Polyglutamine- and temperature-dependent conformational rigidity in mutant huntingtin revealed by immunoassays and circular dichroism spectroscopy

By Fodale, Valentina; Kegulian, Natalie C.; Verani, Margherita; Cariulo, Cristina; Azzollini, Lucia; Petricca, Lara; Daldin, Manuel; Boggio, Roberto; Padova, Alessandro; Kuhn, Rainer; et al
From PLoS One (2014), 9(12), e112262/1-e112262/31, 31 pp.. Language: English, Database: CAPLUS, DOI:10.1371/journal.pone.0112262

In Huntington’s disease, expansion of a CAG triplet repeat occurs in exon 1 of the huntingtin gene (HTT), resulting in a protein bearing>35 polyglutamine residues whose N-terminal fragments display a high propensity to misfold and aggregate. Recent data demonstrate that polyglutamine expansion results in conformational changes in the huntingtin protein (HTT), which likely influence its biol. and biophys. properties. Developing assays to characterize and measure these conformational changes in isolated proteins and biol. samples would advance the testing of novel therapeutic approaches aimed at correcting mutant HTT misfolding. Time-resolved Förster energy transfer (TR-FRET)-based assays represent high-throughput, homogeneous, sensitive immunoassays widely employed for the quantification of proteins of interest. TR-FRET is extremely sensitive to small distances and can therefore provide conformational information based on detection of exposure and relative position of epitopes present on the target protein as recognized by selective antibodies. We have previously reported TR-FRET assays to quantify HTT proteins based on the use of antibodies specific for different amino-terminal HTT epitopes. Here, we investigate the possibility of interrogating HTT protein conformation using these assays. Methodol./Principal Findings: By performing TR-FRET measurements on the same samples (purified recombinant proteins or lysates from cells expressing HTT fragments or full length protein) at different temps., we have discovered a temp.-dependent, reversible, polyglutamine-dependent conformational change of wild type and expanded mutant HTT proteins. CD spectroscopy confirms the temp. and polyglutamine-dependent change in HTTstructure, revealing an effect of polyglutamine length and of temp. on the alpha-helical content of the protein. Conclusions/Significance: The temp.- and polyglutamine-dependent effects obsd. with TR-FRET on HTT proteins represent a simple, scalable, quant. and sensitive assay to identify genetic and pharmacol. modulators of mutant HTT conformation, and potentially to assess the relevance of conformational changes during onset and progression of Huntington’s disease.