TDP-43 post-translational modifications in health and disease

Despite the diverse biological pathways known to be regulated by ubiquitylation, global identification of substrates that are targeted for ubiquitylation has remained a challenge. To globally characterize the human ubiquitin-modified proteome (ubiquitinome), we utilized a monoclonal antibody that recognizes diglycine (diGly)-containing isopeptides following trypsin digestion. We identify ~19,000 diGly-modified lysine residues within ~5000 proteins. Using quantitative proteomics we monitored temporal changes in diGly site abundance in response to both proteasomal and translational inhibition, indicating both a dependence on ongoing translation to observe alterations in site abundance and distinct dynamics of individual modified lysines in response to proteasome inhibition. Further, we demonstrate that quantitative diGly proteomics can be utilized to identify substrates for cullin-RING ubiquitin ligases. Interrogation of the ubiquitinome allows for not only a quantitative assessment of alterations in protein homeostasis fidelity, but also identification of substrates for individual ubiquitin pathway enzymes. Copyright © 2011 Elsevier Inc. All rights reserved.

Post-translational modification of proteins by ubiquitin is a fundamentally important regulatory mechanism. However, proteome-wide analysis of endogenous ubiquitylation remains a challenging task, and almost always has relied on cells expressing affinity tagged ubiquitin. Here we combine single-step immunoenrichment of ubiquitylated peptides with peptide fractionation and high-resolution mass spectrometry to investigate endogenous ubiquitylation sites. We precisely map 11,054 endogenous putative ubiquitylation sites (diglycine-modified lysines) on 4,273 human proteins. The presented data set covers 67% of the known ubiquitylation sites and contains 10,254 novel sites on proteins with diverse cellular functions including cell signaling, receptor endocytosis, DNA replication, DNA damage repair, and cell cycle progression. Our method enables site-specific quantification of ubiquitylation in response to cellular perturbations and is applicable to any cell type or tissue. Global quantification of ubiquitylation in cells treated with the proteasome inhibitor MG-132 discovers sites that are involved in proteasomal degradation, and suggests a nonproteasomal function for almost half of all sites. Surprisingly, ubiquitylation of about 15% of sites decreased more than twofold within four hours of MG-132 treatment, showing that inhibition of proteasomal function can dramatically reduce ubiquitylation on many sites with non-proteasomal functions. Comparison of ubiquitylation sites with acetylation sites reveals an extensive overlap between the lysine residues targeted by these two modifications. However, the crosstalk between these two post-translational modifications is significantly less frequent on sites that show increased ubiquitylation upon proteasome inhibition. Taken together, we report the largest site-specific ubiquitylation dataset in human cells, and for the first time demonstrate proteome-wide, site-specific quantification of endogenous putative ubiquitylation sites.

Inclusions of TAR DNA-binding protein-43 (TDP-43), a nuclear protein that regulates transcription and RNA splicing, are the defining histopathological feature of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-Us) and sporadic and familial forms of amyotrophic lateral sclerosis (ALS). In ALS and FTLD-U, aggregated, ubiquitinated, and N-terminally truncated TDP-43 can be isolated from brain tissue rich in neuronal and glial cytoplasmic inclusions. The loss of TDP-43 function resulting from inappropriate cleavage, translocation from the nucleus, or its sequestration into inclusions could play important roles in neurodegeneration. However, it is not known whether TDP-43 fragments directly mediate toxicity and, more specifically, whether their abnormal aggregation is a cause or consequence of pathogenesis. We report that the ectopic expression of a approximately 25-kDa TDP-43 fragment corresponding to the C-terminal truncation product of caspase-cleaved TDP-43 leads to the formation of toxic, insoluble, and ubiquitin- and phospho-positive cytoplasmic inclusions within cells. The 25-kDa C-terminal fragment is more prone to phosphorylation at S409/S410 than full-length TDP-43, but phosphorylation at these sites is not required for inclusion formation or toxicity. Although this fragment shows no biological activity, its exogenous expression neither inhibits the function nor causes the sequestration of full-length nuclear TDP-43, suggesting that the 25-kDa fragment can induce cell death through a toxic gain-of-function. Finally, by generating a conformation-dependent antibody that detects C-terminal fragments, we show that this toxic cleavage product is specific for pathologic inclusions in human TDP-43 proteinopathies.