Structure, Function, Folding, and Aggregation of a Neuroferritinopathy

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Structure, Function, Folding, and Aggregation of a Neuroferritinopathy-related Ferritin Variant Takumi Kuwata, Yuta Okada, Tomoki Yamamoto, Daisuke Sato, Kazuo Fujiwara, Takuma Fukumura, and Masamichi Ikeguchi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01068 • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 15, 2019

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Biochemistry

Structure, Function, Folding, and Aggregation of a Neuroferritinopathy-related Ferritin Variant Takumi Kuwata1†, Yuta Okada1†, Tomoki Yamamoto1†, Daisuke Sato1, Kazuo Fujiwara1, Takuma Fukumura2 and Masamichi Ikeguchi1* Department of Bioinformatics, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan 2 EM Research and Development Department, JEOL Ltd., 3-1-2 Musashino, Akishima, Tokyo 196-8558, Japan 1

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ABSTRACT: Neuroferritinopathy is a rare, adult-onset, dominantly inherited movement

disorder caused by mutations in ferritin gene. A ferritin light chain variant related to neuroferritinopathy, in which alanine 96 is replaced with threonine (A96T), was expressed in Escherichia coli, purified, and characterized. The circular dichroism spectrum, analytical ultracentrifuge, and small-angle X-ray scattering studies have shown that both subunit structure and assembly of A96T are the same as those of wild-type human ferritin light chain (HuFTL). Iron-incorporation ability was also comparable to that of HuFTL. Although the structural stability against heat, acid, and denaturant was reduced, the structure was sufficiently stable under physiological conditions. The most remarkable defect observed for A96T was lower refolding efficiency and higher propensity to aggregate. The possible relationship between folding deficiency and disease is discussed.


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Biochemistry

INTRODUCTION Ferritin is the ubiquitous iron storage proteins made of 24 subunits. It has a spherical shell-shaped structure and can accommodate up to 4500 iron atoms in its internal cavity.1, 2 Prokaryotes have two different ferritins, heme-binding bacterioferritin and non-heme-binding ferritin. Both ferritins form 24-meric homopolymers. Our time-resolved small-angle X-ray scattering (SAXS) studies have shown that E. coli non-heme-binding ferritin (EcFtnA) dissociates into dimers at acidic pH; however, the dimers maintain their subunit structure and they can reassemble reversibly at neutral pH.3, 4 The studies also revealed that the assembly mechanism of folded dimers was essentially consistent with the mechanism that was previously proposed for horse spleen ferritin assembly, which occurs simultaneously with folding from the unfolded state.5 Mammalian cytosolic ferritin is the heteropolymer composed by heavy (H) and light (L) chains at tissue-specific proportions. The H-chain has the ferroxidase activity which catalyzes the conversion of Fe(II) to Fe(III), whereas the L-chain is thought to contribute to the nucleation of iron core. Both recombinant human H- and L-chains have been expressed in Escherichia coli (E. coli) and can be refolded in vitro.6 The crystal structures of H- and L-chain homopolymers (HuFTH and HuFTL, respectively) have been determined and shown to assume similar quaternary structures and similar subunit structures, which have five -helices labeled A–E.7, 8 The crystal structure of HuFTL is schematically shown in Fig. 1.


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Figure 1. Three-dimensional structure of HuFTL. One subunit is illustrated by a rainbow-colored ribbon model, and the other subunits are represented in wire models. Helices A–E are labeled. Alanine 96 is shown in magenta with its side chain (stick model). This figure was drawn with PyMol based on the atomic coordinates of PDB: 2FFX.8 Approximate positions of 2-fold, 3-fold, and 4-fold symmetry axes are indicated by ellipsoid, triangle, and square, respectively. Neuroferritinopathy is a rare autosomal-dominant disease caused by mutations in the ferritin light chain 1 (FTL1) gene leading to abnormal excessive iron accumulation in the brain, predominantly in the basal ganglia.9, 10 To date, 10 different mutations have been identified, 9 of which are nucleotide insertions (frameshift mutations) in the exon 4, producing mutant ferritins with different amino acid sequences and extension from 4 to 16 residues at the C-terminal region.10, 11 The properties of some of these mutant proteins have been investigated.12-15 In particular, the crystal structure has been solved for a mutant protein, p.Phe167SerfsX26 (this abbreviation denotes a frame shift near the codons encoding Phe167 resulting in replacement with a Ser and an additional 26 residues at the C-terminus of the resulting protein, but here it is simply abbreviated as L167fs)14,15 which is a product of the FTL498-499InsTC mutation.16 The mutation site Phe167 is located in the E-helix of the wild-type structure. The crystal structure showed that L167fs forms a 24-meric shell structure like HuFTL and that the subunit structure


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Biochemistry

also resembles that of HuFTL except for the C-terminal region (residues 156–191). Residues 166–191 were thought to be disordered because no interpretable electron density was observed beyond residue 165. Although some subunits provided interpretable electron density up to residue 165, the structure from residue 156 to 165 was clearly different from that of HuFTL.14 It has been proposed that the disordered C-terminal region protrudes above the spherical shell and mediates aggregation through iron bridges.13 In contrast to the frameshift mutants, structural information on the point mutant A96T has not yet been obtained. A heteromeric ferritin consisting of A96T and H-chain (H/A96T) was produced by coexpressing both proteins in E. coli and its ability to incorporate iron was analyzed.15 The iron-incorporation activity of H/A96T was indistinguishable from that of the wild-type heteromer of L- and H-chains. This result suggests that A96T can assume a structure similar to that of the wild-type L-chain. In this study, we expressed A96T in E. coli, purified it for structural and functional characterization, and established which kinds of defects it has.

MATERIALS AND METHODS Materials. Synthetic DNA encoding HuFTL was obtained from Eurofin Genomics K.K. (Tokyo, Japan). Vector pET-3c was purchased from Novagen (WI, USA). A QuikChange site-directed mutagenesis kit was obtained from Agilent Technologies (CA, USA). Other chemicals are analytical grade from Wako Pure Chemical Industries (Osaka, Japan) or Nacalai Tesque (Kyoto, Japan) unless otherwise noted. Expression and Purification of HuFTL. A synthetic DNA fragment encoding the HuFTL DNA sequence was inserted between the NdeI and BamHI sites of pET-3c, generating pET-HuFTL. E. coli BL21(DE3) harboring pET-HuFTL were grown at 37 C, and protein expression was induced by addition of 0.1 mM isopropyl -D-1-thiogalactopyranoside (IPTG). Cultivation was continued at 25 C for 20 hours. Cells were then harvested by centrifugation, resuspended in phosphate-buffered saline, and lysed by sonication.


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The majority of HuFTL was observed in the supernatant of the lysate. After heat-treatment of the supernatant at 65 °C for 10 min, the solution was centrifuged, and the supernatant was collected. HuFTL was precipitated by adding ammonium sulfate to 50%, and the pellet was dialyzed against 50 mM Tris-HCl (pH 8.0). The solubilized protein solution was applied to Sephacryl S-300 chromatography. The fraction containing HuFTL was further purified by Q-Sepharose chromatography with a linear NaCl concentration gradient of 0–350 mM. Although the sample thus obtained shows a single band in SDS-PAGE, it contained various ferritin oligomers. Therefore, the monomeric ferritin (24-mer) was isolated by gel-filtration chromatography with Superose 6 Increase (1.0  30 cm). Before this chromatography, the sample was treated with 5 mM sodium dithionite and 10 mM EDTA to remove iron. The absence of bound irons from our samples was confirmed by inductively coupled plasma (ICP) spectroscopy. Therefore, HuFTL is isolated as the apoprotein unless otherwise noted. The purity of the final sample was checked by SDS-PAGE, native PAGE, and reverse-phase HPLC. Expression and Purification of A96T. Substitutions of alanine 96 with threonine were performed with a QuikChange site-directed mutagenesis kit to derivate pET-A96T from pET-HuFTL. E. coli BL21(DE3) harboring pET-A96T were grown at 37 C, and protein expression was induced by addition of 0.4 mM IPTG. Cultivation was continued at 37 C overnight, and cells were harvested by centrifugation, resuspended in phosphate-buffered saline, and lysed by sonication. A96T was found in the insoluble fraction (pellet). Although we tried to obtain A96T in the soluble fraction by expressing it under the conditions used for HuFTL expression, we could not obtain the protein in the soluble fraction. We then decided to obtain A96T in the insoluble fraction under conditions that were expected to give a high yield. The pellet was washed with 1% Triton X-100 and then with water, and it was then solubilized with 10 M urea. A96T was refolded by 100-fold dilution with 50 mM Tris-HCl (pH 8.0) and the refolding solution was allowed to stand at room temperature overnight. The refolding solution


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Biochemistry

was centrifuged, and the supernatant was loaded onto a DEAE-Sepharose column. The protein was eluted with a linear gradient of NaCl concentration (0–500 mM), and the fraction containing A96T was further purified by Superdex 200 chromatography. The absence of Prussian blue staining confirmed that A96T thus purified did not contain iron. Furthermore, we confirmed the amount of iron was less than 2 atoms per 24-mer by ICP spectroscopy. A96T was thus obtained as the apoprotein unless otherwise noted. Analytical Ultracentrifuge (AUC) Experiments. Sedimentation velocity experiments were performed with a Beckman XL-I analytical ultracentrifuge and an An-50 Ti rotor at 20 C at 30,000 or 40,000 rpm. The solution conditions were 0.1 M HCl-KCl (pH 1.5– 2.0), 20 mM sodium phosphate containing 100 mM NaCl (pH 2.1–3.0 and pH 7.0) and 50 mM formate (pH 3.5). Sedimentation of the proteins was monitored at 280 nm at 1 min (24-meric form) or 2 min (dimeric form) intervals using a radial step size of 0.003 cm. The protein concentration was 0.3–1.0 mg/mL. Data were analyzed using SEDFIT.17 Circular Dichroism (CD) Measurements. CD measurements were performed with a Chirascan (Applied Photophysics, Leatherhead, U.K.) spectropolarimeter at 25 C with cuvettes of 1 mm or 10 mm path length for far- and near-UV regions. The protein concentration was determined spectrophotometrically, with the extinction coefficient at 280 nm, 280 = 14,650 M–1 cm–1, which was determined by the method of Gill and von Hippel.18 Small-angle X-ray Scattering (SAXS) Measurements. SAXS was measured at BL10C of the Photon Factory at the High Energy Accelerator Research Organization, Tsukuba, and at BL45XU of SPring-8, Hyogo, Japan. The X-ray wavelength was 1.5 Å (Photon Factory) or 1.0 Å (SPring-8). The camera length was 1.5 or 2 m, which was calibrated with a powder diffraction of silver behenate. Scattering profiles were collected by PILATUS3 2M (Dectris, Switzerland). To minimize the effect of X-ray radiation damage on the scattering data, a flow cell was used at BL10C. The temperature was controlled at 25 C by circulating water from a thermostat bath. To obtain one-dimensional scattering data, the scattering intensities on


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two-dimensional images were averaged azimuthally using Sangler.19 The radius of gyration, Rg, was obtained using the Guinier approximation:20

(

𝐼(𝑄) = 𝐼(0)exp ―

𝑅𝑔2𝑄2 3

),

where the scattering vector Q = (4/) sin ( is the wavelength and 2 is the scattering angle), I(Q) is the scattering intensity at a given Q value. Transmission Electron Microscopy (TEM). Sample solutions (3 L) were applied to a carbon-coated copper grid stained with 1.0% (w/v) uranyl acetate. Micrographs were recorded with a JEM-1400Flash transmission electron microscope (JEOL, Tokyo, Japan) operated at 120 kV. Iron-incorporation assay. Iron loading activity of HuFTL and A96T was investigated as described by Baraibar et al.13 Ferrous ammonium sulfate (0–4.5 mM) in 10 mM HCl was added to HuFTL and A96T (1 M) in 100 mM PIPES (pH 7.0). After incubation for 2 h at 25 C, the sample was centrifuged at 13000g for 15 min. Iron incorporations were monitored by measuring the absorbance of the supernatant at 310 nm using a cuvette with 2 mm pathlength or densitometric analysis of Prussian blue stained bands on native PAGE gel.

RESULTS Structure of A96T. When the recombinant proteins were expressed in E. coli, A96T was obtained in the insoluble fraction of cell lysate, while recombinant HuFTL was obtained in the soluble fraction. This suggested that A96T had some defects in its structure or stability. Contrary to this expectation, we could refold A96T in vitro from the insoluble fraction. The structural properties of refolded and purified A96T were compared with those of HuFTL. The elution volume in gel-filtration chromatography and mobility in native PAGE were similar to those of HuFTL (not shown), suggesting that A96T forms a 24-meric cage-like structure. This was confirmed by analytical ultracentrifuge (AUC), small-angle X-ray scattering (SAXS)


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Biochemistry

analysis, and negative-stain transmission electron microscopy (TEM). AUC showed that the sedimentation coefficient and molecular weight of A96T were similar to the corresponding values of HuFTL under the same conditions (Table 1). The SAXS profile of A96T was similar to that of HuFTL (Fig. 2A). From the Guinier region (Q < 0.023) of the profile, the radius of gyration (Rg) was calculated for a series of SAXS data obtained at different protein concentrations (Fig. S1). The Rg values of A96T and HuFTL at infinite dilution were identical within experimental error (Table 1). From the TEM images, it is clear that both HuFTL and A96T assume a spherical shell structure. The circular dichroism (CD) spectrum of A96T was also similar to that of HuFTL (Fig. 2B and 2C), indicating that the secondary and tertiary structures of the subunits of the two proteins are the same. These results showed that the secondary, tertiary, and quaternary structures of A96T are essentially identical to those of HuFTL.

Table 1. Sedimentation coefficients and gyration radii of HuFTL and A96T HuFTL A96T a Sedimentation coefficient 16.3 S 15.9 S Molecular weighta 480,000 490,000 Gyration radiusb 55.0  0.7 Å 54.7  0.7 Å a Conditions were 20 mM phosphate, 0.1 M NaCl (pH 7.0) at 20 C. b The value extrapolated to zero protein concentration (Fig. S1). Conditions were 20 mM sodium phosphate (pH 7.0) and 50 mM Tris-HCl (pH 8.0) for HuFTL and A96T, respectively at 25 C.


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Figure 2. SAXS profiles, CD spectra, and TEM images of HuFTL and A96T. (A) SAXS profiles of HuFTL and A96T. SAXS intensity is shown as a function of scattering vector Q = (4/) sin, where  is the wavelength and 2 is the scattering angle. HuFTL and A96T concentrations are 1.2 and 0.75 mg/mL, respectively. (B) The far-UV CD spectra of HuFTL and A96T. The spectra were measured at protein concentrations of 0.1 mg/mL. (C) The near-UV CD spectra of HuFTL and A96T. The protein concentration was 0.3 mg/mL. In the panels (A)–(C), data for HuFTL and A96T are shown in blue and red, respectively. (D) TEM image of purified HuFTL. (E) TEM image of purified A96T. Iron-incorporation Ability of A96T. The ability of A96T to incorporate iron was assayed according to the methods described by Baraibar et al.13 and compared with that of HuFTL. Freshly prepared ferrous ammonium sulfate was added to the protein solution. After 2 h, the samples were centrifuged, and the quantity of incorporated iron was monitored by the absorbance of the supernatant at 310 nm (Fig. 3A). The result for HuFTL was similar to that reported by Baraibar et al.13 The absorbance at 310 nm increased with iron concentration and reached a plateau value at high iron concentration. The result obtained with A96T was the same as that with HuFTL within the experimental error, indicating that the ability of A96T to incorporate iron is similar to that of HuFTL. Iron-incorporation ability was further assayed by Prussian blue staining of bands corresponding to the 24-mer in the native PAGE. The band


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Biochemistry

intensity also increased with the concentration of added iron and reached a plateau at high iron concentration (Fig. 3B). The results obtained with A96T were also the same as those with HuFTL, within experimental error, indicating that the ability of A96T to incorporate iron is similar to that of HuFTL. This contrasts with the result for another neuroferritinopathy-related mutant protein, L167fs, for which the protein precipitated at an iron concentration higher than 1 mM.13

Figure 3. Comparison of iron loading activity between HuFTL (blue) and A96T (red). (A) Iron incorporation was assayed by absorbance at 310 nm. (B) Iron mineralization in HuFTL and A96T was monitored by Prussian blue staining after separating unmineralized iron from samples by nondenaturing gel (7%) electrophoresis. Error bars represent the standard deviation of three independent experiments.


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Structural Stability of A96T. To address whether the structural stability of A96T is lower than that of HuFTL, the thermal unfolding was followed by monitoring the CD ellipticity change at 222 nm (Fig. 4). The ellipticity of HuFTL did not show abrupt changes within the observed temperature range, although it increased gradually. This indicates that HuFTL is highly stable. It has been reported that HuFTL is stable up to 80 C even in the presence of 4.5 M guanidine hydrochloride (GdnHCl).13 Contrary to this remarkable stability, the ellipticity of A96T increased abruptly at temperatures higher than 70 C, indicating that A96T unfolded at those temperatures.

Figure 4. Thermal stability of HuFTL (blue) and A96T (red). Temperature dependence of CD ellipticity at 222 nm was measured at pH 7. Protein concentrations were 0.1 mg/mL. We further investigated the stability against acid and denaturant. The dissociation at acidic pH was monitored by AUC. For HuFTL, two components with sedimentation coefficients of 16.3 S and 2.5–3.3 S were observed between pH 2.5 and 2.1, and almost no other components were observed (Fig. S2). Below pH 2.1, only the small component was observed. The molecular weight of the 16.3 S component was estimated to be 480,000 and was consistent with a 24-mer. On the other hand, the molecular weight of the small component was estimated to be 30,000–43,000, corresponding to either the monomer or dimer. These results indicated that


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Biochemistry

HuFTL dissociated cooperatively below pH 2.5 (Fig. 5A), which is consistent with the previous gel-electrophoretic analyses.21 The tertiary and secondary structures of HuFTL were also monitored by CD. Fig. 5B and 5C show the dependence of CD ellipticities at 286 and 222 nm on the pH, reflecting the tertiary and secondary structures, respectively (CD spectra are shown in Fig. S3). Both ellipticities at 286 and 222 nm changed abruptly between pH 2.5 and 2, and the spectrum at pH 2 or less showed the absence of tertiary structure and the presence of residual secondary structure, typical for the molten globule state.22 These are reasonably consistent with the previous result of Santambrogio et al.21 For A96T, dissociation and unfolding occurred between pH 3 and 2.5 (Fig. 5), which is a pH range higher than that of HuFTL, indicating that A96T is less stable than HuFTL.

Figure 5. Dissociation and conformational change of HuFTL (blue) and A96T (red) at acidic pH. The fractional population of 24-mer (A) was measured by AUC at 20 C. The pH dependence of CD ellipticities at 286 nm (B) and 222 nm (C) was measured at 25 C. Protein concentrations in CD measurements were 0.1–0.3 mg/mL (B) or 0.2–0.5 mg/mL (C). Data points shown with error bars are the average of 2–6 different samples, and error bars represent the standard deviation. GdnHCl denaturation was examined at pH 3.5 because HuFTL is stable even in the presence of 6 M GdnHCl at pH 7.4.21 Both CD ellipticities at 286 and 222 nm of HuFTL changed between 2 and 4 M GdnHCl (Fig. 6A and 6B), indicating that both tertiary and secondary structures were cooperatively disrupted in this concentration range. For A96T, on the other hand, the ellipticity change occurred at much lower GdnHCl concentrations (Fig. 6A and 6B), again indicating that A96T is less stable than HuFTL.


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In summary, A96T is less stable against heat-, acid-, or GdnHCl-induced unfolding than HuFTL. Under the physiological conditions (pH 7 in the absence of any denaturant), however, A96T is sufficiently stable, and it is unlikely that the lower stability of A96T is the origin of the disease.

Figure 6. GdnHCl-induced unfolding of HuFTL (blue) and A96T (red). GdnHCl concentration dependence of CD ellipticities at 286 nm (A) and 222 nm (B) was measured at pH 3.5 and 25 C. Protein concentrations were 0.3–0.5 mg/mL (A) or 0.1–0.3 mg/mL (B). Data are the average of 2–6 different samples, and error bars represent the standard deviation. Refolding Efficiency of A96T. All denaturations studied above were found to be partially reversible. Therefore, we examined the refolding efficiency of A96T and compared it with that of HuFTL. At first, both A96T and HuFTL were unfolded in 6 M GdnHCl, pH 3.5. A 14 ACS Paragon Plus Environment

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Biochemistry

series of unfolded protein solutions (4–20 mg/mL) were diluted 10-fold with 50 mM Tris-HCl buffer (pH 8.0). The resultant pH was 7.95. The solutions were kept at 4 C for 18 hours, and then centrifuged to remove precipitated proteins. The protein concentration in the supernatant was assayed by UV absorbance measurements. The supernatant was also applied to native PAGE and found to contain 24-mer and a small amount of ferritin oligomer (48-mer and 72-mer). No subunit monomer or dimer was detected. Therefore, we considered that the supernatant contained only refolded and reassembled ferritin and defined the refolding efficiency to be the ratio of the protein concentration in the supernatant against the total protein concentration of the refolding solution. As seen in Fig. 7, the refolding efficiency of HuFTL at 0.4 mg/mL was more than 90%, and it decreased with increasing protein concentration during refolding. It is reasonable to assume that refolding yield decreases with increasing protein concentration given that folding and aggregation are competitive processes, with the former being independent of protein concentration and the latter depending on it. The refolding efficiency of A96T was only 47% even at the lowest protein concentration investigated, and it decreased with the protein concentration. This result shows that the refolding efficiency of A96T is much lower than that of the wild-type L-chain, which is consistent with the observation that expressed A96T was found in the insoluble fraction whereas HuFTL was found in the soluble fraction.


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Figure 7. Refolding efficiency of HuFTL (blue) and A96T (red). Refolding was initiated by 10-fold dilution of unfolded proteins in 6 M GdnHCl (pH 3.5) with 50 mM Tris-HCl buffer (pH 8.0). The solutions were kept at 4 C for 18 hours, and then centrifuged to remove precipitated proteins. The refolding efficiency was evaluated as the ratio of the protein concentration in the supernatant against the total protein concentration. Properties of A96T aggregate. Given that the precipitation of A96T occurred during refolding in the absence of iron, the precipitate of A96T was thought to differ from those observed for L167fs during iron titration.13 It might be amyloid-like fibrils such as amyloid-β that play a significant role in the pathogenesis of Alzheimer’s disease.23 To characterize the aggregate of A96T formed during refolding, we observed the in vitro refolding solution by TEM with negative staining. TEM images of a refolding solution of A96T at protein concentration of 0.4 mg/mL are shown in Fig. 8. At this protein concentration, as seen in Fig. 7, the refolding solution contains both refolded and aggregated proteins. At low magnification (1,000), aggregated proteins were observed as dark materials with the appearance of a tangled thread. Although spherical shell-like structures of refolded A96T were found at high magnification (50,000), we could not identify any amyloid-like fibrils even at the edge of the dark materials. Therefore, the aggregate formed during A96T refolding seems to be amorphous in nature.


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Biochemistry

Figure 8. Negatively stained TEM images of A96T refolding solution. 4 mg/mL A96T unfolded in 6 M GdnHCl (pH 3.5) was diluted 10-fold with 50 mM Tris-HCl buffer (pH 8.0). The solutions were kept at 4 C for 18 hours, and the precipitate was well suspended before sample mounting on the grid. Squares colored white indicate the area expanded in the higher magnification images.

DISCUSSION The present study has shown that A96T can form a structure that is indistinguishable from that of HuFTL and has iron-incorporation ability comparable to that of HuFTL, but its folding efficiency is reduced, which makes A96T prone to aggregation during folding. Alanine 96 is located near the N-terminus of the C-helix as shown in Fig. 1 and seems to be conserved for mammalian ferritins including H-chains.24 Although alanines are also frequently found at the corresponding sites of eukaryote sequences, they are not conserved for prokaryotes.25 Even in eukaryotes, turbot ferritin H-chain and pacific abalone ferritin 1 have leucine and valine as a corresponding residue, respectively.26 Together with these, the present result in which the A96T mutation can also permit successful iron-incorporation indicates that alanine is not essential for this position. The three-dimensional structure of A96T was modelled using UCSF Chimera.27 The Ala96 side chain of the crystal structure of HuFTL was replaced with a threonine side chain, and energy minimization was done by relaxing only the side chain atoms within 5 Å of atoms of the replaced Thr96. In the energy-minimized structure, the relaxed side-chain conformations did not change significantly, suggesting that the threonine side chains can be accommodated within


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the structure of HuFTL (Fig. S4). The question is then: How is the molecular property of A96T related to the disease? The best-characterized mutation of neuroferritinopathy is the FTL498-499InsTC mutation. The protein variant produced by this mutation (L167fs) has been well characterized.13-15 Although L167fs has a 24-meric spherical shell structure, its C-terminal region is disordered,14, 15 and it is proposed to participate in iron-mediated cross-links between ferritin shells.13 Pathological mechanisms were addressed by two groups making model animals (transgenic mice). The first transgenic mouse, which expressed L167fs regulated by the mouse prion protein (MoPrP) promoter, was generated by Vidal and collaborators.28 Immunohistochemical analyses showed age-dependent accumulation of intranuclear and intracytoplasmic inclusion bodies in glia and neurons throughout the central nervous system, which is similar to the observation from autopsies of patients.16 Iron was colocalized with inclusion bodies. The research group analyzed the expression of proteins related to iron metabolism, cellular iron level, and oxidative stress, such as lipid peroxidation, protein carbonyls, and nitrone–protein adducts.29 A similar change in concentrations of proteins, iron, and reactive oxygen species was observed in primary cultures of skin fibroblasts from patients.30 From all of these observations, Vidal and coworkers concluded that pathogenesis is likely to result from a combination of reduction in iron storage function and enhanced toxicity associated with iron-induced ferritin aggregation. The other group generated transgenic mice in which transgene expression was driven by the constitutive and ubiquitous PGK promoter.31 They observed aggregation of transgene product L167fs inside neurons, and iron accumulation was also observed by MRI and histochemical analysis. However, electron spectroscopic imaging showed that iron was accumulated in the osmophilic body in the cytosol, not in the nucleus.31 From the analysis of protein oxidation and ubiquitination, lipid peroxidation, and catalase expression of the transgenic mice, the authors concluded that the pathogenic mechanism of FTL498-499InsTC-derived neuroferritinopathy was based on uncontrolled intracytosolic free


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Biochemistry

iron arising from the inability of ferritin to incorporate iron and that the aggregation of L167fs is a secondary effect. To our knowledge, there is no immunohistochemical observation for patients carrying the mutation that produces A96T. Although T2-weighted MRI images showed hyperintensity in the pallidum, no significant involvement of the putamen, thalamus, substantia nigra, or dentate nuclei was found in the patient.32 Unfortunately, T2*-weighted images, which are sensitive to iron deposition,33 have not been reported. Therefore, it is not yet known whether A96T forms iron-containing intracellular inclusion bodies, as observed for the patients or model animals carrying FTL498-499InsTC mutation. Muhoberac and Vidal9 have pointed out the lack of autosomal dominant transmission, given that the mother of the proband (also a carrier of A96T mutation) is asymptomatic. Thus, it remains to be seen whether A96T is pathogenic and reflects a broader spectrum of the disease or is simply a polymorphic variant of the ferritin L-chain. This study has shown that the ability of A96T to incorporate iron is comparable to that of HuFTL. Even though the A96T mutation is related to the disease, its mechanism may be somewhat different from that followed in the case of the FTL498-499InsTC mutation. A possibility is that the low folding efficiency results in low functional ferritin concentration, which causes misregulation of iron metabolism. However, it remains to be clarified whether the A96T folding efficiency is also lower than that of HuFTL in brain cells. Although the low folding efficiency and aggregation of A96T was observed at higher protein concentrations in vitro and when it was overexpressed in E. coli, the concentration of A96T in brain cells may be sufficiently low to allow proper folding without aggregation. In brain cells, furthermore, there are molecular chaperons that are not present in vitro, and these are different from those present in E. coli cells. To clarify the relationship between the A96T mutation and the disease, further studies using cellular or animal models are necessary.

ASSOCIATED CONTENT


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Supporting Information Protein concentration dependence of Rg (Fig. S1), distribution of the sedimentation coefficients observed at various pH conditions (Fig. S2), CD spectra at various pH conditions (Fig. S3), and comparison of the A96T structural model with the crystal structure of HuFTL (Fig. S4) are supplied as Supporting Information.

AUTHOR INFORMATION Corresponding Author *

E-mail: [email protected]

Author Contributions †

Three authors contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors thank Kaoru Kondo and Yuhei Koyama for their preliminary work on this study. The synchrotron radiation SAXS experiments were performed at BL45XU of SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI), Hyogo, Japan (Proposals 2017A1403) and at BL10C under the approval of the Photon Factory Program Advisory Committee (Proposal No. 2016G049). This research was also supported in part by the Platform for Drug Discovery, Information, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Proposal No. 2016R-62).

ABBREVIATIONS


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Biochemistry

AUC, analytical ultracentrifuge; CD, circular dichroism; GdnHCl, guanidine hydrochloride; HuFTH, homopolymer of human ferritin heavy chains; HuFTL, homopolymer of human ferritin light chains; ICP, inductively coupled plasma; L167fs, human ferritin light chain variant produced by nucleotide insertion at nucleotide 498-499; SAXS, small-angle X-ray scattering; TEM, transmission electron microscope

Accession Codes HuFTL P02792


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Structure, Function, Folding, and Aggregation of a Neuroferritinopathy

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