Journal: Scientific Reports

Article Title: Structure of the glucosyltransferase domain of TcdA in complex with RhoA provides insights into substrate recognition

doi: 10.1038/s41598-022-12909-8

Figure Lengend Snippet: The overall structure of the GTD TcdA –RhoA complex. ( A ) A schematic diagram showing the design of the GTD TcdA –RhoA chimera protein. ( B / C ) Cartoon representations of the GTD TcdA –RhoA complex in two different views. GTD TcdA and RhoA are colored hot pink and cyan, respectively. The W519-loop of GTD TcdA is colored yellow, the lower and upper clamps of GTD TcdA are colored green, the pre-switch I and switch I of RhoA are colored wheat. The α16/17 of GTD TcdA and switch II of RhoA are showed as cylinders in ( C ). ( D / E ) Close-up views into the interface between GTD TcdA and RhoA focusing on the pre-switch I ( E ) and switch I ( D / E ) from two different viewing angles as indicated by purple and green symbols. The interacting residues are colored using the same scheme as that in ( B ). ( F ) Close-up view into the switch II-binding interface, and the interacting residues are colored the same as ( C ).

Article Snippet: The genes encoding RhoA (residues 1–181, Addgene, plasmid #12959) and TcdA GTD (residues 1–542, strain VPI10463) connected by a peptide linker (GGGGSGGGSGTGSGGGGS) were cloned into the pGEX6p-1 vector via BamH I/ Xho I restriction sites.

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: Structure of the glucosyltransferase domain of TcdA in complex with RhoA provides insights into substrate recognition

doi: 10.1038/s41598-022-12909-8

Figure Lengend Snippet: Conformational changes on GTD TcdA and RhoA induced by complex formation. ( A ) Structural superposition of the GTD TcdA –RhoA (hot pink and cyan, respectively) complex, GTD TcdA ·U2F (slate, PDB code: 5UQL), and GTD TcdA ·UDP-glucose (pale yellow, PDB code: 3SRZ) based on the GTD. ( B ) The W519-loop of GTD TcdA adopts a similar conformation in the GTD TcdA ·UDP-glucose–RhoA complex (hot pink) and GTD TcdA ·UDP-glucose (pale yellow), which is drastically different from that of the apo GTD TcdA (slate, PDB code: 4DMV). ( C ) Comparing the conformations of the switch I of RhoA in the GTD TcdA –RhoA·GDP complex (cyan), RhoA·GTPγs (pale yellow, PDB code: 1A2B), and RhoA·GDP (slate, PDB code: 1FTN). ( D ) Comparing the conformations of the switch II of RhoA in the GTD TcdA –RhoA·GDP complex (cyan) and RhoA·GDP (slate, PDB code: 1FTN). ( E ) Superposition of RhoA-bound GTD TcdA ·UDP-glucose (hot pink), GTD TcdA ·U2F (slate) and GTD TcdA ·UDP-glucose (pale yellow) focusing on the α16/17 helixes. ( F ) GTD TcdA adopts conformational changes to accommodate the pre-switch I and switch I of RhoA when compared to the GTD TcdA ·U2F (slate) and GTD TcdA ·UDP-glucose (pale yellow) structures.

Article Snippet: The genes encoding RhoA (residues 1–181, Addgene, plasmid #12959) and TcdA GTD (residues 1–542, strain VPI10463) connected by a peptide linker (GGGGSGGGSGTGSGGGGS) were cloned into the pGEX6p-1 vector via BamH I/ Xho I restriction sites.

Techniques:

Journal: Scientific Reports

Article Title: Structure of the glucosyltransferase domain of TcdA in complex with RhoA provides insights into substrate recognition

doi: 10.1038/s41598-022-12909-8

Figure Lengend Snippet: Amino acid sequence alignment among the GTDs of TcdA and two TcdB variants. Residue numbers and the secondary structures of the RhoA-bound GTD TcdA are shown on the top. The residues on the GTDs of TcdA-VPI10463, TcdB-VPI10463, and TcdB-M68 that interact with RhoA, Cdc42, and R-Ras are highlighted with red triangles, green stars, and black rhombuses, respectively. The green boxes highlight the upper and lower clamps of the GTD.

Article Snippet: The genes encoding RhoA (residues 1–181, Addgene, plasmid #12959) and TcdA GTD (residues 1–542, strain VPI10463) connected by a peptide linker (GGGGSGGGSGTGSGGGGS) were cloned into the pGEX6p-1 vector via BamH I/ Xho I restriction sites.

Techniques: Sequencing, Residue

Journal: Scientific Reports

Article Title: Structure of the glucosyltransferase domain of TcdA in complex with RhoA provides insights into substrate recognition

doi: 10.1038/s41598-022-12909-8

Figure Lengend Snippet: Structural comparison of the GTD TcdA –RhoA and the GTD TcdB-VPI10463 –Cdc42 complexes. ( A ) K448 of GTD TcdA (hot pink) but not the equivalent E449 of GTD TcdB (pale yellow, PDB code: 7S0Y) form a salt bridge with E40 (cyan) of RhoA and potentially D38 of Cdc42 (pale yellow). ( B ) Residues A377 and L378 on the lower clamp of GTD TcdA (hot pink) interact with the switch I of RhoA better than the equivalent N378 and S379 on GTD TcdB-VPI10463 (pale yellow, PDB code: 7S0Y). ( C ) Examining the interactions between the pre-switch I of RhoA (cyan) and the lower clamp of GTD TcdA (hot pink) in the GTD TcdA –RhoA complex when compared to the GTD TcdB-VPI10463 –Cdc42 complex (pale yellow) and RhoA·GDP (slate, PDB code: 1FTN). ( D ) Comparing the interactions in the switch II area for the GTD TcdA –RhoA (hot pink and cyan, respectively) and the GTD TcdB-VPI10463 –Cdc42 (pale yellow) complexes.

Article Snippet: The genes encoding RhoA (residues 1–181, Addgene, plasmid #12959) and TcdA GTD (residues 1–542, strain VPI10463) connected by a peptide linker (GGGGSGGGSGTGSGGGGS) were cloned into the pGEX6p-1 vector via BamH I/ Xho I restriction sites.

Techniques: Comparison

Journal: Cell reports

Article Title: Modeling Patient-Derived Glioblastoma with Cerebral Organoids

doi: 10.1016/j.celrep.2019.02.063

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pLenti-C-tRFP , OriGene , Cat# PS100074.

Techniques: Recombinant, Plasmid Preparation, Imaging, SYBR Green Assay, Luciferase, Construct, Software

Journal: Cell Death Discovery

Article Title: Deubiquitinase USP47-stabilized splicing factor IK regulates the splicing of ATM pre-mRNA

doi: 10.1038/s41420-020-0268-1

Figure Lengend Snippet: a HeLa cells were transfected for 48 h with individual siRNAs from a pool of 76 siRNA targeting deubiquitinases (DUBs). Next, the level of endogenous IK was measured by western blotting and protein bands were quantified using the Image J software. b Cells were transfected with 11 DUB siRNAs involved in DNA Damage Response (DDR), and the level of IK was examined. c Cells were transfected with three different siUSP47s for 48 h, and the levels of USP47 were examined. d Cells were transfected with siUSP47#1 for 48 h, and the levels of IK, SMU1, and USP47 were examined. e Cells were transfected with siUSP47#1 for 48 h, and the levels of IK mRNA were determined. The relative ratios of mRNA were normalized to those of β-actin and graphed. * p < 0.05. f Cells transfected with siUSP47 #1 for 24 h were transfected with Flag-USP47 for an additional 24 h, and the levels of IK and SMU1 were determined. g , h HEK-293T cells transfected with HA-IK or/and Flag-USP47 were immunoprecipitated with an anti-HA or anti-Flag antibody, and the levels of HA and FLAG were examined. i HeLa cells were immunoprecipitated with anti-USP47, and the levels of IK and USP47 were examined.

Article Snippet: Full-length human IK and USP47 cDNAs were purchased from OriGene (OriGene Technologies, Inc., Rockville, MD) and cloned into pcDNA 3.1, pcDNA 3.0, and pCMV-tag-2B vectors.

Techniques: Transfection, Western Blot, Software, Immunoprecipitation

Journal: Cell Death Discovery

Article Title: Deubiquitinase USP47-stabilized splicing factor IK regulates the splicing of ATM pre-mRNA

doi: 10.1038/s41420-020-0268-1

Figure Lengend Snippet: a Cells were transfected with USP47#1 and treated with 40 μg/mL of cycloheximide (CHX) for the indicated times to determine the levels of IK. b Cells were transfected with siUSP47#1 for 24 h and incubated with Flag-IK for an additional 24 h, and the levels of IK were examined. Green-fluorescent protein (GFP) was used as a transfection control. c Cells were transfected with siUSP47#1 for 48 h and treated with 10 μM MG132 for an additional 6 h, and the levels of IK were detected. d Cells transfected with Flag-USP47 for 24 h were treated with 40 μg/mL of CHX for the indicated times and the levels of endogenous IK were examined. e Cells were transfected with siUSP47#1 for 42 h and treated with 10 μM MG132 for 6 h. Cells were lysed and immunoprecipitated with an anti-IK antibody, followed by immunoblotting using an anti-Ub antibody. f Cells were transfected with Flag-USP47 and treated with 10 μM MG132 for 6 h. Cells were lysed and immunoprecipitated using an anti-IK antibody, followed by immunoblotting using an anti-Ub antibody. g Cells were transfected with a GFP-IK and increasing amounts of Flag-USP47, and the levels of GFP-IK and Flag-USP47 were examined.

Article Snippet: Full-length human IK and USP47 cDNAs were purchased from OriGene (OriGene Technologies, Inc., Rockville, MD) and cloned into pcDNA 3.1, pcDNA 3.0, and pCMV-tag-2B vectors.

Techniques: Transfection, Incubation, Control, Immunoprecipitation, Western Blot

Journal: Cell Death Discovery

Article Title: Deubiquitinase USP47-stabilized splicing factor IK regulates the splicing of ATM pre-mRNA

doi: 10.1038/s41420-020-0268-1

Figure Lengend Snippet: a Cells were transfected with siUSP47#1 and the levels of ATM, IK, and USP47 were examined. b Cells were transfected with siUSP47#1 for 48 h, and the levels of ATM E1-E2 mRNAs were determined. c To restoration assay, cells transfected with siUSP47#1 for 24 h were transfected with IK-GFP for an additional 24 h, and the levels of ATM E1-E2 mRNAs were examined. d Cells transfected with siUSP47#1 for 24 h were transfected with IK-GFP for an additional 24 h, and the levels of ATM and IK were examined. e Schematic for the proposed function and relationship between IK and USP47.

Article Snippet: Full-length human IK and USP47 cDNAs were purchased from OriGene (OriGene Technologies, Inc., Rockville, MD) and cloned into pcDNA 3.1, pcDNA 3.0, and pCMV-tag-2B vectors.

Techniques: Transfection