Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Summary

The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing conditions.

Abstract

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate protein. The ubiquitination process occurs intracellularly in eukaryotes and regulates almost all basic cellular biological processes. Purification of ubiquitinated proteins aids the investigation of the role of ubiquitination in controlling the function of substrate proteins. Here, a step-by-step procedure to purify ubiquitinated proteins in mammalian cells is described with the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified under stringent nondenaturing and denaturing conditions. Total cellular Flag-tagged p53 protein was purified with anti-Flag antibody-conjugated agarose under nondenaturing conditions. Alternatively, total cellular His-tagged ubiquitinated protein was purified using nickel-charged resin under denaturing conditions. Ubiquitinated p53 proteins in the eluates were successfully detected with specific antibodies. Using this procedure, the ubiquitinated forms of a given protein can be efficiently purified from mammalian cells, facilitating studies on the roles of ubiquitination in regulating protein function.

Introduction

Ubiquitin is an evolutionarily conserved protein of 76 amino acids^1,2,3. Ubiquitin covalently binds lysine residues on target proteins through cascades involving activating (E1), conjugating (E2), and ligase (E3) enzymes. Ubiquitin is first activated by the E1 enzyme and is then transferred to the E2 conjugating enzymes. Subsequently, E3 ubiquitin ligases interact with both ubiquitin-loaded E2 enzymes and substrate proteins and mediate the formation of an isopeptide bond between the C-terminal of ubiquitin and a lysine residue in the substrate^1,2,3,4,5. Ubiquitination involves the attachment of ubiquitin moieties to lysine residues on substrate proteins or to itself, leading to protein monoubiquitination or polyubiquitination. This ubiquitination process occurs intracellularly in eukaryotes and regulates a large variety of biological processes. Ubiquitination results in the degradation of substrate proteins via the ubiquitin-proteasome system^1,2,3,4,5. In addition, ubiquitination modulates protein subcellular localization, protein complex formation, and protein trafficking in cells^3,5. Ubiquitin moieties ligated to substrate proteins can be removed by deubiquitinating enzymes (DUBs)^6,7. Notably, the different ways in which ubiquitin chains are assembled provide a myriad of means to regulate various biological processes^1,5. The exact roles of ubiquitination in regulating substrate protein function remain incompletely understood till now. The purification of ubiquitinated proteins contributes to the elucidation of the effects of protein ubiquitination on a variety of cellular processes.

The p53 protein is one of the most important tumor suppressor proteins and exhibits genetic mutations or inactivation in almost all human cancers^8,9,10,11. p53 stability and activity are delicately regulated in vivo by posttranslational modifications, including ubiquitination, phosphorylation, acetylation, and methylation^12,13. The p53 protein has a short half-life ranging from 6 min to 40 min in various cells, which results mainly from its polyubiquitination and subsequent proteasomal degradation^10,12. Mouse double minute 2 (Mdm2) is an E3 ubiquitin ligase of p53 that binds to the N-terminus of p53 to inhibit its transcriptional activity^12,14,15. Mdm2 promotes the polyubiquitination and proteasomal degradation of p53 to control its stability and induces monoubiquitination of p53 to facilitate its nuclear export^12,14,15,16. Here, Mdm2-mediated p53 ubiquitination is used as an example to introduce a method for the purification of ubiquitinated proteins from mammalian cells in detail. The regulators that influence the ubiquitination status of target proteins can be identified using this in vivo ubiquitination assay when they are overexpressed or knocked down/knocked out in mammalian cells. In addition, ubiquitinated proteins can be used as substrates for in vitro deubiquitination assay. A high-throughput screening can be performed to identify specific DUBs for target proteins by incubating ubiquitinated substrates with individual DUBs. Ubiquitinated proteins may act as a scaffold to recruit downstream signaling proteins in cells. A ubiquitinated target protein complex can be purified by sequential immunoprecipitation under native purification conditions and identified by mass-spectrometry. The current protocol can be extensively used to investigate the cellular proteins regulated by ubiquitination.

Several methods have been established to purify ubiquitinated proteins, which include the use of affinity-tagged ubiquitin, ubiquitin antibodies, ubiquitin-binding proteins, and isolated ubiquitin-binding domains (UBDs)¹⁷. Here, we provide a protocol using affinity-tagged ubiquitin as a mediator to purify ubiquitinated proteins in mammalian cells. The use of poly-His-tagged ubiquitin offers advantages over the other methods. Ubiquitinated proteins are purified in the presence of strong denaturants, which reduces non-specific binding to nickel-charged resin by linearizing cellular proteins and disrupting protein-protein interactions. In contrast, the use of ubiquitin antibodies, ubiquitin-binding proteins, and isolated UBDs as mediators cannot effectively exclude binding partners from target protein because purification needs to be performed under less stringent conditions. Moreover, purification may also lead to increased binding of unrelated proteins using these mediators. In addition, there is a binding propensity for various ubiquitin linkage types as well as mono- and poly-ubiquitination by ubiquitin-binding proteins or isolated UBDs¹⁷. The use of poly-His-tagged ubiquitin contributes to pull down all cellular ubiquitinated proteins. Alternatively, the use of commercially available anti-Flag or anti-HA antibody-conjugated agarose make it easier to immunoprecipitate large-scale Flag- or HA-tagged target proteins under nondenaturing conditions. A second purification step, for example, by nickel-charged resin targeting poly-His-tagged ubiquitin, can be used to acquire ubiquitinated target proteins with a high purity for downstream experiments. Notably, an epitope tagging purification strategy can be adapted when a specific antibody cannot be acquired to immunoprecipitate target proteins effectively. Finally, purification of ubiquitinated proteins in mammalian cells, in comparison with purification in vitro, retains the ubiquitin linkage mode of target proteins under more physiological conditions.

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Protocol

NOTE: H1299 cells were kindly provided by the Stem Cell Bank, Chinese Academy of Sciences and were proven to be negative for mycoplasma contamination.

1. Cell culture

1. For the initial culture, place 1 x 10⁶ cells of human lung adenocarcinoma cell line, H1299 in a 10 cm Petri dish with 10-12 mL of RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% glutamine additive, 1% sodium pyruvate, and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin). Maintain the cells at 37 °C in a humidified incubator with 5% CO₂. Split the cells every 2-3 days depending upon when they reach 80%-90% confluency.
2. One day before transfection, prepare 6-9 x 10⁶ cells for three Petri dishes and plate 2-3 x 10⁶ cells in a single 10 cm Petri dish containing 10-12 mL of medium to achieve a confluence of 70%-90% during transfection.
   ​NOTE: HEK293T cells can also be used to purify ubiquitinated proteins due to their high transfection efficiency and protein expression level.

2. Plasmid transfection

1. Add 1.5 mL of reduced serum medium in three 15 mL centrifuge tubes.
2. Add plasmid DNA ready for transfection to each tube to make dilutions as follows. Tube 1: 1 µg of Flag-p53 plasmid, 12 µg of empty vector; Tube 2: 1 µg of Flag-p53 plasmid, 3 µg of His-HA-Ub (HH-Ub) plasmid, and 9 µg of empty vector; Tube 3: 1 µg of Flag-p53 plasmid, 3 µg of HH-Ub plasmid, and 9 µg of Mdm2 plasmid. Add a total of 0.2 µg green fluorescent protein (GFP) plasmid to each tube to monitor the transfection efficiency.
   NOTE: A pilot experiment should be performed to determine the optimal amounts of plasmids needed to achieve efficient target protein expression in cells.
3. Add 78 µL of liposome transfection reagent to 4.5 mL of reduced serum medium in another centrifuge tube to dilute liposomes. Mix thoroughly by flicking the tube and allow to stand for 5 min at room temperature (RT).
4. Add 1.5 mL of the diluted liposome solution into each tube from step 2.2 containing 1.5 mL of the diluted DNA solution. Mix thoroughly and crosslink the plasmids with the liposomes for at least 20 min at RT. Use a plasmid DNA: liposome ratio of 1:2 (µg:µL) for each transfection.
5. Discard the original medium from the Petri dishes and add 9 mL of reduced serum medium into each dish. Add 3 mL of the liposome-DNA mixture to each dish and mix the solution by gently shaking the plate back and forth 3x, and then left and right 3x for even distribution of the mixture in the plate.
6. Culture the cells in a humidified incubator at 37 °C with 5% CO₂. Replace the medium after 4-6 h and continue to culture the cells for 24-36 h.

3. Cell collection

1. After 24-36 h, treat the cells with MG132 at a final concentration of 10 µM for 4-6 h.
   NOTE: MG132 is a peptide aldehyde that efficiently blocks the proteolytic activity of the 26S proteasome complex. The amounts of proteins ubiquitinated with lysine-48 (K48)-linked polyubiquitin chains can be increased after cells are treated with MG132 or other proteasome inhibitors.
2. Discard the medium, wash the cells 2x (taking care to not flush out the cells) with ice-cold phosphate buffered saline (PBS), and aspirate the cells with serological pipets.
3. Add 1 mL of PBS to the dish, remove the cells by scraping with a clean scraper, and transfer the cell suspension into microcentrifuge tubes. Centrifuge at 700 x g for 5 min to collect cell pellets.

4. Purification of ubiquitinated proteins under nondenaturing conditions

1. Prepare Flag lysis buffer (50 mM Tris-HCl (pH 7.9), 137 mM NaCl, 10 mM NaF, 1 mM ethylene diamine tetraacetic acid (EDTA), 1% Triton X-100, 0.2% sarkosyl, and 10% glycerol) freshly supplemented with protease inhibitor cocktail before use.
2. Add 800 µL of ice-cold Flag lysis buffer to the cells in each tube, mix the cells using a vortex oscillator or pipette gun, and then incubate the mixture on a rotator at 4 °C for 30 min.
3. Subject the mixture to 5-10 brief pulses of ultrasonication. Perform the ultrasonication on the ice at a sonication frequency of 20 kHZ and 80% amplitude with each pulse lasting for 1 s. Incubate the mixture on a rotator at 40 revolutions per minute (rpm) at 4 °C for 30 min.
4. Centrifuge the ultrasonicated samples at 8,000 x g for 20-30 min at 4 °C. Transfer the supernatant to a new microcentrifuge tube.
5. Aliquot 80 µL (1/10) of the cell extract and mix with 20 µL of 5x sodium dodecyl sulfate (SDS) loading buffer. Boil the samples at 98 °C for 5 min, cool on ice for 2 min, and store at -20 °C until use. Use these samples as the input group to monitor protein expression.
6. Add 30 µL of anti-Flag M2 antibody-conjugated (Flag/M2) beads to the remaining cell extracts and incubate on a rotator at 4 °C for at least 4 h or overnight.
7. Centrifuge at 1,500 x g for 2 min at 4 °C to collect the beads. Add 1 mL of ice-cold Flag lysis buffer to the beads and mix by inverting the tube several times.
8. Centrifuge at 1,500 x g for 2 min at 4 °C to collect the beads. Repeat step 4.7 for 4-6 times.
9. Add 40 µL of Flag peptides at a final concentration of 200 ng/µL to the beads and incubate on a rotator at 4 °C for 2 h to elute the bound proteins.
10. Centrifuge at 1,500 x g for 5 min at 4 °C. Transfer the supernatant to a new microcentrifuge tube.
11. Add 10 µL of 5x SDS loading buffer, boil the mixture at 98 °C for 5 min, cool on ice for 2 min, and store at -20 °C for Western blotting. Alternatively, store the eluate from step 4.10 directly at -80 °C for other downstream experiments.
    ​NOTE: The amount of purified ubiquitinated proteins can be scaled up by increasing the number of cells and amounts of plasmids transfected. An epitope tagging strategy can be adapted to purify cellular complexes that bind specifically to ubiquitinated p53 under native purification conditions. By co-expressing Flag-p53, mdm2, and HA-ubiquitin, the ubiquitinated p53 protein complex may be immunoprecipitated by sequential Flag and HA antibody-conjugated agarose from mammalian cells. To keep binding partners of ubiquitinated p53 proteins, the less stringent BC100 lysis buffer (20 mM Tris-HCl (pH 7.9), 100 mM NaCl, 10% glycerol, 0.2 mM EDTA, 0.2% Triton X-100, and 1x protease inhibitor) should be used during the process of purification. The eluate from HA peptide is subjected to mass spectrometry to identify all partners that bind specifically to ubiquitinated p53.

5. Purification of ubiquitinated proteins under denaturing conditions

1. Add 1 mL of ice-cold PBS to the cell pellets obtained in step 3.3 and mix evenly. Aliquot 100 µL (1/10 volume) of the cell suspension into a microcentrifuge tube as the input sample.
2. Centrifuge at 700 x g for 5 min at 4 °C to collect the cell pellets. Add 80 µL of Flag lysis buffer to the input sample, mix the cells using a vortex oscillator or pipette gun, and lyse the cells on ice for 1 h.
3. Centrifuge at 8,000 x g for 20-30 min at 4 °C. Aliquot 80 µL of the supernatant into a new microcentrifuge tube.
4. Add 20 µL of 5x SDS loading buffer to the supernatant, boil at 98 °C for 5-10 min, cool on ice for 2 min, and store at -20 °C until use.
5. Centrifuge the remaining 900 µL of the cell suspension at 700 x g for 5 min at 4 °C to collect the cell pellet. Add 1 mL of ubiquitin buffer 1 (UB buffer 1; 6 M guanidine-HCI, 0.1 M Na₂HPO₄, 6.8 mM NaH₂PO₄, 10 mM Tris-HCI (pH 8.0), and 0.2% Triton X-100, freshly supplemented with 10 mM β-mercaptoethanol and 5 mM imidazole) to the cell pellets and mix several times by pipetting up and down to evenly distribute the cells.
   NOTE: The concentration of imidazole may vary from 5 mM to 20 mM to decrease nonspecific protein binding on the nickel-charged resin. Ubiquitin buffer contains guanidine hydrochloride, which inactivates both ligases and DUBs. β-mercaptoethanol is a clear, colorless liquid with a strong, unpleasant odor similar to rotten eggs. A high concentration solution will cause serious damage to the mucous membrane, upper respiratory tract, skin, and eyes. Wear gloves and goggles and operate in the fume hood.
6. Subject the cell lysates to 10-20 rounds of ultrasonication until the solution is no longer viscous. Perform the ultrasonication on the ice at a sonication frequency of 20 kHZ and 80% amplitude with each pulse lasting for 1 s. Centrifuge at 8,000 x g for 20-30 min at RT and transfer the supernatant to a new microcentrifuge tube.
7. Add 30 µL of nickel-charged resin to the supernatant and incubate on a rotator set at 15 rpm for 4 h or overnight at RT. Centrifuge at 1,500 x g for 2 min at RT to collect the beads.
8. Add 1 mL of UB buffer 1, incubate with rotation in a shaker for 10 min at RT, and centrifuge at 1,500 x g for 2 min at RT to collect the beads.
9. Add 1 mL of ubiquitin buffer 2 (UB buffer 2; 8 M urea, 0.1 M Na₂HPO₄, 6.8 mM NaH₂PO₄, 10 mM Tris-HCI (pH 8.0), and 0.2% Triton X-100) to the beads, incubate with rotation in a shaker for 10 min at RT, and centrifuge at 1,500 x g for 2 min to collect the beads. Repeat this once more.
10. To the beads, add 1 mL of PBS, incubate with rotation in a shaker for 10 min at RT, and centrifuge at 1,500 x g for 2 min at RT to collect the beads. Repeat this once more.
11. Elute bound proteins by incubating the beads with 40 µL of imidazole at a final concentration of 0.5 M for 1 h at RT. Centrifuge at 1,500 x g for 2 min at RT.
12. Transfer the supernatant to a clean microcentrifuge tube, add 10 µL of 5x SDS loading buffer, boil at 98 °C for 5 min, cool on ice for 2 min, and store at -20 °C for Western blotting. Alternatively, store the unprocessed eluate at -80 °C for other downstream experiments.

6. Detection of purified ubiquitinated proteins by Western blotting

1. Resolve the samples from steps 4.11 and 5.12 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transfer them to a nitrocellulose membrane by Western blotting to detect target proteins using the corresponding antibodies as described¹⁸.
2. In brief, incubate the membrane by immersing in 20 mL of blocking solution containing 5% defat milk powder in TBST buffer (15 mM Tris-HCI; pH = 7.6, 4.6 mM Tris-base, 150 mM NaCI, freshly supplemented with 0.1% Tween-20 before use) at RT for 1 h. Then, incubate the membrane with primary antibodies at RT for 2 h or overnight at 4 °C. Use the following antibodies for each set. All primary antibodies were used at a dilution of 1:1000.
   1. Detect total p53 protein, including ubiquitinated p53, with an anti-p53 monoclonal antibody after immunoprecipitation with Flag/M2 agarose beads.
   2. Detect ubiquitinated p53 proteins with an anti-HA antibody or anti-ubiquitin antibody after immunoprecipitation with Flag/M2 agarose beads.
   3. Detect ubiquitinated p53 proteins with an anti-p53 monoclonal antibody after nickel-charged resin pulldown.
   4. Detect total ubiquitinated protein with an anti-HA antibody or anti-ubiquitin antibody after nickel-charged resin pulldown.
3. Incubate the membrane with horseradish peroxidase (HRP)-conjugated secondary antibodies at RT for 1 h after washing 3x with TBST buffer for 5 min each. All secondary antibodies were used at a dilution of 1:3000. Then, wash the membrane with TBST buffer 3x for 5 min each with gentle agitation.
4. Start chemiluminescence imaging system to detect signals from Western blotting. Prepare the substrate solution for HRP by mixing solutions A and B at a ratio of 1:1.
5. Place the nitrocellulose membrane in the camera obscura face up and coat the substrate solution evenly on the membrane using a pipetting gun. Select the automatic exposure procedure to capture chemiluminescent signals and manually adjust the exposure time until an ideal signal is acquired.

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Results

The schematic diagram shows the Flag-tagged p53 (Flag-p53) and His/HA double-tagged ubiquitin (HH-Ub) proteins (Figure 1A). The procedures utilized to purify ubiquitinated proteins are summarized in Figure 1B. Poly-His-tagged ubiquitin can be ligated to target proteins in mammalian cells. Ubiquitinated proteins can be purified with Flag/M2 beads under nondenaturing conditions or by immobilized metal ion affinity chromatography (IMAC) under denaturing conditions ...

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Discussion

Ubiquitination plays a critical role in almost all physiological and pathological cellular processes². In recent years, great progress has been made in understanding the molecular role of ubiquitin in signaling pathways and how changes in the ubiquitin system lead to different human diseases². The purification of ubiquitinated proteins contributes to providing insight into the exact roles of ubiquitination in these processes. The mixtures of ubiquitin-conjugated proteins ca...

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Materials

Name	Company	Catalog Number	Comments
β-mercaptoethanol	Sangon Biotech	M6250
Amersham ECL Mouse IgG, HRP-linked whole Ab (from sheep)	GE healthcare	NA931	Secondary antibdoy
Amersham ECL Rat IgG, HRP-linked whole Ab (from donkey)	GE healthcare	NA935	Secondary antibdoy
Anti-Flag M2 Affinity Gel	Sigma-Aldrich	A2220	FLAG/M2 beads
Anti-GFP monocolonal antibody	Santa cruz	sc-9996	Primary antibody
Anti-HA High Affinity	Roche	11867423001	Primary antibody
Anti-Mdm2 monocolonal antibody (SMP14)	Santa cruz	sc-965	Primary antibody
Anti-p53 monocolonal antibody (DO-1)	Santa cruz	sc-126	Primary antibody
EDTA	Sigma-Alddich	E5134	solvent
Fetal Bovine Serum	VivaCell	C04001-500	FBS
FLAG Peptide	Sigma-Alddich	F3290	Prepare elution buffer
GlutaMAX	Gibco	35050-061	supplement
Guanidine-HCI	Sangon Biotech	A100287-0500	solvent
H1299	Stem Cell Bank, Chinese Academy of Sciences
Image Lab	Bio-rad		software
Immidazole	Sangon Biotech	A500529-0100	solvent
Immobilon Western Chemiluminescent HRP Substrate	Millipore	WBKLS0500
Lipofectamine 2000 reagents	Invitrogen	11668019	Transfection reagent
MG132	MedChemExpress	HY-13259	Proteasome inhibitor
Na2HPO4	Sangon Biotech	A501727-0500	solvent
NaCl	Sangon Biotech	A610476-0005	solvent
NaF	Sigma-Alddich	201154	solvent
NaH2PO4	Sangon Biotech	A501726-0500	solvent
Ni-NTA Agarose	QIAGEN	30230	nickel-charged resin
Nitrocellulose Blotting membrane	GE healthcare	10600002	0.45 µm pore size
Opti-MEM reduced serum medium	Gibco	31985-070	Transfection medium
PBS	Corning	21-040-cv
Penicillin-Streptomycin Solution	Sangon Biotech	E607011-0100	antibiotic
Protease inhibitor cocktail	Sigma-Aldrich	P8340
RPMI 1640	Biological Industries	01-100-1ACS	medium
Sarkosyl	Sigma-Alddich	L5777	solvent
SDS Loading Buffer	Beyotime	P0015L
Sodium Pyruvate	Gibco	11360-070	supplement
Tris-base	Sangon Biotech	A501492-0005	solvent
Tris-HCI	Sangon Biotech	A610103-0250	solvent
Triton X-100	Sangon Biotech	A110694-0500	reagent
Tween-20	Sangon Biotech	A100777-0500	supplement
Ultra High Sensitive Chemiluminescence Imaging System	Bio-rad		ChemiDoc XRS+
Urea	Sangon Biotech	A510907-0500	solvent