Streich FC, Ronchi VP, Connick JP, Haas AL

Ligation of polyubiquitin chains to proteins is a fundamental post-translational modification, often resulting in targeted degradation of conjugated proteins. Attachment of polyubiquitin chains requires the activities of an E1 activating enzyme, an E2 carrier protein, and an E3 ligase. The mechanism by which polyubiquitin chains are formed remains largely speculative, ...

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especially for RING-based ligases. The tripartite motif (TRIM) superfamily of ligases has functions implicated in many cellular processes, including innate immunity, cellular localization, development and differentiation, signaling, and cancer progression. The present results show that representative TRIM ligases catalyze polyubiquitin chain formation in the absence of substrate, the rates of which can be used as a functional readout of enzyme function. Initial rate studies under biochemically-defined conditions show that TRIM32 and TRIM25 are specific for the Ubc5 family of E2 conjugating proteins and, along with TRIM5α, exhibit cooperative kinetics with respect to Ubc5A concentration, with submicromolar [S](0.5) and Hill coefficients of 3-5, suggesting they possess multiple binding sites for their cognate E2-ubiquitin thiolester. Mutation studies reveal a second, non-canonical binding site encompassing the C-terminal Ubc5 α-helix. Polyubiquitin chain formation requires TRIM subunit oligomerization through the conserved coiled-coil domain, but can be partially replaced by fusing the catalytic domain with GST to promote dimerization. Other results suggest that TRIM32 assembles polyubiquitin chains as a Ubc5-linked thiolester intermediate. These results represent the first detailed mechanistic study of TRIM ligase activity and provide a functional context for oligomerization observed in the superfamily.

Date: Feb. 13, 2013

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