H., C. for full-length TDP-43. We prolonged the mutagenesis study to full-length TDP-43 and performed MS. Ubiquitinylated lysine residues were recognized in the nuclear localization sequence (NLS; Lys-84 and KLRK1 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 impact TDP-43’s nucleo-cytoplasmic distribution, solubility, aggregation, and RNA-processing activities. Nevertheless, the K95A mutant experienced significantly reduced Ser-409/410 phosphorylation, emphasizing the suspected interplay between TDP-43 ubiquitinylation and phosphorylation. Collectively, our analysis of TDP-43 ubiquitinylation sites shows the NLS residues Lys-84 and Lys-95 have more prominent functions in TDP-43 function than the more C-terminal lysines and suggests a link between specific ubiquitinylation events and pathological TDP-43 phosphorylation. phosphorylation, ubiquitinylation, truncation, SUMOylation, acetylation, oxidation, and deamidation (1, 13,C15), have been studied to varying extent. As for phosphorylation, many sites of changes are known (6,C8, 13), and the impact on TDP-43 toxicity and aggregation, mainly of Ser-409/410 phosphorylation, was analyzed to a certain degree (15). In contrast to the varied TDP-43 phosphorylation sites, only few specific ubiquitinylation sites have been identified so far (Table 1), and their unique functions on solubility, localization, and aggregation and also their physiological functions are not recognized. Ubiquitinylations may not only influence protein degradation but can also affect protein localization, endocytosis, DNA restoration, and protein activity and may mediate proteinCprotein relationships (16, 17). Table 1 Published TDP-43 ubiquitinylation sites (30), although their inactivation did not alter the amount and solubility of total ubiquitinylated TDP-43, emphasizing that Hoechst 33258 trihydrochloride further TDP-43 lysine residues are ubiquitinylated under pathological conditions. Consequently, it is important to identify and characterize these specific ubiquitinylated lysine residues and how their changes affects localization, solubility, and aggregation of TDP-43. TDP-43 consists of 20 lysine residues, most of which are located in Hoechst 33258 trihydrochloride the N-terminal section and RRM1, and only few lysine residues reside in TDP-43 domains that are portion of truncated CTFs (Fig. 1His definitely6-ubiquitin was co-expressed with mCherry-control (HEK293E cells were transfected with His6-ubiquitin and mCherry-CTFWT, mCherry-CTFK408R, and mCherry-CTFK2/3/4R. After proteasomal inhibition with MG-132 for 2 h, cells were lysed and analyzed as with sequential extraction of HEK293E cells overexpressing mCherry-CTF lysine mutants and quantification. After 2 days of mCherry-CTF manifestation, RIPA- and urea-soluble fractions were prepared and analyzed by European blotting with antibodies against TDP-43, mCherry, and phospho-TDP-43 (Ser-409/410) and actin as loading control. quantification of protein levels recognized with anti-TDP ( 0.05 compared with mCherry-CTFWT. quantification of the aggregate formation of mCherry-CTF lysine mutants upon proteasomal inhibition (Fig. S1, and and and and and Fig. S1). Moreover, although we recognized decreased ubiquitinylation and/or insolubility of CTFK224R and CTF4KR, the number of cells expressing these mutants with aggregates was not reduced. A possible explanation for this might be that the amount of phosphorylated and aggregated CTF per cell is definitely altered but not the total cell figures that show aggregated TDP-43. In addition, we would like to point out that we also recognized many very tiny inclusions upon MG-132 treatment in cells that indicated the control mCherry protein, which are included in the quantification (Fig. 1and Fig. S1, and and and ?and2).2). Consequently, we asked whether there is a cross-talk between ubiquitinylation at Lys-408 and phosphorylation at Ser-409/410. To study the influence of the Ser-409/410 phosphorylation within the ubiquitinylation of mCherry-CTFK408R, we generated phosphorylation-mimic (SSDD) and phosphorylation-dead (SSAA) mCherry-CTFs in which serine residues 409/410 were mutated to aspartate and alanine residues, respectively. Additionally, the K408R mutation was launched into the Ser-409/410 mutants. Ni-NTA pulldown of ubiquitinylated CTF mutants showed comparably increased levels of ubiquitinylated CTFK408R as well as of CTFSSAA and CTFK408R/SSAA, but we did Hoechst 33258 trihydrochloride not detect a further increase of ubiquitinylation in the double mutant (Fig. 2). The anti-phospho-TDP-43 acknowledged CTFSSDD on Western blots, indicating the appropriateness of the phosphomimic substitution. Interestingly, CTFSSDD and CTFK408R/SSDD showed strongly decreased ubiquitinylation, but the steady-state levels of these phosphomimic CTF mutants were also reduced (Fig. 2). Therefore, Ser-409/410 phosphorylation appears not to be a priming changes enhancing ubiquitinylation of TDP-CTF. Open in a separate window Number 2. Cross-talk between C-terminal ubiquitinylation and phosphorylation of CTF. HEK293E cells were double-transfected with CTFWT, CTF4KR, CTFK408R, and the phospho-mimic- or -lifeless CTFSSDD or CTFSSAA double mutants (all CTF constructs fused to mCherry) or mCherry-control vector (?). After cell lysis with urea buffer, His6-ubiquitinylated proteins were affinity-purified with Ni-NTACagarose. Total protein (and label endogenous TDP-43,.