Fig 2: Molecular modeling indicates NEDD8 binds to parkin in the same position as P-Ub and opens up the UBL domain in a manner similar to P-Ub. a Molecular interactions for P-Ub with parkin at the P-Ub binding helix agree with the previous experimental model of P-Ub interaction with parkin [1] (b). All-atom metadynamics simulation of the P-Ub-parkin complex predicts the movement of the UBL domain exposing the Ser65 from the auto-inhibited form of parkin (c–e). Protein-protein docking predicts NEDD8 interacts with the auto-inhibited form of parkin similar to that of P-Ub (f). Molecular interaction analysis indicates that NEDD8 binds to parkin at the same location as P-Ub, i.e., at the P-Ub binding helix (g). All-atom metadynamics simulation of the NEDD8-parkin complex, like that of P-Ub, also opened the UBL domain, exposing Ser65 (h–j)

Fig 3: Cytoplasmic NEDD8 levels are higher in hippocampal pyramidal neurons in Alzheimer brain than in analogous neurons in control brains, and parkin and NEDD8 are colocalized in a disease-specific manner. a Compared with their age-matched control (AMC) counterparts (n = 5), b nuclei (blue) of neurons in Alzheimer patients (n = 6) have less NEDD8 (green) (**p < 0.005) (c). d Moreover, in representative images of hippocampal pyramidal cells from these AMC and AD patients (e), colocalization of NEDD-8/Parkin (red dots depicted by PLA represent proximity of ≤ 40 nm) is less in AMC than in analogous neurons in Alzheimer patients *p < 0.05 on Wilcoxon rank sum test, n = 3 per group

Fig 4: ATP-depletion resulted in the inhibition of CSN deneddylase activity. a 10 nM His-6-Cul1/ROC1 were pre-neddylated for 30 min at 30°C. Then 10 μg HeLa cells extract were added at 30°C (time plot: lanes 3–6); or alternatively, ATP-trap mix was added for 45 min at 30°C, followed by the addition of 10 μg HeLa cells extract at 30°C (time plot: lanes 8–11). b 3 nM His-6-Cul1/ROC1 was pre-neddylated for 30 min at 30°C. Then 100 nM purified CSN was added at 20°C (time plot: lanes 3–6); or alternatively, ATP-trap mix was added for 45 min at 30°C, followed by the addition of 100 nM purified CSN at 20°C (time plot: lanes 8–11). c 10 nM His-6-Cul1/ROC1 was incubated as described in b; time plots were for 1.5–7.5 min, and not for 5–40 min as in b. Blotting was directed against NEDD8, and not against Cul1, as described elsewhere.

Fig 5: NLRX1-mediated ROS controls NF-?B signalling and affects cullin 1 NEDDylation.Immunofluorescence microscopy of A549 cells stained with antibody for the p65 NF-?B subunit. Cells were pre-treated with mitoTEMPO (10 µM, 2 h pre-infection), or with vehicle control (DMSO) and infected with Kp52145 for 3 h (a). b Cells were transfected with NLRX1 siRNA (50 nM) or a non-silencing control (AS) and infected with Kp52145 for 3 h. c Cells were infected with Kp52145, the vgrG4 mutant (?vgrG4) or the complemented strain (comp, ?vgrG4/pBAD30vgrG4) for 3 h. The percentage of p65 NF-?B localised in the nucleus is represented on the graph in a–c and it is the result of counting of minimum of hundred cells from each of three independent experiments. The total number of counted cells is indicated on top of each bar. ELISA of IL-8 secreted by A549 cells transfected with either NLRX1 siRNA (50 nM) or a non-silencing control (AS) and infected with K. pneumoniae strains. After 3 h of contact, the medium was replaced with medium containing gentamicin (100 µg/mL) to kill extracellular bacteria, and after 2 h the medium was collected (d). Images are representative of three independent experiments. Data in graphs are presented as the mean ± SD of three independent experiments. Two way-ANOVA with Holm-Sidak’s multiple comparisons test was used for statistical significance (p values indicated in the graph). e Immunoblot analysis of total I?Ba and tubulin levels in lysates of A549 cells infected with Kp52145 for the indicated times. f Immunoblot analysis of phosphorylated I?Ba and tubulin levels in lysates of A549 cells infected with Kp52145 for the indicated times. g Levels of phosphorylated I??a/ß and tubulin in cells infected with Kp52145 for the indicated times. I?Ba immunoprecipitation and immunoblot for K48-linkage specific polyubiquitin (Ub48) in cells treated with the proteasome inhibitor MG262 (5 µM, 2 h before infection), and infected with Kp52145 for 3 h or left uninfected (n.i.). Pre-immune mouse IgG was used as a control for immunoprecipitation (h). Immunoblot analysis of Cul-1 and tubulin levels in lysates of A549 cells infected with Kp52145 for 5 h, or treated with H2O2 (5 µM, 5 min). Cul-1 appears as a doublet, with the higher molecular band representing the NEDDylated form of Cul-1 (i). j Immunoblot analysis of Cul-1 and tubulin levels in lysates of A549 cells transfected with NLRX1 siRNA (50 nM) or a non-silencing control (AS), infected with Kp52145 for 5 h. k Cells were infected with Kp52145, the vgrG4 mutant (?vgrG4) or the complemented strain (comp) for 5 h, and the levels of Cul-1 and tubulin assessed by western blot. l Cells were pre-treated with mitoTEMPO (10 µM, 2 h pre-infection) or a vehicle control (DMSO), infected with Kp52145 for 5 h, and Ubc12 was immunoprecipitated followed by detection of NEDD8 and Ubc12 levels by immunoblotting. Images are representative of three independent experiments.