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

PLoS One. 2014 Dec 2;9(12):e112262. doi: 10.1371/journal.pone.0112262. eCollection 2014.

Abstract

Background: 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 biological and biophysical properties. Developing assays to characterize and measure these conformational changes in isolated proteins and biological 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.

Methodology/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 temperatures, we have discovered a temperature-dependent, reversible, polyglutamine-dependent conformational change of wild type and expanded mutant HTT proteins. Circular dichroism spectroscopy confirms the temperature and polyglutamine-dependent change in HTT structure, revealing an effect of polyglutamine length and of temperature on the alpha-helical content of the protein.

Conclusions/significance: The temperature- and polyglutamine-dependent effects observed with TR-FRET on HTT proteins represent a simple, scalable, quantitative and sensitive assay to identify genetic and pharmacological modulators of mutant HTT conformation, and potentially to assess the relevance of conformational changes during onset and progression of Huntington's disease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / chemistry
  • Circular Dichroism
  • Disease Progression
  • Epitopes / chemistry
  • Exons
  • Fluorescence Resonance Energy Transfer
  • HEK293 Cells
  • Humans
  • Huntingtin Protein
  • Immunoassay
  • Mutant Proteins / chemistry*
  • Mutation
  • Nerve Tissue Proteins / chemistry*
  • Nerve Tissue Proteins / genetics*
  • Peptides / chemistry*
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Temperature
  • Thioredoxins / chemistry

Substances

  • Amino Acids
  • Epitopes
  • HTT protein, human
  • Huntingtin Protein
  • Mutant Proteins
  • Nerve Tissue Proteins
  • Peptides
  • Recombinant Proteins
  • polyglutamine
  • Thioredoxins

Grant support

CHDI Foundation is a privately funded nonprofit biomedical research organization exclusively dedicated to discovering and developing therapeutics that slow the progression of Huntington's disease. CHDI Foundation conducts research in a number of different ways; for the purposes of this manuscript, research was conducted at IRBM Promidis and the University of Southern California under a sponsored research agreement. RL, MJI and NCK are also supported by the Hereditary Disease Foundation. LP has received funding from the European Community's Seventh Framework Programme FP7/2010 under grant agreement no. 264508. The authors listed all contributed to the conception, planning, and direction of the research, including generation, analysis, and interpretation of the data.