cbs domain  (Addgene inc)

Journal: bioRxiv

Article Title: Specific proteolysis mediated by a p97-directed proteolysis-targeting chimera (PROTAC)

doi: 10.1101/2024.03.08.584142

Figure Lengend Snippet: A) Diagram of p97 presenting ubiquitinated proteins to the proteasome via a UBX domain contacting adaptor (Top). p97-PROTAC system, consisting of a UBX domain fused to a nanobody (Nb) that recruits substrates for p97-mediated segregation, unfolding and proteasomal mediated degradation (Bottom). B) p97-PROTAC (UBX-Nb (GFP) ) recognizes GFP tagged proteins at different cellular locations. HeLa cells were seeded on coverslip and co-transfected with UBX-Nb (GFP) and GFP-Colin, GFP-Emerin and GFP-ETV1. Cells were fixed and we performed immunofluorescence for anti-myc tag to verify the expression of UBX-Nb (GFP) and evaluated the colocalization. C) Western blot analysis of GFP-Coilin degradation by transfection with p97-PROTAC (UBX-Nb (GFP) ), D) quantification of C;. E) Western blot analysis GFP-Emerin degradation by transfection with p97-PROTAC (UBX-Nb (GFP) ), F) quantification of E;. G) Western blot analysis GFP-ETV1 degradation by transfection with p97-PROTAC (UBX-Nb (GFP) ), D) quantification of G; Western blots were quantified and statistically analyzed using a student’s t-test. P < 0.05 compared to controls. n=3.

Article Snippet: The inhibitors used in this work were: MG132, a proteasome inhibitor from Sigma-Aldrich (catalog number 474790); PYR-41, an E1 ligase inhibitor from Sigma-Aldrich (catalog number N2915); and CB-5085, a D2 domain inhibitor of p97 from Cayman Chemical.

Techniques: Transfection, Immunofluorescence, Expressing, Western Blot

Journal: bioRxiv

Article Title: Specific proteolysis mediated by a p97-directed proteolysis-targeting chimera (PROTAC)

doi: 10.1101/2024.03.08.584142

Figure Lengend Snippet: (A) Strategy for inserting a YFP tag on the N-terminus of the 53BP1 gene in U2OS SEC-C cells using CRISPR/Cas9 D10A. B) Selected Knock-In (KI) YFP-53BP1 clones isolated via flow cytometry. Clones were confirmed via florescence microscopy (GE Deltavision Widefield). C) Super-resolution images obtained with a Delta Vision OMX V4 structured illumination microscope (3D-SIM). D) Immunofluorescence against 53BP1 (red) and colocalization with YFP-53BP1 in Knock-In cells. Images were obtained using a Delta Vision OMX V4 structured illumination microscope (3D-SIM). E) Recruitment of the p97-PROTAC UBX-Nb (GFP) (Red) to YFP-53BP1 (green) within Liquid-liquid phase separation structures. Data were obtained with a High-content Celldiscoverer 7. UBX-Nb (GFP) was detected using its myc-tag. F) Western blot analysis YFP-53BP1 degradation by UBX-Nb (GFP) transfection in the knock-In U2OS cells, G) Quantification of F. Western blots were quantified and statistically analyzed using a student’s t-test. P < 0.05 compared to controls. n=3.

Article Snippet: The inhibitors used in this work were: MG132, a proteasome inhibitor from Sigma-Aldrich (catalog number 474790); PYR-41, an E1 ligase inhibitor from Sigma-Aldrich (catalog number N2915); and CB-5085, a D2 domain inhibitor of p97 from Cayman Chemical.

Techniques: CRISPR, Knock-In, Clone Assay, Isolation, Flow Cytometry, Microscopy, Immunofluorescence, Western Blot, Transfection

Journal: bioRxiv

Article Title: Specific proteolysis mediated by a p97-directed proteolysis-targeting chimera (PROTAC)

doi: 10.1101/2024.03.08.584142

Figure Lengend Snippet: Immunohistochemistry against p97 in brain tissue sections from Non-Human Primates (Nhp) Macaca fascicularis , rat ( Sprague Dawley ) and mouse (C57BL6/C). p97 expression was detected in substantia nigra (SNpc), hippocampal, and cortical neurons. n=4

Article Snippet: The inhibitors used in this work were: MG132, a proteasome inhibitor from Sigma-Aldrich (catalog number 474790); PYR-41, an E1 ligase inhibitor from Sigma-Aldrich (catalog number N2915); and CB-5085, a D2 domain inhibitor of p97 from Cayman Chemical.

Techniques: Immunohistochemistry, Expressing

Journal: bioRxiv

Article Title: Specific proteolysis mediated by a p97-directed proteolysis-targeting chimera (PROTAC)

doi: 10.1101/2024.03.08.584142

Figure Lengend Snippet: A) Model representations of the FAF1 UBX domain (purple), UBX-Nb (GFP) (blue), GFP (green), and the p97 hexamer (light grey cartoon with semi-transparent molecular surface representation). B) A magnified view of the model shown in A. C) GFP monomer was co-transfected with UBX-Nb (GFP) or empty vector in HeLa cells. Protein degradation was analyzed by western blot analysis. D) Quantification of C. E) GFP monomer was co-transfected with UBX-Nb (GFP) or empty vector in HeLa cells, after 24 h were incubated with DMSO or the proteasome inhibitor MG132 (25 uM final concentration) for 4h. Protein degradation was analyzed by western blot. F) Quantification of E. G) p97 was silenced by transfection with p97-siRNA in HeLa cells. Subsequently, the cells were transfected with GFP-Emerin and either the empty vector or UBX-Nb (GFP) vector. Protein degradation was analyzed western blot analysis. H) Quantification of GFP-Emerin in cells treated with the UBX-Nb (GFP) vector in HeLa cells treated with either and siNT control or an SiRNA p97 siRNA. I) Quantification of the UBX-Nb (GFP) in cells treated with the UBX-Nb (GFP) vector in HeLa cells treated with either and siNT control or an SiRNA p97 J) HeLa cells co-transfected with GFP-Emerin and either empty vector or UBX-Nb (GFP) in addition the cells were treated with the E1 ubiquitin inhibitor PYR-41 (50 µM) for 4 hours at 37°C. Subsequently, total proteins were extracted, and protein degradation was analyzed by western blot. K) Quantification of J. L) GFP-Emerin was co-transfected with UBX-Nb (GFP) or empty vector in HeLa cells, after 24 h the cells were incubated with DMSO (as control) or the p97 inhibitor CB-5083 (4 uM final concentration) for 6h. Protein degradation was analyzed by western blot using total proteins. M) Quantification of L. Western blots were quantified and statistically analyzed using a student’s t-test. P < 0.05 compared to controls. n=3.

Article Snippet: The inhibitors used in this work were: MG132, a proteasome inhibitor from Sigma-Aldrich (catalog number 474790); PYR-41, an E1 ligase inhibitor from Sigma-Aldrich (catalog number N2915); and CB-5085, a D2 domain inhibitor of p97 from Cayman Chemical.

Techniques: Transfection, Plasmid Preparation, Western Blot, Incubation, Concentration Assay

Journal: bioRxiv

Article Title: Specific proteolysis mediated by a p97-directed proteolysis-targeting chimera (PROTAC)

doi: 10.1101/2024.03.08.584142

Figure Lengend Snippet: HeLa cells were transiently co-transfected with HTT GFP-tagged plasmids containing either 23 CAG repeats (GFP-HTT Q23 - wild-type HTT) or 74 CAG repeats (GFP-HTT Q74 : mutant HTT) A) HeLa cells co-transfected with GFP-HTT Q23 and increasing amount of the p97 PROTAC UBX-Nb (GFP) , degradation was determined by western blot analysis B) quantification of A; C) colocalization of GFP-HTT Q23 and p97 PROTAC UBX-Nb (GFP) D) HeLa cells were co-transfected with GFP-HTT Q74 and increasing amount of the p97 PROTAC UBX-Nb (GFP) , degradation was determined by western blot analysis E) quantification of D; F) colocalization of GFP-HTT Q23 and p97 PROTAC UBX-Nb (GFP) . Western blots were quantified and statistically analyzed using a student’s t-test. P < 0.05 compared to controls. n=3.

Article Snippet: The inhibitors used in this work were: MG132, a proteasome inhibitor from Sigma-Aldrich (catalog number 474790); PYR-41, an E1 ligase inhibitor from Sigma-Aldrich (catalog number N2915); and CB-5085, a D2 domain inhibitor of p97 from Cayman Chemical.

Techniques: Transfection, Mutagenesis, Western Blot

Journal: bioRxiv

Article Title: Specific proteolysis mediated by a p97-directed proteolysis-targeting chimera (PROTAC)

doi: 10.1101/2024.03.08.584142

Figure Lengend Snippet: A) HeLa cells were co-transfected with a vector expressing αSynuclein mutant A53T fused to GFP (GFP-αSynuclein A53T) and empty or increasing concentrations of UBX-Nb (GFP) . A53T-GFP degradation was determined by Western blot. B) Quantification of A. C) Cells were co-transfected with a vector expressing untagged alpha synuclein mutant A53T and an empty vector or increasing concentrations of UBX-Nb (Syn87) . Untagged αSynuclein A53T degradation was determined by Western blot using an anti αSynuclein antibody . D) Quantification of C. E) Representative model of p97-PROTAC functioning in the degradation of proteins and protein aggregates. Western blots were quantified and statistically analyzed using a student’s t-test. P < 0.05 compared to controls. n=3.

Article Snippet: The inhibitors used in this work were: MG132, a proteasome inhibitor from Sigma-Aldrich (catalog number 474790); PYR-41, an E1 ligase inhibitor from Sigma-Aldrich (catalog number N2915); and CB-5085, a D2 domain inhibitor of p97 from Cayman Chemical.

Techniques: Transfection, Plasmid Preparation, Expressing, Mutagenesis, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Disease-causing cystathionine β-synthase linker mutations impair allosteric regulation

doi: 10.1016/j.jbc.2023.105449

Figure Lengend Snippet: Comparison of L-serine-dependent cystathionine and cysteine-dependent H 2 S synthesis specific activities by wild-type and linker variants of human CBS a

Article Snippet: Human CBS carrying a 10 amino acid deletion (residues 516–525) in the regulatory domain was purchased from GenScript and cloned into the pET28b+ vector incorporating a cleavable C-terminal 6-His tag.

Techniques: Comparison

Journal: The Journal of Biological Chemistry

Article Title: Disease-causing cystathionine β-synthase linker mutations impair allosteric regulation

doi: 10.1016/j.jbc.2023.105449

Figure Lengend Snippet: Structures of dimeric human CBS in the basal and activated conformations. A , scheme showing the intersection between the methionine cycle ( grey ) and the transsulfuration pathway ( black ). For clarity, only the dominant H 2 S-generating reactions catalyzed by CBS and CTH are shown in red . α-KB is α-ketobutyrate. B , modular organization of CBS. C and D , the basal (PDB: 4COO , C ) and activated (PDB: 4PCU , D ) conformations of the CBS Δ516–525 dimer are shown using the same color scheme as in B . The two subunits are shown in dark and light shades. Heme, PLP, and AdoMet (in the activated conformation) are shown in red , yellow , and cyan spheres , respectively. E , schematic representation of the large conformational changes seen in the crystal structure of the CBS Δ516–525 dimer and the cryo-EM structure of the native fibrillar form. AdoMet either triggers or stabilizes the activated conformation in which the regulatory domains dimerize atop the catalytic cores, dramatically changing the organization of the fibrillar form.

Article Snippet: Human CBS carrying a 10 amino acid deletion (residues 516–525) in the regulatory domain was purchased from GenScript and cloned into the pET28b+ vector incorporating a cleavable C-terminal 6-His tag.

Techniques: Cryo-EM Sample Prep

Journal: Molecular Genetics & Genomic Medicine

Article Title: Correlation between large FBN1 deletions and severe cardiovascular phenotype in Marfan syndrome: Analysis of two novel cases and analytical review of the literature

doi: 10.1002/mgg3.2166

Figure Lengend Snippet: Summary of MFS patients with single‐exon deletion in FBN1 gene.

Article Snippet: This specific group had mutations affecting or creating cysteine residues and in‐frame deletion variants in the cb‐EGF domains of exons 25–36 and 43–49 (Takeda, Hara, et al., ; Takeda, Inuzuka, et al., ).

Techniques: Sequencing, Microarray

Journal: Molecular Genetics & Genomic Medicine

Article Title: Correlation between large FBN1 deletions and severe cardiovascular phenotype in Marfan syndrome: Analysis of two novel cases and analytical review of the literature

doi: 10.1002/mgg3.2166

Figure Lengend Snippet: Summary of MFS patients with multiple exon deletions in FBN1 gene.

Article Snippet: This specific group had mutations affecting or creating cysteine residues and in‐frame deletion variants in the cb‐EGF domains of exons 25–36 and 43–49 (Takeda, Hara, et al., ; Takeda, Inuzuka, et al., ).

Techniques: Microarray, Sequencing, Long Range PCR, Southern Blot, Western Blot

Journal: Molecular Genetics & Genomic Medicine

Article Title: Correlation between large FBN1 deletions and severe cardiovascular phenotype in Marfan syndrome: Analysis of two novel cases and analytical review of the literature

doi: 10.1002/mgg3.2166

Figure Lengend Snippet: (a, b) Results of semiquantitative MLPA analyses. Normalized relative peak areas measured with P065 and P066 kits. (a) Reduced relative peak areas of FBN1 exon 46. (b) Reduced relative peak areas of FBN1 exon 47. Combined results from P065 and P066 MLPA kit indicate the heterozygous deletion of exons 46–47.

Article Snippet: This specific group had mutations affecting or creating cysteine residues and in‐frame deletion variants in the cb‐EGF domains of exons 25–36 and 43–49 (Takeda, Hara, et al., ; Takeda, Inuzuka, et al., ).

Techniques:

Journal: Molecular Genetics & Genomic Medicine

Article Title: Correlation between large FBN1 deletions and severe cardiovascular phenotype in Marfan syndrome: Analysis of two novel cases and analytical review of the literature

doi: 10.1002/mgg3.2166

Figure Lengend Snippet: Summary of transcription factor‐binding site analyses of FBN1 gene. TFBS analyses was performed in our patient and published CNV patients carrying single exon deletion with known genomic localization or few exon deletions.

Article Snippet: This specific group had mutations affecting or creating cysteine residues and in‐frame deletion variants in the cb‐EGF domains of exons 25–36 and 43–49 (Takeda, Hara, et al., ; Takeda, Inuzuka, et al., ).

Techniques:

Journal: Molecular Genetics & Genomic Medicine

Article Title: Correlation between large FBN1 deletions and severe cardiovascular phenotype in Marfan syndrome: Analysis of two novel cases and analytical review of the literature

doi: 10.1002/mgg3.2166

Figure Lengend Snippet: Localizations of TFBSs within the deleted region of FBN1 gene as found in our patient. Data illustrated in GRCh37 reference genome. Black rectangles represent exons of the FBN1 gene, amber markings represent regulatory elements.

Article Snippet: This specific group had mutations affecting or creating cysteine residues and in‐frame deletion variants in the cb‐EGF domains of exons 25–36 and 43–49 (Takeda, Hara, et al., ; Takeda, Inuzuka, et al., ).

Techniques:

Journal: Scientific Reports

Article Title: A FRET-based screening method to detect potential inhibitors of the binding of CNNM3 to PRL2

doi: 10.1038/s41598-020-69818-x

Figure Lengend Snippet: CNNM3-YPet binding to CyPet-PRL2 detected by FRET. FRET data regarding the binding of CNNM3-YPet to CyPet-PRL2. R 2 = 0.9588, n = 6.

Article Snippet: The plasmid constructs for the YPet-tagged CNNM3 CBS domain and CyPet-tagged PRL2 have been deposited into AddGene ( https://www.addgene.org/ ) (Addgene IDs: 149686 and 149685).

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: A FRET-based screening method to detect potential inhibitors of the binding of CNNM3 to PRL2

doi: 10.1038/s41598-020-69818-x

Figure Lengend Snippet: Inhibition of the binding of CNNM3-YPet and CyPet-PRL2 using non-YPet-tagged wild-type CNNM3 CBS and mutated CNNM3 CBS. ( A ) Close-up view of the CNNM3-PRL binding site (PDB: 5K22). ( B ) Non-YPet-tagged CNNM3 CBS-based inhibition test of the binding of CNNM3-YPet (200 nM) and CyPet-PRL2 (200 nM) using FRET. All data are expressed as the mean ± SE. * p < 0.05, **** p < 0.0001, by Tukey’s multiple comparison test, n = 6.

Article Snippet: The plasmid constructs for the YPet-tagged CNNM3 CBS domain and CyPet-tagged PRL2 have been deposited into AddGene ( https://www.addgene.org/ ) (Addgene IDs: 149686 and 149685).

Techniques: Inhibition, Binding Assay

Journal: Scientific Reports

Article Title: A FRET-based screening method to detect potential inhibitors of the binding of CNNM3 to PRL2

doi: 10.1038/s41598-020-69818-x

Figure Lengend Snippet: Inhibition of the binding of CNNM3-YPet and CyPet-PRL2 using non-CyPet-tagged PRL1 and PRL2. ( A ) Amino acid sequence alignment of human PRL1 (BAG70128.1) and human PRL2 (NP_001356788.1). Red boxes, identical residues; red letters, similar residues. ( B ) Structural comparison of PRL1 (blue, PDB: 1XM2) and PRL2 (orange, PDB: 5K22). ( C ) Non-CyPet PRL-based inhibition test of the binding of CNNM3-YPet (200 nM) and CyPet-PRL2 (200 nM) using FRET. All data are expressed as the mean ± SE. *** p < 0.001; **** p < 0.0001, by Tukey’s multiple comparison test, n = 6.

Article Snippet: The plasmid constructs for the YPet-tagged CNNM3 CBS domain and CyPet-tagged PRL2 have been deposited into AddGene ( https://www.addgene.org/ ) (Addgene IDs: 149686 and 149685).

Techniques: Inhibition, Binding Assay, Sequencing

Journal: Scientific Reports

Article Title: A FRET-based screening method to detect potential inhibitors of the binding of CNNM3 to PRL2

doi: 10.1038/s41598-020-69818-x

Figure Lengend Snippet: Effect of divalent cations and nucleotides on the FRET intensities. ( A ) Effect of divalent cations on the binding of CNNM3-YPet (200 nM) and CyPet-PRL2 (200 nM) using FRET. Control: No additional divalent cations; Mg 2+ : 10 mM Mg 2+ was added; Zn 2+ : 10 mM Zn 2+ was added. All data are expressed as the mean ± SE. **** p < 0.0001, by Dunnett’s multiple comparison test. n = 6. ( B ) Effect of nucleotides on the binding of CNNM3-YPet (200 nM) and CyPet-PRL2 (200 nM) using FRET. Control: No additional peptide. ATP: 3 mM ATP was added; ADP: 3 mM ADP was added; AMP: 3 mM AMP was added. All data are expressed as the mean ± SE. * p < 0.05, by t -test, n = 6.

Article Snippet: The plasmid constructs for the YPet-tagged CNNM3 CBS domain and CyPet-tagged PRL2 have been deposited into AddGene ( https://www.addgene.org/ ) (Addgene IDs: 149686 and 149685).

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: A FRET-based screening method to detect potential inhibitors of the binding of CNNM3 to PRL2

doi: 10.1038/s41598-020-69818-x

Figure Lengend Snippet: Inhibition of the binding of CNNM3-YPet and CyPet-PRL2 by CNNM peptides. ( A ) Amino acid sequences of synthesized peptides derived from the CNNM CBS loop. Black dots show the residues replaced by cysteine in peptide 2. Red boxes, identical residues; red letters, similar residues. ( B ) Location of CNNM peptides (red) in the CNNM3 structure (PDB: 5K22). V416 and L431 are replaced by cysteine residues in peptide 2. ( C ) CNNM peptide-based inhibition test of the binding of CNNM3-YPet (200 nM) and CyPet-PRL2 (200 nM) using FRET. Control: no additional peptide; peptide 1: 100 μM peptide 1 was added; peptide 2: 100 μM peptide 2 was added. All data are expressed as the mean ± SE. * p < 0.05, by Dunnett’s multiple comparison test. n = 6. ( D ) CNNM peptide 2-based inhibition test of the binding of CNNM3-YPet (200 nM) and CyPet-PRL2 (200 nM) using FRET. Control: no additional peptide. Peptide 2: 500 μM peptide 2 was added. All data are expressed as the mean ± SE. **** p < 0.0001, by t -test, n = 6.

Article Snippet: The plasmid constructs for the YPet-tagged CNNM3 CBS domain and CyPet-tagged PRL2 have been deposited into AddGene ( https://www.addgene.org/ ) (Addgene IDs: 149686 and 149685).

Techniques: Inhibition, Binding Assay, Synthesized, Derivative Assay

Journal: Scientific Reports

Article Title: A FRET-based screening method to detect potential inhibitors of the binding of CNNM3 to PRL2

doi: 10.1038/s41598-020-69818-x

Figure Lengend Snippet: Evaluation of the JMS-053 effect on the interaction between CNNM3 and PRL2. ( A ) Effect of JMS-053 on the binding of CNNM3-YPet (200 nM) and CyPet-PRL2 (200 nM) using FRET. All data are expressed as the mean ± SE. n = 6. ( B , C ) ITC data on the PRL2 with JMS-053 ( B ) and CNNM3 CBS protein ( C ). Measurements were repeated twice, and similar results were obtained.

Article Snippet: The plasmid constructs for the YPet-tagged CNNM3 CBS domain and CyPet-tagged PRL2 have been deposited into AddGene ( https://www.addgene.org/ ) (Addgene IDs: 149686 and 149685).

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: A FRET-based screening method to detect potential inhibitors of the binding of CNNM3 to PRL2

doi: 10.1038/s41598-020-69818-x

Figure Lengend Snippet: FRET binding model of the CNNM3 CBS domain and PRL2. ( A ) Cartoon depiction of construct design. ( B ) FRET model of the binding of CNNM3-YPet and CyPet-PRL2.

Article Snippet: The plasmid constructs for the YPet-tagged CNNM3 CBS domain and CyPet-tagged PRL2 have been deposited into AddGene ( https://www.addgene.org/ ) (Addgene IDs: 149686 and 149685).

Techniques: Binding Assay, Construct