Journal: The EMBO Journal

Article Title: Shared and distinct mechanisms of UBA1 inactivation across different diseases

doi: 10.1038/s44318-024-00046-z

Figure Lengend Snippet: ( A ) Schematic overview of the sequential, three-phase in vitro assay used to measure UBA1 transthiolation. (i) Complete charging of UBA1 by incubation of 250 nM UBA1b with 10 μM FITC-ubiquitin and 5 mM ATP (ii) Quenching of UBA1 charging and single transfer to E2 enzyme by addition of 100 mM EDTA and 1 μM E2 enzyme (iii) Reactivation of UBA1 charging and multi-transfer to E2 enzyme by addition of 100 mM MgCl 2 ( B ) UBA1 WT was subjected to the experiment described in panel ( A ) using UBE2D3. Reactions were subjected to SDS page and analyzed by fluorescence imaging. Upper panel : Fluorescence scan showing UBA1 and UBE2D3 ubiquitin thioester levels after each reaction phase. Lower panel : UBE2D3 ubiquitin thioester levels in reaction phase (iii) were quantified and plotted against the reaction time, revealing that UBE2D3 is maximally charged after 60 min. ( C ) Non-canonical VEXAS mutations (red) are deficient in E2 transthiolation in vitro. Indicated UBA1 proteins were subjected to the experiment described in panel ( A ) using either UBE2D3, UBE2R2, or UBE2S. Reactions were subjected to SDS page and analyzed by fluorescence imaging. Upper panel : Fluorescence scan showing UBA1 charging after reaction phase (i) as control. Lower panel : Fluorescence scan showing UBA1 re-charging and E2 transfer after reaction phase (iii). Quantifications of 3 biological replicates are shown in Fig. . ( D ) Non-canonical VEXAS mutations (red) are impaired in supporting E2 ubiquitin thioester levels in cells. CHO ts20 cells were reconstituted with indicated UBA1 variants and incubated at the permissive temperature for 6 h, followed by immunoblotting using antibodies against indicated E2 enzymes. ( E – G ) Quantification of ubiquitin charging levels of indicated E2s (charged/total) shown in panel ( D ). n = 3 biological replicates, error bars = s.d., * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA.

Article Snippet: Alternatively, 10 µM FITC-ubiquitin was used in the assay and SDS-PAGE gels were analyzed by a fluorescence scanner (Chemidoc MP, Biorad).

Techniques: In Vitro, Incubation, SDS Page, Fluorescence, Imaging, Western Blot