Abstract
TAR DNA-binding protein of 43 kDa (TDP-43) forms pathological aggregates in neurodegenerative diseases, particularly in certain forms of frontotemporal dementia and amyotrophic lateral sclerosis. Pathological modifications of TDP-43 include proteolytic fragmentation, phosphorylation, and ubiquitinylation. A pathognomonic TDP-43 C-terminal fragment (CTF) spanning amino acids 193-414 contains only four lysine residues that could be potentially ubiquitinylated. Here, serial mutagenesis of these four lysines to arginine revealed that not a single residue is responsible for the ubiquitinylation of mCherry-tagged CTF. Removal of all four lysines was necessary to suppress ubiquitinylation. Interestingly, Lys-408 substitution enhanced the pathological phosphorylation of the immediately adjacent serine residues 409/410 in the context of mCherry-CTF. Thus, Lys-408 ubiquitinylation appears to hinder Ser-409/410 phosphorylation in TDP-43 CTF. However, we did not observe the same effect for full-length TDP-43. We extended the mutagenesis study to full-length TDP-43 and performed MS. Ubiquitinylated lysine residues were identified in the nuclear localization sequence (NLS; Lys-84 and Lys-95) and RNA-binding region (mostly Lys-160, Lys-181, and Lys-263). Mutagenesis of Lys-84 confirmed its importance as the major determinant for nuclear import, whereas Lys-95 mutagenesis did not significantly affect TDP-43's nucleo-cytoplasmic distribution, solubility, aggregation, and RNA-processing activities. Nevertheless, the K95A mutant had significantly reduced Ser-409/410 phosphorylation, emphasizing the suspected interplay between TDP-43 ubiquitinylation and phosphorylation. Collectively, our analysis of TDP-43 ubiquitinylation sites indicates that the NLS residues Lys-84 and Lys-95 have more prominent roles in TDP-43 function than the more C-terminal lysines and suggests a link between specific ubiquitinylation events and pathological TDP-43 phosphorylation.
Highlights
TAR DNA-binding protein of 43 kDa (TDP-43) forms pathological aggregates in neurodegenerative diseases, in certain forms of frontotemporal dementia and amyotrophic lateral sclerosis
In our previous study [29], we observed a strong ubiquitinylation of a 35-kDa C-terminal fragment (CTF) of TDP-43 amino acids 193– 414 with an N-terminal mCherry tag
To isolate and detect ubiquitinylated TDP-43, we have previously established a protocol in which His6-ubiquitin– conjugated proteins are isolated with Ni-NTA affinity purification and analyzed by SDS-PAGE/Western blotting [29, 31]
Summary
TAR DNA-binding protein of 43 kDa (TDP-43) forms pathological aggregates in neurodegenerative diseases, in certain forms of frontotemporal dementia and amyotrophic lateral sclerosis. The TAR DNA-binding protein of 43 kDa (TDP-43) is the main protein component of pathological inclusions in the brain and spinal cord of almost all cases of amyotrophic lateral sclerosis (ALS) and in ϳ45% of frontotemporal lobar degeneration (FTLD-TDP) [1,2,3]. This ubiquitously expressed DNA/RNAbinding protein contains two highly conserved RNA recognition motifs (RRM1 and RRM2), a C-terminal low complexity, glycine-rich domain (GRD) that is important for its protein– protein interactions and pathological aggregation. S1–S5 and Table S1. 1 To whom correspondence should be addressed: Laboratory of Functional
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