Journal: Molecular cell

Article Title: An Epstein-Barr virus protein interaction map reveals NLRP3 inflammasome evasion via MAVS UFMylation

doi: 10.1016/j.molcel.2023.05.018

Figure Lengend Snippet:

Article Snippet: Anti-CNOT10 rabbit polyclonal antibody , Proteintech , cat# 15938-1-AP; RRID: AB_2229678.

Techniques: Recombinant, Magnetic Beads, Transfection, Sequencing, Modification, Mass Spectrometry, Produced, Protease Inhibitor, Selection, Purification, Gel Extraction, SYBR Green Assay, Reporter Assay, Protein Quantitation, CRISPR, Amplification, Molecular Cloning, Plasmid Preparation, Software

Journal: Cell reports

Article Title: The human CNOT1-CNOT10-CNOT11 complex forms a structural platform for protein-protein interactions.

doi: 10.1016/j.celrep.2022.111902

Figure Lengend Snippet: Figure 1. Structure of the N-terminal module of the human CCR4-NOT complex: A structured core with an antenna domain (A) Coomassie-stained SDS-PAGE analysis of the purified recombinant sample of the human CNOT1N-CNOT10-CNOT11 complex used for crystal structure determination. The complex corresponds to the N-terminal module of CCR4-NOT and includes only the N-terminal region of the scaffold protein CNOT1N. (B) Schematic representation of the domain organization of human CNOT1N (yellow), CNOT10 (light green), and CNOT11 (blue). The structured domains are indicated as rectangles and linker regions are shown as black lines. Labeling of the different portions of the proteins corresponds to the structural analysis, as discussed in the text. In particular, the N-terminal, middle, and C-terminal region of CNOT11 are indicated with the corresponding suffix N, M, C. HEAT,35 TPR,36

Article Snippet: GFP fusion proteins were revealed with mouse monoclonal antibody anti-GFP (JL-8, Clontech) used at 1/1000 or 1/5000 dilution, endogenous CNOT10, CNOT7 and CNOT11 proteins were revealed with rabbit polyclonal antiCNOT10 antibody (15938-1- AP, Proteintech) used at 1/1000 dilution or with home-made rabbit anti-CNOT7 or anti-CNOT11 antiserum used at 1/1000 dilution, respectively.

Techniques: Staining, SDS Page, Recombinant, Labeling

Journal: Cell reports

Article Title: The human CNOT1-CNOT10-CNOT11 complex forms a structural platform for protein-protein interactions.

doi: 10.1016/j.celrep.2022.111902

Figure Lengend Snippet: Figure 2. The structured core of the CNOT1N-CNOT10-CNOT11 complex is formed by evolutionary conserved interactions (A–F) Zoom-in views of the major structural interactions in the CNOT1N-CNOT10-CNOT11N-M core of the CCR4-NOT N-terminal module. The individual views are also indicated in the context of the entire structure. (A–C) highlight conserved interactions within the inner layer of the complex comprising the extended CNOT11M region and the CNOT10 TPR superhelix. The interactions with the two outer layers are shown in (D and E) (CNOT1N domain 1, CNOT11, and CNOT10) and (F) (CNOT1N domain 2 and CNOT10). The evolutionary conservation of the interactions is shown in Figures S2A–S2C. (G) Biochemical validation of the structural analysis. Co-immunoprecipitation of endogenous CNOT10 with GFP-tagged CNOT11 truncated proteins. HEK293 cells were transfected with plasmids expressing GFP-tagged human CNOT11 fusion proteins or empty GFP expression vector. Proteins were immunoprecip- itated with GFP-Trap magnetic beads and the co-precipitation was analyzed by western blotting. For some constructs, limited CNOT11 degradation during immunoprecipitation generated additional lower molecular-weight bands.

Article Snippet: GFP fusion proteins were revealed with mouse monoclonal antibody anti-GFP (JL-8, Clontech) used at 1/1000 or 1/5000 dilution, endogenous CNOT10, CNOT7 and CNOT11 proteins were revealed with rabbit polyclonal antiCNOT10 antibody (15938-1- AP, Proteintech) used at 1/1000 dilution or with home-made rabbit anti-CNOT7 or anti-CNOT11 antiserum used at 1/1000 dilution, respectively.

Techniques: Biomarker Discovery, Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Magnetic Beads, Western Blot, Construct, Generated, Molecular Weight

Journal: Cell reports

Article Title: The human CNOT1-CNOT10-CNOT11 complex forms a structural platform for protein-protein interactions.

doi: 10.1016/j.celrep.2022.111902

Figure Lengend Snippet: Figure 1. Structure of the N-terminal module of the human CCR4-NOT complex: A structured core with an antenna domain (A) Coomassie-stained SDS-PAGE analysis of the purified recombinant sample of the human CNOT1N-CNOT10-CNOT11 complex used for crystal structure determination. The complex corresponds to the N-terminal module of CCR4-NOT and includes only the N-terminal region of the scaffold protein CNOT1N. (B) Schematic representation of the domain organization of human CNOT1N (yellow), CNOT10 (light green), and CNOT11 (blue). The structured domains are indicated as rectangles and linker regions are shown as black lines. Labeling of the different portions of the proteins corresponds to the structural analysis, as discussed in the text. In particular, the N-terminal, middle, and C-terminal region of CNOT11 are indicated with the corresponding suffix N, M, C. HEAT,35 TPR,36

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-CNOT10 Proteintech Cat# 15938-1-AP; RRID:AB_2229678 Mouse monoclonal anti-GFP (clone JL-8) Takara Cat# 632381; RRID:AB_2313808 Rabbit polyclonal anti-CNOT11 (Mauxion et al.)12 N/A Rabbit polyclonal anti-CNOT7 (Mauxion et al.)12 N/A Bacterial and virus strains Escherichia coli BL21 (DE3) STAR pRARE Stratagene N/A Escherichia coli B834 Sigma-Aldrich Cat# 69041 Chemicals, peptides, and recombinant proteins Protein Assay Dye Reagent Concentrate Bio-Rad Cat# 5000006 ChromoTek GFP-Trap Magnetic Agarose Proteintech Cat# gtma ChromoTek GFP-Trap Magnetic Particles M-270 Proteintech Cat# gtd Immobilon Western HRP Substrate MerckMillipore Cat# WBKLS0100 Luminata Crescendo Western HRP Substrate MerckMillipore Cat# WBLUR0100 IgG Sepharose 6 Fast Flow Affinity Resin Cytiva Cat# 17096901 AcTEV protease Invitrogen Cat# 12575-015 Calmodulin Affinity Resin Agilent Cat# 214303-52 cOmplete protease inhibitor cocktail, EDTA-free Roche SKU# 5056489001 H. sapiens CNOT1N (residues 1-687) + N-terminal His-SUMO-tag fusion protein cleavable with SENP2 This study N/A H. sapiens CNOT10 (residues 1-714) + N-terminal His-Thioredoxin-tag fusion proteins cleavable with 3C protease This study N/A H. sapiens CNOT11 (residues 61-510) + N-terminal His-Thioredoxin-tag fusion proteins cleavable with 3C protease This study N/A H. sapiens CNOT11C (residues 325-510) + N-terminal His-SUMO-tag fusion protein cleavable with SENP2 This study N/A H. sapiens CNOT11C-GGNBP2 (residues 638-673) + N-terminal His-SUMO-tag fusion protein cleavable with SENP2 This study N/A SENP Sigma-Aldrich Cat# SAE0067 3C Protease Sigma-Aldrich Cat# SAE0045 Critical commercial assays Effectene Transfection Reagent Qiagen Cat# 301427 Beta-Glo Assay System Promega Cat# E4720 Deposited data CNOT1N-CNOT10-CNOT11 This paper PDB: 8BFI CNOT11C This paper PDB: 8BFH partial CNOT11C-GGNBP2 (residues 638-673) This paper PDB: 8BFJ Experimental models: Cell lines Human embryonic kidney 293 (HEK293) ATCC CRL1573; RRID:CVCL_0045 (Continued on next page) 14 Cell Reports 42, 111902, January 31, 2023

Techniques: Staining, SDS Page, Recombinant, Labeling

Journal: Cell reports

Article Title: The human CNOT1-CNOT10-CNOT11 complex forms a structural platform for protein-protein interactions.

doi: 10.1016/j.celrep.2022.111902

Figure Lengend Snippet: Figure 2. The structured core of the CNOT1N-CNOT10-CNOT11 complex is formed by evolutionary conserved interactions (A–F) Zoom-in views of the major structural interactions in the CNOT1N-CNOT10-CNOT11N-M core of the CCR4-NOT N-terminal module. The individual views are also indicated in the context of the entire structure. (A–C) highlight conserved interactions within the inner layer of the complex comprising the extended CNOT11M region and the CNOT10 TPR superhelix. The interactions with the two outer layers are shown in (D and E) (CNOT1N domain 1, CNOT11, and CNOT10) and (F) (CNOT1N domain 2 and CNOT10). The evolutionary conservation of the interactions is shown in Figures S2A–S2C. (G) Biochemical validation of the structural analysis. Co-immunoprecipitation of endogenous CNOT10 with GFP-tagged CNOT11 truncated proteins. HEK293 cells were transfected with plasmids expressing GFP-tagged human CNOT11 fusion proteins or empty GFP expression vector. Proteins were immunoprecip- itated with GFP-Trap magnetic beads and the co-precipitation was analyzed by western blotting. For some constructs, limited CNOT11 degradation during immunoprecipitation generated additional lower molecular-weight bands.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-CNOT10 Proteintech Cat# 15938-1-AP; RRID:AB_2229678 Mouse monoclonal anti-GFP (clone JL-8) Takara Cat# 632381; RRID:AB_2313808 Rabbit polyclonal anti-CNOT11 (Mauxion et al.)12 N/A Rabbit polyclonal anti-CNOT7 (Mauxion et al.)12 N/A Bacterial and virus strains Escherichia coli BL21 (DE3) STAR pRARE Stratagene N/A Escherichia coli B834 Sigma-Aldrich Cat# 69041 Chemicals, peptides, and recombinant proteins Protein Assay Dye Reagent Concentrate Bio-Rad Cat# 5000006 ChromoTek GFP-Trap Magnetic Agarose Proteintech Cat# gtma ChromoTek GFP-Trap Magnetic Particles M-270 Proteintech Cat# gtd Immobilon Western HRP Substrate MerckMillipore Cat# WBKLS0100 Luminata Crescendo Western HRP Substrate MerckMillipore Cat# WBLUR0100 IgG Sepharose 6 Fast Flow Affinity Resin Cytiva Cat# 17096901 AcTEV protease Invitrogen Cat# 12575-015 Calmodulin Affinity Resin Agilent Cat# 214303-52 cOmplete protease inhibitor cocktail, EDTA-free Roche SKU# 5056489001 H. sapiens CNOT1N (residues 1-687) + N-terminal His-SUMO-tag fusion protein cleavable with SENP2 This study N/A H. sapiens CNOT10 (residues 1-714) + N-terminal His-Thioredoxin-tag fusion proteins cleavable with 3C protease This study N/A H. sapiens CNOT11 (residues 61-510) + N-terminal His-Thioredoxin-tag fusion proteins cleavable with 3C protease This study N/A H. sapiens CNOT11C (residues 325-510) + N-terminal His-SUMO-tag fusion protein cleavable with SENP2 This study N/A H. sapiens CNOT11C-GGNBP2 (residues 638-673) + N-terminal His-SUMO-tag fusion protein cleavable with SENP2 This study N/A SENP Sigma-Aldrich Cat# SAE0067 3C Protease Sigma-Aldrich Cat# SAE0045 Critical commercial assays Effectene Transfection Reagent Qiagen Cat# 301427 Beta-Glo Assay System Promega Cat# E4720 Deposited data CNOT1N-CNOT10-CNOT11 This paper PDB: 8BFI CNOT11C This paper PDB: 8BFH partial CNOT11C-GGNBP2 (residues 638-673) This paper PDB: 8BFJ Experimental models: Cell lines Human embryonic kidney 293 (HEK293) ATCC CRL1573; RRID:CVCL_0045 (Continued on next page) 14 Cell Reports 42, 111902, January 31, 2023

Techniques: Biomarker Discovery, Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Magnetic Beads, Western Blot, Construct, Generated, Molecular Weight

Journal: Cell Reports

Article Title: The human CNOT1-CNOT10-CNOT11 complex forms a structural platform for protein-protein interactions

doi: 10.1016/j.celrep.2022.111902

Figure Lengend Snippet: Structure of the N-terminal module of the human CCR4-NOT complex: A structured core with an antenna domain (A) Coomassie-stained SDS-PAGE analysis of the purified recombinant sample of the human CNOT1 N -CNOT10-CNOT11 complex used for crystal structure determination. The complex corresponds to the N-terminal module of CCR4-NOT and includes only the N-terminal region of the scaffold protein CNOT1 N . (B) Schematic representation of the domain organization of human CNOT1 N (yellow), CNOT10 (light green), and CNOT11 (blue). The structured domains are indicated as rectangles and linker regions are shown as black lines. Labeling of the different portions of the proteins corresponds to the structural analysis, as discussed in the text. In particular, the N-terminal, middle, and C-terminal region of CNOT11 are indicated with the corresponding suffix N, M, C. HEAT, TPR, MIF4G ; C9BD (CNOT9 binding domain). (C) Crystal structure of the CNOT1 N -CNOT10-CNOT11 complex in two different orientations, colored and labeled as in (B). The model of the structured core (CNOT1 N -CNOT10-CNOT11 M -N ) is refined to 3.1 Å resolution. Due to the apparent flexible connection to the core, the model of the “antenna” domain (CNOT11 C ) was determined separately at 2.2 Å resolution and fitted in the density of the ternary complex.

Article Snippet: GFP fusion proteins were revealed with mouse monoclonal antibody anti-GFP (JL-8, Clontech) used at 1/1000 or 1/5000 dilution, endogenous CNOT10, CNOT7 and CNOT11 proteins were revealed with rabbit polyclonal anti-CNOT10 antibody (15938-1- AP, Proteintech) used at 1/1000 dilution or with home-made rabbit anti-CNOT7 or anti-CNOT11 antiserum used at 1/1000 dilution, respectively.

Techniques: Staining, SDS Page, Purification, Recombinant, Labeling, Binding Assay

Journal: Cell Reports

Article Title: The human CNOT1-CNOT10-CNOT11 complex forms a structural platform for protein-protein interactions

doi: 10.1016/j.celrep.2022.111902

Figure Lengend Snippet: The structured core of the CNOT1 N -CNOT10-CNOT11 complex is formed by evolutionary conserved interactions (A–F) Zoom-in views of the major structural interactions in the CNOT1 N -CNOT10-CNOT11 N -M core of the CCR4-NOT N-terminal module. The individual views are also indicated in the context of the entire structure. (A–C) highlight conserved interactions within the inner layer of the complex comprising the extended CNOT11 M region and the CNOT10 TPR superhelix. The interactions with the two outer layers are shown in (D and E) (CNOT1 N domain 1, CNOT11, and CNOT10) and (F) (CNOT1 N domain 2 and CNOT10). The evolutionary conservation of the interactions is shown in Figures S2 A–S2C. (G) Biochemical validation of the structural analysis. Co-immunoprecipitation of endogenous CNOT10 with GFP-tagged CNOT11 truncated proteins. HEK293 cells were transfected with plasmids expressing GFP-tagged human CNOT11 fusion proteins or empty GFP expression vector. Proteins were immunoprecipitated with GFP-Trap magnetic beads and the co-precipitation was analyzed by western blotting. For some constructs, limited CNOT11 degradation during immunoprecipitation generated additional lower molecular-weight bands.

Article Snippet: GFP fusion proteins were revealed with mouse monoclonal antibody anti-GFP (JL-8, Clontech) used at 1/1000 or 1/5000 dilution, endogenous CNOT10, CNOT7 and CNOT11 proteins were revealed with rabbit polyclonal anti-CNOT10 antibody (15938-1- AP, Proteintech) used at 1/1000 dilution or with home-made rabbit anti-CNOT7 or anti-CNOT11 antiserum used at 1/1000 dilution, respectively.

Techniques: Biomarker Discovery, Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Magnetic Beads, Western Blot, Construct, Generated, Molecular Weight

Journal: Cell Reports

Article Title: The human CNOT1-CNOT10-CNOT11 complex forms a structural platform for protein-protein interactions

doi: 10.1016/j.celrep.2022.111902

Figure Lengend Snippet: The antenna domain of CNOT11 mediates the recruitment of GGNBP2 to CCR4-NOT (A) CNOT11 C protein partners from a human placenta cDNA library identified in a yeast two-hybrid screen. The number of clones encoding fusions with different ORF is shown. The common region found in the different clones encoding CNOT11 C partners is: BRAP (113–490), EDRF1 (283–595), FAM193A (1,155–1,240), FAM193B (680–822), GGNBP2 (483–697), and GPBP1L1 (307–462). An arrow marks GGNBP2 also identified by affinity purification-mass spectrometry. (B) Identification of proteins co-purifying with the N-terminus of CNOT11 by mass spectrometry. HEK293 cells were transfected with plasmids expressing TAP-tagged CNOT11 fusion proteins that were purified by two tandem affinity purification steps. Co-purified proteins were identified by mass spectrometry. Proteins found in control purification (such as keratins) were filtered out. The average number of Peptide-Spectrum Match (PSM) and standard deviation for the top 14 identified proteins in three mass spectrometry replicates for CNOT11 N -M-C and CNOT11 N -M are shown. (C) Identification of proteins co-purifying with the C-terminus of CNOT11 by mass spectrometry. Protocol similar to (B). The average number of Peptide-Spectrum Match (PSM) and standard deviation for the top 6 identified proteins in three mass spectrometry replicates for CNOT11 N -M-C and CNOT11 C are shown. An arrow marks GGNBP2, also identified in the two-hybrid screen. (D) Co-immunoprecipitation of endogenous CNOT7, CNOT10, and CNOT11 with GFP-tagged GGNBP2 proteins. HEK293 cells were transfected with plasmids expressing GFP-tagged human GGNBP2 fusion proteins (numbers in parentheses indicate amino acids included, FL = Full-Length), or empty GFP expression vector. Proteins were immunoprecipitated with GFP-Trap magnetic agarose beads and the co-precipitation was analyzed by western blotting on 8% PAGE. Note that CNOT11 always appears as multiple bands in cell lysates while overexpressed GFP-GGNBP2 often appears slightly degraded. (E) Identification of proteins co-purifying with GFP-GGNBP2 by mass spectrometry. The plot compares the average number of PSM for each protein obtained in purification with GFP-GGNBP2FL (y axis) versus control purifications with GFP alone and GFP-GGNBP2(1-482) (x axis). GGNBP2 is indicated in red while subunits of the CCR4-NOT complex and partners are indicated in blue.

Article Snippet: GFP fusion proteins were revealed with mouse monoclonal antibody anti-GFP (JL-8, Clontech) used at 1/1000 or 1/5000 dilution, endogenous CNOT10, CNOT7 and CNOT11 proteins were revealed with rabbit polyclonal anti-CNOT10 antibody (15938-1- AP, Proteintech) used at 1/1000 dilution or with home-made rabbit anti-CNOT7 or anti-CNOT11 antiserum used at 1/1000 dilution, respectively.

Techniques: cDNA Library Assay, Two Hybrid Screening, Clone Assay, Affinity Purification, Mass Spectrometry, Transfection, Expressing, Purification, Control, Standard Deviation, Immunoprecipitation, Plasmid Preparation, Western Blot

Journal: Cell Reports

Article Title: The human CNOT1-CNOT10-CNOT11 complex forms a structural platform for protein-protein interactions

doi: 10.1016/j.celrep.2022.111902

Figure Lengend Snippet: The CNOT11 antenna domain recognizes the C-terminal segment of GGNBP2 (A) Structure-based sequence alignment of CNOT11-binding domain of GGNBP2. Highlighted in dark and light orange are residues with high and medium conservation across the species shown. A schematic representation shows the position of the two α-helices with respect to the amino acid sequence. (B) Co-immunoprecipitation of endogenous CNOT11 with GFP-tagged truncated GGNBP2 proteins. HEK293 cells were transfected with plasmids expressing GFP-tagged human GGNBP2 fusion proteins (numbers in parentheses indicate amino acids included, FL = Full-Length, GGNBP2 C-terminal residues were replaced by an HA tag in the GFP-GGNBP2(1-625)-HA construction). Proteins were immunoprecipitated with GFP-Trap magnetic agarose beads and the co-precipitation was analyzed by western blotting. Note that CNOT11 always appears as multiple bands in cell lysates. (C) Reconstitution of the CNOT11-GGNBP2 interaction in vitro with recombinant proteins. Chromatogram from size-exclusion chromatography and corresponding Coomassie-stained SDS-PAGE analysis of the peak fraction shows the presence of a complex between CNOT11 C and two C-terminal segments of GGNBP2: residues 626–697 and the truncation mutant encompassing residues 638–673 that was still able to interact with CNOT11 C and that was used for the crystal structure determination of the complex. (D) Overall structure of the complex between CNOT11 C (in blue) and GGNBP2 C (in orange, the atomic model from the crystal structure; in red, superposed the model from AlphaFold predictions, Figure S4 A). The HEAT repeats of CNOT11 C are labeled, as well as the GGNBP2 C helices (α0 and α1). On the right, the CNOT11 C - GGNBP2 C complex (atomic model from the crystal structure) is positioned in the CNOT1 N -CNOT10-CNOT11 module.

Article Snippet: GFP fusion proteins were revealed with mouse monoclonal antibody anti-GFP (JL-8, Clontech) used at 1/1000 or 1/5000 dilution, endogenous CNOT10, CNOT7 and CNOT11 proteins were revealed with rabbit polyclonal anti-CNOT10 antibody (15938-1- AP, Proteintech) used at 1/1000 dilution or with home-made rabbit anti-CNOT7 or anti-CNOT11 antiserum used at 1/1000 dilution, respectively.

Techniques: Sequencing, Binding Assay, Immunoprecipitation, Transfection, Expressing, Western Blot, In Vitro, Recombinant, Size-exclusion Chromatography, Staining, SDS Page, Mutagenesis, Labeling

Journal: Cell Reports

Article Title: The human CNOT1-CNOT10-CNOT11 complex forms a structural platform for protein-protein interactions

doi: 10.1016/j.celrep.2022.111902

Figure Lengend Snippet:

Article Snippet: GFP fusion proteins were revealed with mouse monoclonal antibody anti-GFP (JL-8, Clontech) used at 1/1000 or 1/5000 dilution, endogenous CNOT10, CNOT7 and CNOT11 proteins were revealed with rabbit polyclonal anti-CNOT10 antibody (15938-1- AP, Proteintech) used at 1/1000 dilution or with home-made rabbit anti-CNOT7 or anti-CNOT11 antiserum used at 1/1000 dilution, respectively.

Techniques: Virus, Recombinant, Western Blot, Protease Inhibitor, Transfection, Software

Journal: Cell Reports

Article Title: The human CNOT1-CNOT10-CNOT11 complex forms a structural platform for protein-protein interactions

doi: 10.1016/j.celrep.2022.111902

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-CNOT10 , Proteintech , Cat# 15938-1-AP; RRID: AB_2229678.

Techniques: Virus, Recombinant, Western Blot, Protease Inhibitor, Transfection, Software