Name: method for measuring the activity of deubiquitinating enzymes in cell lines and tissue samples
Uploaded: 2015-05-10T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples at JoVE.com

We would like to thank the Lee lab of the University of Minnesota for providing the mouse spinal cord tissue samples that were used. This work was supported by the Department of Defense Ovarian Cancer Research Program (OCRP) OC093424 to MB, by the Randy Shaver Cancer Research and Community Fund to MB and by the Minnesota Ovarian Cancer Alliance (MOCA) to MB. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

1. Lysis Buffer Preparation
<ol>
	<li>Dissolve sucrose in deionized (DI) water to make a 2 M stock solution. Filter a 2 M solution of sucrose using a vacuum driven 0.22 &#181;m polyvinylidene fluoride (PVDF) filter.</li>
	<li>Dissolve dithiothreitol (DTT) in DI water to make a 500 mM stock solution and store under anaerobic conditions. Dissolve magnesium chloride (MgCl2) in DI water to make a 100 mM stock solution. Dissolve adenosine triphosphate (ATP) disodium hydrate in DI water to make a 50 mM stock solution.</li>
	<li>Make up a Tris solution at pH 7.4 by dissolving Trizma hydrochloride in DI water and adjusting the pH using sodium hydroxide (NaOH).</li>
	<li>Combine portions of the stock solutions in DI water to make a lysis buffer with a sucrose concentration of 250 mM, a DTT concentration of 1 mM, a MgCl2 concentration of 5 mM, an ATP concentration of 1 mM and a Tris concentration of 50 mM. For example, mix 535.3 &#181;l of tris, 500 &#181;l of MgCl2, 1.25 ml of sucrose, 20 &#181;l of DTT, 200 &#181;l of ATP and 7.4947 ml of DI water to make 10 ml of lysis buffer. This can be used for approximately 20 experiments.</li>
</ol>
2. Culturing Cells for the Experiment
<ol>
	<li>Make media by aliquoting 30 ml of DMEM + 10% fetal bovine serum (FBS) into a 50 ml conical tube.</li>
	<li>Transfer frozen M17 or HeLa cells from liquid nitrogen storage to a small beaker with lukewarm water.</li>
	<li>Transfer the cells to the 50 ml conical tube. First, add some media to the cell vial. Then transfer the resuspended cells within 5 sec of thawing.</li>
	<li>Centrifuge the cell suspension at 450 x g for 5 min to obtain a cell pellet. Add 20 ml of media into a T75 flask.</li>
	<li>Remove the media from the cells using suction. Add 5 ml of fresh media to the cell pellet and resuspend.</li>
	<li>Transfer the 5 ml cell suspension to the 20 ml of media in the T75 flask and incubate at 37 &#176;C. Change the media every 3 days.</li>
</ol>
3. Cell Harvesting
<ol>
	<li>Remove the media via suction, being careful not to touch the cells.</li>
	<li>Wash the cells with 5 ml of phosphate buffered saline (PBS). Remove PBS using suction.</li>
	<li>Add 3 ml of trypsin-ethylenediaminetetraacetic acid (EDTA) to the T75 flask and incubate at 37 &#176;C for 3 min.</li>
	<li>Add 6 ml of fresh media to the flask and resuspend the cells.</li>
	<li>Transfer the suspension to a 15 ml conical tube and centrifuge at 290 &#215; g for 5 mins.</li>
	<li>Remove the supernatant and add 5 ml of PBS. Gently tap the bottom of the tube to break up the pellet. Centrifuge at 290 x g for 5 min.</li>
	<li>Repeat step 3.6 three times.</li>
	<li>Remove PBS and resuspend the cells in 1 ml of fresh PBS. Transfer to the Eppendorf tube (1.5 ml). Spin at 290 x g for 5 min.</li>
</ol>
4. Cell Lysis
<ol>
	<li>Measure the approximate volume of the pellet using a pipette and weigh out glass beads in a mass:volume ratio of 1:1. Add twice the volume of the pellet in lysis buffer. 
	Note: Add the buffer before adding the glass beads.</li>
	<li>Lyse the cells in the Eppendorf tube at the maximum agitation for 30 min at 4 &#176;C by vortexing in a cold room.</li>
	<li>Centrifuge briefly at 200 x g for 30 sec to settle the beads. Collect the supernatant.</li>
	<li>Add the same volume of glass beads as before to the supernatant.</li>
	<li>Vortex once again at maximum agitation for 30 min at 4 &#176;C.</li>
	<li>Centrifuge briefly at 200 x g for 30 sec to settle the glass beads. Collect the supernatant.</li>
	<li>Centrifuge at 5,030 x g for 5 min to remove the nuclei, membranes and unbroken cells. Collect the supernatant, which is the cell lysate.</li>
</ol>
5. Tissue Homogenization
<ol>
	<li>Make a mass:volume of 1:9 solution of mouse spinal cord tissue to lysis buffer. 
	Note: This method is applicable to a variety of different tissue samples.</li>
	<li>Homogenize the tissue using a setting of level 2 on the homogenizer for 30 sec to ensure that there are no chunks remaining.</li>
	<li>Spin down at 5,030 x g for 3 min to remove the nuclei, membranes and unbroken cells.</li>
	<li>Collect the supernatant, which is the lysate.</li>
</ol>
6. Sample Derivatization
<ol>
	<li>Perform a bicinchoninic acid (BCA) in a 96-well plate analysis to determine the protein concentration of the tissue lysate using a colorimetric 96-well plate reader as specified by the Pierce BCA Protein Assay kit protocol.</li>
	<li>Bring an aliquot corresponding to 20 &#181;g of total protein to 50 &#181;l using de-ionized (DI) water.</li>
	<li>Add 2 &#181;l of 1.35 &#181;M of HA-Ub-Vinyl sulfone (VS) to the solution and incubate for 1 hr at 37 &#176;C. This results 20 &#181;g of lysate to 50 nM of probe in the final reaction mixture. This is in large molar excess to ensure tagging of functional DUBs.</li>
	<li>Incubate the sample in Laemmli&#8217;s sample buffer for 5 mins at 95 &#176;C. For a 52 &#181;l reaction volume use 26 &#181;l of 2x sample buffer, 13 &#181;l of 4x sample buffer or 8.87 &#181;l of 6x sample buffer.</li>
	<li>Cool on ice before loading the gel.</li>
</ol>
7. Western Blot
<ol>
	<li>Load a 12-well 4-20% tris-glycine gel with 40 &#181;l of the prepared sample</li>
	<li>Run the gel at 95 V until the ion front reaches the bottom.</li>
	<li>Carry out an overnight transfer of the gel onto a polyvinylidene difluoride (PVDF) membrane.</li>
	<li>Incubate the membrane in the amido black stain and scan the membane to obtain an image showing the amount of protein in each lane.</li>
	<li>Destain the membrane and incubate in 5% milk in PBS for 1 hr to block.</li>
	<li>Incubate the membrane in the primary anti-HA antibody at a concentration of 1:10,000 in 5% milk in PBS either overnight at 4 &#176;C or for 4 hr at room temperature.</li>
	<li>Perform 3 washes for 5 min each in PBS with 0.1% Tween-20 detergent.</li>
	<li>Incubate the membrane in a mouse horseradish peroxidase at a concentration of 1:10,000 in 5% milk in PBS for at least 2 hr.</li>
	<li>Perform 3 washes for 5 min each in PBS with 0.1% Tween-20 detergent.</li>
	<li>Perform 1 wash for 5 min in PBS.</li>
	<li>Incubate the membrane in a chemiluminescent detection reagent for 10 min and detect using a chemiluminescent detector. Use the automatic exposure setting on the detector.</li>
</ol>

<ol>
	<li>Ovaa, H., Kessler, B. M., Rol&#233;n, U., Galardy, P. J., Ploegh, H. L., Masucci, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-+based+ubiquitin-specific+(USP)+profiling+of+virus-infected+and+malignant+human+cells.">Activity- based ubiquitin-specific (USP) profiling of virus-infected and malignant human cells.</a> Proc Natl Acad Sci USA. 101 (8), 2253-2258 (2004).</li><li>Wilkinson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitination+and+deubiquitination:+Targeting+of+proteins+for+degradation+by+the+proteasome.">Ubiquitination and deubiquitination: Targeting of proteins for degradation by the proteasome.</a> Semin Cell Dev Biol. 11 (3), 141-148 (2000).</li><li>Ziad, M. E., Wilkinson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+proteolysis+by+human+deubiquitinating+enzymes.">Regulation of proteolysis by human deubiquitinating enzymes.</a> Biochim Biophys Acta. 1843 (1), 114-128 (2014).</li><li>Rol&#233;n, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+Profiling+of+Deubiquitinating+Enzymes+in+Cervical+Carcinoma+Biopsies+and+Cell+Lines.">Activity Profiling of Deubiquitinating Enzymes in Cervical Carcinoma Biopsies and Cell Lines.</a> Mol Carcinog. 45 (4), 260-269 (2006).</li><li>Borodovsky, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry-Based+Functional+Proteomics+Reveals+Novel+Members+of+the+Deubiquitinating+Enzyme+Family.">Chemistry-Based Functional Proteomics Reveals Novel Members of the Deubiquitinating Enzyme Family.</a> Chem Biol. 9 (10), 1149-1159 (2002).</li><li>Andersson, F. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+Parkinson&#8217;s-disease-associated+mutations+on+the+deubiquitinating+enzyme+UCH-L1.">The effect of Parkinson&#8217;s-disease-associated mutations on the deubiquitinating enzyme UCH-L1.</a> J Mol Biol. 407 (2), 261-272 (2011).</li><li>Poon, W. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-Amyloid+(A&#946;)+oligomers+impair+brain-derived+neurotrophic+factor+retrograde+trafficking+by+down-regulating+ubiquitin+C-terminal+hydrolase+UCH-L1.">&#946;-Amyloid (A&#946;) oligomers impair brain-derived neurotrophic factor retrograde trafficking by down-regulating ubiquitin C-terminal hydrolase UCH-L1.</a> J Biol Chem. 288 (23), 16937-16948 (2013).</li><li>Nijman, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+deubiquitinating+enzyme+USP1+regulates+the+Fanconi+anemia+pathway.">The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway.</a> Mol Cell. 17 (3), 331-339 (2005).</li><li>Stevenson, L. F., Sparks, A., Allende-Vega, N., Xirodimas, D. P., Lane, D. P., Saville, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+deubiquitinating+enzyme+USP2a+regulates+the+p53+pathway+by+targeting+Mdm2.">The deubiquitinating enzyme USP2a regulates the p53 pathway by targeting Mdm2.</a> EMBO J. 26 (4), 976-986 (2007).</li><li>Coughlin, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+RA-9+inhibits+proteasome-associated+DUBs+and+ovarian+cancer+in+vitro+and+in+vivo+via+exacerbating+unfolded+protein+responses.">Small-molecule RA-9 inhibits proteasome-associated DUBs and ovarian cancer in vitro and in vivo via exacerbating unfolded protein responses.</a> Clin Cancer Res. 20 (12), 3174-3186 (2014).</li><li>Colla, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+Reticulum+Stress+Is+Important+for+the+Manifestations+of+&#945;-Synucleinopathy+In.">Endoplasmic Reticulum Stress Is Important for the Manifestations of &#945;-Synucleinopathy In.</a> Vivo. J Neurosci. 32 (10), 3306-3320 (2012).</li></ol>

The authors have nothing to disclose.

Since ubiquitination is a fundamental cellular activity, understanding the regulatory mechanisms could be the key to unearthing the processes of disease and pathology. The use of HA tagged Ub-derived active site directed probes reported here provides an easy, but highly applicable method for studying Ub-mediated protein degradation. This method is faster and less expensive than studying each one of the DUBs individually.
In this method, the lysis of the cells is achieved via mechanical means &#8211; using the glass beads. This gentle method of lysis preserves a metabolic intracellular picture. However, a major drawback of this lysis method is its low efficiency of about 60-70%. This means a lot of cells are required to ensure lysates are concentrated enough for experiments. T75 flasks with 80 - 90% confluent cell layers should be used to produce a concentrated lysate. Furthermore, the trypsinization step of the experiment is critical because most of the attached cells must be suspended in solution. Check the flask under a microscope to make sure that a significant portion of the cells in culture are floating before transferring to the 50 ml tube. Failure to properly trypsinize adherent cells will result in a smaller pellet and by extension less lysate at a lower concentration. Lysates that are not in use should be stored at -80 &#176;C, transferred to -20 &#176;C 24 hr before use and thawed on ice the day of use. This allows the lysate to thaw evenly and reduces damage to the enzymes from rapid thawing.
To apply this method to tissues either a dounce or polytron homogenizer can be used in place of the glass beads11. The obtained lysate can be tagged with the probes immediately or stored for use at -80 &#176;C. If the dounce or polytron is used to obtain the lysate then glass beads should not be used. This leads to a reduction in the total protein concentration of the lysate. Alternatively, using primary cell culture methods the solid tissue samples could be cultured first and then lysed with the glass beads.
A major limitation of this method is that even though it allows the user to visualize the activity of the enzymes, it doesn&#8217;t give accurate information on the expression patterns of the individual DUBs. This method uses functional homology in the enzymes not structural homology. Therefore, further experiments with the individual antibodies will have to be done to explain whether the reduction in activity is a result of defects in the active site or an actual loss of the enzyme. Additionally, the lysis method used in this protocol does not break open the nuclei of the cells. DUBs in the nucleus could provide further critical information about disease processes.
Nevertheless this method is unique in its ability to provide information that cannot be obtained from any other source. Both RNA methods and the use of individual antibodies do not provide functional information about enzymes. In the future, the potential exists for the application of this method to immunohistochemical (IHC) staining. IHC staining will not only give information about the activity of the enzymes, but it will also provide insights into the location in tissue where the various DUBs are active. Also, this method could be coupled with a subcellular fractionation technique such as the one detailed by Colla et al. in 2012 to study the activity of the DUBs in membrane bound organelles12. Although this is a relatively new technique in molecular biology, there is a huge potential to expand the applications. Using active site directed molecular probes might hold the key to elucidating the etiology of many diseases.

The ubiquitin-proteasome system (UPS) serves as one of the major degradation pathways in the mammalian cell. Substrates bound for degradation in the proteasome are covalently tagged with polymers of ubiquitin (Ub)1. Before the targeted substrate enters the proteasome for degradation, the poly-ubiquitin tag must be removed. A class of enzymes known as deubiquitinating enzymes (DUBs) is responsible for the removal and recycling of ubiquitin molecules2. It has been predicted from the human genome that there are nearly a hundred DUBs working in the cell3. With such a large number of DUBs controlling Ub-mediated cellular processes, studying these enzymes presents a challenge since mRNA techniques do not give information on activity and western blotting only gives information on expression levels.
The use of influenza hemagglutinin (HA) tagged, Ub-derived active site directed probes allows for a covalent modification of the functional DUBs and therefore gives a direct visualization of the activity of these enzymes on a western blot4. The probes have a C-terminal thiol reactive group that serves as a suicide substrate for the active site cysteine residue5. With these probes, it is possible to study the activity and potential expression of many DUBs under both pathological and physiological states of the cell.
Changes in DUB activity have been implicated in a range of pathological conditions such as Parkinson&#8217;s, Alzheimer&#8217;s, anemia and various cancers6-10. This technique provides a powerful tool for the study of disease. In the present paper, we show the application of this technique in HeLa and M17 cells that have been lysed using glass beads. Additionally, we outline how to use this method in mouse spinal cord tissue samples. The information obtained from this technique can be used as a starting point for identifying therapeutic targets as well as establishing models for the study of different disease conditions. The true utility of this technique lies in its ability to provide information on multiple DUBs in a single assay.

<table><tbody><tr><td>PowerGen 125 (homogenizer)</td><td>Fischer Scientific</td><td>14-261-02P</td><td></td></tr><tr><td>Vacuum-driven filters 0.22 &amp;micro;m</td><td></td><td>BPV2210</td><td></td></tr><tr><td>Glass beads, acid washed &amp;le;106 &amp;micro;m (~140 U.S. sieve)</td><td>Sigma-aldrich</td><td>G4649-10G</td><td></td></tr><tr><td>Sucrose</td><td>Fischer Scientific</td><td>S6-212</td><td></td></tr><tr><td>DL-dithiothreitol</td><td>Sigma-Aldrich</td><td>D0632</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Adenosine 5&#039;-triphosphate disodium salt hydrate</td><td>Sigma-Aldrich</td><td>A26209</td><td></td></tr><tr><td>Trizma hydrochloride</td><td>Sigma-Aldrich</td><td>T3253</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Life Technologies</td><td>11965-092</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Tissue culture flask 75 cm&lt;sup&gt;2&lt;/sup&gt; w/ filter cup 250 ml 120/cs</td><td>Cellstar</td><td>T-3001-2</td><td></td></tr><tr><td>0.05% Trypsin-EDTA (1x)</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>HI FBS</td><td>Life Technologies</td><td>16140-071</td><td></td></tr><tr><td>Monoclonal anti-HA antibody produced in mouse</td><td>Sigma-Aldrich</td><td>H9658</td><td></td></tr><tr><td>Ubiquitin vinyl sulfone (HA-tag)</td><td>Enzo Life Sciences</td><td>BML-UW0155-0025</td><td></td></tr><tr><td>Laemmli&#039;s SDS-Sample Buffer (4x, reducing)</td><td>Boston BioProducts</td><td>BP-110R</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23225</td><td></td></tr></tbody></table>

method for measuring the activity of deubiquitinating enzymes in cell lines and tissue samples

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, techniques for studying the regulatory mechanism of this system are essential to elucidating the cellular and molecular processes of the aforementioned diseases. The use of hemagglutinin derived ubiquitin probes outlined in this paper serves as a valuable tool for the study of this system. This paper details a method that enables the user to perform assays that give a direct visualization of deubiquitinating enzyme activity. Deubiquitinating enzymes control proteasomal degradation and share functional homology at their active sites, which allows the user to investigate the activity of multiple enzymes in one assay. Lysates are obtained through gentle mechanical cell disruption and incubated with active site directed probes. Functional enzymes are tagged with the probes while inactive enzymes remain unbound. By running this assay, the user obtains information on both the activity and potential expression of multiple deubiquitinating enzymes in a fast and easy manner. The current method is significantly more efficient than using individual antibodies for the predicted one hundred deubiquitinating enzymes in the human cell.

Cultured M17 and HeLa cells were harvested using the method detailed in the protocol (3. Cell Harvesting) and mouse spinal cord tissue was obtained. The cell pellet/spinal cord tissue was placed in a tube with the lysis buffer described in the reagent preparation section. Cell pellets were lysed using glass beads (Figure 1A) and mouse spinal cord tissue samples were homogenized using the homogenizer (Figure 1B). After lysis or homogenization, the sample was then centrifuged at 5,030 x g to remove the glass beads and/or the unbroken membranes and organelles (Figure 2). Both of these methods are mechanical means of lysis to preserve enzymatic activity. The protein concentration of the cells was then determined using the BCA method. 20 &#181;g of total protein was incubated with 2 &#181;l of 1.35 &#181;M probe for 1 hr at 37 &#176;C (Figure 3). The samples were then incubated in 4x Laemmli&#8217;s sample buffer at 95 &#176;C to quench the reaction. The prepared samples were loaded on a 4-20% tris-glycine gel and run at 95 V until the ion front reached the bottom. The proteins were transferred overnight onto a PVDF membrane. The protein loading was checked using the amido black stain (Figure 4). This control step ensures that the differences in activity are due to actual cellular processes and not unequal protein loading. Equal protein amounts were used in the first experiment (Figure 4A). In the second experiment, unequal protein amounts were loaded due to the expected differences in the activity profiles of the different cell lines (Figure 4B). This was done to ensure the detection of an activity profile for the M17 cell lysates incubated with N-ethylmaleimide (NEM), which is a cysteine inhibitor and should lead to a reduced signal on the western blot. The membranes were incubated in anti-HA primary antibody, a mouse horseradish peroxidase and then detected using chemiluminescent imaging (Figure 5). Successful tagging of the DUBs using the probes results in a profile in each lane of the membrane (Figure 5A, C). The intensity of the bands at different molecular weights corresponds to the activity level of the DUB enzyme. The differences in activity levels of various DUBs across cell lines become evident when similar proteins such as UCHL1 are compared in the HeLa and M17 cells (Figure 5). The chemiluminescent visualization of the gel is critical because unlike the amido black stain, which shows the amount of protein loaded, this shows the amount of active protein that has reacted with the probes. The light image of the molecular weight standards (Figure 5B) serves as the guide for identifying the various DUBs. The mass of the probe needs to be added to the size of the protein during the analysis of the results to characterize the DUBs at the correct molecular weights.
<img alt="Figure 1" src="/files/ftp_upload/52784/52784fig1.jpg" /> 
Figure 1: Lysis Methods for Cells and Tissue. Picture showing cell lysis using glass beads in (A) versus polytron lysis in (B).
<img alt="Figure 2" src="/files/ftp_upload/52784/52784fig2.jpg" /> 
Figure 2: Centrifugation Products of Cells and Tissue. Cell lysate showing the settled glass beads and unbroken membranes and organelles at the bottom after lysis and centrifugation in (A). Tissue lysate showing the unbroken membranes and organelles at the bottom after homogenization and centrifugation in (B).
<img alt="Figure 3" src="/files/ftp_upload/52784/52784fig3highres.jpg" width="700" /> 
Figure 3: Mechanism for tagging of DUBs. A schematic showing the covalent linkage between the active site cysteine residue and the functional group of the HA-Ub-VS. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52784/52784fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52784/52784fig4highres.jpg" width="400" /> 
Figure 4: Amido black stains of the membranes. Membranes showing the amido black staining done on lysates incubated with N-ethylmaleimide (NEM), tagged with HA-Ub-VinylSulfone (VS) and run on a western blot. The samples are as follows from left to right &#8211; (A) HeLa - NEM, HeLa + NEM; (B) Mr Standards, M17 - NEM, M17 + NEM. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52784/52784fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52784/52784fig5highres.jpg" width="650" /> 
Figure 5: Western blots results. Membranes showing the activity levels of the various DUBs as determined by probing for Anti-HA. The samples are as follows from left to right &#8211; (A) HeLa - NEM, HeLa + NEM; (B) Mr Standards; (C) M17 - NEM, M17 + NEM. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52784/52784fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples at JoVE.com

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic targets.

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

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9.6K Views.  University of Minnesota. The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic targets.

Video: Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

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 ...

Biochemistry

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 ...

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 ...

Measuring Ubiquitylated, SUMOylated, and ADP-Ribosylated DNA-Protein Crosslinks

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

Author Spotlight: Quantitative Detection of DNA Protein Crosslinks and Their Post-Translational Modifications  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

Assessing DUB Variants in Neurodevelopmental Disorders Using a Cleavage Assay

Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay

Neurodevelopmental disorders (NDDs) are associated with impairments in nervous system function but often remain poorly understood at the molecular level. Discrete disorders caused by single genes provide models to investigate mechanisms driving atypical neurodevelopment. Variants of genes encoding deubiquitylating enzyme (DUB) family proteins are associated with several NDDs, but there is a need to determine the pathogenic mechanisms of disorders driven by these gene variants. The impact of gene variants on DUB activity can be experimentally determined using a substrate-independent in vitro ubiquitin cleavage assay. This assay does not require knowledge of downstream substrates to directly measure catalytic activity. Here, the protocol for determining the impact of gene variants on enzymatic activity is modeled using the DUB Ubiquitin Specific Protease 27, X-linked (USP27X), which is mutated in X-linked intellectual disability 105 (XLID105). This experimental pipeline can be used to clarify the mechanisms underlying neurodevelopmental disorders driven by variants in DUB genes.

This protocol explains how to measure the effect of genetic variation associated with neurodevelopmental disorders on deubiquitylating enzyme activity by combining recombinant protein purification with ubiquitin chain cleavage assays.

Neurodevelopmental disorders (NDDs) are associated with impairments in nervous system function but often remain poorly understood at the molecular level. ...

Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain

The ubiquitin-proteasome system is a key regulator of protein degradation and a variety of other cellular processes in eukaryotes. In the brain, increases in ubiquitin-proteasome activity are critical for synaptic plasticity and memory formation and aberrant changes in this system are associated with a variety of neurological, neurodegenerative and psychiatric disorders. One of the issues in studying ubiquitin-proteasome functioning in the brain is that it is present in all cellular compartments, in which the protein targets, functional role and mechanisms of regulation can vary widely. As a result, the ability to directly compare brain ubiquitin protein targeting and proteasome catalytic activity in different subcellular compartments within the same animal is critical for fully understanding how the UPS contributes to synaptic plasticity, memory and disease. The method described here allows collection of nuclear, cytoplasmic and crude synaptic fractions from the same rodent (rat) brain, followed by simultaneous quantification of proteasome catalytic activity (indirectly, providing activity of the proteasome core only) and linkage-specific ubiquitin protein tagging. Thus, the method can be used to directly compare subcellular changes in ubiquitin-proteasome activity in different brain regions in the same animal during synaptic plasticity, memory formation and different disease states. This method can also be used to assess the subcellular distribution and function of other proteins within the same animal.

This protocol is designed to efficiently quantify ubiquitin-proteasome system (UPS) activity in different cellular compartments of the rodent brain. Users are able to examine UPS functioning in nuclear, cytoplasmic and synaptic fractions in the same animal, reducing the amount of time and number of animals needed to perform these complex analyses.

The ubiquitin-proteasome system is a key regulator of protein degradation and a variety of other cellular processes in eukaryotes. In the brain, increases ...

Neuroscience

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 ...

Virtual Screening and Validation of UCHL3 Inhibitors Using Molecular Docking

Screening Traditional Chinese Medicine Compounds for Inhibiting UCHL3 Activity Based on Molecular Docking and Deubiquitinating Enzyme Probe Technology

Deubiquitinating enzymes&#160;(DUBs) play a pivotal role in modulating ubiquitination homeostasis, with UCHL3 being an archetypal cysteine DUB intricately involved in a myriad of physiological and pathological processes. Therefore, developing small molecule inhibitors targeting Ubiquitin C-Terminal Hydrolase L3 (UCHL3) is of great significance. This protocol aims to establish a process for virtual screening and in vitro validation of small molecule inhibitors of cysteine DUB represented by UCHL3. Firstly, potential inhibitors of UCHL3 are virtually screened using molecular docking technology, and the interaction between drugs and protein targets is visualized. Subsequently, the effectiveness of the screened drug, Danshensu, is verified through in vitro activity inhibition assays. Ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) and hemagglutinin-ubiquitin-vinyl sulfone (HA-Ub-VS) are used as probes for in vitro activity testing, as they can competitively bind to DUB with small molecule inhibitors to assess the activity of UCHL3. The results indicate that Danshensu has a good binding affinity with UCHL3 in molecular docking, and it can competitively inhibit the activity of UCHL3 with HA-Ub-VS. These findings provide important references for further research and development of therapeutic drugs targeting UCHL3.

The experiment used here shows a method of molecular docking combined with probe technologies to predict and validate the interaction between small molecules of traditional Chinese medicine and protein targets.

Deubiquitinating enzymes&#160;(DUBs) play a pivotal role in modulating ubiquitination homeostasis, with UCHL3 being an archetypal cysteine DUB intricately ...

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

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Chapters in this video

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

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

Detection of Protein Ubiquitination

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay

Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain

In Vitro Analysis of E3 Ubiquitin Ligase Function

Screening Traditional Chinese Medicine Compounds for Inhibiting UCHL3 Activity Based on Molecular Docking and Deubiquitinating Enzyme Probe Technology