α-Synuclein in central nervous system and from erythrocytes, mammalian cells, and Escherichia coli exists predominantly as disordered monomer.
Since the discovery and isolation of α-synuclein (α-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that α-syn produced in Escherichia coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in α-helical structure (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107-110; Wang, W., Perovic, I., Chittuluru, J., Kaganovich, A., Nguyen, L. T. T., Liao, J., Auclair, J. R., Johnson, D., Landeru, A., Simorellis, A. K., Ju, S., Cookson, M. R., Asturias, F. J., Agar, J. N., Webb, B. N., Kang, C., Ringe, D., Petsko, G. A., Pochapsky, T. C., and Hoang, Q. Q. (2011) Proc. Natl. Acad. Sci. 108, 17797-17802). However, it remains unknown whether or not this putative tetramer is the main physiological form of α-syn in the brain. In this study, we investigated the oligomeric state of α-syn in mouse, rat, and human brains. To assess the conformational and oligomeric state of native α-syn in complex mixtures, we generated α-syn standards of known quaternary structure and conformational properties and compared the behavior of endogenously expressed α-syn to these standards using native and denaturing gel electrophoresis techniques, size-exclusion chromatography, and an oligomer-specific ELISA. Our findings demonstrate that both human and rodent α-syn expressed in the central nervous system exist predominantly as an unfolded monomer. Similar results were observed when human α-syn was expressed in mouse and rat brains as well as mammalian cell lines (HEK293, HeLa, and SH-SY5Y). Furthermore, we show that α-syn expressed in E. coli and purified under denaturing or nondenaturing conditions, whether as a free protein or as a fusion construct with GST, is monomeric and adopts a disordered conformation after GST removal. These results do not rule out the possibility that α-syn becomes structured upon interaction with other proteins and/or biological membranes.
Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Station 19, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
Bruno
B
Fauvet
Martial K
MK
Mbefo
Mohamed-Bilal
MB
Fares
Carole
C
Desobry
Sarah
S
Michael
Mustafa T
MT
Ardah
Elpida
E
Tsika
Philippe
P
Coune
Michel
M
Prudent
Niels
N
Lion
David
D
Eliezer
Darren J
DJ
Moore
Bernard
B
Schneider
Patrick
P
Aebischer
Omar M
OM
El-Agnaf
Eliezer
E
Masliah
Hilal A
HA
Lashuel
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D045744:Cell Line, Tumor; D002490:Central Nervous System / Q000378:metabolism; D002850:Chromatography, Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D051844:alpha-Synuclein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D051844:alpha-Synuclein