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

Southwell, Amber L. and Bugg, Charles W. and Kaltenbach, Linda S. and Dunn, Denise and Butland, Stefanie and Weiss, Andreas and Paganetti, Paolo and Lo, Donald C. and Patterson, Paul H. (2011) Perturbation with Intrabodies Reveals That Calpain Cleavage Is Required for Degradation of Huntingtin Exon 1. PLoS ONE, 6 (1). Art. No. e16676. ISSN 1932-6203. PMCID PMC3031625. http://resolver.caltech.edu/CaltechAUTHORS:20110317-142922950

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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. 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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1371/journal.pone.0016676DOIArticle
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0016676PublisherArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3031625/PubMed CentralArticle
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.
Funders:
Funding AgencyGrant Number
Hereditary Disease FoundationUNSPECIFIED
National Institute of Neurological Disorders and Stroke (NINDS)5RO1NS055298
PubMed Central ID:PMC3031625
Record Number:CaltechAUTHORS:20110317-142922950
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20110317-142922950
Official Citation:Southwell AL, Bugg CW, Kaltenbach LS, Dunn D, Butland S, et al. (2011) Perturbation with Intrabodies Reveals That Calpain Cleavage Is Required for Degradation of Huntingtin Exon 1. PLoS ONE 6(1): e16676. doi:10.1371/journal.pone.0016676
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:22970
Collection:CaltechAUTHORS
Deposited By: Benjamin Perez
Deposited On:17 Mar 2011 23:06
Last Modified:30 Dec 2015 21:55

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