Fig 2: Pervanadate stimulation reduces CRL neddylation.(A) Representative immunoblot and (B) quantification of NEDD8 relative density from primary mouse CD4+ T cells stimulated with 100 µM pervanadate or incubated either with 3 µM MLN4924 for 2 h as a dennedylated control or 2 mM OPT for 30 min as a neddylated control (n = 3). (C) Representative immunoblot and (D) quantification of NEDD8 relative density from primary mouse CD43- B cells stimulated as in (B) (n = 3). (E) Representative immunoblot and quantification (F) of NEDD8 relative density from MEFs stimulated as in (B) (n = 3). (G) Representative immunoblot and (H) quantification of NEDD8 relative density from BT549 cells stimulated as in (B) (n = 3). (I) Representative immunoblot and (J) quantification of NEDD8 relative density from HT29 colon cancer cells stimulated as in (B) (n = 3). Values represent mean ± s.e.m.; *P<0.05, **P<0.01, ***P<0.001.

Fig 3: Abrogation of the COP9 signalosome and other pathways provides resistance to MLN4924. (A) Response to MLNL4924 treatment (72-h) upon CHUK, IRAK1, MIOS, or COPS4 invalidation in MSK-LX227C ex vivo cells. IC50s are shown. Data are means ± SEM, from at least n = 7 biological replicates. (****) P < 0.0001, unpaired Student's t-test. (B) Representative immunoblot analysis for NEDD8, showing the levels of neddylated cullins and for COPS4 in MSK-LX227C cells treated with DMSO or MLN4924 (100 nM) for 96 h. Parental MSK-LX227C ex vivo cells were compared with cells infected with an inducible shRNA targeting GFP or COPS4. On-dox cells were supplemented with 1 µg/mL doxycycline. GAPDH was used as loading control. (C) Quantification of relative levels of neddylated cullin protein abundance from multiple replicate immunoblot experiments described in B, showing combined parental MSK-LX227C and shGFP data compared with the combined data of two independent COPS4 shRNAs. Data are means ± SEM (n = 7 biological replicates). (**) P < 0.01, (****) P < 0.0001, unpaired Student's t-test. (D) Representative immunoblot analysis for the levels of neuroendocrine regulators and pro-apoptotic proteins for samples described in B. GAPDH was used as loading control. (E) Quantification of the relative levels of cleaved Caspase 7, cleaved PARP, INSM1, and ASCL1 protein abundance for multiple replicate immunoblot experiments described in D. Data are means ± SEM (n = 3 biological replicates). (**) P < 0.01, (***) P < 0.001, unpaired Student's t-test. For C and E, relative abundance levels calculated by normalization to individual GAPDH loading controls with the untreated parental MSK-LX227C set to a relative abundance of 1.

Fig 4: Genetic alleles of the NEDD8 pathway do not affect atypical NEDDylation(A) Mature NEDD8 and NEDD8?GG were overexpressed in CHO TS41 cells harbouring a temperature-sensitive allele of NAE, and incubated at the permissive (32°C) and restrictive (39°C) temperature, as indicated. A shift to the restrictive temperature does not affect atypical NEDDylation, suggesting that NEDD8 conjugates are independent of NAE. Importantly, cullin NEDDylation is strongly reduced at the restrictive temperature. Expression of NEDD8?GG does not induce atypical NEDDylation, indicating that smears observed upon mature NEDD8 expression correspond to conjugates to proteins. (B) Atypical NEDDylation by UBE1 is conserved in S. cerevisiae. A Western blot using an anti-HA antibody of cells expressing endogenous levels of untagged or HA-tagged scNEDD8 is shown. The bands detected correspond (by molecular mass) to NEDDylated cullin proteins. The asterisk denotes the antibody cross-reacting band. (C) HA–scNEDD8 forms ectopic conjugates upon overexpression (GAL promoter), whereas the ?GG version does not. (D) Deletions of the scNEDD8 E2 Ubc12 and E1 Uba3 affect cullin NEDDylation but not atypical NEDDylation, showing that atypical NEDDylation is strictly independent of the NEDD8 enzymes. (E) Overexpression of scNEDD8 in a temperature-sensitive UBA1 mutant (uba1–204) at the restrictive temperature diminishes atypical NEDDylation, indicating that the ubiquitin E1 enzyme is mediating the formation of atypical NEDD8 conjugates. Molecular masses are shown in kDa to the left-hand side of the Western blots.

Fig 5: Activation of neddylation in T cells upon TCR ligation and blood-stage P. yoelii 17XNL infection.(A) Immunoblotting of total cell extracts from splenic T cells of C57BL/6 mice stimulated with CD3/CD28 Dynabeads for various times (different parts of the same gel were grouped together with a dividing line). Uba3 and NEDD8-conjugated proteins were detected using anti-Uba3 and anti-NEDD8 antibodies, respectively. (B) Splenic T cells purified from C57BL/6 mice were incubated with MLN4924 (0.1, 0.5 µM) or DMSO (–) for 1h, then left untreated or stimulated with CD3/CD28 Dynabeads for 48h, NEDD8-conjugated cullins were detected by probing with anti-NEDD8 antibody. (C) Immunoblotting for Uba3 and NEDD8-conjugated proteins in splenic T cells isolated from C57BL/6 mice at indicated time points of blood-stage P. yoelii 17XNL infection, Uba3 and neddylated proteins were detected by probing with anti-Uba3 and anti-NEDD8 antibodies. Numbers indicate relative density of the bands quantified by scanning densitometry, normalized to ß-Actin and are presented relative to unstimulated cells (0 h) or uninfected mice (day 0). Data are representative of three independent experiments with similar results.