Abstract Detail

Cell biology

McVey, Sarah [1], Liu, Hongxia [1], Ravichandran, Sridhar [2], Stone, Sophia [1].

Role of RING-type E3 ligase, XBAT35.2, in regulating cell death.

Ubiquitination is a post-translational modification that results in single (monoubiquitination) or multiple (polyubiquitination) ubiquitin molecule(s) being covalently attached to a substrate. A common consequence of ubiquitination is degradation of polyubiquitinated proteins by the 26S proteasome. This ubiquitin proteasome system (UPS) requires the sequential action of three enzymes: E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme), and E3 (ubiquitin ligase). E1 activates ubiquitin and E2 receives the activated ubiquitin from E1. The E2-ubiquitin intermediate interacts with the E3, which recruits the substrate. The E2 and E3 then coordinate the attachment of ubiquitin to lysine residues on the selected protein substrate. Our laboratory focuses on a specific subset of E3 ubiquitin ligase called the Really Interesting New Gene (RING) family. My research focuses on one of the two protein isoforms of XBAT35 RING-type E3 ligase, XBAT35.2. This ligase interacts with Accelerated Cell Death 11 (ACD11) protein based on previous yeast-two hybrid screens and bimolecular fluorescence complementation assays (BiFC). The overall research goal is to determine the biological significance of the interaction between XBAT35.2 E3 ligase and ACD11. Protein interaction between XBAT35.2 and ACD11 in planta has been confirmed using an immunoprecipitation assay. Additional objectives are to demonstrate in vivo ubiquitination of ACD11, determine if ACD11 is turned over by the 26S proteasome and determine the role of XBAT35.2 in ACD11 ubiquitination and proteasomal-dependent degradation. Preliminary results show that ACD11 remains stable in a cell free degradation assay in the presence and absence of a proteasome inhibitor, MG132. When transiently expressed with XBAT35.2, there is protein turnover of ACD11, which suggests that it is being degraded by the 26S proteasome. Protein degradation does not occur when ACD11 is transiently expressed with mutated E3 ligase. Preliminary results also suggest that ACD11 turnover when transiently expressed with XBAT35.2 is specific to the 26S proteasome. We have also shown that XBAT35 plants and transgenic plants overexpressing XBAT35.2 display altered responses to pathogen, suggesting a role for the E3 ligase in pathogen defense. These results correlate with the observation that overexpression of XBAT35.2 in tobacco cells is capable of inducing cell death. A functional RING E3 ligase domain is required for induction of cell death because when we mutated the RING domain, transient overexpression of an inactive E3 ligase did not induce any cell death. Similarly, transient overexpression of XBAT35.1 did not induce cell death, which indicates that this induction is specific to the XBAT35.2 protein isoform.

1 - Dalhousie University, Biology, Halifax, NS, B3H4R2, CAN
2 - Dalhousie University, Environmental Sciences, Truro, NS, B2N5E3, CAN

Keywords:
Ubiquitination 
E3 ligase
XBAT35
ACD11.

Presentation Type: Oral Paper:Papers for Topics
Session: 58
Location: Salon 17/The Shaw Conference Centre
Date: Wednesday, July 29th, 2015
Time: 10:45 AM
Number: 58011
Abstract ID:969
Candidate for Awards:CSPB President's Award for Best Student Presentation