Characterization of the interaction interface between the conjugating enzyme, Ubc12, and the ligase, Rbx1, of the NEDDylation pathway
Date Issued
2009
Date
2009
Author(s)
Lee, Nian-Wei
Abstract
Neural precursor cell expressed, developmentally down-regulated 8 (Nedd8) is a highly conserved eukaryotic ubiquitin-like protein. It is conjugated to its substrates in a process known as NEDDylation which comprises an E1-E2-E3 enzymatic cascade similar to ubiquitination. Dysregulated NEDDylation is implicated in the pathogenesis of many human diseases and the components of the NEDDylation pathway may accordingly be of potential therapeutic importance. ullin–RING ubiquitin E3 ligases (CRLs) are the most prominent class of ubiquitin-ligases. Rbx1/Roc1, a RING finger protein, functions as an important component of CRLs. Modification of cullins by Nedd8 has been shown to activate CRLs and it was suggested that Rbx1 acts as the E3 for cullin NEDDylation. In other words, Rbx1 interacts first with Ubc12, the NEDDylation E2, to neddylate cullins and activate CRLs. Rbx1 then, in the context of activated CRL holo-enzyme, interacts with the ubiquitination E2s to promote substrates ubiqutination. Rbx1 is thus a dual - functioning E3 and switches its role in the processes of CRLs NEDDylation and the following substrates ubiquitination. The role-switching mechanism of Rbx1 as a dual- functioning E3 remains elusive. To answer this question, deciphering the precise surfaces of Rbx1/ubiquitination E2 versus Rbx1/NEDDylation E2 interactions is a top priority.ere we report that His88, Pro121, Val122, and the unique N-terminal extension of Ubc12 are implicated in the interaction with Rbx1 in yeast two-hybrid system. Ile54, Pro95, and Leu96 of Rbx1 are also shown to be important for the interaction between Ubc12 and Rbx1 in yeast two-hybrid system. The implications of Rbx1 I54 / P95 / L96 in cullin NEDDylation were further demonstrated in baculovirus-insect cell system. More work is necessary to reveal the full picture of both Rbx1/ubiquitination E2 and Rbx1/NEDDylation E2 interaction surfaces.
Subjects
NEDDylation
Nedd8
Ubc12
Rbx1
Cullin–RING ubiquitin ligase
SDGs
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