TY  - JOUR
AU  - Hans, Friederike
AU  - Eckert, Marita
AU  - von Zweydorf, Felix
AU  - Gloeckner, Christian Johannes
AU  - Kahle, Philipp J
TI  - Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43).
JO  - The journal of biological chemistry
VL  - 293
IS  - 41
SN  - 0021-9258
CY  - Bethesda, Md.
PB  - Soc.60645
M1  - DZNE-2020-06592
SP  - 16083-16099
PY  - 2018
AB  - 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.
KW  - Active Transport, Cell Nucleus
KW  - Cell Nucleus: metabolism
KW  - DNA-Binding Proteins: chemistry
KW  - DNA-Binding Proteins: genetics
KW  - DNA-Binding Proteins: metabolism
KW  - HEK293 Cells
KW  - Humans
KW  - Lysine: chemistry
KW  - Mutagenesis, Site-Directed
KW  - Mutation
KW  - Phosphorylation
KW  - Protein Processing, Post-Translational
KW  - Solubility
KW  - Ubiquitin: metabolism
KW  - DNA-Binding Proteins (NLM Chemicals)
KW  - TDP-43 protein, human (NLM Chemicals)
KW  - Ubiquitin (NLM Chemicals)
KW  - Lysine (NLM Chemicals)
LB  - PUB:(DE-HGF)16
C6  - pmid:30120199
C2  - pmc:PMC6187624
DO  - DOI:10.1074/jbc.RA118.003440
UR  - https://pub.dzne.de/record/140270
ER  -