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Published January 31, 2011 | Published + Supplemental Material
Journal Article Open

Perturbation with Intrabodies Reveals That Calpain Cleavage Is Required for Degradation of Huntingtin Exon 1


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. Methodology/Principal Findings: 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_L12.3, turnover of soluble mHDx-1 in living cells is blocked. Conclusions/Significance: 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.

Additional Information

© 2011 Southwell et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received October 4, 2010; Accepted December 24, 2010; Published January 31, 2011. Editor: Mel Feany, Brigham and Women's Hospital, Harvard Medical School, United States of America. Funding: This work was funded by the Hereditary Disease Foundation (www.hdfoundation.org) and the NINDS 5RO1NS055298 (www.ninds.nih.gov). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank David Colby and K. Dane Wittrup for VL12.3, Elena Cattaneo for ST14A cells, Pamela Bjorkman for mHDx-1-TRX, Vivian Hook for anti-Htt N1-17 antibody, Christian Essrich for the design and piloting of the brain slice experiments, and Ali Khoshnan and Rona Graham for discussion and support. Author Contributions: Conceived and designed the experiments: ALS CWB PP DCL PHP. Performed the experiments: ALS CWB LSK DD SB AW. Analyzed the data: ALS CWB SB AW. Wrote the paper: ALS DCL PHP.

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Published - Southwell2011p12934PLoS_ONE.pdf

Supplemental Material - journal.pone.0016676.s001.tif

Supplemental Material - journal.pone.0016676.s002.tif


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August 23, 2023
October 23, 2023