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.remotexs.ntu.edu.sg/files/ftp_upload/63561/63561fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Cell culture
<ol>
	<li>Grow HEK293T cell line in a 100 mm TC-treated culture dish to 80%-90% confluence in growth media (Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) high glucose supplemented with 10% fetal bovine serum (FBS) and penicillin (100 units), streptomycin (100 &#181;g) and L-glutamine (0.292 mg/mL)). Incubate the culture at 37 &#176;C in a humidified cell culture incubator at 5% CO2.</li>
	<li>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).</li>
	<li>Detach the cells by adding 1 mL of trypsin/EDTA (ethylenediaminetetraacetic acid) solution. Incubate the dish at 37 &#176;C for 5 min. Resuspend the cells using a serological pipette in 2 mL of growth media.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Cell transfection
NOTE: It is not recommended to transfect the cell culture if the confluence reached is less than 80%.
<ol>
	<li>Before transfection, certify if the cells are free from contamination and at an adequate confluence for transient transfections.</li>
	<li>For each transfection sample, prepare the DNA-Polyethylenimine (PEI 1 &#181;g/&#181;L at pH 7.2) complexes as follows:
	<ol>
		<li>Dilute 3 &#181;g of each plasmid in 100 &#181;L 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 &#181;g 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 &#181;g.</li>
		<li>Thaw the PEI at RT and add it into the solution following a proportion of 3 &#181;L of PEI per 1 &#181;g 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.</li>
	</ol>
	</li>
	<li>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 &#176;C in a humidified cell culture incubator at 5% CO2.</li>
	<li>Replace the growth medium after 5 h, since prolonged PEI exposure can be toxic to HEK293T cells. Incubate the cells at 37 &#176;C in a humidified cell culture incubator at 5% CO2 for 36 h.</li>
</ol>
3. Cell lysis and immunoprecipitation
<ol>
	<li>After the incubation period and 6 h prior to cell lysis, treat the transfected cells with 10 &#181;M of the proteasome inhibitor MG-132. Once again, incubate the cells at 37 &#176;C in a humidified cell culture incubator at 5% CO2.</li>
	<li>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 &#176;C for 5 min. Resuspend the cells in 1 mL of growth media.</li>
	<li>Transfer the cell suspension into a fresh and clean 15 mL tube and centrifuge it at 500 x g for 5 min at RT.</li>
	<li>Remove the supernatant by pouring it out carefully. Gently resuspend the cell pellet in 200 &#181;L 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.</li>
	<li>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 &#176;C.</li>
	<li>Meanwhile, equilibrate the agarose-anti-HA beads with ice-cold NP-40 lysis buffer. Use 15 &#181;L of agarose-anti-HA beads for each sample. Wash the beads with 200 &#181;L of NP-40 lysis buffer by pulsing it in a microcentrifuge tube at 3,000 x g for 1 min at 4 &#176;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.</li>
	<li>After centrifuging the cell lysates, recover the supernatant. Quantify the protein content in the total lysate using the Bradford method16.</li>
	<li>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 &#176;C, which allows the UXT-V2-HA to bind to the agarose-anti-HA beads.</li>
	<li>Collect the agarose-anti-HA beads by pulsing them in a microcentrifuge tube at 3,000 x g for 1 min at 4 &#176;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).</li>
	<li>After the final wash, remove all the supernatant carefully using a pipette and elute the polyubiquitylated protein with HA peptide (300 &#181;g/mL) diluted on FLAG/HA buffer. Incubate the agarose-anti-HA beads with HA peptide for 1 h at 4 &#176;C in a rocking shaker platform.</li>
	<li>Spin down the beads at 3,000 x g for 2 min at 4 &#176;C and carefully pipette the supernatant containing the polyubiquitinated proteins. If necessary, store the eluate in a fresh and clean microtube at -20 &#176;C.</li>
	<li>Resolve the eluates and the cell lysates in 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)17 and immunoblotting.</li>
	<li>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.</li>
	<li>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 &#946;-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&#39;s instructions.</li>
</ol>

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The authors declare that there is no conflict of interest.

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 neoplasms responsible for monoclonal immunoglobulin secretion in multiple myeloma (MM) promotes a pathophysiological pathway due to misfolded and/or unfolded protein accumulation in the endoplasmic reticulum (ER)23. Once this ER stress happens, it activates several processes, including the activation of the ubiquitin-proteasome system. The use of proteasome inhibitors (bortezomib, carfilzomib, and ixazomib) to induce protein accumulation and consequent cell death for multiple myeloma treatment showed promising results as an anti-tumor drug by reducing disease progression in high-risk patients24,25. Moreover, other efforts focused on protein stability are aimed, such as inhibitors for the E3 ubiquitin-ligase MDM2 that showed great ability in avoiding the ubiquitylation of p53, hence preventing its degradation through the proteasome26. Thus, a new era of pharmaceutical/biomedical research centered on understanding and controlling the UPS as a method to combat the disease has arisen.
This study describes a method to analyze the ubiquitylation of a specific target protein by a given E3 ubiquitin-ligase in a cellular environment. In this assay, a significant number of cells are transiently co-transfected with selected plasmids encoding for a tagged protein substrate, an E3 ligase, and a tagged ubiquitin. Before lysis, the cells must be treated with a proteasome inhibitor to allow the accumulation of polyubiquitylated proteins that would undergo degradation in the proteasome. In order to obtain a less complex sample, the target protein was immunoprecipitated, and its polyubiquitylation was analyzed by WB using specific antibodies based on the ubiquitin tag.
The amount of protein in each sample and agarose beads must be normalized to allow the data comparison among those samples. A high-intensity smear signal of UXT-V2 polyubiquitylation in the presence of the wild-type E3 ubiquitin-ligase was detected. Even though the signal obtained from the target protein, the E3 ligase, and the ubiquitin could be from an endogenous origin, the use of recombinant proteins has the advantage of higher yield and signal detection in WB analysis. Furthermore, using the wild-type E3 ligase and its mutant allows the specific analysis of their activity in the substrate, which is an essential control for this experiment. It was observed that the Fbxo7 mutant (Fbxo7-&#916;F-box) could not promote the polyubiquitylation of UXT-V2, suggesting the smear signal detected in the presence of the wild-type Fbxo7 was specific. In addition, it is important to observe that in the absence of the tagged substrate or myc-ub, the polyubiquitylated smear was not detected, indicating the specificity of agarose-anti-HA used. Using an anti-ubiquitin antibody to probe the eluate from substrate IP might detect a polyubiquitylated smear signal even in the negative controls, since unspecific proteins or indirect protein partners of the target can also be eluted together with the substrate. Once these unspecific proteins might be polyubiquitylated, the high sensitivity of anti-ubiquitin can detect them.
There are also some variations for this protocol regarding plasmid transfection, cell lysis buffer, immunoprecipitation antibody, and WB probing. The IP presented here was developed with anti-HA antibody to precipitate the substrate, and the WB membrane was probed with anti-myc antibody to visualize the ubiquitin signal. The transfection of ubiquitin-HA and IP with anti-HA and WB probing the membrane with an anti-substrate or anti-substrate tag is also an alternative for this methodology. However, the immunoprecipitation of ubiquitin might bring a huge number of unspecific polyubiquitylated proteins in addition to ubiquitin-binding proteins. In this case, it is recommended to use RIPA buffer (radioimmunoprecipitation assay buffer) during cell lysis to minimize these undesired proteins27. However, it is essential to evaluate whether the IP protocol is compatible with RIPA buffer. Furthermore, substrate polyubiquitylation might impair the binding of the antibody to this specific target by blocking the conformational epitope. Thus, the application of antibodies with anti- tag is more reliable.
While this approach is very useful for soluble protein purification, it presents some limitations. First, the action of unspecific endogenous E3 ubiquitin-ligases could be observed. To overcome this issue and to confirm the specificity of the target ubiquitylation in cells, it is essential to also perform an in vitro ubiquitylation assay using purified E3 ligase complex and selected substrate in addition to E1 and E2 enzymes, ubiquitin, ubiquitin buffer, and ATP, which are commercially available. Another limitation of this method occurs when the overexpression of the target protein causes a cytotoxic effect that culminates in reduced cellular viability. In this situation, using the endogenous protein target for immunoprecipitation is recommended. Additionally, as mentioned above, this protocol is suitable for soluble protein extraction; therefore, it cannot extract membrane-bound proteins, for instance. Whether the substrate tested is a membrane protein, the RIPA buffer could replace the NP-40 lysis buffer described here if the IP protocol is not compromised by it. In this protocol, the focus of protein clearance is the ubiquitin-proteasome system, which explains that only the proteasome inhibitor was used. However, numerous soluble proteins undergo degradation through autophagy in the lysosome. Consequently, using a lysosome inhibitor, such as bafilomycin A1, could also be necessary to block late-phase autophagy due to vacuolar H+-ATPase28.
Since ubiquitylation is a reversible process, the ubiquitin can be removed from the substrate by the catalytic activity of deubiquitinases (DUBs); this is one of the major obstacles when characterizing E3 ubiquitin-ligase substrates29. The action of DUBs increases the substrate discrimination in this kind of experiment once it decreases polyubiquitylation dwell times on some targets30. Therefore, it would be beneficial to treat the cells with deubiquitinase inhibitors to avoid this problem. Positive or negative crosstalk could also influence the fate of a specific substrate. It is already established that post-translational modifications (PTMs) can crosstalk in various scenarios, and phosphorylation, for example, can regulate ubiquitylation by modulation of E3 ubiquitin-ligase activity, promoting substrate recognition by E3 ligases or through regulating substrate and E3 ligase interaction by affecting its cellular localization31. This could be an issue when choosing the cell line in which the experiment will be executed. If the signals that are prerequisites for ubiquitylation are absent in the desirable cell line, it is required to select an alternative cell line to conduct the analysis or induce the signal in the cell line selected. A common problem that may occur in ubiquitylation assays performed in cells is massive cell death after MG132 treatment, which is mostly caused by the generation of reactive oxygen species (ROS) that induce apoptotic cell death. After several tests to standardize the protocol, it was found an ideal MG132 treatment for HEK293T cells to not exceed 6 h before lysis at 10 &#181;M of concentration. This protocol is also suitable for other mammalian cell types to identify substrate-E3 ubiquitin-ligase pairs; although, it is crucial to standardize the volume of PEI used in the transfection and the MG132 treatment period for the chosen cell line.

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><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 &lt;em&gt;g&lt;/em&gt; 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&amp;rsquo;s Modified Eagle&amp;rsquo;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>Na&lt;sub&gt;3&lt;/sub&gt;VO&lt;sub&gt;4&lt;/sub&gt; (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>&amp;nbsp;Kindly donated by Dr. Marcelo Dam&amp;aacute;rio</td><td></td><td>Tag 2xFlag (N-terminal). Restriction enzymes: EcoRI and XhoI</td></tr><tr><td>pcDNA3-2xFlag-Fbxo7-&amp;Delta;F-box</td><td>&amp;nbsp;Kindly donated by Dr. Marcelo Dam&amp;aacute;rio</td><td></td><td>Tag 2xFlag (N-terminal). Restriction enzymes: EcoRI and XhoI. &amp;Delta;335-367</td></tr><tr><td>pcDNA3-UXTV2-HA</td><td>&amp;nbsp;Kindly donated by Dr. Marcelo Dam&amp;aacute;rio</td><td></td><td>Tag HA (C-terminal). Restriction enzymes: EcoRI and XhoI</td></tr><tr><td>pCMV-6xHis-Myc-Ubiquitin</td><td>&amp;nbsp;Kindly donated by Dr. Marcelo Dam&amp;aacute;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 apoptosis and antiviral signalosome formation19,20. Most research has focused on UXT-V2 that is mainly localized in the nucleus and acts as a co-factor for multiple transcriptional factors involved in cell proliferation regulation, differentiation, and in inflammatory pathways. UXT-V2 is involved in the NF-&#954;B signaling pathway working as an essential coactivator in the NF-&#954;B enhanceosome21. It was demonstrated that UXT-V2 is a canonical substrate of the E3 ubiquitin-ligase SCF(Fbxo7), mediating its polyubiquitylation and degradation by the proteasome, and consequently inhibiting the NF-&#954;B signaling pathway11.
The specific polyubiquitylation of UXT-V2-HA mediated by Fbxo7 in cells was observed after probing the eluate from anti-HA IP with an anti-myc antibody, which detects the myc-ub conjugated to the substrate (Figure 2). The specificity of anti-myc for polyubiquitylated substrate was visualized in lanes 1 and 2 (Figure 2), wherein smear of polyubiquitylated proteins was not detected when cells were co-transfected with Fbxo7 and myc-ub in the absence of UXT-V2-HA plasmid (Figure 2, lane 1). Additionally, no smear corresponding to protein polyubiquitination was visualized in the combination of Fbxo7 and UXT-V2-HA without the myc-ub plasmid (Figure 2, lane 2). To prove that UXT-V2 polyubiquitylation was mediated by Fbxo7, an empty vector pcDNA3, wild type Fbxo7 or Fbxo7-&#916;F-box mutant, which is unable to assemble active SCF complex due to the absence of the F-box domain that is responsible for interaction with SKP1, was transfected in combination with UXT-V2-HA and myc-ub. A strong smear signal of polyubiquitylated UXT-V2 was observed only in the presence of the wild-type Fbxo7 (Figure 2, lane 4), suggesting that UXT-V2 was polyubiquitylated by SCF(Fbxo7) complex in cells compared to the controls (Figure 2, lanes 3 and 5). The presence of a faint smear signal of polyubiquitylated UXT-V2 in the presence of these negative controls might indicate the action of endogenous Fbxo7 or other E3 ubiquitin-ligase activity.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63561/63561fig02.jpg" /> 
Figure 2. Ubiquitylation assay in cells: Transient transfection of HEK293T cells with the indicated plasmids. After cell lysis, the cell extracts (inputs) were immunoprecipitated with agarose-anti-HA beads. The eluted and input samples were resolved by SDS-PAGE, and the western blot was probed with specific antibodies. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63561/63561fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates at JoVE.com

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, ...

evaluation-substrate-ubiquitylation-e3-ubiquitin-ligase-mammalian

Nanyang Technological University

Research

JoVE Journal

Biochemistry

2.5K Views.  Federal University of São Carlos. Dit protocol maakt het mogelijk om de alomtegenwoordigheid van een specifiek substraat door een E3-ligase te evalueren. We weten dat de karakterisering van ligase-substraatinteractie cruciaal is om hun functie in cellen te begrijpen. Deze techniek vereist geen dure reagentia of geavanceerde apparatuur.Het heeft plasmiden nodig en codeert de genen van belang, ontwikkelt een immunoprecipitatietest met cellysaten en een Western blot om substraatubiquitylering te detecteren. Verschillende ...

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

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Biology

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent attachment of 76 amino acid ubiquitin modifiers to a target protein, depending on the length and topology of the polyubiquitin chain, can result in different outcomes ranging from protein degradation to changes in the localization and/or activity of modified protein. Three enzymes sequentially catalyze the ubiquitination process: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. E3 ubiquitin ligase determines substrate specificity and, therefore, represents a very interesting study subject. Here we present a comprehensive approach to study the relationship between the enzymatic activity and function of the RING-type E3 ubiquitin ligase. This four-step protocol describes 1) how to generate an E3 ligase deficient mutant through site-directed mutagenesis targeted at the conserved RING domain; 2&#8211;3) how to examine the ubiquitination activity both in vitro and in planta; 4) how to link those biochemical analysis to the biological significance of the tested protein. Generation of an E3 ligase-deficient mutant that still interacts with its substrate but no longer ubiquitinates it for degradation facilitates the testing of enzyme-substrate interactions in vivo. Furthermore, the mutation in the conserved RING domain often confers a dominant negative phenotype that can be utilized in functional knockout studies as an alternative approach to an RNA-interference approach. Our methods were optimized to investigate the biological role of the plant parasitic nematode effector RHA1B, which hijacks the host ubiquitination system in plant cells to promote parasitism. With slight modification of the in vivo expression system, this protocol can be applied to the analysis of any RING-type E3 ligase regardless of its origins.

The goal of this manuscript is to present an outline for the comprehensive biochemical and functional studies of the RING-type E3 ubiquitin ligases. This multistep pipeline, with detailed protocols, validates an enzymatic activity of the tested protein and demonstrates how to link the activity to function.

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent ...

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.

This method describes the assessment of global changes in ubiquitin chain topology. The assessment is performed by the application of a mass spectrometry-based targeted proteomics approach.

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The ...

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

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.

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.

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

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

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