Our method provides a rapid way to understand the functional impact of variants in Ube3a, a ubiquitin ligase that causes Angelman syndrome and is strongly linked to a genetic form of autism known as Dup15q syndrome. The characterization of ubiquitin ligase activity is traditionally labor intensive and slow, and such methods are difficult and not really amenable to characterize the hundreds of variants identified for Ube3a. Angelman syndrome is caused by the deletion of the maternal Ube3a gene, whereas Dup15q syndrome is caused by the duplication of the maternal Ube3a, so it's important to deduce how missense variants changed the activity of the Ube3a enzyme, and this is something that's difficult to do without empirically testing each variant.
Demonstrating the procedure will be Jalin Stelzer, a research technician in my laboratory. Begin by performing the assays in HEK293T cells, grown in Dulbecco's Modified Eagle Medium at 37 degrees Celsius, with 5%carbon dioxide in a humidified container, as described in the manuscript. In a biosafety cabinet, plate the suspended HEK293T cells onto a tissue-culture-treated 96 well flat-bottomed plate and incubate the cells overnight at 37 degrees.
On the second day, transfect the cells in a biosafety cabinet with Firefly and Renilla luciferase reporter plasmids, and Ube3a plasmids in triplicate. Perform the transfections in a total volume of 10 microliters per well. Create a master mix for the transfections for each variant in a 1.5 milliliter microcentrifuge tube, and incubate at room temperature for 15 minutes.
After the incubation, gently agitate the transfection mixture by tapping the tube, and then, add 10 microliters of the transfection mixture directly to the existing growth media in the wells. Afterward, return the cells to the incubator and allow the plasmids to express for 48 hours. Using a fluorescence microscope, monitor the transfection efficiency by GFP fluorescence.
More than 80%of cells should express GFP after 48 hours. Carefully aspirate the culture media from transfected HEK293T cells and then, wash the cells with cold PBS. Aspirate the PBS.
Add 25 microliters of non-denaturing lysis buffer for cell lysis, and incubate for 15 minutes on ice with gentle rocking. Then, add 20 microliters of the resulting lysate into a new 96-well assay plate containing 100 microliters of a Firefly luciferase substrate reagent, and mix the plate gently by tapping. Load the plate down to a plate reader and measure the luminescence using a top detector with a read height of one millimeter and an integration time of one second.
Immediately after reading the Firefly luciferase luminescence, add 100 microliters of Renilla luciferase substrate to the wells, which quenches the Firefly luciferase activity while assessing Renilla luciferase activity. Mix the samples by gentle plate agitation and measure the luminescence again to assess the Renilla luciferase activity. Compare the relative activity of variant proteins by quantifying the reporter responses and normalizing the Firefly and Renilla value for each variant against the wild type Ube3a.
HEK293T cells transfected with increasing amounts of plasmid encoding wild-type Ube3a show a linear increase in the BAR response. The BAR assay identifies disease-linked variants of the plasmids, demonstrating the accuracy and generality of this method. BAR assay screen for 152 Ube3a variants displays benign loss of function and gain of function mutations relative to wild-type Ube3a.
The gain of function glutamate substitution, loss of function proline substitution, and another loss of function arginine substitution at position 588 of Ube3a produced drastically different effects, thereby underscoring the importance of empirical variant assessment. The heat plot shows normalized BAR results for comprehensive mutational analysis at position 588 in Ube3a. The white shading represents wild-type Ube3a activity levels, the blue shading indicates loss of function, and the red shading indicates gain of function mutations.
Beyond the clinical implications of our work, Ube3a is a member of the HECT domain family of ubiquitin ligases, which all possess the conserved HECT catalytic domain. And I think one of the fascinating aspects of our work is that it provides rich structure function data about Ube3a, and this can be used to gain a deeper mechanistic understanding for this class of enzymes.
