A cellular model to monitor proteasome dysfunction by alpha-synuclein.
Impairment of the ubiquitin-proteasome degradation system has recently been suggested to be related to the onset of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. In this study, we investigated whether intracellular alpha-synuclein affects proteasome activity in SH-SY5Y cells. To monitor intracellular proteasome activity, we used a reporter consisting of a short peptide degron fused to the carboxyl-terminus of green fluorescent protein (GFP-CL1), which is known to be degraded by proteasome. The level of intact GFP-CL1 was dramatically increased by coexpression of GFP-CL1 and alpha-synuclein, as judged by confocal microscopic and immunoblot analyses. Expression of two pathogenic mutants of alpha-synuclein, A30P and A53T, and phosphomimetic S129D mutant increased the intensities of GFP more effectively than did wild-type alpha-synuclein. GFP fluorescence in cells transfected with Delta73-83 mutant or beta-synuclein, which does not assemble into filaments in vitro, was not changed as compared with that in cells expressing GFP-CL1 alone. Thus, the ability of alpha-synuclein to inhibit proteasome activity is related to its propensity to assemble into filaments. Furthermore, we observed that some compounds inhibiting alpha-synuclein filament formation in vitro prevented the alpha-synuclein-mediated proteasome dysfunction in cells transfected with both GFP-CL1 and alpha-synuclein. The cellular model expressing both GFP-CL1 and alpha-synuclein may be a useful tool to screen compounds protecting neurons from alpha-synuclein-mediated proteasome dysfunction.
Nonaka
Takashi
T
Department of Molecular Neurobiology, Tokyo Institute of Psychiatry, Tokyo Metropolitan Organization for Medical Research, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156-8585. nonakat@prit.go.jp
Hasegawa
Masato
M
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D005419:Flavonoids / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010636:Phenols / Q000494:pharmacology; D059808:Polyphenols; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism / Q000502:physiology*; D061988:Proteasome Inhibitors; D017384:Sequence Deletion; D054875:Ubiquitination / Q000235:genetics; D051844:alpha-Synuclein / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*
D005419:Flavonoids; D010636:Phenols; D059808:Polyphenols; D011480:Protease Inhibitors; D061988:Proteasome Inhibitors; D051844:alpha-Synuclein; D049452:Green Fluorescent Proteins; D046988:Proteasome Endopeptidase Complex