[Effect of SUMO-1 on mitochondria subcellular localization of alpha-synuclein and its degradation via ubiquitin-proteasome system].
OBJECTIVE
To investigate the effect of sumoylation of alpha-synuclein by SUMO-1 on the mitochondria subcellular localization of alpha-synuclein and its degradation via ubiquitin-proteasome system.


METHODS
Primers of wild-type, A53T pathogenic mutant and K96R mutant of human alpha-synuclein were designed to amplify the corresponding cDNAs without stop codon. The cDNAs were cloned into pGEM T-easy vector, analyzed by using enzyme mapping and DNA sequencing, and subcloned into pEGFP-N1 vector. The recombinant plasmids of pEGFP-alpha-synuclein-WT, pEGFP-alpha-synuclein-A53T and pEGFP-alpha-synuclein-K96R were transfected into HEK293 cells by lipofectamine method. The expression of the alpha-synuclein protein was measured by immunofluorescence and confocal microscope. Then mitochondria staining as well as immunofluorescence were utilized to investigate the effect of wild-type, A53T mutant and sumoylation of alpha-synuclein on mitochondria subcellular localization of alpha-synuclein. The effect of sumoylation of alpha-synuclein on its degradation via the ubiquitin-proteasome system in the cells was assayed by Western-blot.


RESULTS
The enzyme mapping suggested that the eukaryotic expression plasmids for human wild-type, A53T and K96R mutants of the alpha-synuclein gene were constructed successfully. By immunofluorescence and confocal microscope, it was observed that alpha-synuclein-WT and alpha-synuclein-A53T proteins aggregated in cytoplasm, and alpha-synuclein-K96R protein aggregation was decreased in cytoplasm of cultured cells. The alpha-synuclein proteins of wild-type, A53T and K96R mutants were co-localized with mitochondria. Western-blot analysis revealed that both wild-type and A53T mutant affected the amount of the ubiquitinated proteins.


CONCLUSIONS
Neither overexpression of wild-type and A53T pathogenic mutant alpha-synuclein, nor sumoylation of alpha-synuclein, affected the subcellular localization in the mitochondria. However, overexpression of wild-type and A53T mutant alpha-synuclein affected the amount of the ubiquitinated proteins.
Department of Neurology, Hainan Provincial People's Hospital, Haikou, Hainan, 570311 PR China.
Tao
T
Chen
Xiao-ping
XP
Liao
Guo-qiang
GQ
Wen
Zhi-gang
ZG
Nong
Feng
F
Ouyang
Yi-dong
YD
Deng
Min
M
Guo
Hui-ling
HL
Wu
Peng
P
Zhou
D015153:Blotting, Western; D002460:Cell Line; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D025842:SUMO-1 Protein / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D051844:alpha-Synuclein / Q000378:metabolism*
D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; D025801:Ubiquitin; D051844:alpha-Synuclein; D046988:Proteasome Endopeptidase Complex