Journal: bioRxiv

Article Title: Structural basis of NSD2 degradation via targeted recruitment of SCF-FBXO22

doi: 10.1101/2025.08.29.673087

Figure Lengend Snippet: (a) UNC10088 C -induced conformational change of the loop adjacent to C326. Structural overlay of the SCF FBXO22 -UNC10088 C -NSD2 PWWP1 model (dark gray) and the SCF FBXO22 -BACH1 BTB complex (light gray, PDB: 8UA3) , revealing the displacement of FBXO22 residues Y390. (b) Chemical structures of UNC8732 analogs that contain an aromatic aldehyde in place of the aliphatic amine of UNC8732. (c) Benzaldehyde derivatives UNC10415667 and UNC10415668 promote FBXO22-dependent ubiquitination of *NSD2 PWWP1 when added at 0.5 µM, based on fluorescent scanning of an SDS-PAGE gel. (d) Treatment of U2OS NSD2-HiBit cells with UNC10415667 (1 µM) for 6 hours resulted in a potent reduction of NSD2 levels by about 75%. A more modest effect was observed upon treatment with UNC10415668, and all other compounds had no significant effect. n ≥ 3 independent experiments. Error bars: standard error of the mean. (e) Treatment of U2OS NSD2 HiBit cells with UNC10415667 in a dose response fashion revealed a DC 50 value of 0.46 µM, which is about 2-fold more potent than UNC8732 and UNC10088. n = 3 independent experiments. Error bars: standard error of the mean. (f) UNC10415667 uses C326 to recruit NSD2 to the FBXO22 FIST domain as monitored by size-exclusion chromatography. Chromatograms of different elution profiles between mixtures of WT FBXO22 FIST or a C326A mutant with NSD2 and UNC10415667 (top). Representative Coomassie-stained SDS-PAGE gels of collected fractions. (g) Differential scanning fluorimetry (DSF) was used to monitor the stability of FBXO22 FIST wild type and variants in the presence of UNC10088 or UNC10415667 compared to DMSO, revealing that both compounds stabilize WT FBXO22 FIST and the C326 single cysteine variant (C326 only) as evidenced by an increase in melting temperature (ΔT m ), but not the C326A mutant. n = 3 independent experiments. Error bars: standard error of the mean.

Article Snippet: 9.5 μL of the master mix containing FBXO22 FIST WT or variants, SYPRO Orange Dye (Invitrogen), and DSF buffer (20 mM Tris 7.5, 150 mM NaCl, and 1 mM TCEP) were added to a 384-well qPCR plate (Genesee Scientific).

Techniques: Ubiquitin Proteomics, SDS Page, Size-exclusion Chromatography, Mutagenesis, Staining, Variant Assay