title: Perturbation with intrabodies reveals that calpain cleavage is required for degradation of huntingtin exon 1.
abstract: BACKGROUND
  Proteolytic processing of mutant huntingtin (mHtt), the protein that causes Huntington's disease (HD), is critical for mHtt toxicity and disease progression. mHtt contains several caspase and calpain cleavage sites that generate N-terminal fragments that are more toxic than full-length mHtt. Further processing is then required for the degradation of these fragments, which in turn, reduces toxicity. This unknown, secondary degradative process represents a promising therapeutic target for HD.
  
  
  RESULTS
  We have used intrabodies, intracellularly expressed antibody fragments, to gain insight into the mechanism of mutant huntingtin exon 1 (mHDx-1) clearance. Happ1, an intrabody recognizing the proline-rich region of mHDx-1, reduces the level of soluble mHDx-1 by increasing clearance. While proteasome and macroautophagy inhibitors reduce turnover of mHDx-1, Happ1 is still able to reduce mHDx-1 under these conditions, indicating Happ1-accelerated mHDx-1 clearance does not rely on these processes. In contrast, a calpain inhibitor or an inhibitor of lysosomal pH block Happ1-mediated acceleration of mHDx-1 clearance. These results suggest that mHDx-1 is cleaved by calpain, likely followed by lysosomal degradation and this process regulates the turnover rate of mHDx-1. Sequence analysis identifies amino acid (AA) 15 as a potential calpain cleavage site. Calpain cleavage of recombinant mHDx-1 in vitro yields fragments of sizes corresponding to this prediction. Moreover, when the site is blocked by binding of another intrabody, V(L)12.3, turnover of soluble mHDx-1 in living cells is blocked.
  
  
  CONCLUSIONS
  These results indicate that calpain-mediated removal of the 15 N-terminal AAs is required for the degradation of mHDx-1, a finding that may have therapeutic implications.
authors.affiliation: Division of Biology, California Institute of Technology, Pasadena, California, United States of America.
authors.forename: Amber L
authors.initials: AL
authors.lastname: Southwell
authors.forename: Charles W
authors.initials: CW
authors.lastname: Bugg
authors.forename: Linda S
authors.initials: LS
authors.lastname: Kaltenbach
authors.forename: Denise
authors.initials: D
authors.lastname: Dunn
authors.forename: Stefanie
authors.initials: S
authors.lastname: Butland
authors.forename: Andreas
authors.initials: A
authors.lastname: Weiss
authors.forename: Paolo
authors.initials: P
authors.lastname: Paganetti
authors.forename: Donald C
authors.initials: DC
authors.lastname: Lo
authors.forename: Paul H
authors.initials: PH
authors.lastname: Patterson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002154:Calpain / Q000378:metabolism*; D005091:Exons / Q000235:genetics*; D000071058:Huntingtin Protein; D007128:Immunoglobulin Fragments / Q000378:metabolism*; D051379:Mice; D050505:Mutant Proteins / Q000276:immunology; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D012995:Solubility
chemical_list: C506812:Htt protein, mouse; D000071058:Huntingtin Protein; D007128:Immunoglobulin Fragments; D050505:Mutant Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D002154:Calpain