Detailed Dissection of UBE3A-Mediated DDI1 Ubiquitination

Abstract

The ubiquitin E3 ligase UBE3A has been widely reported to interact with the proteasome, but it is still unclear how this enzyme regulates by ubiquitination the different proteasomal subunits. The proteasome receptor DDI1 has been identified both in Drosophila photoreceptor neurons and in human neuroblastoma cells in culture as a direct substrate of UBE3A. Here, we further characterize this regulation, by identifying the UBE3A-dependent ubiquitination sites and ubiquitin chains formed on DDI1. Additionally, we found one deubiquitinating enzyme that is capable of reversing the action of UBE3A on DDI1. The complete characterization of the ubiquitination pathway of an UBE3A substrate is important due to the role of this E3 ligase in rare neurological disorders as Angelman syndrome.

Dimer Interface Organization is a Main Determinant of Intermonomeric Interactions and Correlates with Evolutionary Relationships of Retroviral and Retroviral-Like Ddi1 and Ddi2 Proteases

“…Our results imply that non-viral retroviral-like PRs potentially have relatively lower dimer stability compared to retroviral counterparts. We assume that getting better insight into the structural requirements for the dimer formation through the protease domain may help understanding the roles of Ddi-like proteins in proteasomal shuttles and ubiquitination pathways [6,7,63,64,67,68,84,85], even in physiological or pathological conditions. Furthermore, future studies are needed to prove our findings by determining the in vitro dimer stabilities, and correlating the features described in our study with those of additional eukaryotic retroviral-like or endogenous retrovirus PRs.…”

“…In Caenorhabditis elegans, Ddi1 expression was found to be induced by proteasome dysfunction; furthermore, results proved that the catalytic activity of Ddi1 PR is necessary for protein activation [65], and involvement of PR activity in Nrf1 processing was also demonstrated for human Ddi2 PR [66,67]. Protease domain of Ddi1-Hs protein was found to undergo post-translational modification, ubiquitination sites were identified in the proximities of the active site (K192) and the dimer interface (K382) [68], but effect of these modifications of protease function has not been elucidated. Among the Ddi-like PRs, proteolytic activity of a recombinant protein was proved in vitro only for Ddi1-Lm [12].…”

The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF

“…GFP pull-downs from HEK293T cells were performed as previously described ( 30 , 31 ). In the case of fly samples, slight modifications were added to adapt the protocol to Drosophila heads.…”

“…Remarkably, this approach can be efficiently applied to identify neuronal E3 ligase substrates ( 28 , 29 ). The second methodology, in contrast, favors the isolation of GFP-tagged proteins under denaturing conditions to further characterize their ubiquitination pattern under the presence or absence of an E3 ligase ( 30 , 31 ).…”

Molecular Evolution, Neurodevelopmental Roles and Clinical Significance of HECT-Type UBE3 E3 Ubiquitin Ligases

“…Other reviews [ 56 ] highlight the mechanistic function of UBE3A, describing its association with several proteasome receptors, like DDI1 [ 57 , 58 ] or PSMD4 but also with proteasome itself, which is thought to control the proteolysis [ 59 ]. AS-associated point mutations in UBE3A N-terminus show impaired PSMD4 binding [ 60 ].…”