Publications in Journal of Biological Chemistry by NOMIS researchers

GGGGCC (G4C2) repeat expansion in the C9orf72 gene has been shown to cause frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Dipeptide repeat proteins produced through repeat-associated non-AUG (RAN) translation are recognized as potential drivers for neurodegeneration. Therefore, selective inhibition of RAN translation could be a therapeutic avenue to treat these neurodegenerative diseases. It was previously known that the porphyrin TMPyP4 binds to G4C2 repeat RNA. However, the consequences of this interaction have not been well characterized. Here, we confirmed that TMPyP4 inhibits C9orf72 G4C2 repeat translation in cellular and in in vitro translation systems. An artificial insertion of an AUG codon failed to cancel the translation inhibition, suggesting that TMPyP4 acts downstream of non-AUG translation initiation. Polysome profiling assays also revealed polysome retention on G4C2 repeat RNA, along with inhibition of translation, indicating that elongating ribosomes stall on G4C2 repeat RNA. Urea-resistant interaction between G4C2 repeat RNA and TMPyP4 likely contributes to this ribosome stalling and thus to selective inhibition of RAN translation. Taken together, our data reveal a novel mode of action of TMPyP4 as an inhibitor of G4C2 repeat translation elongation.

Insoluble, hyperubiquitylated TAR DNA-binding protein of 43 kDa (TDP-43) in the central nervous system characterizes frontotemporal dementia and ALS in many individuals with these neurodegenerative diseases. The causes for neuropathological TDP-43 aggregation are unknown, but it has been suggested that stress granule (SG) formation is important in this process. Indeed, in human embryonic kidney HEK293E cells, various SG-forming conditions induced very strong TDP-43 ubiquitylation, insolubility, and reduced splicing activity. Osmotic stress-induced SG formation and TDP-43 ubiquitylation occurred rapidly and coincided with colocalization of TDP-43 and SG markers. Washout experiments confirmed the rapid dissolution of SGs, accompanied by normalization of TDP-43 ubiquitylation and solubility. Surprisingly, interference with the SG process using a protein kinase R-like endoplasmic reticulum kinase inhibitor (GSK2606414) or the translation blocker emetine did not prevent TDP-43 ubiquitylation and insolubility. Thus, parallel pathways may lead to pathological TDP-43 modifications independent of SG formation. Using a panel of kinase inhibitors targeting signaling pathways of the osmotic shock inducer sorbitol, we could largely rule out the stress-activated and extracellular signal-regulated protein kinase modules and glycogen synthase kinase 3β. For arsenite, but not for sorbitol, quenching oxidative stress with N-acetylcysteine did suppress both SG formation and TDP-43 ubiquitylation and insolubility. Thus, sodium arsenite appears to promote SG formation and TDP-43 modifications via oxidative stress, but sorbitol stimulates TDP-43 ubiquitylation and insolubility via a novel pathway(s) independent of SG formation. In conclusion, pathological TDP-43 modifications can be mediated via multiple distinct pathways for which SGs are not essential.

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.