Asparaginyl endopeptidase cleaves TDP-43 in brain

• Type of Material: Article
• Authors: Jeremy Hartford Herskowitz, Emory University; Yair M. Gozal, Emory University; Duc Duong, Emory University; Eric B Dammer, Emory University; Marla Gearing, Emory University; Keqiang Ye, Emory University; James J Lah, Emory University; Junmin Peng, Emory University; Allan I Levey, Emory University; Nicholas Seyfried, Emory University
• Language: English
• Date: 2012-08
• Publisher: Wiley: 12 months
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
• Final Published Version (URL): http://onlinelibrary.wiley.com/doi/10.1002/pmic.201200006/abstract
• Title of Journal or Parent Work: Proteomics
• ISSN: 1615-9853
• Volume: 12
• Issue: 0
• Start Page: 2455
• End Page: 2463
• Grant/Funding Information: This work was supported by the National Institutes of Health through the Emory Alzheimer’s Disease Center grant (P50AG025688), the Emory Neuroscience NINDS Core Facilities (P30NS055077), and NIH training grant (F32AG032848-02 to N.T.S.).
• Keywords: TAR DNA-binding protein 43; Asparaginyl endopeptidase; degradomics; frontotemporal dementia; neurodegeneration
• Research Categories: Health Sciences, Pathology; Biology, Neuroscience; Biology, Genetics

Abstract

TAR DNA-binding protein 43 (TDP-43) is a nuclear protein involved in RNA splicing and a major protein component in ubiquitin-positive, tau-negative inclusions of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Under disease conditions TDP-43 redistributes to the cytoplasm where it can be phosphorylated, ubiquitinated, and proteolytically cleaved. Enzymes responsible for TDP-43 proteolytic processing in brain remain largely unreported. Using a mass spectrometry approach, we identified two truncated TDP-43 peptides, terminating C-terminal to asparagines 291 (N291) and 306 (N306). The only documented mammalian enzyme capable of cleaving C-terminal to asparagine is asparaginyl endopeptidase (AEP). TDP-43-immunoreactive fragments (~35 and 32 kDa) predicted to be generated by AEP cleavage at N291 and N306 were observed by western blot analyses of post-mortem FTLD brain tissue and cultured human cells over-expressing TDP-43. Studies in vitro determined that AEP can directly cleave TDP-43 at seven sites, including N291 and N306. Western blots of brain homogenates isolated from AEP-null mice and wild-type littermate controls revealed that TDP-43 proteolytic fragments were substantially reduced in the absence of AEP in vivo. Taken together, we conclude that TDP-43 is cleaved by AEP in brain. Moreover, these data highlight the utility of combining proteomic strategies in vitro and in vivo to provide insight into TDP-43 biology that will fuel the design of more detailed models of disease pathogenesis.