Extended Data Fig. 4: Aldehyde degraders bind FBXO22 in a cysteine 326-dependent manner.
From: Recruitment of FBXO22 for targeted degradation of NSD2
(a): Representative BLI sensorgrams upon the addition of increasing concentrations of UNC10088 and a fixed concentration of NSD2-PWWP1 (2 µM). WT or C326A-mutant SKP1-FBXO22 was loaded on SA biosensors to an average response of 1 nm. Curves are shown as the average of three independent experiments. (b): In vitro NSD2-PWWP1 ubiquitination with the indicated sets of substrate priming machinery, UBCH5B or UBCH7 with HHARI. Each reaction also contained CUL1-RBX1 and CDC34B. The reaction mixture was analyzed by SDS-PAGE and fluorescence scanning. NSD2-PWWP1 was subject to a sortase reaction for fluorescent labeling. The gel presented is representative of three independent experiments. (c): Immunoblotting following transfection of empty vector pcDNA, WT FBXO22-HT, or C326A-mutant FBXO22-HT in U2OS cells for 24h. (d): Experimental flow chart for the designed ‘wash’ experiment to test for reversibility of the compound-FBXO22 interaction. *0 hour denotes the estimated concentration based on the series of dilutions performed. (e): Normalized fluorescence data from DSF for samples obtained from the experiment described in panel E. The data represents the mean ± SD of 3 technical replicates from one independent experiment. (f): Schematic of the reaction between the aldehyde-containing degrader compound and the C326 of FBXO22. (g): Percent Deuterium Exchange of C326-spanning peptides of co-purified SKP1/FBXO22 in the presence (orange) and absence (black) of UNC10088 over 12 hours. The data represents the mean ± 3σ (3 × SD) of a triplicate technical measurement (n=3).
