The overall goal of this protocol is to analyze important key aspects of E3 ligase function, including E2 enzyme specificity, substrate selection and ubiquitin transfer. The main advantage of this technique is its wide applicability, as proteins from all eukaryotic sources can be recombinantly expressed and studied in vitro without the need for special equipment. When using the lysine discharge technique for the first time, it is important to optimize assay parameters, such as enzyme concentrations, to identify the ideal discharge conditions for the E3 ligase of choice.
To perform an in vitro auto-ubiquitylation assay, set up a pipetting scheme to test the functional ability of different E2 enzymes and prepare a master mix on ice for all of the ubiquitylation reactions plus one additional reaction. Next, add one micromolar of E2 enzymes to the appropriate tubes, and incubate the samples in a PCR thermal cycler for two hours under the indicated conditions. After the incubation, add SDS sample buffer to each reaction and mix by pipetting several times.
Then immediately boil the samples at 95 degrees Celsius for five minutes and store the denatured proteins at 20 degrees Celsius. To perform an in vitro substrate ubiquitylation assay, prepare the pipetting scheme for all of the reactions. After calculating the amount of master mix required for one reaction, prepare the master mix for all of the reactions on ice and add the master mix to each tube.
Add the respective E3 ligases individually. Then incubate the samples in the PCR thermal cycler for two hours as demonstrated. At the end of the incubation, add two times SDS sample buffer to each reaction, mix several times by pipetting, and boil the samples for five minutes at 95 degrees Celsius.
To perform a lysine discharge assay, set up the pipetting scheme for the charging reaction and incubate the charging reactions for 15 minutes at 37 degrees Celsius. To stop the charging reaction, add apyrase to a final concentration of 1.8 units per milliliter and incubate the reaction for five minutes at room temperature. At the end of the incubation, add EDTA to a final concentration of 30 millimolar and use double-distilled water to adjust the volume of the reaction to 30 microliters.
To discharge E2 ubiquitylation by E3, set up five tubes corresponding to the time points for the experiment, add 6.7 microliters of non-reducing sample buffer to each tube, remove six microliters of the stopped charging reaction from the time point zero tube, and adjust the total volume to 26.7 microliters. To set up the discharge reactions, add double-distilled water, ubiquitylation buffer, BSA, lysine, E3 ligase and the charged E2 to each reaction. After the selected time periods, transfer 20-microliter samples from each discharge reaction to the corresponding sample tube.
Then immediately vortex the samples, and place them at 70 degrees Celsius for 10 minutes. In this representative Western blot analysis, the inactive CHIP was not auto-ubiquitylated. However, the E3-independent ubiquitin products were formed in the presence of inactive CHIP and in the absence of CHIP.
The wild-type CHIP was auto-ubiquitylated when combined with the proteins of the UBE2D and the UBE2E families, whereas the free polyubiquitin chains were produced in cooperation with the proteins of the UBE2D family but not with the proteins of the UBE2E family. The binding of the ubiquitin by UBE2N/V1 directed the formation of free ubiquitin chains. The auto-ubiquitylation and ubiquitylation of UNC-45B was observed with wild-type CHIP but not with the inactive mutant of CHIP, indicating that UNC-45B acts as a conserved substrate for CHIP.
When the catalytic activity of CHIP was analyzed using a lysine discharge assay, the uncharged UBE2D2 enzyme had a molecular weight of 17 kilodaltons and the charged UBE2D2 with a single ubiquitin molecule had a molecular weight of approximately 26 kilodaltons. At time 0, the entire charged E2 yield was observed. In the presence of inactive CHIP, a faint E3 ligase-independent discharge of UBE2D2 but not auto-ubiquitylation of CHIP was detected.
In the presence of wild-type CHIP, the discharge of UBE2D2 was fast and completed within 60 minutes, and the auto-ubiquitylation of CHIP indicated a transfer of ubiquitin onto its own lysine residues. Due to the limited E2 ability, work as quickly as possible and immediately proceed with the discharge reaction followed by SDS-PAGE and Western blotting. Following these in vitro ubiquitylation protocols, the specific ubiquitylation linkage types can be identified by probing the Western blots with linkage-specific antibodies.
