title: Polyglutamine- and temperature-dependent conformational rigidity in mutant huntingtin revealed by immunoassays and circular dichroism spectroscopy.
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.
  
  
  RESULTS
  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
  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.
authors.affiliation: IRBM Promidis, Pomezia, Rome, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Fodale
authors.affiliation: Department of Biochemistry and Molecular Biology, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.
authors.forename: Natalie C
authors.initials: NC
authors.lastname: Kegulian
authors.affiliation: IRBM Promidis, Pomezia, Rome, Italy.
authors.forename: Margherita
authors.initials: M
authors.lastname: Verani
authors.affiliation: IRBM Promidis, Pomezia, Rome, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Cariulo
authors.affiliation: IRBM Promidis, Pomezia, Rome, Italy.
authors.forename: Lucia
authors.initials: L
authors.lastname: Azzollini
authors.affiliation: IRBM Promidis, Pomezia, Rome, Italy.
authors.forename: Lara
authors.initials: L
authors.lastname: Petricca
authors.affiliation: IRBM Promidis, Pomezia, Rome, Italy.
authors.forename: Manuel
authors.initials: M
authors.lastname: Daldin
authors.affiliation: IRBM Promidis, Pomezia, Rome, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Boggio
authors.affiliation: IRBM Promidis, Pomezia, Rome, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Padova
authors.affiliation: IRBM Promidis, Pomezia, Rome, Italy.
authors.forename: Rainer
authors.initials: R
authors.lastname: Kuhn
authors.affiliation: CHDI Management/CHDI Foundation, Los Angeles, California, United States of America.
authors.forename: Robert
authors.initials: R
authors.lastname: Pacifici
authors.affiliation: CHDI Management/CHDI Foundation, Los Angeles, California, United States of America.
authors.forename: Douglas
authors.initials: D
authors.lastname: Macdonald
authors.affiliation: CHDI Management/CHDI Foundation, Los Angeles, California, United States of America.
authors.forename: Ryan C
authors.initials: RC
authors.lastname: Schoenfeld
authors.affiliation: CHDI Management/CHDI Foundation, Los Angeles, California, United States of America.
authors.forename: Hyunsun
authors.initials: H
authors.lastname: Park
authors.affiliation: Department of Biochemistry and Molecular Biology, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.
authors.forename: J Mario
authors.initials: JM
authors.lastname: Isas
authors.affiliation: Department of Biochemistry and Molecular Biology, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.
authors.forename: Ralf
authors.initials: R
authors.lastname: Langen
authors.affiliation: IRBM Promidis, Pomezia, Rome, Italy.
authors.forename: Andreas
authors.initials: A
authors.lastname: Weiss
authors.affiliation: IRBM Promidis, Pomezia, Rome, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Caricasole
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D002942:Circular Dichroism; D018450:Disease Progression; D000939:Epitopes / Q000737:chemistry; D005091:Exons; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D000071058:Huntingtin Protein; D007118:Immunoassay; D050505:Mutant Proteins / Q000737:chemistry*; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics*; D010455:Peptides / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature; D013879:Thioredoxins / Q000737:chemistry
chemical_list: D000596:Amino Acids; D000939:Epitopes; C086055:HTT protein, human; D000071058:Huntingtin Protein; D050505:Mutant Proteins; D009419:Nerve Tissue Proteins; D010455:Peptides; D011994:Recombinant Proteins; C097188:polyglutamine; D013879:Thioredoxins