cul5 rbx2 complex  (Bio-Techne corporation)

Journal: Nature

Article Title: The CRL5–SPSB3 ubiquitin ligase targets nuclear cGAS for degradation

doi: 10.1038/s41586-024-07112-w

Figure Lengend Snippet: a , Representative image sequence demonstrating cGAS chromosome attachment in mitosis followed by intranuclear redistribution and degradation in cGAS–GFP-expressing HeLa cells (Supplementary Video ). Scale bars, 10 μm. b , The relative nuclear cGAS–GFP mean fluorescence intensity (MFI) in post-mitotic HeLa cells. n = 29. c , QIBC analysis of endogenous nuclear (left) and cytosolic (right) cGAS levels in HeLa cells. d , The relative nuclear cGAS–GFP MFI in post-mitotic HeLa cells that were treated with epoxomicin ( n = 31), bortezomib ( n = 30) or DMSO ( n = 31). e , Focused RNAi-based screen of UPS factors regulating nuclear cGAS–GFP abundance. Each dot shows the cGAS–GFP mean intensity blotted against integrated intensity. Knockdowns of genes that yielded increased cGAS–GFP levels (>3 s.d.) relative to the control siRNA are shown in yellow. Genes encoding cGAS (dark blue), proteasomal subunits (light blue), CRL5 complex components (dark green) and SPSB3 (light green) are highlighted. f , Schematic of CRL5–SPSB3-directed cGAS ubiquitylation. g , The relative nuclear cGAS–GFP MFI in post-mitotic HeLa cells treated with siRNA against SPSB3 (siS PSB3 ; n = 18) or CUL5 ( n = 14), non-targeting control siRNA ( n = 16) or epoxomicin ( n = 14). h , QIBC analysis of nuclear cGAS levels in HeLa cells that were treated with siRNA against SPSB3 or CUL5 or control siRNA. i , j , Nuclear (Nuc.) and cytosolic (Cyto.) cGAS–GFP was analysed using immunoblotting in cGAS–GFP-expressing HeLa cells that were treated with siRNA against SPSB3 or control siRNA ( i ) or MLN4924 ( j ) and incubated with cycloheximide (CHX) for the indicated time courses. H2B and GAPDH were used as the loading controls. For b , d and g , data are mean ± s.d. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Šídák’s multiple-comparison test ( c and h ) and two-way ANOVA with Tukey’s multiple-comparison test ( d and g ). For a , i and j , one representative experiment of three independent experiments is shown.

Article Snippet: Recombinant proteins required for in vitro ubiquitylation assays were expressed in BL21(DE3) cells, except for UBE1 and the neddylated CUL5–RBX2 complex (purchased from Bio-Techne, E-305-025 and E3-451-025).

Techniques: Sequencing, Expressing, Fluorescence, Western Blot, Incubation, Comparison

Journal: Nature

Article Title: The CRL5–SPSB3 ubiquitin ligase targets nuclear cGAS for degradation

doi: 10.1038/s41586-024-07112-w

Figure Lengend Snippet: a , Results from the siRNA screen highlighting cGAS-GFP nuclear abundance by mean fluorescence intensity and integrated fluorescence intensity in cells treated with siRNAs against SPSB family members ( n = 3). b , Nuclear cGAS measurements by confocal microscopy in HeLa cells (endogenous cGAS) or U2OS cells (cGAS-GFP expression) transfected with siRNAs against CUL5 , SPSB3 and PSMA5 or control siRNA or treated with epoxomicin or DMSO ( n = 2). c , Relative nuclear cGAS-GFP MFI measurement in post-mitotic U2OS cells treated with siRNA against SPSB3 ( n = 15) or CUL5 ( n = 15), control siRNA ( n = 14) or epoxomicin ( n = 15). d , Nuclear cGAS-GFP measurements by confocal microscopy in HeLa cells treated with siRNA against SPSB3 ( n = 31) or CUL5 ( n = 33) or control siRNA ( n = 22). e , Whole cell lysates, cytosolic and nuclear fractions were extracted from HeLa cells transfected with non-targeting control siRNA of siRNAs targeting SPSB3 or CUL5 and analysed by immunoblot. Vinculin and H2B were used as loading control of whole cell lysates, cytosolic and nuclear fractions, respectively. f , mRNA levels of cGAS were measured by RT-qPCR in HeLa cells treated with non-targeting control siRNA or siRNAs against SPSB3 or CUL5 for 5 days. Ratios of relative cGAS mRNA levels normalized to the control are shown ( n = 3). g , QIBC analysis of endogenous cytosolic cGAS level in HeLa cells treated with siRNA against SPSB3 , CUL5 or control siRNA. h , Nuclear cGAS-GFP measurements by confocal microscopy in HeLa cells treated with epoxomicin ( n = 32), MLN4924 ( n = 29), or DMSO ( n = 32). i , QIBC analysis of endogenous cGAS levels in HeLa cells treated with MLN4924 or DMSO. j , mRNA levels of cGAS were measured by RT-qPCR in HeLa cells treated with MLN4924 or DMSO. Ratios of relative cGAS mRNA levels normalized to the control are shown ( n = 6). k , Cytosolic and nuclear fractions were extracted from HeLa cells expressing doxycycline (Dox)-inducible SPSB3 that were treated or not with Dox for 4 days). Immunoblots probing cGAS and SPSB3 (FLAG) are shown. Vinculin and H2B were used as loading control for cytosolic and nuclear fractions, respectively. l , Whole cell lysates and nuclear fractions collected from primary endothelial cells, BJ-5ta fibroblasts or differentiated THP-1 cells treated with non-targeting control siRNA or siRNA against SPSB3 for 5 days were analysed by immunoblot. Vinculin and H2B were used as loading control for whole cell lysates and nuclear fractions, respectively. Numbers indicate individual cells ( c , d , h ) or technical replicates( a , f , j ). Data are mean ± SD ( a , c , d , f , g , h , j ) or mean ± SEM ( i ). P values were obtained by two-way ANOVA with Tukey’s multiple comparison test ( c ), one-way ANOVA with Dunnett’s multiple comparison test ( d , h ), one-way ANOVA ( f ), one-way ANOVA with Šídák’s multiple comparison test ( g ) or two-tailed Student’s t -test ( j ). One representative of three ( e , f , k - l ) independent experiments is shown.

Article Snippet: Recombinant proteins required for in vitro ubiquitylation assays were expressed in BL21(DE3) cells, except for UBE1 and the neddylated CUL5–RBX2 complex (purchased from Bio-Techne, E-305-025 and E3-451-025).

Techniques: Fluorescence, Confocal Microscopy, Expressing, Transfection, Western Blot, Quantitative RT-PCR, Comparison, Two Tailed Test

Journal: Nature

Article Title: The CRL5–SPSB3 ubiquitin ligase targets nuclear cGAS for degradation

doi: 10.1038/s41586-024-07112-w

Figure Lengend Snippet: a , IP of Flag-tagged cGAS from HEK293T cells transfected with cGAS-Flag and HA-ubiquitin and treated with indicated siRNAs. Samples were analysed by immunoblot. Vinculin was used as a loading control. b , c , Representative confocal microscopy images of HeLa cells stained for SPSB3 ( b ) or CUL5 ( c ) and DAPI (blue). Scale bars, 20 μm. d , QIBC analysis of endogenous nuclear (left) or cytosolic (right) SPSB3 levels in HeLa cells. e , IP of GFP-tagged NLS-cGAS from HEK293T cells transfected with constructs for GFP-tagged NLS-cGAS, HA-tagged ubiquitin with or without SPSB3. Samples were analysed by immunoblot. f , Size-exclusion chromatography (SEC) and SDS–PAGE of an assembled Halo-cGAS-SPSB3-ELOBC complex. g , SEC and SDS–PAGE of an assembled cGAS-SPSB3-ELOBC complex. Data are mean ± SD ( d ). P values were obtained by one-way ANOVA with Šídák’s multiple comparison test ( d ). One representative of three ( a - d ) or two ( e - g ) independent experiments is shown.

Article Snippet: Recombinant proteins required for in vitro ubiquitylation assays were expressed in BL21(DE3) cells, except for UBE1 and the neddylated CUL5–RBX2 complex (purchased from Bio-Techne, E-305-025 and E3-451-025).

Techniques: Transfection, Western Blot, Confocal Microscopy, Staining, Construct, Size-exclusion Chromatography, SDS Page, Comparison

Journal: Nature

Article Title: The CRL5–SPSB3 ubiquitin ligase targets nuclear cGAS for degradation

doi: 10.1038/s41586-024-07112-w

Figure Lengend Snippet: a , A composite cryo-EM density map of the nucleosome–cGAS–SPSB3–ELOBC complex, assembled from two focused-refinement maps (nucleosome–cGAS and cGAS–SPSB3–ELOBC). Different contour levels were used for optimal visualization using UCSF ChimeraX . cGAS(site C), mutated in the DNA-binding C site, was used to obtain this dataset. b , Ribbon representation of the nucleosome–cGAS–SPSB3–ELOBC complex structure. The arrows indicate the B30.2/SPRY and SOCS box domains of SPSB3. c , Detailed view of the binding interface between cGAS and SPSB3. d , Sequence logo analysis of the cGAS and SPSB3 interface derived from 150 vertebrate species. e , Model of nucleosome-bound cGAS targeted by an activated (neddylated) CRL5–SPSB3 complex with RBR E3 ligase ARIH2 priming polyubiquitylation by transferring the first ubiquitin onto cGAS. Lysine residues that have been identified as cGAS ubiquitylation sites by MS and are essential for SPSB3-mediated degradation of cGAS are coloured red; the catalytic Cys310 of ARIH2 is coloured gold. The model was built by docking the nucleosome–cGAS–SPSB3–ELOBC model into a model comprising the ELOBC–CUL5 (Protein Data Bank (PDB): 4JGH ) , CUL5–NEDD8–RBX2–ARIH2 (PDB: 7ONI ) and ARIH1–UB (PDB: 7B5M ) complexes.

Article Snippet: Recombinant proteins required for in vitro ubiquitylation assays were expressed in BL21(DE3) cells, except for UBE1 and the neddylated CUL5–RBX2 complex (purchased from Bio-Techne, E-305-025 and E3-451-025).

Techniques: Cryo-EM Sample Prep, Binding Assay, Sequencing, Derivative Assay, Transferring

Journal: Nature

Article Title: The CRL5–SPSB3 ubiquitin ligase targets nuclear cGAS for degradation

doi: 10.1038/s41586-024-07112-w

Figure Lengend Snippet: a , Right: Ribbon diagram and a 3D reconstruction of the nucleosome-cGAS siteC -SPSB3-ELOBC complex. cGAS in blue, SPSB3 in green, nucleosomal DNA in lilac, histones in grey, ELOB in yellow, ELOC in antique white. Left: EM densities (shown as mesh at 5σ) for cGAS residues interacting with the SPSB3. Shown are cGAS N513, N514 and R512/D465 interactions with SPSB3 respectively. b , The cGASSPSB3 model is aligned with the VASA-SPSB1 model (PDB: 3F2O) , individual interacting residues are shown by sticks. c , Sequence alignment of SPSB1/2/3/4. Residues used for substrate recognition are highlighted. d , Percent identity matrix of human SPSB1/2/3/4. e , Model of the nucleosome/cGAS 2:2 sandwich targeted by an activated (neddylated) CRL5-SPSB3 complex with RBR E3 ligase ARIH2 priming polyubiquitylation by transferring the first ubiquitin onto cGAS. cGAS K427/K428 is competitively targeted by either nucleosomal DNA or the E3 ligase. The model is built by docking the nucleosome-cGAS-SPSB3-ELOBC model into a model composed of the nucleosome/cGAS 2:2 complex (PDB: 6Y5D) , ELOBC-CUL5 (PDB: 4JGH) , CUL5-NEDD8-RBX2-ARIH2 (PDB: 7ONI) , and ARIH1-UB (PDB: 7B5M) complexes.

Article Snippet: Recombinant proteins required for in vitro ubiquitylation assays were expressed in BL21(DE3) cells, except for UBE1 and the neddylated CUL5–RBX2 complex (purchased from Bio-Techne, E-305-025 and E3-451-025).

Techniques: Sequencing, Transferring

Journal: Nature

Article Title: The CRL5–SPSB3 ubiquitin ligase targets nuclear cGAS for degradation

doi: 10.1038/s41586-024-07112-w

Figure Lengend Snippet: a , mRNA levels of cGAS were measured by RT-qPCR in HeLa cGAS KO cells reconstituted with cGAS (WT), cGAS(NN), or cGAS(KK). Ratios relative to WT levels are shown ( n = 3). b , Expression levels of cGAS , ISG15 or IFNB1 were assessed by RT-qPCR in HeLa cGAS KO cells reconstituted with doxycycline-inducible cGAS (WT) or a cGAS mutant defective in DNA binding and ubiquitylation (KRKKNN (K173E/R176E/K407E/K411A/N513/514A)) after 4 days of doxycycline treatment. Ratios of relative cGAS , ISG15 and IFNB1 mRNA levels normalized to the control are shown ( n = 3). c , Cells from ( b ) were lysed and analysed by immunblot for cGAS and ISG15 levels. Vinculin was used as a loading control. d , cGAS and ISG15 measurements by immunoblot in HeLa cGAS KO cells transfected with mRNA of encoding for human cGAS or cGAS N513A/N514A (NN). Cells were harvested on day 0, 1, 2, or 4 after transfection. Vinculin was used as a loading control. e , cGAS and ISG15 measurements by immunoblot in CT26 cGas KO cells transfected with mRNA of encoding for mouse cGas or cGAS N498A/N499A (NN). Cells were harvested on day 0, 1, 2, or 4 after transfection. Vinculin was used as a loading control. f , Expression of SPSB3 , ISG15 or IFNB1 were assessed by RT-qPCR 3 days after enforced expression or not of SPSB3 with doxycycline in HeLa cells. Ratios of relative SPSB3 , ISG15 and IFNB1 mRNA levels normalized to the control are shown ( SPSB3 , n = 3; ISG15 , IFNB1 , n = 4). g - i , mRNA expression levels of genes as indicated were assessed by RT-qPCR in primary endothelial cells ( n = 3) ( g ), BJ-5ta fibroblast cells ( n = 3) ( h ), and differentiated THP-1 cells ( n = 3) or THP-1 cGAS KO cells ( n = 3) ( i ) treated with non-targeting control siRNAs or siRNAs against SPSB3 for 5 days. Ratios of the relative expression of each transcript compared to the controls are shown. j , Induction of IFNB1 mRNA was measured by RT-qPCR in HeLa cGAS KO cells reconstituted with cGAS (WT), cGAS(NN), or cGAS(KK) infected with HSV-1 (MOI = 0.1) or VACV (MOI = 1) for 18 h ( n = 4). k , HeLa cells treated with non-targeting control siRNAs or siRNAs against SPSB3 or CUL5 for 5 days were infected with HSV-1-GFP (left, MOI = 0.01) or VACV-GFP (right, MOI = 0.5) for 24 h ( n = 3). GFP + cells were quantified by flow cytometry. Data are mean ± SD, numbers indicate the number of technical replicates ( a - b , f - k ). P values were obtained by one-way ANOVA ( a , f , g - k ) or two-tailed Student’s t -test ( b , f ). One representative of two ( a ) or three ( b - k ) independent experiments is shown.

Article Snippet: Recombinant proteins required for in vitro ubiquitylation assays were expressed in BL21(DE3) cells, except for UBE1 and the neddylated CUL5–RBX2 complex (purchased from Bio-Techne, E-305-025 and E3-451-025).

Techniques: Quantitative RT-PCR, Expressing, Mutagenesis, Binding Assay, Western Blot, Transfection, Infection, Flow Cytometry, Two Tailed Test