THE HECT DOMAIN LIGASE HERC5 EHIBITS INTRINSIC UBIQUITIN LIGASE ACTIVITY: IMPLICATIONS FOR THE TYPE1 INTERFERON RESPONSE
Document Type
Presentation
Start Date
22-10-2010 10:45 AM
End Date
22-10-2010 12:00 PM
Description
The HECT domain ubiquitin ligases share 350 residue C-terminal domain containing an active cysteine to which is formed a ubiquitin thiolester intermediate in target protein conjugation. Recent kinetic studies with E6AP (UBE3A), the founding member of this superfamily, demonstrate the ligase possesses two binding sites for E2-125I-ubiquitin thiolester substrate: one site from which the initial HECT domain-ubiquitin thiolester is formed (Site 1) and one required for subsequent polyubiquitin chain elongation (Site 2) prior to stochastic transfer to target protein. HERC5 is a Type1 interferon-induced HECT ligase having an essential role in cellular innate immune response to viral and inflammatory signals. Recent studies suggest HERC5 is the principal ligase for conjugation of interferon-induced ISG15 ubiquitin-like protein to a broad subset of cellular targets. Exploiting the intrinsic ability of ligases to form polyubiquitin chains in the absence of target proteins, we now show that HERC5 has intrinsic ubiquitin ligase activity and exhibits a broad specificity for members of the Ubc2-and Ubc4/5-like E2 clades in a semiquantitative screen of 125I-polyubiquitin chain formation. Detailed kinetic studies show cooperative sigmoidal kinetics with respect to [E2-125-ubiquitin thiolester]0 , as evidenced by linear Hill plots (n~2). These studies show HERC5 is specific for the Ubc5 family of E2 carrier proteins, which possess similar kinetics (Ka.?300 nM, kcar 0.05 s·\ Less active E2 paralogs exhibited hyperbolic kinetics in parallel assays. The inactive Ubc5A-ubiquitin oxyester substrate analog was an activator at low concentrations, consistent with cooperativity, but an inhibitor at high concentrations (K1 116 nM). Isotope dilution assays of 125I-wild type polyubiquitin chain formation using unlabeled K>R point mutants at each of the seven ubiquitin lysines showed that HERC5 forms K48-linked chains capable for targeting proteins for 26S proteasome-dependent degradation. Together with early observations that HERC5 binds polysomes and target nascent polypeptides for ISG15 conjugation, the present studies suggest interferon induction of HERC5 functions to upregulate ubiquitin-dependent antigen presentation (Funded by GM34009, GM47426 to A.L.H.).
Recommended Citation
Ronchi, Virginia P.; Streich, F.; LeCompte, K.; and Haas, A. L., "THE HECT DOMAIN LIGASE HERC5 EHIBITS INTRINSIC UBIQUITIN LIGASE ACTIVITY: IMPLICATIONS FOR THE TYPE1 INTERFERON RESPONSE" (2010). Dr. Joseph M. Moerschbaecher, III Graduate Research Day. 20.
https://digitalscholar.lsuhsc.edu/grad_rs/2010/poster1/20
THE HECT DOMAIN LIGASE HERC5 EHIBITS INTRINSIC UBIQUITIN LIGASE ACTIVITY: IMPLICATIONS FOR THE TYPE1 INTERFERON RESPONSE
The HECT domain ubiquitin ligases share 350 residue C-terminal domain containing an active cysteine to which is formed a ubiquitin thiolester intermediate in target protein conjugation. Recent kinetic studies with E6AP (UBE3A), the founding member of this superfamily, demonstrate the ligase possesses two binding sites for E2-125I-ubiquitin thiolester substrate: one site from which the initial HECT domain-ubiquitin thiolester is formed (Site 1) and one required for subsequent polyubiquitin chain elongation (Site 2) prior to stochastic transfer to target protein. HERC5 is a Type1 interferon-induced HECT ligase having an essential role in cellular innate immune response to viral and inflammatory signals. Recent studies suggest HERC5 is the principal ligase for conjugation of interferon-induced ISG15 ubiquitin-like protein to a broad subset of cellular targets. Exploiting the intrinsic ability of ligases to form polyubiquitin chains in the absence of target proteins, we now show that HERC5 has intrinsic ubiquitin ligase activity and exhibits a broad specificity for members of the Ubc2-and Ubc4/5-like E2 clades in a semiquantitative screen of 125I-polyubiquitin chain formation. Detailed kinetic studies show cooperative sigmoidal kinetics with respect to [E2-125-ubiquitin thiolester]0 , as evidenced by linear Hill plots (n~2). These studies show HERC5 is specific for the Ubc5 family of E2 carrier proteins, which possess similar kinetics (Ka.?300 nM, kcar 0.05 s·\ Less active E2 paralogs exhibited hyperbolic kinetics in parallel assays. The inactive Ubc5A-ubiquitin oxyester substrate analog was an activator at low concentrations, consistent with cooperativity, but an inhibitor at high concentrations (K1 116 nM). Isotope dilution assays of 125I-wild type polyubiquitin chain formation using unlabeled K>R point mutants at each of the seven ubiquitin lysines showed that HERC5 forms K48-linked chains capable for targeting proteins for 26S proteasome-dependent degradation. Together with early observations that HERC5 binds polysomes and target nascent polypeptides for ISG15 conjugation, the present studies suggest interferon induction of HERC5 functions to upregulate ubiquitin-dependent antigen presentation (Funded by GM34009, GM47426 to A.L.H.).