Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Podsumowanie

We provide a detailed protocol for a ubiquitylation assay of a specific substrate and an E3 ubiquitin-ligase in mammalian cells. HEK293T cell lines were used for protein overexpression, the polyubiquitylated substrate was purified from cell lysates by immunoprecipitation, and resolved in SDS-PAGE. Immunoblotting was used to visualize this post-translational modification.

Streszczenie

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, including cell survival, proliferation, and differentiation. It is a reversible process that consists of a covalent attachment of ubiquitin to the substrate through a cascade reaction of at least three different enzymes, composed of E1 (Ubiquitin-activation enzyme), E2 (Ubiquitin-conjugating enzyme), and E3 (Ubiquitin-ligase enzyme). The E3 complex plays an important role in substrate recognition and ubiquitylation. Here, a protocol is described to evaluate substrate ubiquitylation in mammalian cells using transient co-transfection of a plasmid encoding the selected substrate, an E3 ubiquitin ligase, and a tagged ubiquitin. Before lysis, the transfected cells are treated with the proteasome inhibitor MG132 (carbobenzoxy-leu-leu-leucinal) to avoid substrate proteasomal degradation. Furthermore, the cell extract is submitted to small-scale immunoprecipitation (IP) to purify the polyubiquitylated substrate for subsequent detection by western blotting (WB) using specific antibodies for ubiquitin tag. Hence, a consistent and uncomplicated protocol for ubiquitylation assay in mammalian cells is described to assist scientists in addressing ubiquitylation of specific substrates and E3 ubiquitin ligases.

Wprowadzenie

Post-translational modifications (PTMs) are an important mechanism regarding protein regulation, which is essential for cell homeostasis. Protein ubiquitylation is a dynamic and intricate modification that creates an assortment of different signals resulting in several cellular outcomes in eukaryotic organisms. Ubiquitylation is a reversible process consisting in the attachment of a ubiquitin protein containing 76 amino acids to the substrate, occurring in an enzymatic cascade composed by three distinct reactions¹. The first step is characterized by ubiquitin activation, which depends on an ATP hydrolysis to form a high-energy thioester-linked ubiquitin between the ubiquitin C-terminus and the cysteine residue present in the active site of the E1 enzyme. Subsequently, the ubiquitin is transferred to the E2 enzyme forming a thioester-liked complex with the ubiquitin. Afterward, the ubiquitin is covalently attached to the substrate by the E2, or more often, by the E3 enzyme, which recognizes and interacts with the substrate^2,3. Occasionally, E4 enzymes (Ubiquitin-chain elongation factors) are necessary to promote multiubiquitin chain assembly³.

Ubiquitin has seven lysine residues (K6, K11, K27, K29, K33, K48, and K63), allowing the formation of polyubiquitin chains that generate distinct linkages to produce different tridimensional structures that are going to be recognized by several effector proteins^4,5. Hence, the kind of polyubiquitin chain introduced in the substrate is essential to decide its cell fate^6,7,8. Moreover, the substrate could also be ubiquitinated through its N-terminal residues called N-degrons. Specific E3 ubiquitin-ligases are responsible for N-degron recognition, allowing the polyubiquitylation of nearby lysine residue⁹.

Nowadays, there are more than 40 different SCF-specific substrates characterized. Among those, key regulators of several biological pathways, including cell differentiation and development as well as cell survival and death, can be found^10,11,12,13. Thus, the identification of specific substrates of each E3 ubiquitin-ligase is essential to design a comprehensive map of various biological events. Even though the identification of true substrates is biochemically challenging, the use of biochemistry-based methods is very suitable to evaluate chain specificity and the distinction between mono- and polyubiquitylation¹⁴. This study describes a complete protocol for ubiquitylation assay using the mammalian cell line HEK293T overexpressing the substrate UXT-V2 (Ubiquitously expressed prefoldin-like chaperone isoform 2) with the E3 ubiquitin-ligase complex SCF(Fbxo7). UXT-V2 is an essential co-factor for NF-κB signaling, and once this protein is knocked down in cells, it inhibits TNF-α-induced NF-κB activation¹¹. Thus, to detect polyubiquitylated UXT-V2, the proteasome inhibitor MG132 is used since it has the ability to block the proteolytic activity of the 26S subunit of the proteasome complex¹⁵. Furthermore, the cell extract is submitted to a small-scale IP to purify the substrate, utilizing a specific antibody immobilized to agarose resin for subsequent detection by WB using selected antibodies. This protocol is very useful to validate substrate ubiquitylation in the cellular environment, and it can also be adapted for different types of mammalian cells and other E3 ubiquitin-ligase complexes. However, it is necessary to validate the substrate tested through an in vitro ubiquitylation assay as well, since both protocols complement each other regarding the identification of true substrates.

Protokół

NOTE: An overview of ubiquitylation assay protocol in mammalian cells is represented in Figure 1.

Figure 1. Overview of the ubiquitylation assay procedure. Please click here to view a larger version of this figure.

1. Cell culture

1. Grow HEK293T cell line in a 100 mm TC-treated culture dish to 80%-90% confluence in growth media (Dulbecco's Modified Eagle's Medium (DMEM) high glucose supplemented with 10% fetal bovine serum (FBS) and penicillin (100 units), streptomycin (100 µg) and L-glutamine (0.292 mg/mL)). Incubate the culture at 37 °C in a humidified cell culture incubator at 5% CO₂.
2. To passage the cells, aspirate the media from the culture dish using a serological pipette. Wash the cells once with 1 mL of sterilized 1x phosphate-buffered saline (PBS 1x).
3. Detach the cells by adding 1 mL of trypsin/EDTA (ethylenediaminetetraacetic acid) solution. Incubate the dish at 37 °C for 5 min. Resuspend the cells using a serological pipette in 2 mL of growth media.
4. Transfer the cell suspension to a fresh and clean 15 mL tube and centrifuge at 500 x g for 5 min at room temperature (RT). Remove the supernatant by pouring it out carefully. Gently resuspend the cell pellet in 3 mL of growth media by pipetting up and down to obtain a homogenous cell suspension.
5. Transfer 1 mL of the cell suspension to a 100 mm TC-treated culture dish containing 9 mL of growth media.
   NOTE: If the cells are in good conditions, each culture dish of HEK293T, wherein the confluence is 80%-90%, can generate three culture dishes with 80% confluence 2 days after the passage.

2. Cell transfection

NOTE: It is not recommended to transfect the cell culture if the confluence reached is less than 80%.

1. Before transfection, certify if the cells are free from contamination and at an adequate confluence for transient transfections.
2. For each transfection sample, prepare the DNA-Polyethylenimine (PEI 1 µg/µL at pH 7.2) complexes as follows:
   1. Dilute 3 µg of each plasmid in 100 µL of opti-MEM I reduced serum medium without supplementation and mix it gently by pipetting the solution up and down.
      NOTE: Here, the cells were transfected with 4 µg of each plasmid: Empty vector (pcDNA3) or FLAG-Fbxo7 constructs and UXT-V2-HA, with or without 6xHis-myc-ubiquitin. The total DNA content was 12 µg.
   2. Thaw the PEI at RT and add it into the solution following a proportion of 3 µL of PEI per 1 µg of DNA. Homogenize the solution by pipetting up and down. Then incubate it for 15 min at RT to allow the formation of DNA-PEI complexes.
      NOTE: The optimal proportion of PEI volume per DNA quantity differs according to the cell line selected.
3. Add the total volume of DNA-PEI complexes to each dish containing the cell culture and mix it gently by rocking the plate back and forth. Incubate the cells at 37 °C in a humidified cell culture incubator at 5% CO₂.
4. Replace the growth medium after 5 h, since prolonged PEI exposure can be toxic to HEK293T cells. Incubate the cells at 37 °C in a humidified cell culture incubator at 5% CO₂ for 36 h.

3. Cell lysis and immunoprecipitation

1. After the incubation period and 6 h prior to cell lysis, treat the transfected cells with 10 µM of the proteasome inhibitor MG-132. Once again, incubate the cells at 37 °C in a humidified cell culture incubator at 5% CO₂.
2. Aspirate the media from each culture dish using a serological pipette and wash it once with 1 mL of 1x PBS. Detach the cells by adding 1 mL of trypsin and incubating the dish at 37 °C for 5 min. Resuspend the cells in 1 mL of growth media.
3. Transfer the cell suspension into a fresh and clean 15 mL tube and centrifuge it at 500 x g for 5 min at RT.
4. Remove the supernatant by pouring it out carefully. Gently resuspend the cell pellet in 200 µL of ice-cold NP-40 lysis buffer (50 mM Tris-HCl pH 7.2, 225 mM KCl, and 1% NP-40), supplemented with the protease and phosphatase inhibitors cocktail (10 mM NaF and 1 mM Na₃VO₄), and transfer the solution into a clean 1.5 mL microtube.
5. Incubate the cell lysate for 30 min on ice. After incubation, centrifugate the cell lysates at 16,900 x g for 20 min at 4 °C.
6. Meanwhile, equilibrate the agarose-anti-HA beads with ice-cold NP-40 lysis buffer. Use 15 µL of agarose-anti-HA beads for each sample. Wash the beads with 200 µL of NP-40 lysis buffer by pulsing it in a microcentrifuge tube at 3,000 x g for 1 min at 4 °C. With a pipette, aspirate and discard the supernatant very carefully; repeat this process three times. Afterward, keep the beads equilibrated on ice until use.
7. After centrifuging the cell lysates, recover the supernatant. Quantify the protein content in the total lysate using the Bradford method¹⁶.
8. Ensure that each sample subjected to immunoprecipitation presents an equal amount of protein. Incubate the necessary volume of cell lysate with the equilibrated agarose-anti-HA beads for 4 h, gently rotating in a rotating incubator at 4 °C, which allows the UXT-V2-HA to bind to the agarose-anti-HA beads.
9. Collect the agarose-anti-HA beads by pulsing them in a microcentrifuge tube at 3,000 x g for 1 min at 4 °C. Carefully aspirate and discard the supernatant. Wash the beads three times with ice-cold NP-40 cell lysis buffer and twice with ice-cold FLAG/HA buffer (10 mM Hepes pH 7.9, 15 mM MgCl2, 225 mM KCl, and 0.1% NP-40).
10. After the final wash, remove all the supernatant carefully using a pipette and elute the polyubiquitylated protein with HA peptide (300 µg/mL) diluted on FLAG/HA buffer. Incubate the agarose-anti-HA beads with HA peptide for 1 h at 4 °C in a rocking shaker platform.
11. Spin down the beads at 3,000 x g for 2 min at 4 °C and carefully pipette the supernatant containing the polyubiquitinated proteins. If necessary, store the eluate in a fresh and clean microtube at -20 °C.
12. Resolve the eluates and the cell lysates in 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)¹⁷ and immunoblotting.
13. In this study, a wet transfer WB was performed. For this kind of transfer, place the gel in a transfer sandwich composed of filter paper-gel-membrane-filter paper, cushion it with pads, and press it together by a support grid. Place this system vertically in a tank filled with transfer buffer and between stainless steel/platinum wire electrodes. The transfer occurs for 90 min at 150 V in wet transfer buffer (glycine 192 mM, tris-base 25 mM, 0.025% SDS, 20% Methanol).
    NOTE: Since the cell extracts were quantified (step 3.7), run an SDS-PAGE with an equal quantity of protein for each sample. Also, to resolve the eluate, run equal volumes of each sample.
14. Probe the immunoblot membrane using selected antibodies ¹¹. Ensure that a smear signal is detected in the eluate from the polyubiquitylated substrate pulled in the IP process by using anti-myc antibody in the samples containing the wild-type E3 ligase, the substrate, and myc-ub. In the cell lysate (input), ensure that the signal from the chosen substrate, the Fbxo7 protein, ubiquitylated proteins, and a housekeeping protein (e.g., GAPDH and β-actin) is detected to guarantee the same quantity of proteins in each lane.
    NOTE: The dilution for each antibody used was prepared according to the manufacturer's instructions.

Wyniki

UXT (ubiquitously expressed transcript) is a prefoldin-like protein that forms ubiquitously expressed protein-folding complexes in mouse and human tissues such as heart, brain, skeletal muscle, placenta, pancreas, kidney, and liver¹⁸. Two splicing isoforms of UXT, which are named UXT-V1 and UXT-V2, have been described performing distinct functions and subcellular locations. UXT-V1 is predominantly localized in the cytoplasm and inside the mitochondria, and it is implicated in TNF-α-induced ap...

Dyskusje

Ubiquitylation is an essential post-translational modification that regulates the levels of several proteins and plays a crucial role in many signaling pathways and biological processes, ensuring a healthy intracellular environment. The ubiquitin-proteasome system (UPS) is one of the main focuses of recent pharmaceutical research, providing the possibility of stabilizing tumor suppressors or inducing the degradation of oncogenic products²². For instance, the aberrant proliferation of plasma cell n...

Materiały

Name	Company	Catalog Number	Comments
1.5 mL microtube	Axygen	PMI110-06A
100 mm TC-treated culture dish	Corning	430167
15 mL tube	Corning	430766
96-well plate	Cralplast	655111
Agarose-anti-HA beads	Sigma-Aldrich	E6779
Anti Mouse antibody	Seracare	5220-0341	Goat anti-Mouse IgG
Anti Rabbit antibody	Seracare	5220-0337	Goat anti-Rabbit IgG
Anti-Actin antibody	Sigma-Aldrich	A3853	Dilution used: 1:2000
Anti-Fbxo7 antibody	Sigma-Aldrich	SAB1407251	Dilution used: 1:1000
Anti-HA antibody	Sigma-Aldrich	H3663	Dilution used: 1:1000
Anti-Myc antibody	Cell Signalling	2272	Dilution used: 1:1000
Bradford reagent	Sigma-Aldrich	B6916-500ML
BSA	Sigma-Aldrich	A9647-100G	Bovine Serum Albumin
Cell incubator	Nuaire	NU-4850
Centrifuge	Eppendorf	5804R	500 x g for 5 min
ChemiDoc	BioRad
Digital pH meter	Kasvi	K39-2014B
Dulbecco’s Modified Eagle’s Medium	Corning	10-017-CRV	High glucose
Fetal bovine serum	Gibco	F4135	Filtrate prior use
HA peptide	Sigma-Aldrich	I2149
HEK293T cells	ATCC	CRL-3216
Hepes	Gibco	15630080
KCl	VWR Life Science	0365-500G
Kline rotator	Global Trade Technology	GT-2OIBD
MG-132	Boston Biochem	I-130
Microcentrifuge	Eppendorf	5418R
Na3VO4 (Ortovanadato)
NaF
Nitrocellulose blotting membrane	GE Healthcare	10600016
NP40 (IGEPAL CA-630)	Sigma-Aldrich	I8896-100ML
Optical microscope	OPTIKA microscopes	SN510768
Opti-MEM	Gibco	31985-070
pcDNA3	Invitrogen	V79020	For mammalian expression
pcDNA3-2xFlag-Fbxo7	Kindly donated by Dr. Marcelo Damário		Tag 2xFlag (N-terminal). Restriction enzymes: EcoRI and XhoI
pcDNA3-2xFlag-Fbxo7-ΔF-box	Kindly donated by Dr. Marcelo Damário		Tag 2xFlag (N-terminal). Restriction enzymes: EcoRI and XhoI. Δ335-367
pcDNA3-UXTV2-HA	Kindly donated by Dr. Marcelo Damário		Tag HA (C-terminal). Restriction enzymes: EcoRI and XhoI
pCMV-6xHis-Myc-Ubiquitin	Kindly donated by Dr. Marcelo Damário		Tag 6x-His-Myc (N-terminal). Restriction enzymes: EcoRI and KpnI
Pen Strep Glutamine 100x	Gibco	10378-016
Phosphate buffered saline 10x	AccuGENE	51226	To obtain a 1x PBS, dilute the 10x PBS into ultrapure water
Polyethylenimine (PEI)	Sigma-Aldrich	9002-98-6
Ponceau S	VWR Life Science	0860-50G
Protease inhibitor cocktail SIGMAFAST	Sigma-Aldrich	S8820
Rocking Shaker	Kasvi	19010005
SDS-PAGE system	BioRad	165-8004
Solution Homogenizer	Phoenix Luferco	AP-22
Trizma base	Sigma-Aldrich	T6066-500G
Trypsine (TrypLe Express)	Gibco	12605-028
Western Blotting Luminol Reagent	Santa Cruz Biotechnology	SC-2048