Converging Small Ubiquitin-like Modifier (SUMO) and Ubiquitin Signaling: Improved Methodology Identifies Co-modified Target Proteins
2017; Elsevier BV; Volume: 16; Issue: 12 Linguagem: Inglês
10.1074/mcp.tir117.000152
ISSN1535-9484
AutoresSabine A.G. Cuijpers, Edwin Willemstein, Alfred C.O. Vertegaal,
Tópico(s)Peptidase Inhibition and Analysis
ResumoPost-translational protein modifications (PTMs) including small chemical groups and small proteins, belonging to the ubiquitin family, are essential for virtually all cellular processes. In addition to modification by a single PTM, proteins can be modified by a combination of different modifiers, which are able to influence each other. Because little is known about crosstalk among different ubiquitin family members, we developed an improved method enabling identification of co-modified proteins on a system-wide level using mass spectrometry. We focused on the role of crosstalk between SUMO and ubiquitin during proteasomal degradation. Using two complementary approaches, we identified 498 proteins to be significantly co-modified by SUMO and ubiquitin upon MG132 treatment. These targets included many enzymatic components of PTM machinery, involved in SUMOylation and ubiquitylation, but also phosphorylation, methylation and acetylation, revealing a highly complex interconnected network of crosstalk among different PTMs. In addition, various other biological processes were found to be significantly enriched within the group of co-modified proteins, including transcription, DNA repair and the cell cycle. Interestingly, the latter group mostly consisted of proteins involved in mitosis, including a subset of chromosome segregation regulators. We hypothesize that group modification by SUMO-targeted ubiquitin ligases regulates the stability of the identified subset of mitotic proteins, which ensures proper chromosome segregation. The mitotic regulators KIF23 and MIS18BP1 were verified to be co-modified by SUMO and ubiquitin on inhibition of the proteasome and subsequently identified as novel RNF4 targets. Both modifications on MIS18BP1 were observed to increase simultaneously during late mitosis, whereas the total protein level decreased immediately afterward. These results confirm the regulation of MIS18BP1 via SUMO-ubiquitin crosstalk during mitosis. Combined, our work highlights extensive crosstalk between SUMO and ubiquitin, providing a resource for further unraveling of SUMO-ubiquitin crosstalk. Post-translational protein modifications (PTMs) including small chemical groups and small proteins, belonging to the ubiquitin family, are essential for virtually all cellular processes. In addition to modification by a single PTM, proteins can be modified by a combination of different modifiers, which are able to influence each other. Because little is known about crosstalk among different ubiquitin family members, we developed an improved method enabling identification of co-modified proteins on a system-wide level using mass spectrometry. We focused on the role of crosstalk between SUMO and ubiquitin during proteasomal degradation. Using two complementary approaches, we identified 498 proteins to be significantly co-modified by SUMO and ubiquitin upon MG132 treatment. These targets included many enzymatic components of PTM machinery, involved in SUMOylation and ubiquitylation, but also phosphorylation, methylation and acetylation, revealing a highly complex interconnected network of crosstalk among different PTMs. In addition, various other biological processes were found to be significantly enriched within the group of co-modified proteins, including transcription, DNA repair and the cell cycle. Interestingly, the latter group mostly consisted of proteins involved in mitosis, including a subset of chromosome segregation regulators. We hypothesize that group modification by SUMO-targeted ubiquitin ligases regulates the stability of the identified subset of mitotic proteins, which ensures proper chromosome segregation. The mitotic regulators KIF23 and MIS18BP1 were verified to be co-modified by SUMO and ubiquitin on inhibition of the proteasome and subsequently identified as novel RNF4 targets. Both modifications on MIS18BP1 were observed to increase simultaneously during late mitosis, whereas the total protein level decreased immediately afterward. These results confirm the regulation of MIS18BP1 via SUMO-ubiquitin crosstalk during mitosis. Combined, our work highlights extensive crosstalk between SUMO and ubiquitin, providing a resource for further unraveling of SUMO-ubiquitin crosstalk. The limited capacity of our genome is compensated for by the processes of alternative splicing and post-translational modification (PTM) 1The abbreviations used are: PTM; post-translational modification; ABC, ammonium bicarbonate; ACN, acetonitrile; CAA, chloroacetamide; CD, catalytic domain; DMEM, dulbecco's modified eagle's medium; DMSO, dimethyl sulphoxide; DTT, dithiothreitol; FA, formic acid; FC, fold change; FCS, fetal calf serum; GOBP, gene ontology based biological process; HEK293T, human embryonic kidney 293 cell line; IP, immunoprecipitation; LDS, Lithium Dodecyl Sulfate; NanoLC-MS/MS, nanoflow liquid chromatography-tandem mass spectrometry; P/S, penicillin/streptomycin; PBS/T, PBS with 0.05% Tween-20; PD, pulldown; PEI, polyethyleneimine; RT, room temperature; SIM, SUMO interaction motif; STUbL, SUMO-targeted ubiquitin ligase; SUMO, small ubiquitin-like modifier; TFA, trifluoroacetic acid; U2OS, human bone osteosarcoma cell line. 1The abbreviations used are: PTM; post-translational modification; ABC, ammonium bicarbonate; ACN, acetonitrile; CAA, chloroacetamide; CD, catalytic domain; DMEM, dulbecco's modified eagle's medium; DMSO, dimethyl sulphoxide; DTT, dithiothreitol; FA, formic acid; FC, fold change; FCS, fetal calf serum; GOBP, gene ontology based biological process; HEK293T, human embryonic kidney 293 cell line; IP, immunoprecipitation; LDS, Lithium Dodecyl Sulfate; NanoLC-MS/MS, nanoflow liquid chromatography-tandem mass spectrometry; P/S, penicillin/streptomycin; PBS/T, PBS with 0.05% Tween-20; PD, pulldown; PEI, polyethyleneimine; RT, room temperature; SIM, SUMO interaction motif; STUbL, SUMO-targeted ubiquitin ligase; SUMO, small ubiquitin-like modifier; TFA, trifluoroacetic acid; U2OS, human bone osteosarcoma cell line.. Especially the latter adds an essential additional layer of complexity to our proteome, which is necessary to provide the cell with sufficient functionally different protein states that are needed for efficient regulation of cellular processes and pathways. In addition, PTMs provide the cell with a rapid response mechanism to deal with changing intracellular or environmental conditions. Modification by a PTM can affect the function of a protein in various ways, for example by changing its conformation, localization, binding partners or half-life. Proteins can be modified by chemical groups (including phosphorylation, acetylation and methylation) or by covalent attachment of small proteins (such as ubiquitin, small ubiquitin-like modifier (SUMO), and NEDD8) (1.Deribe Y.L. Pawson T. Dikic I. Post-translational modifications in signal integration.Nat. Struct. Mol. Biol. 2010; 17: 666-672Crossref PubMed Scopus (533) Google Scholar, 2.Seet B.T. Dikic I. Zhou M.M. Pawson T. Reading protein modifications with interaction domains.Nat. Rev. Mol. Cell Biol. 2006; 7: 473-483Crossref PubMed Scopus (532) Google Scholar). Ubiquitin and ubiquitin-like proteins have similar modification cascades, consisting of family-member specific activating E1, conjugating E2, and ligating E3 enzymes (3.Van der Veen A.G. Ploegh H.L. Ubiquitin-like proteins.Annu. Rev. Biochem. 2012; 81: 323-357Crossref PubMed Scopus (258) Google Scholar). In addition, each modification can be removed by specific proteases (4.Komander D. Clague M.J. Urbe S. Breaking the chains: structure and function of the deubiquitinases.Nat. Rev. Mol. Cell Biol. 2009; 10: 550-563Crossref PubMed Scopus (1451) Google Scholar). The interesting phenomenon of crosstalk among post-translational modifications is increasingly receiving more attention (5.Hunter T. The age of crosstalk: phosphorylation, ubiquitination, and beyond.Mol. Cell. 2007; 28: 730-738Abstract Full Text Full Text PDF PubMed Scopus (681) Google Scholar). Various crosstalk mechanisms are known that provide an additional layer of fine tuning protein functionality. For example, a first modification can influence a second modification on the same target, as is the case for phosphorylation-dependent ubiquitylation (6.Petroski M.D. Deshaies R.J. Function and regulation of cullin-RING ubiquitin ligases.Nat. Rev. Mol. Cell Biol. 2005; 6: 9-20Crossref PubMed Scopus (1678) Google Scholar, 7.Koepp D.M. Schaefer L.K. Ye X. Keyomarsi K. Chu C. Harper J.W. Elledge S.J. Phosphorylation-dependent ubiquitination of cyclin E by the SCFFbw7 ubiquitin ligase.Science. 2001; 294: 173-177Crossref PubMed Scopus (658) Google Scholar) and phosphorylation-dependent SUMOylation (8.Hietakangas V. Anckar J. Blomster H.A. Fujimoto M. Palvimo J.J. Nakai A. Sistonen L. PDSM, a motif for phosphorylation-dependent SUMO modification.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 45-50Crossref PubMed Scopus (379) Google Scholar). In addition, modifications can affect the function of the PTM machinery, as exemplified by Neddylation of Cullin components in ubiquitin E3 ligases (9.Osaka F. Kawasaki H. Aida N. Saeki M. Chiba T. Kawashima S. Tanaka K. Kato S. A new NEDD8-ligating system for cullin-4A.Genes Dev. 1998; 12: 2263-2268Crossref PubMed Scopus (225) Google Scholar) and acetylation of the SUMO E2 UBC9 (10.Hsieh Y.L. Kuo H.Y. Chang C.C. Naik M.T. Liao P.H. Ho C.C. Huang T.C. Jeng J.C. Hsu P.H. Tsai M.D. Huang T.H. Shih H.M. Ubc9 acetylation modulates distinct SUMO target modification and hypoxia response.EMBO J. 2013; 32: 791-804Crossref PubMed Scopus (48) Google Scholar). Finally, proteins can be modified by specific crosstalk machinery which recognize proteins with a specific PTM and subsequently modify these targets with a second and different PTM, including SUMO-targeted ubiquitin ligases (STUbLs) like RNF4 (11.Sriramachandran A.M. Dohmen R.J. SUMO-targeted ubiquitin ligases.Biochim. Biophys. Acta. 2014; 1843: 75-85Crossref PubMed Scopus (184) Google Scholar, 12.Tatham M.H. Geoffroy M.C. Shen L. Plechanovova A. Hattersley N. Jaffray E.G. Palvimo J.J. Hay R.T. RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation.Nat. Cell Biol. 2008; 10: 538-546Crossref PubMed Scopus (659) Google Scholar, 13.Lallemand-Breitenbach V. Jeanne M. Benhenda S. Nasr R. Lei M. Peres L. Zhou J. Zhu J. Raught B. De The H. Arsenic degrades PML or PML-RARalpha through a SUMO-triggered RNF4/ubiquitin-mediated pathway.Nat. Cell Biol. 2008; 10: 547-555Crossref PubMed Scopus (562) Google Scholar, 14.Perry J.J. Tainer J.A. Boddy M.N. A SIM-ultaneous role for SUMO and ubiquitin.Trends Biochem. Sci. 2008; 33: 201-208Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 15.Prudden J. Pebernard S. Raffa G. Slavin D.A. Perry J.J. Tainer J.A. McGowan C.H. Boddy M.N. SUMO-targeted ubiquitin ligases in genome stability.EMBO J. 2007; 26: 4089-4101Crossref PubMed Scopus (280) Google Scholar, 16.Sun H. Leverson J.D. Hunter T. Conserved function of RNF4 family proteins in eukaryotes: targeting a ubiquitin ligase to SUMOylated proteins.EMBO J. 2007; 26: 4102-4112Crossref PubMed Scopus (242) Google Scholar). Studying crosstalk among different PTMs can reveal essential information about protein function that would have been missed by focusing on single modifications. Currently, crosstalk between ubiquitin and ubiquitin-like PTMs is mostly studied by using targeted approaches, which for example recently identified an important role for crosstalk between SUMO and ubiquitin in meiotic recombination among chromosomes (17.Rao H.B. Qiao H. Bhatt S.K. Bailey L.R. Tran H.D. Bourne S.L. Qiu W. Deshpande A. Sharma A.N. Beebout C.J. Pezza R.J. Hunter N. A SUMO-ubiquitin relay recruits proteasomes to chromosome axes to regulate meiotic recombination.Science. 2017; 355: 403-407Crossref PubMed Scopus (92) Google Scholar). However, addressing arising questions about crosstalk on an unbiased proteome-wide level is challenging, because proper purification methods are missing due to technical challenges and low stoichiometry of modified proteins (18.Olsen J.V. Mann M. Status of large-scale analysis of post-translational modifications by mass spectrometry.Mol. Cell. Proteomics. 2013; 12: 3444-3452Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). Here, we have developed an improved strategy to purify and identify proteins co-modified by two different small protein PTMs, SUMO, and ubiquitin. This improved method is generic and can be applied to different combinations of these PTMs and will thereby enable us to study the phenomenon of crosstalk on a more comprehensive PTM-wide level. U2OS and HEK293T cells were cultured at 5% CO2 and 37 °C in DMEM (Thermo Fisher Scientific, Bremen, Germany) including 10% FCS (Thermo Fisher Scientific), 100 U/ml penicillin, and 100 mg/ml streptomycin (Thermo Fisher Scientific). When indicated cells were selected with 2.5 μm puromycin (Calbiochem, Darmstadt, Germany) to obtain stable co-expressing cell lines, treated with 10 μm MG132 (Sigma, Saint Louis, MO) for 6 h to inhibit the proteasome or infected with lentivirus encoding shRNAs at an MOI of 3 to obtain protein knockdown. Cell synchronization was achieved by incubation with 4 mm thymidine (Sigma) or 0.1 μg/ml nocodazole (Sigma) and confirmed by flow cytometry using propidium iodide (Sigma) to visualize cellular DNA content. Purification of His10-SUMO2 conjugates was performed as described before (19.Hendriks I.A. D'Souza R.C. Yang B. Verlaan-de Vries M. Mann M. Vertegaal A.C. Uncovering global SUMOylation signaling networks in a site-specific manner.Nat. Struct. Mol. Biol. 2014; 21: 927-936Crossref PubMed Scopus (336) Google Scholar). In short, cell lysates were incubated with Ni-NTA beads (Qiagen, Hilden, Germany) overnight at 4 °C, washed and eluted for 30 min at room temperature (RT). When indicated, eluted samples were diluted and treated with the catalytic domain (CD) of USP2 (Boston Biochem, Cambridge, MA) and/or SENP2 (Boston Biochem) for 3 h at RT to deconjugate ubiquitin and/or SUMO respectively from its target proteins. Cells were lysed according to our His10-pulldown protocol (19.Hendriks I.A. D'Souza R.C. Yang B. Verlaan-de Vries M. Mann M. Vertegaal A.C. Uncovering global SUMOylation signaling networks in a site-specific manner.Nat. Struct. Mol. Biol. 2014; 21: 927-936Crossref PubMed Scopus (336) Google Scholar) and samples were incubated with Ni-NTA beads, washed and eluted. Upon concentration and stepwise dilution, samples were incubated with anti-FLAG-M2 beads (Sigma). Subsequently, samples were washed and prepared for immunoblotting or mass spectrometry analysis. Proteins were separated on Novex 4–12% Bis-Tris Plus gradient gels (Life Technologies, Carlsbad, CA) in MOPS buffer for 45 min at 165 Volt and transferred onto Hybond nitrocellulose membranes (GE Healthcare, Chicago, IL) in cold transfer buffer at 25 V for 3 h. Membranes were blocked in PBS containing 0.05% Tween-20 (Merck, Darmstadt, Germany) and 8% milk powder, followed by incubation with primary antibodies. After washing three times in PBS with 0.05% Tween-20 (PBS/T), the membranes were incubated with secondary antibodies and washed another three times in PBS/T. Pierce ECL 2 immunoblotting substrate (Life Technologies) was used to visualize the signal on RX Medical films (Fuji, Tokyo, Japan). After digestion with trypsin (Promega, Madison, WI), samples were acidified by trifluoroacetic acid (Sigma). Stage tips containing C18 (Sigma) were activated by passing HPLC-grade methanol (Sigma), washed with 80% acetonitrile (ACN, Sigma) in 0.1% formic acid (FA, Sigma) and equilibrated with 0.1% FA. Upon loading the samples and washing twice with 0.1% FA, the stage tips were dried completely and eluted twice with 80% ACN. The samples were vacuum dried using a SpeedVac RC10.10 (Jouan, Nantes, France), redissolved in 0.1% FA and transferred to autoloader vials before measurement by mass spectrometry. For each experimental condition at least four biological replicates were performed to allow detection of significant differences, which were all measured in technical triplicate by nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). Samples were measured on an EASY-nLC 1000 system (Proxeon, Odense, Denmark) connected to an Orbitrap Q-Exactive (Thermo Fisher Scientific) through a nano-electrospray ion source. Peptides were separated in a 13 cm analytical column with an inner-diameter of 75 μm, which was packed in-house with 1.8 μm C18 beads (Reprospher, Ammerbuch-Entringen, Germany). A gradient length was used of 60 min from 2% to 95% ACN in 0.1% FA with a flow rate of 200 nl/minute. The data-dependent acquisition mode with a top 10 method was used to operate the mass spectrometer. Full-scan MS spectra were acquired at a target value of 3 × 106 with a resolution of 70,000. The higher-collisional dissociation tandem mass spectra were recorded at a target value of 1 × 105 and a resolution of 17,500 with a normalized collision energy of 25%. The maximum injection times for MS1 and MS2 were respectively 20 ms and 100 ms. For 60 s, the precursor ion masses of scanned ions were dynamically excluded from MS/MS analysis. Ions with a charge of 1 or greater than 6 were excluded from triggering MS2 events. Subsequently, the raw data analysis was performed using MaxQuant Software version 1.5.3.30 with its integrated search engine Andromeda. A first search was carried out with 20 ppm for precursor ions, followed by a main search using 4.5 ppm. To search against the in silico digested proteome containing 92,180 entries of Homo sapiens from UniProt (24 March 2016), the mass tolerance of MS/MS spectra were set to 20 ppm. In addition, MS/MS data were searched by Andromeda for potential common mass spectrometry contaminants. Trypsin/P specificity was used to perform database searches, allowing four missed cleavages. In addition, carbamidomethylation of cysteine residues was considered as a fixed modification, whereas oxidation of methionines, N-terminal carbamylation and acetylation, and diGly modification on lysines were considered as variable modifications. Match between runs was performed with a 20 min alignment time window and a 0.7 min match time window, while a minimum peptide length of 7 was used. To consider proteins for quantification, at least two identified peptides were required, including unique and razor peptides. Proteins and peptides were identified using a false discovery rate of 1% (20.Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9150) Google Scholar). Finally, label-free quantification was performed using LFQ settings with fast LFQ disabled to quantify all identified peptides (supplemental Table S1). Because substantial differences among conditions were expected, LFQ normalization by MaxQuant was skipped to prevent undesirable correction among these samples. Proteins identified by the same set of peptides were combined to a single protein group by MaxQuant (supplemental Table S2). The proteins identified in each sample were further analyzed using Perseus Software version 1.5.2.4. Samples from DMSO and MG132 treated cells were analyzed separately to prevent incorrect imputation. Both data sets were filtered for potentially improper protein identifications by removing proteins that would fit the categories "potential contaminant," "reverse," or "only identified by site." Subsequently, all LFQ intensities were log2 transformed and all experimental replicates for each condition were assigned together in four groups per treatment for the main analysis. Finally, all proteins were removed that were not identified in at least four experimental replicates in at least one of these four groups. For an additional tailored analysis in the supplementary data, the experimental conditions of both approaches were pooled together and assigned to two groups per treatment to increase the statistical power. Proteins that were not identified in at least eight pooled replicates of these two groups were removed. For both analyses missing values were imputed based on the total matrix of each data set, using normally distributed values with a randomized 0.3 width (log2) and a 1.8 down shift (log2). Two-sample Student T-tests were performed between the SUMO and ubiquitin expressing cell line samples and their corresponding U2OS control samples to obtain p values, their FDR corrected q values and differences for each protein. Finally, four volcano plots were created showing these p values (as -Log10(p)) on the y axis and differences (as Log2FC (fold change)) on the x axis for the His10-SUMO2/FLAG-ubiquitin purification under DMSO and MG132 conditions, and for the His10-ubiquitin/FLAG-SUMO2 purification under DMSO and MG132 conditions. To identify significantly co-modified proteins, a false discovery rate of 3% was accepted and all proteins with a q value over 0.03 were removed. To increase the reliability of our data set, we overlaid the co-modified proteins identified by both independent purification approaches and thereby obtained two robust lists of proteins co-modified by SUMO and ubiquitin upon DMSO or MG132 treatment. Significance was determined similarly for the additional tailored analysis described above. However, because samples of both approaches were pooled, this analysis directly resulted in one list of proteins co-modified upon DMSO treatment and a second one containing co-modified proteins upon MG132 treatment. Subsequently, the proteins of each list were annotated using the gene ontology annotation of biological processes (GOBP). Enrichment of specific processes was determined by comparison with the Human proteome obtained from Uniprot containing 20577 proteins. Fisher exact tests were performed and the enrichment of a biological process was considered to be significant if its Benjamini-Hochberg FDR value was below 0.03. Additionally, interactions among co-modified proteins were identified using the STRING database version 10.0 with a medium confidence of 0.400. Subsequently this interconnected network and the data from Perseus were imported in Cytoscape version 3.5.0 to visualize the interaction among proteins of specific biological processes and their individual values as a co-modified target. Although the samples were not specifically enriched for modified peptides, a search was performed by MaxQuant to identify peptides modified by a diGly motif (supplemental Table S3). Subsequently, all peptides modified by a diGly motif that were identified equally or more in the parental control samples, compared with the samples from cell lines expressing both SUMO2 and ubiquitin, were considered as ubiquitylated background binders and therefore removed from the list. In addition, all peptides assigned to the ubiquitin precursor UBA52 instead of to ubiquitin, or with lower quality spectra were removed to obtain a list containing peptides modified by a diGly motif that were specifically identified in the samples containing co-modified proteins. For each peptide the best localization evidence spectrum was retrieved from MaxQuant (supplemental PDF S1). Manual inspection of MS/MS spectra following the Andromeda search was performed to remove potential false positive identifications. A His10-pulldown was performed and the samples were diluted to enable protein renaturing as described above. Samples were incubated with control GST or recombinant GST-RNF4 bound Glutathione Sepharose 4 Fast Flow beads (GE Healthcare) for 2 h at 4 °C while moving. Unbound samples were taken, followed by washing four times with wash buffer containing 50 mm Tris (pH 7.5), 150 mm NaCl, 1% Triton X-100 and protease inhibitors without EDTA. Samples were eluted for 30 min at 1200 rpm in wash buffer supplemented with 20 mm glutathione (Sigma). We have developed an improved method that enables enrichment of proteins simultaneously modified by two different small protein PTMs. Many technical challenges, especially for ubiquitin and ubiquitin-like PTMs, prevented system-wide approaches to uncover novel crosstalk on a comprehensive and proteome-wide level. Our improved method makes use of two consecutive purifications, namely enrichment for a specific His10-tagged protein modifier followed by immunoprecipitation (IP) of a different FLAG-tagged protein modifier. As an example, Fig. 1A shows the experimental workflow of this method applied on a sample obtained from cells expressing His10-SUMO2 and FLAG-ubiquitin. By expressing a differentially tagged version of both protein modifiers of interest at close to endogenous levels, subsequent purifications enabled enrichment of co-modified proteins. In our approach we focused on co-modification of target proteins by two key PTMs, namely ubiquitin and SUMO. However, this method could be used to study crosstalk among many different ubiquitin-like PTMs by simply changing the expressed modifiers. First, two novel cell lines were created to enable two complementary experimental approaches which would increase the reliability of our data. For the first approach, U2OS cells stably expressing His10-SUMO2 (19.Hendriks I.A. D'Souza R.C. Yang B. Verlaan-de Vries M. Mann M. Vertegaal A.C. Uncovering global SUMOylation signaling networks in a site-specific manner.Nat. Struct. Mol. Biol. 2014; 21: 927-936Crossref PubMed Scopus (336) Google Scholar) were infected with lentivirus encoding a FLAG-ubiquitin construct. Upon selection with puromycin, a stable cell line was created, expressing both His10-tagged SUMO2 and FLAG-tagged ubiquitin (Fig. 1B). For the complementary approach, an additional cell line was made which expressed both His10-tagged ubiquitin and FLAG-tagged SUMO2. To obtain this cell line, U2OS cells stably expressing FLAG-SUMO2 (21.Schimmel J. Eifler K. Sigurðsson J.O. Cuijpers S.A. Hendriks I.A. Verlaan-de Vries M. Kelstrup C.D. Francavilla C. Medema R.H. Olsen J.V. Vertegaal A.C. Uncovering SUMOylation Dynamics during Cell-Cycle Progression Reveals FoxM1 as a Key Mitotic SUMO Target Protein.Mol. Cell. 2014; 53: 1053-1066Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) were infected with lentivirus encoding a His10-ubiquitin construct and selected with puromycin (Fig. 1C). Upon co-purification, the second purification step would enrich co-modified proteins from the pool of SUMOylated target proteins (Approach 1) or from the pool of ubiquitylated target proteins (Approach 2). The overlap between both approaches would be considered as highly reliable co-modified target proteins. Because our improved method would purify proteins modified directly by SUMO and ubiquitin as well as proteins modified by chains consisting of both SUMO and ubiquitin, an experiment was performed to determine the fraction of proteins modified by such potential mixed chains. A His10-pulldown was performed from cells expressing His10-SUMO2 and the sample was treated with the catalytic domain (CD) of SENP2 and/or USP2. If both PTMs would be covalently attached directly and independently to their target proteins, the SENP2CD treatment should not affect the ubiquitin signal and the USP2CD treatment should not affect the SUMO2/3 signal (supplemental Fig. S1A top). However, if these target proteins would be modified by any form of mixed SUMO-ubiquitin chains, these treatments should co-decrease the SUMO2/3 and/or the ubiquitin signal (supplemental Fig. S1A bottom). Immunoblot analysis showed no decrease in the SUMO2/3 signal on USP2CD treatment and no decrease in the ubiquitin signal on SENP2CD treatment, revealing limited purification of target proteins modified by mixed SUMO-ubiquitin and/or mixed ubiquitin-SUMO chains (supplemental Fig. S1B). Similar results were obtained from cells treated with MG132, indicating that the role for mixed chains of SUMO and ubiquitin is also limited upon inhibition of the proteasome (supplemental Fig. S1C). As shortly mentioned above, two complementary experiments were performed to identify co-modified proteins (Fig. 2 top). For the first approach, we used parental U2OS cells as a negative control and U2OS cells that expressed His10-SUMO2 and FLAG-ubiquitin. The second approach made use of parental U2OS cells and U2OS cells expressing His10-ubiquitin and FLAG-SUMO2. Because several targeted approaches studying co-modification of single proteins indicated a potential important role for crosstalk between SUMO and ubiquitin in regulating the half-life of proteins by affecting their degradation by the proteasome, we decided to purify co-modified proteins from cells treated with either DMSO as a control or MG132 to inhibit the proteasome. This resulted in four experimental conditions per approach. Upon His10-pulldown, SUMOylated targets were purified from the samples in the first approach, followed by enrichment of proteins comodified by both SUMO2 and ubiquitin. Upon His10-pulldown from the samples of the second approach, ubiquitylated targets were purified, followed by enrichment of co-modified proteins. In addition to analysis by mass spectrometry, a fraction of each sample was saved for analysis by immunoblotting to control for purification efficiencies. Equal amounts of starting material were loaded for the samples taken after the first purification (PD) and for the samples taken after double purifications (PD+IP). Immunoblot analysis using an antibody against polyHistidine revealed a decrease in SUMOylated target proteins upon the second purification of the first experimental approach, indicating that only a fraction of the SUMOylated proteins is simultaneously ubiquitylated (supplemental Fig. S2A). Analysis of the same samples using an antibody against FLAG revealed limited loss of co-modified t
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