955C Poster - 15. Models of human disease
Saturday April 09, 1:30 PM - 3:30 PM

Authors:
Benjamin Geier ¹; David Kakhniashvili ¹; Daniel Johnson ¹; Jon Huibregtse ²; Lawrence Reiter ¹

Affiliations:
1) University of Tennessee Health Science Center; 2) University of Texas at Austin

Keywords:
e. epilepsy; n. proteomics

Proteolytic activity via the ubiquitin-proteasome system (UPS) serves a crucial role in regulating proteins for degradation and redistribution in the cell. Several human disorders directly result from dysregulation of the UPS. Mutations that affect expression levels of the ubiquitin protein ligase E3A (UBE3A/E6AP) can result in Duplication 15q or Angelman syndrome. While many researchers have paired cell culture methods with immunoprecipitation and blotting techniques to identify UBE3A interactors, to date, only a handful of validated UBE3A substrates have been identified. Through the implementation of a new biochemical method known as a ubiquitin activated interaction trap (UBAIT), we can now covalently link ubiquitin substrates to UBE3A in vivo. We generated two upstream activator sequence (UAS) lines that incorporate a 6x polyhistidine-tagged Dube3a UBAIT construct for expression experiments in all cells (actin-GAL4), glial cells (repo-GAL4), and neurons (elav-GAL4). The first UBAIT is a K48R wild-type ubiquitin line that actively binds ubiquitin substrates and covalently attaches them to His-tagged Dube3a for isolation. The second UBAIT employs a non-functional ubiquitin with a DGG mutation that cannot covalently attach substrates to His-tagged Dube3a, but can serve as a control for other proteins stuck to the expressed UBAITs. Using Ni-NTA column purification combined with unbiased whole proteome analysis we identified 27 proteins using actin-GAL4 and 59 proteins using repo-GAL4 that appear to be bonafide Dube3a substrates and were not present in the DGG control UBAIT group. These hits included the Na/K pump ATPalpha, which our group previously showed is regulated by Dube3a in glial cells. Other hits include trehalose transporter 1-1(Tret1-1), wings up A(wupA), and rolled(rl). This work marks the first successful implementation of the UBAIT system in a model organism, opening room for a multitude of successive in vivo UBE3A proteomic experiments.