Asparaginyl endopeptidase cleaves TDP-43 in brain

Authors

  • Jeremy H. Herskowitz,

    1. Department of Neurology, Center for Neurodegenerative Diseases, Emory University School of Medicine, Atlanta, GA, USA
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    • These authors contributed equally to this work.

  • Yair M. Gozal,

    1. Department of Neurology, Center for Neurodegenerative Diseases, Emory University School of Medicine, Atlanta, GA, USA
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  • Duc M. Duong,

    1. Department of Neurology, Center for Neurodegenerative Diseases, Emory University School of Medicine, Atlanta, GA, USA
    2. Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA
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  • Eric B. Dammer,

    1. Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA
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  • Marla Gearing,

    1. Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA
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  • Keqiang Ye,

    1. Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA
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  • James J. Lah,

    1. Department of Neurology, Center for Neurodegenerative Diseases, Emory University School of Medicine, Atlanta, GA, USA
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  • Junmin Peng,

    1. Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA
    2. Emory Proteomics Service Center, Emory University School of Medicine, Atlanta, GA, USA
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  • Allan I. Levey,

    Corresponding author
    • Department of Neurology, Center for Neurodegenerative Diseases, Emory University School of Medicine, Atlanta, GA, USA
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    • These authors contributed equally to this work.

  • Nicholas T. Seyfried

    Corresponding author
    1. Emory Proteomics Service Center, Emory University School of Medicine, Atlanta, GA, USA
    2. Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA
    • Department of Neurology, Center for Neurodegenerative Diseases, Emory University School of Medicine, Atlanta, GA, USA
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    • These authors contributed equally to this work.


Correspondence: Dr. Nicholas T. Seyfried, Department of Neurology, Center for Neurodegenerative Diseases, Emory University School of Medicine, Atlanta, Georgia 30322, USA

E-mail: nseyfri@emory.edu

Fax: 404-727-3728

Additional corresponding author: Dr. Allan I. Levey

E-mail: alevey@emory.edu

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 and amyotrophic lateral sclerosis. 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 MS 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 postmortem frontotemporal lobar degeneration 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.

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