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

We're interested in defining the molecular basis of neurodevelopmental disorders associated with gene variants in enzymes of the ubiquitin system. We use cutting edge techniques to dissect the signaling pathways controlled by those enzymes and to determine the impact of gene variants on protein biology. The advantage of this method is that it is a substrate-independent technique that does not require previous knowledge of the substrates that are regulated by a specific DUB.

This way we can directly measure the impact of neurodevelopmental disorder-associated gene variance on DUB catalytic activity and suggest possible pathogenic mechanisms. Because the cellular functions of most DUBs are poorly understood, our lab focus will be on identifying the signaling pathways that these enzymes mediate and how gene variants associated with genetic disease affect these processes. To begin, thaw 20 microliters of chemically competent Rosetta 2 Escherichia coli cells on ice immediately before use.

Add one to 10 nanograms of the pGEX6P1 USP27X expression plasmid and incubate for five minutes on ice. Heat shock the cells for 30 seconds at 42 degrees Celsius in a dry bath. Then incubate the cells on ice for two minutes.

Add 80 microliters of room temperature SOC medium to the cells. Incubate for 60 minutes at 37 degrees Celsius with 200 RPM rotation in a 19-millimeter orbit benchtop temperature controlled shaker. Then plate 50 microliters of the culture on an LB agar plate, supplemented with chloramphenicol and ampicillin.

Incubate for 20 hours at 37 degrees Celsius, lid side down, in a temperature controlled incubator. Pick a single colony of transformed bacteria and add it to 10 milliliters of sterile LB medium supplemented with chloramphenicol and ampicillin. Incubate for 20 hours as shown previously.

Then add 10 milliliters of the overnight culture to one liter of TB medium supplemented with chloramphenicol and ampicillin. Incubate until the optical density at 600 nanometers reaches 0.5 to 0.6. Next, add 50-micromolar isopropyl beta-d-1-thiogalactopyranoside to the culture to induce expression.

After cooling the sample to 16 degrees Celsius, incubate for 20 hours at 16 degrees Celsius with 200 RPM rotation. Then centrifuge the cells for 20 minutes at 3000 G or higher at four degrees Celsius to pellet the cells from the expression culture. Finally, store the pellet at minus 80 degrees Celsius for at least one hour.

To begin, secure the empty gravity flow column in a retort stand. Fill the column with two to three milliliters of gluthathione agarose resin to purify a cell pellet collected from one liter of expression culture. Wash the column with one resin bed volume of 20%ethanol.

After washing the column, thaw the cell pellet at four degrees Celsius. Add 30 milliliters of MS500 buffer to the thawed pellet and incubate with gentle end-over-end rotation. Now sonicate the lysed cells in a 50-milliliter centrifuge tube on ice until the lysate flows freely when dispensed from a pipette tip.

Centrifuge for 30 minutes at four degrees Celsius and 20, 000 G or higher to clear the supernatant. Then decant the supernatant into a beaker and load the cleared lysate onto the column. Run the lysate through the column by gravity flow while collecting the flow-through.

Afterward, wash the column with at least two resin bed volumes of MS500 wash buffer and collect the wash flow-through in five milliliter fractions. Next, add one microliter of each fraction to 100 microliters of Bradford reagent to check for protein presence. Recover the protein by running MS500 elusion buffer through the column and collecting five milliliter elution fractions.

To precipitate the protein, add two volumes of four-molar ammonium sulfate to the eluate and gently invert the tube until it becomes cloudy. Centrifuge for 30 minutes at four degrees Celsius and 20, 000 G or higher. Remove the supernatant after each centrifugation without disturbing the protein pellet.

Then re-dissolve the protein in MS500 buffer supplemented with 25%glycerol before storing it. Prepare 10 microliters of purified GST-tagged USP27X in deubiquitylating enzyme activation buffer for each time point for each deubiquitylating enzyme. Before adding ubiquitin chains, add seven microliters of SDS-PAGE loading buffer to the time zero sample to prevent the deubiquitylation reaction from starting.

To each time point for each deubiquitylating enzyme, add 375 nanograms of K63-linked di-ubiquitin chains diluted in 10 microliters of enzyme activation buffer. Incubate the tubes at 30 degrees Celsius, stopping each time point by adding seven microliters of SDS-PAGE loading buffer. Perform SDS-PAGE using a four to 12%gradient gel.

Wild type USP27X cleaved K63-linked di-ubiquitin chains into mono-ubiquitin after one hour of incubation. The XLID-105 associated variance F313V, Y381H, and S404N did not cleave K63-linked di-ubiquitin chains after one hour of incubation.