Multiplex SILAC analysis of a cellular TDP-43 proteinopathy model reveals protein inclusions associated with SUMOylation and diverse polyubiquitin chains.
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.
Seyfried
Nicholas T
NT
Department of Human Genetics, School of Medicine, Emory University, Atlanta, Georgia 30322, USA.
Gozal
Yair M
YM
Dammer
Eric B
EB
Xia
Qiangwei
Q
Duong
Duc M
DM
Cheng
Dongmei
D
Lah
James J
JJ
Levey
Allan I
AI
Peng
Junmin
J
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004622:Embryo, Mammalian; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry* / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008962:Models, Theoretical; D009474:Neurons / Q000378:metabolism / Q000473:pathology; D025821:Polyubiquitin / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology; D040901:Proteomics / Q000379:methods*; D025842:SUMO-1 Protein / Q000378:metabolism*; D057177:TDP-43 Proteinopathies / Q000378:metabolism* / Q000473:pathology; D054875:Ubiquitination
D004268:DNA-Binding Proteins; D025842:SUMO-1 Protein; D025821:Polyubiquitin