SENP8 limits aberrant neddylation of NEDD8 pathway components to promote cullin-RING ubiquitin ligase function
- New York University School of Medicine, United States
- Memorial Sloan Kettering Cancer Center, Unites States
Figures
Figure 1
Expression of a deconjugation-resistant NEDD8 mutant (L73P) stabilizes neddylation of cullins and other non-cullin substrates.
(A) Schematics of the regulation of NEDD8 substrates by modification with either WT- (left panel) or L73P-Nedd8 (right panel), and deneddylation by NEDD8-specific proteases. CSN is the deneddylase …
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Figure 1—source data 1
NEDD8- modified peptides identified by MS analysis of FLAG-NEDD8 IP samples.
- https://doi.org/10.7554/eLife.24325.003
Figure 2
Nedd8 L73P is conjugated to Ubc12 in vitro and in cells.
(A) An HCD MS2 spectrum of the doubly charged Ubc12 N-terminal peptide, MIKLFSLK, N-terminally acetylated and K-ε-GG modified on Lys3 (top) and sequence alignment of Ubc12 N-termini from the …
Figure 3 with 2 supplements
SENP8 regulates Ubc12 deneddylation.
(A) HeLa cells were treated with control, CSN5, or SENP8 siRNAs for 48 hr prior to harvesting. Whole-cell lysates were analyzed by immunoblotting for the indicated proteins. (B) SENP8 knockout …
Figure 3—figure supplement 1
Detection of NEDD8-modified Ubc12 in SENP8-deficient cells.
(A) Whole-cell lysates of parental and SENP8-deficient HEK 293 T cells were incubated with or without Ubc12 antibody, and immune complexes were captured using Protein G beads. Input and IP samples …
Figure 3—figure supplement 2
Modification of Ubc12 with NEDD8-L73P is dependent on Ubc12 catalytic activity.
(A) In vitro neddylation reactions were performed with WT, NK0, or catalytically inactive (C111A) Ubc12 in the presence of E1, WT NEDD8, and ATP. As controls, reactions were performed with Ubc12 …
Figure 4 with 1 supplement
Identification of endogenous neddylation substrates in SENP8-deficient cells.
(A) Lysates from parental and CRISPR-generated SENP8 knockout HEK293T cells were immunoblotted for the indicated proteins. For this figure and all subsequent figures, MCM7 serves as a loading …
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Figure 4—source data 1
SILAC analysis of K-ε-GG remnant-containing peptides detected in untreated parental and SENP8 knockout cell lysates.
- https://doi.org/10.7554/eLife.24325.009
Figure 4—figure supplement 1
Statistical information related to K-ε-GG MS screen comparing untreated parental and SENP8 knockout cell lysates (Figure 4B).
(A) Quantification of K-ε-GG sites, peptides, proteins, and peptide spectrum matches (PSMs) in replicates 1 and 2 of Figure 4B. (B) Venn diagram showing degree of overlap between K-ε-GG remnant …
Figure 5 with 1 supplement
Loss of SENP8 leads to aberrant neddylation of NEDD8 pathway components.
(A) Schematic of the NEDD8 conjugation pathway. Dotted arrows represent potential points of regulation by SENP8-mediated deneddylation. Orange circle represents NEDD8. (B and C) Cellular extracts …
Figure 5—figure supplement 1
Correction of DCN1 binding and cullin neddylation defects in SENP8-deficient cells.
(A) SENP8-deficient HEK293T cells were transfected with either EV or SENP8 WT plasmids, harvested, and endogenous Ubc12 was immunoprecipitated from lysates using anti-Ubc12 antibody. Relative …
Figure 6
SENP8 depletion contributes to altered cell cycle progression and cell growth.
(A) SENP8 knockout (lanes 2–4) and SENP8-rescued (lanes 5–6) HeLa cell lines were generated as specified in Materials and methods. Whole-cell lysates were analyzed by immunoblotting with the …
Figure 7 with 2 supplements
Loss of SENP8 contributes to decreased ubiquitylation and increased stability of CRL substrates.
(A) Workflow for identification of K-ε-GG-modified peptides in MG132-treated WT and SENP8 knockout cells by antibody-based enrichment and quantitative SILAC-MS. Light- and Heavy-labeled HEK293T …
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Figure 7—source data 1
SILAC analysis of K-ε-GG remnant-containing peptides detected in MG132-treated parental and SENP8 knockout cell lysates.
- https://doi.org/10.7554/eLife.24325.015
Figure 7—figure supplement 1
Statistical information related to K-ε-GG MS screen comparing MG132-treated parental and SENP8 knockout cell lysates (Figure 7B).
(A) Quantification of K-ε-GG sites, peptides, proteins, and PSMs in replicates 1 and 2 of Figure 7B. (B) Venn diagram showing degree of overlap between K-ε-GG remnant peptides quantified in …
Figure 7—figure supplement 2
SENP8-deficient cells show increased stability and reduced ubiquitylation of CRL substrates.
(A) Parental and SENP8 knockout HeLa cells were treated with 30 µg/ml cycloheximide (CHX) for the indicated times and subjected to immunoblotting analysis for Set8 and UHRF1 levels. As a control, …
Figure 8
Model.
A model depicting the role of SENP8 in regulating reversible neddylation of NEDD8 conjugation pathway components.
