Familial Mutants of α-Synuclein with Increased Neurotoxicity Have a Destabilized Conformation
2005; Elsevier BV; Volume: 280; Issue: 35 Linguagem: Inglês
10.1074/jbc.c500288200
ISSN1083-351X
AutoresCarlos W. Bertoncini, Claudio O. Fernández, Christian Griesinger, Thomas M. Jovin, Markus Zweckstetter,
Tópico(s)Nuclear Receptors and Signaling
ResumoA30P and A53T mutations of the presynaptic proteinα-synuclein are associated with familial forms of Parkinson disease. NMR spectroscopy demonstrates that Parkinsonism-linked mutations greatly perturb specific tertiary interactions essential for the native state of α-synuclein. However, α-synuclein is not completely unfolded but exhibits structural fluctuations on the time scale of secondary structure formation and loses its native conformation gradually when protein stability decreases. The redistribution of the ensemble of α-synuclein conformers may underlie toxic gain-of-function by fostering self-association and altered binding affinity to ligands and receptors. A30P and A53T mutations of the presynaptic proteinα-synuclein are associated with familial forms of Parkinson disease. NMR spectroscopy demonstrates that Parkinsonism-linked mutations greatly perturb specific tertiary interactions essential for the native state of α-synuclein. However, α-synuclein is not completely unfolded but exhibits structural fluctuations on the time scale of secondary structure formation and loses its native conformation gradually when protein stability decreases. The redistribution of the ensemble of α-synuclein conformers may underlie toxic gain-of-function by fostering self-association and altered binding affinity to ligands and receptors. Parkinson disease (PD) 1The abbreviations used are: PD, Parkinson disease; αS, α-synuclein; RDC, residual dipolar coupling; wt, wild type; NAC, non-Aβ component of Alzheimer disease amyloid; PRE, paramagnetic relaxation enhancement; MTSL, 1-oxy-2,2,5,5-tetramethyl-d-pyrroline-3-methyl methanethiosulfonate; HSQC, heteronuclear single quantum coherence. is the most common neurodegenerative movement disorder and is characterized by the loss of dopaminergic neurons in the substantia nigra and the deposition of eosinophilic fibrillar proteinaceous aggregates known as Lewy bodies (1Goedert M. Nat. Rev. Neurosci. 2001; 2: 492-501Crossref PubMed Scopus (1099) Google Scholar). A central role in this neurological disorder is played by the abundant, presynaptic 140-residue proteinα-synuclein (αS). αS is the major component of Lewy bodies (2Spillantini M.G. Schmidt M.L. Lee V.M. Trojanowski J.Q. Jakes R. Goedert M. Nature. 1997; 388: 839-840Crossref PubMed Scopus (6177) Google Scholar), and locus triplication causing an increased dosage of the wild type (wt) αS gene potentiates PD (3Singleton A.B. Farrer M. Johnson J. Singleton A. Hague S. Kachergus J. Hulihan M. Peuralinna T. Dutra A. Nussbaum R. Lincoln S. Crawley A. Hanson M. Maraganore D. Adler C. Cookson M.R. Muenter M. Baptista M. Miller D. Blancato J. Hardy J. Gwinn-Hardy K. Science. 2003; 302: 841Crossref PubMed Scopus (3510) Google Scholar). Two autosomal dominantly inherited forms of PD are caused by Ala53 to Thr (A53T) and Ala30 to Pro (A30P) mutations in the αS gene, which were identified in Italian (4Polymeropoulos M.H. Lavedan C. Leroy E. Ide S.E. Dehejia A. Dutra A. Pike B. Root H. Rubenstein J. Boyer R. Stenroos E.S. Chandrasekharappa S. Athanassiadou A. Papapetropoulos T. Johnson W.G. Lazzarini A.M. Duvoisin R.C. Di Iorio G. Golbe L.I. Nussbaum R.L. Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6667) Google Scholar) and German kindreds (5Kruger R. Kuhn W. Muller T. Woitalla D. Graeber M. Kosel S. Przuntek H. Epplen J.T. Schols L. Riess O. Nat. Genet. 1998; 18: 106-108Crossref PubMed Scopus (3321) Google Scholar), respectively. Both αS mutants display an impaired degradation by chaperone-mediated autophagy (6Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1572) Google Scholar) and share an increased tendency to form soluble oligomeric intermediates, with the A53T mutant fibrillating even faster than the wt protein (7Conway K.A. Lee S.J. Rochet J.C. Ding T.T. Williamson R.E. Lansbury P.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 571-576Crossref PubMed Scopus (1341) Google Scholar). Recent reports suggest that these soluble oligomers, or protofibrils, may constitute the major toxic species, whereas mature fibrils may account for a protective function (8Volles M.J. Lansbury P.T. Biochemistry. 2003; 42: 7871-7878Crossref PubMed Scopus (405) Google Scholar). As the loss of αS appears to have minimal effects on development (9Abeliovich A. Schmitz Y. Farinas I. Choi-Lundberg D. Ho W.H. Castillo P.E. Shinsky N. Verdugo J.M.G. Armanini M. Ryan A. Hynes M. Phillips H. Sulzer D. Rosenthal A. Neuron. 2000; 25: 239-252Abstract Full Text Full Text PDF PubMed Scopus (1403) Google Scholar), the pathogenic effects of mutant αS are attributed to a toxic gain-offunction. These findings are intriguing inasmuch as αS has generally been considered as an intrinsically unstructured protein (10Weinreb P.H. Zhen W. Poon A.W. Conway K.A. Lansbury Jr., P.T. Biochemistry. 1996; 35: 13709-13715Crossref PubMed Scopus (1321) Google Scholar). Why and how single missense mutations in disordered αS accelerate its oligomerization and contribute to the degeneration of dopamine nerve cells are questions fundamental to the understanding and treatment of PD. Recently, it was shown that native αS adopts an ensemble of conformations that are stabilized by long range interactions (11Hoyer W. Cherny D. Subramaniam V. Jovin T.M. Biochemistry. 2004; 43: 16233-16242Crossref PubMed Scopus (276) Google Scholar, 12Dedmon M.M. Lindorff-Larsen K. Christodoulou J. Vendruscolo M. Dobson C.M. J. Am. Chem. Soc. 2005; 127: 476-477Crossref PubMed Scopus (570) Google Scholar, 13Bertoncini C.W. Jung Y.S. Fernandez C.O. Hoyer W. Griesinger C. Jovin T.M. Zweckstetter M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1430-1435Crossref PubMed Scopus (630) Google Scholar, 14Rasia R.M. Bertoncini C.W. Marsh D. Hoyer W. Cherny D. Zweckstetter M. Griesinger C. Jovin T.M. Fernandez C.O. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4294-4299Crossref PubMed Scopus (340) Google Scholar). The latter form an intricate network involving the N (residues 1–60) and C termini (residues 109–140), which shields the highly amyloidogenic, non-Aβ component of Alzheimer disease amyloid (NAC) (residues 61–95) from the solvent (Fig. 1A). Polyamine binding and temperature increase release the intrinsic tertiary structure of αS, leading to a completely unfolded conformation (13Bertoncini C.W. Jung Y.S. Fernandez C.O. Hoyer W. Griesinger C. Jovin T.M. Zweckstetter M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1430-1435Crossref PubMed Scopus (630) Google Scholar). The relevance of these conditions, which cause aggregation in vitro, for the development of PD is, however, unclear. On the other hand, very strong support for the importance of αS in PD comes from the A30P and A53T mutations in the αS gene linked to familial forms of the disease. Using NMR residual dipolar couplings (RDCs) and parmaagnetic relaxation enhancement (PRE) we demonstrate here that Parkinsonism-linked mutations greatly perturb the ensemble of α-synuclein conformers. This conformationally altered αS may constitute a common mediator in the induction of PD by both environmental and genetic conditions. Protein Preparation—PT7 plasmids containing wt, A30P, and A53T αS sequences were a gift from the Lansbury Laboratory, Harvard Medical School, Cambridge, MA. The following αS cysteine-containing mutants were constructed using the QuikChange site-directed mutagenesis kit (Stratagene) on the above mentioned plasmids: Ala18Cys/A30P, Ala90Cys/A30P, Ala140Cys/A30P, Ala18Cys/A53T, Ala90Cys/A53T, and Ala140Cys/A53T. The introduced modifications were further verified by DNA sequencing. The positions of the cysteine replacements was distributed along the sequence of αS and their introduction did not cause chemical shift changes in residues other than those surrounding the point of mutation. 15N-labeled αS, wt, and mutants were expressed in Escherichia coli grown in M9 minimal medium supplemented with15NH4Cl (Cambridge Isotope Laboratories) and purified as described previously (15Hoyer W. Antony T. Cherny D. Heim G. Jovin T.M. Subramaniam V. J. Mol. Biol. 2002; 322: 383-393Crossref PubMed Scopus (419) Google Scholar). The final protein working solutions were dialyzed against buffer A (20 mm Tris-HCl buffer, pH 7.4, 100 mm NaCl). Spin Labeling of αS—The nitroxide spin label chosen for reaction with the cysteine-containing mutants was MTSL (1-oxy-2,2,5,5-tetramethyl-d-pyrroline-3-methyl)-methanethiosulfonate (Toronto Research Chemicals, Toronto, Ontario, Canada). MTSL had already proven to efficiently react with αS cysteine mutants, and the reaction was carried out as described previously (13Bertoncini C.W. Jung Y.S. Fernandez C.O. Hoyer W. Griesinger C. Jovin T.M. Zweckstetter M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1430-1435Crossref PubMed Scopus (630) Google Scholar). Paramagnetic samples were measured at 100 μm MTSL-labeled protein concentration, and diamagnetic experiments were performed with 100 μm protein in the presence of 0.5 mm dithiotreithol (Sigma) to counteract undesirable intensity changes in certain amide resonances of αS caused by addition of the oxidant ascorbic acid. Alignment of αS in Anisotropic Media—RDCs were measured in αS variants aligned in 5% w/v n-octyl-penta(ethylene glycol)/octanol (C8E5) (Sigma) (16Rückert M. Otting G. J. Am. Chem. Soc. 2000; 122: 7793-7797Crossref Scopus (540) Google Scholar). Formation of the anisotropic, dilute liquid crystalline phase was monitored by the splitting of the deuterium signal, which was 20 ± 2 Hz. Alternatively, alignment was achieved by addition of 10 mg/ml of Pf1 bacteriophage (Asla, Riga, Latvia) with deuterium splittings of 9 ± 1 Hz (17Hansen M.R. Mueller L. Pardi A. Nat. Struct. Biol. 1998; 5: 1065-1074Crossref PubMed Scopus (690) Google Scholar). NMR Measurements—NMR spectra were acquired at 15 °C on Bruker Avance 600 and 700 NMR spectrometers on a 100 μm sample of αS variants in buffer A. Measurement conditions of low temperature and absence of stirring were chosen to suppress oligomerization. PRE profiles were derived from the measurement of the peak intensity ratios from two two-dimensional 15N-1H heteronuclear single quantum coherence (HSQC) NMR spectra aquiered in the presence and absence of the nitroxide radical. Spectra were recorded using 256 × 1024 complex data points in F1 and F2 dimensions with 16 scans per increment and a relaxation delay of 1.2 s. The spectral widths were 1650 and 5500 Hz in the 15N and 1H dimensions, respectively. One-bond N-H residual dipolar couplings (DNH) were acquired using the two-dimensional inphaseantiphase-HSQC sequence (18Ottiger M. Delaglio F. Bax A. J. Magn. Res. 1998; 131: 373-378Crossref PubMed Scopus (842) Google Scholar) under both isotropic and anisotropic conditions. Spectra were processed and analyzed using nmrPipe (19Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11570) Google Scholar) and NMRView (20Johnson B.A. Blevins R.A. J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2678) Google Scholar). DNH values were calculated as the difference between splittings measured in an aligned sample and those measured in the isotropic phase (RDCs were not corrected for the negative gyromagnetic ratio of 15N). RDCs observed under different conditions were normalized based on the size of the splitting of the deuterium signal (relative to RDCs for wt αS in buffer A). Familial Mutations Do Not Completely Unfold αS—Paramagnetic enhancement of nuclear spin relaxation by nitroxide spin labels is a useful technique for deriving distance restrains for unfolded states of proteins (21Dyson H.J. Wright P.E. Chem. Rev. 2004; 104: 3607-3622Crossref PubMed Scopus (531) Google Scholar). A nitroxide radical causes broadening of the NMR signals of nearby protons, an effect that extends as far as 20 Å from modified cysteine residues. As αS lacks cysteine, three different double mutants containing variable single Ala to Cys replacements were constructed for both the A30P and A53T mutant αS. Attachment of the spin label MTSL to residue 90 greatly attenuated NMR signals of amino acids in the C terminus of A30P mutant αS with peak intensity ratios between the paramagnetic and the diamagnetic states (Iparam/Idiam) in the range of 0.5 to 0.9 (Fig. 1B, panel II). This result indicates that the C-terminal domain of A30P αS is compact, as had been observed previously for wt αS (13Bertoncini C.W. Jung Y.S. Fernandez C.O. Hoyer W. Griesinger C. Jovin T.M. Zweckstetter M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1430-1435Crossref PubMed Scopus (630) Google Scholar) and also holds for the A53T mutant (Fig. 1B, panel V). When the spin label was attached to residue 140 in the wt protein, the NMR signals of residues located in the NAC region were considerably broadened, providing support for a back-folding of the C terminus on this region in wt αS (13Bertoncini C.W. Jung Y.S. Fernandez C.O. Hoyer W. Griesinger C. Jovin T.M. Zweckstetter M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1430-1435Crossref PubMed Scopus (630) Google Scholar). There was no evidence for such a long range interaction in either the A30P or the A53T mutants (Fig. 1B, panels III and VI). In the A18C spin-labeled mutant αS, intensity ratios as low as 0.5 were observed in the C terminus, similar to those of wt αS (13Bertoncini C.W. Jung Y.S. Fernandez C.O. Hoyer W. Griesinger C. Jovin T.M. Zweckstetter M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1430-1435Crossref PubMed Scopus (630) Google Scholar). In contrast, the relaxation enhancement of residues 40–60 was decreased by about 20% for both missense mutants compared with the wt protein (Fig. 1B, panels I and IV). The reduced paramagnetic broadening of residues 40–60 suggests that the compaction of the N-terminal region is reduced in mutant αS, in agreement with recent tryptophan fluorescence energy transfer measurements (22Lee J.C. Langen R. Hummel P.A. Gray H.B. Winkler J.R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16466-16471Crossref PubMed Scopus (128) Google Scholar). Parkinsonism-linked Mutations Greatly Perturb Specific Tertiary Interactions Essential for the Native State of αS—Residual dipolar couplings are exquisitely sensitive to bond vector orientation and can be measured in a weakly aligned protein, for which the large internuclear dipolar interactions no longer average to zero (23Bax A. Grzesiek S. Acc. Chem. Res. 1993; 26: 131-138Crossref Scopus (795) Google Scholar). Recently, we reported that RDCs identify five different domains in wt αS (domain I, residues 1–28; domain II, residues 33–65; domain III, residues 70–88; domain IV, residues 95–105; and domain V, the C terminus) and probe a hydrophobic core comprising the C terminus and domain III and IV (13Bertoncini C.W. Jung Y.S. Fernandez C.O. Hoyer W. Griesinger C. Jovin T.M. Zweckstetter M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1430-1435Crossref PubMed Scopus (630) Google Scholar). RDCs in PD-linked mutant αS were observed with the A30P and A53T αS mutants dissolved in a dilute liquid crystalline phase of 5% C8E5/octanol. No significant differences in the chemical shifts of spectra in C8E5/octanol and free in solution were observed, suggesting that the anisotropic phase did not appreciably perturb the ensemble of αS conformations. The RDCs in A30P and A53T mutant αS were predominantly positive, with regions displaying relatively large RDCs separated by residues with RDCs close to zero (Fig. 2, A and B). Slightly negative couplings were observed for some residues. The mutant dipolar couplings correlate well with the RDC domain pattern of the wt protein. In contrast to wt αS, however, the RDCs for domain II (residues 50–62) and domain III (residues 70–94) were strongly reduced. Moreover, residues 20–44 displayed much smaller RDCs in the A30P mutant than in both wt and A53T mutant αS, likely due to the missense mutation in this region. In the C-terminal domain, the magnitudes of RDCs were comparable with those in the rest of the protein, in contrast to the RDC profile of wt αS, in which the effect was higher by ∼ 100%. The dipolar couplings of A30P αS were similar to those of wt αS in the presence of 8 m urea (Fig. 2C), whereas the magnitude of RDCs of A53T αS was comparable with that of wt αS in the presence of 4 m urea (Fig. 2D). In addition, the RDC profile of A18C αS, a non-amyloidogenic mutant of αS with an unaltered time course of aggregation (data not shown), was nearly identical to that of wt αS (supplemental Fig. S1). The results presented in Fig. 2 were reproduced in a second alignment medium and at 37 °C (supplemental Fig. S2). We previously showed that polyamine binding induces a complete unfolding of wt αS. In the A30P and A53T mutants, however, the strong paramagnetic broadening exerted by a spin label attached to either residue 18 or 90 on the C-terminal domain (Fig. 1B, panels I, II, IV, and V) indicate that long range interactions are not completely abolished. This interpretation is compatible with measurements of the radius of gyration, which did not differ significantly between wt and mutant αS, but were smaller than for wt αS in 8 m urea (24Li J. Uversky V.N. Fink A.L. Neurotoxicology. 2002; 23: 553-567Crossref PubMed Scopus (127) Google Scholar, 25Morar A.S. Olteanu A. Young G.B. Pielak G.J. Protein Sci. 2001; 10: 2195-2199Crossref PubMed Scopus (157) Google Scholar). Moreover, 15N relaxation time measurements did not indicate any major long range effects of A30P and A53T replacements on the backbone flexibility of αS (26Bussell Jr., R. Eliezer D. J. Biol. Chem. 2001; 276: 45996-46003Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Residual dipolar couplings, as reported in this study, were considerably reduced in the vicinity of both missense mutations. For the A30P mutation, the increased flexibility is in agreement with the reduced heteronuclear15N R ρ1 relaxation rates of backbone amide groups and a reduced helical propensity for residues 18–31 (26Bussell Jr., R. Eliezer D. J. Biol. Chem. 2001; 276: 45996-46003Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Besides these local changes, however, a very strong reduction of dipolar couplings in the C-terminal domain of αS was observed (Fig. 2). As only RDCs and not 15N relaxation time measurements are sensitive to motions from nano- to microseconds, this suggests that the PD-linked mutations strongly increase structural fluctuations on this time scale, one in which secondary structures form during protein folding (27Kubelka J. Hofrichter J. Eaton W.A. Curr. Opin. Struct. Biol. 2004; 14: 76-88Crossref PubMed Scopus (700) Google Scholar). Additional long range effects were observed for the hydrophobic NAC region, in which the magnitude of RDCs was considerably decreased (Figs. 2, A and B) and in which no paramagnetic broadening was detected by a spin label attached to residue 140 (Fig. 1B, panels III and VI). These results point toward a reduced shielding of the hydrophobic NAC region in the A30P and A53T mutant αS. In summary, the dipolar couplings demonstrate that the intricate network of long-range interactions is perturbed in PD-linked mutant αS. The protein is more flexible and able to sample a large range of conformations. Thus, both A30P and A53T mutant αS can overcome more easily the energetic barrier for self-association, leading to an increased tendency to oligomerize (7Conway K.A. Lee S.J. Rochet J.C. Ding T.T. Williamson R.E. Lansbury P.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 571-576Crossref PubMed Scopus (1341) Google Scholar). Formation of mature fibrils, on the other hand, requires an incorporation of the N terminus in a β-sheet structure, a process hindered by the presence of proline and thus potentially accounting for a slower rate of fibril formation by the A30P αS mutant (7Conway K.A. Lee S.J. Rochet J.C. Ding T.T. Williamson R.E. Lansbury P.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 571-576Crossref PubMed Scopus (1341) Google Scholar). αS Gradually Loses Its Native Conformation—RDCs and PREs probe different structural features of αS. Backbone RDCs report on local, backbone conformational propensities (such as transient elements of secondary structure) in unfolded states of proteins (28Mohana-Borges R. Goto N.K. Kroon G.J.A. Dyson H.J. Wright P.E. J. Mol. Biol. 2004; 340: 1131-1142Crossref PubMed Scopus (162) Google Scholar), whereas PREs are sensitive to average, long range intramolecular distances (21Dyson H.J. Wright P.E. Chem. Rev. 2004; 104: 3607-3622Crossref PubMed Scopus (531) Google Scholar). Therefore, NMR dipolar couplings and paramagnetic relaxation enhancement by nearby spin labels correspond to two other techniques commonly used to characterize unfolding of proteins: far-ultraviolet CD to monitor secondary structure content, and fluorescence resonance energy transfer to evaluate intramolecular distances. Although differential scanning calorimetry did not reveal any thermal transition for either wt or mutant αSinthe range 15–110 °C (29Syme C.D. Blanch E.W. Holt C. Jakes R. Goedert M. Hecht L. Barron L.D. Eur. J. Biochem. 2002; 269: 148-156Crossref PubMed Scopus (224) Google Scholar), backbone RDCs in the C terminus of αS were greatly reduced upon heating to 37 °C (13Bertoncini C.W. Jung Y.S. Fernandez C.O. Hoyer W. Griesinger C. Jovin T.M. Zweckstetter M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1430-1435Crossref PubMed Scopus (630) Google Scholar) or by the introduction of missense mutations (Fig. 2). As the sites of mutation are far from the C terminus, the detrimental effects of the mutations must be transmitted through the network of transient long range interactions to the C-terminal domain of αS, such that conformational restrictions of the backbone are reduced. However, the retention of strong PRE broadening at 37 °C or in the presence of missense mutations indicates that dipolar couplings and PREs have different thermal transition midpoints. Redistribution of the Ensemble of αS Conformers May Underlie Toxic Gain-of-Function—Several biological and biochemical properties underlying the toxic gain-of-function of the familial mutants of αS can now be rationalized in terms of the different intrinsic structural features of these proteins (Table I). Recently, it was shown that C-terminal truncation of αS is a normal cellular process and that the generation and accumulation of truncated αS may be involved in the initiation and progression of αS aggregation in vivo. Residues 119–123 were identified as potential cleavage sites and the accumulation of truncated αS was enhanced by the expression of mutant αS (30Li W. West N. Colla E. Pletnikova O. Troncoso J.C. Marsh L. Dawson T.M. Jäkälä P. Hartmann T. Price D.L. Lee K.L. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2162-2167Crossref PubMed Scopus (373) Google Scholar). These findings may be explained by the results of the present study. Although the truncation site is far from the A30P and A53T missense mutations, the destabilization of αS tertiary structure may allow proteases to degrade the αS C terminus more efficiently. This destabilization may also be the source for the increased affinity of lysosomal chaperone-mediated autophagy receptors for mutant αS, recognizing the sequence 95VKKDQ99 present in the C terminus of αS and thereby causing an impairment in protein catabolism (6Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1572) Google Scholar). Regarding self-association, an increased tendency to form dimers, oligomers, and annular protofibrills has been evidenced for both PD-linked mutants (7Conway K.A. Lee S.J. Rochet J.C. Ding T.T. Williamson R.E. Lansbury P.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 571-576Crossref PubMed Scopus (1341) Google Scholar, 31Krishnan S. Chi E.Y. Wood S.J. Kendrick B.S. Li C. Garzon-Rodriguez W. Wypych J. Randolph T.W. Narhi L.O. Biere A.L. Citron M. Carpenter J.F. Biochemistry. 2003; 42: 829-837Crossref PubMed Scopus (167) Google Scholar, 32Lashuel H.A. Hartley D. Petre B.M. Walz T. Lansbury Jr., P.T. Nature. 2002; 418: 291Crossref PubMed Scopus (1128) Google Scholar), which may as well be explained in terms of our findings. The decreased shielding of the highly hydrophobic NAC region due to the reduction of long range interactions allows A30P and A53T mutant αS to preferentially expose this region to the solvent, facilitating self-association.Table IDistinctive features of αS-mediated pathogenesis correlate with the properties of A30P and A53T mutantsCharacteristics associated with the increased neurotoxicity of A30P and A53TBiological featuresReduced cell viability in cell culture models (33Dev K.K. Hofele K. Barbieri S. Buchman V.L. van der Putten H. Neuropharmacology. 2003; 45: 14-44Crossref PubMed Scopus (230) Google Scholar)Increased inhibition of proteasomal activity (34Tanaka Y. Engelender S. Igarashi S. Rao R.K. Wanner T. Tanzi R.E. Sawa A. Dawson V.L. Dawson T.M. Ross C.A. Hum. Mol. Genet. 2001; 10: 919-926Crossref PubMed Google Scholar, 35Stefanis L. Larsen K.E. Rideout H.J. Sulzer D. Greene L.A. J. Neurosci. 2001; 21: 9549-9560Crossref PubMed Google Scholar)Impaired protein degradation via chaperone-mediated autophagy (6Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1572) Google Scholar)Enhanced in vivo C-terminal truncation (30Li W. West N. Colla E. Pletnikova O. Troncoso J.C. Marsh L. Dawson T.M. Jäkälä P. Hartmann T. Price D.L. Lee K.L. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2162-2167Crossref PubMed Scopus (373) Google Scholar)Biochemical featuresFacilitated formation of dimers (31Krishnan S. Chi E.Y. Wood S.J. Kendrick B.S. Li C. Garzon-Rodriguez W. Wypych J. Randolph T.W. Narhi L.O. Biere A.L. Citron M. Carpenter J.F. Biochemistry. 2003; 42: 829-837Crossref PubMed Scopus (167) Google Scholar)Acceleration of oligomerization (7Conway K.A. Lee S.J. Rochet J.C. Ding T.T. Williamson R.E. Lansbury P.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 571-576Crossref PubMed Scopus (1341) Google Scholar)Presence of annular protofibrills (32Lashuel H.A. Hartley D. Petre B.M. Walz T. Lansbury Jr., P.T. Nature. 2002; 418: 291Crossref PubMed Scopus (1128) Google Scholar)Rapid fibrillation (only A53T) (7Conway K.A. Lee S.J. Rochet J.C. Ding T.T. Williamson R.E. Lansbury P.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 571-576Crossref PubMed Scopus (1341) Google Scholar)Higher vesicle permeabilization activity (36Volles M.J. Lansbury P.T. Biochemistry. 2002; 41: 4595-4602Crossref PubMed Scopus (423) Google Scholar)Structural featuresPerturbed autoinhibitory long range interactions (this work)Increased backbone flexibility in the nano- to microsecond time scale (this work)Increased exposure of NAC region (this work) Open table in a new tab We conclude that the redistribution of the structural ensemble of the A30P and A53T αS mutants potentiates oligomerization and modulates binding to ligands, proteases, and receptors. This effect may account for the toxic gain-of-function of familial Parkinsonism-linked mutant αS. Conformationally altered αS may constitute a general molecular mechanism underlying the induction of PD by environmental and genetic conditions (Fig. 3). Thus, agents specifically designed to stabilize the native state of αS may prove useful in impeding or reversing its pathologic aggregation in both idiopathic and familial forms of Parkinson disease. Download .pdf (.7 MB) Help with pdf files
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