p416 25q gpd  (Addgene inc)

Journal: bioRxiv

Article Title: Crystal structure of a novel guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

doi: 10.1101/2020.06.04.133876

Figure Lengend Snippet: (a) Inputs and anti-Flag immunoprecipitates of lysates from HeLa cell ectopically expressing the indicated Flag-tagged OtDUB fragments. Proteins were resolved by SDS-PAGE and immunoblotted for Rho GTPases (Rac1,2,3; Cdc42; RhoA). (b) FLAG (top) and reciprocal GST (bottom) pulldown experiments between purified recombinant FLAG-tagged OtDUB fragments and GST-tagged Rac1 or Cdc42. Proteins were resolved by SDS-PAGE and stained with Coomassie Blue. (c) Time course of the dissociation of BODIPY-GDP from Rac1 or Cdc42 (9 μM) in the presence of OtDUB 275-1369 (0, 50, or 250 nM) as measured by loss of BODIPY-GDP fluorescence. Excitation and emission wavelength were 488 nm and 535 nm, respectively.

Article Snippet: DNA encoding residues 1-177 of both human RAC1 and CDC42 were cloned into an unmodified pET-28a (Addgene) vector that encodes an N-terminal TEV-cleavable 6xHis-tag using the NdeI/XhoI sites.

Techniques: Expressing, SDS Page, Purification, Recombinant, Staining, Fluorescence

Journal: bioRxiv

Article Title: Crystal structure of a novel guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

doi: 10.1101/2020.06.04.133876

Figure Lengend Snippet: (a) OtDUB fragments expressed in yeast. Transformants were grown for 4 h in minimal liquid medium containing galactose. After induction, equivalent optical density units (ODU) were processed and extracts from 0.25 ODU were resolved by SDS-PAGE and immunoblotted with anti-Flag antibody, then Ponceau stained (PonS) for loading comparison. Absorbance by dead cells results in reduced Ponceau signal for C135A and 275-1369. (b) Fluorescent nucleotide exchange assays demonstrate absence of activity against RhoA in the OtDUB 275-1369 fragment (left), and absence of activity against Rac1 in truncation construct OtDUB 657-1369 . (c) Input proteins from pulldown experiment in .

Article Snippet: DNA encoding residues 1-177 of both human RAC1 and CDC42 were cloned into an unmodified pET-28a (Addgene) vector that encodes an N-terminal TEV-cleavable 6xHis-tag using the NdeI/XhoI sites.

Techniques: SDS Page, Staining, Comparison, Activity Assay, Construct

Journal: bioRxiv

Article Title: Crystal structure of a novel guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

doi: 10.1101/2020.06.04.133876

Figure Lengend Snippet: (a) OtDUB truncations used for mapping studies (top) and GST pulldown experiment using GST-OtDUB fragments as bait and Rac1 as prey (bottom). Gel was stained with Coomassie Blue. N-terminal truncation to residue 580 abolished binding, whereas the 548-759 fragment (OtDUB GEF domain) retained full binding capacity. (b) Size exclusion chromatography of OtDUB GEF :Rac1 mixtures demonstrates stable complex formation (top). Column fractions were evaluated by SDS-PAGE and protein staining (bottom). (c) Time course of the dissociation of BODIPY-GDP from Rac1 in the presence of increasing amounts of OtDUB GEF (residues 548-759). Raw fluorescence curves were fit to a single exponential decay (top), and initial rates were plotted against Rac1 concentration (bottom). Linear transformation of the titration yielded a k cat /K M of 2.6 ± 0.3 ×10 5 M -1 s -1 .

Article Snippet: DNA encoding residues 1-177 of both human RAC1 and CDC42 were cloned into an unmodified pET-28a (Addgene) vector that encodes an N-terminal TEV-cleavable 6xHis-tag using the NdeI/XhoI sites.

Techniques: Staining, Residue, Binding Assay, Size-exclusion Chromatography, SDS Page, Fluorescence, Concentration Assay, Transformation Assay, Titration

Journal: bioRxiv

Article Title: Crystal structure of a novel guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

doi: 10.1101/2020.06.04.133876

Figure Lengend Snippet: (a) Size exclusion chromatography (SEC) of OtDUB GEF :Cdc42 mixtures, as in . (b) A 260/280 ratios for fractions in each SEC run. OtDUB GEF alone and GTPase alone runs have A 260/280 of 0.6-0.9, whereas complex peaks formed after mixing are devoid of nucleotide ( A 260/280 ∼ 0.5). Exchanged nucleotide is also observed at late elution volumes (triangle). (c) Isothermal titration calorimetry quantification of binding affinity for OtDUB GEF and Rac1 (K d ∼ 5 μM) or Cdc42 (K d ∼ 16 μM). (d) Fluorescent nucleotide exchange assays as in , except with Cdc42 instead of Rac1, with increasing concentrations of OtDUB GEF . (e) Linear transformation and calculated k cat / K M values for Rac1 (2.6 ± 0.3 ×10 5 M -1 s -1 ) and Cdc42 (1.7 ± 0.1 ×10 4 M -1 s -1 ).

Article Snippet: DNA encoding residues 1-177 of both human RAC1 and CDC42 were cloned into an unmodified pET-28a (Addgene) vector that encodes an N-terminal TEV-cleavable 6xHis-tag using the NdeI/XhoI sites.

Techniques: Size-exclusion Chromatography, Isothermal Titration Calorimetry, Binding Assay, Transformation Assay

Journal: bioRxiv

Article Title: Crystal structure of a novel guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

doi: 10.1101/2020.06.04.133876

Figure Lengend Snippet: (a) Three orthogonal views of the complex with OtDUB GEF in purple and Rac1 in cyan. Switch I and II loops are in salmon and yellow, respectively, and the β2-3 hairpin selectivity interface is green. (b) Close-up views of each key interface between OtDUB GEF and Rac1 showing selected residues, with hydrogen bonds and electrostatic interactions shown as dashes, water molecules as red spheres. (c) GST pulldown assays (left) of GST-OtDUB GEF (WT or charge-neutralizing mutations at each interface) incubated with Rac1 and analyzed by SDS-PAGE and Coomassie Blue staining. Corresponding BODIPY-GDP release assays are shown on the right. (d) Close-up views of each interface comparing the OtDUB GEF residues interacting with Rac1 (thick sticks) to the interactions made by other bacterial GEFs (thin sticks).

Article Snippet: DNA encoding residues 1-177 of both human RAC1 and CDC42 were cloned into an unmodified pET-28a (Addgene) vector that encodes an N-terminal TEV-cleavable 6xHis-tag using the NdeI/XhoI sites.

Techniques: Incubation, SDS Page, Staining

Journal: bioRxiv

Article Title: Crystal structure of a novel guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

doi: 10.1101/2020.06.04.133876

Figure Lengend Snippet: (a) Overall crystal structure of OtDUB GEF resolved to 3.0 Å, with six copies in the asymmetric unit (ASU), with the 2Fo-Fc electron density map (mesh) contoured at 1.0 σ. Pairwise Cα RMSDs between copies in the ASU ranges from 0.36 to 1.9 Å. (b) Overall crystal structure of OtDUB GEF in complex with Rac1 resolved to 1.7 Å, with the 2Fo-Fc electron density map (mesh) contoured at 1.0 σ. The asymmetric unit contains two copies each of OtDUB GEF and of Rac1. Each complex is virtually identical, with Cα RMSD between complexes of 0.1 Å. Inset: detailed view of the nucleotide-binding region of Rac1 reveals absence of electron density for GDP (modeled, grey). (c) Size exclusion chromatography of OtDUB GEF/Δlever :Rac1 mixtures. The late eluting peak representing exchanged nucleotide, which was observed with wild-type OtDUB GEF , is absent. (d) Structural comparison of six copies of OtDUB GEF from the apo structure (green shades) and two copies of OtDUB GEF from the Rac1 complex structure (purple shades). Inset: detailed view of the catalytic lever region reveals a rigid conformation in both apo and complex OtDUB GEF molecules.

Article Snippet: DNA encoding residues 1-177 of both human RAC1 and CDC42 were cloned into an unmodified pET-28a (Addgene) vector that encodes an N-terminal TEV-cleavable 6xHis-tag using the NdeI/XhoI sites.

Techniques: Binding Assay, Size-exclusion Chromatography, Comparison

Journal: bioRxiv

Article Title: Crystal structure of a novel guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

doi: 10.1101/2020.06.04.133876

Figure Lengend Snippet: (a) β2-3 hairpin “interswitch” regions from Rac1, Cdc42, and RhoA are shown as cartoon and transparent surface, with surface-exposed residues as sticks. (b) Structural alignment of the selectivity patches of Cdc42 and RhoA reveal steric clashes with RhoA that likely prevent binding with OtDUB GEF (purple surface and sticks)

Article Snippet: DNA encoding residues 1-177 of both human RAC1 and CDC42 were cloned into an unmodified pET-28a (Addgene) vector that encodes an N-terminal TEV-cleavable 6xHis-tag using the NdeI/XhoI sites.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Crystal structure of a novel guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

doi: 10.1101/2020.06.04.133876

Figure Lengend Snippet: Structural alignment by GTPase (surface) of available bacGEFs (cylinders): SopE (red), Map (yellow), IpgB2 (green), and OtDUB GEF (purple) highlight convergence on a conserved V-shaped fold on the same location on Rac1.

Article Snippet: DNA encoding residues 1-177 of both human RAC1 and CDC42 were cloned into an unmodified pET-28a (Addgene) vector that encodes an N-terminal TEV-cleavable 6xHis-tag using the NdeI/XhoI sites.

Techniques:

Journal: bioRxiv

Article Title: Crystal structure of a novel guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

doi: 10.1101/2020.06.04.133876

Figure Lengend Snippet: (a) Top: Structural overlay of GTPases from GDP-bound (PDB: 5N6O, grey) and GEF-bound structures: OtDUB GEF (cyan), SopE (red), Map (yellow), and TIAM1 (slate). GEFs are removed for clarity. Switch I residues Glu 62 , Ala 59 , and Lys 16 are shown as sticks. Arrows indicate the previously observed motions of the residues upon GEF binding. Bottom: BODIPY-GDP exchange assay reveals that Glu 62 is not required for efficient exchange catalyzed by OtDUB GEF . (b) Top: A loop of OtDUB GEF that interrupts α2 acts as a “catalytic lever” to promote release of GDP. OtDUB GEF is shown as cartoon (purple), and Rac1 is shown as transparent surface (cyan). GDP (not present in our structure) is modeled based on PDB 5N6O for reference. The steric clash and electrostatic repulsion are indicated by the red lines. Bottom: Nucleotide exchange assay reveals a strict requirement in the lever segment for exchange of BODIPY-GDP. (c) Calculated electrostatic surface potential map (unit k B T/e) shows high negative charge in the OtDUB GEF lever formed by a tri-carbonyl motif at the vicinity of the diphosphate group of GDP.

Article Snippet: DNA encoding residues 1-177 of both human RAC1 and CDC42 were cloned into an unmodified pET-28a (Addgene) vector that encodes an N-terminal TEV-cleavable 6xHis-tag using the NdeI/XhoI sites.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Crystal structure of a novel guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

doi: 10.1101/2020.06.04.133876

Figure Lengend Snippet: (a) Lysates from HeLa cells carrying empty vector or plasmids expressing wild-type or E572A OtDUB were subjected to GST-Pak1PBD pulldowns to enrich active Rac1 and Cdc42. Representative experiment (left) from 4 independent experiments that were quantified (right) relative to input levels and the empty vector control. Bars represent mean and S.D. P value determined from a two-tailed unpaired Student t-test. Outlier from Rac1 wild-type (5.4 fold increase in activity) excluded one trial. Outlier was identified using Grubbs algorithm with alpha = 0.05. (b) Representative epifluorescence images of fibroblasts carrying the vector expressing only GFP or plasmids expressing wild-type or E572A OtDUB GEF . (c) Quantification of cell area, perimeter, and major axis length.

Article Snippet: DNA encoding residues 1-177 of both human RAC1 and CDC42 were cloned into an unmodified pET-28a (Addgene) vector that encodes an N-terminal TEV-cleavable 6xHis-tag using the NdeI/XhoI sites.

Techniques: Plasmid Preparation, Expressing, Control, Two Tailed Test, Activity Assay

Journal: Frontiers in Genetics

Article Title: SUMO-Targeted Ubiquitin Ligases (STUbLs) Reduce the Toxicity and Abnormal Transcriptional Activity Associated With a Mutant, Aggregation-Prone Fragment of Huntingtin

doi: 10.3389/fgene.2018.00379

Figure Lengend Snippet: STUbL subunits Slx5 and Slx8 alleviate toxicity of poly-Q expanded Htt. (A) WT, slx5 Δ, and slx8 Δ strains expressing Htt-25Q, Htt-103Q, or empty vector (EV) were grown to mid-logarithmic phase and 5 μl of 10-fold serial dilutions of each culture were spotted on SC-URA medium. Plates were incubated at 30°C for 3 days. (B) Yeast transformants in A and the indicated controls were grown overnight in 5 ml of SC-URA medium. Ten OD 600 readings of cultures were recorded every hour until the OD 600 reached ∼2.0. The average doubling times of four independent experiments were graphed with +/− standard error. EV (empty vector) (C) A shuffle strain, slx5 Δ with SLX5/TRP plasmid (YOK 2990), was transformed with either Htt-25Q or Htt-103Q constructs. Transformants were patched in duplicate on selective medium (SC-TRP URA) and rich medium (YPD). Patches were then replica plated on SC-URA medium with 5FAA to counter-select against the TRP1 marked plasmid.

Article Snippet: Yeast plasmids expressing 25Q and 103Q Htt were purchased from Addgene.org (Addgene plasmid # 1177 (GPD-25Q-GFP Htt in p416), # 1180 (GPD-103Q-GFP Htt in p416)).

Techniques: Expressing, Plasmid Preparation, Incubation, Transformation Assay, Construct

Journal: Frontiers in Genetics

Article Title: SUMO-Targeted Ubiquitin Ligases (STUbLs) Reduce the Toxicity and Abnormal Transcriptional Activity Associated With a Mutant, Aggregation-Prone Fragment of Huntingtin

doi: 10.3389/fgene.2018.00379

Figure Lengend Snippet: Plasmid-borne SLX5 reduces aggregates of poly-Q expanded huntingtin. (A) Representative images of Htt-103Q in WT cells with and without an SLX5/CEN plasmid. Example of aggregates (clearly defined, bright cytoplasmic structures – red arrow-heads) and speckles (multiple small, not clearly defined cytoplasmic granules – blue arrow-heads) (B) Quantitation of phenotypes observed in 2A. WT strain expressing Htt-103Q-GFP alone (YOK 2842) or Htt-103Q-GFP with SLX5 (YOK 2843) were grown to mid-logarithmic phase in selective medium. Images of 103Q diffuse staining, aggregates, and speckles in the indicated strains were recorded and then quantitated. Average counts for three independent experiments were graphed +/− standard deviation. Y-axis: percent of cells ( n = 100/experiment). Y-axis: phenotypes scored (C) Aggregates of poly-Q expanded Htt are localized in the cytoplasm. WT and slx5 Δ strains transformed with GAL-Htt-97Q-DsRed (YOK 3112 and YOK 3114) were grown overnight in SC-TRP medium with 2% raffinose. Cultures were diluted to ∼0.2 OD in a fresh medium with 2% galactose and incubated for an additional 16 h for expression of Htt-97Q-DsRed prior to imaging Htt aggregates using a fluorescence microscope. Nuclei were stained with Hoechst dye. Merged images indicate the absence of Htt-97Q aggregates in nuclei (yellow arrow-heads).

Article Snippet: Yeast plasmids expressing 25Q and 103Q Htt were purchased from Addgene.org (Addgene plasmid # 1177 (GPD-25Q-GFP Htt in p416), # 1180 (GPD-103Q-GFP Htt in p416)).

Techniques: Plasmid Preparation, Quantitation Assay, Expressing, Staining, Standard Deviation, Transformation Assay, Incubation, Imaging, Fluorescence, Microscopy

Journal: Frontiers in Genetics

Article Title: SUMO-Targeted Ubiquitin Ligases (STUbLs) Reduce the Toxicity and Abnormal Transcriptional Activity Associated With a Mutant, Aggregation-Prone Fragment of Huntingtin

doi: 10.3389/fgene.2018.00379

Figure Lengend Snippet: Slx5 modulates the transcriptional activity due to expression of Htt-103Q-NLS. Depiction of 398 SLX5 modulated genes identified by RNA sequencing. (A) RNA-seq analysis shows that Htt103Q-NLS leads to a global effect on the transcriptome as it affects the expression of 3438 genes (25Q [V] vs. 103Q [V]). Plasmid-borne SLX5 affects the expression of 398 genes in Htt103Q-NLS cells (103Q [V] vs. 103Q [ SLX5 ]) as shown in Supplementary Table . The overlap between Htt25Q-NLS and Htt103Q-NLS (25Q [V] vs. 103Q [V]) is 363 genes. The expanded view of A shows that expression of most of the 363 genes (99.4%) inversely correlates between 25Q [V] vs. 103Q [V] and 103Q [V] vs. 103Q [ SLX5 ]. Expression of 247 of the 363 genes (68.0%) is down-regulated by Htt103Q, and this effect is reversed by plasmid-borne SLX5 (Down – Up). Expression of 114 of the 363 genes (31.4%) is up-regulated by the Htt103Q, and this effect is reversed by plasmid-borne SLX5 (Up – Down). (B) Subcellular localization of differentially expressed genes. The localization of proteins encoded by the 398 genes indicated in Supplementary Table was analyzed using cellular components assignment from the PANTHER Classification System and the Saccharomyces Genome Database. Pie chart shows a ratio of the genes placed into cellular component categories. Individual genes are listed in Supplementary Table . (C) Schematic of gene expression in Htt25Q-NLS and Htt103Q-NLS cells and the effect of plasmid-borne SLX5 on the transcriptome of Htt103Q-NLS cells. Expression of genes A and B are downregulated and upregulated in Htt103Q-NLS cells relative to Htt25Q-NLS cells, respectively. Slx5 reduces the association of Htt with chromatin and this contributes to the reversal in gene expression such that gene A is upregulated and gene B is downregulated. (D) RT-PCR validation of gene expression analysis. Total RNAs were purified from strains expressing either Htt25Q-NLS or Htt103Q-NLS from a GAL promoter for 4 h with or without plasmid-borne SLX5 . RT-PCR analyses was performed using the same samples used for the RNA-seq. Relative intensities are reported as the mean ± SD of three biological repeats. Reactions for YDL223C/HBT1 and YPL186C/UIP4 were performed in duplicate. N = 3 for YOR339C/UBC11 and YFL039C/ACT1 , N = 6 for YDL223C/HBT1 and YPL186C/UIP4 .

Article Snippet: Yeast plasmids expressing 25Q and 103Q Htt were purchased from Addgene.org (Addgene plasmid # 1177 (GPD-25Q-GFP Htt in p416), # 1180 (GPD-103Q-GFP Htt in p416)).

Techniques: Activity Assay, Expressing, RNA Sequencing, Plasmid Preparation, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Purification