Identification of wild-type and mutant huntingtin interaction partners by cross-linking immunoprecipitation and LFQ-based mass spectrometry

Karen A. Sap1, Arzu Tugce Guler1, Aleksandra Bury1, Dick Dekkers2, Jeroen Demmers2, Eric A. Reits1

1 Department of Medical Biology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands (presenting author: k.a.sap@amsterdamumc.nl).
2 Department of Biochemistry, Proteomics Center, Erasmus University Medical Center, Rotterdam, The Netherlands 

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. 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. 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). 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.

Furthermore, we performed global proteome analyses in different mouse brain regions expressing fl Htt Q20, Q50, and Q140. We observed differential protein abundances between the samples, such as in proteins involved in oxidative phosphorylation, ionotropic glutamate receptors, cholinergic and dopaminergic synapse, and clathrin-mediated endocytosis.

These findings contribute to understanding the roles that wild-type and mutant huntingtin play in various cellular processes in normal conditions and Huntington’s disease pathology.

Funded by Campagneteam Huntington and CHDI