Fig 2: Evolutionary analyses of UBA1 and UBA6 protein sequences. (A) Convergence of distributions of sampled parameter values of the evolutionary model across the four independent MrBayes runs. The effective sample size (ESS) value per parameter and run is in all cases much greater than the usually suggested minimum value of 200, indicating excellent parameter sampling. (B) Comparison of split frequencies among the four MrBayes runs. The Pearson's correlation coefficient values of 1 and the average standard deviations of split frequencies (ASDSF) that are well below 0.01 are evidence of sufficient topological convergence. (C) Evolution of cumulative average change in split frequencies during the course of each MrBayes run. Dots indicate the average change, whereas ribbons show the 100%, 95%, and 75% quantiles of the change in split frequencies. As each run independently converges on a solution, the change in split frequencies drops to nearly zero. (D) Two-dimensional projection of tree topologies explored by each MrBayes run. The high degree of similarity among plots constitutes another proof of topological convergence. (E) Heatmap of Matching Split distances (Bogdanowicz & Giaro, 2012a) among the three resulting trees and a non-phylogenetic star topology as a reference point. Overall, there are very few differences among the three trees, with the maximum likelihood ones being barely more similar to each other compared with the Bayesian tree. (F) Topological congruence among the trees produced by the three algorithms. The topology shown here is that of the MrBayes tree, as the Bayesian algorithm performed a more thorough exploration of parameter and tree space. Branches are coloured based on the taxonomic group that they belong to. Statistical support—in terms of posterior probabilities or bootstrap values—for each node across all trees is denoted with black, grey, or white circles. Nodes with black or grey circles are considered robust.

Fig 3: Model for the formation of ATR-signalling bodies.Upon addition of a gapped DNA substrate in human cell-free extracts, poly(ADP-ribosyl)ation accelerates the recruitment of DNA damage signalling proteins, including the ubiquitin-activating enzyme UBA1. The coupling of pADPr-seeded protein assembly with protein ubiquitylation and protein phosphorylation prevents the formation of nonfunctional protein aggregates and promotes dedicated protein–protein interactions required for ATR signalling.

Fig 4: Identification of the molecular target of TAK-243 affecting F508del-CFTR cellular amount. A F508del-CFTR expressing CFBE41o- cells were transfected with non-specific siRNA (Scr), or UBA1 or UBA6-specific siRNAs for 48 h and lysed. Lysate proteins were analyzed by western blot with the indicated antibodies. Calnexin (Clxn) was used as loading control (n = 4) (left panel). Evaluation of F508del-CFTR activity was performed by HS-YFP assay in F508del-CFTR expressing CFBE41o- cells treated as indicated above. Centre panel exhibits representative traces measuring YFP quenching (n = 8); right panel shows the CFTR activity as a percentage of control (Scr) (means ± SD values, n = 8; *p < 0.05 vs Ctrl). B Immunoblot analysis of CFTR and NEDD8-Cullin in whole lysates from F508del-CFTR expressing CFBE41o- cells treated with DMSO (Ctrl) or increasing concentrations of Pevonedistat for 24 h. a-tubulin was used as loading control (n = 3) (left panel). Assessment of F508del-CFTR activity was carried out by HS-YFP assay in F508del-CFTR expressing CFBE41o- cells treated as indicated above or with double-corrector treatment (DCT = 10 µM VX-661 + 3 µM VX-445) for 24 h. Centre panel exhibits representative traces measuring YFP quenching (n = 8); right panel shows the CFTR activity as a percentage of control (Ctrl) (means ± SD values, n = 8; *p < 0.05 vs Ctrl). C F508del-CFTR expressing CFBE41o- cells were treated with DMSO (Ctrl) or TAK-243 (200 nM) for 24 h. Subsequently, protein synthesis was inhibited by adding 100 µg/ml cycloheximide (CHX) and cells were harvested after 2, 4, and 8 h. Protein lysates were analyzed by western blot with anti-CFTR antibody. Calnexin (Clxn) was used as a loading control. The rightmost panel represents the densitometric quantification of the immunostained bands of F508del-CFTR (band B) normalized by the value at time = 0 (means ± SD values, n = 4; *p < 0.05 vs the value of the same time-point of Ctrl). D F508del-CFTR expressing CFBE41o- cells were treated with DMSO or 200 nM TAK-243 and lysate after 24 h. 300 µg of lysate proteins were immunoprecipitated with a control antibody from the same class (Ctrl) or anti-CFTR (CFTR) antibody. The immunocomplexes were analyzed by western blot with the indicated antibodies (left panel). The figure panel is representative of four independent experiments. A long exposition of CFTR detection is also shown (i.e., long exposition). The central panels show representative density profiles of CFTR and ubiquitin in the CFTR-immunoprecipitated samples. Quantification of the density profiles was performed in the right panels by integrating the profile curves in the indicated intervals of molecular weight (Ubiquitin: 220–350 kDa; Ub-CFTR: 220–350 kDa; CFTR band B: 130–150 kDa) (means ± SD; n = 4; *p < 0.05 vs Ctrl). E Immunoblot analysis of CFTR in whole lysates derived from F508del-CFTR expressing CFBE41o- cells treated with DMSO (Ctrl, black) or TAK-243 (200 nM) (orange) or VX-809 (3 µM) (green), following heat-denaturation at indicated temperatures (left panels and central panel). Right panel reports the densitometric quantification of aggregation-resistant F508del-CFTR band B, normalized by HSP90 expression (means ± SD; n = 3; *p < 0.05 vs the value of the same temperature-point of Ctrl). CFTR melting temperature (mean ± SD): Ctrl = 41.96 ± 2.35 °C; TAK-243 = 42.64 ± 2.60 °C; VX-809 = 49.73 ± 3.47 °C

Fig 5: Analysis of UBA1 and UBA6 protein sequences.(A) The phylogenetic tree of UBA1 and UBA6 protein sequences, as inferred with MrBayes. The clustering of UBA1 orthologues is in good agreement with the phylogeny of eukaryotic subgroups. Note that evolutionary distance separates UBA1 sequences from the UBA6 out-group. (B) Alignment of the region 646–703 from the H. sapiens UBA1, and the corresponding regions of ScUba1 and H. sapiens UBA6, extracted from a larger alignment of 110 UBA1 and UBA6 sequences. The stars correspond to identical residues. The positions of nonidentical residues that were selected for mutagenesis are highlighted in yellow. (C) Structural comparison of a fragment of the Mus musculus UBA1 (H. sapiens UBA1 crystal structure is not available) and the S. cerevisiae orthologue. The seven amino acids that were chosen for mutagenesis are explicitly shown in space-filling representation. The superposition of the two proteins (bottom) highlights their close structural similarity.