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Characterization of Trim32‐catalyzed polyubiquitin chain formation
Author(s) -
Streich Frederick C,
Haas Arthur L
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.848.8
Subject(s) - ubiquitin , chemistry , cooperativity , protein subunit , ubiquitin ligase , proteasome , microbiology and biotechnology , deubiquitinating enzyme , coiled coil , ubiquitin protein ligases , superfamily , biochemistry , biophysics , stereochemistry , biology , receptor , gene
Trim32, a member of the Trim superfamily of ubiquitin ligases defined by a conserved tripartite RING‐β box‐coiled coil motif, was originally discovered as an HIV‐I Tat interacting protein and has been implicated in limb‐girdle muscular dystrophy, Bardet‐Biedl syndrome, and displays anti‐apoptotic activity associated with carcinoma. Trim32‐catalyzed polyubiquitination of target proteins is a critical mechanism by which proteins are marked for 26S proteasome‐dependent degradation; however, little is known regarding the mechanism of conjugation or chain elongation among members of this superfamily. Kinetic studies show, contrary to the literature, that the Ubc5 but not UbcH6 family is the cognate E2 supporting Trim32 conjugation. Trim32 catalyzes a slow rate of free polyubiquitin chain formation in the absence of substrate and demonstrates cooperativity with respect to [Ubc5A] o (n=3.4), suggesting four tightly coupled E2 binding sites. Cooperativity requires subunit association through the βbox‐coiled coil motif since the RING domain alone is incapable of polyubiquitin chain assembly. Ring domain dimerization through N‐terminal GST‐mediated association reconstitutes ubiquitin chain formation. Characterizing the mechanism of polyubiquitin chain formation will help describe why mutants of Trim32 and other Trim proteins are associated with debilitating diseases. [Funded by GM340092 to ALH]