Project: Investigation of putative HTT interacting proteins Experiment: Analysis of HTT BioID datasets Date completed:­ 2019/04/09 Rationale: BioID technology employs a promiscuous biotin ligase (BirA) fused to the terminus of the target protein, huntingtin, allowing proximal proteins to be biotinylated and then subsequently identified through mass spectrometry experiments. This technique has not been applied to assess huntingtin interactors to date in the published literature, so will provide a novel methodology to characterize the huntingtin interactome. As huntingtin is a large protein molecule and the precise location of the N and C-termini remain unresolved due to their flexible nature, both N and C terminally BirA-tagged constructs for full-length huntingtin will be generated for overexpression as well as a truncated construct spanning amino acids 80-3100, the region of the protein resolved in the recent cryo-electron microscopy structure which omits the flexible termini. Huntingtin fusion proteins will be overexpressed in cells subjected to different ROS stresses as well as control conditions. Resultant cell lysates will be analysed through collaboration with Prof. Anne-Claude Gingras (Lunenfeld Tanenbaum Research Institute, University of Toronto). From this work, we hope to obtain a list of putative huntingtin interactors which will be compared to previously published findings and assessed for stable complex formation with huntingtin

Dr. Harding is the recipient of the Huntington's Disease Society of America Berman Topper Career Development Fellowship which funds and supports this research, in addition to generous funding from the CHDI Foundation and the Huntington Society of Canada. The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada through Ontario Genomics Institute [OGI-055], Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck KGaA, Darmstadt, Germany, MSD, Novartis Pharma AG, Ontario Ministry of Research, Innovation and Science (MRIS), Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and Wellcome.