Name: evaluation of substrate ubiquitylation by e3 ubiquitin-ligase in mammalian cell lysates
Uploaded: 2022-05-10T14:00:00
Description: Watch this Scientific Journal Video about Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates at JoVE.com

F.R.T is supported by FAPESP grant number 2020/15771-6 and CNPq Universal 405836/2018-0. P.M.S.P and V.S are supported by CAPES. C.R.S.T.B.C was supported by FAPESP scholarship number 2019/23466-1. We thank Sandra R. C. Maruyama (FAPESP 2016/20258-0) for the material support.

NOTE: An overview of ubiquitylation assay protocol in mammalian cells is represented in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63561/63561fig01.jpg" /> 
Figure 1. Overview of the ubiquitylation assay procedure. <a href="https://www.jove.com/files/ftp_upload/63561/63561fig01large.jpg" target="_blank">Please click here to view a larger version...

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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...

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 substrate2,3. 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 proteins4,5. Hence, the kind of polyubiquitin chain introduced in the substrate is essential to decide its cell fate6,7,8. 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 found10,11,12,13. 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-&#954;B signaling, and once this protein is knocked down in cells, it inhibits TNF-&#945;-induced NF-&#954;B 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microtube</td>
			<td>Axygen</td>
			<td>PMI110-06A</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm TC-treated culture dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tube</td>
			<td>Corning</td>
			<td>430766</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Cralplast</td>
			<td>655111</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose-anti-HA beads</td>
			<td>Sigma-Aldrich</td>
			<td>E6779</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti Mouse antibody</td>
			<td>Seracare</td>
			<td>5220-0341</td>
			<td>Goat anti-Mouse IgG</td>
		</tr>
		<tr>
			<td>Anti Rabbit antibody</td>
			<td>Seracare</td>
			<td>5220-0337</td>
			<td>Goat anti-Rabbit IgG</td>
		</tr>
		<tr>
			<td>Anti-Actin antibody</td>
			<td>Sigma-Aldrich</td>
			<td>A3853</td>
			<td>Dilution used: 1:2000</td>
		</tr>
		<tr>
			<td>Anti-Fbxo7 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>SAB1407251</td>
			<td>Dilution used: 1:1000</td>
		</tr>
		<tr>
			<td>Anti-HA antibody</td>
			<td>Sigma-Aldrich</td>
			<td>H3663</td>
			<td>Dilution used: 1:1000</td>
		</tr>
		<tr>
			<td>Anti-Myc antibody</td>
			<td>Cell Signalling</td>
			<td>2272</td>
			<td>Dilution used: 1:1000</td>
		</tr>
		<tr>
			<td>Bradford reagent</td>
			<td>Sigma-Aldrich</td>
			<td>B6916-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A9647-100G</td>
			<td>Bovine Serum Albumin</td>
		</tr>
		<tr>
			<td>Cell incubator</td>
			<td>Nuaire</td>
			<td>NU-4850</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5804R</td>
			<td>500 x g for 5 min</td>
		</tr>
		<tr>
			<td>ChemiDoc</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital pH meter</td>
			<td>Kasvi</td>
			<td>K39-2014B</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td>Corning</td>
			<td>10-017-CRV</td>
			<td>High glucose</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>F4135</td>
			<td>Filtrate prior use</td>
		</tr>
		<tr>
			<td>HA peptide</td>
			<td>Sigma-Aldrich</td>
			<td>I2149</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>VWR Life Science</td>
			<td>0365-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Kline rotator</td>
			<td>Global Trade Technology</td>
			<td>GT-2OIBD</td>
			<td></td>
		</tr>
		<tr>
			<td>MG-132</td>
			<td>Boston Biochem</td>
			<td>I-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5418R</td>
			<td></td>
		</tr>
		<tr>
			<td>Na3VO4 (Ortovanadato)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaF</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose blotting membrane</td>
			<td>GE Healthcare</td>
			<td>10600016</td>
			<td></td>
		</tr>
		<tr>
			<td>NP40 (IGEPAL CA-630)</td>
			<td>Sigma-Aldrich</td>
			<td>I8896-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical microscope</td>
			<td>OPTIKA microscopes</td>
			<td>SN510768</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3</td>
			<td>Invitrogen</td>
			<td>V79020</td>
			<td>For mammalian expression</td>
		</tr>
		<tr>
			<td>pcDNA3-2xFlag-Fbxo7</td>
			<td>&#160;Kindly donated by Dr. Marcelo Dam&#225;rio</td>
			<td></td>
			<td>Tag 2xFlag (N-terminal). Restriction enzymes: EcoRI and XhoI</td>
		</tr>
		<tr>
			<td>pcDNA3-2xFlag-Fbxo7-&#916;F-box</td>
			<td>&#160;Kindly donated by Dr. Marcelo Dam&#225;rio</td>
			<td></td>
			<td>Tag 2xFlag (N-terminal). Restriction enzymes: EcoRI and XhoI. &#916;335-367</td>
		</tr>
		<tr>
			<td>pcDNA3-UXTV2-HA</td>
			<td>&#160;Kindly donated by Dr. Marcelo Dam&#225;rio</td>
			<td></td>
			<td>Tag HA (C-terminal). Restriction enzymes: EcoRI and XhoI</td>
		</tr>
		<tr>
			<td>pCMV-6xHis-Myc-Ubiquitin</td>
			<td>&#160;Kindly donated by Dr. Marcelo Dam&#225;rio</td>
			<td></td>
			<td>Tag 6x-His-Myc (N-terminal). Restriction enzymes: EcoRI and KpnI</td>
		</tr>
		<tr>
			<td>Pen Strep Glutamine 100x</td>
			<td>Gibco</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline 10x</td>
			<td>AccuGENE</td>
			<td>51226</td>
			<td>To obtain a 1x PBS, dilute the 10x PBS into ultrapure water</td>
		</tr>
		<tr>
			<td>Polyethylenimine (PEI)</td>
			<td>Sigma-Aldrich</td>
			<td>9002-98-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau S</td>
			<td>VWR Life Science</td>
			<td>0860-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail SIGMAFAST</td>
			<td>Sigma-Aldrich</td>
			<td>S8820</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocking Shaker</td>
			<td>Kasvi</td>
			<td>19010005</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE system</td>
			<td>BioRad</td>
			<td>165-8004</td>
			<td></td>
		</tr>
		<tr>
			<td>Solution Homogenizer</td>
			<td>Phoenix Luferco</td>
			<td>AP-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma-Aldrich</td>
			<td>T6066-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsine (TrypLe Express)</td>
			<td>Gibco</td>
			<td>12605-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Western Blotting Luminol Reagent</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-2048</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of substrate ubiquitylation by e3 ubiquitin-ligase in mammalian cell lysates

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.

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-&#945;-induced ap...

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

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.

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, ...

This protocol allows to evaluate the ubiquitylation of a specific substrate by an E3 ligase. We know that the characterization of ligase-substrate interaction is crucial to understanding their function in cells. This technique does not require expensive reagents or sophisticated equipment.It needs plasmids and coding the genes of interest, developing an immunoprecipitation assay with cell lysates, and a Western blot to detect substrate ubiquitylation. Several human disease like cancer and neurological disorders are associated with dysregulation of E3 ligase. Hence, the identification of substrate ubiquitylation by specific E3 ligase could assist in treating or diagnosing those diseases.Demonstrating the procedure will be by Valentine Spagnol. To begin, grow HEK293T cell line to 80 to 90%confluence in Dulbecco's modified Eagles medium supplemented with 10%fetal bovine serum and 100 units penicillin, 100 micrograms streptomycin, and 0.292 milligrams per milliliters L-glutamine. Then, incubate this culture at 37 degrees Celsius in a humidified cell culture incubator at 5%carbon dioxide.Passage the cells by aspirating the media from the culture dish using a serological pipette and wash them once with one milliliter of sterilized 1X PBS. Next, detach the cells by adding one milliliter of trypsin ethylenediaminetetraacetic acid solution and after five minutes of incubation at 37 degrees Celsius, resuspend these cells in two milliliters of growth media using a serological pipette. Transfer the cell suspension to a fresh and clean 15-milliliter tube and centrifuge at 500 times G for five minutes at room temperature.Then, gently remove the supernatant and resuspend the cell pellet in three milliliters of growth media by pipetting up and down to obtain a homogenous cell suspension. Then, transfer one milliliter of this cell suspension to a 100-millimeter TC-treated culture dish containing nine milliliters of growth media. Before transfection, certify if the cells are free from contamination and at an adequate confluence for transient transfections.For each transfection sample, prepare the DNA-polyethyleneimine complexes by diluting three micrograms of each plasmid in 100 microliters of Opti-MEM 1 reduced serum medium without supplementation and mixing it gently by pipetting the solution up and down. Next, thaw the DNA-polyethyleneimine at room temperature and add it into the solution following a proportion of three microliters of DNA-polyethyleneimine per one microgram of DNA. Homogenize the solution by pipetting up and down.Then, incubate it for 15 minutes at room temperature to allow the formation of DNA-polyethyleneimine complexes. Add the total volume of DNA-polyethyleneimine complexes to each dish containing the cell culture and mix it gently by rocking the plate back and forth. Then, incubate these cells at 37 degrees Celsius in a humidified cell culture incubator.After the incubation and six hours prior to cell lysis, treat the transfected cells with 10-micromolar proteasome inhibitor MG132 and incubate the cells in the humidified cell culture incubator. Next, aspirate the media from each culture dish using a serological pipette and wash the cells once with one milliliter of 1X PBS. Then, detach the cells by adding one milliliter trypsin and incubate the dish at 37 degrees Celsius for five minutes.Resuspend the cells in one milliliter of growth media and transfer the cell suspension into a fresh and clean two-milliliter microtube and centrifuge it at 500 times G for five minutes at room temperature. Once centrifugation is over, remove the supernatant by pouring it out carefully and gently resuspend the cell pellet in 200 microliters of ice-cold MP40 lysis buffer supplemented with the protease and phosphatase inhibitors cocktail. Then transfer this solution into a clean 1.5-milliliter microtube.Incubate this cell lysate for 30 minutes on ice, and after the incubation, centrifuge the cell lysates at 16, 900 times G for 20 minutes at four degrees Celsius. Meanwhile, equilibrate the agarose anti-HA beads with ice-cold MP40 lysis buffer. Use 15 microliters of agarose anti-HA beads for each sample.Wash the beads with 200 microliters of MP40 lysis buffer by pulsing them in a micro-centrifuge tube at 3, 000 times G for one minute at four degrees Celsius. Next, aspirate and discard the supernatant carefully with a pipette and repeat this process three times. Afterward, keep the beads equilibrated on ice until use.After centrifuging the cell lysates, recover the supernatant and quantify the protein content in the total lysate using the Bradford method to 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 four hours, gently rotating in a rotating incubator at four degrees Celsius, which allows the UXT-V2-HA to bind to the agarose anti-HA beads. Collect the agarose anti-HA beads by pulsing them in a microcentrifuge tube at 3, 000 times G for one minute at four degrees Celsius.Carefully aspirate and discard the supernatant. Wash the beads three times with ice-cold MP40 cell lysis buffer and twice with ice-cold FLAG HA buffer. After the final wash, remove all the supernatant carefully using a pipette and elute the poly-ubiquitylated protein with 300 micrograms per milliliter of HA peptide diluted on FLAG HA buffer.Incubate the agarose anti-HA beads with HA peptide for one hour at four degrees Celsius in a rocking shaker platform. Next, spin down the beads at 3, 000 times G for two minutes at four degrees Celsius and carefully remove the supernatant containing the poly-ubiquitinated proteins. If necessary, store the eluent in a fresh and clean microtube at minus 20 degree Celsius.Resolve the eluents and the cell lysates in 10%sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting. To perform a wet transfer Western blotting, 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. Then, place this system vertically in a tank filled with transfer buffer in between stainless steel or platinum wire electrodes.The transfer occurs for 90 minutes at 150 volts in a wet transfer buffer. Finally, probe the immunoblot membrane using anti-myc antibody. The specific poly-ubiquitylation of UXT-V2-HA mediated by F-box protein 7 in cells was observed after probing the eluent from anti-HA immunoprecipitation with an anti-myc antibody, which detects the myc-ub conjugated to the substrate.The specificity of anti-myc for poly-ubiquitylated substrate was visualized in lanes one and two, wherein a smear of poly-ubiquitylated proteins was not detected when cells were co-transfected with F-box protein 7 and myc-ub in the absence of UXT-V2-HA plasmid. Additionally, no smear corresponding to protein poly-ubiquitination was visualized in the combination of F-box protein 7 and UXT-V2-HA without the myc-ub plasmid. When an empty-vector wild-type F-box protein 7 or F-box protein 7 mutant unable to assemble active SCF was transected in combination with UXT-V2-HA and myc-ub, a strong smear signal of poly-ubiquitylated UXT-V2 was observed only for wild-type F-box protein 7, suggesting that UXT-V2 was poly-ubiquitylated by SCF F-box protein 7 complex and cells compared to the controls.The mono-precipitation steps, including incubation of cell lysates with the equilibrated beads and elution of immunoprecipitated proteins are essential to achieve the aiming of this protocol. After this procedure, in vitro ubiquitylation assay should be performed to confirm the specificity of the substrate ubiquitylation by the E3 ligase.

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