Given the complexity of the ubiquitination and deubiquitination reactions inside the cells it is important to establish in vitro assays with defined purified components. This helps to understand the mechanisms of ubiquitination and deubiquitination and also to determine how mutations associated with genes that encode the enzymes responsible for this reaction can cause human disease. We can identify the domains and motifs in these components that drive these reactions.
We can also develop high throughput screen assays that can be used to screen for small molecule inhibitors that can be developed as potential therapeutics. To begin bacteria lysis, add PMSF and antiprotease cocktail into the ice cold lysis buffer A.Add 25 millilters of the lysis buffer A into the bottle containing bacteria pellet and resuspend the pellet. Transfer the lysate containing bacteria in a tube for sonication.
Use a probe sonicator to sonicate the lysate at 70%output amplitude for 30 seconds, four to five times. Then centrifuge at 21, 000 times g for 20 minutes at four degrees Celsius. After incubating the bacteria lysate with GSH beads for five hours according to the manuscript, transfer the beads along with 10 millilters of buffer into an empty chromatography column.
Then, add 1.5 millilters of buffer A containing 25 millimolar glutathione into the column and collect the elution by gravity into 1.5 milliliter microtubes. Repeat the elution procedure four times with fresh buffer A an continue treatment of the eluted fractions according to the manuscript. To begin, resuspend the His-BAP1 and the MBP-DEUBAD bacteria pellets in 25 millilters of ice cold lysis buffer B.Mix 25 millilters of each lysate and incubate on ice for 15 minutes.
Sonicate and then centrifuge the lysate at 21, 000 times g for 20 minutes. After lysate centrifugation, filter the lysate through a 0.45 micrometer pore syringe filter into a bottle and mix it with the Nickel NTA beads. Incubate at four degrees Celsius with shaking for five hours.
After that, spin down the beads at 25, 000 times g for three minutes. Use a pipette to save the supernatant in a 50 milliliter tube as the flow through. Wash the beads with five millilters of buffer B, containing 20 millimolar imidazole, six times with three minute centrifuge.
After the last wash, transfer the beads with 10 millilters of buffer B into an empty chromatography column and add one milliliter of buffer B containing 200 millimolar imidazole. Collect the elution by gravity into 1.5 milliliter microtubes containing DTT and EDTA. Repeat the elution procedure four times and pool the desired eluted fractions into a 15 milliliter tube.
Add buffer C to dilute the eluted complex three times. Then transfer the diluted elution to the prepared amylose beads in tubes and incubate overnight at four degrees Celsius with shaking. In the morning, centrifuge the tubes at 2, 500 times g for three minutes and keep the supernatant in a 15 milliliter tube as flow through.
Wash the protein bound beads with five millilters of buffer C for five to six times. Then add 500 microliters of buffer D to the purified His-BAP1/MBP-DEUBAD complex on the amylose agarose beads to make a 50%bead solution. Transfer 20 microliters of the 50%bead solution to a microcentrifuge tube and add 20 microliters of two times Laemmli Sample Buffer for SDS-PAGE and Coomassie blue analysis.
Store the remaining bead solution at minus 80 degrees Celsius for future usage. After culture the HEK293T cells, plate 12 million cells in 20 millilters of complete media in a 15 centimeter culture dish, one day before the transfection. After one one day, under the cell culture hood, aspirate media and supply with 12 millilters of serum free media.
Transfect the cells with 21 micrograms of pCDNA flat-H2A using 63 microliters of PEI at one milligram per milliliter. After 12 hours, change to complete medium. After harvesting the cells according the manuscript, resuspend the cell pellet in about 10 volumes of buffer E containing NEM to inhibit deubiquitinases and you incubate on ice for 20 minutes.
After centrifugation at 3, 000 times g for five minutes, discard the supernatant. Wash the chromatin pellet twice with 10 volumes of buffer E first and then wash twice with buffer F.Resuspend the chromatin in five millilters of buffer F and treat the pellet with micrococcal nuclease at the concentration of three units per milliliter for 10 minutes at room temperature. After incubation, transfer a 40 microliter aliquot of the mixture to a 1.5 milliliter tube for analysis of nucleosomal DNA fragments.
Add 40 microliters of phenol-chloroform and 20 microliters of six times DNA loading buffer. Vortex and spin at 18, 000 times g for two minutes. Then transfer 15 microliters of the aqueous phase in a 2%agarose gel to load the DNA.
After stopping the reaction and centrifugation according to the manuscript, incubate the soluble chromatin fraction with anti-flag resin beads in a 15 milliliter tube overnight at four degrees Celsius with shaking. Wash the beads six times with buffer G.Transfer the beads with five millilters of buffer G into an empty chromatography column. Dilute the bead's bound nucleosomes with 260 microliters of buffer G containing 200 micrograms per milliliter of flag elution peptide and one to five one molar Tris at pH eight.
Elute the nucleosomes three times, one hour in the cold room. To perform ubiquitination assay, first add one microgram of the purified nucleosomes in 40 microliters of buffer H.After that, add an enzyme solution mix containing 250 nanograms of UBE1, 672 nanograms of UB and E2 UB conjugating enzymes and one microgram of BMI1-RING1B E3 ubiquitin ligase complex into the tube. Incubate the reaction for three hours at 37 degrees Celsius with occasional shaking.
Stop the reaction by adding 40 microliters of two times Laemmli Sample Buffer and analyze histone H2AK119 ubiquitination by western blotting. To perform the in vitro deubiquitination assay resuspend the purified nucleosomes in 40 microliters of buffer I and one microgram of bacteria purified His-BAP1/MBP-DEUBAD beads. Place the tube in an incubator at 37 degrees Celsius for three hours to carry out the deubiquitination reaction with occasional shaking.
Then stop the reaction by adding 40 microliters of two times Laemmli buffer and analyze by immunoblotting. In this experiment, Coomassie blue staining for a typical purification of the GST-BM1-RING1B complex using GST affinity beads, shows that the bands migrate at expected molar weight around 45 kilodaltons and 13 kilodaltons respectively. Similarly the purification of His-BAP1 MBP-DEUBAD complex also resulted in relatively high homogenous preparations with bands around 90 kilodaltons and 55 kilodaltons respectively.
On the other hand the purified nucleosomal fraction is essentially composed by a 147 base pair band indicating the presence of highly enriched mononucleosomal fraction. Coomassie blue staining of purified nucleosomes shows a typical band pattern of the four histone subunits with stoichiometric amounts. Ubiquitination of histone H2A with the BMI1-RING1B complex shows a time dependent increase of the ubiquitinated form of the protein while the levels of the nonmodified form are concomitantly decreased.
The ubiquitination reaction is total as virtually all H2A proteins are transformed to H2A K119ub. On the other hand, deubiquitination assay was conducted native nucleosomes. Following incubation of these nucleosomes with BAP1-DEUBAD a time dependent reduction of H2A K119ub signal was observed.
The success of ubiquitination and deubiquitination reactions depend on the manner the protein are purified. We need a good yield and the buffer use for protein resuspension and purification should be compatible with biochemical reactions. When this ubiquitination and deubiquitination assays are well established we can test the affect activators and inhibitors as well as the impact of disease associated gene mutations.
This method have been established by the contribution of several laboratories and are very useful to understand the mechanisms of ubiquitination and deubiquitination. Several chemicals are hazardous and need to be manipulated in the hood. This include phenol-chloroform, acids, bases, and reducing agents.
