Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43)

Hans, Friederike; Eckert, Marita; Zweydorf, Felix von; Gloeckner, Christian Johannes; Kahle, Philipp J.

doi:10.1074/jbc.ra118.003440

Cited by 32 publications

(47 citation statements)

References 67 publications

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“…In addition, we confirm reduced binding of wild‐type but not K95A to importin‐α upon poly‐GA expression. Consistent with the data by Hans et al (), K84 mutants completely block NLS activity even in the absence of poly‐GA, and it is conceivable that ubiquitination at K84 may also inhibit nuclear import (Kim et al , ; Lumpkin et al , ; Akimov et al , ). However, removing K95 largely prevented ubiquitination in our assays, suggesting that K95 is the main ubiquitination site within the TDP‐43 NLS.…”

Section: Discussionsupporting

confidence: 82%

“…However, removing K95 largely prevented ubiquitination in our assays, suggesting that K95 is the main ubiquitination site within the TDP‐43 NLS. Interestingly, a recent study linked K95 ubiquitination to pathological phosphorylation at S409/410 (Hans et al , ). Boosting proteasome activity in poly‐GA‐expressing cells may allow more efficient degradation of TDP‐43 ubiquitinated at K95 (and other sites) and thus prevent accumulation of cytoplasmic TDP‐43.…”

Section: Discussionmentioning

confidence: 99%

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Cell‐to‐cell transmission of C9orf72 poly‐(Gly‐Ala) triggers key features of ALS / FTD

et al. 2020

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The C9orf72 repeat expansion causes amyotrophic lateral sclerosis and frontotemporal dementia, but the poor correlation between C9orf72-specific pathology and TDP-43 pathology linked to neurodegeneration hinders targeted therapeutic development.Here, we addressed the role of the aggregating dipeptide repeat proteins resulting from unconventional translation of the repeat in all reading frames. Poly-GA promoted cytoplasmic mislocalization and aggregation of TDP-43 non-cell-autonomously, and anti-GA antibodies ameliorated TDP-43 mislocalization in both donor and receiver cells. Cell-to-cell transmission of poly-GA inhibited proteasome function in neighboring cells. Importantly, proteasome inhibition led to the accumulation of TDP-43 ubiquitinated within the nuclear localization signal (NLS) at lysine 95. Mutagenesis of this ubiquitination site completely blocked poly-GA-dependent mislocalization of TDP-43. Boosting proteasome function with rolipram reduced both poly-GA and TDP-43 aggregation. Our data from cell lines, primary neurons, transgenic mice, and patient tissue suggest that poly-GA promotes TDP-43 aggregation by inhibiting the proteasome cell-autonomously and non-cell-autonomously, which can be prevented by inhibiting poly-GA transmission with antibodies or boosting proteasome activity with rolipram.

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Section: Discussionsupporting

confidence: 82%

Section: Discussionmentioning

confidence: 99%

Cell‐to‐cell transmission of C9orf72 poly‐(Gly‐Ala) triggers key features of ALS / FTD

et al. 2020

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“…In addition to Znf179, CUL2 ubiquitin ligase can modify misfolded TDP-43 with poly-ubiquitin, coordinately with von Hippel Lindau protein (VHL) [ 95 ]. Mass spectrometry analysis identified TDP-43 ubiquitination sites to be Lys-84, Lys-95 Lys-160, Lys-181, and Lys-263 residues and that its poly-ubiquitin chains link via Lys-48 and Lys-63 [ 96 ]. SOD1 is targeted by NEDL1 and gp78 ubiquitin ligases.…”

Section: Ubiquitination Of Neurodegenerative Disease-associated Prmentioning

confidence: 99%

Ubiquitin, Autophagy and Neurodegenerative Diseases

Watanabe

Taguchi

Tanaka

2020

Cells

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Ubiquitin signals play various roles in proteolytic and non-proteolytic functions. Ubiquitin signals are recognized as targets of the ubiquitin–proteasome system and the autophagy–lysosome pathway. In autophagy, ubiquitin signals are required for selective incorporation of cargoes, such as proteins, organelles, and microbial invaders, into autophagosomes. Autophagy receptors possessing an LC3-binding domain and a ubiquitin binding domain are involved in this process. Autophagy activity can decline as a result of genetic variation, aging, or lifestyle, resulting in the onset of various neurodegenerative diseases. This review summarizes the selective autophagy of neurodegenerative disease-associated protein aggregates via autophagy receptors and discusses its therapeutic application for neurodegenerative diseases.

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“…1b). Thus, acetylation of K84 partially reduced nuclear import of TDP-43 whereas the acetyl-mimic K79Q substitution did not show evident changes in nucleocytoplasmic distribution of TDP-43, likely because K79 is localized just outside the NLS, whereas K84 is at the core of the NLS 6,15 .…”

Section: Resultsmentioning

confidence: 91%

“…While investigating the sites of TDP-43 lysine ubiquitinations 15 , we detected in the mass spectrometry (MS) measurements another lysine modification, namely acetylation (Supplementary Table 1). In transfected HEK293E cells, 6xHis-tagged wtTDP-43 was acetylated at lysine residues K79 and K84 around the NLS and K121 and K136 in RRM1 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Sirtuin-1 Sensitive Lysine-136 Acetylation Drives Phase Separation and Pathological Aggregation of TDP-43

Morato

Hans

Zweydorf

et al. 2020

Preprint

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The trans-activation response DNA-binding protein TDP-43 regulates RNA processing and forms neuropathological aggregates in patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Investigating TDP-43 post-translational modifications, we discovered that K84 acetylation reduced nuclear import whereas K136 acetylation impaired RNA binding and splicing capabilities of TDP-43. Such failure of RNA interaction triggered TDP-43 phase separation mediated by the C-terminal low complexity domain, leading to the formation of insoluble aggregates with pathologically phosphorylated and ubiquitinated TDP-43. Confirming the results from site-directed mutagenesis, we succeeded to introduce authentic acetyl-lysine at the identified sites via amber suppression.[AcK84]TDP-43 showed cytoplasmic mislocalization and the aggregation propensity of [acK136]TDP-43 was confirmed. With newly developed antibodies, we found that the nuclear sirtuin SIRT1 can potently deacetylate [acK136]TDP-43. Moreover, SIRT1 reduced the aggregation propensity of [acK136]TDP-43. Thus, distinct lysine acetylations modulate nuclear import, RNA binding and phase separation of TDP-43, suggesting novel regulatory mechanisms for TDP-43 pathogenesis.the biochemical assay (compare Fig. 1d with Fig. 1b). Thus, acetylation of K84 partially reduced nuclear import of TDP-43 whereas the acetyl-mimic K79Q substitution did not show evident changes in nucleocytoplasmic distribution of TDP-43, likely because K79 is localized just outside the NLS, whereas K84 is at the core of the NLS 6,15 . The RRM1 mutants K136R and K136Q were predominantly localized in the nucleus, but a significant number of cells displayed a droplet-like pattern of nuclear aggregates (Fig. 1b,c). This droplet-like nuclear distribution was reminiscent of the RNA-binding deficient F147L/F149L mutant TDP-43 27 . For comparison, we generated the previously described 17,18 acetyl-mimic K145Q mutant in RRM1, which showed the expected stippled distribution (Fig. 1b). As K136 is in direct contact with bound nucleic acids 28,29 , we assume that modifications at K136 disrupt nucleic acid binding and therefore disengage TDP-43 from hnRNP complex localizations. Such dissociated K136-modified TDP-43 may be free to self-aggregate into inclusions. K136 mutant TDP-43 is pathologically ubiquitinated, phosphorylated, and insolubleWe examined if the K136 mutant TDP-43 inclusions in cell culture also showed pathological features described for human patients 4,5,13,30 . 6xHis-tagged TDP-43 variants were transiently transfected into HEK293E cells with stable knockdown of endogenous TDP-43 (sh TDP-43 ) 31 to minimize interference from the endogenous wtTDP-43. To assess protein solubility, RIPA-urea solubility fractionation assays were performed. While most of the 6xHis-tagged wtTDP-43 was RIPA-soluble, a larger portion of both K136R and K136Q TDP-43 mutants shifted into the RIPA-insoluble fraction (Fig. 2a). Although [F147L/F149L]TDP-43 formed similar intranuclear patches 27 , this designed RNA-...

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Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43)

Cited by 32 publications

References 67 publications

Cell‐to‐cell transmission of C9orf72 poly‐(Gly‐Ala) triggers key features of ALS / FTD

Cell‐to‐cell transmission of C9orf72 poly‐(Gly‐Ala) triggers key features of ALS / FTD

Ubiquitin, Autophagy and Neurodegenerative Diseases

Sirtuin-1 Sensitive Lysine-136 Acetylation Drives Phase Separation and Pathological Aggregation of TDP-43

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