title: Cysteine proteases bleomycin hydrolase and cathepsin Z mediate N-terminal proteolysis and toxicity of mutant huntingtin.
abstract: N-terminal proteolysis of huntingtin is thought to be an important mediator of HD pathogenesis. The formation of short N-terminal fragments of huntingtin (cp-1/cp-2, cp-A/cp-B) has been demonstrated in cells and in vivo. We previously mapped the cp-2 cleavage site by mass spectrometry to position Arg167 of huntingtin. The proteolytic enzymes generating short N-terminal fragments of huntingtin remain unknown. To search for such proteases, we conducted a genome-wide screen using an RNA-silencing approach and an assay for huntingtin proteolysis based on the detection of cp-1 and cp-2 fragments by Western blotting. The primary screen was carried out in HEK293 cells, and the secondary screen was carried out in neuronal HT22 cells, transfected in both cases with a construct encoding the N-terminal 511 amino acids of mutant huntingtin. For additional validation of the hits, we employed a complementary assay for proteolysis of huntingtin involving overexpression of individual proteases with huntingtin in two cell lines. The screen identified 11 enzymes, with two major candidates to carry out the cp-2 cleavage, bleomycin hydrolase (BLMH) and cathepsin Z, which are both cysteine proteases of a papain-like structure. Knockdown of either protease reduced cp-2 cleavage, and ameliorated mutant huntingtin induced toxicity, whereas their overexpression increased the cp-2 cleavage. Both proteases partially co-localized with Htt in the cytoplasm and within or in association with early and late endosomes, with some nuclear co-localization observed for cathepsin Z. BLMH and cathepsin Z are expressed in the brain and have been associated previously with neurodegeneration. Our findings further validate the cysteine protease family, and BLMH and cathepsin Z in particular, as potential novel targets for HD therapeutics.
authors.affiliation: Division of Neurobiology, Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA. tratovi1@jhmi.edu
authors.forename: Tamara
authors.initials: T
authors.lastname: Ratovitski
authors.forename: Ekaterine
authors.initials: E
authors.lastname: Chighladze
authors.forename: Elaine
authors.initials: E
authors.lastname: Waldron
authors.forename: Ricky R
authors.initials: RR
authors.lastname: Hirschhorn
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Ross
mesh_terms: D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D056663:Cathepsin Z / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering
chemical_list: C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D056663:Cathepsin Z; D053148:Caspase 3; D003546:Cysteine Endopeptidases; C031262:bleomycin hydrolase