title: PA700, the regulatory complex of the 26S proteasome, interferes with alpha-synuclein assembly.
abstract: 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 alpha-synuclein forms the major component of Lewy bodies. We reported a specific interaction between rat alpha-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 wild-type and A53T mutant alpha-synuclein. PA700 inhibits both wild-type and A53T alpha-synuclein fibril formation as measured by Thioflavin T fluorescence. Using size exclusion chromatography, we present evidence for a stable PA700-alpha-synuclein complex. Sedimentation analyses reveal that PA700 sequesters alpha-synuclein in an assembly incompetent form. Analysis of the morphology of wild-type and A53T alpha-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 alpha-synuclein assembled in the presence of PA700 revealed a decrease in the overall amount of assembled alpha-synuclein with no significant change in protein conformation. Thus, PA700 acts on alpha-synuclein assembly and not on the structure of fibrils. We hypothesize that PA700 sequesters alpha-synuclein oligomeric species that are the precursors of the fibrillar form of the protein, thus preventing its assembly into fibrils.
authors.affiliation: Laboratoire de Génétique Moléculaire de la Neurotransmission et des Processus Neurodégénératifs, Centre National de la Recherche Scientifique, Hôpital de la Pitié Salpêtrière, Paris, France. mghee@infobiogen.fr
authors.forename: Medeva
authors.initials: M
authors.lastname: Ghee
authors.forename: Ronald
authors.initials: R
authors.lastname: Melki
authors.forename: Nadine
authors.initials: N
authors.lastname: Michot
authors.forename: Jacques
authors.initials: J
authors.lastname: Mallet
mesh_terms: D001704:Biopolymers; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046529:Microscopy, Electron, Transmission; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D051843:Synucleins; D051844:alpha-Synuclein
chemical_list: D001704:Biopolymers; D009419:Nerve Tissue Proteins; C085834:PA700 proteasome activator; D011994:Recombinant Proteins; D051843:Synucleins; D051844:alpha-Synuclein; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease