Proteasomal Inhibition by α-Synuclein Filaments and Oligomers
2004; Elsevier BV; Volume: 279; Issue: 13 Linguagem: Inglês
10.1074/jbc.m306390200
ISSN1083-351X
AutoresEvo Lindersson, Rasmus Beedholm, Peter Højrup, Torben Moos, Wei‐Ping Gai, Klavs B. Hendil, Poul Henning Jensen,
Tópico(s)Alzheimer's disease research and treatments
ResumoA unifying feature of many neurodegenerative disorders is the accumulation of polyubiquitinated protein inclusions in dystrophic neurons, e.g. containing α-synuclein, which is suggestive of an insufficient proteasomal activity. We demonstrate that α-synuclein and 20 S proteasome components co-localize in Lewy bodies and show that subunits from 20 S proteasome particles, in contrast to subunits of the 19 S regulatory complex, bind efficiently to aggregated filamentous but not monomeric α-synuclein. Proteasome binding to insoluble α-synuclein filaments and soluble α-synuclein oligomers results in marked inhibition of its chymotrypsin-like hydrolytic activity through a non-competitive mechanism that is mimicked by model amyloid-Aβ peptide aggregates. Endogenous ligands of aggregated α-synuclein like heat shock protein 70 and glyceraldehyde-6-phosphate dehydrogenase bind filaments and inhibit their anti-proteasomal activity. The inhibitory effect of amyloid aggregates may thus be amenable to modulation by endogenous chaperones and possibly accessible for therapeutic intervention. A unifying feature of many neurodegenerative disorders is the accumulation of polyubiquitinated protein inclusions in dystrophic neurons, e.g. containing α-synuclein, which is suggestive of an insufficient proteasomal activity. We demonstrate that α-synuclein and 20 S proteasome components co-localize in Lewy bodies and show that subunits from 20 S proteasome particles, in contrast to subunits of the 19 S regulatory complex, bind efficiently to aggregated filamentous but not monomeric α-synuclein. Proteasome binding to insoluble α-synuclein filaments and soluble α-synuclein oligomers results in marked inhibition of its chymotrypsin-like hydrolytic activity through a non-competitive mechanism that is mimicked by model amyloid-Aβ peptide aggregates. Endogenous ligands of aggregated α-synuclein like heat shock protein 70 and glyceraldehyde-6-phosphate dehydrogenase bind filaments and inhibit their anti-proteasomal activity. The inhibitory effect of amyloid aggregates may thus be amenable to modulation by endogenous chaperones and possibly accessible for therapeutic intervention. The development of polyubiquitin-containing intracellular inclusions in cell bodies and dystrophic neurites of nerve cells is a key feature of several neurodegenerative disorders, e.g. in Alzheimer's disease, Parkinson's disease (PD), 1The abbreviations used are: PD, Parkinson's disease; AS, α-synuclein; DLB, dementia with Lewy bodies; GAPDH, glyceraldehyde-6-phosphate dehydrogenase; HSP70, heat shock protein-70; PBS, phosphate-buffered saline; SNpc, substantia nigra pars compacta; TBS, Tris-buffered saline; Z, benzyloxycarbonyl; MES, 4-morpholineethanesulfonic acid. dementia with Lewy bodies (DLB), and Huntington's disease (1Ellison D. Love S. Ellison D. Love S. Neuropathology. Mosby International Ltd., London, UK1998: 28.1-28.6Google Scholar). The polyubiquitination of proteins represents the result of the cellular system that recognizes misfolded or unwanted proteins and tags them for degradation by the proteasome via the sequential action of three enzymes (for a recent review on the ubiquitin-proteasomal system, see Ref. 2Glickman M.H. Ciechanover A. Physiol. Rev. 2001; 82: 373-428Crossref Scopus (3405) Google Scholar). First, the ubiquitin-activating enzyme activates ubiquitin in an ATP-dependent process, after which the activated ubiquitin is passed onto the ubiquitin carrier protein class of ubiquitin conjugates. Then ubiquitin-protein isopeptide ligases recognize the misfolded proteins and mediate covalent binding of ubiquitin to them. Normally, polyubiquitinated proteins are recognized by the 19 S regulatory complex of the 26 S proteasome that unfolds the substrates and feeds them to the 20 S proteolytic core particle of the 26 S proteasome. The proteasome comprises three hydrolytic activities: the trypsin-like, the chymotrypsin-like, and the caspase-like hydrolytic activities that combined ensure the complete degradation of substrates. Accumulation of ubiquitinated proteins is an indicator for an imbalance between the production and ubiquitin labeling of misfolded proteins and the catabolic capacity of the proteasome. A group of neurodegenerative disorders, e.g. PD and DLB, is characterized by the filamentous Lewy body type of cytoplasmic inclusions in the degenerating cells (1Ellison D. Love S. Ellison D. Love S. Neuropathology. Mosby International Ltd., London, UK1998: 28.1-28.6Google Scholar). Aggregates of misfolded α-synuclein (AS) form the filamentous core of the Lewy bodies and Lewy neurites (3Goedert M. 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Accordingly, AS aggregates bear the characteristics of amyloid-type filaments accumulating in other diseases, e.g. formed from tau peptides in Alzheimer's disease and huntingtin in Huntington's disease (3Goedert M. Spillantini M.G. Davies S.W. Curr. Opin. Neurobiol. 1998; 8: 619-632Crossref PubMed Scopus (229) Google Scholar). The employment of animal models corroborates the involvement of aggregated AS in the neurodegenerative process where overexpression of AS leads to abnormal AS accumulation, misfolding, neuronal dysfunction, and overt degeneration (10Kahle P.J. Neumann M. Ozmen L. Muller V. Odoy S. Okamoto N. Jacobsen H. Iwatsubo T. Trojanowski J.Q. Takahashi H. Wakabayashi K. Bogdanovic N. Riederer P. Kretzschmar H.A. Haass C. Am. J. Pathol. 2001; 159: 2215-2225Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 14Masliah E. Rockenstein E. Veinbergs I. Mallory M. Hashimoto M. Takeda A. Sagara Y. Sisk A. Mucke L. Science. 2000; 287: 1265-1269Crossref PubMed Scopus (1577) Google Scholar, 15Feany M.B. Bender W.W. Nature. 2000; 404: 394-398Crossref PubMed Scopus (1740) Google Scholar). A compromised proteasomal function in the substantia nigra in PD has been reported (16McNaught K.S. Jenner P. Neurosci. Lett. 2001; 297: 191-194Crossref PubMed Scopus (560) Google Scholar, 17McNaught K.S. Olanow C.W. Halliwell B. Isacson O. Jenner P. Nat. Rev. Neurosci. 2001; 2: 589-594Crossref PubMed Scopus (458) Google Scholar, 18McNaught K.S. Belizaire R. Jenner P. Olanow C.W. Isacson O. Neurosci. Lett. 2002; 326: 155-158Crossref PubMed Scopus (203) Google Scholar, 19McNaught K.S. Belizaire R. Isacson O. Jenner P. Olanow C.W. Exp. Neurol. 2003; 179: 38-46Crossref PubMed Scopus (512) Google Scholar, 20Tofaris G.K. Razzaq A. Ghetti B. Lilley K. Spillantini M.G. J. Biol. 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Chem. 2003; 278: 11753-11759Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Such a proteasomal inhibition may have an impact on the dopaminergic neurons that seem to be particularly vulnerable to the stress of unfolded proteins (23Petrucelli L. O'Farrell C. Lockhart P.J. Baptista M. Kehoe K. Vink L. Choi P. Wolozin B. Farrer M. Hardy J. Cookson M.R. Neuron. 2002; 36: 1007-1019Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar). We demonstrate that AS, ubiquitin, and 20 S proteasomal components co-localize in Lewy bodies, and we show that aggregated but not monomeric AS binds efficiently to the 20 S proteasome part of the 26 S proteasome. The proteasome binding results in an efficient and selective non-competitive inhibition of the chymotrypsin-like proteasomal activity of the 20 S proteolytic particle. This inhibition, which is mimicked by Aβ amyloid filaments, is reverted by the amyloid targeting substances thioflavin S and Congo Red. Moreover, heat shock protein 70 (HSP70) that protects neurons toward AS-mediated toxicity in transgenic models (25Auluck P.K. Chan H.Y. Trojanowski J.Q. Lee V.M. Bonini N.M. Science. 2002; 295: 865-868Crossref PubMed Scopus (1080) Google Scholar) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) bind to the AS filaments and abrogate their anti-proteasomal activity. Finally, subjecting cells to heat shock renders their cytosolic proteasomes less sensitive to the toxicity of aggregated AS thus demonstrating a potential for modulation of this toxicity by endogenous factors. The proteasomal inhibition by aggregated AS may represent a common neurodegenerative mechanism in diseases like PD, DLB, and multiple system atrophy where the degenerating cells accumulate AS aggregates. Abolition of the inhibition may open for new neuroprotective strategies. Miscellaneous and Proteins—All chemicals were of analytical grade if not otherwise stated. Thioflavin S and Congo Red were from Sigma. Human 20 S proteasome was isolated from human erythrocytes by affinity chromatography on immobilized MCP21 monoclonal antibody (26Hendil K.B. Uerkvitz W. J. Biochem. Biophys. Methods. 1991; 22: 155-165Crossref Scopus (41) Google Scholar). The specific activities of the 20 S proteasome are as follows: chymotrypsin-like activity, 4.0 nmol/min/mg protein using succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin; trypsin-like activity, 5.1 nmol/min/mg protein using Z-Ala-Ala-Arg-7-amido-4-methylcoumarine; caspase-like activity, 1.9 nmol/min/mg protein using Z-Leu-Leu-Glu-β-naphthylamide. The Aβ-(1-40) peptide was from (Schaefer-N., Copenhagen, Denmark). Recombinant human HSP70 was from Stressgen, (Victoria, British Columbia, Canada), and GAPDH was from Sigma. Both GAPDH and HSP70 were centrifuged for 30 min at 120,000 rpm at 4 °C in a TLA 120.1 rotor in a Optima TLX centrifuge (Beckman Instruments) to remove any aggregated materials prior to pull-down experiments. Recombinant human full-length AS-(1-140), C-terminally truncated AS-(1-95), and β-synuclein were expressed in Escherichia coli and purified as described previously (27Jensen P.H. Højrup P. Hager H. Nielsen M.S. Jakobsen L. Olesen O.F. Gliemann J. Jakes R. Biochem. J. 1997; 323: 539-546Crossref PubMed Scopus (139) Google Scholar, 28Jensen P.H. Islam K. Kenney J. Nielsen M.S. Power J. Gai W.P. J. Biol. Chem. 2000; 275: 21500-21507Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), followed by an additional reverse phase-high pressure liquid chromatography purification step on a Jupiter C18 column (Phenomenex) in 0.1% trifluoroacetic acid with an acetonitrile gradient. The proteins were subsequently aliquoted, lyophilized, and stored at -80 °C. Primary Antibodies—Sheep anti-AS was affinity-purified as described (29Gai W.P. Power J.H.T. Blumbergs P.C. Jensen P.H. J. Neurochem. 1999; 73: 2093-2100PubMed Google Scholar) and obtained from Abcam, UK. Rabbit ASY-1 IgG was raised against full-length human recombinant AS (28Jensen P.H. Islam K. Kenney J. Nielsen M.S. Power J. Gai W.P. J. Biol. Chem. 2000; 275: 21500-21507Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Rabbit FILA-1 IgG, specific for aggregated AS, was prepared by immunizing rabbits with sucrose density gradient-purified AS-(1-140) filaments. The immune serum was subsequently passed several times through a column with immobilized AS. This ensured the removal of all detectable immunoreactivity toward monomeric AS as determined by dot blotting. The serum lacking the monomer binding antibodies was then incubated with density gradient purified AS filaments, and the aggregate-binding antibodies were isolated by gradient co-sedimentation with the filaments. The antibodies were subsequently eluted from the filaments by incubation in 100 mm glycine, pH 2.5, followed by gradient centrifugation to separate the insoluble AS filaments from the FILA-1 IgG. The IgG was finally concentrated by protein A chromatography. The monoclonal antibodies, used to detect 20 S proteasome subunits, are as follows: MCP72 (α7 subunit); MCP196 (α5 subunit); MCP20 (α6 subunit); MCP231 (α2, α3, α6, α7 subunits); MCP421 (β1 subunit) (30Kristensen P. Johnsen A.H. Uerkvitz W. Tanaka K. Hendil K.B. Biochem. Biophys. Res. Commun. 1995; 207: 1059Crossref PubMed Scopus (1) Google Scholar). The monoclonal antibodies to subunits of the 19 S regulatory proteasome complex are as follows: p31-38 (S14/Rpn12 subunit); S5a-18 (S5a/Rpn10 subunit); TBP1-19 (S6′/Rpt5 subunit) (31Hendil K.B. Hartmann-Petersen R. Tanaka K. J. Mol. Biol. 2002; 315: 627-636Crossref PubMed Scopus (53) Google Scholar). A rabbit polyclonal antibody to S6′ (Affinity Bioreagents) was also used as were affinity-purified rabbit anti-ubiquitin (Dako, Denmark) and monoclonal antibodies against HSP70 (clone BRM-22) and β-actin (both from Sigma). Immunohistochemistry—Samples of substantia nigra from sporadic PD (n = 4), DLB (n = 3), and control subjects (n = 4) were obtained from the Netherlands Brain Bank (Amsterdam, The Netherlands) and fixed in 10% neutral buffered formalin and embedded in paraffin. Control samples were obtained among patients without neurological disease and with no pathological changes in sections of the substantia nigra pars compacta (SNpc). The diagnosis of PD in the human autopsies was based on loss of neuromelanin pigment and the excessive number of Lewy bodies in the remaining SNpc neurons in the eosin-stained section. The paraffin sections were cut at 2 μm and subjected to immunohistochemistry as described previously (32Mirza B. Hadberg H. Thomsen P. Moos T. Neuroscience. 2000; 95: 425-432Crossref PubMed Scopus (278) Google Scholar). Complementary sections were demelanized using successive incubations with 0.25% potassium permanganate for 20 min followed by 1% oxalic acid and 1% potassium bisulfite for 4 min (33Leveugle B. Faucheux B.A. Bouras C. Nillesse N. Spik G. Hirsch E.C. Agid Y. Hof P.R. Acta Neuropathol. 1996; 91: 566-572Crossref PubMed Scopus (109) Google Scholar). The sections were incubated overnight at 4 °C with polyclonal rabbit anti-human ubiquitin diluted 1:500 (Dako, DK), sheep anti-human AS diluted 1:200 (Abcam, UK), or anti-human 20 S proteasome antibody MCP20. The sections were developed using 3,3-diaminobenzidine tetrahydrochloride as chromogen. To examine the extent of nonspecific binding, non-immune sera were substituted for the primary antibody. The relative ratio between the presence of AS and 20 S proteasome in Lewy bodies was estimated by staining four pairs of sections per autopsy for AS and 20 S proteasome, which were counted for their content of positive Lewy bodies. Immunolabeling of Isolated Lewy Bodies—Immunohistochemical staining was conducted in isolated Lewy bodies from fresh frozen brains of patients with DLB. The cingulate cortex from five DLB cases were analyzed. The isolation procedure was modified from Refs. 28Jensen P.H. Islam K. Kenney J. Nielsen M.S. Power J. Gai W.P. J. Biol. Chem. 2000; 275: 21500-21507Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 29Gai W.P. Power J.H.T. Blumbergs P.C. Jensen P.H. J. Neurochem. 1999; 73: 2093-2100PubMed Google Scholar, and 34Gai W.P. Yuan H.X. Li X.Q. Power J.T.H. Blumbergs P.C. Jensen P.H. Exp. Neurol. 2000; 166: 324-333Crossref PubMed Scopus (220) Google Scholar with the Lewy body smears prepared by washing the Lewy body enriched fraction three times in Tris-buffered saline (TBS, 0.1 m Tris-HCl, 0.9% NaCl, 5 mm EDTA, and protease inhibitors) followed by smearing on gelatin-coated glass slides and air-drying for 2 h at room temperature. The smears were fixed in 4% formaldehyde in TBS for 10 min and then incubated with 3% H2O2 in 50% methanol/TBS for 10 min to bleach endogenous peroxidase activity. Following three 5-min rinse in TBS, the smears were blocked with 20% normal horse serum for 30 min and then incubated for 60 min in primary antibody solution containing monoclonal antibody MCP72 (1:300) or MCP196 (1:300). To facilitate recognizing Lewy bodies, the primary antibody solution also included sheep anti-AS (1:300) or anti-ubiquitin (1:300). The smears were rinsed three times in TBS and then labeled for 60 min with Cy2-conjugated donkey anti-mouse IgG, in combination with Cy3-conjugated donkey anti-sheep IgG or Cy3-conjugated anti-rabbit IgG (all used 1:100 dilution, all from Jackson ImmunoResearch). The primary and secondary antibody dilutions used above were predetermined by serial titrations before formal experiments. Controls for antibody specificity included omitting primary or secondary antibodies and preabsorption the primary antibody with ubiquitin or AS. Lewy body staining was not detected in these control experiments. The smears were examined using a Bio-Rad confocal laser scanning microscope and software package (Bio-Rad MRC 1024) (34Gai W.P. Yuan H.X. Li X.Q. Power J.T.H. Blumbergs P.C. Jensen P.H. Exp. Neurol. 2000; 166: 324-333Crossref PubMed Scopus (220) Google Scholar) with the scanned image representing an ∼1-μm thick slice through the center or equatorial plane of the Lewy body. Preparation of AS Aggregates—AS aggregates were made by resuspending lyophilized AS in 20 mm Tris, 150 mm NaCl, pH 7.5, 0.02% NaN3 at 7 mg/ml followed by ultracentrifugation to remove insoluble aggregates (28Jensen P.H. Islam K. Kenney J. Nielsen M.S. Power J. Gai W.P. J. Biol. Chem. 2000; 275: 21500-21507Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Incubation of β-synuclein (5.5 mg/ml) and Aβ-(1-40) peptide (4 mg/ml) was performed by analogous procedures. The supernatant was incubated at 37 °C on a shaker for ∼14 days. Insoluble AS aggregates were isolated for proteasome activity assays and electron microscopy by sedimentation through a 40% sucrose cushion (28Jensen P.H. Islam K. Kenney J. Nielsen M.S. Power J. Gai W.P. J. Biol. Chem. 2000; 275: 21500-21507Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) followed by resuspension in the buffer of choice. Soluble AS aggregates were isolated by gel filtration of the soluble supernatant, obtained after ultracentrifugation of the aggregated AS sample, on a 24 × 1-cm Bio-Gel A 1.5M column (Bio-Rad) in 150 mm NaCl, 10 mm NaH2PO4, pH 7.4 (PBS), at 0.5 ml/min. The buffer was changed to 1 m Hepes, pH 8.0, when isolating soluble oligomers for the proteasome activity assay. The elution of the aggregated oligomers was determined by dot-blotting of the eluted fractions using the FILA-1 antibody, and the total AS was monitored by probing the membrane with the ASY-1 antibody. Electron Microscopy—Electron microscopy was used to analyze the insoluble AS filaments and to visualize the binding of purified 20 S proteasomes to the filaments. Filaments isolated from 30 μl of aggregated AS (7 mg/ml) were resuspended in 30 μl of PBS supplemented 20 mm NaN3 after pelleting through the sucrose cushion as described above. Purified 20 S proteasome was centrifuged for 10 min at 100,000 rpm at 4 °C in a TLA 120.1 rotor in an Optima TLX centrifuge (Beckman Instruments) to remove aggregated materials. Soluble 20 S proteasome (40 μl at 0.18 mg/ml) was incubated together with 5 μl of resuspended AS filaments (5 mg/ml) for 2 h at 25 °C. Non-bound proteasome was removed by sucrose gradient centrifugation of the filaments with bound 20 S proteasome. The sedimented filaments with attached 20 S proteasome was resuspended in 70 μl of distilled water. Filaments not incubated with 20 S proteasomes before the centrifugation and soluble 20 S proteasomes diluted to 0.16 mg/ml in PBS before direct application to the grid were used as control. All samples were pipetted onto carbon-coated nickel grids (3 μl for each grid) and allowed to stand for 2 min. The samples were stained with 1% aqueous uranyl acetate for 1 min. The grids were then air-dried and examined in a Morgagni 268, 80-kV electron microscope, and photographs were taken at 14,000, 18,000, or 22,000 times magnification. For immunogold labeling, grids with samples were blocked with blocking buffer (PBS with 0.05 m glycine and 0.1% milk protein) for 10 min and incubated with the primary antibody MCP72 (0.07 mg/ml) for 1 h. The grids were then washed three times for 5 min with blocking buffer and then incubated with goat anti-rabbit IgG conjugated to 5-nm diameter gold particles (Amersham Biosciences) diluted 1:100 in PBS with 1% fish gelatin, 0.06% polyethylene glycol, and 0.1% milk protein for 1 h. The grids were then washed twice for 5 min in PBS with 0.1% milk protein and then twice for 5 min in water. The grids were finally stained and examined as the non-immunolabeled samples. Filament Pull-down Assay—Cellular cytosol was prepared from primary cultures of normal human skin fibroblasts (35Rattan S.I. Biochem. Mol. Biol. Int. 1998; 45: 753-759PubMed Google Scholar) and from rat brain (36Jensen P.H. Nielsen M.S. Jakes R. Dotti C.G. Goedert M. J. Biol. Chem. 1998; 273: 26292-26294Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). Cells, at confluence, were trypsinized and collected by centrifugation (1000 × g, 10 min, 4 °C). The cells were sonicated in 50 mm NaCl, 10 mm Hepes, pH 8, 0.5 m sucrose, 1 mm EDTA, 0.2% (v/v) Triton X-100, 0.2 mm phenylmethanesulfonyl fluoride, 0.05% β-mercaptoethanol (5 ml for three 75-cm2 flasks) on ice. The supernatant was collected after centrifugation at 108,000 × g for 30 min at 4 °C giving a cytosol of 1 mg of protein/ml. To demonstrate the binding to AS filaments, 250 μl of cytosol were incubated with 70 μg of AS filaments for 1.5 h at 37 °C; 30 μg of GAPDH were incubated with 9 μg of AS filaments for 16 h at 4 °C; HSP70 (1.2 μg) were incubated with 21 μg of AS filaments for 2 h at 25 °C; purified 20 S (5.4 μg) were incubated with 70 μg of AS filaments for 16 h at 4 °C. All volumes were adjusted to 300 μl with 20 mm Tris and 150 mm NaCl, pH 7.4, except for the HSP70, where the buffer was 50 mm Tris, 100 mm NaCl, 1 mm dithioerythritol and 0.1 mm phenylmethanesulfonyl fluoride, pH 7.2. As negative controls cytosol, GAPDH, HSP70, 20 S proteasomes, or filaments were treated as above but without mixing the individual proteins. As positive controls, cytosol, GAPDH, HSP70, 20 S proteasome, or the filaments were loaded directly on the SDS-PAGE. Excess of purified monomeric AS was supplemented to the ligand-filament samples to determine whether this would inhibit the association of the ligands to the filaments or the interaction displayed a selectivity for the filaments as demonstrated by an unchanged amount of ligand in the pellet. The filament pull-down assay was performed by placing the protein samples on a cushion of 1.6 ml of 40% sucrose, 13 mm MES, and 1 mm EDTA, pH 7.0, followed by centrifugation at 55,000 rpm for 30 min in a TLS 55 swing out rotor at 4 °C in a Optima TLX centrifuge (Beckman Instruments). The pellets were either recovered for the proteasome assay by resuspension in 1 m Hepes, pH 8.0, or solubilized in 30 μl of 8 m urea with 4% SDS for 16 h in 37 °C. The latter was used to ensure complete depolymerization of the filaments. Non-bound proteins, remaining in the supernatant, were analyzed by the precipitation of 500 μl of the supernatant in trichloroacetic acid. The solubilized pellets and the precipitated supernatants were dissolved in dithioerythritol-containing SDS-PAGE loading buffer, heated 95 °C for 3 min, and subjected to SDS-PAGE followed by analysis with staining of the gel by silver or Coomassie Blue or by immunoblotting (28Jensen P.H. Islam K. Kenney J. Nielsen M.S. Power J. Gai W.P. J. Biol. Chem. 2000; 275: 21500-21507Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Proteasome Assay—Approximately 80% confluent fibroblasts were scraped into 100 mm Tris-HCl buffer, pH 7.5 (1 ml per 75-cm2 flask). The cells were placed on ice for 15 min to allow lysis. The cytosolic extract was subsequently harvested as the supernatant after a centrifugation at 11,500 × g for 10 min at 4 °C. For the proteasome assay, the cytosol was used at a concentration of 100 μg of protein/ml and the purified human erythrocyte 20 S proteasome at a concentration of 2 μg of protein/ml. The hydrolytic activity for the chymotrypsin-like, trypsin-like, and caspase-like hydrolytic activities were determined as described previously (37Fonager J. Beedholm R. Clark B.F. Rattan S.I. Exp. Gerontol. 2002; 37: 1223-1228Crossref PubMed Scopus (93) Google Scholar). The coefficient of variation for the proteasome assay ranged from 0.8 to 6.3% in six independent experiments with the mean being 3%. Proteasome enzyme activities were calculated as the difference in activity measured in the absence and in the presence of the proteasome inhibitor, lactacystin, and in some experiments the peptide aldehyde Z-Leu-Leu-leucinal (MG132), both being potent inhibitors of primarily of the chymotrypsin-like activity. All the activity measurements were done in duplicate or triplicate from independent samples, as stated in the legends. Fluorescence of cleavage products from peptide substrate was measured on a Kontron SFM 25 spectrofluorometer. The effect of aggregated and non-aggregated AS on the proteasomal activity was determined by incubating proteasomes with AS for 60 min at 37 °C prior to the addition of the fluorogenic substrate. Localization of 20 S Components in Lewy Bodies—Lewy bodies denote the characteristic inclusion bodies of PD and DLB (38McKeith I.G. Galasko D. Kosaka K. Perry E.K. Dickson D.W. Hansen L.A. Salmon D.P. Lowe J. Mirra S.S. Byrne E.J. Lennox G. Quinn N.P. Edwardson J.A. Ince P.G. Bergeron C. Burns A. Miller B.L. Lovestone S. Collerton D. Jansen E.N. Ballard C. de Vos R.A. Wilcock G.K. Jellinger K.A. Perry R.H. Neurology. 1996; 5: 1113-1124Crossref Scopus (3661) Google Scholar); they contain abundant ubiquitinated material indicative of an insufficient proteasomal function (Fig. 1A). AS in a filamentous form comprise a main component of Lewy bodies (Fig. 1B) (39Spillantini M.G. Schmidt M.L. Lee V.M. Trojanowski J.Q. Jakes R. Goedert M. Nature. 1997; 388: 839-840Crossref PubMed Scopus (6342) Google Scholar). 20 S proteasomes were observed in the cytoplasm of both control and brain disease cases, and the α6 subunit-specific antibody MCP20 also labeled Lewy bodies of both PD and DLB cases (Fig. 1C). Labeling of Lewy bodies by the anti-ubiquitin, anti-AS, and anti-proteasome 20 S antibodies was mainly confined to their peripheral zone (Fig. 1, A-C). Immunolabeling was not observed in nigral neurons when the primary antibody was omitted from the immunoreaction (Fig. 1D). The number of stained Lewy bodies was lower with 20 S proteasome antibodies than with AS. We also used
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