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Accelerated formation of α-synuclein oligomers by concerted action of the 20S proteasome and familial Parkinson mutations

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Abstract

A hallmark of Parkinson disease (PD) is the formation of intracellular protein inclusions called Lewy bodies that also contain mitochondria. α-Synuclein (αSyn) is a major protein component of Lewy bodies, where it is in an amyloid conformation and a significant fraction is truncated by poorly understood proteolytic events. Previously, we demonstrated that the 20S proteasome cleaves αSyn in vitro to produce fragments like those observed in Lewy bodies and that the fragments accelerate the formation of amyloid fibrils from full-length αSyn. Three point mutations in αSyn are associated with early-onset familial PD: A30P, E46K, and A53T. However, these mutations have very different effects on the amyloidogenicity and vesicle-binding activity of αSyn, suggesting neither of these processes directly correlate with neurodegeneration. Here, we evaluate the effect of the disease-associated mutations on the fragmentation, conformation, and association reactions of αSyn in the presence of the 20S proteasome and liposomes. The 20S proteasome produced the C-terminal fragments from both the mutant and wildtype αSyn. These truncations accelerated fibrillization of all α-synucleins, but again there was no clear correlation between the PD-associated mutations and amyloid formation in the presence of liposomes. Recent data suggests that cellular toxicity is caused by a soluble oligomeric species, which is a precursor to the amyloid form and is immunologically distinguishable from both soluble monomeric and amyloid forms of αSyn. Notably, the rate of formation of the soluble, presumptively cytotoxic oligomers correlated with the disease-associated mutations when both 20S proteasome and liposomes were present. Under these conditions, the wildtype protein was also cleaved and formed the oligomeric structures, albeit at a slower rate, suggesting that 20S-mediated truncation of αSyn may play a role in sporadic PD as well. Evaluation of the biochemical reactions of the PD-associated α-synuclein mutants in our in vitro system provides insight into the possible pathogenetic mechanism of both familial and sporadic PD.

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References

  • Alim MA, Ma QL, Takeda K, Aizawa T, Matsubara M, Nakamura M, Asada A, Saito T, Kaji H, Yoshii M et al (2004) Demonstration of a role for alpha-synuclein as a functional microtubule-associated protein. J Alzheimers Dis 6:435–442, discussion 443–439

    Google Scholar 

  • Amici M, Sagratini D, Pettinari A, Pucciarelli S, Angeletti M, Eleuteri AM (2004) 20S proteasome mediated degradation of DHFR: implications in neurodegenerative disorders. Arch Biochem Biophys 422:168–174

    Article  CAS  Google Scholar 

  • Baba M, Nakajo S, Tu PH, Tomita T, Nakaya K, Lee VM, Trojanowski JQ, Iwatsubo T (1998) Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson’s disease and dementia with Lewy bodies. Am J Pathol 152:879–884

    CAS  Google Scholar 

  • Bedford L, Hay D, Devoy A, Paine S, Powe DG, Seth R, Gray T, Topham I, Fone K, Rezvani N et al (2008) Depletion of 26S proteasomes in mouse brain neurons causes neurodegeneration and Lewy-like inclusions resembling human pale bodies. J Neurosci 28:8189–8198

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Bernado P, Bertoncini CW, Griesinger C, Zweckstetter M, Blackledge M (2005) Defining long-range order and local disorder in native alpha-synuclein using residual dipolar couplings. J Am Chem Soc 127:17968–17969

    Article  CAS  Google Scholar 

  • Brooks P, Fuertes G, Murray RZ, Bose S, Knecht E, Rechsteiner MC, Hendil KB, Tanaka K, Dyson J, Rivett J (2000) Subcellular localization of proteasomes and their regulatory complexes in mammalian cells. Biochem J 346(Pt 1):155–161

    Article  CAS  Google Scholar 

  • Campbell BC, McLean CA, Culvenor JG, Gai WP, Blumbergs PC, Jakala P, Beyreuther K, Masters CL, Li QX (2001) The solubility of alpha-synuclein in multiple system atrophy differs from that of dementia with Lewy bodies and Parkinson’s disease. J Neurochem 76:87–96

    Article  CAS  Google Scholar 

  • Chandra S, Chen X, Rizo J, Jahn R, Sudhof TC (2003) A broken alpha -helix in folded alpha -Synuclein. J Biol Chem 278:15313–15318

    Article  CAS  Google Scholar 

  • Chandra S, Gallardo G, Fernandez-Chacon R, Schluter OM, Sudhof TC (2005) Alpha-synuclein cooperates with CSPalpha in preventing neurodegeneration. Cell 123:383–396

    Article  CAS  Google Scholar 

  • Choi W, Zibaee S, Jakes R, Serpell LC, Davletov B, Crowther RA, Goedert M (2004) Mutation E46K increases phospholipid binding and assembly into filaments of human alpha-synuclein. FEBS Lett 576:363–368

    Article  CAS  Google Scholar 

  • Cole NB, Murphy DD, Grider T, Rueter S, Brasaemle D, Nussbaum RL (2002) Lipid droplet binding and oligomerization properties of the Parkinson’s disease protein alpha-synuclein. J Biol Chem 277:6344–6352

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Conway KA, Lee SJ, Rochet JC, Ding TT, Harper JD, Williamson RE, Lansbury PT Jr (2000a) Accelerated oligomerization by Parkinson’s disease linked alpha-synuclein mutants. Ann N Y Acad Sci 920:42–45

    Article  CAS  Google Scholar 

  • Conway KA, Lee SJ, Rochet JC, Ding TT, Williamson RE, Lansbury PT Jr (2000b) Acceleration of oligomerization, not fibrillization, is a shared property of both alpha-synuclein mutations linked to early-onset Parkinson’s disease: implications for pathogenesis and therapy. Proc Natl Acad Sci U S A 97:571–576

    Article  CAS  Google Scholar 

  • Daher JP, Ying M, Banerjee R, McDonald RS, Hahn MD, Yang L, Flint Beal M, Thomas B, Dawson VL, Dawson TM et al (2009) Conditional transgenic mice expressing C-terminally truncated human alpha-synuclein (alphaSyn119) exhibit reduced striatal dopamine without loss of nigrostriatal pathway dopaminergic neurons. Mol Neurodegener 4:34

    Article  CAS  Google Scholar 

  • Danzer KM, Krebs SK, Wolff M, Birk G, Hengerer B (2009) Seeding induced by alpha-synuclein oligomers provides evidence for spreading of alpha-synuclein pathology. J Neurochem.

  • David DC, Layfield R, Serpell L, Narain Y, Goedert M, Spillantini MG (2002) Proteasomal degradation of tau protein. J Neurochem 83:176–185

    Article  CAS  Google Scholar 

  • Davidson WS, Jonas A, Clayton DF, George JM (1998) Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes. J Biol Chem 273:9443–9449

    Article  CAS  Google Scholar 

  • Dedmon MM, Lindorff-Larsen K, Christodoulou J, Vendruscolo M, Dobson CM (2005a) Mapping long-range interactions in alpha-synuclein using spin-label NMR and ensemble molecular dynamics simulations. J Am Chem Soc 127:476–477

    Article  CAS  Google Scholar 

  • Dedmon MM, Lindorff-Larsen K, Christodoulou J, Vendruscolo M, Dobson CM (2005b) Mapping long-range interactions in alpha-synuclein using spin-label NMR and ensemble molecular dynamics simulations. J Am Chem Soc 127:476–477

    Article  CAS  Google Scholar 

  • Di Noto L, Whitson LJ, Cao X, Hart PJ, Levine RL (2005) Proteasomal degradation of mutant superoxide dismutases linked to amyotrophic lateral sclerosis. J Biol Chem 280:39907–39913

    Article  CAS  Google Scholar 

  • Duda JE, Giasson BI, Mabon ME, Lee VM, Trojanowski JQ (2002) Novel antibodies to synuclein show abundant striatal pathology in Lewy body diseases. Ann Neurol 52:205–210

    Article  CAS  Google Scholar 

  • Eliezer D, Kutluay E, Bussell R Jr, Browne G (2001) Conformational properties of alpha-synuclein in its free and lipid-associated states. J Mol Biol 307:1061–1073

    Article  CAS  Google Scholar 

  • Greenbaum EA, Graves CL, Mishizen-Eberz AJ, Lupoli MA, Lynch DR, Englander SW, Axelsen PH, Giasson BI (2005) The E46K mutation in alpha-synuclein increases amyloid fibril formation. J Biol Chem 280:7800–7807

    Article  CAS  Google Scholar 

  • Hague S, Rogaeva E, Hernandez D, Gulick C, Singleton A, Hanson M, Johnson J, Weiser R, Gallardo M, Ravina B et al (2003) Early-onset Parkinson’s disease caused by a compound heterozygous DJ-1 mutation. Ann Neurol 54:271–274

    Article  CAS  Google Scholar 

  • Hardy J, Cai H, Cookson MR, Gwinn-Hardy K, Singleton A (2006) Genetics of Parkinson’s disease and parkinsonism. Ann Neurol 60:389–398

    Article  CAS  Google Scholar 

  • Hoyer W, Cherny D, Subramaniam V, Jovin TM (2004) Impact of the acidic C-terminal region comprising amino acids 109–140 on alpha-synuclein aggregation in vitro. Biochemistry 43:16233–16242

    Article  CAS  Google Scholar 

  • Jensen PH, Nielsen MS, Jakes R, Dotti CG, Goedert M (1998) Binding of alpha-synuclein to brain vesicles is abolished by familial Parkinson’s disease mutation. J Biol Chem 273:26292–26294

    Article  CAS  Google Scholar 

  • Jo E, Fuller N, Rand RP, St George-Hyslop P, Fraser PE (2002) Defective membrane interactions of familial Parkinson’s disease mutant A30P alpha-synuclein. J Mol Biol 315:799–807

    Article  CAS  Google Scholar 

  • Jo E, Darabie AA, Han K, Tandon A, Fraser PE, McLaurin J (2004) Alpha-synuclein-synaptosomal membrane interactions: implications for fibrillogenesis. Eur J Biochem 271:3180–3189

    Article  CAS  Google Scholar 

  • Kahle PJ, Neumann M, Ozmen L, Muller V, Jacobsen H, Schindzielorz A, Okochi M, Leimer U, van Der Putten H, Probst A et al (2000) Subcellular localization of wild-type and Parkinson’s disease-associated mutant alpha -synuclein in human and transgenic mouse brain. J Neurosci 20:6365–6373

    CAS  Google Scholar 

  • Kayed R, Head E, Thompson JL, McIntire TM, Milton SC, Cotman CW, Glabe CG (2003) Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300:486–489

    Article  CAS  Google Scholar 

  • Kayed R, Sokolov Y, Edmonds B, McIntire TM, Milton SC, Hall JE, Glabe CG (2004) Permeabilization of lipid bilayers is a common conformation-dependent activity of soluble amyloid oligomers in protein misfolding diseases. J Biol Chem 279:46363–46366

    Article  CAS  Google Scholar 

  • Kayed R, Head E, Sarsoza F, Saing T, Cotman CW, Necula M, Margol L, Wu J, Breydo L, Thompson JL et al (2007) Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers. Mol Neurodegener 2:18

    Article  CAS  Google Scholar 

  • Kitada T, Asakawa S, Hattori N, Matsumine H, Yamamura Y, Minoshima S, Yokochi M, Mizuno Y, Shimizu N (1998) Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392:605–608

    Article  CAS  Google Scholar 

  • Klucken J, Ingelsson M, Shin Y, Irizarry MC, Hedley-Whyte ET, Frosch MP, Growdon JH, McLean PJ, Hyman BT (2006) Clinical and biochemical correlates of insoluble alpha-synuclein in dementia with Lewy bodies. Acta Neuropathol (Berl) 111:101–108

    Article  CAS  Google Scholar 

  • Kostka M, Hogen T, Danzer KM, Levin J, Habeck M, Wirth A, Wagner R, Glabe CG, Finger S, Heinzelmann U et al (2008) Single particle characterization of iron-induced pore-forming alpha-synuclein oligomers. J Biol Chem 283:10992–11003

    Article  CAS  Google Scholar 

  • 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 alpha-synuclein in Parkinson’s disease. Nat Genet 18:106–108

    Article  CAS  Google Scholar 

  • Lansbury PT Jr, Brice A (2002) Genetics of Parkinson’s disease and biochemical studies of implicated gene products. Curr Opin Cell Biol 14:653–660

    Article  CAS  Google Scholar 

  • Leroy E, Boyer R, Auburger G, Leube B, Ulm G, Mezey E, Harta G, Brownstein MJ, Jonnalagada S, Chernova T et al (1998) The ubiquitin pathway in Parkinson’s disease. Nature 395:451–452

    Article  CAS  Google Scholar 

  • Li J, Uversky VN, Fink AL (2001) Effect of familial Parkinson’s disease point mutations A30P and A53T on the structural properties, aggregation, and fibrillation of human alpha-synuclein. Biochemistry 40:11604–11613

    Article  CAS  Google Scholar 

  • Li J, Uversky VN, Fink AL (2002) Conformational behavior of human alpha-synuclein is modulated by familial Parkinson’s disease point mutations A30P and A53T. Neurotoxicology 23:553–567

    Article  CAS  Google Scholar 

  • Li W, Lesuisse C, Xu Y, Troncoso JC, Price DL, Lee MK (2004) Stabilization of alpha-synuclein protein with aging and familial parkinson’s disease-linked A53T mutation. J Neurosci 24:7400–7409

    Article  CAS  Google Scholar 

  • Li W, West N, Colla E, Pletnikova O, Troncoso JC, Marsh L, Dawson TM, Jakala P, Hartmann T, Price DL et al (2005) Aggregation promoting C-terminal truncation of alpha-synuclein is a normal cellular process and is enhanced by the familial Parkinson’s disease-linked mutations. Proc Natl Acad Sci U S A 102:2162–2167

    Article  CAS  Google Scholar 

  • Liu Y, Fallon L, Lashuel HA, Liu Z, Lansbury PT Jr (2002) The UCH-L1 gene encodes two opposing enzymatic activities that affect alpha-synuclein degradation and Parkinson’s disease susceptibility. Cell 111:209–218

    Article  CAS  Google Scholar 

  • Liu CW, Corboy MJ, DeMartino GN, Thomas PJ (2003) Endoproteolytic activity of the proteasome. Science 299:408–411

    Article  CAS  Google Scholar 

  • Liu CW, Giasson BI, Lewis KA, Lee VM, Demartino GN, Thomas PJ (2005) A precipitating role for truncated alpha-synuclein and the proteasome in alpha-synuclein aggregation: implications for pathogenesis of Parkinson disease. J Biol Chem 280:22670–22678

    Article  CAS  Google Scholar 

  • Liu CW, Li X, Thompson D, Wooding K, Chang TL, Tang Z, Yu H, Thomas PJ, DeMartino GN (2006) ATP binding and ATP hydrolysis play distinct roles in the function of 26S proteasome. Mol Cell 24:39–50

    Article  CAS  Google Scholar 

  • McGuire MJ, DeMartino GN (1986) Purification and characterization of a high molecular weight proteinase (macropain) from human erythrocytes. Biochim Biophys Acta 873:279–289

    CAS  Google Scholar 

  • Mishizen-Eberz AJ, Guttmann RP, Giasson BI, Day GA 3rd, Hodara R, Ischiropoulos H, Lee VM, Trojanowski JQ, Lynch DR (2003) Distinct cleavage patterns of normal and pathologic forms of alpha-synuclein by calpain I in vitro. J Neurochem 86:836–847

    Article  CAS  Google Scholar 

  • Murray IV, Giasson BI, Quinn SM, Koppaka V, Axelsen PH, Ischiropoulos H, Trojanowski JQ, Lee VM (2003) Role of alpha-synuclein carboxy-terminus on fibril formation in vitro. Biochemistry 42:8530–8540

    Article  CAS  Google Scholar 

  • Narhi L, Wood SJ, Steavenson S, Jiang Y, Wu GM, Anafi D, Kaufman SA, Martin F, Sitney K, Denis P et al (1999) Both familial Parkinson’s disease mutations accelerate alpha-synuclein aggregation. J Biol Chem 274:9843–9846

    Article  CAS  Google Scholar 

  • Necula M, Chirita CN, Kuret J (2003) Rapid anionic micelle-mediated alpha-synuclein fibrillization in vitro. J Biol Chem 278:46674–46680

    Article  CAS  Google Scholar 

  • Necula M, Kayed R, Milton S, Glabe CG (2007) Small molecule inhibitors of aggregation indicate that amyloid beta oligomerization and fibrillization pathways are independent and distinct. J Biol Chem 282:10311–10324

    Article  CAS  Google Scholar 

  • Nuscher B, Kamp F, Mehnert T, Odoy S, Haass C, Kahle PJ, Beyer K (2004) Alpha-synuclein has a high affinity for packing defects in a bilayer membrane: a thermodynamics study. J Biol Chem 279:21966–21975

    Article  CAS  Google Scholar 

  • Paisan-Ruiz C, Jain S, Evans EW, Gilks WP, Simon J, van der Brug M, Lopez de Munain A, Aparicio S, Gil AM, Khan N et al (2004) Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease. Neuron 44:595–600

    Article  CAS  Google Scholar 

  • Pawar AP, Dubay KF, Zurdo J, Chiti F, Vendruscolo M, Dobson CM (2005) Prediction of “aggregation-prone” and “aggregation-susceptible” regions in proteins associated with neurodegenerative diseases. J Mol Biol 350:379–392

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Shringarpure R, Grune T, Mehlhase J, Davies KJ (2003) Ubiquitin conjugation is not required for the degradation of oxidized proteins by proteasome. J Biol Chem 278:311–318

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Sokolov Y, Kozak JA, Kayed R, Chanturiya A, Glabe C, Hall JE (2006) Soluble amyloid oligomers increase bilayer conductance by altering dielectric structure. J Gen Physiol 128:637–647

    Article  CAS  Google Scholar 

  • Tanahashi N, Murakami Y, Minami Y, Shimbara N, Hendil KB, Tanaka K (2000) Hybrid proteasomes. Induction by interferon-gamma and contribution to ATP-dependent proteolysis. J Biol Chem 275:14336–14345

    Article  CAS  Google Scholar 

  • Tofaris GK, Layfield R, Spillantini MG (2001) alpha-synuclein metabolism and aggregation is linked to ubiquitin-independent degradation by the proteasome. FEBS Lett 509:22–26

    Article  CAS  Google Scholar 

  • Tofaris GK, Garcia Reitbock P, Humby T, Lambourne SL, O’Connell M, Ghetti B, Gossage H, Emson PC, Wilkinson LS, Goedert M et al (2006) Pathological changes in dopaminergic nerve cells of the substantia nigra and olfactory bulb in mice transgenic for truncated human alpha-synuclein(1–120): implications for Lewy body disorders. J Neurosci 26:3942–3950

    Article  CAS  Google Scholar 

  • Touitou R, Richardson J, Bose S, Nakanishi M, Rivett J, Allday MJ (2001) A degradation signal located in the C-terminus of p21WAF1/CIP1 is a binding site for the C8 alpha-subunit of the 20S proteasome. Embo J 20:2367–2375

    Article  CAS  Google Scholar 

  • Ulmer TS, Bax A, Cole NB, Nussbaum RL (2005) Structure and dynamics of micelle-bound human alpha-synuclein. J Biol Chem 280:9595–9603

    Article  CAS  Google Scholar 

  • van Duijn CM, Dekker MC, Bonifati V, Galjaard RJ, Houwing-Duistermaat JJ, Snijders PJ, Testers L, Breedveld GJ, Horstink M, Sandkuijl LA et al (2001) Park7, a novel locus for autosomal recessive early-onset parkinsonism, on chromosome 1p36. Am J Hum Genet 69:629–634

    Article  Google Scholar 

  • Wakamatsu M, Ishii A, Iwata S, Sakagami J, Ukai Y, Ono M, Kanbe D, Muramatsu SI, Kobayashi K, Iwatsubo T et al. (2006) Selective loss of nigral dopamine neurons induced by overexpression of truncated human alpha-synuclein in mice. Neurobiol Aging.

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

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  1. Karen A. Lewis

    Present address: Department of Chemistry and Biochemistry, University of Colorado at Boulder, UCB 215, Boulder, CO, 80309, USA

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  1. Department of Physiology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX, 75390-9040, USA

    Karen A. Lewis, Arynn Yaeger, George N. DeMartino & Philip J. Thomas

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Lewis, K.A., Yaeger, A., DeMartino, G.N. et al. Accelerated formation of α-synuclein oligomers by concerted action of the 20S proteasome and familial Parkinson mutations. J Bioenerg Biomembr 42, 85–95 (2010). https://doi.org/10.1007/s10863-009-9258-y

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