Phosphorylated alpha-synuclein is ubiquitinated in alpha-synucleinopathy lesions.
alpha-Synuclein is one of the major components of intracellular fibrillary aggregates in the brains of a subset of neurodegenerative disorders, including Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, and Hallervorden-Spatz disease, which are referred to as alpha-synucleinopathies. We have shown previously (Fujiwara, H., Hasegawa, M., Dohmae, N., Kawashima, A., Masliah, E., Goldberg, M. S., Shen, J., Takio, K., and Iwatsubo, T. (2002) Nat. Cell Biol. 4, 160-164) that alpha-synuclein deposited in synucleinopathy brains is extensively phosphorylated at Ser-129 and migrates at 15 kDa. Here we examined the biochemical characteristics of the additional, higher molecular mass species of phosphorylated alpha-synuclein-positive polypeptides that also are recovered in the Sarkosyl-insoluble fraction of synucleinopathy and migrate at about 22 and 29 kDa. These 22 and 29 kDa bands were positive for three different anti-ubiquitin antibodies and comigrated perfectly with in vitro ubiquitinated alpha-synuclein that may correspond to mono- and diubiquitinated alpha-synuclein, respectively. Furthermore, cyanogen bromide cleavage of the 22 and 29 kDa polypeptides shifted the mobility to 19 and 26 kDa, respectively, and they retained immunoreactivity for both ubiquitin and alpha-synuclein. Finally, protein sequence analysis showed that the 19 kDa band contained two amino-terminal sequences of alpha-synuclein and ubiquitin. These results strongly suggest that phosphorylated alpha-synuclein is targeted to mono- and diubiquitination in synucleinopathy brains, which may have implications for mechanisms of these diseases.
Hasegawa
Masato
M
Department of Molecular Neurobiology, Tokyo Institute of Psychiatry, Tokyo Metropolitan Organization for Medical Research, 2-1-8 Kamikitazawa, Setagaya-ku, Japan. masato@prit.go.jp
Fujiwara
Hideo
H
Nonaka
Takashi
T
Wakabayashi
Koichi
K
Takahashi
Hitoshi
H
Lee
Virginia M-Y
VM
Trojanowski
John Q
JQ
Mann
David
D
Iwatsubo
Takeshi
T
D000368:Aged; D000595:Amino Acid Sequence; D001921:Brain / Q000378:metabolism* / Q000473:pathology; D003488:Cyanogen Bromide / Q000737:chemistry; D005260:Female; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D019636:Neurodegenerative Diseases / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation; D051843:Synucleins; D025801:Ubiquitin / Q000378:metabolism*; D051844:alpha-Synuclein
D009419:Nerve Tissue Proteins; C497604:SNCA protein, human; D051843:Synucleins; D025801:Ubiquitin; D051844:alpha-Synuclein; D003488:Cyanogen Bromide