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. 2017 Jan 18:8:14109.
doi: 10.1038/ncomms14109.

Uncovering the SUMOylation and ubiquitylation crosstalk in human cells using sequential peptide immunopurification

Frédéric Lamoliatte  1   2 , Francis P McManus  1 , Ghizlane Maarifi  3 , Mounira K Chelbi-Alix  3 , Pierre Thibault  1   2   4
Affiliations

Uncovering the SUMOylation and ubiquitylation crosstalk in human cells using sequential peptide immunopurification

Frédéric Lamoliatte et al. Nat Commun. .

Abstract

Crosstalk between the SUMO and ubiquitin pathways has recently been reported. However, no approach currently exists to determine the interrelationship between these modifications. Here, we report an optimized immunoaffinity method that permits the study of both protein ubiquitylation and SUMOylation from a single sample. This method enables the unprecedented identification of 10,388 SUMO sites in HEK293 cells. The sequential use of SUMO and ubiquitin remnant immunoaffinity purification facilitates the dynamic profiling of SUMOylated and ubiquitylated proteins in HEK293 cells treated with the proteasome inhibitor MG132. Quantitative proteomic analyses reveals crosstalk between substrates that control protein degradation, and highlights co-regulation of SUMOylation and ubiquitylation levels on deubiquitinase enzymes and the SUMOylation of proteasome subunits. The SUMOylation of the proteasome affects its recruitment to promyelocytic leukemia protein (PML) nuclear bodies, and PML lacking the SUMO interacting motif fails to colocalize with SUMOylated proteasome further demonstrating that this motif is required for PML catabolism.

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Figures

Figure 1
Figure 1. Optimization of a SUMO remnant immunoaffinity purification strategy.
(a) Protein sequences of the endogenous ubiquitin, endogenous SUMO3 and SUMO3m. (b) Overview of the remnant immunoaffinity purification. Cell lysates are subjected to a NiNTA column to enrich SUMOylated proteins before tryptic digestion. Peptides containing the SUMO3m remnant are enriched using the anti-K-(NQTGG) antibody. Subsequent peptides are injected on a Tribrid Fusion. Peptide identification is performed using MaxQuant.
Figure 2
Figure 2. Motif analysis of SUMO-modified lysine residues.
(a) Pie chart distribution of identified SUMO3 sites based on sequence motif. SUMO3 sites located within a full consensus sequence are represented in red, partial in blue, inverted in green and non-consensus in grey. (b) Distribution of the identified SUMO motifs with regards to the intensity of their respective peptides. High abundance peptides are mostly represented by full consensus sequences while low abundance peptides show partial or non-canonical consensus sequences. (c) Distribution of the phosphorylated residues with respect to the SUMOylation sites identified on a peptide. (d) Phospho-dependent motif identified by Motif X using all the phosphorylated SUMOylated peptides.
Figure 3
Figure 3. Temporal profiling of the SUMOylome and ubiquitylome in response to MG132 treatment.
(a) Overview of the strategy used to enrich SUMOylated and ubiquitylated peptides. Peptides arising from the digestion of SUMOylated proteins were subjected to a first peptide IP using the cross-linked anti-K-(GG) antibody. The flow through of the first IP was subjected to SUMOylated peptide enrichment with the anti-K-(NQTGG) antibody. The two resulting eluates were injected separately on the LC-MS/MS setup. (b) Heat map of normalized intensity for SUMOylated peptides as a function of MG132 treatment period. 1620 SUMO sites were identified with a localization confidence >0.75. Peptide identifications were clustered using Fuzzy-C-means clustering. 676 sites were regulated and were divided in five groups based on their kinetic profiles. (c) Distribution of the proportion of SUMO motifs over time. A depletion of both non-consensus and consensus motifs and an increase of non-canonical motif over the 8 h time period was observed. (d) Heat map of normalized intensity for ubiquitylated peptides as a function of MG132 treatment period. 349 Ubi sites were identified with a localization confidence >0.75. A total of 114 sites were regulated and were divided in 3 groups based on their kinetic profile.
Figure 4
Figure 4. Crosstalk between SUMOylation and ubiquitylation levels on the histone deubiquitinase USP22 in response to MG132.
(a) Distribution of SUMOylation and ubiquitylation sites identified on the histone deubiquitinase USP22 and its substrate Histone H2B. The Zinc Finger domain of USP22 is shown in green, the potential nuclear localization sequence (NLS) in red, the catalytic domain in blue. The Histone core is depicted in orange. The lysine residues shown in bold depict the modified residues identified in this study. (b) Temporal profiles of the modified peptides of USP22 and H2B identified in the MG132 kinetic study.
Figure 5
Figure 5. SUMOylated and ubiquitylated proteins show a high degree of interconnectivity.
Protein interaction network of all identified SUMOylation and ubiquitylation sites identifies in the kinetic experiment. Protein identified as only SUMOylated are represented in yellow, only ubiquitylated in bleu and identified as both in green. The size of each node represents the number of neighbours.
Figure 6
Figure 6. Immunofluorescence of 20S Proteasome and PML or PML-SIM under MG132 stress.
(a) HEK293-SUMO3m cells were transfected with PML IV or PML IV-SIM. Two days later they were treated with 10 μM MG132 for 6 h. Double immunofluorescence analyses were performed using monoclonal mouse anti-PML (red) and rabbit anti-20S (green) antibodies. (b) PML and 20S proteasome co-localization for PML and PML-SIM cells treated with MG132 from (a). The bar charts represent the ratio of the PML co-localized with the 20S proteasome, where error bars represent the standard deviation from 3 biological replicates for n=30 cells. Experiments for (c) and (d) were performed as for (a) and (b) in PML −/− MEFs co-transfected with SUMO3 and PML IV or PML IV-SIM, where error bars represent the s.d. from 5 biological replicates for n=60 cells. Scale bars, 10 μm.
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