Mutant huntingtin N-terminal fragments of specific size mediate aggregation and toxicity in neuronal cells

Tamara Ratovitski, Marjan Gucek, Haibing Jiang, Ekaterine Chighladze, Elaine Waldron, James D'Ambola, Hou Zhipeng, Liang Yideng, Michelle A. Poirier, Ricky R. Hirschhorn, Rona Graham, Michael R. Hayden, Robert N. Cole, Christopher A. Ross

Research output: Contribution to journalArticle

Abstract

Huntingtin proteolysis is implicated in Huntington disease pathogenesis, yet, the nature of huntingtin toxic fragments remains unclear. Huntingtin undergoes proteolysis by calpains and caspases within an N-terminal region between amino acids 460 and 600. We have focused on proteolytic steps producing shorter N-terminal fragments, which we term cp-1 and cp-2 (distinct from previously described cp-A/cp-B). We used HEK293 cells to express the first 511 residues of huntingtin and further define the cp-1 and cp-2 cleavage sites. Based on epitope mapping with huntingtin-specific antibodies, we found that cp-1 cleavage occurs between residues 81 and 129 of huntingtin. Affinity and size exclusion chromatography were used to further purify huntingtin cleavage products and enrich for the cp-1/cp-2 fragments. Using mass spectrometry, we found that the cp-2 fragment is generated by cleavage of huntingtin at position Arg167. This site was confirmed by deletion analysis and specific detection with a custom-generated cp-2 site neo-epitope antibody. Furthermore, alterations of this cleavage site resulted in a decrease in toxicity and an increase in aggregation of huntingtin in neuronal cells. These data suggest that cleavage of huntingtin at residue Arg167 may mediate mutant huntingtin toxicity in Huntington disease.

Original languageEnglish (US)
Pages (from-to)10855-10867
Number of pages13
JournalJournal of Biological Chemistry
Volume284
Issue number16
DOIs
StatePublished - Apr 17 2009

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Ratovitski, T., Gucek, M., Jiang, H., Chighladze, E., Waldron, E., D'Ambola, J., Zhipeng, H., Yideng, L., Poirier, M. A., Hirschhorn, R. R., Graham, R., Hayden, M. R., Cole, R. N., & Ross, C. A. (2009). Mutant huntingtin N-terminal fragments of specific size mediate aggregation and toxicity in neuronal cells. Journal of Biological Chemistry, 284(16), 10855-10867. https://doi.org/10.1074/jbc.M804813200