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Author Notes:

Correspondence: Junmin Peng, Department of Human Genetics, School of Medicine, Emory University, Atlanta, Georgia 30322. Telephone: 404-712-8510. Email: jpeng@genetics.emory.edu.

Nicholas T. Seyfried and Yair M. Gozal contributed equally to this work.

We thank Drs. Jonathan Glass and Ping Xu for discussion and critically reading the manuscript.

Subjects:

Research Funding:

This work was supported, in whole or in part, by the National Institutes of Health through the Emory Alzheimer's Disease Center (Grant P50AG025688), the Emory Neuroscience NINDS Core Facilities (Grant P30NS055077), and Training Grants T32NS007480 (to N. S.) and F30NS057902 (to Y. G.).

This work was also supported by Research Scholar Grant RSG-09-181-01 from the American Cancer Society.

Multiplex SILAC Analysis of a Cellular TDP-43 Proteinopathy Model Reveals Protein Inclusions Associated with SUMOylation and Diverse Polyubiquitin Chains

Journal Title:

Molecular & Cellular Proteomics

Volume:

Volume 9, Number 4

Publisher:

, Pages 705-718

Type of Work:

Article | Final Publisher PDF

Abstract:

Transactive response (TAR) DNA-binding protein 43 (TDP-43) is a major protein component within ubiquitin-positive inclusions of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Although TDP-43 is a nuclear DNA/RNA-binding protein, in pathological conditions, TDP-43 has been reported to redistribute to the cytoplasm where it is cleaved and forms insoluble, ubiquitinated, and phosphorylated inclusions. Here we present a cellular model in which full-length human TDP-43 or a splicing isoform (TDP-S6) that lacks the C terminus is overexpressed in a human cell line and mouse primary neurons. Whereas recombinant and endogenous TDP-43 was primarily localized in the nucleus, the shorter TDP-S6 formed highly insoluble cytoplasmic and nuclear inclusions reminiscent of disease-specific pathology. Western blot analysis of detergent-insoluble extracts showed an increase in high molecular weight immunoreactive species for TDP-S6 compared with TDP-43, consistent with ubiquitination or ubiquitin-like modifications. We used a multiplex stable isotope labeling with amino acids in cell culture approach to compare the detergent-insoluble proteome from mock-, TDP-43-, and TDP-S6-transfected cells. TDP-S6 overexpression caused a concomitant increase in both ubiquitin (Ub) and the small Ub-like modifier-2/3 (SUMO-2/3) within the insoluble proteome. Similarly, full-length TDP-43 overexpression also resulted in the elevation of SUMO-2/3. Immunofluorescence showed strong co-localization of endogenous Ub with both cytoplasmic and nuclear TDP-S6 inclusions, whereas SUMO-2/3 was co-localized mainly with the nuclear inclusions. Quantitative mass spectrometry further revealed that mixed Lys-48 and Lys-63 polyUb linkages were associated with the TDP insoluble fractions. Together our data indicate that expression of a TDP-43 splice variant lacking a C terminus recapitulates many of the cellular and biochemical features associated with disease pathology and that the interplay of ubiquitination and SUMOylation may have an important role in TDP-43 regulation.

Copyright information:

© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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