Identification of novel potentially toxic oligomers formed in vitro from mammalian-derived expanded huntingtin exon-1 protein

J Biol Chem. 2012 May 4;287(19):16017-28. doi: 10.1074/jbc.M111.252577. Epub 2012 Mar 20.


Huntington disease is a genetic neurodegenerative disorder that arises from an expanded polyglutamine region in the N terminus of the HD gene product, huntingtin. Protein inclusions comprised of N-terminal fragments of mutant huntingtin are a characteristic feature of disease, though are likely to play a protective role rather than a causative one in neurodegeneration. Soluble oligomeric assemblies of huntingtin formed early in the aggregation process are candidate toxic species in HD. In the present study, we established an in vitro system to generate recombinant huntingtin in mammalian cells. Using both denaturing and native gel analysis, we have identified novel oligomeric forms of mammalian-derived expanded huntingtin exon-1 N-terminal fragment. These species are transient and were not previously detected using bacterially expressed exon-1 protein. Importantly, these species are recognized by 3B5H10, an antibody that recognizes a two-stranded hairpin conformation of expanded polyglutamine believed to be associated with a toxic form of huntingtin. Interestingly, comparable oligomeric species were not observed for expanded huntingtin shortstop, a 117-amino acid fragment of huntingtin shown previously in mammalian cell lines and transgenic mice, and here in primary cortical neurons, to be non-toxic. Further, we demonstrate that expanded huntingtin shortstop has a reduced ability to form amyloid-like fibrils characteristic of the aggregation pathway for toxic expanded polyglutamine proteins. Taken together, these data provide a possible candidate toxic species in HD. In addition, these studies demonstrate the fundamental differences in early aggregation events between mutant huntingtin exon-1 and shortstop proteins that may underlie the differences in toxicity.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • Cells, Cultured
  • Electrophoresis, Polyacrylamide Gel
  • Exons / genetics*
  • Glutathione Transferase / genetics
  • Glutathione Transferase / metabolism
  • HEK293 Cells
  • Humans
  • Huntingtin Protein
  • Huntington Disease / genetics
  • Huntington Disease / metabolism
  • Mice
  • Microscopy, Atomic Force
  • Microscopy, Confocal
  • Microscopy, Electron, Transmission
  • Mutation
  • Nerve Tissue Proteins / chemistry
  • Nerve Tissue Proteins / genetics*
  • Nerve Tissue Proteins / metabolism
  • Neurons / cytology
  • Neurons / metabolism
  • Peptides / genetics
  • Protein Conformation
  • Protein Multimerization
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Time Factors
  • Trinucleotide Repeat Expansion / genetics*


  • HTT protein, human
  • Huntingtin Protein
  • Nerve Tissue Proteins
  • Peptides
  • Recombinant Fusion Proteins
  • polyglutamine
  • Glutathione Transferase