Fig. 2: BI8626 ubiquitination requires the catalytic cascade, including HUWE1HECT.
From: Selective ubiquitination of drug-like small molecules by the ubiquitin ligase HUWE1
a SEC-based fractionation of HUWE1HECT-driven ubiquitination reactions ± ATP, without inhibitor (top). The assignment of the elution peaks is based on SDS-PAGE analyses, as shown in Supplementary Fig. 9a, b. The asterisk marks buffer components. SDS-PAGE analyses of the indicated elution fractions, focusing on the Ub-sized bands (bottom). b SEC-based fractionation of HUWE1HECT-driven ubiquitination reactions ± ATP in the presence of 20 μM BI8626 (top). SDS-PAGE analyses of the indicated elution fractions, focusing on the Ub-sized bands (bottom). Note that additional, delayed Ub-containing fractions are observed in the presence of ATP, which are not seen in inhibitor-free conditions. For the full SDS-PAGE, see Supplementary Fig. 9d. The lower benzyl moiety of BI8626, including the critical primary amino group is shown. Intact protein MS (c) and deconvoluted neutral MW spectrum (d) of the major BI8626-containing elution peak (orange; V ~ 1.8 mL) from (b). The calculated molecular weight (MW) of BI8626-Ub is specified. Intact protein MS (e) and deconvoluted neutral MW spectrum (f) of the minor BI8626-containing elution peak (orange; V ~ 1.4 mL) from (b). g SEC-based fractionation (top) and SDS-PAGE analysis (bottom) of a HUWE1HECT-driven ubiquitination reaction, containing 20 μM derivative 1, analogous to (b). The lower benzyl moiety of derivative 1, including the critical amino group is shown. h SEC-based fractionation (top) and SDS-PAGE analysis (bottom) of a HUWE1HECT-driven ubiquitination reaction, containing 20 μM derivative 2, analogous to (b). The lower benzyl moiety of derivative 2 that does not contain a primary amino group is shown. i SEC-based fractionation (top) and SDS-PAGE analysis (bottom) of a HUWE1HECT-driven ubiquitination reaction, containing 20 μM BI8626, analogous to (b), but employing UbΔGG instead of WT Ub. j SEC-based fractionation of two independent HUWE1HECT-driven ubiquitination reactions, containing 20 μM BI8626, analogous to (b), but devoid of the E1 or E2. k SEC-based fractionation (top) (bottom) of two independent ubiquitination reactions, containing 20 μM BI8626, analogous to (b), but devoid of HUWE1HECT or employing a catalytically inactive HUWE1HECT variant (C4341A) instead of the WT. Source data are provided as a Source Data file.
