Journal: Open Biology

Article Title: Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity

doi: 10.1098/rsob.130213

Figure Lengend Snippet: PINK1-dependent phosphorylation of Parkin Ser 65 leads to activation of Parkin E3 ligase activity and multi-monoubiquitylation of Miro1. Wild-type (WT) ( a ) but not kinase-inactive (KI) ( b ) PINK1 activates wild-type Parkin E3 ligase activity leading to Miro1 multi-monoubiquitylation, an effect that is blocked by mutant Parkin Ser65Ala (S65A) ( c ). Two micrograms of wild-type or S65A Parkin were incubated with indicated amounts of wild-type or kinase-inactive (D359A) MBP-TcPINK in a kinase reaction (50 mM Tris–HCl (pH 7.5), 0.1 mM ethylene glycol tetra-acetic acid (EGTA), 10 mM MgCl 2 , 0.1% 2-mercaptoethanol and 0.1 mM ATP) for 60 min. The ubiquitylation reaction was then initiated by addition of ubiquitylation assay components (50 mM Tris–HCl (pH 7.5), 0.05 mM EGTA, 10 mM MgCl 2 , 0.5% 2-mercaptoethanol, 0.12 μM human recombinant E1 purified from Sf21 insect cell line, 1 μM human recombinant UbcH7 purified from E. coli , 0.05 mM Flag-ubiquitin (Boston Biochem) and 2 mM ATP) and 2 μg of His-Sumo-Miro1. Reactions were terminated after 60 min by addition of SDS–PAGE loading buffer and resolved by SDS–PAGE. Miro1, ubiquitin, Parkin and PINK1 were detected using anti-SUMO, anti-FLAG, anti-Parkin and anti-MBP antibodies, respectively. Representative of three independent experiments.

Article Snippet: The ubiquitylation reaction was then initiated by addition of ubiquitylation assay components (50 mM Tris–HCl (pH 7.5), 0.05 mM EGTA, 10 mM MgCl 2 , 0.5% 2-mercaptoethanol, 0.12 μM human recombinant E1 purified from Sf21 insect cell line, 1 μM human recombinant UbcH7 purified from E. coli , 0.05 mM Flag-ubiquitin (Boston Biochem) and 2 mM ATP) and 2 μg of His-Sumo-Miro1.

Techniques: Activation Assay, Activity Assay, Mutagenesis, Incubation, Ubiquitin Assay, Recombinant, Purification, SDS Page

Journal: Open Biology

Article Title: Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity

doi: 10.1098/rsob.130213

Figure Lengend Snippet: PINK1-dependent phosphorylation of Parkin Ser 65 is required for discharge of ubiquitin from E2. Parkin was phosphorylated using wild-type (WT) or kinase-inactive (KI) MBP-TcPINK1. An E2 discharge assay was established by incubation of this mixture with 2 μg of UbcH7 that had been pre-incubated with 0.5 µg of E1 and FLAG-ubiquitin in the presence of ATP for 60 min. Reactions were allowed to continue for 15 min ( a,c,d ) or as indicated ( b ) and stopped using SDS–PAGE loading buffer in absence of reducing agent. Samples were resolved by SDS–PAGE and proteins detected by Colloidal Coomassie staining. ( a ) Ubiquitin-loaded UbcH7 (UbcH7-Ub) was observed in the absence of Parkin (lanes 1,2). WT Parkin only in the presence of WT MBP-TcPINK1 was able to efficiently discharge UbcH7-Ub (lanes 5,6). No discharge was observed with WT Parkin alone (lanes 3,4) or WT Parkin in the presence of KI MBP-TcPINK1 (lanes 7,8). ( b ) Time course of E2 discharge after addition of activated WT Parkin in the presence of WT MBP-TcPINK1 demonstrated rapid and maximal discharge of UbcH7-Ub at 4 min. ( c ) Abrogation of UbcH7-Ub discharge by Parkin Ser65Ala (S65A; lanes 5,6) in contrast to WT Parkin in the presence of WT PINK1 (lanes 3,4). ( d ) Comparison of the effects Parkin disease mutations on ubiquitin discharge from UbcH7. Red dotted line indicates the WT activity. K27N, R33Q, R42P, K161N, G430D and G328E mutants showed no significant changes in activity. A46P, S65A, K211N, R275W, T415N and C431F displayed markedly decreased E2-ubiquitin discharge ability. Asterisk indicates the R33Q Parkin–ubiquitin thioester. Representative of three independent experiments.

Article Snippet: The ubiquitylation reaction was then initiated by addition of ubiquitylation assay components (50 mM Tris–HCl (pH 7.5), 0.05 mM EGTA, 10 mM MgCl 2 , 0.5% 2-mercaptoethanol, 0.12 μM human recombinant E1 purified from Sf21 insect cell line, 1 μM human recombinant UbcH7 purified from E. coli , 0.05 mM Flag-ubiquitin (Boston Biochem) and 2 mM ATP) and 2 μg of His-Sumo-Miro1.

Techniques: Incubation, SDS Page, Staining, Comparison, Activity Assay

Journal: Frontiers in Immunology

Article Title: Foot-and-Mouth Disease Virus Counteracts on Internal Ribosome Entry Site Suppression by G3BP1 and Inhibits G3BP1-Mediated Stress Granule Assembly via Post-Translational Mechanisms

doi: 10.3389/fimmu.2018.01142

Figure Lengend Snippet: Confirmation of differentially expressed proteins and phosphoproteins by western blotting and Phos-tag western Blotting. (A) Analysis of ubiquitin conjugating enzyme E2 I, ubiquitin conjugating enzyme E2 L3, glyceraldehyde-3-phosphate dehydrogenase, β-actin expression levels in foot-and-mouth disease virus (FMDV)-infected and control cells by western blotting. SILAC-ratios and immunoblotting ratios (infection/control) were shown on the right side. (B) Analysis of the dynamic phosphorylation alterations of the three differentially phosphoproteins (ribosomal protein L15, chromosome 5 open reading frame 24, and FOS-like 2) in FMDV-infected and control cells by Phos-tag western blotting.

Article Snippet: To confirm the expression levels of GAPDH, ubiquitin conjugating enzyme E2 I (UBE2I), ubiquitin conjugating enzyme E2 L3 (UBE2L3), ribosomal protein L15 (RPL15), chromosome 5 open reading frame 24 (C5ORF24) and FOS-like 2 (FOSL2), anti-GAPDH antibody (Beyotime, China), anti-UBE2I antibody (Proteintech, China), anti-UBE2L3 antibody (Proteintech, China), anti-RPL15 antibody (Proteintech, China), anti-C5ORF24 antibody (Proteintech, China), and anti-FOSL2 antibody (Proteintech, China) were used for immunoblotting.

Techniques: Western Blot, Ubiquitin Proteomics, Expressing, Virus, Infection, Control, Multiplex sample analysis, Phospho-proteomics

Journal: Cell reports

Article Title: MYO10 regulates genome stability and cancer inflammation through mediating mitosis

doi: 10.1016/j.celrep.2023.112531

Figure Lengend Snippet:

Article Snippet: Anti-UbcH7 , Bio-Techne/NOVUS Biologicals , #NB100-2265, rabbit polyclonal, RRID: AB_2304026.

Techniques: Staining, Recombinant, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Knock-Out, Software, Fluorescence, Microscopy

Journal: Structure(London, England:1993)

Article Title: Structure of the Human FANCL RING-Ube2T Complex Reveals Determinants of Cognate E3-E2 Selection

doi: 10.1016/j.str.2013.12.004

Figure Lengend Snippet: Overall Structure of FANCL-Ube2T Complex (A) The overall structure of the RING domain of FANCL (magenta) bound to Ube2T (blue) is shown in cartoon representation. Gray spheres represent zinc ions. A gold star represents the position of Ube2T’s catalytic cysteine. (B) RING domain of FANCL (magenta) overlain with c-cbl RING domain (green; PDB ID code 1FBV ). (C) Ube2T (blue) overlain with Ube2L3 (orange; PDB ID code 1FBV ) showing the structural conservation of the UBC fold, comprising a four-stranded β-meander flanked by an N-terminal helix (helix1) and two C-terminal helices (helixes 2 and 3). A gold star represents the position of the catalytic cysteine. The gray oval shows the E3 binding interface of E2s. (D) Top left panel: The pi stacking in the binding interface between Y311 of FANCL and R6 and R9 of Ube2T. Top right panel: The hydrophobic binding interface of the RING domain (magenta) and Ube2T (blue). Bottom panels: The electrostatic and hydrogen bonding network of the RING-Ube2T interface. Interactions are represented by dashed lines. See also Figure S1 .

Article Snippet: Human Ube2D3 (UbcH5c), Ube2L3 (UbcH7), Ube2K, Ube2B, and Ube2R1 were cloned from I.M.A.G.E. clones (Geneservice) and inserted into the pDEST17 (Invitrogen) and pET RSF vectors containing an N-terminal 6xHis tag and a TEV cleavage site.

Techniques: Binding Assay

Journal: Structure(London, England:1993)

Article Title: Structure of the Human FANCL RING-Ube2T Complex Reveals Determinants of Cognate E3-E2 Selection

doi: 10.1016/j.str.2013.12.004

Figure Lengend Snippet: Structural Comparison of the FANCL RING Domain-Ube2t Complex with Other RING-E2 Complexes (A) A structure-based sequence alignment of E2s. PDB ID codes of E2s, as listed in the figure: 1FBV , 3RPG , 4AP4 , 4AUQ , 3RZ3 , 2YB6 , 3K9O , 3H8K , 2Z5D , 2F4W , 3HCT , and 3BZH . (B) A structure-based sequence alignment of RING and Ubox domains. Ubox domains are highlighted by a cyan box. PDB ID codes used of RING and Ubox domains, as listed in the figure: 1FBV , 4F52 , 4AUQ , 3HCT , 2C2V , 3LIZ , 3RPG , 4AP4 , 4EPO , 2YHO , 2Y43 , 4KBL , and 4K7D . Residues shaded in red to yellow colors indicate conserved residues, where red corresponds to strict conservation. Gray bars indicate zinc coordinating atoms. Green circles highlight residues involved in the hydrophobic interface between FANCL and Ube2T. Purple circles denote residues involved in hydrogen bonding and electrostatic interactions in the FANCL Ube2T interface. (C) Superpositions of the FANCL RING-Ube2T complex (colored pink and blue, respectively), with c-cbl RING-Ube2L3 complex (left) shaded gray (PDB ID code 1FBV ), idol-Ube2D1 complex (middle) shaded gray (PDB ID code 2YHO ), and ring1b-Ube2D3 complex (right) shaded gray (PDB ID code 3RPG ). Numbered residues are the same as the FANCL RING-Ube2T complex, with dashed lines showing interactions.

Article Snippet: Human Ube2D3 (UbcH5c), Ube2L3 (UbcH7), Ube2K, Ube2B, and Ube2R1 were cloned from I.M.A.G.E. clones (Geneservice) and inserted into the pDEST17 (Invitrogen) and pET RSF vectors containing an N-terminal 6xHis tag and a TEV cleavage site.

Techniques: Sequencing

Journal: Structure(London, England:1993)

Article Title: Structure of the Human FANCL RING-Ube2T Complex Reveals Determinants of Cognate E3-E2 Selection

doi: 10.1016/j.str.2013.12.004

Figure Lengend Snippet: Conserved Hydrophobic RING Residues Are Required for Ube2T Binding and FANCL Selects Solely Ube2T In Vitro (A) Size-exclusion chromatogram profiles of wild-type (WT) or mutant RING domains (green dashed line) and WT Ube2T (blue dotted line) overlaid with profiles from binding experiments in which WT Ube2T has been incubated with WT or mutant RING domains (pink line) and subjected to size-exclusion chromatography. Binding was assessed by complex formation, which is indicated by a peak shift to the left labeled complex. (B) Size-exclusion chromatogram of FANCL RING domain incubated with an E2 mix consisting of Ube2T, Ube2D3, and Ube2L3 (pink line). Chromatograms of Ube2T (blue dotted line) and the RING domain (green dashed line) are also overlaid. A peak shift to the left is observed, indicating complex formation. SDS-PAGE gel of the fractions collected from the size-exclusion experiment and stained with Coomassie Brilliant Blue. The E2 gel bands found in the shifted peak were assessed by mass spectrometry for protein identification and confirmed as exclusively Ube2T. See also Figure S2 .

Article Snippet: Human Ube2D3 (UbcH5c), Ube2L3 (UbcH7), Ube2K, Ube2B, and Ube2R1 were cloned from I.M.A.G.E. clones (Geneservice) and inserted into the pDEST17 (Invitrogen) and pET RSF vectors containing an N-terminal 6xHis tag and a TEV cleavage site.

Techniques: Binding Assay, In Vitro, Mutagenesis, Incubation, Size-exclusion Chromatography, Labeling, SDS Page, Staining, Mass Spectrometry