Synphilin-1 degradation by the ubiquitin-proteasome pathway and effects on cell survival.
Parkinson's disease is characterized by loss of nigral dopaminergic neurons and the presence of cytoplasmic inclusions known as Lewy bodies. alpha-Synuclein and its interacting partner synphilin-1 are among constituent proteins in these aggregates. The presence of ubiquitin and proteasome subunits in these inclusions supports a role for this protein degradation pathway in the processing of proteins involved in this disease. To begin elucidating the kinetics of synphilin-1 in cells, we studied its degradation pathway in HEK293 cells that had been engineered to stably express FLAG-tagged synphilin-1. Pulse-chase experiments revealed that this protein is relatively stable with a half-life of about 16 h. Treatment with proteasome inhibitors resulted in attenuation of degradation and the accumulation of high molecular weight ubiquitinated synphilin-1 in immunoprecipitation/immunoblot experiments. Additionally, proteasome inhibitors stimulated the formation of peri-nuclear inclusions which were immunoreactive for synphilin-1, ubiquitin and alpha-synuclein. Cell viability studies revealed increased susceptibility of synphilin-1 over-expressing cells to proteasomal dysfunction. These observations indicate that synphilin-1 is ubiquitinated and degraded by the proteasome. Accumulation of ubiquitinated synphilin-1 due to impaired clearance results in its aggregation as peri-nuclear inclusions and in poor cell survival.
Lee
Gwang
G
Genetic Pharmacology Unit, Experimental Therapeutics Branch, NINDS, National Institutes of Health, Bethesda, Maryland 20892, USA.
Junn
Eunsung
E
Tanaka
Mikiei
M
Kim
Yong Man
YM
Mouradian
M Maral
MM
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007668:Kidney / Q000737:chemistry / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D046911:Macromolecular Substances; D051379:Mice; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009447:Neuroblastoma / Q000378:metabolism; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology*; D051843:Synucleins; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*; D051844:alpha-Synuclein
D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D007976:Leupeptins; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D009419:Nerve Tissue Proteins; C497604:SNCA protein, human; C118958:SNCAIP protein, human; C497605:Snca protein, mouse; C498464:Sncaip protein, mouse; D051843:Synucleins; D025801:Ubiquitin; D051844:alpha-Synuclein; C067713:lactacystin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine; D004121:Dimethyl Sulfoxide