title: N-terminal proteolysis of full-length mutant huntingtin in an inducible PC12 cell model of Huntington's disease.
abstract: Proteolytic cleavage of mutant huntingtin may play a key role in the pathogenesis of Huntington's disease; however the steps in huntingtin proteolysis are not fully understood. Huntingtin was shown to be cleaved by caspases and calpains within a region between 460-600 amino acids from the N-terminus. Two smaller N-terminal fragments produced by unknown protease have been previously described as cp-A and cp-B. To further investigate the huntingtin proteolytic pathway, we used an inducible PC12 cell model expressing full-length huntingtin with either normal or expanded polyglutamine. This cell model recapitulates several steps of huntingtin proteolysis: proteolysis mediated by caspases within the region previously mapped for caspase cleavage, and cleavage generating two novel N-terminal fragments (cp-1 approximately 90-105 residues long and cp-2 extending beyond 115-129 epitope of huntingtin). Interestingly, the deletion of amino acids 105-114 (mapped previously as a cleavage site for cp-A) failed to affect the production of cp-1 or cp-2. Therefore, we conclude that these new fragments are distinct from previously described cp-A and cp-B. We demonstrate that cp-1 and cp-2 fragments are produced and accumulate within nuclear and cytoplasmic inclusions prior to huntingtin-induced cell toxicity, and these fragments can be formed by caspase-independent proteolytic cleavage of huntingtin in PC12 cells. In addition, inhibition of calpains leads to decreased subsequent degradation of cp-1 and cp-2 fragments, and accelerated formation of inclusions. Further delineation of huntingtin cleavage events may lead to novel therapeutic targets for HD.
authors.affiliation: Division of Neurobiology, Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. tratovi1@jhmi.edu
authors.forename: Tamara
authors.initials: T
authors.lastname: Ratovitski
authors.forename: Masayuki
authors.initials: M
authors.lastname: Nakamura
authors.forename: James
authors.initials: J
authors.lastname: D'Ambola
authors.forename: Ekaterine
authors.initials: E
authors.lastname: Chighladze
authors.forename: Yideng
authors.initials: Y
authors.lastname: Liang
authors.forename: Wenfei
authors.initials: W
authors.lastname: Wang
authors.forename: Rona
authors.initials: R
authors.lastname: Graham
authors.forename: Michael R
authors.initials: MR
authors.lastname: Hayden
authors.forename: David R
authors.initials: DR
authors.lastname: Borchelt
authors.forename: Ricky R
authors.initials: RR
authors.lastname: Hirschhorn
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Ross
mesh_terms: D000818:Animals; D010450:Endopeptidases / Q000378:metabolism*; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000235:genetics*; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D016716:PC12 Cells; D010446:Peptide Fragments; D051381:Rats
chemical_list: C499524:Htt protein, rat; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D010450:Endopeptidases