An iTRAQ Proteomics Screen Reveals the Effects of the MDM2

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An iTRAQ Proteomics Screen Reveals The Effects Of The Mdm2 Binding Ligand Nutlin-3 On Cellular Proteostasis Judith Nicholson, Kalainanghi Neelagandan, Anne Sophie HUart, Kathryn Ball, Mark Molloy, and Ted Hupp J. Proteome Res., Just Accepted Manuscript • Publication Date (Web): 08 Oct 2012 Downloaded from http://pubs.acs.org on October 9, 2012

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Journal of Proteome Research

An iTRAQ Proteomics Screen Reveals The Effects Of The Mdm2 Binding Ligand Nutlin-3 On Cellular Proteostasis

Judith Nicholson1^, Kalainanghi Neelagandan2, Anne-Sophie Huart1, Kathryn Ball2, Mark P. Molloy3, Ted Hupp1* 1.

Cell Signalling Unit, p53 Signal Transduction Laboratories, Edinburgh Cancer Research

Centre, University of Edinburgh, Crewe Road South, Edinburgh, EH4 2XR 2.

Cell Signalling Unit, Interferon and Cell Signalling Laboratories, Edinburgh Cancer

Research Centre, University of Edinburgh, Crewe Road South, Edinburgh, EH4 2XR 3.

Australian Proteome Analysis Facility, Macquarie University, Sydney, New South Wales

2109, Australia

KEYWORDS: MDM2, Nutlin-3, iTRAQ, Interactome, NPM, proteostasis, protein-protein interactions, quantitative proteomics

ACS Paragon Plus Environment

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ABSTRACT MDM2 participates in protein synthesis, folding, and ubiquitin-mediated degradation and is therefore a proteostasic hub protein. The MDM2 interactome contains over 100 proteins, yet stratification of dominant MDM2-interacting proteins has not been achieved. 8-plex iTRAQ (nanoLC-MS/MS) of MCF7 cells treated with the MDM2-binding ligand Nutlin-3 identified the most abundant cellular protein changes over early time points, 1,323 unique proteins were identified including 35 with altered steady-state levels within 2 hours of Nutlin-3 treatment identifying a core group of MDM2 related proteins. Six of these proteins were previously identified MDM2 interactors and the effects of Nutlin-3 on the MDM2-nucleophosmin interaction (NPM), was further validated. This revealed Nutlin-3 mediates the in vivo conversion of NPM from an oligomer to a monomer as an MDM2 dependent phenomenon, with Nutlin-3 stimulating MDM2 binding to a peptide motif derived from the oligomerization interface of NPM. These data form the first proteomic screen of Nutlin-3 in cells whereby we (i) identify the most abundant MDM2 interacting proteins whose steady-state levels change early after Nutlin-3 treatment; (ii) identify the first protein apart from p53, NPM, whose interaction with MDM2 can be stimulated allosterically by Nutlin-3 and (iii) raise the possibility that Nutlin-3 might act as a general agonist of other MDM2 protein-protein interactions.


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INTRODUCTION MDM2 has been well characterized as one of a group of pro-oncogenic E3 ubiquitin ligases which ubiquitinate the tumour suppressor p531. Although this function is integral to the role of MDM2 in attenuating p53 function, MDM2 is a multi-tasking protein participating in varied biochemical processes not necessarily linked to this ubiquitin ligase function. MDM2 has been shown to control ribosomal biogenesis pathways2, function as a molecular chaperone3 and plays a surprising role in polypeptide translation by binding the internal ribosomal entry site sequence of mRNA4. These functions link MDM2 with the maintenance of the steady-state levels of cellular proteins, or proteostasis. The almost 150 reported protein-protein interactions of MDM25 have not been stratified with respect to affinity, dominance, or cell-specificity and it remains a significant challenge to annotate this knowledge. Viewing MDM2 as a factor involved in the newly formed concept of “proteostasis” might facilitate rationalizing these many MDM2 protein-protein interactions. Proteostasis is the balance between protein synthesis, folding and degradation6 that is important for normal cell homeostasis and whose dysregulation is thought to contribute to age related protein misfolding diseases. As MDM2 has been shown to participate in protein synthesis, protein folding, and protein degradation it is therefore a proteostatic hub protein. In particular MDM2 participates widely in ribosomal protein interactions, and is present in the nucleolus when bound to ARF7 representing a potential interface or switch between MDM2 protein synthesis and degradation pathways. Proteostasis pathways are vital in cancer because cancer cells have a higher rate of protein synthesis and misfolding, and therefore a greater burden is placed on the core molecular chaperone machinery and degradation pathways8. An example of a promising drug targeting these functions is geldanamycin9 and the derivative 17-AAG, which


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target the molecular chaperone Hsp90 and preferentially kill cancer cells as they degrade oncogenic proteins and accumulate toxic misfolded proteins. Further investigation of the core and wider interactome of MDM2 is a route to identifying new targets for drug discovery, as well as further understanding the role of MDM2 and proteostasis in cancer. Annotating the dominant interactors with MDM2 in the basal/induced state can define the hierarchy of each interactor within the MDM2 hub and begin to construct a map of the proteostasis network. Nutlin-3 is a small molecule ligand that binds to the hydrophobic pocket of MDM2 within a peptide binding groove and which competes with p53 binding to the protein; however MDM2 interaction with Nutlin-3 which has been shown to increase p53 protein levels in cells also paradoxically increases p53 ubiquitination10 thus highlighting a property of Nutlin-3 to act as either an agonist or antagonist of MDM2. The increase in p53 ubiquitination is due to a dual-site allosteric mechanism of MDM2 binding to p53 in which the binding of Nutlin-3 to the Nterminal hydrophobic pocket of MDM2 stabilizes an interaction of the acidic domain of MDM2 to a second MDM2 binding site in the p53 DNA-binding domain11. Nutlin-3 therefore acts as an allosteric agonist affecting multiple MDM2 binding sites and may affect other MDM2 binding partners as either a competitor or enhancer of binding. An example of this is the transcription factor E2F1, which is known to be stabilized by MDM2 binding and destabilized by Nutlin-3 treatment as the loss of the MDM2-E2F1 interaction allows other E3 ligases to target E2F1 for degradation12, 13. This highlights the dual roles of MDM2 as a mediator of protein degradation or protein stabilization; i.e. “proteostasis” and Nutlin-3 is therefore useful as a tool to begin to dissect dominant MDM2 interactors that change in cells following Nutlin-3 treatment Quantitative proteomics can provide detail about the interactome of a protein by measuring the change in multiple protein levels in parallel after stimulus with a stress or a drug, and is uniquely


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appropriate for this task due to the ability to capture dynamic changes in the proteome. In this study we targeted the MDM2 interactome by treating cells with Nutlin-3, and carried out a global proteomics screen to identify changes in protein levels, quantified by iTRAQ. Acute time points were chosen to focus on because the primary response proteins are likely to represent the core MDM2 interactome rather than secondary messenger effects. The immediate proteome change in response to Nutlin-3 may be directly linked to altered MDM2 protein-protein interactions, and therefore fluctuations in the MDM2 interactome. In particular, the proteins identified by this method may be relevant to the proteostasis function of MDM2, as regulation of this process is primarily on the protein level. In this report we define this primary response of the proteome to Nutlin-3, identifying six core previously identified MDM2-binding proteins. We further validated one of these identified proteins; the nucleolar shuttle protein nucleophosmin (NPM), which binds MDM214 and ARF15 and can regulate ribosomal assembly and export. We provide evidence that oligomeric NPM can be converted to monomers by Nutlin-3 through a direct agonist effect on MDM2. The benefits of validating in detail a hit (i.e NPM) from a proteomics screen in this way are that it highlights the breadth of information contained in a single proteomics experiment. It is not possible to validate every proteomic hit, yet the information contained in the increasing amount of proteomics data being produced may generate new hypothesis and back up or refute existing hypotheses, particularly concerning signaling pathways and interaction networks. Crosstalk between proteomics and molecular biology initiatives can contribute to improving both approaches to biological questions. The approach in this study (i) forms the first proteomics screen on the MDM2 binding drug Nutlin-3; (ii) identifies dominant MDM2-interactors that change very early after Nutlin-3 treatment thus allowing us to begin to stratify the MDM2 interactome; and (iii) highlights the ability of Nutlin-3 to act as an allosteric


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agonist of MDM2 towards another protein apart from p53, i.e NPM. This approach may also represent a novel route to understanding the non-ubiquitination roles of MDM2, and therefore lead to a greater understanding of MDM2 function as a proteostasis hub in human biology. MATERIALS AND METHODS Cell culture and drug treatment MCF7, H1299 or HeLa human breast adenocarcinoma cells were seeded on 20 cm plates with 20 ml DMEM growth medium (Invitrogen) supplemented with 10% foetal calf serum (FCS, Biosera) and 1% penicillin and streptomycin (PenStrep) (Invitrogen). Cells were incubated at 37°C with humidity and a CO2 level of 10%. Cells were treated with either Nutlin-3 (40 µM, Alexis Biochemicals) or DMSO only and harvested at 0, 1, 2, 4, 8 or 12 hours after treatment. HeLa cells were also treated with 0-20 µM campothecin or troglitazone. Cells were then harvested and lysed (Lysis buffer - 0.15% SDS, 20 mM HEPES pH 8.0, 10 mM NaF, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 x protease inhibitor (Roche Complete)) and briefly sonicated. After centrifuging at 10 000 rpm to remove insoluble material total protein concentration for each lysate was obtained by Bradford assay. iTRAQ Sample Preparation and nanoLC-MS/MS 80 µg of each cell lysate was reduced and alkylated with Applied Biosystems iTRAQ reagents, then mixed with three volumes of 0.5 M TEAB pH 8.0. Samples were then incubated overnight at 37°C with trypsin, with trypsin:protein 1:20 (Promega Gold). Digested samples were lyophilized in a vacuum centrifuge and resuspended in 30 µl 0.5 M TEAB, then labeled with 8plex iTRAQ reagents according to manufacturer’s instructions (Applied Biosystems). After


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iTRAQ labeling 2 µl of each of the eight samples were combined, de-salted by C18 zip-tip (Millipore) and checked by MALDI-TOF/TOF to confirm the presence of all the iTRAQ labels. The remainder of labeled digested samples were combined and separated into 30 fractions by strong cation exchange, dried and resuspended in 0.1% TFA, 2% acetonitrile with several fractions with lower protein content pooled resulting in 20 samples for MS analysis overall. Samples were analyzed by nanoLC-MS/MS using a QStar XL mass spectrometer (Applied Biosystems) with positive nanoflow electrospray analysis and a data-dependent acquisition mode. Protein identification and quantitation Peak lists were generated and proteins were identified using ProteinPilot (Applied Biosystems, Version 3.0) to submit a query via the MASCOT (Matrix Science, 9th March 2010) search engine. Using a 95% ProteinPilot confidence cut off (FDR < 5%, Score > 1.3) 49980 spectra were identified from a total of 58119 spectra searched. FDR analysis was carried out in ProteinPilot using the PSPEP algorithm and a reversed decoy database. Precursor mass tolerance was set at 1.2 Da and fragment mass tolerance at 0.6 Da, with one missed cleavage allowed. Parameters for searching were: Database – SwissProt (Ver. 54.6), Labeling - iTRAQ, Fixed modifications - cysteine alkylation, Variable modifications – methionine oxidation, Enzyme trypsin, Instrument - Qstar, Species - Homo sapiens (20248 entries in SwissProt). Peptides were quantified based on the intensity of iTRAQ reporter ions via the Paragon algorithm (Ver. 3.0.0.0) in the ProteinPilot programme after background correction and bias correction by the software (Applied Biosciences). Proteins were grouped using the ProGroup algorithm included in the ProteinPilot program to discriminate between similar isoforms. Complete details of peptides used for identification and quantitation are listed in Supplemental Table 1.


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Immunoblotting SDS-PAGE and western blots were carried out with detection by horseradish peroxidase conjugated secondary antibodies and X-ray film exposures. Antibodies used were – monoclonal anti-p53 (DO1), monoclonal anti-MDM2 (2A10), polyclonal anti-NPM (Cell Signalling Technologies), anti-NPM monoclonal (Cell Signalling Technologies), monoclonal anti-DNA-PK (Santa Cruz), monoclonal anti-β-actin (Sigma-Aldrich), monoclonal anti-E2F1 (Santa Cruz). Secondary antibodies used - HRP conjugated rabbit anti-mouse or swine anti-rabbit (Dako). Enzyme-linked immunosorbent assays (ELISA) Full-length (FL) and N-terminal (NT) MDM2 were purified as previously published11 from the pGEX-6p vector and cleaved from the GST-tag at the PreScission protease site with 3C express protease (Expedeon). To measure protein-protein interactions using the ELISA, white 96-well plates were coated with either streptavidin or His-tagged NPM overnight. For peptide ELISA plates were incubated with biotinylated peptides, blocked, then incubated with 0 – 10 µg/well MDM2 (FL or NT), followed by primary antibody and secondary antibody for one hour each and washed well with PBS-T between steps. After incubation with secondary antibody and washing plates were incubated with ECL and read immediately on a luminometer. For protein ELISA the same protocol was followed without the peptide incubation step. MDM2 was incubated with ligand (Nutlin-3 or peptide) for 30 mins prior to addition to plate. Alpha Screen Proximity Ligation Assay Alpha screens were carried out as previously described11. Briefly, streptavidin coated donor beads were incubated with biotinylated BOX-I peptide (ETFSDLWKLLPENN) or NPM6


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peptide (AGAKDELHIVEAEAM) for 20 mins and protein A acceptor beads incubated with anti-MDM2 (2A10) for 20 mins. Beads were combined with MDM2 in half area 96-well plates, incubated for 1-2 hr and quantitated using EnVision plate reader (Perkin Elmer) after excitation at 680 nm. siRNA Treatment of cells Cells were lysed as described and analyzed by immunoblot. Dharmacon (Thermo) siRNA targeting MDM2 was transfected into MCF7 cells using Dharmafect transfection reagent, and cells were lysed as before with the insoluble fraction also obtained by urea lysis, then analyzed by immunoblot. Co-immunoprecipitations 100 ml protein G sepharose beads (Sigma-Aldrich or Expedeon) were washed with 1 ml cold lysis buffer three times by resuspending and centrifuging at 5000 rpm for 1 min, discarding the supernatant. Cell lysates were then pre-cleared of proteins which bind to protein G or sepharose beads by incubating lysate with 100 µl protein G beads for 40 mins on a rotary shaker at 4°C. This mixture was centrifuged at 13 000 rpm for 2 mins and supernatant transferred to a fresh eppendorf. 2 µg of anti-MDM2 (2A10) was added to the lysate and incubated overnight at 4°C with rotation. 15 µl protein G beads which had been washed as before were added to the lysateantibody mixture and incubated for 1 hr at 4°C with rotation. Beads were washed by centrifuging at 5000 rpm for 2 min at 4°C, discarding supernatant and resuspending in fresh lysis buffer for each wash. Antibody-protein complexes were eluted by boiling for 3-5 min with SDS sample loading buffer, then centrifuging at 5000 rpm for 5 min to obtain supernatant. Supernatant, load and wash fractions were then analyzed by SDS-PAGE and western blot.


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RESULTS Dual proteostasic function of MDM2 Nutlin-3 treatment of A375 human cancer cells was carried out to demonstrate that MDM2 can function as both a mediator of target protein stabilization or degradation (Fig. 1, left). The treatment of cells with 5-10 µM Nutlin-3 (Figure 1, left, lanes 3 and 4 vs 1 and 2) resulted in p53 protein stabilization and E2F1 protein destabilization. Both of these proteins were shown to be direct MDM2 target proteins as a result of an interaction of MDM2 with the FxxxWxxL motif within the transactivation domain of both proteins16. E2F1 is also downregulated and p53 protein stabilized when cells are treated with Nutlin-3 or siRNA targeting MDM2 in MCF7 cells confirming the MDM2-dependence in the changes in p53 and E2F1 (data not shown). This is consistent with reports that although p53 protein accumulates by destabilization of the MDM2:p53 complex, disruption of the MDM2:E2F1 complex increases the accessibility of E2F1 to other E3 ubiquitin ligases17. These results show that Nutlin-3 can alter both stabilization and destabilization pathways regulated by MDM2 (right panel).

Nutlin-3 responsive proteins determined by iTRAQ LC-MS/MS mass spectrometry To identify potential MDM2-protein complexes which are disrupted in cells by Nutlin-3 leading to changes in protein stability an 8-plex iTRAQ proteomic mass spectrometry screen was carried out to detect proteins which have altered levels after Nutlin-3 treatment over a course of early and later time points. A limitation of this approach is that full coverage of the proteome is not possible therefore model MDM2 binding proteins such as p53 and E2F1 may not be quantified.


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This limitation however allows us to identify and focus abundant and dominant MDM2 interactors. Eight cell lysates from MCF7 cells harvested at different time points after Nutlin-3 treatment were labeled with iTRAQ reagents after digestion and were separated by strong cation exchange (SCX) prior to nanoLC MS/MS (Fig. 2). With a confidence cut-off applied of 95% 1323 proteins were identified after grouping and 85% of total spectra were assigned using ProteinPilot software. Overall 78 unique proteins were detected which were altered at some point over the whole time course, with 38 proteins identified up or downregulated at multiple time points (Table 1). Biological replicates of the 2 and 12 hour time points which were included in iTRAQ experiment and 32 proteins were detected in more than one biological replicate. In the majority of cases the two replicates were in agreement regarding whether a protein is up or downregulated, exceptions include keratin which may represent contamination at the digest stage. VDAC1 and Histone H4 also display this at the earlier time-point, and this highlights the necessity of validating further quantitation obtained with large scale proteomics experiments, which are highly useful as hypothesis generating tools, but currently require biological follow up or further MS such as MRM to confirm quantitation. Several proteins fluctuated in level over the time course, including fatty acid synthase which is initially downregulated then upregulated at later time points. The dominant proteins Nutlin-3 responsive proteins included the known MDM2 binding proteins NPM, DNA-PK, hnRNPK, nucleolin, XRCC6 and TIF-1 beta (Figure 3a). A cluster of ribosomal proteins were detected as upregulated 12 hours post Nutlin-3 treatment, providing a connection to MDM2 function as a regulator of ribosomal pathways and internal validation that the proteins detected are in the MDM2 interactome. Many of the identified proteins display consistent responses over time, as each time-point represents a separate digestion and labeling step this builds greater confidence to


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the quantitation results. Proteins whose apparent steady-state levels are affected by Nutlin-3 treatment and have not previously been shown to interact with MDM2 may be newly identified MDM2 related or indirectly regulated proteins. The protein NONO for example, which is also known as p54nrb and has been shown to bind to IRES sequence for c-myc, and IRES binding of p53 mRNA is amongst the known functions of MDM24, 18. The presence of both up and downregulated proteins is consistent with the model that MDM2 has a proteostasic character whose equilibrium can be perturbed by the ligand Nutlin-3.

Focusing on the very acute effects of Nutlin-3 there are 4 dominant proteins with downregulated levels at one hour after treatment (e.g. “E2F1-like”), 15 proteins are upregulated (“p53-like”) and 20 downregulated (e.g. “E2F1-like”) at two hours after treatment. The most upregulated proteins include DNA-PK and NPM, and the most downregulated proteins include prohibitin-2, snRNPK, annexin A5 and fatty acid synthase (Table 2). NPM and DNA-PK are known MDM2 interacting proteins and typical MDM2 substrate proteins such as p53 and APE1 have been shown previously to increase in levels at this early time point, along with increased ubiquitination19. p53 was not detected in this screen, which is likely due to the low abundance of this protein, however the proteins which are detected as changing may be links to p53 and MDM2 related pathways. Focusing on those proteins that are altered after two hours treatment, IPA Ingenuity pathway analysis generated through the use of IPA (Ingenuity Systems, www.ingenuity.com) shows potential connections via p53, RNA polymerase and E2F1 related pathways (Fig. 4). Comparison of this to the known MDM2 interactome reveals p53, NPM and histone H4 are present in the network calculated based on proteins altered after 2 hours Nutlin-3 treatment as well as being previously published MDM2 interacting proteins 5, 20. This both


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supports the role of the newly identified proteins in the MDM2 interactome and categorizes those previously known MDM2 proteins as core MDM2 interactors. Further pathway analysis indeed reveals that later time points in this experiment gives rise to distinct predicted and expansive networks, with different members of the known MDM2 interactome becoming active at each stage (Supplemental Fig. 1).

MDM2 regulates oligomerization of NPM To further validate in detail the iTRAQ quantitation results, and investigate the effects of Nutlin3 on non-p53 MDM2 related proteins, immunoblots were used to validate the iTRAQ data for the MDM2 binding protein NPM (Fig. 5). NPM was detected at elevated levels after early Nutlin-3 treatment (Tables 1 and 2) and therefore may be integral to the cellular response to Nutlin-3. The role of NPM in ribosomal assembly and nucleolar shutting makes this an attractive target as a mediator of the proteostasis functions of MDM2. Immunoblots detected NPM mainly as SDS resistant oligomers, primarily pentamers21. Surprisingly, the level of these oligomers was decreased after Nutlin-3 treatment, however this corresponded to an increase in the level of NPM monomers detected over the Nutlin-3 time course as the level of NPM oligomers decreased (Fig. 5a), suggesting that Nutlin-3 may mediate the de-oligomerization of NPM. The increase in “monomeric” NPM in the lysate from cells treated with Nutlin-3 (Figure 5a, lanes 1-5) may contribute to the elevated levels of NPM observed in the iTRAQ analysis (Tables 1 and 2). It is possible that the oligomerization status changes may be detected by the proteomics experiment rather than an overall increase or decrease in protein levels, this reflects a limitation of current proteomic screens: detection of proteomic changes could reflect localization shifts rather than


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changes in the total protein level22. The validation of NPM protein changes in the western blot has revealed a novel aspect in the control of NPM protein levels and we focused on validating this MDM2-NPM interaction as an unexpected way in which perturbation of MDM2 can alter proteostasis; and investigated the mechanism by which Nutlin-3 stimulates the deoligomerization of NPM. To further investigate the role of MDM2 in NPM oligomerization MDM2 was transfected into H1299 cells, which have low levels of MDM2 and undetectable levels of p53. Transfection of MDM2 had the effect of decreasing NPM oligomerization similar to the effect of Nutlin-3 treatment, indicating increased levels of MDM2 result in NPM deoligomerization (Fig. 5b). Treating cells with siRNA to MDM2 in H1299 cells had the opposite effect, resulting in a reduction in NPM monomers and an increase in NPM oligomers (Fig. 5c). Intermediate bands between the pentamer and monomer are also visible in the H1299 cells (Figure 5b), although they are not well resolved, which may indicate sub-pentamer complexes are forming or that NPM exists in a series of conformations.

Nutlin—MDM2 specificity in de-oligomerization of NPM In order to determine whether Nutlin-3 can also function as an MDM2 agonist with respect to NPM oligomerization in another cell line, we evaluated the effects of Nutlin-3 on NPM oligomerization in HeLa cells. This cancer cell does not have a well-defined and “model” wt-p53 pathway like MCF7 cells, as it is derived from HPV mediated transformation. Nevertheless, HeLa cells express large amounts of MDM2 and larger amounts of NPM relative to MCF7 cells. When HeLa cells were treated with Nutlin-3, a similar de-oligomerization of NPM is observed (Figure 5d). This NPM pentamer perturbation is specific to Nutlin-3 effects, as other p53-


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“activating” drugs do not similarly perturb NPM despite the fact that they induce MDM2 and p53 (Figure 5e from left, lanes 1-6). To summarize, increased levels of MDM2 result in increased de-oligomerization of NPM in cells, and Nutlin-3 appears to act as an agonist of MDM2 function with regards to NPM de-oligomerization. This is contrary to the antagonist effect of Nutlin-3 when related to the p53 degradation function of MDM2.

Potential MDM2 binding interface in NPM oligomerization domain There three two distinct possibilities regarding the regulation of NPM de-oligomerization induced by Nutlin-3 in cells. Firstly, Nutlin-3 could inhibit the binding of MDM2 to the NPM oligomer and resulting in spontaneous disassembly of the NPM oligomer into monomeric state. This result implies that MDM2 normally binds to some motif on NPM and stabilizes the NPM oligomeric state. Alternatively, Nutlin-3 treatment cause the increased binding of MDM2 to the NPM monomer and prevent oligomerization by competing with other NPM oligomers for binding in which case Nutlin-3 acts as an agonist of MDM2 function relative to NPM. Lastly, there is no direct effect of Nutlin-3 on NPM oligomerization and the changes detected reflect a secondary effect of Nutlin-3 on the MDM2 interactome. To distinguish between these possibilities, we mapped the NPM binding site for MDM2 using overlapping 15-mer polypeptide library fragments of full-length NPM (Fig 6a) to determine whether MDM2 binds to a specific motif on NPM and if so how this interaction is effected directly by Nutlin-3. One dominant peptide derived from NPM (NPM6) binds full-length MDM2 (Fig 6b) with a high specific activity (in RLU). This binding to the NPM6 peptide can be attributed to the isolated N-terminal peptide domain MDM2 (aa 1-126) (Fig. 6c), although with a lower specific activity than full-


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length MDM2 protein. Interestingly, this NPM6 peptide is located in the NPM oligomerization domain (Fig 7e), the structure of which has previously been solved by X-ray crystallography23. Thus, we can determine whether MDM2-binding to this oligomerization motif on NPM is stabilized or inhibited by Nutlin-3. The interaction between full-length MDM2 and NPM can be reconstituted using purified proteins (Fig 7a). A titration of NPM6 greatly enhances this interaction (Fig. 7b) with a linear dose dependence from 1-10 µM, suggestive of a relative strong protein-peptide interaction. An explanation for this effect is that the NPM6 peptide lies at the oligomerization interface of NPM (Fig.7e), and may therefore cause the de-oligomerization of NPM thus leaving a greater amount of the peptide-interface in an accessible position on the surface of the protein for MDM2 protein binding. If the NPM6 peptide forms the major MDM2 docking interface, this would be more available in the monomeric form of NPM. Coimmunoprecipitation of NPM from cell lysates using MDM2 as bait results in both monomers and oligomers binding to MDM2 (Fig. 7c), however after Nutlin-3 treatment MDM2 binds a greater amount of NPM monomer and no oligomer is visible (Fig. 7c), further supporting a model where Nutlin-3 induces MDM2 binding to NPM monomers along with resulting deoligomerization of NPM and increased accessibility of the MDM2-NPM binding site, as MDM2 can pull down oligomeric NPM this indicates Nutlin-3 shifts the equilibrium towards an MDM2 state which disrupts oligomers, whereas non-Nutlin-3 bound MDM2 is capable of binding NPM without disrupting oligomerisation entirely, perhaps due to a second NPM oligomerisation interface (Fig. 7c).

Taken together these data show the dependence of NPM oligomerization on MDM2, and support a model where MDM2 binding to oligomerization interface of NPM is enhanced in the presence


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of Nutlin-3. We evaluated whether Nutlin-3 can directly stabilize the MDM2 protein-NPM6 peptide complex using the Alpha Screen proximity ligation assay that measures real-time binding of a protein-protein interaction. Donor beads were assembled with biotinylated BOX-I peptide derived from p53 or a biotinylated NPM6 derived from the oligomerizatiom domain of NPM (Fig 7d). The addition of Nutlin-3 de-stabilized the binding of the BOX-I peptide (Fig 7d), as expected24. By contrast, Nutlin-3 enhanced NPM peptide binding to full-length MDM2 by more than 50% (Fig. 7d). These data suggest that Nutlin-3 can allosterically activate an interface that binds the NPM peptide on MDM2, and the crystal structure of NPM shows the peptide NPM-6 is more exposed in the monomer than the oligomer (Fig. 7e). NPM-6 is located at the binding interface between oligomers in the NPM oligomerization domain, and adjacent to the binding site of NSC348884 the NPM oligomerization disruption drug25, which further implicates this MDM2 binding peptide in the de-oligomerization of NPM. A model can be proposed based on the data in which MDM2 mediates NPM deoligomerization and Nutlin-3 stimulates this process in which Nutlin-3 activates a binding site in MDM2 for NPM, and monomers preferentially display this site (Fig. 8).

Discussion A system wide-understanding of kinase, acetylation, methylation, and ubiquitin signalling networks and their dynamics is the new challenge of molecular cell biology that aims to build on the iterative science that has defined protein-protein interactions, often one-by-one over a time frame of decades. For example, it has taken over twenty years to define the currently known interactome of the p53 tumour suppressor protein that now comprises almost 400 proteins5. The


17


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Page 18 of 43

use of mass-spectrometry, by contrast, has allowed the rapid annotation of large interactomes in one “snap-shot”. An example of this is the use of phospho-specific antibodies as an affinity matrix to directly enrich for substrates has revealed that the interactome of the ATM kinase, identifying over 700 proteins; a far greater number of target proteins than anticipated26. In the case of ubiquitin ligases that mediate proteostasis, mass spectrometry could also provide a tool to examine an interactome of a target protein as this approach can quantitatively evaluate dynamic changes in protein levels in the cell after specific perturbation of the ubiquitin ligase. There is intense interest in understanding the function and regulation of the oncoprotein and ubiquitin ligase MDM2 and how this knowledge can be exploited to develop better anti-cancer therapeutics. Annotating dominant MDM2 interacting proteins in a cell using a systems level approach might define the dominant changes that can explain how disturbing proteostasis might better kill cancer cells. It is known that the binding of MDM2 to its intracellular target proteins can stabilize as well as destabilize protein levels (Figure 1), due to the role of MDM2 in both protein degradation and protein synthesis pathways. MDM2 can also stimulate p53 protein synthesis through interaction with mRNA stem loops4, showing it does not just function as a ubiquitin ligase. An annotation defining the dominance of the greater than one hundred MDM2 binding proteins has not been constructed, neither has the cell-specificity of such interactions, nor has been provided a rationalization of how it is possible for many proteins to interact with MDM2 at any one given time. As MDM2 can bind multiple proteins it is therefore useful to consider the consequences of both of these mechanisms after Nutlin-3 modulation of MDM2 at a systems biology scale. Although Nutlin-3 has primarily been characterized as a p53-MDM2 interaction inhibitor, it is also known that drug binding at the MDM2 hydrophobic pocket can make other binding sites available for protein-protein interactions11; for example Nutlin-3 can


18

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stabilize the acidic domain of MDM2 to interact with the DNA-binding domain of p53 and stimulate p53 ubiquitination leading to p53 protein degradation. This means that although Nutlin-3 “inhibits” MDM2 function with regards to overall p53 activity, at the mechanistic level Nutlin-3 can enhance the enzymatic function of MDM2 with regards to p53 ubiquitination. This might also result in the transfer of proteins to other regulatory pathways as has been observed for E2F112, 17. Acute time points after Nutlin-3 treatment are integral to understanding the mechanism of the MDM2 response, illustrated by the very early increase in p53 levels after the drug treatment (Fig 1a). It has been shown that Nutlin-3 can stabilize p53 as early as 15 minutes after treatment in contrast to other MDM2 ligands27, and this immediate response on the proteome level can be described as the first stage in the wider, cascading cellular response to Nutlin-3. Proteomics can therefore increase our knowledge of the MDM2 interactome by identifying which proteins take part in the core Nutlin-3 response. Capturing this early response is a prime goal for proteomic studies, as protein conformation changes and protein-protein interactions trigger the beginning of widespread cellular signaling cascades. The two contrasting Nutlin-3 responses in relation to regulating protein levels support a proteostatic role for MDM2 and the mass spectrometry results that were obtained here support this. Ribosomal proteins and initiation factors are present as well as a range of both up and downregulated proteins at an early time point, and these proteins may be targets of MDM2 mediated ubiquitination, highlighting another role for time resolved proteomics experiments as a means to solving the problem of finding novel E3 ligase substrates. The time course design of this experiment allows insights into the dynamic nature of the proteome, for example the levels of fatty acid synthase fluctuate over time and further


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investigation into these mechanisms may be important for understanding the MDM2 interactome. Other intriguing proteins were identified as being Nutlin-3 responsive. It is interesting that importin is a highly upregulated protein at 2 hours after Nutlin-3 treatment, as NPM and MDM2 have been shown to shuttle between the nucleolus, nucleoplasm and cytoplasm potentially cooperating to transport ribosomal proteins15 and importin is involved in nuclear transport28. Other relevant proteins which participate in the Nutlin-3 regulated MDM2 interactome include apoptosis markers VDAC1 and VDAC229, and the known cancer marker pyruvate kinase30. Our preliminary data indicate that pyruvate kinase can indeed bind MDM2 (manuscript in preparation) and builds on the cancer related interacting proteins for pyruvate kinase including βcatenin31 and HIF-132. Indeed, many of the proteomic hits identified in this study could constitute interesting leads for MDM2 related studies. Validation of one of the proteomic screen hits in detail, NPM, was carried out based on our validation that showed total NPM protein levels do not necessarily change. Rather, the oligomerization state of NPM changes after Nutlin-3 treatment (Figure 5) and we evaluated how Nutlin-3 changes NPM oligomerization. NPM has been shown to bind MDM2, and oligomerization of NPM is vital for NPM function25. NPM can exist as a fusion protein with ALK and this functions as an oncoprotein associated with lymphoma33 and has been linked with a change in MDM2 phosphorylation status34. ARF has previously been shown to be an NPM and MDM2 binding partner and these proteins participate in a regulatory pathway with ribosomal protein L11 which mediates MDM2 localization and p53 ubiquitination function35. The nucleolar location and functions of NPM constitute a link between MDM2 and ribosomal pathways. These pathways may be related to proteostasis roles of MDM2 by bringing it into proximity with the protein synthesis and folding machinery via interactions


20

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with ribosomal proteins. The effect Nutlin-3 has to stabilize levels of NPM as detected using iTRAQ (Tables 1 and 2) and change NPM oligomerization status (Figure 4 and 5) suggests MDM2 is a key regulator of NPM, and Nutlin-3 can modulate this regulation. A drug, NSC348884 has been developed which targets the NPM oligomerization domain and this has been shown to cause apoptosis in cancer cells, validating the importance of this oligomerization state as a drug target25. The data from this study demonstrates that as well as being an antagonist of p53-MDM2, Nutlin-3 is an agonist of MDM2 mediated de-oligomerization of NPM. It is therefore possible that Nutlin-3 may be an agonist for other MDM2 functions and this is significant for ‘non-ubiquitination’ or “non-p53” effects of Nutlin-3. This study highlights similarities between the effect of Nutlin-3 on NPM and p53, both have stabilized levels after Nutlin-3 treatment, and peptide binding sites which exhibit altered MDM2 binding in response to the ligand, which significantly may represent a more widespread mechanism of MDM2 proteinprotein interactions. The modulation of nucleolar and ribosomal MDM2 functions of Nutlin-3 highlights a potential role for the molecule to target non-p53 pathways linked to MDM2. The primary effects of Nutlin-3 in p53 mutant cancer may even be due to the activation of other pathways, rather than direct effects on p5336. Some studies have demonstrated p53 independent Nutlin-3 responses, for example in DNA damage repair37. NPM was detected in this screen due to increased levels in the mass-spectrometric compatible lysis buffer used at an early time-point after Nutlin-3 treatment that may be due to a feedback effect as MDM2 binds NPM monomers and the level of oligomers decreases (Figure 8). MDM2 has been shown to bind peptides at the hydrophobic pocket, and this domain is inherently flexible38. Regulation of the Nutlin-3 binding site is mediated by a flexible lid domain, and phosphorylation of Serine 1724. The MDM2 binding NPM peptide identified in this study bound


21


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strongly to full-length MDM2, and it is intriguing that this binding was enhanced by the addition of either the NPM6 peptide or Nutlin-3 (Figure 5). This result suggests an additional MDM2NPM interface may be made available when Nutlin-3 binds MDM2, with a mechanism similar to the MDM2-p53 allosteric model. This is supported by the decreased ability of MDM2 to coimmunoprecipitate NPM oligomers after Nutlin-3 treatment (Figure 7), suggesting a binding site for monomeric NPM is available or activated. Previous work has suggested the regulation of NPM is p53 dependent34, however we have demonstrated here that MDM2 also actively affects the oligomerization status of NPM. Just as Nutlin-3 disrupts NPM oligomerisation by stimulating the MDM2-NPM binding, it is possible that p53 could have the same effect by occupying the NPM binding site on MDM2. As p53 function is dependent on tetramerization it would also be intriguing to determine whether Nutlin-3 binding to MDM2 changes p53 oligomerisation. Further work would be required to extensively map and confirm this interface, as it is known that the acidic domain of MDM2 can bind peptides when Nutlin-3 is present and the disorder of this domain may mean it can bind a wide range of peptides and this cannot be ruled out as an NPM binding site. NPM also has a large disordered region, and this can be a factor in mediating protein-protein interactions. CONCLUSIONS In this study we have used a quantitative proteomic screen to investigate the core interactome of MDM2 using Nutlin-3 as a tool to perturb the equilibrium of MDM2. This led to the discovery of the MDM2 mediated regulation of NPM oligomerization, and mapping of a potential interface for NPM binding to MDM2. The NPM-MDM2 relationship is interesting with regards to the proteostasis function of MDM2 that could be exploited as a route to targeting a broad range of cancer cells, which does not necessarily depend on p53 status. This is foreshadowed by the


22

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ability if Nutlin-3 to induce apoptosis in p53 mutant cells39, suggesting a p53-independent effect of Nutlin-3 on cellular growth control. Interestingly this was in combination with a proteasome inhibitor, again alluding to the proteostasis role of MDM2. Here we have defined an agonist role for the drug Nutlin-3, and this highlights the importance of combining proteomic mass spectrometry results with biological validation. By focusing on a dominant hit from the proteomic screen we identified and characterized a new consequence of Nutlin-3 binding MDM2. The dynamic and acute responses of the interactome of a protein to a drug are vital to understanding the way a drug elicits a response, and proteomic mass spectrometry an extremely powerful tool for identifying new substrates and mechanisms for a protein, as well as generating a means for choosing future protein-protein interactions to investigate based on quantitative mass spectrometric evidence rather than the low throughput methods which studies on the MDM2 interactome have until now relied on.

FIGURES LEGENDS

Figure 1. Nutlin-3 alters the steady-state levels of two core MDM2-binding proteins. A) left, immunoblot of cells treated with Nutlin-3 to evaluate changes in steady-state levels of two key MDM2-interacting proteins, p53 and E2F1. The treatment of cells with Nutlin-3 (µM) displaces MDM2 from p53 (stabilizing p53) and displaces MDM2 from E2F1 (destabilizing E2F1). C is control untreated and D is DMSO control treatment. B) right, Model depicting the effects of MDM2 inhibition by Nutlin-3 on the stability and degradation of its interacting proteins. Although MDM2 mediates degradation of p53 and stabilization of E2F1 through binding to peptide docking sites in the N-terminus of p53 or the C-terminus of E2F1, the number and identity of all cellular proteins which are affected by Nutlin-3 are undefined. Treatment of cells with Nutlin-3 would result in either stabilization (p53-like proteins) or destabilization (E2F1-like proteins) of MDM2 interacting proteins, stratify the dominant MDM2 interacting proteins, and begin to define the role of MDM2 in proteostasis.


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Figure 2. Summary of iTRAQ labeling scheme. – A) iTRAQ labeling scheme for cells harvested after different periods of time post-Nutlin-3 treatment. B) Workflow for preparation of iTRAQ labeled samples. C) Summary of MS identifications and spectra identified at various confidence cut-offs, cut-off used highlighted in bold. Figure 3. Proteins detected in iTRAQ analysis which are known members of the MDM2 interactome and cluster analysis – A) Overlap between proteins identified in iTRAQ proteomics screen and known MDM2 interactome with change in protein level detected relative to untreated cells and hours detected as changed after Nutlin treatment. B) Number of proteins detected up and downregulated at each time point after Nutlin treatment. C) Hierarchical cluster analysis of proteins detected as up or downregulated after Nutlin treatment (created using multiple experiment viewer, MEV40)

Figure 4 - Ingenuity pathway analysis of proteins with altered levels after 2 hours Nutlin-3 treatment. The color codes highlight: downregulated proteins (Green), upregulated proteins (Red), and members of the previously published known MDM2 interactome (Orange). Grey shapes (e.g. XRCC5) represent proteins in the dataset (that form interactions with the proteins that are changing) but which are not up or downregulated. White shapes (e.g. RB1) represent proteins (that form interactions with the proteins that are changing) but are not detected in this dataset. Figure 5. Immunoblots reveal NPM oligomerisation response to Nutlin-3. A) Effect of Nutlin-3 treatment on NPM levels over time, with monomers detected running at approximately 40 kDa, main oligomers (pentamer) detected at approximately 200 kDa. As level of MDM2 increases NPM oligomers are reduced and monomers increased in level. The “increase” in NPM protein detected using iTRAQ (Tables 1 and 2) may reflect an overall increase in NPM as well as an increase in “soluble” monomeric NPM protein, consistent with the immunoblot. B) Transfected MDM2 decreases the amount of higher order NPM oligomers (lanes indicate µg MDM2 transfected). C) siRNA to MDM2 increases the amount of NPM oligomers (nmol siRNA added indicated above lanes). D) Reduction of NPM pentamers in HeLa cells (no wt p53, high levels of MDM2) stimulated by Nutlin-3 treatment (Nutlin-3 concentration in µM indicted above lanes). E) p53 activating drugs campothecin and troglitazone do not cause a reduction in NPM oligomerization contrary to Nutlin-3 (Concentration of drugs in µM indicated above lanes) indicating the deoligomerisation effect depends on MDM2.


24

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Figure 6. Identification of a high affinity binding peptide for MDM2 in the NPM oligomerization domain. – A) Sequence of NPM as overlapping biotinylated peptides; those which exhibit binding to MDM2 highlighted in bold. B) Full-length MDM2 binding to immobilized NPM peptides was measured by ELISA. Biotinylated peptides were captured on streptavidin-coated solid phase and after the addition of full-length MDM2, binding was measured with the anti-MDM2 MAB (2A10) and peroxidase labeled 2nd antibody followed by enhanced chemiluminescence. The binding activity of MDM2 is plotted as a function of peptide number. C) N-terminal domain MDM2 (aa1-126) binding to immobilized NPM peptides. Biotinylated peptides were captured on streptavidin-coated solid phase and after the addition of full-length MDM2, binding was measured with the anti-MDM2 MAB (4B2) and peroxidase labeled 2nd antibody followed by enhanced chemiluminescence. The binding activity of MDM2 is plotted as a function of peptide number.

Figure 7 - Nutlin-3 stimulates MDM2 binding to the oligomerization interface peptide from NPM. A) Full-length MDM2 binding to immobilized full-length His-NPM. Solid phase was coated with NPM or non-MDM2 binding protein control (AGR2) and MDM2 was titrated into reactions to measure maximal binding with the anti-MDM2 MAB (4B2) and peroxidase labeled 2nd antibody followed by enhanced chemiluminescence. B) Enhancement of protein-protein interactions between MDM2 and NPM by the addition of NPM6 peptide 6. Full-length NPM was coated onto the solid phase and after the addition of MDM2 protein, the indicated amounts of peptide 6 were added and protein complex stability was measured with the anti-MDM2 MAB (4B2) and peroxidase labeled 2nd antibody followed by enhanced chemiluminescence. C) Co-immunoprecipitation of NPM oligomers with MDM2 at 0 and 2 hours after Nutlin-3 treatment followed by immunoblotting to measure the ratios of oligomeric and monomeric NPM. No oligomers are detected binding to MDM2 after Nutlin-3 treatment. D) Competition of MDM2 from BOX-I peptide with Nutlin-3 and enhancement of NPM6 peptide by Nutlin-3 detected by AlphaScreen. E) (left) PDB:2P1B NPM oligomerization domain pentamer, monomer highlighted in yellow, peptide highlighted in pink (middle) Different view of NPM oligomer, (right) Two subunits of NPM oligomer with NPM6 peptide highlighted in pink.

Figure 8. Model of MDM2 activation by Nutlin-3 and regulation of NPM oligomerization (i-iii) MDM2 is an allosterically regulated protein with various peptide-binding domains; the most well-characterized being the N-terminal p53-peptide binding groove in which fits the small molecule Nutlin-3. The top figure depicts three allosteric effects of Nutlin-3 on MDM2: (i) Nutlin-3 allosterically increases binding of the central acidic domain of MDM2 to the central domain of p53 resulting in enhanced p53 ubiquitination in cells (17); (ii) Nutlin-3 displaces proteins that bind to the N-terminus of MDM2 (such as p53 itself, E2F1, DNA-PK, and proteins in Table 1; 2) that results in changes in steady state levels of the


25


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Page 26 of 43

protein; and (iii) Nutlin-3 allosterically stimulates binding of MDM2 to the oligomerization interface of NPM and stabilizes the monomeric form (Figure 5-7). This latter effect (iii) is similar to (i); where Nutlin3 acts as an agonist of MDM2 functions; the difference being the outcome-in the case of p53 this results in increased ubiquitination and in the case of NPM; (iv) a shift in equilibrium from oligomer to monomeric conformation upon addition of Nutlin-3.

Table 1 - List of proteins up and downregulated at various time points detected in iTRAQ screen.* 1hr Accession (Uniprot)

P80404

Protein 4-aminobutyrate aminotransferase, mitochondrial precursor

% Cov

113: 114

P-val

115: 114

P-val

8

33.4

0.506

0.048

0.535

0.043

29

72.7

0.679

0.025

9

41.6

0.673

0.010

0.515 0.809

0.048

0.619

0.000

0.692

0.000

P08195

O75369

Filamin-B

47

32.6

P62805

Histone H4

37

79.6

P23246 P55786

P21796

P20700

O00629

Splicing factor, proline- and glutamine-rich Puromycin-sensitive aminopeptidase

Voltage-dependent anionselective channel protein 1

Lamin-B1

Importin subunit alpha-4

4hr

Pep. (95%)

ATP synthase subunit alpha, mitochondrial precursor 4F2 cell-surface antigen heavy chain

P25705

2hr

0.738

0.002

0.200

0.049

3.251

0.046

31.5

0.540

0.044

3

15.8

0.667

0.043

0.692

0.032

0.334

0.038

1.600

0.045

0.575

0.038

1.706

0.020

5.916

0.035

4.875

0.038

21

2

59.7

51.2

11.9

P-val

0.001

12

16

116: 114

8hr

P27797

Calreticulin precursor

29

80.6

1.406

0.033

Q99623

Prohibitin-2

10

65.6

0.581

0.028

P40926

Malate dehydrogenase, mitochondrial precursor

25

75.4

1.803

0.018

34

76.4

2.512

0.017

37

73.8

3.105

0.007

0.453

0.000

117: 114

12hr

P-val

118: 114

P-val

0.063

0.040

0.511

0.028

0.305

0.013

0.637

0.008

0.759

0.021

0.698

0.004

0.679

0.000

0.649

0.000

4.286

0.010

2.032

0.008

0.655

0.020

0.655

0.020

1.820

0.021

1.888

0.012

1.820

0.005

1.906

0.001

3.565

0.040

4.786

0.038

5.495

0.037

0.213

0.004

2.606

0.032

2.355

0.023

7.244

0.036

P14618

Protein disulfide-isomerase precursor Pyruvate kinase isozymes M1/M2

Q08211

ATP-dependent RNA helicase A

39

41.1

2.377

0.006

P30101

Protein disulfide-isomerase A3 precursor

35

65.4

2.109

0.006

0.353

0.020

Q9Y4L1

Hypoxia up-regulated protein 1 precursor

13

29.5

1.644

0.003

1.406

0.043

P07237


2.109

0.032 2.938

2.109

0.013

0.024

26

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P06748

Nucleophosmin

42

88.4

3.532

0.003

2.992

0.015

P10809

60 kDa heat shock protein, mitochondrial precursor

78

81.2

1.871

0.001

P08727

Keratin, type I cytoskeletal 19

92

86.8

0.353

0.000

1.871

0.000

2.109

0.000

P02545

Lamin-A/C

38

55.7

0.637

0.000

P05787

Keratin, type II cytoskeletal 8

176

93.8

0.096

0.000

3.192

0.000

2.168

P05783

Keratin, type I cytoskeletal 18

147

95.1

3.767 0.070

0.000 0.000

3.404

0.000

2.249

3.733

0.000

1.380

0.631

0.044

2.443

0.000

3.698

0.000

0.000

4.325

0.000

0.000

6.427 4.055

0.000 0.000

5.970

0.000

12hr 118: 114

P-val

1.514

0.029

0.603

0.049

0.011

Table 1 Cont. Accession (Uniprot)

Protein

Pep. (95%)

% Cov

1hr 113: 114

P-val

2hr 115: 114

P-val

P08758

Annexin A5

0.299

0.041

P21266

Glutathione S-transferase Mu 3

0.515

0.020

P11413

Glucose-6-phosphate 1dehydrogenase

26

68.4

0.692

0.009

Q01105

Protein SET

16

47.2

Q9Y224

Protein C14orf166

6

36.1

P29401

24

P05023

Transketolase Sodium/potassium-transporting ATPase subunit alpha-1 precursor

9

P04843

Dolichyldiphosphooligosaccharide-protein glycosyltransferase 67 kDa subunit precursor

24

P60174

Triosephosphate isomerase

20

P05455

Lupus La protein

13

P23284

Peptidyl-prolyl cis-trans isomerase B precursor

P45880

Voltage-dependent anionselective channel protein 2

0.047

50.7

0.592

0.046

21

1.393

0.043

52.6

2.512

0.043

85.1

0.344

0.042

47.3

0.565

0.042

11

62.5

1.660

0.042

8

48.3

1.614

0.038

Leucine-rich repeat-containing protein 59

11

P06744

Glucose-6-phosphate isomerase

22

O14745

Ezrin-radixin-moesin-binding phosphoprotein 50

12

34.4

Q9H993

UPF0364 protein C6orf211

8

32.9

0.667

0.011

Q15233

Non-POU domain-containing octamer-binding protein

12

41.6

2.355

0.038

3

15.4

40.4

3.500

0.029

52.5

0.286

0.021

0.449

0.017

0.453

0.016

2.089

0.010

58

79.4

21

90.9

Alpha-enolase

34

82.7

Core histone macro-H2A.1

14

48.4

18

0.033

0.038

P06733

Phosphoglycerate kinase 1

2.938

3.020

O75367

P00558

0.048

0.046

0.047

Q96AG4

P62937

0.692

0.619

0.692

Heterogeneous nuclear ribonucleoprotein G

Glyceraldehyde-3-phosphate dehydrogenase Peptidyl-prolyl cis-trans isomerase A

P-val

0.619

P38159

P04406

4hr 116: 114

0.483

1.675

0.029

0.004

51.8


0.283

0.010

0.437

0.008

0.433

0.007

1.888

0.003

0.387

8hr 117: 114

1.803

1.432

P-val

0.024

0.021

1.923

0.030

2.630

0.005

3.873

0.031

0.560

0.026

2.148

0.020

2.938

0.003

1.820

0.025

2.168

0.003

0.002

27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

P78527

DNA-dependent protein kinase catalytic subunit

23

8.166

18.3

0.000

Page 28 of 43

9.818

0.000

7.447

0.000

9.290

0.000

7.516

0.000

P06576

ATP synthase subunit beta, mitochondrial precursor

57

76.9

0.698

0.037

0.429

0.005

P22626

Heterogeneous nuclear ribonucleoproteins A2/B1

31

65.7

0.724

0.026

0.074

0.040

P61978

Heterogeneous nuclear ribonucleoprotein K

33

66.1

0.603

0.026

0.413

0.010

% Cov

8hr 117: 114

P-val

12hr 118: 114

P-val

2.168

0.006

1.459

0.005

2.188

0.006

1.995

0.008

Table 1 Cont. Accession (Uniprot)

Protein

Pep. (95%)

P49327

Fatty acid synthase

62

38.1

P13639

Elongation factor 2

27

49.5

P36578

60S ribosomal protein L4

12

43.3

P21333

Q09666 P27635

Filamin-A

Neuroblast differentiationassociated protein AHNAK

39

1hr 113: 114

P-val

2hr 115: 114

P-val

0.597

0.015

2.051

0.015

27.9

4hr 116: 114

Pval

1.871

0.047

3.664

0.000

1.406

0.034

1.432

0.012

43

44.8

2.312

0.000

8

49.5

1.331

0.028

6

17.6

0.501

0.050

3

17.9

0.698

0.045

Q02218

60S ribosomal protein L10 2-oxoglutarate dehydrogenase E1 component, mitochondrial precursor

Q92820

Gamma-glutamyl hydrolase precursor

P54886

Delta-1-pyrroline-5-carboxylate synthetase

11

26.4

0.679

0.042

P07355

Annexin A2

27

74.3

1.959

0.034

P14625

Endoplasmin precursor

41

58.3

0.535

0.033

P39023


11

27.5

1.331

0.032

Q9BZZ5

Apoptosis inhibitor 5

3

20.2

0.731

0.029

P13010

ATP-dependent DNA helicase 2 subunit 2

38

65.9

0.302

0.029

Q99729

Heterogeneous nuclear ribonucleoprotein A/B

13

32.8

0.094

0.028

57.2

0.497

0.021 0.020

P19338

Nucleolin

32

P16401

Histone H1.5

15

59.3

2.070

P62424

60S ribosomal protein L7a

8

31.2

2.704

0.010

P02786

Transferrin receptor protein 1

18

30.9

0.128

0.007

P12956

ATP-dependent DNA helicase 2 subunit 1

24

45.2

0.236

0.007

P46781

40S ribosomal protein S9

5

54.6

2.070

0.006

Q13263

Transcription intermediary factor 1-beta

29

40

0.316

0.006

P18124


5

49.2

2.535

0.005

Q02878

7

50.4

1.977

0.004

P51572

60S ribosomal protein L6 B-cell receptor-associated protein 31

7

38.6

2.128

0.003

P26599

Polypyrimidine tract-binding protein 1

20

54.6

0.187

0.001


28

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P42704

Leucine-rich PPR motifcontaining protein, mitochondrial precursor

37

0.211

40.1

*Bold values indicate second biological replicate (2 hr – ratio of 119:114, 12 hr ratio of 121:114 as indicated in Figure 2). % Cov - sequence coverage, Pept. (95%) – Number of peptides identified with >95% confidence used for quantitation, P-val – P-value (cut-off

An iTRAQ Proteomics Screen Reveals the Effects of the MDM2

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