Identification of Full-Length Wild-Type and Mutant Huntingtin Interacting Proteins by Crosslinking Immunoprecipitation in Mice Brain Cortex

J Huntingtons Dis. 2021;10(3):335-347. doi: 10.3233/JHD-210476.


Background: Huntington's disease is a neurodegenerative disorder caused by a CAG expansion in the huntingtin gene, resulting in a polyglutamine expansion in the ubiquitously expressed mutant huntingtin protein.

Objective: Here we set out to identify proteins interacting with the full-length wild-type and mutant huntingtin protein in the mice cortex brain region to understand affected biological processes in Huntington's disease pathology.

Methods: Full-length huntingtin with 20 and 140 polyQ repeats were formaldehyde-crosslinked and isolated via their N-terminal Flag-tag from 2-month-old mice brain cortex. Interacting proteins were identified and quantified by label-free liquid chromatography-mass spectrometry (LC-MS/MS).

Results: We identified 30 interactors specific for wild-type huntingtin, 14 interactors specific for mutant huntingtin and 14 shared interactors that interacted with both wild-type and mutant huntingtin, including known interactors such as F8a1/Hap40. Syt1, Ykt6, and Snap47, involved in vesicle transport and exocytosis, were among the proteins that interacted specifically with wild-type huntingtin. Various other proteins involved in energy metabolism and mitochondria were also found to associate predominantly with wild-type huntingtin, whereas mutant huntingtin interacted with proteins involved in translation including Mapk3, Eif3h and Eef1a2.

Conclusion: Here we identified both shared and specific interactors of wild-type and mutant huntingtin, which are involved in different biological processes including exocytosis, vesicle transport, translation and metabolism. These findings contribute to the understanding of the roles that wild-type and mutant huntingtin play in a variety of cellular processes both in healthy conditions and Huntington's disease pathology.

Keywords: Huntington’s disease; cerebral cortex; cross-linking reagents; huntingtin protein; immunoprecipitation; protein interaction mapping; proteomics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brain / metabolism
  • Chromatography, Liquid
  • Huntingtin Protein / genetics*
  • Huntingtin Protein / metabolism
  • Huntington Disease* / genetics
  • Immunoprecipitation
  • Mice
  • Mutant Proteins / metabolism
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism
  • Synaptotagmin I
  • Tandem Mass Spectrometry


  • Htt protein, mouse
  • Huntingtin Protein
  • Mutant Proteins
  • Nerve Tissue Proteins
  • Synaptotagmin I
  • Syt1 protein, mouse