Journal: PLoS Genetics

Article Title: A HECT Ubiquitin-Protein Ligase as a Novel Candidate Gene for Altered Quinine and Quinidine Responses in Plasmodium falciparum

doi: 10.1371/journal.pgen.1004382

Figure Lengend Snippet: A. The PfUT HECT domain/GFP fusion protein catalyzes self polyubiquitination. The PfUT HECT domain/GFP fusion protein was isolated from the corresponding transfected Dd2 line and the catalytic activity of the PfUT HECT domain/GFP fusion protein was tested in an in vitro assay reconstituted with the components indicated. The left scheme indicates the origin of the components and their function. The human components ubiquitin (Ub), E1 activating enzyme (E1; UBA), and the E2 conjugating enzymes (E2; UBCH5a or UBCH13) are highlighted in blue. The PfUT HECT domain/GFP fusion protein (PfUT) is indicated in green. The reactions were examined by Western analysis using SDS PAGE on a 4 -12% gradient gel under non-reducing conditions and an antiserum specific to ubiquitin (αUb, dilution 1∶2000). The asterisks mark ubiquitin intermediate adducts generated by UBCH13 and UBCH5a. High molecular weight ubiquitinated products are indicated. A molecular weight marker is indicated in kDa. A representative example of at least three biological replicates is shown. The supplementary shows an independent biological replicate and supplementary shows absence of enzymatic activity when parasite purified GFP was used in the assay instead of the PfUT HECT domain/GFP fusion. B. The PfUT HECT domain/GFP fusion protein catalyzes ubiquitination of substrate proteins. In vitro ubiquitination assays were reconstituted using the components indicated, including a PfCRT/GFP fusion protein isolated from the corresponding transfected Dd2 line. The reactions were examined by Western analysis using SDS PAGE on a 4–12% gradient gel under non-reducing conditions and antisera specific to the PfUT HECT domain (αPfUT, dilution 1∶5000), ubiquitin (αUb, dilution 1∶2000), and PfCRT (αPfCRT, dilution 1∶1000). In addition to PfCRT/GFP, the immunoglobulin heavy (53 kDa plus ubiquitin) and light chains (25 kDa plus ubiquitin) (present in the reaction because the conditions required to elute PfCRT/GFP also eluted immunoglobulins from the column) were ubiqutinated and are indicated by asterisks.

Article Snippet: The following reagents purchased from Boston Biochem were used in the ubiquitination assay: 70 μM human recombinant ubiquitin, 200 nM human recombinant ubiquitin activating enzyme UBA, 5 μM human recombinant ubiquitin conjugating enzyme UbcH5a or UbcH13, and 1 x energy regeneration solution.

Techniques: Isolation, Transfection, Activity Assay, In Vitro, Ubiquitin Proteomics, Western Blot, SDS Page, Generated, High Molecular Weight, Molecular Weight, Marker, Purification

Journal: Cell Death and Differentiation

Article Title: CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

doi: 10.1038/s41418-020-00635-5

Figure Lengend Snippet: CIB1 and CHIP specifically interact. A SDS-PAGE gel of proteins bound to IgG (left lane) or CIB1 (right lane). Protein marker stands stand for (from top to bottom): 170, 130, 100, 70 (Red), 55, 40, 35, 25, 15/10 kDa. B Endogenous interaction between CIB1 and CHIP in PC-9 cells. C PC-9 cells were transiently transfected for 36 h with CHIP plasmid, and cell lysates were immunoblotted with indicated antibodies. D HEK293 cells were transiently transfected for 36 h with Flag-CIB1 and Myc-CHIP, and cell lysates were immunoblotted with indicated antibodies. E Representative immunohistochemically stained images of LAC tissues using the anti-CHIP and anti-CIB1 antibodies. Areas in the black squares are magnified in the right slide panels. F Pearson analysis correlation between CIB1 and CHIP expression in LAC tissues ( r = −0.3582, P = 0.0049) G HEK293 cells were transfected with Flag-CIB1 (1 μg) and various concentrations of Myc-CHIP plasmids (0, 0.2, 0.5, and 1 μg). And after 36 h, cell lysates were immunoblotted with anti-Flag, anti-Myc, and anti-Tublin antibodies. H Representative confocal images of immunostaining for CHIP (green) and CIB1 (red) in PC-9 and A549 cells. Scale bar, 50 μm. I Cells were treated with 40 μg/ml cycloheximide for the indicated times, and cell lysates were immunoblotted with indicated antibody. J HEK293 cells were transfected for 36 h with Flag-CIB1 alone or together with Myc-CHIP and treated with MG-132 (10 μM), or leupeptin (100 μM) for 4 h. Cell lysates were analyzed by western blotting with the indicated antibodies.

Article Snippet: 2.5 μl 10× E3 Ligase Reaction Buffer (B-71, Boston Biochem), 1 μl Recombinant Human Ubiquitin Protein (U-100H-10M, Boston Biochem), 10 mM MgATP Solution (B-20, Boston Biochem), 100 nM Recombinant Human His6-Ubiquitin E1 Enzyme (E-304–05, Boston Biochem), 1 μM Recombinant Human UbcH5a/UBE2D1 Protein (E2–616–100, Boston Biochem), 1 μM Recombinant Human CHIP/STUB1 Protein (E3–220–050, Boston Biochem), 5 μM CIB1 Fusion Protein (Ag2391, Protein Tech) was added in a microcentrifuge tube and be incubated in a 37 °C water bath for 60 min. Then the lysis was added by SDS-PAGE sample buffer and followed by electrophoresis.

Techniques: SDS Page, Marker, Transfection, Plasmid Preparation, Staining, Expressing, Immunostaining, Western Blot

Journal: Cell Death and Differentiation

Article Title: CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

doi: 10.1038/s41418-020-00635-5

Figure Lengend Snippet: A PC-9 were transfected with CHIP plasmid for 36 h. Cell extracts were immunoprecipitated with the anti-CIB1 antibody, followed by immunoblotting with the anti-Ubiquitin antibody. B HEK293 cells were transfected with HA-ubiquitin, Flag-CIB1 with or without Myc-CHIP. Cell lysates were immunoprecipitated with the anti-HA antibody, followed by immunoblotting with anti-flag antibodies. C PC-9 cells were transfected with CHIP or CHIP-ΔUbox plasmid. Cell extracts were immunoprecipitated with the anti-ubiquitin antibody, followed by immunoblotting with the anti-CIB1 antibody. D HEK293 cell were transfected with Flag-CIB1, HA-ubiquitin, with or without Myc-CHIP or Myc -CHIP-ΔUbox. Denatured cell lysates were immunoprecipitated with the anti-HA antibody, followed by immunoblotting with Flag antibodies. E Where specified, purified E1, E2, ubiquitin, CHIP and CIB1 proteins were incubated with in vitro ubiquitination buffers. Reaction samples were analyzed by SDS-polyacrylamide gel electrophoresis, followed by Sliver staining or immunoblotting with the indicated antibodies. F HEK293 cells were transfected for 36 h with Flag-CIB1, Myc-CHIP, HA-Ubiquitin or HA-K48R-ubiquitin or HA-K63R-ubiquitin. Cell extracts were immunoprecipitated with the anti-HA antibody followed by immunoblotting with the anti-Flag antibody.

Article Snippet: 2.5 μl 10× E3 Ligase Reaction Buffer (B-71, Boston Biochem), 1 μl Recombinant Human Ubiquitin Protein (U-100H-10M, Boston Biochem), 10 mM MgATP Solution (B-20, Boston Biochem), 100 nM Recombinant Human His6-Ubiquitin E1 Enzyme (E-304–05, Boston Biochem), 1 μM Recombinant Human UbcH5a/UBE2D1 Protein (E2–616–100, Boston Biochem), 1 μM Recombinant Human CHIP/STUB1 Protein (E3–220–050, Boston Biochem), 5 μM CIB1 Fusion Protein (Ag2391, Protein Tech) was added in a microcentrifuge tube and be incubated in a 37 °C water bath for 60 min. Then the lysis was added by SDS-PAGE sample buffer and followed by electrophoresis.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Purification, Incubation, In Vitro, Polyacrylamide Gel Electrophoresis, Staining

Journal: Cell Death and Differentiation

Article Title: CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

doi: 10.1038/s41418-020-00635-5

Figure Lengend Snippet: A HEK293 cells were co-transfected with HA-ubiquitin and Flag-CIB1 and its corresponding mutated plasmids for 36 h. Cell lysates were subjected to immunoprecipitation with HA antibody, followed by Flag immunoblotting. B HEK293 cells were co-transfected with HA-ubiquitin with HA-ubiquitin, and Flag-CIB1 or Flag-K10R-CIB1 or Flag-K65R-CIB1 or Flag-K10R+K65R-CIB1. Cell lysates were subjected to immunoprecipitation with HA antibody, followed by Flag immunoblotting. C HEK293 cells were co-transfected with Flag-CIB1, Flag-K10R-CIB1, Flag-K65R-CIB1, Flag-K10R+K65R-CIB1 and Myc-CHIP. Cell lysates were analyzed by immunoblotting with MYC, Flag and α-Tublin antibodies. D Computational modeling results show multiple binding sites for ubiquitin on CIB1. The red region is the binding site for CIB1.

Article Snippet: 2.5 μl 10× E3 Ligase Reaction Buffer (B-71, Boston Biochem), 1 μl Recombinant Human Ubiquitin Protein (U-100H-10M, Boston Biochem), 10 mM MgATP Solution (B-20, Boston Biochem), 100 nM Recombinant Human His6-Ubiquitin E1 Enzyme (E-304–05, Boston Biochem), 1 μM Recombinant Human UbcH5a/UBE2D1 Protein (E2–616–100, Boston Biochem), 1 μM Recombinant Human CHIP/STUB1 Protein (E3–220–050, Boston Biochem), 5 μM CIB1 Fusion Protein (Ag2391, Protein Tech) was added in a microcentrifuge tube and be incubated in a 37 °C water bath for 60 min. Then the lysis was added by SDS-PAGE sample buffer and followed by electrophoresis.

Techniques: Transfection, Immunoprecipitation, Western Blot, Binding Assay

Journal: Cell Death and Differentiation

Article Title: CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

doi: 10.1038/s41418-020-00635-5

Figure Lengend Snippet: A , B Representative images and quantification of the Transwell assays using transfected A549/H1299 cells. C , D Quantification of the wound healing assays using transfected A549/H1299 cells. E Western blot assay showing the phosphorylation of AKT and EMT enhanced by CIB1. Restoration of CHIP attenuated this elevation in LAC cells.

Article Snippet: 2.5 μl 10× E3 Ligase Reaction Buffer (B-71, Boston Biochem), 1 μl Recombinant Human Ubiquitin Protein (U-100H-10M, Boston Biochem), 10 mM MgATP Solution (B-20, Boston Biochem), 100 nM Recombinant Human His6-Ubiquitin E1 Enzyme (E-304–05, Boston Biochem), 1 μM Recombinant Human UbcH5a/UBE2D1 Protein (E2–616–100, Boston Biochem), 1 μM Recombinant Human CHIP/STUB1 Protein (E3–220–050, Boston Biochem), 5 μM CIB1 Fusion Protein (Ag2391, Protein Tech) was added in a microcentrifuge tube and be incubated in a 37 °C water bath for 60 min. Then the lysis was added by SDS-PAGE sample buffer and followed by electrophoresis.

Techniques: Transfection, Western Blot

Journal: Cell Death and Differentiation

Article Title: CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

doi: 10.1038/s41418-020-00635-5

Figure Lengend Snippet: A Representative luciferase images and quantification of average luciferase intensity of lungs in the i.v. metastasis assay. B , C Representative photographs and quantification of metastatic tumor nodes in mouse lungs from the i.v. metastasis assay. D Representative immunohistochemically stained images of lung tissue using anti-CIB1, anti-E-cadherin, and N-cadherin antibody. E CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal and tumor metastasis in lung adenocarcinoma through AKT pathway. * P < 0.05 vs negative control (NC) group, ** P < 0.01 vs NC group.

Article Snippet: 2.5 μl 10× E3 Ligase Reaction Buffer (B-71, Boston Biochem), 1 μl Recombinant Human Ubiquitin Protein (U-100H-10M, Boston Biochem), 10 mM MgATP Solution (B-20, Boston Biochem), 100 nM Recombinant Human His6-Ubiquitin E1 Enzyme (E-304–05, Boston Biochem), 1 μM Recombinant Human UbcH5a/UBE2D1 Protein (E2–616–100, Boston Biochem), 1 μM Recombinant Human CHIP/STUB1 Protein (E3–220–050, Boston Biochem), 5 μM CIB1 Fusion Protein (Ag2391, Protein Tech) was added in a microcentrifuge tube and be incubated in a 37 °C water bath for 60 min. Then the lysis was added by SDS-PAGE sample buffer and followed by electrophoresis.

Techniques: Luciferase, Staining, Negative Control

Journal: Cell

Article Title: Phosphoribosylation of Ubiquitin Promotes Serine Ubiquitination and Impairs Conventional Ubiquitination.

doi: 10.1016/j.cell.2016.11.019

Figure Lengend Snippet: Figure 3. SdeA Phosphoribosylates Ubiquitin and Inactivates the Ubiquitin System (A) After modification reactions with SdeA WT, with SdeA H284A, and from control samples, lacking either SdeA or NAD+, ubiquitin was isolated and analyzed by ESI mass spectrometry. Differentially modified ubiquitin as well as unmodified ubiquitin were found in different charge states, so the spectra were deconvoluted with Xtract for further analysis. After treatment with SdeA-WT, 15% of ubiquitin was ADP-ribosylated (ADPR-Ubiquitin), and the rest was phosphoribosylated (P-Rib-Ubiquitin). In contrast, after incubation with PDE mutant SdeA H284A, only 1% of ADPR-Ub was processed to P-Rib-Ub. In control samples, no modified Ub was detectable. (See also Figure S1B). (B) Quantitative analysis of in vitro Ub modification assays. Indicated SdeA proteins were incubated with excess amounts of ubiquitin in the presence or absence of Rab33b. The reaction mixtures were subjected to SDS-PAGE (upper panel) and native-PAGE (lower panel). Ubiquitin modification can be monitored through the altered electrophoretic mobility of modified ubiquitin in native-PAGE. (C) The activity of Parkin was tested in an in vitro ubiquitination assay using either WT ubiquitin, phosphoribosylated ubiquitin (produced by treatment with WT SdeA), or ADP-ribosylated ubiquitin (produced by treatment with H284A SdeA). Reaction mixtures were separated by SDS-PAGE, and poly-Ub chain formation was visualized by Coomassie (SDS gel, upper panel) and western blotting using an anti-ubiquitin antibody (lower panel). See also Figures S3A and S3B. (D) Effect of SdeA on an ongoing ubiquitination reaction was tested. SdeA was added after 10 min of ubiquitination reaction. E1-Ube1, E2-UbcH5a, E3-Trim56 were used in the in vitro ubiquitination reaction. The reaction mixtures were subjected to SDS-PAGE/western blotting and probed with Ub antibody.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Ubiquitin Ubi-1 abcam ab7254; RRID: AB_305802 Ubiquitin Cell Signaling Technology 3936S; RRID: AB_10691572 Actin Sigma-Aldrich A4700; RRID: AB_476730 Vinculin Sigma-Aldrich V4505; RRID: AB_477617 HA Cell Signaling 2367S; RRID: AB_10691311 GFP (B-2) Santa Cruz Biotechnology sc-9996; RRID: AB_627695 K63-Chain Genentech APU3.A8 K48-Chain Genentech APU2.07 Streptavidin-HRP ThermoFischer Scientific 21130 Mono- and poly-ubiquitinylated proteins (FK2) Biomol BML-PW8810; RRID: AB_10541840 Ubiquityl-Histone H2A Millipore 05-678; RRID: AB_309899 Histone H2A Abcam ab18255; RRID: AB_470265 HIF-1a R&D systems MAB1536; RRID: AB_2116983 Chemicals, Peptides, and Recombinant Proteins b-Nicotinamide adenine dinucleotide sodium salt (NAD) Sigma-Aldrich N0632 Biotin-NAD TREVIGEN 4670-500-01 Adenosine 50-triphosphate (ATP) Roth K054.4 Mitotracker Red Deep Red FM ThermoFischer Scientific M22426 Pro-Q Diamond phosphoprotein gel stain ThermoFischer Scientific P33300 Penicillin-streptomycin (10,000 U/mL) ThermoFischer Scientific 15140122 Hygromycin B Invivogen ant-hm-1 Doxycyclin Sigma-Aldrich D9891 Inositol hexakisphosphate Sigma-Aldrich P8810 Isopropyl-b-D-thiogalactopyranoside Roth CN08.4 MBP-PINK1 The University of Dundee DU34701 PARKIN The University of Dundee DU40847 Ubiquitin N-terminal Fluorescein Boston Biochem U-580 UbcH5a (human) Enzo Lifesciences BML-UW9050-0100 Critical Commercial Assays Enzcheck Phosphate assay kit ThermoFischer Scientific E6646 AMP glow assay Promega V5011 Deposited Data Structure of SdeA-modified ubiquitin This study PDB: 5M93 Mass spectrometry data (ubiquitin modification, ubiquitin linkage to substrate serines) This study ProteomeXchange: PXD005240 Experimental Models: Cell Lines HeLa ATCC CCL-2 HEK293T ATCC CRL-3216 Flp-In T-Rex HeLa-HA-Parkin Dikic lab: Richter et al., 2016 N/A Experimental Models: Organisms/Strains Escherichia coli T7 express New England Biolabs C2566H Escherichia coli NEB Turbo New England Biolabs C2984H (Continued on next page) e1 Cell 167, 1636–1649.e1–e7, December 1, 2016

Techniques: Ubiquitin Proteomics, Control, Isolation, Mass Spectrometry, Incubation, Mutagenesis, In Vitro, SDS Page, Clear Native PAGE, Activity Assay, Produced, SDS-Gel, Western Blot

Journal: Cell

Article Title: Phosphoribosylation of Ubiquitin Promotes Serine Ubiquitination and Impairs Conventional Ubiquitination.

doi: 10.1016/j.cell.2016.11.019

Figure Lengend Snippet: Figure 4. Crystal Structure of SdeA-Modified Ubiquitin and Its Implications (A) The crystal structure of phosphoribosylated ubiquitin (shown in yellow) overlaid onto the WT ubiquitin structure (shown in green, PDB: 1UBQ). Phosphor- ibosylated Arg42 and the interacting Arg72 of the modified ubiquitin structure are shown. See also Figure S4A. (B) Purified ubiquitin mutants and WT were incubated with SdeA WT and H284A mutant in the presence of biotin-NAD+. The reaction mix was subjected to SDS- PAGE followed by Coomassie staining (upper-most panel), phosphoprotein staining with Pro-Q Diamond stain (middle panel), and streptavidin-HRP blotting (lower most panel). See also Figure S4C. (C) A pyrophosphate (PPi) release assay was performed with ubiquitin-activating enzyme E1 and WT ubiquitin or modified ubiquitin. The plot shows nanomoles of PPi released over time. See also Figure S4D and S4E. (D) Effect of SdeA-mediated ubiquitin modification on ubiquitin transfer between E1 and E2. E1, E2, and WT ubiquitin or phosphoribosylated ubiquitin were incubated with ATP and NAD+. Transfer of ubiquitin to the catalytic site of E2 was monitored by SDS-PAGE of reaction mixture components followed by Coomassie staining. (E) E2 (HIS-UbcH5a) discharge assay in presence of either unmodified ubiquitin or phosphoribosylated ubiquitin. E2-Ub discharge was induced by addition of the E3 ligase Trim56. The reaction was stopped at the indicated time points and reaction mixtures were analyzed by SDS-PAGE and western blotting.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Ubiquitin Ubi-1 abcam ab7254; RRID: AB_305802 Ubiquitin Cell Signaling Technology 3936S; RRID: AB_10691572 Actin Sigma-Aldrich A4700; RRID: AB_476730 Vinculin Sigma-Aldrich V4505; RRID: AB_477617 HA Cell Signaling 2367S; RRID: AB_10691311 GFP (B-2) Santa Cruz Biotechnology sc-9996; RRID: AB_627695 K63-Chain Genentech APU3.A8 K48-Chain Genentech APU2.07 Streptavidin-HRP ThermoFischer Scientific 21130 Mono- and poly-ubiquitinylated proteins (FK2) Biomol BML-PW8810; RRID: AB_10541840 Ubiquityl-Histone H2A Millipore 05-678; RRID: AB_309899 Histone H2A Abcam ab18255; RRID: AB_470265 HIF-1a R&D systems MAB1536; RRID: AB_2116983 Chemicals, Peptides, and Recombinant Proteins b-Nicotinamide adenine dinucleotide sodium salt (NAD) Sigma-Aldrich N0632 Biotin-NAD TREVIGEN 4670-500-01 Adenosine 50-triphosphate (ATP) Roth K054.4 Mitotracker Red Deep Red FM ThermoFischer Scientific M22426 Pro-Q Diamond phosphoprotein gel stain ThermoFischer Scientific P33300 Penicillin-streptomycin (10,000 U/mL) ThermoFischer Scientific 15140122 Hygromycin B Invivogen ant-hm-1 Doxycyclin Sigma-Aldrich D9891 Inositol hexakisphosphate Sigma-Aldrich P8810 Isopropyl-b-D-thiogalactopyranoside Roth CN08.4 MBP-PINK1 The University of Dundee DU34701 PARKIN The University of Dundee DU40847 Ubiquitin N-terminal Fluorescein Boston Biochem U-580 UbcH5a (human) Enzo Lifesciences BML-UW9050-0100 Critical Commercial Assays Enzcheck Phosphate assay kit ThermoFischer Scientific E6646 AMP glow assay Promega V5011 Deposited Data Structure of SdeA-modified ubiquitin This study PDB: 5M93 Mass spectrometry data (ubiquitin modification, ubiquitin linkage to substrate serines) This study ProteomeXchange: PXD005240 Experimental Models: Cell Lines HeLa ATCC CCL-2 HEK293T ATCC CRL-3216 Flp-In T-Rex HeLa-HA-Parkin Dikic lab: Richter et al., 2016 N/A Experimental Models: Organisms/Strains Escherichia coli T7 express New England Biolabs C2566H Escherichia coli NEB Turbo New England Biolabs C2984H (Continued on next page) e1 Cell 167, 1636–1649.e1–e7, December 1, 2016

Techniques: Ubiquitin Proteomics, Incubation, Mutagenesis, TNKS1 Histone Ribosylation Assay, SDS Page, Staining, Release Assay, Western Blot