritin
(<xref rid="fig9" ref-type="fig">Fig. 9<italic>B</italic></xref><italic>,
FAC/Phen</italic>). No significant differences with the control were observed when
only the chelator was added to the cell culture
(<xref rid="fig9" ref-type="fig">Fig. 9</xref>, <italic>Phen</italic>).</p><p><fig position="float" id="fig5"><label>FIGURE 5.</label><caption><p><bold>Binding of ANS to MT-FTL apoferritin homopolymers.</bold> <italic>A,</italic> ANS
fluorescence emission enhancement and wavelength shift caused by titration of
MT-FTL homopolymers with increasing concentrations of ANS. <italic>B,</italic>background and dilution corrected fluorescence emission intensity at 460 nm as
a function of ANS concentration. Titration was performed on 1.5
μ<sc>m</sc> MT-FTL homopolymer at pH 7.4 and 25 °C in 50
m<sc>m</sc> potassium phosphate buffer. The <italic>dotted line</italic> corresponds
to 100 μ<sc>m</sc> ANS in potassium phosphate buffer.</p></caption><graphic xlink:href="zbc0460854930005"></graphic></fig></p><p><fig position="float" id="fig6"><label>FIGURE 6.</label><caption><p><bold>Decrease in thermal stability of MT- <italic>versus</italic> WT-FTL apoferritin
homopolymers.</bold> <italic>A,</italic> temperature dependence of fluorescence emission
spectra of MT- and WT-FTL homopolymers destabilized by 4.0 <sc>m</sc>GdnHCl. The <italic>red trace</italic> represents denatured ferritin (<italic>den</italic>)
induced by incubation at pH 2.0 in 4.5 <sc>m</sc> GdnHCl. <italic>B,</italic>fluorescence emission intensity ratio (of 355 over 330 nm) to determine
denaturation temperature of both apoferritin homopolymers. Scans were
performed on 1.5 μ<sc>m</sc> ferritin homopolymer at pH 7.4 in potassium
phosphate buffer with excitation at 295 nm. Homopolymers were incubated 12 h
in 4.5 <sc>m</sc> GdnHCl before beginning temperature dependence experiment.
<italic>Errors bars</italic> represent the standard deviation of three independent
experiments.</p></caption><graphic xlink:href="zbc0460854930006"></graphic></fig></p><p><fig position="float" id="fig7"><label>FIGURE 7.</label><caption><p><bold>Thermolysin treatment of WT- and MT-FTL apoferritin homopolymers.</bold>Recombinant MT- and WT-FTL homopolymers (1 μ<sc>m</sc>) were incubated
with thermolysin (0.15 units) in Hepes buffer (0.1 <sc>m</sc>) (pH 7.0), 60
m<sc>m</sc> NaCl, 1 m<sc>m</sc> CaCl<sub>2</sub>. After 10 min at 37
°C, the reaction was quenched with 10 μl of EDTA (50 m<sc>m</sc>).
Sample buffer was added, and the samples treated with thermolysin (+), and
controls without thermolysin (-) were loaded onto 4–20% SDS-PAGE. Gels
were stained with Coomassie Blue (<italic>Total protein</italic>) or blotted using
antibodies specific for the N terminus of FTL or the C terminus of MT-FTL or
WT-FTL.</p></caption><graphic xlink:href="zbc0460854930007"></graphic></fig></p></sec><sec><title>DISCUSSION</title><p>In this work we investigate the protein structure and iron storage function
of ferritin homopolymers formed from a light chain variant
<italic>p.Phe167SerfsX26</italic> that causes HF
(<xref ref-type="bibr" rid="ref9">9</xref>). Both wild type FTH1 and
FTL polypeptide subunits are important for the iron storage function of
ferritin, with the former containing the ferroxidase site for ferrous iron
oxidation and the latter containing the iron nucleation site
(<xref ref-type="bibr" rid="ref2">2</xref>,
<xref ref-type="bibr" rid="ref3">3</xref>). However, homopolymers
composed of FTL subunits have additional properties such as resistance to
precipitation under iron loading and being significantly more stable to
denaturation by heat and solvent
(<xref ref-type="bibr" rid="ref30">30</xref>,
<xref ref-type="bibr" rid="ref36">36</xref>), and apparently FTL
subunits are able to confer these properties upon the heteropolymer. Thus as
the first approach to elucidating the effects of the mutation, we compared and
contrasted wild type with MT-FTL homopolymers, which have a C terminus of
altered length and composition
(<xref ref-type="bibr" rid="ref9">9</xref>).</p><p>Gel filtration and nondenaturing electrophoretic analyses showed that at
physiological pH, recombinant MT-FTL polypeptides were able to assemble as
24-mer homopolymers, which were ultrastructurally indistinguishable from
homopolymers of recombinant WT-FTL polypeptide. However, homopolymers made of
the mutant subunit showed a smaller retention time and a slower
electrophoretic mobility compared with those of the WT-FTL polypeptide,
consistent with the molecular mass difference. Homopolymers of recombinant WT-
and MT-FTL polypeptides were able to incorporate iron <italic>in vivo</italic> in
preparations obtained from <italic>E. coli</italic>, suggesting that both are
functional proteins. <italic>In vitro</italic>, recombinant WT- and MT-apoferritin
homopolymers incorporated similar amounts of iron up to 1000:1 iron:ferritin
molar ratio over a 2-h incubation. However, at higher iron:ferritin ratios,
WT-FTL homopolymers continued incorporating iron, whereas MT-FTL homopolymers
began to precipitate, limiting their iron storage function.</p><p>To understand this functional difference, MT- and WT-FTL apoferritins were
analyzed by spectroscopic techniques. Such techniques are particularly useful
in revealing differences between similarly structured proteins. In the far-UV,
the CD spectra of the MT-FTL homopolymers showed a decrease (∼15%) in
total α-helical content and an increase in turns and unordered
structures compared with the WT-FTL. Such a 15% decrease could be accounted
for precisely by complete unraveling of the shortest α-helical segment
of ferritin (the E helix, located at the C terminus) or alternatively some
fraction of portions of the longer helical segments A–D. The E helix
involvement is supported by the length and position of the mutation itself
(<xref ref-type="bibr" rid="ref9">9</xref>,
<xref ref-type="bibr" rid="ref12">12</xref>). The CD data are in
agreement with secondary structural prediction analysis (Jpred)
(<xref ref-type="bibr" rid="ref37">37</xref>) and analysis for
α-helical context (ProtScale)
(<xref ref-type="bibr" rid="ref38">38</xref>), both of which are
consistent with a loss of the E helical domain in the MT-FTL polypeptide.</p><p><fig position="float" id="fig8"><label>FIGURE 8.</label><caption><p><bold>Iron-induced aggregation of MT-FTL homopolymers and its reversal by the
iron chelator deferroxamine.</bold> MT-FTL homopolymers were incubated in a molar
ratio 1:3500 with ferrous ammonium sulfate as in
<xref rid="fig3" ref-type="fig">Fig. 3</xref>. After centrifugation,
the pellet was resuspended in a solution containing 6 m<sc>m</sc> DFX, 0.1
<sc>m</sc> Hepes (pH 7.4) and incubated for 2 h at 24 °C. After a second
centrifugation, soluble fractions were run in native gels and stained with
Coomassie Blue for protein and with Prussian blue for iron. Densitometric
analysis of the protein bands was performed, and the values are shown as
relative densitometric units (<italic>RDU</italic>) as a percentage of the control
without iron for each protein. All data are expressed as the mean ±
S.D. of three independent experiments. WT-FTL homopolymers did not precipitate
after iron treatment (not shown).</p></caption><graphic xlink:href="zbc0460854930008"></graphic></fig></p><p>The CD spectra of the MT- and WT-FTL homopolymers in the near-UV region,
which reflects the packing environment of the tyrosines and tryptophans, were
very similar. This similarity suggests that the mutation in the FTL
polypeptide introduces only a minor change in tertiary and/or quaternary
structures of ferritin, which is in agreement with the ability to form the
assembled state observed by gel filtration chromatography and nondenaturing
PAGE. The minor spectral difference at 280 nm is likely produced by structural
perturbation around tyrosine. One out of the six tyrosines in FTL is located
in its C-terminal sequence (amino acid 165) and is present in the WT- and
MT-FTL polypeptides. No additional tyrosines or tryptophans are introduced by
the mutation to contribute spectral intensity at 280–300 nm. Routine
scans of the endogenous protein fluorescence with excitation at 280 and 295 nm
gave no difference between WT- and MT-FTL homopolymers (data not shown).
However, a structural difference between WT- and MT-FTL homopolymers was
revealed by titration with the exogenous fluorophore ANS, which increases its
fluorescence and shifts its emission maximum upon interaction with a nonpolar
environment. ANS fluorescence was enhanced in a manner indicating equilibrium
binding to a hydrophobic pocket that is formed in the mutant. Taken together,
the spectroscopic studies are consistent with no changes in the helix content
and packing of the 4-helix bundle (A–D) or in the majority of the
intersubunit interactions, but with an unraveling of the E helix in the mutant
such as to create hydrophobic binding sites for ANS.</p><p><fig position="float" id="fig9"><label>FIGURE 9.</label><caption><p><bold>Iron-induced aggregation of cellular ferritin and its reversal by the
chelator phenanthroline.</bold> <italic>A,</italic> immunoblot for MT-FTL ferritin from
total cell extracts and their SDS-insoluble and SDS-soluble fractions.
Transgenic astrocytes were exposed to water (<italic>CT</italic>); 50 μ<sc>m</sc>ferric ammonium citrate for 72 h (<italic>FAC</italic>); 50 μ<sc>m</sc>phenanthroline for 72 h (<italic>Phen</italic>); or 50 μ<sc>m</sc> ferric
ammonium citrate for 72 h following 72 h of the iron chelator phenanthroline
(50 μ<sc>m</sc>) (<italic>FAC/Phen</italic>). To verify similar loading,
membranes were reprobed with anti-actin antibodies. <italic>B,</italic> confocal
immunofluorescence microscopy of cultured MT-FTL-transgenic astrocytes treated
as in <italic>A</italic>. Cells were immunostained with anti MT-FTL antibody
(<italic>green</italic>) and anti-GFAP antibody (<italic>red</italic>). Note the increase in
MT-FTL signal after iron treatment (FAC). The signal was greatly decreased
after treatment with the chelator (FAC/Phen). <italic>Bar,</italic> 20 μm.</p></caption><graphic xlink:href="zbc0460854930009"></graphic></fig></p><p>A feature of ferritin 24-mers in general is their high stability to heat
and to urea or guanidinium chloride exposure
(<xref ref-type="bibr" rid="ref2">2</xref>). We found that in the
presence of 4.5 <sc>m</sc> GdnHCl, WT-FTL homopolymers denatured with the
higher <italic>T<sub>m</sub></italic> ∼90 °C and MT-FTL at ∼45 °C.
Without addition of the destabilizing agent, MT-FTL denatured near 75 °C,
which is reduced considerably from that reported in the literature
(<xref ref-type="bibr" rid="ref30">30</xref>,
<xref ref-type="bibr" rid="ref32">32</xref>) for the wild type, and
again points toward significant destabilization of the mutant. For comparison,
FTH1 homopolymers are less stable to heat denaturation than FTL homopolymers,
perhaps because of residues located at the intersubunit contacts along the 3-
and 4-fold channels and by salt bridges within the 4-helix bundles themselves
between Lys-62 and Glu-107
(<xref ref-type="bibr" rid="ref24">24</xref>,
<xref ref-type="bibr" rid="ref32">32</xref>). Indeed, native E helix
conformation appears to help stabilize the subunit structure of the FTL
homopolymer by making several hydrophobic contacts with apolar side chains
near the start of helix B and the end of D as well as being linked by hydrogen
bonds to the N-terminal ends of helices B and C
(<xref ref-type="bibr" rid="ref2">2</xref>,
<xref ref-type="bibr" rid="ref6">6</xref>,
<xref ref-type="bibr" rid="ref7">7</xref>,
<xref ref-type="bibr" rid="ref39">39</xref>). Given the spectroscopic
results, the difference in thermal stability observed between the WT and
mutant homopolymers can be attributed to the loss of the interactions around
the E helix in the MT-FTL subunit. Although the E helices are known to
contribute to the exceptional thermostability of ferritin, they are not
essential in the pattern of ferritin assembly
(<xref ref-type="bibr" rid="ref40">40</xref>).</p><p>The data presented so far suggest that ferritin accommodates the extensive
sequence alteration present in the <italic>p.Phe167SerfsX26</italic> mutant without
disturbing its assembly/folding pathway or spherical shell structure.
Susceptibility to thermal denaturation was enhanced by the mutation, but not
in the physiological range of temperature where it may be directly causative
of precipitation. In WT-FTL, the helical C terminus is not accessible, being
accommodated inside the spherical surface (as part of the shell structure) and
forming the 4-fold pore. However, the disposition of the unraveled C terminus
in the mutant was not clearly delineated by spectroscopy or denaturation
studies in that it may be contained within the interior, be totally exterior
to the shell, or have some intermediate conformation. To investigate the
difference in C-terminal conformation between WT and mutant homopolymers, both
were subjected to thermolysin proteolysis and the products analyzed by gel
electrophoresis and Western blot. The results showed that the C terminus of
the MT-FTL homopolymer, but not the WT, was susceptible to proteolysis with
∼75% of the sample exhibiting loss. The N terminus was, however, not
susceptible to cleavage nor were there other proteolysis products evident.
These results strongly support a conformation in which at least part of the C
terminus sequence of the mutant extends into the solvent far enough above the
well formed, spherical exterior surface to be approached and cleaved by
thermolysin for a large fraction of ferritin subunits. Alternatively, a
distribution between completely external and completely internal C termini
could occur. Thus, not only is the 4-fold pore disrupted, but ferritin has an
amino acid sequence protruding external to the shell to interact with its
surroundings (including iron). The lack of multiple thermolysin cleavage bands
also attests to the intact nature of the mutant homopolymer, paralleling the
wild type intactness.</p><p>Changes in the C terminus in different ferritin polypeptides exhibit a
variety of documented effects. C-terminal deletion of a comparable sequence in
the FTH1 polypeptide caused the protein to form oligomers that were unable to
incorporate and keep iron in solution
(<xref ref-type="bibr" rid="ref14">14</xref>,
<xref ref-type="bibr" rid="ref40">40</xref>), whereas lengthening of
the FTH1 polypeptide by addition of various amino acid sequences did not
modify ferritin assembly. Interestingly, when large peptide sequences were
added, they were found to be exposed outside the ferritin shell
(<xref ref-type="bibr" rid="ref14">14</xref>,
<xref ref-type="bibr" rid="ref40">40</xref>,
<xref ref-type="bibr" rid="ref42">42</xref>–<xref ref-type="bibr" rid="ref44">44</xref>).
Insertional mutations of a mouse <italic>Fth1</italic> cDNA using nucleotide sequences
similar to those associated with HF produced mutant polypeptides of different
lengths. In these experiments, the mutant polypeptides showed a significant
alteration in protein folding, assembly, and function, which was correlated
with the loss of the last helical domain that existed in the WT protein
(<xref ref-type="bibr" rid="ref45">45</xref>). Studies using
recombinant mutant FTL (<italic>p.Arg154LysfsX26</italic>), corresponding to the
460–461InsA variant (<xref ref-type="bibr" rid="ref8">8</xref>)
suggested that the recombinant polypeptide was able to assemble into ferritin
shells with low efficiency and that the C terminus was exposed outside the
shell (<xref ref-type="bibr" rid="ref46">46</xref>). Analysis of FTL
polypeptides with the <italic>p.Phe167SerfsX26</italic> mutation suggest that this
mutation is apparently not severe enough to prevent iron incorporation, but it
has a C terminus exposed external to the protein shell similar to that
associated with the <italic>FTL460–461InsA</italic> mutation and the extended
Fth1 polypeptides. Although ferritin formed from our MT-FTL polypeptide
precipitated at iron concentration significantly below that of wild type, such
precipitation was at least partially reversible. Approximately 50% of ferritin
precipitated at a loading of 3500:1 iron:ferritin ratio was resolubilized
using the iron chelator DFX, and that resolubilized protein contained iron.
Iron-induced aggregation was also seen <italic>in vivo</italic>, but was substantially
reversed by the iron chelator Phen. These results argue against large scale
structural disruption of the ferritin shells and are in agreement with the
spectroscopic and TEM data, which showed that the precipitates had the
spherical shape of ferritin.</p><p>Significant mutant C-terminal sequence exposure external to a well formed
ferritin shell, differential iron-induced precipitation between the mutant and
wild type, and reversibility of aggregation by chelation of iron lead to the
model of iron-induced aggregation shown in
<xref rid="fig10" ref-type="fig">Fig. 10</xref>. Specifically, iron
can bind in widely varying affinities to several groups on the exposed C
terminus of MT-FTL ferritin. The C-terminal carboxylate, the glutamates, the
tyrosinate, and perhaps even the serine hydroxyl, threonine hydroxyl, and
peptide backbone groups all can bind iron. The precipitation process appears
to begin when an iron (or iron nucleation complexes) binds to groups on the
exposed C-terminal regions of two mutant 24-mers preventing their
translational motion. Additional C-terminal iron bridging (from other mutant
subunits) and/or surface carboxylate (glutamate and aspartate) bridging may
occur as the two ferritin shells come together tightening their interaction
producing dimers. There is independent evidence of the existence of ferritin
dimers in the literature (<xref ref-type="bibr" rid="ref16">16</xref>,
<xref ref-type="bibr" rid="ref47">47</xref>), which would agree with
involvement of iron-bound carboxylate enhancing bridging. Furthermore,
calorimetry studies provide evidence of a large number of weakly bound irons
independent of those more tightly bound at the ferroxidase center
(<xref ref-type="bibr" rid="ref41">41</xref>). The process then reaches
out to more homopolymers producing precipitation. This process is greatly
affected by the redox state of the iron, not just with respect to hydrolysis
and formation of precipitated hydroxide complexes, which are part of the
bridging and matrix of the ferritin aggregate, but also because the binding
strengths of the various groups mentioned above are strongly redox-dependent
with ferric iron preferring to bind to hard ligands and ferrous iron generally
considered a weaker binder. Finally, the C terminus of the mutant ferritin has
hydrophobic patches, which may augment the strength of the bridging
interaction and perhaps hinder reversibility by iron chelation. It should be
noted that this model is not limited to mutant homopolymers in that
heteropolymers with a fraction of MT-FTL polypeptide subunits are not
precluded from undergoing iron-induced aggregation. The details of the
aggregation process are currently under investigation.</p><p><fig position="float" id="fig10"><label>FIGURE 10.</label><caption><p><bold>Simplified model describing the steps in the iron-induced aggregation
process of mutant ferritin.</bold> <italic>A,</italic> C termini of MT-FTL polypeptides
extend above the spherical surface of ferritin exposing this sequence of the
peptide to solvent and iron. <italic>B,</italic> sequence binds iron (or iron
nucleation complexes) and through it the C terminus of a second MT-FTL
polypeptide, reducing the translational motion of both 24-mers. <italic>C,</italic>additional cross-linking occurs (through iron bridges) between C termini, a C
terminus and surface carboxylate, and/or eventually through carboxylates on
both 24-mers forming ferritin dimers. <italic>D,</italic> dimers aggregate
further.</p></caption><graphic xlink:href="zbc0460854930010"></graphic></fig></p><p>We observed that ferritin precipitates obtained <italic>in vitro</italic> were
composed of fully assembled 24-mers, similar to what has been reported in
inclusions in patients with HF
(<xref ref-type="bibr" rid="ref9">9</xref>,
<xref ref-type="bibr" rid="ref12">12</xref>) and in transgenic mice
expressing the MT-FTL polypeptide
(<xref ref-type="bibr" rid="ref29">29</xref>). At iron: ferritin ratios
above 2000:1, under the same experimental conditions in which we analyzed
WT-and MT-FTL, the majority of FTH1 but not WT-FTL ferritins precipitate
(<xref ref-type="bibr" rid="ref16">16</xref>). The precipitation of
FTH1 has been suggested to be related to extra-cavity iron hydrolysis
(<xref ref-type="bibr" rid="ref16">16</xref>). Iron-induced aggregation
of FTH1 (<xref ref-type="bibr" rid="ref16">16</xref>) and MT-FTL
homopolymers was not irreversible because pellets could be resolubilized by
the addition of iron chelators. <italic>In vivo</italic>, we found that the addition
of iron led to intracellular accumulation of ferritin in astrocytes from
transgenic mice expressing the MT-FTL polypeptide
(<xref ref-type="bibr" rid="ref29">29</xref>). Detergent-insoluble
ferritin is typically seen in brain extracts from patients with HF and
transgenic mice expressing the MT-FTL polypeptide
(<xref ref-type="bibr" rid="ref29">29</xref>). After iron addition,
astrocytic ferritin was found mostly in the detergent-insoluble fraction,
indicating that iron-induced ferritin aggregation occurs <italic>in vivo</italic> and
may be the mechanism underlying ferritin aggregation in patients with HF. The
quantity of detergent-insoluble ferritin was significantly reduced by the
addition of the iron chelator Phen to the astrocyte culture, in agreement with
the <italic>in vitro</italic> studies.</p><p>Our data show that the <italic>FTL498–499InsTC</italic> mutation leads to the
generation of FTL polypeptides that are able to assemble into ferritin
24-mers. However, MT-FTL homopolymers have a diminished ability to sequester
iron and aggregate well before wild type homopolymers as iron levels are
increased. <italic>In vivo</italic>, the MT-FTL polypeptide may act as a dominant
negative mutant, leading to a failure of ferritin in its iron storage function
and an increase in the levels of intracellular iron. Intracellular free iron
generates a positive feedback loop, in which it promotes the release of the
iron regulatory proteins from the ferritin iron-responsive elements
(<xref ref-type="bibr" rid="ref2">2</xref>), overexpression of ferritin
polypeptides, and the aggregation of mutant-containing ferritins as observed
in patients with HF (<xref ref-type="bibr" rid="ref12">12</xref>) and
in transgenic mice (<xref ref-type="bibr" rid="ref29">29</xref>). Thus
we propose that deregulation of cellular iron metabolism and formation of
ferritin aggregates, which may physically interfere with normal cellular
functions (causing a gain of a toxic function), may be the key pathological
mechanisms eventually leading to HF. It should be mentioned that this
aggregation mechanism does not exclude other levels of iron mismanagement
operating more subtly in addition to it, <italic>e.g.</italic> iron-induced oxidative
stress. Considering the numerous hydrophobic amino acids in the mutant C
terminus, a peptide-based iron chelator containing one or more hydrophobic
groups may more effectively hinder ferritin aggregation than a simple chelator
alone.</p></sec></body><back><ack><p>We are thankful to Dr. B. Ghetti for supportive comments. We gratefully
acknowledge the Harvard Microchemistry and Proteomics Analysis Facility for
the mass spectrometry and protein sequencing analysis of recombinant proteins
and the Indiana Center for Biological Microscopy at Indiana University School
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ferritin light chain; MT-FTL, mutant (<italic>p.Phe167SerfsX26</italic>) ferritin
light chain; FTH1, ferritin heavy chain; ANS,
8-anilino-1-naphthalenesulfonate; TEM, transmission electron microscopy;
GdnHCl, guanidine hydrochloride; FAC, ferric ammonium citrate; Phen, 1,
10-phenanthroline; DFX, deferroxamine; IREs, iron-responsive elements; HF,
hereditary ferritinopathy; GFAP, glial fibrillary acidic protein; PBS,
phosphate-buffered saline.</p></fn></fn-group></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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