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| Home > Publications Database > Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43). |
| Home > Publications Database > Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43). |
| Journal Article | DZNE-2020-06592 |
; ; ; ;
2018
Soc.60645
Bethesda, Md.
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Please use a persistent id in citations: doi:10.1074/jbc.RA118.003440
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.
Keyword(s): Active Transport, Cell Nucleus (MeSH) ; Cell Nucleus: metabolism (MeSH) ; DNA-Binding Proteins: chemistry (MeSH) ; DNA-Binding Proteins: genetics (MeSH) ; DNA-Binding Proteins: metabolism (MeSH) ; HEK293 Cells (MeSH) ; Humans (MeSH) ; Lysine: chemistry (MeSH) ; Mutagenesis, Site-Directed (MeSH) ; Mutation (MeSH) ; Phosphorylation (MeSH) ; Protein Processing, Post-Translational (MeSH) ; Solubility (MeSH) ; Ubiquitin: metabolism (MeSH) ; DNA-Binding Proteins ; TDP-43 protein, human ; Ubiquitin ; Lysine
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