Báo cáo khoa học: PA700, the regulatory complex of the 26S proteasome, interferes with a-synuclein assembly pptx

PA700, the regulatory complex of the 26S proteasome, interferes with a-synuclein assembly Medeva Ghee1, Ronald Melki2, Nadine Michot3 and Jacques Mallet1 ´ ´ ´ ´ ´ ´ Laboratoire de Genetique Moleculaire de la Neurotransmission et des Processus Neurodegeneratifs, Centre National de la Recherche ˆ ˆ ` ´ Scientifique, Hopital de la Pitie Salpetriere, Paris, France Laboratoire d’Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, France Protein Production, Aventis Pharma, Vitry, France Keywords a-synuclein; aggregation; PA700; proteasome; tat binding protein Correspondence ´ ´ M Ghee, Laboratoire de Genetique ´ Moleculaire de la Neurotransmission et des ´ ´ ´ Processus Neurodegeneratifs, Centre National de la Recherche Scientifique, ˆ ˆ UMR7091, Batiment CERVI, Hopital de la ˆ ` ˆ ´ Pitie Salpetriere, 83, boulevard de l’Hopital, 75013 Paris, France Fax: +33 42 17 75 33 Tel: + 33 42 17 75 42 Email: mghee@infobiogen.fr R Melki, Laboratoire d’Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, 91198 Gif-surYvette Cedex, France Fax: +33 69 82 35 04 Tel: + 33 69 82 35 03 E-mail: melki@lebs.cnrs-gif.fr (Received 28 February 2005, revised 20 April 2005 accepted 16 May 2005) Parkinson’s disease is characterized by the loss of dopaminergic neurons in the nigrostriatal pathway accompanied by the presence of intracellular cytoplasmic inclusions, termed Lewy bodies Fibrillized a-synuclein forms the major component of Lewy bodies We reported a specific interaction between rat a-synuclein and tat binding protein 1, a subunit of PA700, the regulatory complex of the 26S proteasome It has been demonstrated that PA700 prevents the aggregation of misfolded, nonubiquinated substrates In this study, we examine the effect of PA700 on the aggregation of wildtype and A53T mutant a-synuclein PA700 inhibits both wild-type and A53T a-synuclein fibril formation as measured by Thioflavin T fluorescence Using size exclusion chromatography, we present evidence for a stable PA700–a-synuclein complex Sedimentation analyses reveal that PA700 sequesters a-synuclein in an assembly incompetent form Analysis of the morphology of wild-type and A53T a-synuclein aggregates during the course of fibrillization by electron microscopy demonstrate the formation of amyloid-like fibrils Secondary structure analyses of wild-type and A53T a-synuclein assembled in the presence of PA700 revealed a decrease in the overall amount of assembled a-synuclein with no significant change in protein conformation Thus, PA700 acts on a-synuclein assembly and not on the structure of fibrils We hypothesize that PA700 sequesters a-synuclein oligomeric species that are the precursors of the fibrillar form of the protein, thus preventing its assembly into fibrils doi:10.1111/j.1742-4658.2005.04776.x The abnormal accumulation of insoluble cytoplasmic protein aggregates is a factor common to neurodegenerative diseases, including Parkinson’s disease (PD) PD is clinically characterized by resting tremor, bradykinesia and muscular rigidity Neuropathologically, it is defined by Lewy bodies (LBs), neuronal proteinaceous cytoplasmic inclusions, which accompany a selective degeneration of the dopaminergic neurons of the substantia nigra [1] Although the majority of PD cases are sporadic, rare familial forms of PD have been reported with the identification of mutations in the genes encoding parkin, ubiquitin C-terminal hydrolase L1, DJ-1, a-synuclein and most recently PINK-1 that result in impairment of the ubiquitin–proteasome system, mitochondrial impairment, oxidative stress and protein misfolding [2] Whereas altered expression of these proteins contribute to the pathogenesis of PD, a recent report indicates that overexpression of wild-type Abbreviations AAA, ATPase-associated-with-different-cellular-activities family; HIF1a, hypoxia-inducible factor alpha; LB, Lewy body; PD, Parkinson disease; TBP1, Tat binding protein 1; pVHL, Von Hippel–Landau; WT, wild type FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS 4023 PA700 interacts with a-synuclein oligomers (WT) a-synuclein resulting from a genomic triplication of the region containing a-synuclein in an Iowan kindred is also responsible for PD pathology [3] a-Synuclein is an abundant brain presynaptic protein consisting of 140 amino acid residues It has been established that the WT a-synuclein assembles in vitro into elongated filaments [4] Moreover, the two a-synuclein mutations associated with PD, Ala53Thr [5] and Ala30Pro [6], further accelerate the aggregation process [7–9] A third a-synuclein variant was recently described with the substitution Glu46 fi Lys [10] Its assembly properties are not yet fully documented Biophysical studies analyzing the in vitro aggregation behavior of a-synuclein suggests that fibril formation occurs via a nucleation-dependent mechanism [11] with the rate-limiting step being the transformation of the protein from the monomer to a prefibrillar oligomer It has been suggested that the prefibrillar oligomer, or protofibril, may be the toxic species of the protein Protofibrillar forms of a-synuclein may transiently permeabilize vesicular membranes, predisposing these cells to undergo apoptosis [12] Moreover, it has been reported that a-synuclein forms adducts with dopamine in vitro, stabilizing potential toxic a-synuclein protofibrils [13] The finding that filamentous a-synuclein is the major component of LBs suggests that protein aggregation and ⁄ or dysfunction of the ubiquitin ⁄ proteasomal system play a role in the development of familial PD Aberrant aggregation of proteins is one of many signals that activates the ubiquitin–proteasomal system to process damaged and toxic proteins Elimination of proteins targeted for degradation is mediated by the 26S proteasome, a multisubunit, intracellular protease [14] It contains a proteolytic core complex, The 20S proteasome, a cylinder-shaped particle formed by the axial stacking of four rings of seven a and b subunits, and one or two 19S regulatory complexes (PA700) which associate with the termini of the 20S core The PA700 complex can be dissociated into two subcomplexes called the lid and the base The lid serves to recognize ubiquinated target proteins The base consists of six ATPases that belong to the [15] The functions proposed for the ATPases include: (a) the hydrolysis of ATP to promote the assembly of the 26S proteasome from the PA700 complex and 20S proteasome; (b) the opening of the channel leading to the 20S proteasome; and (c) the binding and unfolding of substrate proteins before translocating them into the 20S central chamber for subsequent proteolysis [14] This latter function is reminiscent of molecular chaperones Indeed, it has been reported that PA700 has chaperone-like activity [16] Moreover, PA700 has been 4024 M Ghee et al shown to recognize and interact with misfolded, nonubiquinated substrates and inhibit their aggregation [17] Nonubiquinated a-synuclein can be degraded by proteasomes in a pathway which does not have an absolute requirement for ubiquination [18] We first provided evidence that a-synuclein is a substrate of PA700 via a direct interaction with Tat Binding Protein (TBP1), a subunit of the base complex [19] The interaction between a-synuclein and TBP1 led us to investigate whether PA700 was capable of inhibiting a-synuclein aggregation In the present study, we analyze the effect of PA700 on WT and A53T a-synucleins assembly in vitro We demonstrate that PA700 prevents fibril formation of both WT and A53T a-synuclein We characterize the a-synuclein oligomeric species that form in the absence and the presence of PA700 and show that PA700 sequesters a-synuclein in an assembly incompetent form These findings suggest a mechanism by which a component of the 26S proteasome may contribute to the processing and eventual degradation of misfolded proteins Results The effects of PA700 on WT and A53T mutant a-synuclein assembly into fibrils To determine whether PA700 affects the fibrillation properties of both WT and A53T, a-synucleins, we designed an in vitro assembly assay in which purified recombinant a-synuclein was incubated in the presence or absence of PA700 for 24 h at 37 °C under continuous shaking The kinetics of fibril formation was monitored by the use of Thioflavin T fluorescence As shown in Fig 1A and C, respectively, both WT and A53T a-synucleins assemble into fibrils in a concentration-dependent manner The aggregation kinetics is triphasic, with an inital lag phase, followed by an exponential growth phase and ending with a steady state phase [4] Figure 1A and C illustrate that a decrease in protein concentration is accompanied by an increase in the lag phase, a decrease in the slope of the exponential growth phase and of fluorescence intensity at steady state, which reflects a decrease in fibril formation The effect of PA700 on the kinetics of a-synuclein fibrillation was analyzed Addition of increasing concentrations of PA700 to either WT or A53T a-synuclein decreased Thioflavin T fluorescence intensity at steady state (Figs 1B and D, respectively) At the highest PA700 concentration used (218 nm) we observed an approximate twofold decrease in Thioflavin T fluoresFEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS M Ghee et al PA700 interacts with a-synuclein oligomers A B C D Fig PA700 inhibits a-synuclein fibril formation The assembly of WT and A53T a-synucleins were monitored by Thioflavin T binding WT (A) and A53T (C) a-synucleins at 280 lM, 210 lM, 140 lM, 70 lM and 35 lM were assembled at 37 °C Kinetics of fibril formation of WT (B) and A53T (D) a-synucleins in the absence or presence of 109 nM PA700 (2570 : 1), 218 nM PA700 (1284 : 1) Error bars indicate the standard deviation in triplicate samples Similar results were obtained in independent experiments FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS 4025 PA700 interacts with a-synuclein oligomers cence intensity at steady state for the WT and a threefold decrease for the A53T mutant The fluorescence intensities at steady states of the assembly reaction of WT and A53T a-synucleins (280 lm) in the presence of 218 nm PA700 (i.e a 1284 : ratio) is equivalent to that of (140 lm) WT and A53T a-synuclein The latter results strongly suggest that PA700 inhibits a-synuclein assembly by binding and sequestering the a-synuclein species that are the precursors of the fibrillar form of the proteins M Ghee et al A Evidences for a soluble, assembly incompetent a-synuclein-PA700 complex To determine whether PA700 forms a stable complex with a-synuclein, samples containing WT and A53T a-synucleins (280 lm) in the absence or the presence of PA700 (218 nm) were incubated at 37 °C with orbital shaking for h and analyzed by size exclusion chromatography as described in the Experimental procedures Figure 2A, showing data obtained for WT a-synuclein, illustrates our findings In the absence of PA700, WT a-synuclein emerges from the column in a single peak centered at 300 kDa In the presence of PA700, WT a-synuclein emerges from the column with a molecular mass of 670 kDa, indicating a colocalization with PA700 Identical results were obtained using the A53T variant (data not shown) This finding strongly suggests that WT and A53T a-synucleins interact with PA700 within stable soluble high molecular mass complexes The interaction of PA700 with WT and A53T a-synucleins was further documented using sedimentation analysis Aliquots were withdrawn during the lag (time h) and steady state (time 18 h) phases from assembly reactions and subjected to ultracentrifugation Both WT a-synuclein and PA700 are found in the supernatant of the sample corresponding to the lag phase (Fig 2B) Following assembly and in the absence of PA700, WT a-synuclein is found in the pellet, whereas over 80% of WT a-synuclein is in the supernatant together with PA700 in samples where assembly was carried out in the presence of PA700 (Fig 2B) Identical results were obtained for A53T a-synuclein (data not shown) This clearly indicates that PA700 sequesters a-synuclein in a soluble, assembly incompetent state Characterization of WT and A53T a-synuclein oligomeric species in the absence and the presence of PA700 by transmission electron microscopy WT and A53T a-synuclein oligomeric species that are generated in the absence or presence of PA700 were 4026 B Fig PA700 forms stable assembly incompetent complexes with WT a-synuclein (A) Size exclusion chromatography elution profiles of WT a-synuclein in the absence (n) and the presence (m) of PA700 are shown PA700 is detected using its intrinsic fluorescence (d) The immunoreactivity (n,m) of WT a-synuclein was monitored by dot-blot as this polypeptide lacks tryptophan residues Arrows show the location of molecular size markers (thyroglobulin, 670 kDa; immunoglobulin G, 158 kDa; ovalbumin, 44 kDa and myoglobin, 17 kDa) run under identical conditions on the same column (B) Sedimentation behavior of WT a-synuclein in the absence and the presence of PA700 Aliquots of WT a-synuclein assembly reactions in the absence (–) or the presence (+) of PA700, h and 18 h after the onset of the assembly reaction (lag and steady state phases, respectively) were centrifuged (160 000 g) for 20 The protein content of the supernatant (S) and pellet (P) fractions was analyzed by SDS ⁄ PAGE (12% polyacrylamide gels) The molecular mass markers (Mw) are shown Identical results were obtained for A53T a-synuclein further characterized by electron microscopy Aliquots of WT and A53T a-synuclein (280 lm) assembly reactions in the presence or absence of (218 nm) PA700 were withdrawn at time intervals, diluted in the assemFEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS M Ghee et al PA700 interacts with a-synuclein oligomers bly buffer and processed for electron microscopy analysis In the lag phase preceeding assembly (time h), globular and short curved oligomers are the unique constituents of both WT and A53T a-synuclein solutions (Fig 3A and B) In the elongation phase (time h), semiflexible, unbranched fibrils are observed They coexist with the globular and the short curved oligomers observed at the earlier stages of assembly (Fig 3C and D) At late stages in WT and A53T a-synuclein assembly reactions (time 18 h), long helical fibrils are observed with very few oligomers remaining in the solution (Fig 3E and F) We next characterized WT and A53T a-synuclein oligomeric species in the presence of PA700 The oligomers that form in the lag phase upon addition of PA700 to WT and A53T a-synuclein are indistinguishable from that observed in the absence of PA700 (data not shown) WT a-synuclein oligomeric species (globular, curved oligomers and fibrils) that form in the absence (Fig 3C and E) or the presence (Fig 4A and C) of PA700 are indistinguishable In contrast, a change in the morphology of A53T a-synuclein oligomeric species that form upon assembly is observed upon addition of PA700 Compare Fig 3D and F to Fig 4B and D Indeed, very few fibrils are present in the solution and the vast majority of the high molecular mass oligomers that form are globular and short curved oligomers (Fig 4B) In addition, the few fibrils observed after examining multiple fields on the electron microscopy grids appear less structured (Fig 4D) than those obtained in the absence of PA700 (Fig 3F) This result further suggests, particularly in the case of the A53T variant, that PA700 sequesters a-synuclein oligomers that are the precursors of the fibrils Secondary structure and quantitative analysis of WT and A53T a-synuclein oligomers in the absence and presence of PA700 by FTIR Spectroscopy To assess whether the characteristic polypeptide chain arrangement of WT and A53T a-synuclein fibrils is WT α-synuclein A53T α-synuclein A B C D E F Lag phase Time 1h Elongation phase Time 5h Fig Electron micrographs of WT and A53T a-synuclein The oligomeric species of WT and A53T a-synucleins (280 lM) were analyzed at the early stages of assembly, i.e the lag phase (A, B, respectively), during the elongation phase (C, D, respectively) and at steady state (E, F, respectively) Representative fields on the grids are depicted (scale bar, 0.5 lm) FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS Steady state phase Time 18h 4027 PA700 interacts with a-synuclein oligomers WT α-synuclein+PA700 M Ghee et al A53T α-synuclein+PA700 A B C D Elongation phase Time 5h Fig Electron micrographs of WT and A53T a-synuclein incubated in the presence of PA700 Recombinant WT and A53T a-synuclein (280 lM) oligomers were imaged during the elongation (time h) and steady state (time 18 h) phases (A, B and C, D, respectively) in the presence of PA700 (218 nM) Representative fields on the grids are depicted (scale bar, 0.5 lm) Steady State phase Time18h affected by PA700 and to quantify the amount of fibrils formed in the absence and the presence of PA700, the FTIR spectra of WT, A53T a-synuclein (280 lm) assembled in the absence or presence of 218 nm PA700 in buffer A and subsequently extensively dialyzed against D2O were recorded The spectra presented in Fig showed very similar amide I regions dominated by an absorption maxima at 1624 cm)1, demonstrating the presence of aggregated b-sheets Fourier deconvolution and curve fitting of the spectra permitted the quantitative analysis of the secondary structure content of the different samples The results are summarized in Table We observed no significant change in the secondary structure content of the fibrils upon addition of PA700 to WT or A53T a-synucleins Interestingly, however, the absorption intensities of the samples assembled in the presence of PA700 are lower than those assembled in the absence of PA700 at constant WT and A53T a-synuclein concentrations (compare the absorption intensities in Fig 5A,C, and Fig 5B,D, respectively) FTIR spectra of PA700 alone reveals a secondary structure composed primarily of a-helices (Fig 1D, inset) These data strongly suggest that PA700 does not change the conformation of a-synuclein within the fibrils but that it sequesters the precursors of the fibrils, thus leading to a smaller amount of polymers at steady state 4028 Discussion The aggregation of either WT or mutant a-synuclein proteins in dopaminergic neurons of the substantia nigra pars compacta is thought to be responsible for the subsequent neurodegeneration of these neurons in PD The process through which a-synuclein is transformed from a disordered monomer into a stable amyloid fibril involves several aggregation states, including the natively unfolded protein, which oligomerizes to form a partially folded, b-sheet rich oligomeric intermediate This aggregation-competent oligomer, or protofibril, has been proposed to be an important precursor that favors the formation of a-synuclein fibrils We show that PA700 interacts with a-synuclein, thereby generating a PA700–a-synuclein species that is unable to polymerize into fibrils Four observations support this finding First, the ability of PA700 to inhibit a-synuclein assembly into fibrils as witnessed by the decrease of the overall amount of fibrillar a-synuclein at steady state in the presence of increasing concentrations of PA700 Second, the existence of a high molecular mass, stable PA700–a-synuclein complex as demonstrated by size exclusion chromatography Third, the presence of increased amounts of soluble a-synuclein in the presence of PA700 in sedimentation experiments Finally, the decrease in the amount of fibrils in the presence of PA700 as measured by quantitative FTIR spectroscopy FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS M Ghee et al PA700 interacts with a-synuclein oligomers A 0.5 0.007 0.4 Abs 0.004 0.002 0.3 Abs 1700 1680 1660 1640 1620 1600 Wavenumber [cm-1] 0.2 0.1 1700 B 1680 1660 1640 1620 1600 0.6 0.009 0.006 Abs 0.004 0.4 0.002 1700 Abs 1680 1660 1640 1620 1600 Wavenumber [cm-1] 0.2 1700 0.21 C Abs Fig Secondary structure and quantitative analysis of WT and A53T a-synuclein oligomers in the absence and presence of PA700 FTIR spectra of fibrillar WT and A53T a-synucleins (280 lM) in the absence (A, B, respectively) or the presence of PA700 (218 nM) (C, D, respectively) The FTIR spectra of the soluble forms of WT, A53T and PA700 are shown as insets in A, B and D, respectively Curve fit spectra are presented in each case as dotted lines Absorption intensities (Abs) and wavenumbers are indicated D 1660 1640 1620 1600 1680 1660 1640 1620 1600 0.1 1700 0.2 0.15 0.004 0.003 Abs 0.002 0.001 1689.34 Abs 0.1 1660 1640 1610.27 Wavenumber [cm-1] 0.05 1700 Addition of increasing concentrations of PA700 to either WT or A53T a-synuclein significantly decreases Thioflavin T fluorescence intensity at steady state, suggesting that PA700 inhibits a-synuclein assembly by binding and sequestering a-synuclein oligomeric species that are the precursors of the fibrillar form of the proteins Strikingly, however, the lag phases of both WT and A53T a-synucleins and the elongation rates remain constant One expects a dramatic increase of the lag FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS 1680 1680 1640 1660 Wavenumber [cm-1] 1620 1600 phase and a significant decrease of the elongation rate if PA700 only sequesters assembly competent a-synucleins leading to the decrease of the latter concentration as shown in Fig 1A,C Thus, PA700 appears to have a complex effect on the assembly reaction It sequesters a-synuclein oligomers in an assembly incompetent form and at the same time favors the formation of oligomeric species that act as nuclei in fibrils assembly This complex observation can be accounted for by 4029 PA700 interacts with a-synuclein oligomers M Ghee et al Table Secondary structure content of WT and A53T a-synuclein oligomers in the absence and presence of PA700 estimated from the deconvolution of the FTIR spectroscopy measurements presented in Fig b-Sheet (%) Structural Assignment WT a-synuclein WT a-synuclein + PA700 A53T a-synuclein A53T a-synuclein + PA700 Disordered (%) 1647 cm)1 19 24 23 29 Fibrils Intermediate oligomer Loops (%) 1624 cm)1 65 61 61 58 A 1663 cm)1 11 8.5 8 B the following It is reasonable to envisage that the affinity of PA700 for a-synuclein oligomers is not infinite A proportion of a-synuclein oligomers is therefore released in solution where they can assemble into fibrils Thus, our experimental observations may result from the PA700 sequestering activity, on the one hand, and of the PA700 mediated a-synuclein oligomerization activity, on the other This is shown schematically in Fig Alternatively, our experimental observations could be accounted for by PA700 chaperone activity [16,17] Indeed, following the interaction of native unfolded, assembly incompetent, a-synuclein with PA700, partially folded, assembly competent, a-synuclein may be produced This favors nucleation and assembly PA700 could therefore bind a subset of a-synuclein oligomers in an assembly incompetent, native state, thus keeping off assembly track these oligomers and reducing the amount of fibrils formed at steady state A recent report by Dedmon and colleagues demonstrate that the molecular chaperone, Hsp70, preferentially binds to cytotoxic prefibrillar a-synuclein species and consequently inhibits its fibril formation [20] The a-synuclein species that binds to PA700 is a high molecular mass species Indeed, the assembly reactions of WT and A53T a-synuclein (280 lm) in the presence of PA700 at a ratio of : 1284 are superimposable to that of a-synucleins in the absence of PA700 at 140 lm If PA700 binds monomeric a-synuclein, the assembly kinetics in the absence or the presence of PA700 would be superimposable as the concentration of free monomeric a-synuclein in the presence of PA700 would be at least 279.5 lm Our data clearly indicate that 140 lm a-synuclein are sequestered by 218 nm PA700, suggesting that the PA700 particle binds an a-synuclein oligomeric form This species is unable to assemble into fibrils as witnessed by the increased amount of high molecular mass PA700–a-synucleinin the supernatants of assembly reactions containing WT or A53T a-synuclein and 4030 PA700 Fig PA700 promotes a-synuclein oligomerization and sequesters soluble oligomeric species in an assembly incompetent form In the absence of PA700 (A), monomeric a-synuclein oligomerizes probably in an isodesmic manner These soluble oligomers are the precursors of the fibrillar form of the protein PA700 interacts with a subset of soluble a-synuclein oligomers (B) This interaction shifts the equilibria between monomeric and ⁄ or low molecular mass a-synuclein oligomers, a subset of which are the precursors of the fibrils toward the formation of a PA700–a-synuclein species This prevents a-synuclein fibril formation However, binding of a-synuclein to PA700 favors the oligomerization of a-synuclein As the affinity of PA700 for a-synuclein oligomers is not infinite, the oligomers are released in solution where they can elongate Thus, PA700 facilitates the rate-limiting nucleation phase and at the same time limits assembly by sequestering a proportion of soluble a-synuclein oligomers PA700 and by the lower amounts of fibrils formed at steady state as measured by FTIR spectroscopy The affinity of PA700 for a-synucleins is not infinite as the complex dissociates on sizing columns (Fig 2A) Furthermore, the PA700–a-synuclein complex neither binds Thioflavin T, nor has an increased b-sheet content as measured by FTIR Indeed, in our kinetic measurements, the formation of soluble high molecular mass a-synuclein species that has an apparent molecular mass of 300 kDa is not accompanied by an increased Thioflavin T binding Similarly, the PA700– a-synuclein complex that forms in the lag phase and in the presence of PA700 does not influence Thioflavin T fluorescence The FTIR measurements demonstrate that the amount of fibrillar a-synuclein (280 lm) (e.g FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS M Ghee et al b-sheet rich form) in the presence of PA700 (218 nm) is equivalent to half of that in the absence of PA700, consistent with the sequestering of 140 lm a-synuclein in a form lacking b-sheets The latter may be assembly incompetent because of its secondary structure The PA700-mediated inhibition of a-synuclein assembly may be the consequence of a physical interaction between a-synuclein oligomers and PA700, i.e a basic sequestering effect The ability of PA700 to inhibit a-synuclein fibril formation in vitro may shed more light on how a-synuclein is degraded in vivo It has been previously reported that a-synuclein is degraded by the 26S proteasome [21], a process which does not seem to require ubiquitination [18] and via autophagy [22] We first observed an interaction between a-synuclein and TBP1, an ATPase residing within the PA700 base subcomplex [19] TBP1 possesses a coiled-coil domain, a mitochondrial energy transfer domain and an ATPase domain that is highly conserved among all members of the AAA family [15] Consistent with these functions, TBP1 would participate in the unfolding of a-synuclein and its translocation into the 20S proteolytic core for rapid hydrolysis Additional evidence supporting this hypothesis demonstrates that TBP1 contributes to the E3 ubiquitin ligase activity of the von Hippel–Lindau (pVHL) protein in order to promote the degradation of the hypoxia-inducible factor alpha (Hif1a) for oxygen-dependent proteolysis [23] The authors suggested that TBP1 may function as a chaperone, tethering pVHL–Hif1a complexes to the proteasome To date, no direct evidence of PA700 functioning in the absence of the 20S proteasome in the cell has been reported Therefore, the chaperone-like properties of the PA700 complex may play an important role in the degradation of misfolded proteins The question as to how a-synuclein escapes the ubiquitin–proteasome system has yet to be elucidated It has been reported that a-synuclein is capable of inhibiting the 26S proteasome via its interaction with the TBP1 subunit [24] Alternatively, PA700 complexes could be sequestered in LB resulting in a depletion of PA700 in the cells Our data show unequivocally that PA700 binds a-synuclein, in particular its oligomeric forms A significant decrease in PA700 concentration could therefore be responsible for the malfunction of the 26S proteasome in a-synuclein degradation In vivo ubiquination of the proteasomal clients may affect their binding and degradation properties Thus, therapeutic approaches to synucleinopathies having as a target PA700 or the proteasome should not only integrate our findings but also the behavior of ubiquitinated a-synuclein FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS PA700 interacts with a-synuclein oligomers Experimental procedures Materials PA700 was purified from bovine red blood cells as described [25,26] Thioflavin T was obtained from ICN Biochemicals (Aurora, OH) Expression and purification of recombinant a-synuclein proteins The bacterial expression construct pRK172 encoding WT human a-synuclein was a gift from R Jakes and M Goedert (MRC Cambridge, UK) The QuikChange site-directed mutagenesis protocol (Stratagene Europe, Amsterdam, The Netherlands) was used to generate the mutant construct, pRK172 a-synuclein A53T Mutagenesis was confirmed by DNA sequencing The expression constructs were transformed into the BL21 (DE3) Escherichia coli strain, grown to an A600 of 0.6–0.8, induced with 0.44 mm isopropyl-1-thio-b-d-galactopyranoside and harvested h later The pellet was resuspended in 10 mm Tris, pH 8, mm EDTA, mm phenylmethanesulfonyl fluoride, and lysed by freezing in liquid nitrogen followed by thawing and probe sonication Cell lysate was precipitated at °C by addition of ammonium sulfate to a final concentration of 30% Following centrifugation, the ammonium sulfate concentration was adjusted to 50% at °C and the solution centrifuged at 5000 g The resulting pellet was resuspended in 10 mm Tris pH 7.5 and the solution loaded onto a DEAE column eluted by a gradient of 0–500 mm NaCl The fractions containing a-synuclein, eluted at 200 mm NaCl, were concentrated in an Ultrafree-15, 5K MWCO filter (Millipore Corp., Bedford, MA, USA), loaded onto a Superdex 75 HiLoad 26 ⁄ 60 column (Amersham Biosciences Europe GmbH, Orsay, France), equilibrated and eluted in 100 mm NH4HCO3 Eluates containing a-synuclein were incubated with m (NH4)2SO4 at °C, loaded onto a butyl-sepharose column in Buffer A (50 mm K2HPO4, KH2PO4, pH 7.1) and eluted in Buffer B (100 mm Buffer A; m (NH4)2SO4, pH 7) Purification of monomeric a-synuclein was confirmed by SDS ⁄ PAGE Purified WT and A53T a-synuclein samples were concentrated using the Ultrafree-15, 5-K MWCO filter Proteins were filtered through Microcon 100-kDa cutoff filters to remove any oligomeric material that could have formed during the concentration Protein concentration was determined using the bicinchoninic acid protein assay (Pierce, Rockford, IL) and BSA as a standard Aggregation assays Fibril formation of WT and A53T mutant a-synuclein recombinant proteins was performed using a GENIOS multidetection microplate reader (TECAN) The aggregation 4031 PA700 interacts with a-synuclein oligomers reaction mixture consists of 280 lm of either WT or A53T recombinant a-synuclein proteins, 10 lm thioflavin T in buffer H (20 mm Tris ⁄ HCl pH 7.5; 20 mm NaCl; mm EDTA, mm 2-mercaptoethanol) and increasing concentrations of PA700 as indicated A total volume of 100 lL was aliquotted per well of a 96-well plate containing a Teflon sphere in each well The samples were incubated at 37 °C with orbital shaking A total of 97 fluorescence measurements were taken at 15-min intervals resulting in a 24-h incubation with excitation at 450 nm and emission at 485 nm Each experiment was performed in triplicate Measurements were corrected by subtracting the background fluorescence M Ghee et al and assembled WT and A53T a-synuclein and PA700 were extensively dialyzed against D2O The spectra of the soluble and fibrillized forms of the aforementioned samples were recorded on a JASCO 660 Plus FTIR spectrometer equipped with an MCT detector using attenuated total reflectance mode The background consisted of D2O A total of 1024 interferograms were collected with a resolution of cm)1 Second derivatives were calculated from smoothed primary spectra The data were fitted using a Gaussian species model centered at 1624, 1647, 1655, 1663, 1677 and 1692 cm)1 [28] Acknowledgements Size exclusion chromatography and sedimentation analysis WT and A53T mutant a-synuclein (280 lm) were incubated at 37 °C with orbital shaking for h in the absence and the presence of PA700 (218 nm) The different samples were loaded on a Superose HR10-30 gel filtration column (Amersham) equilibrated and run at °C in buffer H The column was eluted at a flow rate of 0.5 mLỈmin)1 The presence of PA700 and a-synuclein in the fractions (0.5 mL) emerging from the column was monitored using PA700 intrinsic fluorescence (excitation, 280 nm, emission 340 nm) and a-synuclein immunoreactivity using a dot-blot assay, respectively The column was calibrated with the molecular size markers (thyroglobulin, 670 kDa; immunoglobulin G, 158 kDa; ovalbumin, 44 kDa; myoglobin, 17 kDa and vitamin B-12, 1.35 kDa, Bio-Rad Laboratories, Inc., Hercules, CA, USA) Sedimentation analysis was carried out using a Beckman TL100 ultracentrifuge Aliquots (100 lL) of WT and A53T a-synuclein (280 lm) in the absence or the presence of PA700 (218 nm) were removed at time h (lag phase) and 18 h (steady state) from the reaction mixture incubated with orbital shaking at 37 °C and centrifuged for 20 at 160 000 g, 30 °C The resulting supernatants and pellets were analyzed by SDS ⁄ PAGE [27] Transmission electron microscopic analysis of a-synuclein filaments assembled in vitro from recombinant proteins Aliquots of soluble (280 lm) and assembled WT and A53T a-synuclein in the presence or absence of 218 nm PA700 were deposited on carbon-coated copper grids (200 mesh) The grids were then negatively stained with 1% uranyl acetate, and examined in a Philips EM 410 transmission electron microscope FTIR spectroscopy WT and A53T a-synuclein were assembled into fibrils in the presence or the absence of PA700 in buffer H Soluble 4032 We thank G DeMartino for the generous gift of PA700 and P Thomas and C Liu for their helpful discussions and critical review of the manuscript We thank R Jakes and M Goedert for the pRK172 wildtype a-synuclein plasmid This work was supported by the Fondation de France, Centre National de la Recherche Scientifique, the Association Francaise con´ tre les Myopathies, Universite Pierre et Marie Curie Paris VI and Aventis Pharma References Forno LS (1996) Neuropathology of Parkinson’s disease J Neuropathol Exp Neurol 55, 259–272 Greenamyre JT & Hastings TG (2004) Biomedicine Parkinson’s – divergent causes, convergent mechanisms Science 304, 1120–1122 Singleton AB, Farrer M, Johnson J, Singleton A, Hague S, Kachergus J, Hulihan M, Peuralinna T, Dutra A, Nussbaum R, et al (2003) alpha-Synuclein locus triplication causes Parkinson’s disease Science 302, 841 Uversky VN, Li J & Fink AL (2001) Evidence for a partially folded intermediate in alpha-synuclein fibril formation J Biol Chem 276, 10737–10744 Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, Pike B, Root H, Rubenstein J, Boyer R, et al (1997) Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease Science 276, 2045–2047 Kruger R, Kuhn W, Muller T, Woitalla D, Graeber M, Kosel S, Przuntek H, Epplen JT, Schols L & Riess O (1998) Ala30Pro mutation in the gene encoding alphasynuclein in Parkinson’s disease Nat Genet 18, 106–108 Giasson BI, Uryu K, Trojanowski JQ & Lee VM (1999) Mutant and wild type human alpha-synucleins assemble into elongated filaments with distinct morphologies in vitro J Biol Chem 274, 7619–7622 Narhi L, Wood SJ, Steavenson S, Jiang Y, Wu GM, Anafi D, Kaufman SA, Martin F, Sitney K, Denis P, FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS M Ghee et al 10 11 12 13 14 15 16 17 18 et al (1999) Both familial Parkinson’s disease mutations accelerate alpha-synuclein aggregation J Biol Chem 274, 9843–9846 Conway KA, Harper JD & Lansbury PT (1998) Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease Nat Med 4, 1318–1320 Zarranz JJ, Alegre J, Gomez-Esteban JC, Lezcano E, Ros R, Ampuero I, Vidal L, Hoenicka J, Rodriguez O, Atares B, et al (2004) The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia Ann Neurol 55, 164–173 Wood SJ, Wypych J, Steavenson S, Louis JC, Citron M & Biere AL (1999) alpha-synuclein fibrillogenesis is nucleation-dependent Implications for the pathogenesis of Parkinson’s disease J Biol Chem 274, 19509–19512 Volles MJ & Lansbury PT Jr (2003) Zeroing in on the pathogenic form of alpha-synuclein and its mechanism of neurotoxicity in Parkinson’s disease Biochemistry 42, 7871–7878 Conway KA, Rochet JC, Bieganski RM & Lansbury PT Jr (2001) Kinetic stabilization of the alpha-synuclein protofibril by a dopamine-alpha-synuclein adduct Science 294, 1346–1349 Voges D, Zwickl P & Baumeister W (1999) The 26S proteasome: a molecular machine designed for controlled proteolysis Annu Rev Biochem 68, 1015–1068 Hoyle J, Tan KH & Fisher EM (1997) Localization of genes encoding two human one-domain members of the AAA family: PSMC5 (the thyroid hormone receptorinteracting protein, TRIP1) and PSMC3 (the Tat-binding protein, TBP1) Hum Genet 99, 285–288 Braun BC, Glickman M, Kraft R, Dahlmann B, Kloetzel PM, Finley D & Schmidt M (1999) The base of the proteasome regulatory particle exhibits chaperone-like activity Nat Cell Biol 1, 221–226 Strickland E, Hakala K, Thomas PJ & DeMartino GN (2000) Recognition of misfolding proteins by PA700, the regulatory subcomplex of the 26 S proteasome J Biol Chem 275, 5565–5572 Tofaris GK, Layfield R & Spillantini MG (2001) alphasynuclein metabolism and aggregation is linked to ubi- FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS PA700 interacts with a-synuclein oligomers 19 20 21 22 23 24 25 26 27 28 quitin-independent degradation by the proteasome FEBS Lett 509, 22–26 Ghee M, Fournier A & Mallet J (2000) Rat alpha-synuclein interacts with Tat binding protein 1, a component of the 26S proteasomal complex J Neurochem 75, 2221–2224 Dedmon MM, Christodoulou J, Wilson MR & Dobson CM (2005) Hsp70 inhibits a-synuclein fibril formation via preferential binding to prefibrillar species J Biol Chem 280, 14733–14740 Bennett MC, Bishop JF, Leng Y, Chock PB, Chase TN & Mouradian MM (1999) Degradation of alpha-synuclein by proteasome J Biol Chem 274, 33855–33858 Webb JL, Ravikumar B, Atkins J, Skepper JN & Rubinsztein DC (2003) Alpha-Synuclein is degraded by both autophagy and the proteasome J Biol Chem 278, 25009–25013 Corn PG, McDonald ER, 3rd Herman JG & El-Deiry WS (2003) Tat-binding protein-1, a component of the 26S proteasome, contributes to the E3 ubiquitin ligase function of the von Hippel-Lindau protein Nat Genet 35, 229–237 Snyder H, Mensah K, Theisler C, Lee J, Matouschek A & Wolozin B (2003) Aggregated and monomeric alphasynuclein bind to the S6¢ proteasomal protein and inhibit proteasomal function J Biol Chem 278, 11753– 11759 Ma CP, Slaughter CA & DeMartino GN (1992) Identification, purification, and characterization of a protein activator (PA28) of the 20 S proteasome (macropain) J Biol Chem 267, 10515–10523 McGuire MJ, McCullough ML, Croall DE & DeMartino GN (1989) The high molecular weight multicatalytic proteinase, macropain, exists in a latent form in human erythrocytes Biochim Biophys Acta 995, 181–186 Laemmli U.K (1970) Cleavage of structural proteins during the assembly of the head of bacteriphage T4 Nature 227, 680–685 Seshadri S, Khurana R & Fink AL (1999) Fourier transform infrared spectroscopy in analysis of protein deposits Methods Enzymol 309, 559–576 4033 ... behavior of WT a-synuclein in the absence and the presence of PA700 Aliquots of WT a-synuclein assembly reactions in the absence (–) or the presence (+) of PA700, h and 18 h after the onset of the assembly. .. proteins The base consists of six ATPases that belong to the [15] The functions proposed for the ATPases include: (a) the hydrolysis of ATP to promote the assembly of the 26S proteasome from the PA700... In the absence of PA700, WT a-synuclein emerges from the column in a single peak centered at 300 kDa In the presence of PA700, WT a-synuclein emerges from the column with a molecular mass of