Amyloid-like features of polyglutamine aggregates and their assembly kinetics.
The repeat length-dependent tendency of the polyglutamine sequences of certain proteins to form aggregates may underlie the cytotoxicity of these sequences in expanded CAG repeat diseases such as Huntington's disease. We report here a number of features of various polyglutamine (polyGln) aggregates and their assembly pathways that bear a resemblance to generally recognized defining features of amyloid fibrils. PolyGln aggregation kinetics displays concentration and length dependence and a lag phase that can be abbreviated by seeding. PolyGln aggregates exhibit classical beta-sheet-rich circular dichroism spectra consistent with an amyloid-like substructure. The fundamental structural unit of all the in vitro aggregates described here is a filament about 3 nm in width, resembling the protofibrillar intermediates in amyloid fibril assembly. We observed these filamentous structures either as isolated threads, as components of ribbonlike sheets, or, rarely, in amyloid-like twisted fibrils. All of the polyGln aggregates described here bind thioflavin T and shift its fluorescence spectrum. Although all polyGln aggregates tested bind the dye Congo red, only aggregates of a relatively long polyGln peptide exhibit Congo red birefringence, and this birefringence is only observed in a small portion of these aggregates. Remarkably, a monoclonal antibody with high selectivity for a generic amyloid fibril conformational epitope is capable of binding polyGln aggregates. Thus, polyGln aggregates exhibit most of the characteristic features of amyloid, but the twisted fibril structure with Congo red birefringence is not the predominant form in the polyGln repeat length range studied here. We also find that polyGln peptides exhibit an unusual freezing-dependent aggregation that appears to be caused by the freeze concentration of peptide and/or buffer components. This is of both fundamental and practical significance. PolyGln aggregation is revealed to be a highly specific process consistent with a significant degree of order in the molecular structure of the product. This ordered structure, or the assembly process leading to it, may be responsible for the cell-specific neuronal degeneration observed in Huntington's and other expanded CAG repeat diseases.
Chen
Songming
S
Graduate School of Medicine, University of Tennessee Medical Center, 1924 Alcoa Highway, Knoxville, TN 37920, USA.
Berthelier
Valerie
V
Hamilton
J Bradley
JB
O'Nuallain
Brian
B
Wetzel
Ronald
R
D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D000911:Antibodies, Monoclonal / Q000378:metabolism; D052160:Benzothiazoles; D001666:Binding Sites, Antibody; D005456:Fluorescent Dyes / Q000378:metabolism; D005615:Freezing; D005973:Glutamine / Q000138:chemical synthesis / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D006816:Huntington Disease / Q000378:metabolism; D007700:Kinetics; D008854:Microscopy, Electron; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D011485:Protein Binding; D012995:Solubility; D013844:Thiazoles / Q000378:metabolism
D016229:Amyloid beta-Peptides; D000911:Antibodies, Monoclonal; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D010455:Peptides; D013844:Thiazoles; D005973:Glutamine; C009462:thioflavin T; C097188:polyglutamine