Fig 2: Validating target engagement and selectivity of EN67 in cells. (A) Synthetic route for making the alkyne-functionalized probe of EN67. (B) Gel-based ABPP of alkyne-functionalized EN67 probes against pure recombinant human UBE2D2 C85S protein and corresponding silver staining. (C) NF363C engagement and enrichment of UBE2D2 in HEK293T cells. Cells were treated with dimethyl sulfoxide (DMSO) or NF36C (50 μM) for 24 h. Probe-modified proteins were subsequently appended with an azide-functionalized biotin handle by CuAAC, and proteins were avidin-enriched and eluted for detection of UBE2D2 and an unrelated negative control protein GAPDH by Western blotting. Both input and pulldown of UBE2D2 and GAPDH are shown. (D) isoDTB-ABPP cysteine chemoproteomic profiling of EN67 in HEK293T cells. HEK293T cells were treated with DMSO vehicle or EN67 (50 μM) for 4 h. Lysates were labeled with IA-alkyne (200 μM) for 1 h, and isotopic desthiobiotin tags were appended by CuAAC and taken through the isoDTB-ABPP procedure. Shown are ratios of control/EN67-treated probe-modified peptide ratios and adjusted p-values from n = 3 biological replicates/group. Data are shown in Table S2. Data in panels (B) and (C) are representative of n = 3 biological replicates/group.

Fig 3: Discovering a covalent recruiter for E2 ubiquitin conjugating enzyme UBE2D. (A) Gel-based ABPP screen of cysteine-reactive covalent ligands against human pure UBE2D2 C85S protein showing EN67 as the top hit. (B) Structure of EN67 with the cysteine-reactive acrylamide warhead highlighted in red. Gel-based ABPP showing competition of EN67 against IA-rhodamine binding to UBE2D2 C85S pure protein and silver staining of protein showing equal protein loading. (C) TP53 ubiquitination activity by E1 UBE1, E2 UBE2D2, E3 MDM2, FLAG-ubiquitin, and ATP showing that EN67 does not inhibit TP53 ubiquitination activity. (D) Mapping of EN67 site of modification on human pure UBE2D2 C111 by LC-MS/MS. Experiments in (B), (C), and (D) are representative of n = 3 biological replicates/group.

Fig 4: Phosphorylation of Trim25 at S100 is required for ITPKB ubiquitination. a Phosphorylation proteomics analysis of Trim25 in primary and recurrence GBM. b The phosphorylation level of Trim25 S100 site in GBM tissue. c Dephosphorylation of the E3 ligase Trim25 resulted in a more pronounced alleviation of ITPKB ubiquitination in a cell-free system compared to mock-treated Trim25. Dephosphorylated or mock-treated Trim25 was incubated with immunopurified Flag-ITPKB, ubiquitin, recombinant E1 (Uba1), and E2 (UbcH5b). ITPKB ubiquitination was then determined using a ubiquitination assay. d The interaction between ITPKB and Trim25 was affected by the Trim25 S100D phosphomimetic mutant and S100A dephosphorylation mutant. e The ubiquitination of ITPKB was altered when the S100 site was mutated to Asp (S100D) or Ala (S100A). HEK293T cells were co-transfected with the respective plasmids and treated with MG132 for 3 h after 48 h of transfection. Cell lysates were immunoprecipitated and blotted with specific antibodies for analysis. Statistical significance is shown as: *p < 0.01