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. 2018 May 17;8(1):7754.
doi: 10.1038/s41598-018-25150-z.

Quantitative SUMO proteomics reveals the modulation of several PML nuclear body associated proteins and an anti-senescence function of UBC9

Francis P McManus  1 , Véronique Bourdeau  2 , Mariana Acevedo  2 , Stéphane Lopes-Paciencia  2 , Lian Mignacca  2 , Frédéric Lamoliatte  1   3 , John W Rojas Pino  2 , Gerardo Ferbeyre  4 , Pierre Thibault  5   6
Affiliations

Quantitative SUMO proteomics reveals the modulation of several PML nuclear body associated proteins and an anti-senescence function of UBC9

Francis P McManus et al. Sci Rep. .

Abstract

Several regulators of SUMOylation have been previously linked to senescence but most targets of this modification in senescent cells remain unidentified. Using a two-step purification of a modified SUMO3, we profiled the SUMO proteome of senescent cells in a site-specific manner. We identified 25 SUMO sites on 23 proteins that were significantly regulated during senescence. Of note, most of these proteins were PML nuclear body (PML-NB) associated, which correlates with the increased number and size of PML-NBs observed in senescent cells. Interestingly, the sole SUMO E2 enzyme, UBC9, was more SUMOylated during senescence on its Lys-49. Functional studies of a UBC9 mutant at Lys-49 showed a decreased association to PML-NBs and the loss of UBC9's ability to delay senescence. We thus propose both pro- and anti-senescence functions of protein SUMOylation.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Method for SUMO Site Identification. (a) Amino acid sequence of human SUMO3 and the modified SUMO3 (SUMO3m) used in this work. A 6xHis tag was introduced at the protein N-terminus for Ni-NTA purification. Q87R and Q88N alterations were introduced for trypsin cleavage and peptide identification purposes, respectively. (b) Western blots for 6xHis, SUMO2/3, histone H3 or tubulin of total cell extract (TCE), cytoplasmic fraction (CYT) and nuclear enriched fraction (NUC) from U2OS cells expressing SUMO3m and an empty vector (Control) or H-RAS-G12V (RAS). (c) Workflow adopted for SUMO3 site identification and proteome quantification. U2OS cells expressing SUMO3m were transduced with either an empty or RAS expressing vector. Cells were collected 10 days after transduction and fractionated under hypotonic conditions. For SUMO site identification, the nuclear enriched fractions were subjected to Ni-NTA purification, followed by trypsin digestion and finally subjected to immunopeptide enrichment with an antibody recognizing the NQTGG SUMO remnant left on the peptide backbone. For proteome analysis, the nuclear proteins were digested with trypsin, desalted and fractionated by PGC. The resulting peptides were analyzed on a Q-exactive plus mass spectrometer and data analyzed using MaxQuant.
Figure 2
Figure 2
Western blot validation of SUMO targets regulated in senescence. (a) Ponceau-S staining of 10 μg of nuclear enriched proteins (Crude) transferred on nitrocellulose showing the constant loading between U2OS expressing SUMO3m plus a control vector (Control) or a vector expressing H-RAS-G12V (RAS) to induce senescence. Extracts from both conditions were subjected to Ni-NTA purification to purify proteins SUMOylated with SUMO3m and used for blotting in panels c through g. (b) Anti-RAS blotting depicting the increased levels of steady state RAS in the senescent cells (crude extracts as in a). (c,d) Immunoblots showing an increase in total protein and in SUMOylated PML (c) and SP100 (d) in the senescent samples (cells as in a). (e) Anti-UBC9 blot showing an increased SUMOylated UBC9 in the senescent samples (cells as in a). (f–g) Immunoblots showing a decrease in SUMOylated TRIM28 (f) and in SUMOylated HDAC1 (g) in the senescent samples (cells as in a). Histone H4 serves as a loading control for crude extracts in (b–g).
Figure 3
Figure 3
SUMOylated Protein Network. (a) STRING network of all SUMOylated proteins with a high interaction confidence (0.7 or greater). Proteins whose SUMOylation sites were statistically increased upon RAS-mediated senescence are represented in green and those whose SUMOylation sites were decreased are shown in red (p-value < 0.05). The size of the nodes correlate to the number of interactors while the size of the edge depicts the confidence of the interactions. Highlighted in grey is the highest scoring cluster extracted using MCODE. (b) Confocal immunofluorescence with anti-SP100 and anti-PML antibodies to show colocalization and induction of PML-NBs upon senescence in U2OS cells expressing SUMO3m plus a control vector (control) or a vector expressing RAS (scale bar, 10 μm). (c) Confocal immunofluorescence with anti-HSPA5 and anti-PML antibodies to probe for colocalization upon senescence of cells as in (b) (scale bar, 10 μm).
Figure 4
Figure 4
Post-Translational Modifications on UBC9 in U2OS cells Identified by IP and LC-MS/MS. (a) UBC9 is a heavily post-translationally modified protein as shown by the distribution of SUMOylation, Ubiquitylation and Acetylation sites identified on UBC9 from our IP and LC-MS/MS experiments. (b) Modified sites depicted on the UBC9 crystal structure (3UIP), using the color scheme from (a). (c) Model of the interaction brought about by the SUMOylation of UBC9 at Lys-49 and PML as an example of a protein with both SUMOylated site and SIM (SUMO interacting motif). (d) Quantification for the colocalization of UBC9 and PML were obtained from immunofluorescence analyses using monoclonal mouse anti-Flag and rabbit anti-PML antibodies in U2OS cells stably expressing SUMO3m with wild type UBC9 (F-Ubc9 WT) or its K49R variant (F-Ubc9K49R) and with an empty vector (Vector) or a vector expressing RAS. Cells were fixed for immunofluorescence ten days after transduction. (e) Quantification of the number of PML-NBs in U2OS cells when expressing SUMO3m with wild type UBC9 (F-Ubc9 WT) or its K49R variant (F-Ubc9K49R) and with an empty vector (Vector) or a vector expressing RAS.
Figure 5
Figure 5
UBC9 Can Exhibit Anti-senescent Properties when Forced to PML-NBs. (a) Growth curves of U2OS cells expressing SUMO3m and an Er-H-RAS-G12V construct (fusion of the ligand-binding domain of the estrogen receptor with H-RAS-G12V to control its activity) (Er-RAS) and transduced with a control vector, Flag-Ubc9 wild type (F-Ubc9 WT) or Flag-Ubc9 with the K49R mutation (F-Ubc9K49R). Cells grew normally in the absence of the inducer for Er-RAS (control, grey curves) but enter senescence upon Er-RAS activation with 4-hydroxytamoxifen (OHT, 100 nM changed every two days, black curves). (b) Growth curves of U2OS cells expressing SUMO3m and an Er-H-RAS-G12V (Er-RAS) construct and transduced with a control vector, a GFP-PML fusion, a fusion of PML with wild type Ubc9 (F-Ubc9 WT-PML) or its K49R variant (F-Ubc9K49R-PML). Cells were either control treated (grey curves) to observe growth effect of the fusions alone or treated with 4-hydroxytamoxifen (OHT, 100 nM changed every two days, black curves) to observe the effects of the fusion in the context of RAS-induced senescence. (c) Growth curves of U2OS cells transduced with SUMO3m and a control vector, a GFP-PML fusion, a fusion of PML with wild type Ubc9 (F-Ubc9 WT-PML), its K49R variant (F-Ubc9K49R-PML) or the catalytic mutant C93S (F-Ubc9C93S-PML).
Figure 6
Figure 6
SUMOylation of UBC at Lys-49 favors it relocalization to PML-NBs and promotes the translocation of target protein(s) to the nuclear bodies, conveying an anti-senescence phenotype. Whereas SUMOylation of proteins by the non-SUMOylated UBC9 promotes senescence.
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