The E46K Mutation in α-Synuclein Increases Amyloid Fibril Formation
2005; Elsevier BV; Volume: 280; Issue: 9 Linguagem: Inglês
10.1074/jbc.m411638200
ISSN1083-351X
AutoresEric A. Greenbaum, Charles L. Graves, Amanda J. Mishizen-Eberz, Michael A. Lupoli, David R. Lynch, S. Walter Englander, Paul H. Axelsen, Benoit I. Giasson,
Tópico(s)Alzheimer's disease research and treatments
ResumoThe identification of a novel mutation (E46K) in one of the KTKEGV-type repeats in the amino-terminal region of α-synuclein suggests that this region and, more specifically, Glu residues in the repeats may be important in regulating the ability of α-synuclein to polymerize into amyloid fibrils. It was demonstrated that the E46K mutation increased the propensity of α-synuclein to fibrillize, but this effect was less than that of the A53T mutation. The substitution of Glu46 for an Ala also increased the assembly of α-synuclein, but the polymers formed can have different ultrastructures, further indicating that this amino acid position has a significant effect on the assembly process. The effect of residue Glu83 in the sixth repeat of α-synuclein, which lies closest to the amino acid stretch critical for filament assembly, was also studied. Mutation of Glu83 to a Lys or Ala increased polymerization but perturbed some of the properties of mature amyloid. These results demonstrated that some of the Glu residues within the repeats can have significant effects on modulating the assembly of α-synuclein to form amyloid fibrils. The greater effect of the A53T mutation, even when compared with what may be predicted to be a more dramatic mutation such as E46K, underscores the importance of protein microenvironment in affecting protein structure. Moreover, the relative effects of the A53T and E46K mutations are consistent with the age of onset of disease. These findings support the notion that aberrant α-synuclein polymerization resulting in the formation of pathological inclusions can lead to disease. The identification of a novel mutation (E46K) in one of the KTKEGV-type repeats in the amino-terminal region of α-synuclein suggests that this region and, more specifically, Glu residues in the repeats may be important in regulating the ability of α-synuclein to polymerize into amyloid fibrils. It was demonstrated that the E46K mutation increased the propensity of α-synuclein to fibrillize, but this effect was less than that of the A53T mutation. The substitution of Glu46 for an Ala also increased the assembly of α-synuclein, but the polymers formed can have different ultrastructures, further indicating that this amino acid position has a significant effect on the assembly process. The effect of residue Glu83 in the sixth repeat of α-synuclein, which lies closest to the amino acid stretch critical for filament assembly, was also studied. Mutation of Glu83 to a Lys or Ala increased polymerization but perturbed some of the properties of mature amyloid. These results demonstrated that some of the Glu residues within the repeats can have significant effects on modulating the assembly of α-synuclein to form amyloid fibrils. The greater effect of the A53T mutation, even when compared with what may be predicted to be a more dramatic mutation such as E46K, underscores the importance of protein microenvironment in affecting protein structure. Moreover, the relative effects of the A53T and E46K mutations are consistent with the age of onset of disease. 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The A53T mutation within the amino-terminal region has been consistently reported to increase the propensity of fibril formation (35Giasson B.I. Uryu K. Trojanowski J.Q. Lee V.M.-Y. J. Biol. Chem. 1999; 274: 7619-7622Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 36Conway K.A. Harper J.D. Lansbury P.T. Nat. Med. 1998; 4: 1318-1320Crossref PubMed Scopus (1233) Google Scholar, 38Narhi L. Wood S.J. Steavenson S. Jiang Y. Wu G.M. Anafi D. Kaufman S.A. Martin F. Sitney K. Denis P. Louis J.C. Wypych J. Biere A.L. Citron M. J. Biol. Chem. 1999; 274: 9843-9846Abstract Full Text Full Text PDF PubMed Scopus (611) Google Scholar). Further support for the important role of the amino-terminal region of α-syn comes from fibrillogenesis experiments in which the number of repeats was modified. Deletion of repeats 1 and 2 in the amino terminus of α-syn accelerates filament formation, whereas duplication of these repeats inhibits this process (43Kessler J.C. Rochet J.C. Lansbury Jr., P.T. Biochemistry. 2003; 42: 672-678Crossref PubMed Scopus (97) Google Scholar). The recent identification of the new (E46K) amino-terminal mutation in α-syn (18Zarranz J.J. Alegre J. Gomez-Esteban J.C. Lezcano E. Ros R. Ampuero I. Vidal L. Hoenicka J. Rodriguez O. Atares B. Llorens V. Gomez T.E. del Ser T. Munoz D.G. de Yebenes J.G. Ann. Neurol. 2004; 55: 164-173Crossref PubMed Scopus (2097) Google Scholar) in the fourth repeat of α-syn (Fig. 1) further suggests that the amino-terminal region may be important in modulating filament formation; however, the effect of this mutation had not been studied. Whereas our initial goal was to study the effect that this mutation had on the polymerization of α-syn, it was also noted that a Glu residue similar to Glu46 is present in five of the six degenerative repeats in α-syn. The only repeat that does not have such a residue (repeat 2) has Glu residues adjacent to each side of the repeat (see Fig. 1). The possibility that Glu residues in the repeats are important in modulating the ability of α-syn to polymerize was further investigated by generating the mutant E46A. The Ala mutation results in the loss of one negative charge, instead of a complete change in charge from negative to positive. The effects of the mutants E83A and E83K in the sixth repeat, closest to the amino acid stretch critical for polymerization, were also studied to assess the role of Glu residues. Expression and Purification of α-Syn—Human α-syn cDNA was cloned into the NdeI and HindIII restriction sites of the bacterial expression vector pRK172. The cDNAs coding for the mutant α-syn protein E46A, E46K, E83A, and E83K in the same vector were engineered by creating the corresponding nucleotide substitutions in the wild-type cDNA using complementary sets of synthetic single-stranded DNA containing the mutant sequence and the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). All mutations were confirmed by DNA sequencing. α-Syn proteins were expressed in Escherichia coli BL21(DE3). Bacterial pellets harvested by centrifugation were re-suspended in high-salt buffer (0.75 m NaCl, 50 mm Tris, pH 7.4, 1 mm EDTA) containing a mixture of protease inhibitors, heated to 100 °C for 10 min, and centrifuged at 70,000 × g for 30 min. Supernatants were dialyzed into 100 mm NaCl, 20 mm Tris, pH 7.5 and applied onto a Superdex 200 gel filtration column (Amersham Biosciences) and separated by size exclusion. The fractions were assayed for the presence of the α-syn proteins by SDS-PAGE followed by Coomassie Blue R-250 staining. Proteins were concentrated using Centriprep-10 units (Millipore); dialyzed against 10 mm Tris, pH 7.5; applied to a Mono Q column (Amersham Biosciences); and eluted with a 0–0.5 m NaCl gradient. Protein concentrations were determined using the bicinchoninic acid protein assay (Pierce) and bovine serum albumin as a standard. Filament Assembly and Centrifugal Sedimentation—α-Syn proteins were assembled into filaments by incubation at 37 °C in 100 mm sodium acetate, pH 7.0, with continuous shaking. A fraction of each sample was set aside for K114 fluorometry and electron microscopy (EM) analysis. The remainder of each sample was centrifuged at 100,000 × g for 20 min, and SDS sample buffer (10 mm Tris, pH 6.8, 1 mm EDTA, 40 mm dithiothreitol, 1% SDS, and 10% sucrose) was added to pellets and supernatants, which were heated to 100 °C for 15 min. α-Syn proteins in the supernatants and pellets were quantified by densitometry of Coomassie Blue-stained SDS-polyacrylamide gels or 125I quantitative Western blotting analysis (40Giasson B.I. Murray I.V. Trojanowski J.Q. Lee V.M.-Y. J. Biol. Chem. 2001; 276: 2380-2386Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar, 44Giasson B.I. Duda J.E. Quinn S.M. Zhang B. Trojanoswki J.Q. Lee V.M.-Y. Neuron. 2002; 34: 521-533Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar). K114 Fluorometry—α-Syn can form amyloid fibrils (45Conway K.A. Harper J.D. Lansbury P.T. Biochemistry. 2000; 39: 2552-2563Crossref PubMed Scopus (678) Google Scholar), which can be quantified using the fluorescent amyloid binding dye K114 (46Crystal A.S. Giasson B.I. Crowe A. Kung M.P. Zhuang Z.P. Trojanowski J.Q. Lee V.M.-Y. J. Neurochem. 2003; 86: 1359-1368Crossref PubMed Scopus (102) Google Scholar). This dye derived from the structure of Congo Red is soluble in aqueous buffers, and it demonstrates a tremendous increase in fluorescence upon binding to amyloid fibrils (46Crystal A.S. Giasson B.I. Crowe A. Kung M.P. Zhuang Z.P. Trojanowski J.Q. Lee V.M.-Y. J. Neurochem. 2003; 86: 1359-1368Crossref PubMed Scopus (102) Google Scholar). This assay was conducted, as described previously (46Crystal A.S. Giasson B.I. Crowe A. Kung M.P. Zhuang Z.P. Trojanowski J.Q. Lee V.M.-Y. J. Neurochem. 2003; 86: 1359-1368Crossref PubMed Scopus (102) Google Scholar), by incubating a fraction of each sample with K114 (50 μm) in 100 mm glycine, pH 8.5, and measuring fluorescence (λex = 380 nm, λem = 550 nm, cutoff = 530 nm) with a SpectraMax Gemini fluorometer and SoftMax Pro 4.0 software. CD Spectrometry—α-Syn proteins prepared at concentrations of 2.5 or 5 mg/ml in 100 mm phosphate buffer, pH 7.0, were analyzed by CD immediately upon or after incubation at 37 °C for 24, 48, or 96 h with constant shaking. Proteins were diluted to a final concentration of 5 μm into 5 mm phosphate buffer, pH 7.0. After dilution, the protein was transferred into a 1-mm quartz cuvette, and the CD spectra between 190 and 260 nm were recorded using an Aviv model 202 spectrophotometer (Lakewood, NJ.) Attenuated Total Internal-Reflection Fourier Transform Infrared (ATR-FTIR) Spectroscopy—Monomeric α-syn samples were dialyzed against 30 mm HEPES buffer in D2O at pD = 7.4 (meter reading 7.0), adjusted to a protein concentration of 10 mg/ml, and drawn into a 15-μm flow cell with CaF2 windows. This technique, employing ordinary transmission mode spectroscopy, was the best way to characterize the protein in solution and avoid consequences of any changes in physical condition. 512 co-added interferograms were collected on a Digilab FTS-60A equipped with a wide band MCT detector. All spectra were collected at room temperature at 20 kHz with a resolution of 2 cm–1, undersampling ratio of 2, one level of zero-filling, and triangular apodization. The ratio of detector response to that obtained with protein-free buffer was calculated to yield the absorption spectrum. A flat baseline was subtracted from each spectrum, but no water vapor subtraction or smoothing operations were performed. Assembled α-syn samples were dialyzed against 5 mm HEPES buffer in D2O at pD = 7.4 and adjusted to a protein concentration of 5 mg/ml, and ∼3 μl of this solution were placed on a 2 × 10 × 52-mm germanium internal reflection crystal with 45° facets and evaporated to dryness at room temperature. 1024 co-added interferograms were collected before and after application of each sample and processed as described above to yield the absorption spectrum. This technique, involving drying of the sample and probing with an evanescent field, was necessary because the assembled protein did not readily pass through the flow cell and would not otherwise remain in place throughout the 10-min period required to collect the spectrum. The six spectra of monomeric α-syn were analyzed simultaneously using Irfit (47Silvestro L. Axelsen P.H. Biochemistry. 1999; 38: 113-121Crossref PubMed Scopus (41) Google Scholar), as were the six spectra of aggregated α-syn in a separate operation. Both sets of spectra are well fit by three components. Within each set, each component has the same frequency, half-width, and shape in each experimental spectrum, with only the amplitude of each component adjusted to fit the data. Negative Staining EM—Assembled α-syn filaments were absorbed onto 300 mesh carbon-coated copper grids, stained with 1% uranyl acetate, and visualized with a Joel 1010 transmission electron microscope (Peabody, MA). Images were captured with a Hamamatsu digital camera (Bridgewater, MA) using AMT software (Danvers, MA). For diameter determination, the width of 100–120 filaments was measured using Image-Pro Plus software (Media Cybernetics, Del Mar, CA). Calpain I Cleavage of α-Syn Proteins—Calpain I (Calbiochem) cleavage of purified recombinant human α-syn proteins was carried out using methods similar to those described previously (48Mishizen-Eberz A.J. Guttmann R.P. Giasson B.I. Day III, G.A. Hodara R. Ischiropoulos H. Lee V.M.-Y. Trojanowski J.Q. Lynch D.R. J. Neurochem. 2003; 86: 836-847Crossref PubMed Scopus (126) Google Scholar). Briefly, α-syn protein was incubated with calpain I in buffer containing 40 mm HEPES, pH 7.5, and 5 mm dithiothreitol at 37 °C. Calpain cleavage was initiated by the addition of calcium (1 mm final concentration). Aliquots were removed from the reaction mixture and added to an equal volume of 2× Invitrogen SDS-stop buffer at various time points, heated in a boiling water bath, and stored at –20 °C. Western Blotting Analysis—Proteins were resolved on slab gels by SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes (Schleicher and Schuell, Keene, NH) in buffer containing 48 mm Tris, 39 mm glycine, and 10% methanol. Membranes were blocked with a 1% solution of powdered skimmed milk dissolved in Tris buffered saline-Tween 20 (50 mm Tris, pH 7.6, 150 mm NaCl, and 0.1% Tween 20), incubated with anti-α-syn antibodies Syn 303, SNL-1, or Syn 102 (49Duda J.E. Giasson B.I. Mabon M.E. Lee V.M.-Y. Trojanoswki J.Q. Ann. Neurol. 2002; 52: 205-210Crossref PubMed Scopus (265) Google Scholar, 50Giasson B.I. Jakes R. Goedert M. Duda J.E. Leight S. Trojanowski J.Q. Lee V.M.-Y. J. Neurosci. Res. 2000; 59: 528-533Crossref PubMed Scopus (186) Google Scholar). After incubation with either goat anti-mouse or a goat anti-rabbit antibodies conjugated to horseradish peroxidase, the blots were developed with Renaissance Enhanced Luminol Reagents (PerkinElmer Life Sciences) and exposed onto X-Omat Blue XB-1 films (Eastman Kodak Co.). The effects of two α-syn mutations causal of disease (A53T and E46K) and three synthetic mutations (E46A, E83A, and A83K) were studied. The ability of these proteins to polymerize was compared with that of wild-type (WT) α-syn as assayed by sedimentation analysis and K114 fluorometry (Fig. 2, A and B). At the highest concentration (5 mg/ml) of α-syn proteins used for these analyses, all α-syn proteins readily formed polymers that could be sedimented at 100,000 × g. Whereas all mutant α-syn proteins tested formed amyloid structures as revealed by K114 fluorometry, the E83A and E83K mutants generated much less K114 fluorescence. Experiments using a low concentration of proteins (2.5 or 1 mg/ml) showed that the E46K mutant polymerized more readily than the WT protein but that the effect was not as dramatic as the change seen in the A53T mutant. As quantified by sedimentation assay, the synthetic mutants E46A, E83A, and E83K also showed an increased propensity to polymerize. To determine the conformational changes associated with the polymerization of α-syn proteins, CD and ATR-FTIR spectrometry were performed. CD spectrometry on monomeric α-syn proteins showed a minimum between 195 and 200 nm, indicating predominantly random coil or unstructured conformation (Fig. 3A). After incubation under assembly conditions, WT, A53T, E46K, and E83K α-syn proteins demonstrated CD spectra characterized by negative deflection at 215–225 nm and positive signal at 195–205, indicating that they had acquired predominantly β-pleated sheet conformation (Fig. 3B). Despite positive results in sedimentation assays (Fig. 2) and EM (see Fig. 5), the assembled proteins E46A and E83A yielded relatively low CD signal compared with the other mutants tested.Fig. 5EM analysis of α-syn filaments assembled in vitro. WT, E46A, E46K, E83A, and E83K α-syn proteins at 5 mg/ml were incubated under assembly conditions and analyzed by negative staining EM as described under "Experimental Procedures." Scale bar, 200 nm.View Large Image Figure ViewerDownload (PPT) The infrared spectra of monomeric α-syn proteins exhibit a maximum at 1646.2 cm–1, with a predominant component comprising 60–70% of the signal that is centered at 1643.9 cm–1. This suggests that the proteins are unstructured under these conditions (Fig. 3C). Most of the remaining signal, ∼30%, is accounted for by a broad component centered at 1668.1 cm–1; however, conformational assignment of this component is not reliable. The five mutant proteins were indistinguishable from WT protein. The spectra for the aggregated α-syn proteins were quite distinct from those of monomeric proteins (Fig. 3D). The maximum was at 1627.9 cm–1, with the dominant component centered at 1625.0 cm–1 comprising 44–55% of the signal. The position of this component suggests that substantial amounts of β-sheet structure have formed in the course of aggregation. The component at 1643.9 cm–1 in monomeric proteins appears to have shifted slightly to 1641.4 cm–1 and decreased to between 7% and 24% of the signal. As with the monomeric proteins, ∼30% of the signal is accounted for by a high frequency component centered, in this case, at 1661.4 cm–1. Again, spectra from the five mutated proteins were similar to that of the WT protein. The difference between A53T and E46K α-syn and the other proteins is minimal and therefore difficult to interpret as a significant conformational change. In order to obtain more detailed information regarding the conformational changes accompanying the fibrillization of WT and E46K α-syn, samples prepared at 2.5 mg/ml were allowed to fibrillize for 0, 24, 48, or 96 h. At each time point, CD spectra were collected for WT and E46K α-syn. The changes in CD spectra over time indicated that E46K α-syn converts from random coil conformation to β-sheet more readily than the WT protein (Fig. 4, A and B). These changes can be more easily compared by plotting assembly time versus molar ellipticity at 200 nm. The signal at this wavelength is important for differentiating between random coil and β-sheet because they generate negati
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