Journal: Nature Communications

Article Title: Structural basis for substrate recognition and chemical inhibition of oncogenic MAGE ubiquitin ligases

doi: 10.1038/s41467-020-18708-x

Figure Lengend Snippet: a Sequence conservation of MAGE-A genes mapped onto MAGE-A11:PCF11 structure. b Comparison of MAGE-A3 and -A11 SBC reveals key sequence differences in critical substrate binding residues. c In vitro GST-pulldown assays reveals that GST-PCF11 binds in vitro translated Myc-MAGE-A11, but no other Myc-MAGE-A protein. Source data are provided as a Source Data file. d – g Mutation of MAGE-A3 or -F1 SBC disrupts binding to their respective substrates AMPK and MMS19, not the E3 ligases TRIM28 and NSE1. Surface representations of MAGE-A3 (protomer model formed by PDB ID 4V0P for residues 1-296 and MAGE-A11 based homology model for C-terminal residues 297–311) ( d ) and MAGE-F1 (SWISS-MODEL based on PDB:4V0P) ( f ) show positioning of SBC residues mutated. Consistent with Fig. , MAGE atoms within 5 Å of modeled PCF11 (based on superimposition of MAGE-A11:PCF11) are colored cyan. Residues in the SBC that are mutated are labeled and colored yellow. Co-IP of MAGEs with substrates and ligases were determined in HeLa cells ( e ) or HEK293FT cells ( g ) upon transfection of the indicated constructs for 48 h, IP with anti-Myc, SDS-PAGE, and immunoblotting for indicated proteins. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies were: anti-Myc (Sigma, #C3956), anti-FLAG (Sigma, #F3165), anti-HUWE1 (Novus Biologicals, #NB 100-652), anti-PCF11 (Bethyl Laboratories, #A303-706A), anti-GAPDH (Cell Signaling Technology, #2118), anti-TRIM28 (Abcam, #ab22553), anti-AMPKα1 (Cell Signaling Technology, #2795), and anti-MMS19 (Proteintech, #16015-1-AP) and Tb-anti-GST (Invitrogen, #PV3551).

Techniques: Sequencing, Binding Assay, In Vitro, Mutagenesis, Labeling, Co-Immunoprecipitation Assay, Transfection, Construct, SDS Page, Western Blot

Journal: Nature Communications

Article Title: Structural basis for substrate recognition and chemical inhibition of oncogenic MAGE ubiquitin ligases

doi: 10.1038/s41467-020-18708-x

Figure Lengend Snippet: a 500 analogs from 9 scaffolds were tested in dose response TR-FRET assays for disruption of MAGE-A11 binding to PCF11 identified the quinoline scaffold. b Chemical structure of SJ521054 hit. c TR-FRET competition assay using indicated concentrations of quinoline series compounds for disruption of MAGE-A11:PCF11 binding with n = 2 biologically independent experiments with triplicates per compound. Data are mean ± SD. d SJ521054 blocks PCF11 degradation by MAGE-A11. HEK293FT cells stably expressing FLAG-vector or FLAG-MAGE-A11 were treated with indicated concentrations of SJ521054 for 24 h before immunoblotting. Source data are provided as a Source Data file. e SJ521054 reduces viability of MAGE-A11 expressing DAOY cancer cells. MAGE-A11-knockout DAOY cells or those reconstituted with MAGE-A11 were treated with indicated concentrations of SJ521054 and cell viability was measured by alamarBlue assay 24 h later ( n = 3 biologically independent experiments per group). Data are mean ± SD. Black circle, Vector; Red square, MAGE-A11 WT #1; and Blue triangle, MAGE-A11 WT #2. f Derivatives of SJ521054 were synthesized and tested by TR-FRET competition assays with SJ1008066 showing improved activity ( n = 2 biologically independent experiments with triplicates per compound). Data are mean ± SD. g Direct ligand binding of S J521054 and SJ1008066 were evaluated using NanoDSF using the intrinsic fluorescence of His-SUMO-MAGE-A11 MHD at 350 nm and 330 nm with n = 3 biologically independent experiments per compound. Both compounds showed stabilization of MAGE-A11 in a six-point dose response confirming on-target activity of each compound. Data are mean ± SD. Blue circle, SJ521054 and red square, SJ1008066 . h SJ521054 and SJ1008066 inhibit MAGE-A11 binding to PCF11 in cell lysate. FLAG-MAGE-A11 stably expressing HEK293FT cell lysates were incubated with 100 µM of each compound for 24 h and then subjected to pull-down with anti-FLAG followed by SDS-PAGE and immunoblotting for endogenous PCF11, and quantitated ( n = 4 for SJ521054 , n = 3 for SJ1008066 , biologically independent experiments per compound, two-tailed unpaired Student’s t test, *** P < 0.001). Data are mean ± SEM. Source data are provided as a Source Data file. i SJ521054 and SJ1008066 do not disrupt interaction of MAGE-A3:AMPKα1 or MAGE-F1:MMS19 in cell lysate. HeLa cells or HEK293FT cells were transfected with the indicated constructs for 48 h and cell lysates were incubated with 100 µM of each compound for 24 h and then subjected to IP with anti-Myc followed by SDS-PAGE and immunoblotting for indicated proteins. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies were: anti-Myc (Sigma, #C3956), anti-FLAG (Sigma, #F3165), anti-HUWE1 (Novus Biologicals, #NB 100-652), anti-PCF11 (Bethyl Laboratories, #A303-706A), anti-GAPDH (Cell Signaling Technology, #2118), anti-TRIM28 (Abcam, #ab22553), anti-AMPKα1 (Cell Signaling Technology, #2795), and anti-MMS19 (Proteintech, #16015-1-AP) and Tb-anti-GST (Invitrogen, #PV3551).

Techniques: Binding Assay, Competitive Binding Assay, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Knock-Out, Alamar Blue Assay, Synthesized, Activity Assay, Ligand Binding Assay, Nano Differential Scanning Fluorimetry, Fluorescence, Incubation, SDS Page, Two Tailed Test, Transfection, Construct