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

• Authors: Nicholas Seyfried, Emory University; Yair M. Gozal, Emory University; Eric B Dammer, Emory University; Qiangwei Xia, Emory University; Duc Duong, Emory University; Dongmei Cheng, Emory University; James J Lah, Emory University; Allan I Levey, Emory University; Junmin Peng, Emory University
• Journal Title: Molecular & Cellular Proteomics
• Volume: Volume 9, Number 4
• Publisher: The American Society for Biochemistry and Molecular Biology | 2010-04, Pages 705-718
• Type of Work: Article | Final Publisher PDF
• Publisher DOI: http://doi.org/10.1074/mcp.M800390-MCP200
• Subjects: Biology, Genetics; Biology, Neuroscience

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.