Abstract
Members of the tripartite motif family of E3 ubiquitin ligases are characterized by the presence of a conserved N-terminal module composed of a RING domain followed by one or two B-box domains, a coiled-coil and a variable C-terminal region. The RING and B-box are both Zn-binding domains but, while the RING is found in a large number of proteins, the B-box is exclusive to the tripartite motif (TRIM) family members in metazoans. Whereas the RING has been extensively characterized and shown to possess intrinsic E3 ligase catalytic activity, much less is known about the role of the B-box domains. In this study, we adopted an in vitro approach using recombinant point- and deletion-mutants to characterize the contribution of the TRIM32 Zn-binding domains to the activity of this E3 ligase that is altered in a genetic form of muscular dystrophy. We found that the RING domain is crucial for E3 ligase activity and E2 specificity, whereas a complete B-box domain is involved in chain assembly rate modulation. Further, in vitro, the RING domain is necessary to modulate TRIM32 oligomerization, whereas, in cells, both the RING and B-box cooperate to specify TRIM32 subcellular localization, which if altered may impact the pathogenesis of diseases.
Highlights
Ubiquitination is a versatile form of post-translational modification that regulates a large number of processes inside the cell through the regulation of protein turnover and activity
In the following step of the cascade, ubiquitin is transferred to the E2-conjugating enzyme, which cooperates with an E3 ubiquitin ligase to transfer ubiquitin to the final target substrate [1]
In the case of TRIM32, the role of the tripartite motif domains is still unclear and the available data on their contribution to E3 activity mainly rely on experiments using short protein fragments [7,31]
Summary
Ubiquitination is a versatile form of post-translational modification that regulates a large number of processes inside the cell through the regulation of protein turnover and activity. The ubiquitination reaction is a three-step process beginning with the ATP-dependent activation of ubiquitin, a 76-amino-acid peptide, by the E1 activating enzyme. In the following step of the cascade, ubiquitin is transferred to the E2-conjugating enzyme, which cooperates with an E3 ubiquitin ligase to transfer ubiquitin to the final target substrate [1]. The E3 ubiquitin ligase is able to provide substrate specificity to the ubiquitination cascade. Among the RING-type E3 ligases, the tripartite motif (TRIM) family represents one of the largest groups with over 70 members in humans. This class of E3 ligases is characterized by the presence of a well-conserved N-terminal motif comprising the previously mentioned RING domain, one or two
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