Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet: ( A ) Schematic showing the design of dual-gRNA directed CRISPR screen of E2s regulating the CM-induced degradation of ePL-tagged IKZF1 in 384-well array format. ( B ) Chemiluminescent measurement of ePL-IKZF1 protein expression level in U937_Cas9_ePL-IKZF1 parental cells or cells expressing UBE2G1 -specfic sgRNA alone or in combination with non-targeting or UBE2D3 -specific sgRNA. Cells were treated with POM at the indicated concentrations for 16 hr. Data are presented as mean ± SD, n = 4 technical replicates. ( C ) Immunoblot analysis of U937-Cas9 parental cells or cells expressing non-targeting sgRNA, UBE2G1 -specific sgRNA, UBE2D3 -specfic sgRNA, or both UBE2G1 and UBE2D3 sgRNAs. Cells were treated with POM at the indicated concentrations for 16 hr. SE, short exposure; LE, long exposure. Result is representative of three independent experiments. 10.7554/eLife.40958.012 Figure 2—source data 1. ePL luminescence signal shown in .

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

Techniques: CRISPR, Expressing, Western Blot

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet: ( A ) Sequence alignment of human UBE2G1, human UBE2G2 and human CDC34 using Clustal W 2.1. The acidic loops indispensable for the assembly of K48-linked ubiquitin chains are highlighted with red, and the catalytic cysteines are highlighted with blue. ( B ) Immunoblot analysis of 293T parental or UBE2G1-/- cells transduced with lentiviral vectors expressing FLAG-tagged UBE2G1 wild-type or C90S mutant, or FLAG-tagged UBE2D3 wild-type or C85S mutant. Cells were treated with CC-885 at the indicated concentrations for 4 hr. Note that overexpression of wild-type FLAG-UBE2G1 or FLAG-UBE2D3 partially rescued the GSPT1 degradation defect caused by UBE2G1 deficiency, while overexpression of catalytically-dead mutant FLAG-UBE2G1-C90S or FLAG-UBE2D3-C85S further blocked the degradation of GSPT1. ( C ) In vitro ubiquitination of GSPT1 by CRL4 CRBN with or without CC-885 and indicated E2 variants. Consistent with results observed with bacterial recombinant UBE2G1 and UBE2D3 proteins, FLAG-UBE2G1 and FLAG-UBE2D3 proteins purified from human cells acted in concert to promote the ubiquitination of GSPT1. Results shown in ( B ) and ( C ) are representative of three independent experiments.

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

Techniques: Sequencing, Western Blot, Transduction, Expressing, Mutagenesis, Over Expression, In Vitro, Recombinant, Purification

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet: ( A– D ) In vitro ubiquitination of IKZF1 ( A and C ) and GSPT1 ( B and D ) MBP fusion proteins by recombinant CRL4 CRBN complex. Recombinant protein products as indicated were incubated with or without 80 µM POM ( A and C ) or 80 µM CC-885 ( B and D ) in the ubiquitination assay buffer containing 80 mM ATP at 30°C for 2 hr, and then analyzed by immunoblotting. ( E ) Sequential in vitro ubiquitination of GSPT1 by recombinant CRL4 CRBN complex. MBP-GSPT1 recombination protein was incubated with Ube1, UBE2D3, Cul4-Rbx1, DDB1-cereblon, Ubiquitin, ATP and CC-885 in the ubiquitination assay at 30°C for 4 hr. After purification over size-exclusion chromatography, pre-ubiquitinated MBP-GSPT1 protein was then incubated with Ube1, DDB1-cereblon, Ubiquitin, ATP and UBE2G1 with or without CC-885 or Cul4A-Rbx1 in the ubiquitination assay at 30°C for 2 hr, followed by immunoblot analysis. ( F ) Schematic showing the sequential ubiquitination of CRBN neomorphic substrates by UBE2D3 and UBE2G1. Results shown in ( A–E ) are representative of three independent experiments.

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

Techniques: In Vitro, Recombinant, Incubation, Ubiquitin Assay, Western Blot, Purification, Size-exclusion Chromatography

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet: ( A and B ) 293T parental and UBE2G1-/-;UBE2D3-/- ( clone 4) cells were transiently transfected with plasmids expressing cereblon, V5-tagged IKZF1 and 8xHis-Ub with or without UBE2G1, UBE2D3 or both. ( C ) 293T parental and UBE2G1-/- (clone 13) cells were transiently transfected with plasmids expressing cereblon, IKZF1-V5, 8xHis-Ub with or without UBE2G1 wild-type or C90S mutant. In ( A ), ( B ) and ( C ), 48 hr after transfection, cells were treated with MG-132 (10 µM) and POM at the indicated concentrations for additional 8 hr. Ubiquitinated protein products enriched with magnetic nickel sepharose were subjected to immunoblot analysis. Immunoblot analysis of whole cell extracts showing equal input proteins is shown in . All results shown in this figure are representative of three independent experiments.

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

Techniques: Transfection, Expressing, Mutagenesis, Western Blot

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet: ( A ) Total input for . Immunoblot analysis of 293T parental and UBE2G1-/-;UBE2D3-/- (Clone 4) cells transfected to produce 8xHis-Ubiquitin, cereblon and IKZF1-V5. ( B ) Total input for . Immunoblot analysis of 293T parental and UBE2G1-/-;UBE2D3-/- (Clone 4) cells transfected to produce 8xHis-Ubiquitin, CRBN, IKZF1-V5 with or without UBE2G1 and/or UBE2D3. ( C ) Total input for . Immunoblot analysis of 293T parental and UBE2G1-/- (Clone 13) cells transfected to produce 8xHis-Ubiquitin, CRBN, IKZF1-V5 with or without UBE2G1 wildtype or C90S mutant. In ( A ), ( B ) or ( C ), 48 hr after transfection, cells were treated with 10 µM MG-132 and POM at the indicated concentrations for additional 8 hr. All results shown in this figure are representative of three independent experiments.

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

Techniques: Western Blot, Transfection, Mutagenesis

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet: ( A ) Sequence alignment of human UBE2D family proteins using Clustal W 2.1. Note that the amino acid sequence identity among all four family proteins is close to 90%. ( B ) In vitro ubiquitination of GSPT1 MBP fusion protein by recombinant CRL4 CRBN complex in the presence of UBE2G1, UBE2D1, UBE2D2, or UBE2D3, alone or in combination. Recombinant protein products as indicated were incubated with or without 80 µM CC-885 in the ubiquitination assay buffer at 30°C for 2 hr, and then analyzed by immunoblotting. SE, short exposure; LE, long exposure. Result is representative of three independent experiments.

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

Techniques: Sequencing, In Vitro, Recombinant, Incubation, Ubiquitin Assay, Western Blot

Journal: eLife

Article Title: UBE2G1 governs the destruction of cereblon neomorphic substrates

doi: 10.7554/eLife.40958

Figure Lengend Snippet:

Article Snippet: Rabbit anti-human CRBN65 monoclonal antibody (mAb) (Celgene, San Diego, CA); rabbit anti-human GSPT1 polyclonal antibody (pAb; Abcam, #ab49878), rabbit anti-human IKZF1 mAb (Cell Signaling, #14859), rabbit anti-human IKZF3 mAb (Cell Signaling, #15103), rabbit anti-human CK1α pAb (Abcam, #ab108296), rabbit anti-human ZFP91 pAb (LifeSpan Biosciences, #LS-B14788), mouse anti-human UBE2G1 mAb (Santa Cruz, #SC-100619), rabbit anti-human UBE2G1 pAb (Abcam, #SC-101371), mouse anti-human UBE2D3 mAb (Abcam, #ab58251), rabbit anti-human UBE2D3 pAb (Sigma, #SAB2102622), rabbit anti-human Cul4A pAb (Cell Signaling, #2699), rabbit anti-human DDB1 pAb (Cell Signaling, #5428), rabbit anti-human Rbx1 pAb (Cell Signaling, #4397), rabbit anti-human Cdt1 mAb (Cell Signaling, #8064), rabbit anti-human Cdt2 pAb (Cell Signaling, #A300-948A), rabbit anti-human Set8 pAb (Cell Signaling, #2996), rabbit anti-human RBM39 pAb (Sigma, #HPA001591), rabbit anti-human p21 mAb (Cell Signaling, #2947), rabbit anti-human p27 mAb (Cell Signaling, #3686), rabbit anti-human c-Myc mAb (Cell Signaling, #5605), rabbit anti-human BRD4 pAb (Abcam, #ab128874), mouse anti-penta-HIS mAb (Qiagen, #34660), mouse anti-human Actin mAb (Sigma, #A5316) and mouse anti-human Tubulin mAb (Sigma, #T9026)(Sigma) were used as primary antibodies.

Techniques: Recombinant, Plasmid Preparation, Transfection, Transduction, Magnetic Beads

Journal: bioRxiv

Article Title: Shared and Distinct Mechanisms of UBA1 Inactivation Across Different Diseases

doi: 10.1101/2023.10.10.561769

Figure Lengend Snippet: A ) UBA1 p.S621C and p.A478S form aberrant ubiquitin conjugates in cells that are reducible by NH 2 OH but not β-ME, indicative of oxyesters. Indicated UBA1 HA variants were expressed in HEK 293T cells and lysates were either not treated (non-reducing, NR) or treated with indicated reducing agents and subjected to anti-HA immunoblotting. B ) UBA1 p.S621C- and p.A478S-dependent oxyester formation requires the catalytic cysteine residue C632. Indicated UBA1b HA variants were expressed in HEK 293T cells and lysates were either not treated (non-reducing, NR) or treated with indicated reducing agents and subjected to anti-HA immunoblotting. C ) UBA1 p.S621C, but not UBA1 p.A478S, forms aberrant ubiquitin conjugates in vitro . Indicated recombinant UBA1b proteins (500 nM) were incubated with 10 μM ubiquitin and 5 mM ATP for 10 min at 37°C followed by anti-UBA1 immunoblot analysis. D ) Crystal structure of S. cerevisiae UBA1 (grey) bound to Ub-AMP (Ub A , PDB: 4NNJ) was modeled with the p.A478S mutation ( S. cerevisiae p.A444S), showing that the introduced hydroxyl-group is in optimal position to attack the phospho-anhydride bond of the ubiquitin adenylate (cyan). Human numbering is displayed in bold with S. cerevisiae in parentheses. All stick representations: carbon (grey), oxygen (red), nitrogen (blue), and phosphorus (orange). E ) UBA1 p.A478S forms an oxyester at the mutation site. C-terminally FLAG tagged UBA1 p.A478S was expressed in HEK293T cells, followed by anti-FLAG immunoprecipitation and mass spectrometry analysis. The ubiquitylated residue is colored in orange and labeled with a diGly remnant (GG). Detected b and y ions for the oxyester-containing peptide are highlighted in red and dark blue, respectively. F ) UBA1 p.A478C does not support formation of aberrant ubiquitin conjugates in cells. Indicated UBA1 HA variants were expressed in HEK 293T cells and lysates were either not treated (non-reducing, NR) or treated with indicated reducing agents and subjected to anti-HA immunoblotting. G ) UBA1 p.A478S forms oxyesters in vitro in the presence of an E2 enzyme. (i) 500nM UBA1b WT or p.A478S were incubated with 10μM ubiquitin and 5mM ATP, followed by (ii) addition of 100mM EDTA and 2μM E2 enzyme (UBE2D3). Reactions were either not treated (non-reducing, NR) or treated with indicated reducing agents and subjected to anti-UBA1 immunoblotting. H ) Model of how UBA1 p.A478S forms oxyesters. Recruitment of an E2 enzyme to a doubly loaded UBA1 complex reduces intramolecular movements in the domains of UBA1 that bind the ubiquitin in the adenylation site (Ub A ), thereby promoting the attack of the phospho-anhydride bond of the ubiquitin adenylate by the hydroxyl-group of A478S.

Article Snippet: Primary antibodies for UBA1a/b (Cell Signaling, 4891S), Poly-ubiquitin (Cell Signaling, 3936S), UBE2C (Proteintech, 66087-1-Ig), UBE2D3 (Cell Signaling,4330S), and UBE2R2 (Proteintech,14077-1-AP), and β-actin (Cell Signaling, 4970) were used at a concentration of 1:1000 and visualized using HRP-conjugated secondary antibodies (anti-rabbit [Cell Signaling, 7074S] or anti-mouse [Cell Signaling, 7076S]) at a concentration of 1:1000.

Techniques: Western Blot, Residue, In Vitro, Recombinant, Incubation, Mutagenesis, Immunoprecipitation, Mass Spectrometry, Labeling

Journal: bioRxiv

Article Title: Shared and Distinct Mechanisms of UBA1 Inactivation Across Different Diseases

doi: 10.1101/2023.10.10.561769

Figure Lengend Snippet: A ) Schematic overview of the sequential, three-phase in vitro assay used to measure UBA1 transthiolation. (i) Charging of UBA1 by incubation of 250nM UBA1b with 10μM ubiquitin and 5mM ATP (ii) Quenching of UBA1 charging and single transfer to E2 enzyme by addition of 100mM EDTA and 1μM UBE2D3 (iii) Reactivation of UBA1 charging and multi transfer to E2 enzyme by addition of 100mM MgCl 2 . B ) Non-canonical VEXAS mutations are impaired in E2 transthiolation in vitro . Immunoblot analysis of the sequential, three-phase in vitro assay described in panel A using antibodies against UBA1 (left panel) or UBE2D3 (right panel). C ) Non-canonical VEXAS mutations are impaired in E2 transthiolation in vitro , as revealed by quantifications of relative E2 thioester levels (UBE2D3∼Ub/total signal) of multi turnover reactions (phase iii) depicted in panel B. n = 5 – 7 biological replicates as indicated, error bars = s.d., **** = p < 0.0001, one-way ANOVA. D ) Some non-canonical VEXAS mutations exhibit ∼2-fold defects in UBA1 re-charging, as revealed by quantifications of relative UBA1 thioester levels (UBA1∼Ub/total signal) of multi turnover reactions (phase iii) depicted in panel B. n = 5 – 7 biological replicates as indicated, error bars = s.d., *** = p < 0.001, **** = p < 0.0001, one-way ANOVA. E ) Non-canonical VEXAS mutations most strongly affect the E2 transthiolation step in vitro , as revealed by quantification of the relative E2 transthiolation over the relative UBA1 re-charging defects of multi turnover reactions (phase iii) depicted in panel B. n = 5 – 7 biological replicates as indicated, error bars = s.d., * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, one-way ANOVA. F ) Non-canonical VEXAS mutations are defective in charging different classes of E2s in vitro . Heatmap depicting the relative log 2 -fold changes in E2 ubiquitin thioester formation measured by multi turnover reactions (phase iii) using FITC-labeled ubiquitin (for details refer to ). For quantifications, E2 ubiquitin thioester levels in phase (iii) were first normalized to the total FITC signal in phase (i) and then normalized to WT. n = 1-3 biological replicates per condition as indicated. G ) CHO cells with UBA1a, UBA1c, or non-canonical VEXAS mutants as sole source of UBA1 generally exhibit lower ubiquitin thioester levels for select E2 enzymes as compared to CHO cells with WT UBA1. CHO rescue assays were performed as described in , followed by anti-E2 immunoblotting. Heatmap depicts the relative log 2 -fold changes in ubiquitin thioester levels of UBE2C, UBE2D3, and UBE2R2. n = 3 biological replicates per condition. H ) CHO cells with the p.S621C mutant as sole source of UBA1 exhibit lower ubiquitin thioester levels for UBE2S and UBE2L3 as compared to CHO cells with WT UBA1. CHO rescue assays were performed as described in , followed by anti-UBE2L3 and anti-UBE2S-immunoblotting and quantification. n = 3 biological replicates per condition. I ) Schematic representation of our in vitro findings, revealing that non-canonical VEXAS mutations inactivate UBA1 by most prominently affecting ubiquitin transfer to E2 enzymes.

Article Snippet: Primary antibodies for UBA1a/b (Cell Signaling, 4891S), Poly-ubiquitin (Cell Signaling, 3936S), UBE2C (Proteintech, 66087-1-Ig), UBE2D3 (Cell Signaling,4330S), and UBE2R2 (Proteintech,14077-1-AP), and β-actin (Cell Signaling, 4970) were used at a concentration of 1:1000 and visualized using HRP-conjugated secondary antibodies (anti-rabbit [Cell Signaling, 7074S] or anti-mouse [Cell Signaling, 7076S]) at a concentration of 1:1000.

Techniques: In Vitro, Incubation, Western Blot, Labeling, Mutagenesis

Journal: bioRxiv

Article Title: Shared and Distinct Mechanisms of UBA1 Inactivation Across Different Diseases

doi: 10.1101/2023.10.10.561769

Figure Lengend Snippet: A ) Schematic overview of the sequential, three-phase in vitro assay used to measure UBA1 transthiolation. (i) Complete charging of UBA1 by incubation of 250nM UBA1b with 10μM FITC-ubiquitin and 5mM ATP (ii) Quenching of UBA1 charging and single transfer to E2 enzyme by addition of 100mM EDTA and 1μM E2 enzyme (iii) Reactivation of UBA1 charging and multi transfer to E2 enzyme by addition of 100mM MgCl 2 B ) UBA1 WT was subjected to the experiment described in panel A using UBE2D3. Reactions were subjected to SDS page and analyzed by fluorescence imaging. Upper panel : Fluorescence scan showing UBA1 and UBE2D3 ubiquitin thioester levels after each reaction phase. Lower panel : UBE2D3 ubiquitin thioester levels in reaction phase (iii) were quantified and plotted against the reaction time, revealing that UBE2D3 is maximally charged after 60min. C ) Non-canonical VEXAS mutations are deficient in E2 transthiolation in vitro . Indicated UBA1 proteins were subjected to the experiment described in panel A using either UBE2D3, UBE2R2, or UBE2S. Reactions were subjected to SDS page and analyzed by fluorescence imaging. Upper panel : Fluorescence scan showing UBA1 charging after reaction phase (i) as control. Lower panel : Fluorescence scan showing UBA1 re-charging and E2 transfer after reaction phase (iii). Quantifications of 3 biological replicates are shown in . D ) Non-canonical VEXAS mutations are impaired in supporting E2 ubiquitin thioester levels in cells. CHO ts20 cells were reconstituted with indicated UBA1 variants and incubated at the permissive temperature for 6h, followed by immunoblotting using antibodies against indicated E2 enzymes. E-G ) Quantification of E2 charging levels (charged/total) shown in panel D. E) UBE2D3, F) UBE2C, G) UBE2R2. n=3 biological replicates, error bars = s.d., * = p < 0.05, ** = p < 0.01, *** = p < 0.001, one-way ANOVA.

Article Snippet: Primary antibodies for UBA1a/b (Cell Signaling, 4891S), Poly-ubiquitin (Cell Signaling, 3936S), UBE2C (Proteintech, 66087-1-Ig), UBE2D3 (Cell Signaling,4330S), and UBE2R2 (Proteintech,14077-1-AP), and β-actin (Cell Signaling, 4970) were used at a concentration of 1:1000 and visualized using HRP-conjugated secondary antibodies (anti-rabbit [Cell Signaling, 7074S] or anti-mouse [Cell Signaling, 7076S]) at a concentration of 1:1000.

Techniques: In Vitro, Incubation, SDS Page, Fluorescence, Imaging, Western Blot

Journal: bioRxiv

Article Title: Shared and Distinct Mechanisms of UBA1 Inactivation Across Different Diseases

doi: 10.1101/2023.10.10.561769

Figure Lengend Snippet: A ) Schematic overview of the assay used to determine ubiquitin thioester formation activity of UBA1. B ) Non-reducing immunoblot analysis of in vitro ubiquitin thioester formation reactions carried out at 37°C (left panel) or on ice (right panel), revealing that UBA1 p.L904R is deficient in thiolation and that UBA1 p.H643Y and p.Q724P form aberrant thioesters at 37°C. C ) Quantification of thioester formation (UBA1∼Ub/total UBA1 signal) of the experiments shown in panel B. n = 5 – 8 biological replicates as indicated, error bars = s.d., **** = p < 0.0001, one-way ANOVA. D ) Aberrant thioester formation of UBA1 p.H643Y and p.Q724P depends on the catalytic cysteine residue C632. Denoted recombinant UBA1b proteins (500 nM) were incubated with 10 μM ubiquitin and 5 mM ATP for 10 or 60 min at 37°C followed by anti-UBA1 immunoblot analysis. E ) The average alpha helicity of residues in the helix containing Q724 (Q690) calculated over a 100 ns molecular dynamics trajectory for S. cerevisiae UBA1 (PDB: 4NNJ). Residues N-terminal to Q724 (Q690) in the wild-type protein (grey) maintain an average alpha helical content of 50% or greater. When the Q690P mutation (green) is modeled into the structure, the proline breaks the helix and residues (H686-I688) lose their alpha helicity. F ) Schematic overview of the sequential, three-phase in vitro assay used to measure UBA1 transthiolation. (i) Complete charging of UBA1 by incubation of 250nM UBA1b with 10μM FITC-ubiquitin and 5mM ATP (ii) Quenching of UBA1 charging and single transfer to E2 enzyme by addition of 100mM EDTA and 1μM E2 enzyme (iii) Reactivation of UBA1 charging and multi transfer to E2 enzyme by addition of 100mM MgCl 2 G ) LCINS mutations p.H643Y and p.Q724P but not p.Q469P or P.D555Y are deficient in E2 transthiolation in vitro . Indicated UBA1 proteins were subjected to the experiment described in panel F using either UBE2D3, UBE2R2, or UBE2S. Reactions were subjected to SDS page and analyzed by fluorescence imaging. Upper panel : Fluorescence scan showing UBA1 charging after reaction phase (i) as control. Lower panel : Fluorescence scan showing UBA1 re-charging and E2 transfer after reaction phase (iii). H ) Quantifications of UBA1 ubiquitin thioester levels ( upper panel ) and E2 ubiquitin thioester levels ( lower panel ) after phase (iii) shown in panel G. E2 ubiquitin thioester levels in phase (iii) were first normalized to the total FITC signal in phase (i) and then normalized to WT. n = 1-3 biological replicates per condition as indicated.

Article Snippet: Primary antibodies for UBA1a/b (Cell Signaling, 4891S), Poly-ubiquitin (Cell Signaling, 3936S), UBE2C (Proteintech, 66087-1-Ig), UBE2D3 (Cell Signaling,4330S), and UBE2R2 (Proteintech,14077-1-AP), and β-actin (Cell Signaling, 4970) were used at a concentration of 1:1000 and visualized using HRP-conjugated secondary antibodies (anti-rabbit [Cell Signaling, 7074S] or anti-mouse [Cell Signaling, 7076S]) at a concentration of 1:1000.

Techniques: Activity Assay, Western Blot, In Vitro, Residue, Recombinant, Incubation, Mutagenesis, SDS Page, Fluorescence, Imaging

Journal: bioRxiv

Article Title: Shared and Distinct Mechanisms of UBA1 Inactivation Across Different Diseases

doi: 10.1101/2023.10.10.561769

Figure Lengend Snippet: A) UBA1 p.H643Y and p.Q724P form aberrant ubiquitin thioester species in vitro . Denoted recombinant UBA1b proteins (500 nM) were incubated with 10 μM ubiquitin and 5 mM ATP for 10 or 60 min at 37°C. Reactions were treated with reducing agents (β-ME or NH 2 OH) as indicated, followed by anti-UBA1 immunoblot analysis. B ) Ribbon diagram of an expanded region of S. cerevisiae UBA1 (PDB: 4NNJ) wild-type protein (upper panel) and with H643Y substitution (lower panel). Labeling for this panel and panel C follows . The mutation disrupts hydrogen bonds (red dashed lines) from H643 to N639 and a bound water molecule (red sphere) and forces rearrangements to prevent steric clashes between Y643 and T633/I629 (cyan dashed lines). Stick representation is used to display sidechain heavy atoms of UBA1 residue I629, C632, T633, N639, A640, and H643. In this panel and panel C, oxygen, nitrogen, and sulfur are colored red, blue and yellow, respectively. C ) Ribbon diagram of an expanded region of S. cerevisiae UBA1 (grey, PDB: 4NNJ) wild-type protein (upper panel) with a thioester-linked ubiquitin (light green) and the final frame of a 100 ns molecular dynamics simulation of UBA1 Q724P mutant (lower panel). A hydrogen bond (red dashed line) between the N721 backbone oxygen atom and Q724 (orange) backbone nitrogen atom is disrupted in the Q724P mutant by kinking of the helix (noted in red). The thioester bond between UBA1 C632 and ubiquitin G76, as well as the sidechain heavy atoms of UBA1 residue F637, N721, Q724, F729, F741, W742 and L814 are displayed by stick representation. D ) Model of how UBA1 p.H643Y and p.Q724P lead to the formation of aberrant UBA1 ubiquitin thioesters via intramolecular transthiolation reactions. E ) Aberrant thioester-forming (p.H643Y, p.Q724P) but not thermolabile (p.Q469P, p.D555Y) LCINS mutations are impaired in E2 transthiolation in vitro. Immunoblot analysis of the sequential, three-phase in vitro assay described in panel 5A using antibodies against UBA1 (left panel) or UBE2D3 (right panel). F ) Quantifications of relative UBA1 re-charging (UBA1∼Ub/total signal) and relative E2 thioester levels (UBE2D3∼Ub/total signal) of multi turnover reactions (phase iii) depicted in panel E. UBA1 p.H643Y and p.Q724P significantly reduce transthiolation while not markedly affecting thiolation, revealing an E2 bottleneck. n = 5 biological replicates, error bars = s.d., **** = p < 0.0001, one-way ANOVA. G ) CHO cells with E2 bottleneck but not with thermolabile disease mutants as sole source of UBA1 exhibit markedly lower ubiquitin thioester levels for select E2 enzymes and reduced polyubiquitylation as compared to CHO cells with WT UBA1. CHO rescue assays were performed as described in , followed by anti-E2 and anti-ubiquitin immunoblotting. Heatmap depicts the relative log 2 -fold changes in polyubiquitylation or ubiquitin thioester levels of UBE2C, UBE2D3, and UBE2R2. n = 3 biological replicates per condition.

Article Snippet: Primary antibodies for UBA1a/b (Cell Signaling, 4891S), Poly-ubiquitin (Cell Signaling, 3936S), UBE2C (Proteintech, 66087-1-Ig), UBE2D3 (Cell Signaling,4330S), and UBE2R2 (Proteintech,14077-1-AP), and β-actin (Cell Signaling, 4970) were used at a concentration of 1:1000 and visualized using HRP-conjugated secondary antibodies (anti-rabbit [Cell Signaling, 7074S] or anti-mouse [Cell Signaling, 7076S]) at a concentration of 1:1000.

Techniques: In Vitro, Recombinant, Incubation, Western Blot, Labeling, Mutagenesis, Residue

Journal: bioRxiv

Article Title: Shared and Distinct Mechanisms of UBA1 Inactivation Across Different Diseases

doi: 10.1101/2023.10.10.561769

Figure Lengend Snippet: A ) Schematic overview of the in vitro assay used to determine the efficiency of the first adenylation reaction of UBA1. The assay measures the production of pyrophosphate (PP i ) and the reaction is carried out in presence of NH 2 OH to reduce the ubiquitin thioester. B ) Amongst LCINS mutations only UBA1 p.Q469P confers a significant but small impairment of adenylation activity in vitro at saturating ATP and ubiquitin conditions. n = 2-3 biological replicates with 3 technical replicates each, error bars = s.d., **** = p < 0.0001, one-way ANOVA. C ) SMA-causing UBA1 mutations have no obvious effect on adenylation activity in vitro at saturating ATP and ubiquitin conditions. n = 2-4 biological replicates with 3 technical replicates each, error bars = s.d., no significant changes observed, one-way ANOVA. D ) Schematic overview of the assay used to determine ubiquitin thioester formation activity of UBA1. E ) Non-reducing immunoblot analysis of in vitro ubiquitin thioester formation reactions carried out at 37°C (left panel) or on ice (right panel) and containing 500nM indicated UBA1b proteins, 10μM ubiquitin, and 5mM ATP, revealing that SMA-causing mutations have no obvious impact on UBA1 thiolation in vitro . F ) Quantification of thioester formation (UBA1∼Ub/total UBA1 signal) of the experiments shown in panel E. n = 3 – 10 biological replicates as indicated, error bars = s.d., **** = p < 0.0001, one-way ANOVA. G ) Schematic overview of the sequential, three-phase in vitro assay used to measure UBA1 transthiolation. (i) Complete charging of UBA1 by incubation of 250nM UBA1b with 10μM ubiquitin and 5mM ATP (ii) Quenching of UBA1 charging and single transfer to E2 enzyme by addition of 100mM EDTA and 1 μM UBE2D3 (iii) Reactivation of UBA1 charging and multi transfer to E2 enzyme by addition of 100mM MgCl 2 . H ) SMA-causing mutations have no obvious impact on E2 transthiolation in vitro . Immunoblot analysis of the sequential, three-phase in vitro assay described in panel G using antibodies against UBA1 (left) or UBE2D3 (right). I ) Quantifications of relative UBA1 re-charging (UBA1∼Ub/total signal, left graph) and E2 thioester levels (UBE2D3∼Ub/total signal, right graph) of multi turnover reactions (phase iii) depicted in panel H. n = 3 biological replicates as indicated, error bars = s.d., no significant changes detected, one-way ANOVA. J ) SMA-causing UBA1 mutations are not defective in E2 transthiolation in vitro . Indicated UBA1 proteins were subjected to the experiment described in panel G using either UBE2D3, UBE2R2, or UBE2S. Reactions were run on SDS page and analyzed by fluorescence imaging. Left panel : Fluorescence scan showing UBA1 charging after reaction phase (i) as control. Right panel : Fluorescence scan showing UBA1 re-charging and E2 transfer after reaction phase (iii). K ) Quantifications of E2 ubiquitin thioester levels after phase (iii) shown in panel G. E2 ubiquitin thioester levels in phase (iii) were first normalized to the total FITC signal in phase (i) and then normalized to WT. n = 1-3 biological replicates per condition as indicated.

Article Snippet: Primary antibodies for UBA1a/b (Cell Signaling, 4891S), Poly-ubiquitin (Cell Signaling, 3936S), UBE2C (Proteintech, 66087-1-Ig), UBE2D3 (Cell Signaling,4330S), and UBE2R2 (Proteintech,14077-1-AP), and β-actin (Cell Signaling, 4970) were used at a concentration of 1:1000 and visualized using HRP-conjugated secondary antibodies (anti-rabbit [Cell Signaling, 7074S] or anti-mouse [Cell Signaling, 7076S]) at a concentration of 1:1000.

Techniques: In Vitro, Activity Assay, Western Blot, Incubation, SDS Page, Fluorescence, Imaging

Journal: bioRxiv

Article Title: Shared and Distinct Mechanisms of UBA1 Inactivation Across Different Diseases

doi: 10.1101/2023.10.10.561769

Figure Lengend Snippet: A ) Immunoblots of CHO ts20 cells reconstituted with indicated UBA1 variants and incubated at the permissive temperature for 6h, revealing that a subset of LCINS mutations are impaired in their ability to support ubiquitylation in cells. B ) Quantification of global polyubiquitylation levels shown in panel A. n=7 biological replicates, error bars = s.d., ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, one-way ANOVA. C) Immunoblots of CHO ts20 cells reconstituted with indicated UBA1 variants and incubated at the permissive temperature for 6h, revealing that a subset of LCINS mutations are impaired in their ability to support E2 ubiquitin thioester levels in cells. D-F ) Quantification of E2 charging levels (charged/total) shown in panel C. D) UBE2R2, E) UBE2C, F) UBE2D3. n=3 biological replicates, error bars = s.d., * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, one-way ANOVA.

Article Snippet: Primary antibodies for UBA1a/b (Cell Signaling, 4891S), Poly-ubiquitin (Cell Signaling, 3936S), UBE2C (Proteintech, 66087-1-Ig), UBE2D3 (Cell Signaling,4330S), and UBE2R2 (Proteintech,14077-1-AP), and β-actin (Cell Signaling, 4970) were used at a concentration of 1:1000 and visualized using HRP-conjugated secondary antibodies (anti-rabbit [Cell Signaling, 7074S] or anti-mouse [Cell Signaling, 7076S]) at a concentration of 1:1000.

Techniques: Western Blot, Incubation