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

Summary

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.

Abstract

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.

Introduction

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’s, Alzheimer’s, anemia and various cancers^6-10. 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.

Protocol

1. Lysis Buffer Preparation

1. 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 µm polyvinylidene fluoride (PVDF) filter.
2. 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.
3. Make up a Tris solution at pH 7.4 by dissolving Trizma hydrochloride in DI water and adjusting the pH using sodium hydroxide (NaOH).
4. 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 µl of tris, 500 µl of MgCl₂, 1.25 ml of sucrose, 20 µl of DTT, 200 µl of ATP and 7.4947 ml of DI water to make 10 ml of lysis buffer. This can be used for approximately 20 experiments.

2. Culturing Cells for the Experiment

1. Make media by aliquoting 30 ml of DMEM + 10% fetal bovine serum (FBS) into a 50 ml conical tube.
2. Transfer frozen M17 or HeLa cells from liquid nitrogen storage to a small beaker with lukewarm water.
3. 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.
4. 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.
5. Remove the media from the cells using suction. Add 5 ml of fresh media to the cell pellet and resuspend.
6. Transfer the 5 ml cell suspension to the 20 ml of media in the T75 flask and incubate at 37 °C. Change the media every 3 days.

3. Cell Harvesting

1. Remove the media via suction, being careful not to touch the cells.
2. Wash the cells with 5 ml of phosphate buffered saline (PBS). Remove PBS using suction.
3. Add 3 ml of trypsin-ethylenediaminetetraacetic acid (EDTA) to the T75 flask and incubate at 37 °C for 3 min.
4. Add 6 ml of fresh media to the flask and resuspend the cells.
5. Transfer the suspension to a 15 ml conical tube and centrifuge at 290 × g for 5 mins.
6. 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.
7. Repeat step 3.6 three times.
8. 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.

4. Cell Lysis

1. 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.
2. Lyse the cells in the Eppendorf tube at the maximum agitation for 30 min at 4 °C by vortexing in a cold room.
3. Centrifuge briefly at 200 x g for 30 sec to settle the beads. Collect the supernatant.
4. Add the same volume of glass beads as before to the supernatant.
5. Vortex once again at maximum agitation for 30 min at 4 °C.
6. Centrifuge briefly at 200 x g for 30 sec to settle the glass beads. Collect the supernatant.
7. 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.

5. Tissue Homogenization

1. 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.
2. Homogenize the tissue using a setting of level 2 on the homogenizer for 30 sec to ensure that there are no chunks remaining.
3. Spin down at 5,030 x g for 3 min to remove the nuclei, membranes and unbroken cells.
4. Collect the supernatant, which is the lysate.

6. Sample Derivatization

1. 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.
2. Bring an aliquot corresponding to 20 µg of total protein to 50 µl using de-ionized (DI) water.
3. Add 2 µl of 1.35 µM of HA-Ub-Vinyl sulfone (VS) to the solution and incubate for 1 hr at 37 °C. This results 20 µg of lysate to 50 nM of probe in the final reaction mixture. This is in large molar excess to ensure tagging of functional DUBs.
4. Incubate the sample in Laemmli’s sample buffer for 5 mins at 95 °C. For a 52 µl reaction volume use 26 µl of 2x sample buffer, 13 µl of 4x sample buffer or 8.87 µl of 6x sample buffer.
5. Cool on ice before loading the gel.

7. Western Blot

1. Load a 12-well 4-20% tris-glycine gel with 40 µl of the prepared sample
2. Run the gel at 95 V until the ion front reaches the bottom.
3. Carry out an overnight transfer of the gel onto a polyvinylidene difluoride (PVDF) membrane.
4. Incubate the membrane in the amido black stain and scan the membane to obtain an image showing the amount of protein in each lane.
5. Destain the membrane and incubate in 5% milk in PBS for 1 hr to block.
6. 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 °C or for 4 hr at room temperature.
7. Perform 3 washes for 5 min each in PBS with 0.1% Tween-20 detergent.
8. 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.
9. Perform 3 washes for 5 min each in PBS with 0.1% Tween-20 detergent.
10. Perform 1 wash for 5 min in PBS.
11. 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.

Results

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 ...

Discussion

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 &...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PowerGen 125 (homogenizer)	Fischer Scientific	14-261-02P
Vacuum-driven filters 0.22 µm		BPV2210
Glass beads, acid washed ≤106 µm (~140 U.S. sieve)	Sigma-aldrich	G4649-10G
Sucrose	Fischer Scientific	S6-212
DL-dithiothreitol	Sigma-Aldrich	D0632
Magnesium chloride	Sigma-Aldrich	M8266
Adenosine 5'-triphosphate disodium salt hydrate	Sigma-Aldrich	A26209
Trizma hydrochloride	Sigma-Aldrich	T3253
Dulbecco's Modified Eagle Medium	Life Technologies	11965-092
Phosphate buffered saline	Life Technologies	10010-023
Tissue culture flask 75 cm2 w/ filter cup 250 ml 120/cs	Cellstar	T-3001-2
0.05% Trypsin-EDTA (1x)	Life Technologies	25300-054
HI FBS	Life Technologies	16140-071
Monoclonal anti-HA antibody produced in mouse	Sigma-Aldrich	H9658
Ubiquitin vinyl sulfone (HA-tag)	Enzo Life Sciences	BML-UW0155-0025
Laemmli's SDS-Sample Buffer (4x, reducing)	Boston BioProducts	BP-110R
Pierce BCA Protein Assay Kit	Thermo Scientific	23225