Journal: The Journal of Biological Chemistry

Article Title: Stereochemical Features of Glutathione-dependent Enzymes in the Sphingobium sp. Strain SYK-6 β-Aryl Etherase Pathway *

doi: 10.1074/jbc.M113.536250

Figure Lengend Snippet: The proposed β-etherase pathway in Sphingobium sp. strain SYK-6. Panels ( A ) and ( B ) show pathways for metabolism of β( R )- and β( S )-configured β-aryl ether model compounds, with Lig dehydrogenases (LigD, LigL, LigN, and LigO) catalyzing oxidation of the benzylic alcohol in model substrates β( R )- and β( S )-guaiacyl-α-veratrylglycerol, producing β( R )GVG and β( S )GVG. In the presence of GSH, βGVG enantiomers are cleaved to guaiacol and one of two GS-conjugated β-epimers, GS-β( S )VG or GS-β( R )VG, by either LigE, LigP, or LigF. Subsequent GSH-dependent cleavage of GS-β( R )VG by LigG generates β-deoxy-α-veratrylglycerone and GS-SG. C shows the reaction products of Lig enzymes when the achiral model compound βGVE is used as a substrate. In this case, the product of β-etherase activity is GS-βVE, and the product of GS-βVE thioether cleavage by LigG is α-veratrylethanone.

Article Snippet: The LigE and LigP reaction products eluted from the Biotage KP-C18 column with t R = 30.9 and t R = 31.0 min, respectively.

Techniques: Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Stereochemical Features of Glutathione-dependent Enzymes in the Sphingobium sp. Strain SYK-6 β-Aryl Etherase Pathway *

doi: 10.1074/jbc.M113.536250

Figure Lengend Snippet: HPLC chromatogram traces of pre-enzyme addition (black), LigE ( red ), LigP ( orange ), and LigF ( blue ) enzyme assay samples. A , C 18 -reversed phase chromatography of βGVE prior to the addition of and after a 1-h incubation with GSH and either LigE, LigP, or LigF. B , chiral chromatography of racem -βGVG prior to the addition of and after a 1-h incubation with GSH and either LigE, LigP, or LigF.

Article Snippet: The LigE and LigP reaction products eluted from the Biotage KP-C18 column with t R = 30.9 and t R = 31.0 min, respectively.

Techniques: Enzymatic Assay, Reversed-phase Chromatography, Incubation, Chiral Chromatography

Journal: The Journal of Biological Chemistry

Article Title: Stereochemical Features of Glutathione-dependent Enzymes in the Sphingobium sp. Strain SYK-6 β-Aryl Etherase Pathway *

doi: 10.1074/jbc.M113.536250

Figure Lengend Snippet: Aligned 1 H NMR partial spectra (2.60–4.65 ppm) of the GS conjugate, GS-βVE, in D 2 O. Proton assignment labels correspond with the carbon to which the proton is bound. Alphabetical subscripts differentiate two non-identical geminal protons. Proton peaks that did not integrate as expected are denoted with an asterisk (see “Results”). A , compound GS-βVE obtained via chemical synthesis. B–D , reaction product GS-βVE isolated from LigE ( B ), LigP ( C ), or LigF ( D ) activities using the achiral model compound GβVE as a substrate.

Article Snippet: The LigE and LigP reaction products eluted from the Biotage KP-C18 column with t R = 30.9 and t R = 31.0 min, respectively.

Techniques: Isolation

Journal: The Journal of Biological Chemistry

Article Title: Stereochemical Features of Glutathione-dependent Enzymes in the Sphingobium sp. Strain SYK-6 β-Aryl Etherase Pathway *

doi: 10.1074/jbc.M113.536250

Figure Lengend Snippet: Aligned 1 H NMR partial spectra (2.60–4.65 ppm) of both β-epimers of β- S -glutathionyl-α-veratrylglycerone (GS-β( S )VG and GS-β( R )VG) and both β-epimers of γ,γ-dimethoxy-β- S -(methyl N -acetyl-cysteinyl)-α-veratrylpropanone (CS-β( S )VP and CS-β( R )VP) in D 2 O. Proton assignment labels correspond with the carbon to which the proton is bound. Alphabetical subscripts differentiate two non-identical geminal protons or methoxyls. Red labels and shading are used for the two β( S )-configured compounds, GS-β( S )VG and CS-β( S )VP; blue labels and shading are used for the two β( R )-configured compounds, GS-β( R )VG and CS-β( R )VP; black labels are used for overlapped regions. Regions that differentiate β( S )- and β( R )-configurations ( i.e. protons at β, 2′, and 3′) are shaded across panels. A , chemically synthesized mixture (1:1) of GS-β( S )VG and GS-β( R )VG. B , GS-β( S )VG from LigE. C , GS-β( S )VG from LigP. D , GS-β( R )VG from LigF. E , residual GS-β( S )VG not degraded by LigG. F , chemically synthesized CS-β( S )VP. G , Chemically synthesized CS-β( R )VP.

Article Snippet: The LigE and LigP reaction products eluted from the Biotage KP-C18 column with t R = 30.9 and t R = 31.0 min, respectively.

Techniques: Synthesized

Journal: International journal of biological sciences

Article Title: Rac GTPase activating protein 1 promotes gallbladder cancer via binding DNA ligase 3 to reduce apoptosis.

doi: 10.7150/ijbs.58857

Figure Lengend Snippet: Figure 3. RACGAP1 binds with LIG3 in GBC cells. (A) A Venn diagram of potential RACGAP1 binding targets, as predicted using the STRING database and IP-MS. (B) Immunoblotting using antibodies as indicated after endogenous Co-IP with anti-RACGAP1 or anti-LIG3 antibody. (C) IF images of RACGAP1, LIG3 and DAPI in NOZ and GBC-SD cells. Scale bars represent 10 μm.

Article Snippet: Western blotting analysis was performed using antiRACGAP1, LIG3, γH2A.X, p-ATM, p-ATR, p-CHEK1 and p-CHEK2, PARP1, caspase3 and c-caspase3, PAR, GAPDH and Histone H3 antibodies (p-ATM, p-ATR, p-CHEK1, p-CHEK2 and PARP1 were supplied by ABclonal, MA, USA, others were from Abcam, MA, USA).

Techniques: Binding Assay, Protein-Protein interactions, Western Blot, Co-Immunoprecipitation Assay

Journal: International journal of biological sciences

Article Title: Rac GTPase activating protein 1 promotes gallbladder cancer via binding DNA ligase 3 to reduce apoptosis.

doi: 10.7150/ijbs.58857

Figure Lengend Snippet: Figure 4. RACGAP1 regulates LIG3 expression independent of RhoA activity. (A) LIG3 expression in GBC cells and normal gallbladder epithelium cells. (B) Effects of LIG3 knockdown on GBC cells viability by CCK-8 assays. (C) Effects of RACGAP1 and LIG3 knockdown or overexpression on protein levels of each other in GBC cells. (D) Pulldowns in NOZ lines and immunoblotting assays of Cdc42, Rac1, and RhoA GTPase activity after RACGAP1 knockdown or overexpression. (E) Western blotting analysis of LIG3 protein level after RACGAP1 overexpression and Rho inhibitor I (CT04) treatment. ***P < 0.001.

Article Snippet: Western blotting analysis was performed using antiRACGAP1, LIG3, γH2A.X, p-ATM, p-ATR, p-CHEK1 and p-CHEK2, PARP1, caspase3 and c-caspase3, PAR, GAPDH and Histone H3 antibodies (p-ATM, p-ATR, p-CHEK1, p-CHEK2 and PARP1 were supplied by ABclonal, MA, USA, others were from Abcam, MA, USA).

Techniques: Expressing, Activity Assay, Knockdown, CCK-8 Assay, Over Expression, Western Blot

Journal: International journal of biological sciences

Article Title: Rac GTPase activating protein 1 promotes gallbladder cancer via binding DNA ligase 3 to reduce apoptosis.

doi: 10.7150/ijbs.58857

Figure Lengend Snippet: Figure 5. RACGAP1 downregulation suppresses LIG3-dependent DNA repair which increases DNA damage and apoptosis. (A) DNA damage after RACGAP knockdown in NOZ and GBC-SD cells was identified by the comet assay and observed with a fluorescence microscope. Scale bars represent 100 μm. (B) IF images of DSBs marker (γH2A.X) and DAPI after RACGAP1 knockdown. Scale bars represent 50 μm. (C) Western blotting detected the levels of γH2A.X, DDR (p-ATM, p-ATR, p-CHEK1 and p-CHEK2) and apoptosis (PARP1, c-PARP1, caspase3 and c-caspase3) related proteins, PAR signal in GBC cells after RACGAP1 knockdown. (D) Efficiency of Alt-NHEJ repair was evaluated by EJ2-GFP assay in NOZ and GBC-SD cells after RACGAP1 knockdown. (E) Cell viability and colony formation ability were evaluated in GBC cells transfected with siNC plus vector or siRACGAP1 plus vector or siRACGAP1 plus LIG3. (F) Western blotting analysis of γH2A.X, p-ATM, p-ATR, p-CHEK1, p-CHEK2, PARP1, c-PARP1, caspase3 and c-caspase3, PAR in GBC cells after transfection as indicated. (G) LIG3, γH2A.X, TUNEL and PAR staining of the subcutaneous tumors from xenograft models constructed with NOZ cells transfected with lentiviruses as indicated. Scale bars represent 50 μm.*P < 0.05, **P< 0.01, ***P < 0.001.

Article Snippet: Western blotting analysis was performed using antiRACGAP1, LIG3, γH2A.X, p-ATM, p-ATR, p-CHEK1 and p-CHEK2, PARP1, caspase3 and c-caspase3, PAR, GAPDH and Histone H3 antibodies (p-ATM, p-ATR, p-CHEK1, p-CHEK2 and PARP1 were supplied by ABclonal, MA, USA, others were from Abcam, MA, USA).

Techniques: Knockdown, Single Cell Gel Electrophoresis, Fluorescence, Microscopy, Marker, Western Blot, Transfection, Plasmid Preparation, TUNEL Assay, Staining, Construct

Journal: International journal of biological sciences

Article Title: Rac GTPase activating protein 1 promotes gallbladder cancer via binding DNA ligase 3 to reduce apoptosis.

doi: 10.7150/ijbs.58857

Figure Lengend Snippet: Figure 6. RACGAP1 is dispensable for localization of LIG3 but increases LIG3 stability. (A) Subcellular LIG3 expressions in NOZ and GBC-SD cells were assessed using western blotting. (B) IF images of RACGAP1, LIG3, γH2A.X and DAPI in NOZ and GBC-SD cells after etoposide treatment. Scale bars represent 10 μm. (C) LIG3 mRNA expressions were detected by qRT-PCR in NOZ and GBC-SD cell lines transfected with siNC or siRNA against RACGAP1. (D) LIG3 protein level analyzed by western blotting after knocking down or overexpressing RACGAP1 and treatment with CHX at the indicated times. NS, no significance; ***P < 0.001.

Article Snippet: Western blotting analysis was performed using antiRACGAP1, LIG3, γH2A.X, p-ATM, p-ATR, p-CHEK1 and p-CHEK2, PARP1, caspase3 and c-caspase3, PAR, GAPDH and Histone H3 antibodies (p-ATM, p-ATR, p-CHEK1, p-CHEK2 and PARP1 were supplied by ABclonal, MA, USA, others were from Abcam, MA, USA).

Techniques: Western Blot, Quantitative RT-PCR, Transfection