Oral Presentation 50th Lorne Proteins Conference 2025

Deciphering Dysregulated Molecular Pathways in Huntington's Disease: The Role of HTT in DNA Mismatch Repair (113618)

Christian Makhoul 1 , Terence Gall-Duncan 2 , Mustafa Mehkary 2 , Eduardo S Ramos 3 , Rachel Harding 4 , Erich E Wanker 3 , Christopher E Pearson 2 , Danny M Hatters 1
  1. Biochemistry and Pharmacology, University of Melbourne, Melbourne, Victoria, Australia
  2. Program of Genetics & Genome Biology, The Hospital for Sick Children, Toronto, Ontario, Canada
  3. Neuroproteomics, Max Delbrück Centre for Molecular Medicine, Berlin, Germany
  4. Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada

Huntington’s disease (HD) is a progressive and fatal neurodegenerative disease that is caused by a CAG triplet expansion mutation in the Huntingtin gene of affected individuals. Therapeutically targetable molecular mechanisms that underly Huntington’s disease remain elusive. To identify novel cellular targets involved in HD pathology, we used a biochemical approach that we have previously developed, known as “protein painting”, to discover changes in protein structure and multimeric complex assemblies. This method utilizes a cell-permeable chemical reagent, tetraphenylethene maleimide (TPE-MI) to react with surface-exposed cysteine residues of proteins. Changes in cysteine thiol reactivity can be monitored on a proteome-wide scale to capture alterations in protein structure or ligand interactions and we previously showed that these changes can cast light on the functional status of proteins. We applied this method to a stem cell-derived neuronal model of Huntington’s disease to identify novel targets that are associated with MAP2+ cortical neurons carrying 81 CAG repeats by comparing cysteine reactivity with isogenic controls that only contain 27 CAG repeats in their huntingtin gene. Strikingly, this approach uncovered proteins that are known interactors of huntingtin or that are linked to molecular pathways demonstrated to modify disease progression. Most interestingly, we identified altered cysteine reactivity in proliferating cell nuclear antigen (PCNA), an integral component of the DNA mismatch repair pathway. Our preliminary investigations have uncovered several PCNA interacting motifs on the huntingtin protein and suggest that huntingtin can interact directly with PCNA. Our objective is to further characterize this interaction and to elucidate the functional consequence of poly-glutamine expansion of the mutant huntingtin protein in the context of DNA mismatch repair.