Detection of polyglutamine protein oligomers in cells by fluorescence correlation spectroscopy.
Abnormal aggregation of misfolded proteins and their deposition as inclusion bodies in the brain have been implicated as a common molecular pathogenesis of neurodegenerative diseases including Alzheimer, Parkinson, and the polyglutamine (poly(Q)) diseases, which are collectively called the conformational diseases. The poly(Q) diseases, including Huntington disease and various types of spinocerebellar ataxia, are caused by abnormal expansions of the poly(Q) stretch within disease-causing proteins, which triggers the disease-causing proteins to aggregate into insoluble beta-sheet-rich amyloid fibrils. Although oligomeric structures formed in vitro are believed to be more toxic than mature amyloid fibrils in these diseases, the existence of oligomers in vivo has remained controversial. To explore oligomer formation in cells, we employed fluorescence correlation spectroscopy (FCS), which is a highly sensitive technique for investigating the dynamics of fluorescent molecules in solution. Here we demonstrate direct evidence for oligomer formation of poly(Q)-green fluorescent protein (GFP) fusion proteins expressed in cultured cells, by showing a time-dependent increase in their diffusion time and particle size by FCS. We show that the poly(Q)-binding peptide QBP1 inhibits poly(Q)-GFP oligomer formation, whereas Congo red only inhibits the growth of oligomers, but not the initial formation of the poly(Q)-GFP oligomers, suggesting that FCS is capable of identifying poly(Q) oligomer inhibitors. We therefore conclude that FCS is a useful technique to monitor the oligomerization of disease-causing proteins in cells as well as its inhibition in the conformational diseases.
Takahashi
Yasuo
Y
Laboratory of Supramolecular Biophysics, Research Institute for Electronic Science, Hokkaido University, Sapporo, Hokkaido 060-0812, Japan.
Okamoto
Yuma
Y
Popiel
H Akiko
HA
Fujikake
Nobuhiro
N
Toda
Tatsushi
T
Kinjo
Masataka
M
Nagai
Yoshitaka
Y
D000682:Amyloid / Q000737:chemistry*; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins; D006801:Humans; D019636:Neurodegenerative Diseases / Q000209:etiology; D009842:Oligopeptides / Q000032:analysis*; D010455:Peptides / Q000032:analysis*; D017510:Protein Folding; D013050:Spectrometry, Fluorescence / Q000379:methods*
D000682:Amyloid; D009842:Oligopeptides; D010455:Peptides; D049452:Green Fluorescent Proteins; C097188:polyglutamine