cdnas 
  containing the sequence of human wt - ftl and human mutant 
  ftl498499instc 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 his6 tag . to eliminate the his6 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 cat cga agg 
  tcg tat 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 ) escherichia coli ( invitrogen ) . 
 
transformed cells were grown in luria broth medium ( lb ) containing 30 g / ml 
  kanamycin ( invitrogen ) at 37 c up to an absorbance of 0.91.0 at 
  600 nm .
bacteria were induced to overexpress recombinant proteins by adding 1 
  mm isopropyl thio--d - galactopyranoside ( icn 
  biotechnologies ) for 12 h at 25 c .
purification of recombinant wt- and mt - ftl 
  homopolymers  cells were harvested by centrifugation and frozen at 
  -80 c .
the cell pellets were suspended in 50 mm sodium 
  phosphate , 500 mm 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  g 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 
  mm imidazole in 50 mm sodium phosphate ( ph 7.4 ) , 0.5 
  m nacl .
recombinant proteins were diluted with 50 mm 
  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 
  mm 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 
  mm 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 .
gel filtration chromatography  size exclusion chromatography 
  was performed on a superose 6 10/300 gl column ( ge healthcare ) equilibrated 
  with 50 mm tris , 150 mm 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 
  kav values were evaluated using the unicorn 5.1 software 
  ( ge healthcare ) .
transmission electron microscopy ( tem)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-l 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 .
preparation of apoferritins  recombinant ftl homopolymers 
  were treated for iron removal as described previously 
  ( 14 ) .
briefly , recombinant 
  ferritins were incubated with 1% thioglycolic acid ( ph 5.5 ) and 
  2,2-bipyridine , followed by dialysis against 0.1 m 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 
  ( 15 ) .
iron loading of apoferritins  freshly prepared ferrous 
  ammonium sulfate ( 0.54.5 mm ) in 10 mm hcl was 
  added to mt- and wt - ftl apoferritin homopolymers ( 1 m ) in 0.1 
  m hepes buffer ( ph 7.4 ) at room temperature 
  ( 16 ) .
after 2 h , the samples 
  were centrifuged at 14,000  g for 15 min .
iron incorporation 
  was initially monitored by measuring absorbance of the supernatants at 310 nm 
  ( 14 , 
  17 ) .
iron incorporation into 
  ferritin was more precisely determined by densitometric analysis of prussian 
  blue staining of supernatants run on nondenaturing gel electrophoresis . 
 
apoferritins were also incubated in a 
  molar ratio 1:3500 with ferrous ammonium sulfate and centrifuged at 14,000 
   g for 15 min .
pellets were resuspended in a solution 
  containing 6 mm deferroxamine ( dfx ) , 0.1 m hepes ( ph 
  7.4 ) and incubated for 2 h at 24 c .
cd spectra of recombinant 
  apoferritin homopolymers were obtained in 50 mm 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 m 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 .
cd spectra of the buffer / cuvette 
  were recorded and subtracted from the protein spectra before averaging . 
 
secondary structure analyses were performed using dichroweb 
  ( 18 , 
  19 ) , which allows secondary 
  structure analyses via the software package cdpro 
  ( 20 ) .
selcon3 
  ( 21 ) , continll 
  ( 22 ) , and cdsstr 
  ( 23 ) 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
intrinsic protein fluorescence and thermal stability studies of 
  homopolymers  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 m protein 
  in 1-cm path length cells and with 0.1 m phosphate ( ph 7.4 ) .
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 m phosphate buffer ( ph 7.4 ) 
  containing 4.0 m guanidine hydrochloride ( gdnhcl ) .
homopolymer 
  stability was monitored using the ratio of intrinsic fluorescence emission of 
  355 over 330 nm with excitation at 295 nm 
  ( 24 , 
  25 ) with a maximum at 330 nm 
  signifying native ferritin ( mt and wt ) and 355 nm , denatured ferritin .
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 m in 0.05 m 
  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 m cm .
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 
  ( 26 ) .
thermolysin treatment of wt- and mt - ftl apoferritin 
  homopolymers  proteolysis of recombinant mt- and wt - ftl homopolymers 
  was initiated by adding to 10 g of ferritin a 10-fold concentrated stock 
  solution ( 36.5 units / mg ) of thermolysin ( fluka ) in hepes ( 0.1 m ) 
  ( ph 7.0 ) , 10 mm cacl2 to a final concentration of 0.2 
  mg / ml . the reaction was stopped by the addition of edta ( 50 mm ) and 
  laemmli sample buffer .
samples treated with thermolysin and controls without 
  thermolysin were boiled and loaded onto sds - polyacrylamide gels ( 420% ) 
  ( pierce ) .
gels were stained with coomassie blue ( total protein ) or blotted 
  against the c - terminal antibodies ( mt-1283 or wt-1278 ) 
 
( 9 ) or against the n - terminal 
  antibody d18 ( santa cruz biotechnology , inc ) , which recognized both 
  polypeptides . astrocyte cell cultures and iron / chelator treatment 
primary 
  cortical astrocyte cultures were prepared from 1-day - old mouse pups according 
  to the procedures of saneto and de vellis 
  ( 27 ) and cassina et 
  al .
pups were obtained from transgenic dams homozygous for the 
  ftl498499instc mutation in c57bl/6j genetic background 
  ( 29 ) .
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 cells in 
  25-cm flasks ( corning glass ) in dulbecco 's modified eagle 's medium 
  supplemented with 10% fetal bovine serum , hepes ( 25 mm ) , penicillin 
  ( 100 iu / ml ) , and streptomycin ( 100 g / ml ) ( invitrogen ) .
when confluent , 
  cultures were shaken for 48 h at 250 rpm at 37 c , incubated for another 
  48 h with 10 m cytosine arabinoside , and then amplified to 2.5 
   10 cells / cm in 75-cm 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 mm ) 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 : ( a ) vehicle ( water ) 
  as control group ; ( b ) phen at 100 m during 48 h 
  followed by 24 h at 50 m;(c ) fac 50 m 
  during 4 days ; ( d ) fac treatment as in c followed by phen 
  treatment as in b in the absence of iron .
characterization of detergent - insoluble mt - ftl ferritin from astrocyte 
  cultures  cerebral cortical astrocytes cultures were homogenized in 
  lysis buffer ( 3 ml of 50 mm tris - hcl ( ph 7.4 ) , 1% sds , 30 units / ml 
  benzonase , 2 mm mgcl2 ) 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 420% gradient 
  sds - page ( pierce ) and transferred to nitrocellulose membranes ( amersham 
  biosciences ) .
membranes were blocked for1 h in 70 mm 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 ( 9 , 
  29 ) 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 ) .
the wild 
  type ftl polypeptide ( wt - ftl ) consists of 175 amino acids .
the 
  p.phe167serfsx26 mutant polypeptide ( mt - ftl ) has 191 amino acids and 
  a different c - terminal sequence ( underlined ) .
the boxes 
  indicate the five -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 .
the wild 
  type ftl polypeptide ( wt - ftl ) consists of 175 amino acids .
the 
  p.phe167serfsx26 mutant polypeptide ( mt - ftl ) has 191 amino acids and 
  a different c - terminal sequence ( underlined ) .
the boxes 
  indicate the five -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 .
figure 2.mt-ftl polypeptides assemble into 24-mer homopolymers . a , 
  elution profiles of purified wt- and mt - ftl apoferritin homopolymers from a 
  superose 6 column at ph 7.4 in 0.05 m tris , 0.15 m nacl . 
 
the dark cores most 
  likely represent nanovan that has penetrated in some cases the interior of the 
  24-mers .
c , native page ( 38% ( ph7.4 ) ) of 
  0.5 m wt- and mt - ftl proteins loaded before the removal of iron 
  and stained with coomassie blue ( protein staining ) and with prussian blue 
  ( iron staining ) .
a , 
  elution profiles of purified wt- and mt - ftl apoferritin homopolymers from a 
  superose 6 column at ph 7.4 in 0.05 m tris , 0.15 m nacl . 
 
the dark cores most 
  likely represent nanovan that has penetrated in some cases the interior of the 
  24-mers .
c , native page ( 38% ( ph7.4 ) ) of 
  0.5 m wt- and mt - ftl proteins loaded before the removal of iron 
  and stained with coomassie blue ( protein staining ) and with prussian blue 
  ( iron staining ) .
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 g / ml ; molecular probes ) . 
 
images were captured with a zeiss lsm-510 confocal scanner attached to a zeiss 
  axiovert 100 m inverted microscope .
recombinant mt - ftl polypeptides assemble into 24-mer 
  homopolymers  recombinant wt- and mt - ftl polypeptides 
  ( fig .
1 ) were expressed in 
  e. coli 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 ( fig .
ultrastructural 
  analysis by tem showed that both recombinant ferritins had spherical shape and 
  a size ( diameter 110  ) similar to that of human ferritin 
  ( fig .
nondenaturing page showed that both wt- and mt - ftl homopolymers , examined 
  before iron removal , were able to assemble and incorporate iron in 
  vivo during expression in e. coli 
  ( fig . 2c ) . 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 ) 
  ( fig . 2c ) .
enhanced precipitation of mt - ftl homopolymers  iron loading 
  of apoferritin homopolymers was examined by an often used and well described 
  procedure ( 14 , 
  16 , 
  17 ) . 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 .
3a ) 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 
  ( fig .
3b ) . 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 versus wild type at 1000:1 iron : ferritin 
  loading ( fig .
3c ) , 
  which emphasizes iron mishandling through mutant ferritin precipitation . by 
  tem , mt - ftl
ferrous ammonium sulfate ( 0.54.5 mm ) was added 
  to 1 m of mt- and wt - ftl homopolymers in 0.1 m hepes 
  ( ph 7.0 ) for 2 h at 24 c .
samples were centrifuged for 15 min at 10,000 
   g to separate into soluble and insoluble fractions . 
 
a , iron uptake / hydrolysis was monitored in the soluble fractions by 
  measuring absorbance at 310 nm .
b , soluble and insoluble 
  fractions were loaded into native gels sds - page , respectively , and stained 
  with coomassie blue .
c , 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 ( 38% ) .
ferrous ammonium sulfate ( 0.54.5 mm ) was added 
  to 1 m of mt- and wt - ftl homopolymers in 0.1 m hepes 
  ( ph 7.0 ) for 2 h at 24 c .
samples were centrifuged for 15 min at 10,000 
   g to separate into soluble and insoluble fractions . 
 
a , iron uptake / hydrolysis was monitored in the soluble fractions by 
  measuring absorbance at 310 nm .
b , soluble and insoluble 
  fractions were loaded into native gels sds - page , respectively , and stained 
  with coomassie blue .
c , 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 ( 38% ) .
far - uv ( a ) and near - uv ( b ) spectra were 
  recorded at homopolymer concentrations of 0.12 and 1.5 m , 
  respectively , at ph 7.4 and 25 c in 50 mm potassium phosphate 
  buffer .
far - uv ( a ) and near - uv ( b ) spectra were 
  recorded at homopolymer concentrations of 0.12 and 1.5 m , 
  respectively , at ph 7.4 and 25 c in 50 mm potassium phosphate 
  buffer .
spectroscopic comparison of mt - ftl apoprotein homopolymers versus 
  wt  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 191193 nm 
  ( fig .
the profile 
  obtained was typical of a protein containing predominantly -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 
  -helical content and an increase in turns and unordered structures 
  ( table 1 ) .
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 ( fig . 
 
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 ) .
table 1deconvolution of far - uv cd spectra for wt- and mt - ftl apoferritin 
  homopolymers into percent secondary structural contributions the analysis 
  was performed using fitting programs contnnll , selcon3 , and cdsstr available 
  at the website dichroweb as described under 
experimental 
  procedures.-helix-sheetturnsothermt-ftl  continnll 
   0.55 
   0 
   0.1 
   0.36 
   selcon3 
   0.58 
   0 
   0.1 
   0.32 
 
  cdsstr 
 
  0.57 
 
  0 
 
  0.1 
 
  0.34 
  wt - ftl  continnll 
   0.72 
   0 
   0 
   0.28 
   selcon3 
   0.69 
   0 
   0.1 
   0.3 
 
  cdsstr 
 
  0.72 
 
  0 
 
  0 
 
  0.28 
  aregular fraction is indicated deconvolution of far - uv cd spectra for wt- and mt - ftl apoferritin 
  homopolymers into percent secondary structural contributions the analysis 
  was performed using fitting programs contnnll , selcon3 , and cdsstr available 
  at the website dichroweb as described under  experimental 
  procedures . 
regular fraction is indicated ans binding to mt - ftl homopolymers  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 
  ( 24 , 
  30 ) .
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 
  ( fig .
a 
  saturation curve exhibiting noncooperative binding was observed following 
  titration of ans into mt - ftl homopolymers 
  ( fig .
5b ) . scatchard 
  analyses resulted in a linear plot with an apparent dissociation constant in 
  the micromolar range . decreased thermal stability of mt - versus wt - ftl apoferritin 
  homopolymers  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 
  ( 31 , 
  32 ) .
using established 
  procedures ( 24 , 
  25 ) the protein was excited at 
  295 nm in the presence of 4.0 m 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 
  ( fig .
both 
  transitions were two - state , and transition midpoint temperatures 
  ( tm ) were calculated from the curves 
  ( fig .
wt - ftl 
  homopolymers had the highest tm ( 90 c ) , which 
  decreased markedly with mt - ftl ( 45 c ) .
without addition of 
  denaturant , mt - ftl exhibited a tm near 75 c .
differential proteolysis of wt - versus mt - ftl homopolymers  in 
  general , limited proteolysis does not usually occur with -helices , but 
  largely at loops and disordered protein sequences 
  ( 33 , 
  34 ) . to investigate the 
  possibility of exposed disordered motifs in mt - ftl homopolymers , we conducted 
  limited proteolysis using thermolysin , which displays broad substrate 
  specificity ( 35 ) .
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 
  ( fig .
chelation of iron reverses iron - induced aggregation in 
  vitro  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 ( fig .
chelation of iron reverses iron - induced aggregation in 
  vivo  to investigate iron - mediated aggregation in vivo , 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 
  ( 29 ) .
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 
  ( fig .
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 ( fig . 
 
for these in vivo experiments , phen was 
  preferred over the weakly cell - permeant dfx that was used for the in 
  vitro 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 ( fig .
importantly , addition of fac to astrocytes expressing mt - ftl led 
  to an increase in the signal for mt - ftl 
  ( fig .
9b , 
  fac ) , 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 ( fig . 
  9b , fac ) as described previously 
  ( 28 ) .
addition of phen after 
  incubation with fac partially reversed the astrocytic reactive phenotype and 
  decreased the cellular immunolabeling for ferritin 
  ( fig .
no significant differences with the control were observed when 
  only the chelator was added to the cell culture 
  ( fig . 9 , phen ) .
. a , ans 
  fluorescence emission enhancement and wavelength shift caused by titration of 
  mt - ftl homopolymers with increasing concentrations of ans .
b , 
  background and dilution corrected fluorescence emission intensity at 460 nm as 
  a function of ans concentration .
titration was performed on 1.5 
  m mt - ftl homopolymer at ph 7.4 and 25 c in 50 
  mm potassium phosphate buffer .
binding of ans to mt - ftl apoferritin homopolymers . a , ans 
  fluorescence emission enhancement and wavelength shift caused by titration of 
  mt - ftl homopolymers with increasing concentrations of ans .
b , 
  background and dilution corrected fluorescence emission intensity at 460 nm as 
  a function of ans concentration .
titration was performed on 1.5 
  m mt - ftl homopolymer at ph 7.4 and 25 c in 50 
  mm potassium phosphate buffer .
a , temperature dependence of fluorescence emission 
  spectra of mt- and wt - ftl homopolymers destabilized by 4.0 m 
  gdnhcl .
the red trace represents denatured ferritin ( den ) 
  induced by incubation at ph 2.0 in 4.5 m gdnhcl .
b , 
  fluorescence emission intensity ratio ( of 355 over 330 nm ) to determine 
  denaturation temperature of both apoferritin homopolymers .
scans were 
  performed on 1.5 m ferritin homopolymer at ph 7.4 in potassium 
  phosphate buffer with excitation at 295 nm .
homopolymers were incubated 12 h 
  in 4.5 m gdnhcl before beginning temperature dependence experiment . 
 
a , temperature dependence of fluorescence emission 
  spectra of mt- and wt - ftl homopolymers destabilized by 4.0 m 
  gdnhcl .
the red trace represents denatured ferritin ( den ) 
  induced by incubation at ph 2.0 in 4.5 m gdnhcl .
b , 
  fluorescence emission intensity ratio ( of 355 over 330 nm ) to determine 
  denaturation temperature of both apoferritin homopolymers .
scans were 
  performed on 1.5 m ferritin homopolymer at ph 7.4 in potassium 
  phosphate buffer with excitation at 295 nm .
homopolymers were incubated 12 h 
  in 4.5 m gdnhcl before beginning temperature dependence experiment . 
 
recombinant mt- and wt - ftl homopolymers ( 1 m ) were incubated 
  with thermolysin ( 0.15 units ) in hepes buffer ( 0.1 m ) ( ph 7.0 ) , 60 
  mm nacl , 1 mm cacl2 . after 10 min at 37 
  c ,
sample buffer was added , and the samples treated with thermolysin ( + ) , and 
  controls without thermolysin ( - ) were loaded onto 420% sds - page .
gels 
  were stained with coomassie blue ( total protein ) or blotted using 
  antibodies specific for the n terminus of ftl or the c terminus of mt - ftl or 
  wt - ftl .
recombinant mt- and wt - ftl homopolymers ( 1 m ) were incubated 
  with thermolysin ( 0.15 units ) in hepes buffer ( 0.1 m ) ( ph 7.0 ) , 60 
  mm nacl , 1 mm cacl2 . after 10 min at 37 
  c ,
sample buffer was added , and the samples treated with thermolysin ( + ) , and 
  controls without thermolysin ( - ) were loaded onto 420% sds - page .
gels 
  were stained with coomassie blue ( total protein ) or blotted using 
  antibodies specific for the n terminus of ftl or the c terminus of mt - ftl or 
  wt - ftl .
in this work we investigate the protein structure and iron storage function 
  of ferritin homopolymers formed from a light chain variant 
  p.phe167serfsx26 that causes hf 
  ( 9 ) .
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 
  ( 2 , 
  3 ) .
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 
  ( 30 , 
  36 ) , 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 
  ( 9 ) .
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 in vivo in 
  preparations obtained from e. coli , suggesting that both are 
  functional proteins . in vitro ,
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 . to understand this functional difference ,
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 -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 -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 
  ( 9 , 
  12 ) .
the cd data are in 
  agreement with secondary structural prediction analysis ( jpred ) 
  ( 37 ) and analysis for 
  -helical context ( protscale ) 
  ( 38 ) , both of which are 
  consistent with a loss of the e helical domain in the mt - ftl polypeptide .
figure 8.iron-induced aggregation of mt - ftl homopolymers and its reversal by the 
  iron chelator deferroxamine .
mt - ftl homopolymers were incubated in a molar 
  ratio 1:3500 with ferrous ammonium sulfate as in 
  fig .
after centrifugation , 
  the pellet was resuspended in a solution containing 6 mm dfx , 0.1 
  m 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 ( rdu ) as a percentage of the control 
  without iron for each protein .
wt - ftl homopolymers did not precipitate 
  after iron treatment ( not shown ) .
iron - induced aggregation of mt - ftl homopolymers and its reversal by the 
  iron chelator deferroxamine .
mt - ftl homopolymers were incubated in a molar 
  ratio 1:3500 with ferrous ammonium sulfate as in 
  fig .
after centrifugation , 
  the pellet was resuspended in a solution containing 6 mm dfx , 0.1 
  m 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 ( rdu ) as a percentage of the control 
  without iron for each protein .
wt - ftl homopolymers did not precipitate 
  after iron treatment ( not shown ) .
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 280300 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 .
a , immunoblot for mt - ftl ferritin from 
  total cell extracts and their sds - insoluble and sds - soluble fractions . 
 
transgenic astrocytes were exposed to water ( ct ) ; 50 m 
  ferric ammonium citrate for 72 h ( fac ) ; 50 m 
  phenanthroline for 72 h ( phen ) ; or 50 m ferric 
  ammonium citrate for 72 h following 72 h of the iron chelator phenanthroline 
  ( 50 m ) ( fac / phen ) . to verify similar loading
b , confocal 
  immunofluorescence microscopy of cultured mt - ftl - transgenic astrocytes treated 
  as in a. cells were immunostained with anti mt - ftl antibody 
  ( green ) and anti - gfap antibody ( red ) .
note the increase in 
  mt - ftl signal after iron treatment ( fac ) .
the signal was greatly decreased 
  after treatment with the chelator ( fac / phen ) .
iron - induced aggregation of cellular ferritin and its reversal by the 
  chelator phenanthroline .
a , immunoblot for mt - ftl ferritin from 
  total cell extracts and their sds - insoluble and sds - soluble fractions . 
 
transgenic astrocytes were exposed to water ( ct ) ; 50 m 
  ferric ammonium citrate for 72 h ( fac ) ; 50 m 
  phenanthroline for 72 h ( phen ) ; or 50 m ferric 
  ammonium citrate for 72 h following 72 h of the iron chelator phenanthroline 
  ( 50 m ) ( fac / phen ) . to verify similar loading , 
 
b , confocal 
  immunofluorescence microscopy of cultured mt - ftl - transgenic astrocytes treated 
  as in a. cells were immunostained with anti mt - ftl antibody 
  ( green ) and anti - gfap antibody ( red ) .
note the increase in 
  mt - ftl signal after iron treatment ( fac ) .
the signal was greatly decreased 
  after treatment with the chelator ( fac / phen ) .
a feature of ferritin 24-mers in general is their high stability to heat 
  and to urea or guanidinium chloride exposure 
  ( 2 ) .
we found that in the 
  presence of 4.5 m gdnhcl , wt - ftl homopolymers denatured with the 
  higher tm 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 
  ( 30 , 
  32 ) 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 
  ( 24 , 
  32 ) .
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 
  ( 2 , 
  6 , 
  7 , 
  39 ) . 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 
  ( 40 ) .
the data presented so far suggest that ferritin accommodates the extensive 
  sequence alteration present in the p.phe167serfsx26 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 .
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 
  ( 14 , 
  40 ) , 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 
  ( 14 , 
  40 , 
  4244 ) . 
 
insertional mutations of a mouse fth1 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 
  ( 45 ) .
studies using 
  recombinant mutant ftl ( p.arg154lysfsx26 ) , corresponding to the 
  460461insa variant ( 8) 
  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 ( 46 ) .
analysis of ftl 
  polypeptides with the p.phe167serfsx26 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 ftl460461insa 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 in vivo , 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 .
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 
  fig . 10 .
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 ( 16 , 
  47 ) , 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 
  ( 41 ) .
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 .
figure 10.simplified model describing the steps in the iron - induced aggregation 
  process of mutant ferritin . a , c termini of mt - ftl polypeptides 
  extend above the spherical surface of ferritin exposing this sequence of the 
  peptide to solvent and iron .
b , 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 .
c , 
  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 .
simplified model describing the steps in the iron - induced aggregation 
  process of mutant ferritin .
a , c termini of mt - ftl polypeptides 
  extend above the spherical surface of ferritin exposing this sequence of the 
  peptide to solvent and iron .
b , 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 .
c , 
  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 .
we observed that ferritin precipitates obtained in vitro were 
  composed of fully assembled 24-mers , similar to what has been reported in 
  inclusions in patients with hf 
  ( 9 , 
  12 ) and in transgenic mice 
  expressing the mt - ftl polypeptide 
  ( 29 ) . 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 
  ( 16 ) .
the precipitation of 
  fth1 has been suggested to be related to extra - cavity iron hydrolysis 
  ( 16 ) .
iron - induced aggregation 
  of fth1 ( 16 ) and mt - ftl 
  homopolymers was not irreversible because pellets could be resolubilized by 
  the addition of iron chelators .
in vivo , we found that the addition 
  of iron led to intracellular accumulation of ferritin in astrocytes from 
  transgenic mice expressing the mt - ftl polypeptide 
  ( 29 ) . detergent - insoluble 
  ferritin
is typically seen in brain extracts from patients with hf and 
  transgenic mice expressing the mt - ftl polypeptide 
  ( 29 ) .
after iron addition , 
  astrocytic ferritin was found mostly in the detergent - insoluble fraction , 
  indicating that iron - induced ferritin aggregation occurs in vivo 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 in vitro studies .
our data show that the ftl498499instc 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 . in vivo
, 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 
  ( 2 ) , overexpression of ferritin 
  polypeptides , and the aggregation of mutant - containing ferritins as observed 
  in patients with hf ( 12 ) and 
  in transgenic mice ( 29 ) .
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 , e.g. 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 .
 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 p.phe167serfsx26 ( 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 
  -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 in vitro 
  and in vivo . 
 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 . 