Abstract
E1 and E2 enzymes coordinate the first steps in conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubls). ISG15 is an interferon-alpha/beta-induced Ubl, and the E1 and E2 enzymes for ISG15 conjugation are Ube1L and UbcH8, respectively. UbcH7 is the most closely related E2 to UbcH8, yet it does not function in ISG15 conjugation in vivo, while both UbcH7 and UbcH8 have been reported to function in Ub conjugation. Kinetic analyses of wild-type and chimeric E2s were performed to determine the basis for preferential activation of UbcH8 by Ube1L and to determine whether UbcH8 is activated equally well by Ube1L and E1(Ub) (Ube1). K(m) determinations confirmed the strong preference of Ube1L for UbcH8 over UbcH7 (a 29-fold K(m) difference), similar to the preference of E1(Ub) for UbcH7 over UbcH8 (a 36-fold K(m) difference). Thioester assays of chimeric E2s identified two structural elements within residues 1-39 of UbcH8 that play a major role in defining Ube1L-UbcH8 specificity: the alpha1-helix and the beta1-beta2 region. The C-terminal ubiquitin fold domain (UFD) of Ube1L was required for transfer of ISG15 to UbcH8 and for binding of Ube1L to UbcH8. Replacement of the Ube1L UFD with that from E1(Ub) resulted in preferential transfer of ISG15 to UbcH7. Together, these results indicate that Ube1L discriminates between UbcH8 and closely related Ub E2s based on specific interactions between the Ube1L UFD and determinants within the N-terminal region of UbcH8.
Highlights
Cooperatively in reactions that involve enzyme-bound thioester intermediates [1]
UbcH8 has been reported in several cases to function in Ub conjugation pathways (10 –13), often in a manner that is redundant with UbcH7 (5, 14 –20), suggesting that UbcH8 might function in both the Ub and ISG15 conjugation systems
E1Ub was found to discriminate against activation of UbcH8 to a similar degree as Ube1L discriminated against UbcH7, suggesting that UbcH8 may be limited in its capacity to function in Ub conjugation in vivo
Summary
Plasmids and Mutagenesis—Plasmids containing Ube1L, UbcH8, Herc, and ISG15 were described previously [5, 7, 9, 27]. E2 so that the E1 concentration and incubation time resulted in linear product formation, where less than 10% of the E2 was converted to E2ϳUbl. Preliminary Km assays using 0.23 nM E1Ub/3.4 nM Ube1L, and the proper incubation time were performed to determine the appropriate range of E2 concentrations for each wild-type or chimeric E2 protein. Plasmids and in vivo [11], while only UbcH8 functions in ISG15 conjugaexpressing Herc (0.5 g), 3ϫ FLAG-ISG15 (0.5 g), and UbcH8 tion in vivo [5] To determine if these results are consistent with (.25 g) were transfected with HA-Ube1L, HA-Ube1L⌬UFD Similar results were seen with antibody (Sigma) to detect ISG15-conjugated proteins and ISG15 thioester assays, where UbcH8 activation was detected at anti-HA antibody (Covance) to detect E1 expression
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
