Cul3 / BACURD Ubiquitin E3 Ligase Complexes On Ubiquitination Of RhoA And The Role Of N-terminal Regulatory ETP2 In The Process Of Lethal Toxin Activation Of Nalplb

The first part of this study foucuses on the ubiquitination and degradation of RhoA mediated by Cu13/BACRUDs E3liagse complex. Cullin3is a member of Cullin E3ligase protein family. Like the other family member, Cu13is thought to mediate the assembly of a large number of SCF(Skpl-Cullinl-F-box protein)-like ubiquitin ligase complexes through BTB domain substrate-recruiting adaptors. Previous work has shown that Cu13is essential for early embryonic development in all tested genetic models, including C. elegans, Drosophila melanogaster, mouse, and even Arabidopsis thaliana. However, very few functional substrates for Cu13have been identified and its physical function remains unclear.We found that inhibition of Cu13expression by RNAi would lead to strong stress fibers due to ubiquitination and degradation impairment of small GTPase RhoA. RNAi screen of BTB domain-containing protein in Drosophila S2cell lines has identified a protein family, designated BACURDs. BACURDs form E3ligase complex with Cu13and mediate RhoA ubiquitination and degradation. Dysfunction of the Cu13/BACURD complex impairs RhoA-mediated convergent extension movements during Xenopus gastrulation.The second part of this study focuses on the Nalplb inflammasome activation process induced by anthrax lethal toxin. Anthrax lethal toxin possesses potent effect on the activation of Nalplb inflammasome in mouse macrophages. However, the underlying molecular mechanism remains unclear. To further investigate how lethal toxin activates Nalplb inflammasome, we generate293T recombinant system and RAW264.7cells stably expressing RFP-ASC fusion protein. Previous work indicates that N-end rule specificity within the ubiquitin/proteasome pathway plays a major role in Nalplb inflammasome activation. To find the E3ligase involved in this process, we reduce the expression of UBR E3ligase family and monitor the activation of lethal toxin induced Nalplb inflammasome activation. Finally we identify UBR2play an essential role in the Nalplb activation, which is consistent with the result of mouse genome-wide siRNA screen in RAW264.7stable cell line.