SUMO-binding entities (SUBEs) are useful tools for the enrichment, isolation, identification and characterization of the SUMO proteome from liver cancer mouse model

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SUMO-binding entities (SUBEs) are useful tools for the

enrichment, isolation, identification and characterization

of the SUMO proteome from liver cancer mouse model

Fernando Lopitz-Otsoa, Sofia Lachiondo-Ortega, Teresa Delgado, Mikel

Azkargorta, Felix Elortza, Manuel Rodríguez, María Luz Martínez-Chantar

To cite this version:

Fernando Lopitz-Otsoa, Sofia Lachiondo-Ortega, Teresa Delgado, Mikel Azkargorta, Felix Elortza, et al.. SUMO-binding entities (SUBEs) are useful tools for the enrichment, isolation, identification and characterization of the SUMO proteome from liver cancer mouse model. Journal of visualized experiments : JoVE, JoVE, 2019. �hal-03024432�

SUMO-binding entities (SUBEs) are useful tools for the enrichment, isolation, identification and characterization of the SUMO proteome from liver cancer mouse model

Fernando Lopitz-Otsoa1_{, Sofia Lachiondo-Ortega}1_{, Teresa C Delgado}1_{, Mikel Azkargorta}2_{, Felix Elortza}2_,

Manuel S Rodríguez3,*_{, María Luz Martínez-Chantar}1,*

1_{Liver Disease and Liver Metabolism Lab, CIC bioGUNE, Centro de Investigación Biomédica en Red de}

Enfermedades Hepáticas y Digestivas (CIBERehd), 48160 Derio, Bizkaia, Spain.

2_{Proteomics Platforms,CIC bioGUNE. Carlos III Networked Proteomics Platform (ProteoRed-ISCIII).} 3_{UbiCARE, Advanced Technology Institute in Life Sciences (ITAV)-CNRS-IPBS, 31106 Toulouse, France.}

*Joint Corresponding authors: Manuel S Rodríguez, UbiCARE, Advanced Technology Institute in Life Sciences (ITAV)-CNRS-IPBS, 31106 Toulouse, France. manuel.rodriguez@itav.fr; Tel:+33-582-991026 . María Luz Martínez-Chantar, CIC bioGUNE, Ed. 801A Parque Tecnológico de Bizkaia, 48160 Derio, Bizkaia, Spain. mlmartinez@cicbiogune.es; Tel: +34-944-061318; Fax: +34-944-061301.

Email Addresses of Co-authors:

Fernando Lopitz-Otsoa (flopitz@cicbiogune.es) Sofia Lachiondo-Ortega (slachiondo@cicbiogune.es) Teresa Cardoso Delgado (tcardoso@cicbiogune.es) Mikel Azkargorta (mazkargorta@cicbiogune.es) Felix Elortza (felortza@cicbiogune.es)

KEYWORDS: SUMO, SUBEs, Liver cancer, Hepatocellular carcinoma, hepatoma, Mass-Spectrometry.

SUMMARY

SUMO-binding entities (SUBEs) can be used to enrich, isolate, identify and characterize proteins modified by SUMO in vivo both from hepatoma cells and liver tumors obtained from mice models of hepatocellular carcinoma.

ABSTRACT

Post-translational modifications are key mechanisms regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins, particular attention has been given to the modification by SUMO (Small Ubiquitin MOdifier) in liver cancer. Isolating endogenous SUMOylated proteins in vivo is challengingly due to the presence of active SUMO-specific proteases. Initial studies of SUMOylation in

cases antibodies generally made with non-modified recombinant protein, do not immunoprecipitate SUMOylated forms of the protein of interest. Another approach to study SUMOylation has been the nickel chromatography to capture histidinylated versions of SUMO molecules. This approach can only be used with cells stably expressing or transiently transfected with His-SUMO molecules. To overcome these limitations, SUMO-binding entities (SUBEs) were developed to isolate endogenous SUMOylated proteins. Herein, we describe all the steps required for the enrichment, isolation and identification of SUMOylated substrates using SUBEs from human hepatoma cells or liver cancer mouse model. Firstly, we describe the methods involved in the preparation and lysis of the human hepatoma cells and liver tumor tissue samples. Then, a thorough explanation of the preparation of SUBEs and controls is detailed as well as the protocol for the protein pull-down assays. Finally, some examples are provided regarding the options available for the identification and characterization of the SUMOylated proteome, namely the use of Western-blot analysis for the detection of a specific SUMOylated substrate from liver tumors or the use of Proteomics for a high-throughput characterization of the SUMOyloma and interactome associated from hepatoma cells.

INTRODUCTION

Liver cancer is the sixth most prevailing cancer worldwide (1). Hepatocellular Carcinoma (HCC) is the most common form of primary liver cancer (70-85% of total liver cancer). Common risk factors for the development of HCC include chronic hepatitis B or C infection, metabolic syndrome, Type 2 diabetes and alcohol consumption (2). HCC is considered phenotypically and genetically a very heterogeneous cancer being HCC pathogenesis characterized by disruption of a complex network of signaling pathways. Although our knowledge of the major molecular pathways implicated in the pathogene sis of HCC has increased dramatically in the last years, one of the main difficulties when treating HCC is that many pathways are activated and inhibiting one generally drives compensation by other pathways. Thus, a more global mechanism, such as post-translational modifications of proteins that can simultaneously regulate multiple disrupted signaling pathways may provide a valuable therapeutic approach for liver cancer clinical management.

Post-translational modifications are considered key mechanisms regulating protein homeostasis and function in eukaryotic cells. These modifications extend the diversity of the proteome by inducing structural and functional changes in proteins through different mechanisms like covalent binding of functional groups, cleavage of regulatory subunits and degradation of other proteins. The most common post-translational modifications include phosphorylation, methylation, acetylation, glycosylation, ubiquitination and ubiquitin-like proteins (UbLs). Among all UbLs, protein modification by SUMO (Small Ubiquitin MOdifier) has attracted particular attention due to its critical roles in a variety of cellular processes, including transcription, cellular localization, DNA repair, and cell cycle progression (REF). In liver cancer, SUMOylation appears to be altered (3-6), and changes in the SUMOylation of specific proteins has been identified to play a role in the progression of the cancer related diseases (6).

In mammals, there are five SUMO paralogues, being SUMO-1, -2 and -3 more studied, compared to SUMO-4 and -5 (7-9). SUMOylation in mammals is carried out by an enzymatic thiol-ester cascade involving three enzymes, the heterodimeric SUMO activating enzyme SEA1/SEA2 or E1, the SUMO conjugating enzyme Ubc9 or E2 and a SUMO-E3-ligase specific for each target protein. Several families of SUMO E3s have been reported whose action appears to be in a dynamic equilibrium with hyperactive SUMO-specific proteases known as SUSPs or SENPs (REF). Indeed, isolating endogenous SUMOylated proteins in vivo is rather challengingly mainly due to the presence of these active SENPs which remove SUMO from protein substrates making this reaction with high reversibility (REF).

Initial studies to study SUMOylation in vivo were based on the detection by Western-blot of specific SUMOylated proteins using antibodies against the protein of interest. First, the protein was immunoprecipitated using specific antibodies, and then analyzed by PAGE-Western blot detection with anti-SUMO antibodies. However, antibodies generally made with non-modified recombinant protein, in many cases, do not immunoprecipitate the SUMOylated form of a protein. Another approach to study SUMOylation has been the nickel chromatography using the different Histidinylated (His6) versions of SUMO molecules. Preliminary experiments can be set up by transiently expressing His6-SUMO molecules together with target proteins of interest. However, it will be more convenient to detect SUMO-modified forms from cells stably expressing His6-SUMO (REF). Even though this approach is attractive, it is limited to cultured cells and cannot be applied for example to the study of the SUMOylated proteome in tissue biopsies. To overcome these limitations, SUMO-binding entities (SUBEs), also known as SUMO traps, were developed (REF). Briefly, SUBEs are recombinant proteins that comprise tandem repeats of SUMO-interacting motifs (SIMs) thereby recognizing SUMO molecules on modified proteins with an increase in the overall affinity for SUMO substrates. SUMO-traps were engineered by introducing RNF4-derived SIM2 and SIM3 motifs in tandem, into a glutathione S-transferase (GST) vector tag, an heterologous carrier protein (REF). Overall, this method provides a tool to facilitate the purification and identification of SUMO target proteins in liver cancer samples that include both hepatoma cells and mouse liver biopsies.

PROTOCOL

An overall scheme of the protocol described in this manuscript is sown in Figure 1.

1. Cell Preparation and Lysis

1.1) Maintain cells in P100 plates in standard grown media at 37 °C in a humidified atmosphere of 5% CO2-95% humidity. Huh-7 (human hepatoma cell lines) and THLE2 (human hepatic cell line), were used.

Huh-7 was cultured in DMEM supplemented with 10% FBS, 1% PSA and 1% glutamine. THLE2 cells should be grown in BEBM supplemented with 10% FBS, 5 ng/ml EGF and 70 ng/ml phosphoetanolamine. 1.2) Grow cells in P100 plates to a density of approximately 1.2–1.5 x 106 _{cells per time/condition point.}

SUMOylated proteins (and loose the low abundant ones).

1.3) Using a sterile tissue-culture flow hood, aspirate the media from the flask and wash cells with 5 mL of sterile 1x phosphate-buffered saline (PBS).

1.4) Lyse cells directly on the plate (which is placed on ice). For one P100 plates, use 500 μL lysis buffer. Using cell scraper, gently scrape the cells off the bottom of the plate into the Lysis media. Check all the cells have come off by inspecting the base of the plate before moving on. Alternatively, cells may be harvested by trypsination and pelleted in order to reduce the volume of lysis buffer and amount of SUBEs and GST. In the case of trypsination, aspirate the PBS and add 1 mL of 1x (0.05%) trypsin-EDTA to the plate, making sure that cells are covered by trypsin. Place plate in the incubator maintained at 37°C, 5% CO2, and 95% humidity until all cells have detached from the plate (approximately 5 min). Add 2 mL of pre-warmed growth media to the plate to deactivate the trypsin-EDTA.

1.5) Clarify the lysate by centrifugation in a microfuge at 15500g and 4°C for 10 min. NOTE: The protocol can be paused here.

2. Tissue Preparation and Lysis

All experiments were approved by the respective institutional committees for animal care and handling. All efforts were made to minimize animal suffering and to reduce the number of animals used.

2.1) For the extraction in tissues, wash with cold PBS, and freeze immediately in liquid nitrogen. All samples are stored at −80°C.

2.2) Approximately, 75 mg fragments from snap-frozen or fresh livers were homogenized in 1 mL of ice-cold lysis buffer with the Precellys 24 homogenizer. Run the Precellys 24 at 6500 rpm, 2x60 sec each, 30 sec pause. Transfer the supernatant to another tube and discard the pellet. Alternatively, triturate frozen tissues in liquid nitrogen and recover in 1 mL of lysis buffer.

2.3) Clarify the lysate by centrifugation in a microfuge at 15500g and 4°C for 10 min. NOTE: The protocol can be paused here.

3. Binding of GST-SUBEs or GST control to glutathione-agarose beads

3.1) Preparation of glutathione beads: Reconstitute 10 mL lyophilized glutathione-agarose beads in de-ionized water overnight at 4°C (or minimally for 30 min at room temperature). After swelling, the agarose has to be washed thoroughly with ten volumes of de-ionized water or PBS three times by centrifugation at 1000 RPM during 5 min to remove lactose or ethanol (present in the lyophilized powder to preserve agarose beads). At the end, beads are suspended in PBS to obtain 50% (v/v) slurry.

3.2) Add 100 μg of GST-SUBEs or GST control to 100μL glutathione beads slurry. This reaction take place in a tube with PBS (500µl).

NOTE: The amount of GST-SUBEs used for pull-downs depends on the relative abundance of the SUMOylated proteins of interest. It is recommended to set up conditions analyzing by Western blot the input, bound, and unbound material using anti-SUMO2/3 antibodies or against your proteins of interest.

3.3) Incubate GST-SUBEs or GST control with beads, slowly rotating (EQUIPMENT NAME AND BRANDT) at 4°C for at least 2 h (slow binding reaction).

NOTE: Adding 1 mM dithiothreitol (DTT) improves GST binding to glutathione column.

3.4) After incubation, recover the agarose beads by centrifugation at 1000 RPM during 5 min. At the end, the beads are suspended in PBS to obtain 50% (v/v) slurry.

NOTE: The protocol can be paused here.

4. GST Pull Down Assay

4.1) Following step 1.5 or 2.3, take 1/10 of total volume (e.g., 50 μL) and dilute in equal volume of 3X boiling buffer. This fraction is considered as INPUT.

4.2) Add 450 μL of clarified lysate to 100 μ L glutathione beads slurry. Incubate lysate with beads, slowly rotating at 4°C for at least 2 h (slow binding reaction).

4.3) Spin down beads in a microfuge (1000 RPM for 5 min) and collect supernatant for analysis. take 1/10 of total volume (e.g., 50 μL) and dilute in equal volume of 3X boiling buffer. This fraction is considered as the flow-through (FT) fraction.

4.4) Wash three times with 1 mL ice-cold PBS, 0.05% Tween 20 (MARCA), spin down in a microfuge at 4°C and 1000 RPM for 1 min. Aspirate carefully until no liquid remains. The beads correspond to SUBEs BOUND (SB) fraction.

4.5) Elute the sample with 30 μL 1:1 3X boiling buffer and the respective lysis buffer. This is called the BOUND Fraction.

5. Identification and characterization of SUMO targets and/or SUMOylated proteome

5.1)

Perform Western blot analysis using an anti-SUMO2/3 antibody or the specific antibody of your

choice (Fig.2B).

5.2) In the case of

described by Wisniewski et al. [REF]. Peptides were further desalted using stage-tip C18 microcolumns

(Zip-tip, Millipore) and resuspended in 0.1% FA prior to MS analysis. Samples were loaded onto a novel

timsTOF Pro with PASEF mass spectrometer (Bruker Daltonics) [REF] coupled online to a nanoElute liquid

chromatograph (Bruker), and analysed in triplicate.

REPRESENTATIVE RESULTS

Identification of a specific SUMOylation substrate in liver tumor biopsies

Liver Kinase B1 (LKB1) SUMOylation has been recently shown to be an important oncogenic driver in liver cancer (6). SUBEs were used to enrich and isolate the SUMOylated proteins both in glycine N-methyltransferase (Gnmt) deficient (Gnmt−/−) liver cancer mice and wild type (Gnmt+/+_{) mice. Figure 2}

shows an example of images and results obtained from the SUBEs capture in non-tumor and tumor liver of wild type and Gnmt−/− hepatocellular carcinoma mice. In Figure 2a are included (Ponceau S staining),

the three different fractions (input, FT and BOUND) obtained in the SUBEs pull-down assay. A Ponceau S stain is useful to control a possible deleterious effect on the charge of blotted proteins to be evaluated by Western blots. In Figure 2b a Western blot analysis of LKB1 by using SUBEs to capture endogenous SUMOylated LKB1 is shown. The levels of the SUMOylation of LKB1 are augmented in liver tumors. In the case of Western blot analysis, equal charge and transferred proteins was observed by Ponceau staining in the input fraction and was not significantly altered after washes (flow through fraction). The amount of protein captured with SUBEs was significantly higher, in particular in the tumors. (Figure 2a). Alternatively, Coomassie staining of a duplicated gel can provide similar information. Sticky proteins such as p53 or SUMOylated forms of some proteins might bind to the GST control. To remove background, low-density agarose beads, BSA coating, or additional washes can be considered. However, this could affect applications such as mass spectrometry and might result in loss of information.

Characterization of the SUMOylated interactome in human hepatoma cells

To investigate the capacity of the SUMO-trap to interact with naturally SUMOylated proteins Huh-7 (human hepatoma) and THLE2 (human hepatic) cell lines, were used. Normally, in the case of specific protein SUMOylation, the modification process is induced by challenging/treating cells with a specific agent and/or condition. Depending on the cellular context, the use of SUBEs resolve this problem, since it captures SUMOylated proteins (SUMOylome) at basal level Figure3. The first step is the visualization of the total material captured with SUBEs and using GST as negative control. For this purpose we can use conventional protein staining protocols Figure3a.

Protein identification and abundance calculation were

carried out using PEAKS software (Bioinformatics Solutions), and data was further loaded onto Perseus

platform (REF) for statistical analysis and heatmap generation. Proteins identified with at least two

different peptides were considered in the final analysis. A permutation-based FDR-corrected T-test was

applied for the comparison of the abundances, and proteins with a q < 0.05 and a SUBE/GST ratio greater

than 2 were considered as enriched and kept for further discussion Figure3b.

FIGURES AND TABLES

FIGURE AND TABLE LEGENDS:

Figure 1. Schematic diagram of the protocol flow chart used for the enrichment, isolation and identification and characterization of the SUMOylated proteome in vivo for the study of liver cancer.

Figure 2. Liver Kinase B1 (LKB1) is modified by SUMO-2 in mouse models of Hepatocellular Carcinoma. (A) Ponceau S staining of the three different fractions (input, FT and BOUND) obtained in the

SUBEs pull-down assay. (B)Western blot analysis of LKB1 by using SUMO binding entities (SUBEs) to capture endogenous SUMOylated LKB1; GAPDH is used like load control.

Figure 3. Heatmap depicting the differentially enriched proteins in Huh7 and Thle2 SUBE samples. (A) Protein stain of captured material, with GST (Negative control) and SUBEs. (B) Heatmap depicting the differentially enriched proteins in Huh7 and Thle2 SUBE samples.

DISCUSSION

JoVE is a methods-based journal. Thus, the Discussion section of the article should be focused on the

protocol and not the representative results.

This section should discuss the following with citations:

• Critical steps in the protocol

• Modifications and troubleshooting of the method

• Limitations of the method

• The significance of the method with respect to existing/alternative methods

• Future applications or directions of the method

Herein, we have provided a complete and detailed description of the methodology reporting the use of SUBEs for the enrichment, isolation and identification and characterization of the SUMOylated proteins in

in vivo models of liver cancer. Both in mouse liver tumors and human hepatoma cells, we were able to

correctly isolate and identify SUMOylated proteins of interest or to perform a high-throughput characterization of the SUMOylated proteome and interactome. Even though the synthesis of SUBEs is outside the scope of this manuscript, for further information the following references should be looked at (REFs). The protocol described is fast and very sensitive and the critical step of the protocol include the use of SENPs inhibitors (PR619? Any other?) or chemical isopeptidase inhibitors such as NEM (N-Ethylmaleimide) and IAA (2-Iodoacetamide) in the lysis buffer. OTHER CRITICAL STEPS

Do you see SUMO signatures? (SUMO chains?) You have to check this in the MS data.

SUBEs are recombinant proteins that comprise tandem repeats of SIMs thereby recognizing SUMO molecules on modified proteins with an increase in the overall affinity for SUMO substrates. Due to its high specificity and sensitivity, the use of SUBEs for the isolation of the SUMOylated proteome is advantageous relative to other approaches in literature such as the detection by Western-blot of specific SUMOylated proteins using antibodies against the protein of interest or the nickel chromatography using the different histidinylated versions of SUMO molecules. However, it must be taken into consideration that as the SUBEs protocol is performed under non denaturing conditions, maintaining the interaction between SUMOylated proteins and interactors. Therefore we obtain information about the SUMO interactome

rather than only a list of SUMOylated target proteins. Thus, further experiments are necessary to confirm if a protein identified is a target of SUMO or an interacting factor. Other limitation of the SUBEs is the fact that control GST-tramps used are able to capture many background proteins related to oxidative stress. This issue is especially relevant during the Mass-Spectrometry analysis due to high sensitivity of the technique. In order to overcome these limitations, biotinylated SUMO-traps (bioSUBEs) have been developed (REF).

Other applications of this technology include the combination of SUBEs technology with Real-time Surface Plasmon Resonance (SPR) allowing the real-time interactions with SUMOylated proteins from cell extracts (REF) MORE APLLICATIONS

The bioSUBE version can also be used to detect by florescence SUMOylated proteins in living cells since streptavidin-labelled with distinct florescent dies can be combined with distinct antibodies for specific target proteins.

Also methods for detection and quantification of SUMOylated proteins can be considered with both GST and bioSUBEs versions such as it was done with the TUBEs (REF).

Overall, the use of SUBEs for the isolation and characterization of the SUMOylated proteome relevant in liver cancer is a fast and sensitive method providing vast information on the still rather unknown role of SUMOylation pathway in liver cancer.

ACKNOWLEDGMENTS:

This work was supported by grants from Institut National du Cancer, FRANCE, INCa grant PLBIO16-251 (PLBIO16-251), CONACyT-SRE (Mexico) grant 0280365 and the REPERE program of Occitanie, France (M.S.R.). Also, NIH (US Department of Health and Human services)-R01AR001576-11A1, Gobierno Vasco-Departamento de Salud 2013111114 (to M.L.M.-C), ELKARTEK 2016, Departamento de Industria del Gobierno Vasco, MINECO: SAF2017-87301-R integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación 2013-2016 cofinanciado con Fondos FEDER, BIOEF (Basque Foundation for Innovation and Health Research): EITB Maratoia BIO15/CA/014; Instituto de Salud Carlos III:PIE14/00031, integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación 2013-2016 cofinanciado con Fondos FEDER (to M.L.M.-C), Asociación Española contra el Cáncer (T.C.D, M.L.M-C), Daniel Alagille award from EASL (to T.C.D), Fundación Científica de la Asociación Española Contra el Cancer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M), La Caixa Foundation Program (to M.L.M). We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE

(SEV-2016-0644).

DISCLOSURES

Dr. Martínez-Chantar advises for Mitotherapeutix LLC.

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Stimuli

XRPM/4ºC

2h/4ºC

3 Times/XRPM/4ºC

!

Treatment

Collect supernatant

(INPUT)

GST binding

reaction

Washes

Laemmli

boiling buffer

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P

:Centrifugation

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WB

Elution

GS

T

PU

LL

DO

W

N

SU

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Binding of GST-SUBEs or GST control to

glutathione-agarose beads

SIM SIM SIM

SIM

SIM SIM SIM

b

a

Huh7

250

150

100

75

50

37

Thle2

250

150

100

75

50

37

GS

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GS

T

SU

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Huh7

GST

SUBEs

Thle2

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SUBEs

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