Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 1. GRB2 complexes with AGO2 under non-stimulated conditions. Schematic diagram of, (a) AGO2 and, (b) GRB2 domain structures. Domains are named and colour coded and attributed amino acid sequence number. Red arrows indicate positions of PXXP motifs investigated in this work. (c) Western blot of AGO2 co-immunoprecipitated with GRB2 in serum starved HEK293T, A498 and PC3 cells. A longer exposure was used to capture AGO2 bands than for GRB2 and GAPDH. All images are taken from the same western blot. (d) Fluorescence and fluorescence resonance energy transfer signals of RFP-tagged GRB2 and GFP-tagged AGO2. HEK293T cells overexpressing fluorescent proteins were serum-starved before imaging. N = 3. Scale bars are 10 μm.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Sequencing, Western Blot, Immunoprecipitation, Fluorescence, Förster Resonance Energy Transfer, Imaging

Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 2. Binding of GRB2 to AGO2 is mediated by GRB2 NSH3 and a PXXP motif in AGO2 PAZ domain. (a) Isothermal titration calorimetry (ITC) of a peptide spanning the proline-rich motif 323PHLP326 in AGO2 PAZ domain. (KD = 4.27 ± 1.17 µM). (b, c) ITC of MBP-tagged AGO2 PAZ domain titrated into GRB2. (b) PAZ WT (KD = 585 ± 61 nM). (c) No binding observed for mutation of PXXP (MBP-PAZ 4A). N = 2. (d) Fluorescence resonance energy transfer (FRET) between wild type (WT) and 323AAAA326 (4A) mutant GFP-tagged AGO2 and RFP-tagged GRB2 in HEK293T cells under conditions of serum starvation. White arrows indicate intracellular puncta which show increased FRET when WT AGO2 is expressed. N = 2. Scale bars are 10 μm. (e) Fluorescence lifetime imaging microscopy of RFP-tagged GRB2 proteins and GFP-AGO2 overexpressed in serum-starved HEK293T cells. The formation of a protein complex results in a reduction in fluorescent lifetime represented by a shift to the left of the population of fluorophores (measured in number of pixels). Lifetime population distribution shown by red line on graphs. x = Lifetime (ns), y = number of pixels. Solid black line corresponds to average fluorescent lifetime for GFP, 2.1 ns. Scale bars 25 μm. (f) Expanded region of interest (ROI) further exemplifying left-shift for AGO2/NSH3-SH2 interaction.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Binding Assay, Isothermal Titration Calorimetry, Mutagenesis, Fluorescence, Förster Resonance Energy Transfer, Imaging, Microscopy

Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 3. Impact of GRB2-AGO2 complex on interaction with DICER1 and miRNA. (a) Western blot of AGO2 and DICER1 pulldown by GST-GRB2 in HEK293T cells. HEK293T cells were serum-starved before lysis. Bands captured with both a long and short exposure are shown for DICER1, whereas only the image captured with a short exposure is shown for AGO2. GST proteins were detected by ponceau stain. All images are taken from the same western blot. N = 3. (b–d) MST of AGO2 binding to DICER1 C-terminal region, upon pre-incubation of AGO2 with increasing concentrations of GRB2. The difference in binding affinity was negligible. (e) MST of GRB2 with DICER1 C-terminal region. No binding is observed within a physiologically relevant range hence the two do not interact directly. (f) Expanded ribbon model of molecular docking of GRB2 (green; PDB: 1GRI77) to AGO2 PAZ domain (cyan; red and blue indicate positive and negative charges respectively; PDB: 6RA478). The 323PHLP326 sequence is shown (yellow). GRB2 W36 (magenta) interacts with AGO2 P249 (red). Other residues in GRB2 which may contribute towards the interaction are shown in orange. Also shown is space-filling representation of AGO2 PAZ domain with PRM shown (below); and ribbon model of PAZ domain rotated by 90° to highlight juxtaposition of GRB2 binding site PRM and docking site for miRNA (right). Figures generated using PyMOL.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Western Blot, Lysis, Staining, Binding Assay, Incubation, Sequencing, Generated

Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 4. GRB2 regulates miRNA expression in HEK293T cells. (a) Western blot of GRB2 expression in wild type (293 T) and depleted (G1) HEK293T clones 1 (G1.1) and 2 (G1.2). While G1.1 is a complete knockout, G1.2 contains a deletion and large insertion in the N-terminal SH3 domain. GRB2 was blotted with an antibody which recognised the C-terminal SH3 domain. Both long and short exposures were used to capture the GRB2 bands, whereas the GAPDH image was captured using a short exposure only. All images are taken from the same western blot. N = 3. (b) Heat plot highlighting miRNAs which show significant log2(fold changes) in expression (p < 0.05) between wild type HEK293T and G1 cells, measured by small RNA sequencing. Cells were deprived of growth factor. miRNAs demonstrated positive (red) and negative (blue) expression changes. N = 2. (c, d) RT-qPCR analysis of fold-change in mean expression of precursor miRNA transcripts (precursor and primary, pre-mir-, hashed bars) and mature miRNA (miR-, plain bars) derived from serum-starved G1 or wild type HEK293T cells. Two groups of miRNAs were observed: (c) miRNAs which diminished at both the level of the precursor and mature transcripts and, (d) miRNAs which were enhanced as mature transcripts but not as precursors. Comparisons were made using a two-tailed Student’s t-test and error bars show standard error of mean. N = 4. ns = not significant.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Expressing, Western Blot, Clone Assay, Knock-Out, RNA Sequencing, Quantitative RT-PCR, Derivative Assay, Two Tailed Test

Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 5. The GRB2-let-7 axis regulates oncogene expression. (a) RT-qPCR measurement of fold change in mean expression of let-7 g-5p miRNA and five target mRNAs in serum-starved GRB2 knockout cells (G1) compared to wild type HEK293T (293 T). Comparisons were made using a two-tailed Student’s t-test and error bars show standard error of mean. N = 3. (b) Western blot and (c) quantification of mean protein expression of let-7 targets in growth-factor-deprived G1 and HEK293T cells. The higher molecular band detected by the GRB2 antibody in G1 corresponds to an NSH3-mutated GRB2 polypeptide. For blot 1, a longer exposure was used to capture the DICER1 and GRB2 bands than was used for LIN28B and α-Tubulin. For blot 2, HMGA2 bands were captured using a longer exposure than that required for GRB2 and GAPDH. (d) Quantification of the area covered by migration of HEK293T cells expressing GFP-tagged wild type AGO2 (WT) or an AGO2 mutant which is incapable of binding GRB2 (4A), under conditions of reduced growth factor. Comparisons were made using a two-tailed Student’s t-test and error bars show standard error of mean. N = 3.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Expressing, Quantitative RT-PCR, Knock-Out, Two Tailed Test, Western Blot, Migration, Mutagenesis, Binding Assay

Journal: Veterinary Sciences

Article Title: VEGF-B , VEGF-A , FLT-1 , KDR , ERBB2 , EGFR , GRB2 , RAC1 , CDH1 and HYAL-1 Genes Expression Analysis in Canine Mammary Gland Tumors and the Association with Tumor ClinicoPathological Parameters and Dog Breed Assessment

doi: 10.3390/vetsci8100212

Figure Lengend Snippet: Expression levels of 10 analyzed genes ( VEGF-B , VEGF-A , FLT-1 , KDR , ERBB2 , EGFR , GRB2 , RAC1 , CDH1 and HYAL-1 ) in canine mammary tumors (CMTs) and tumor adjacent tissues in all studied dogs. qRT-PCR data are represented as delta Ct values. Dots indicate outliers. * p < 0.05 measured with Wilcoxon or t -tests.

Article Snippet: The expression level of genes were measured using TaqMan TM Gene Assays with FAM dye ( VEGF-B Cf02721109_u1, VEGF-A Cf02674018_m1, FLT-1 Cf02696454_g1, KDR Cf02627749_m1, ERBB2 Cf02621873_g1, EGFR Cf02626541_m1, GRB2 Cf02667172_m1, RAC1 Cf02699525_m1, CDH1 Cf02624268_m1, HYAL-1 Cf02718719_s1) and TaqMan Universal Master Mix (Applied Biosystems, Carlsbad, CA, USA) on the 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA), according to the manufacturer‘s protocol.

Techniques: Expressing, Quantitative RT-PCR

Journal: Veterinary Sciences

Article Title: VEGF-B , VEGF-A , FLT-1 , KDR , ERBB2 , EGFR , GRB2 , RAC1 , CDH1 and HYAL-1 Genes Expression Analysis in Canine Mammary Gland Tumors and the Association with Tumor ClinicoPathological Parameters and Dog Breed Assessment

doi: 10.3390/vetsci8100212

Figure Lengend Snippet: Expression levels of analyzed genes ( VEGF-B , FLT-1 , EGFR , GRB2 and ERBB2 ), considering clinical parameters in CMTs and tumor adjacent tissues of different dog breeds. Expression levels of VEGF-B , and FLT genes in metastasis-free carcinoma samples ( A , B ), VEGF-B in groups with carcinoma stage I ( C ), EGFR in group with grade I ( G ), VEGF-B , GRB2 and ERBB2 in groups with carcinoma stage III ( E –G). Dots indicate outliers. * p < 0.05, ** p < 0.01 measured with Wilcoxon or t -tests.

Article Snippet: The expression level of genes were measured using TaqMan TM Gene Assays with FAM dye ( VEGF-B Cf02721109_u1, VEGF-A Cf02674018_m1, FLT-1 Cf02696454_g1, KDR Cf02627749_m1, ERBB2 Cf02621873_g1, EGFR Cf02626541_m1, GRB2 Cf02667172_m1, RAC1 Cf02699525_m1, CDH1 Cf02624268_m1, HYAL-1 Cf02718719_s1) and TaqMan Universal Master Mix (Applied Biosystems, Carlsbad, CA, USA) on the 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA), according to the manufacturer‘s protocol.

Techniques: Expressing

Journal: bioRxiv

Article Title: Global mapping of the energetic and allosteric landscapes of protein binding domains

doi: 10.1101/2021.09.14.460249

Figure Lengend Snippet: a. ddPCA uses two protein fragment complementation (PCA) selection assays to quantify the effects of mutations on the abundance ( abundancePCA ) of a protein of interest A and its binding to an interaction partner B ( bindingPCA ) based on growth in the presence of methotrexate. b. Reproducibility of fitness estimates from ddPCA for single and double AA substitutions between all biological replicates. Pearson correlations are indicated in red. c. Comparison of individually measured growth rates (slope of a linear fit of the log 10 (OD 600nm ) against time during the exponential phase) of GRB2-SH3 variants to growth rates inferred from deep sequencing data (see Methods). The dashed line corresponds to the linear regression model. R=Pearson correlation. d. 3D structures of the SH3 domain of GRB2 bound to the ligand peptide of GAB2 (PDB entry 2VWF) and the third PDZ domain of PSD95 bound to the ligand peptide of CRIPT (PDB entry 1BE9). e. Fitness density distributions. Total variant counts for singles (red) and doubles (blue) are indicated (purple indicates overlapping density distributions). Vertical dashed lines indicate the median fitness of STOP codon mutations in the central 50% of the coding sequence. f-g. Heatmaps of the fitness effects of single AA substitutions for GRB2-SH3 ( f ) and PSD95-PDZ3 ( g ) corresponding to the bindingPCA (upper panel) and abundancePCA (lower panel) assays. The final row in each heatmap indicates the minimal distance between domain and ligand side chain heavy atoms (or alpha carbon atoms in the case of glycine). Amino acid labels are coloured in red or blue for positive or negatively charged residues respectively, green for aromatic amino acids and highlighted in yellow for hydrophobic residues. Fitness values more extreme than ±1.5 were set to this limit. h. Scatterplots comparing abundance and binding fitness of single AA substitutions. Variants are coloured by the corresponding residue position in the domain structure: core (relative solvent accessible surface area, RSASA<0.25), surface (RSASA≥0.25) or ligand binding interface (minimal side chain heavy atom distance<5Å).

Article Snippet: To construct these plasmids, 7 gene blocks containing the mutated versions of GRB2-SH3 were synthesized with HindIII and NheI flanking restriction sites (gbGJJ002-8, Twist Bioscience, Table S2).

Techniques: Selection, Binding Assay, Sequencing, Variant Assay, Ligand Binding Assay

Journal: bioRxiv

Article Title: Global mapping of the energetic and allosteric landscapes of protein binding domains

doi: 10.1101/2021.09.14.460249

Figure Lengend Snippet: a-b. Heatmaps showing inferred changes in free energies of binding and folding for GRB2-SH3 ( a ) and PSD95-PDZ3 ( b). The final row in each heatmap indicates the ligand distance and amino acid labels are coloured according to the chemical structure and properties of their side-chains (see ). Free energy changes of ligand-proximal residues (ligand distance<5Å) are boxed and asterisks indicate major allosteric positions. Lower confidence estimates are indicated with dots (95% confidence interval ≥1kcal/mol). Free energy changes more extreme than ±2.5kcal/mol were set to this limit. c. Scatterplots comparing confident binding and folding free energy changes of mutations in the core, surface and binding interface. Contours indicate estimates of 2D densities using 6 contour bins. Axis limits were adjusted to include the largest contour bin (more extreme data points are not shown). d. Distributions of confident binding (red) and folding (blue) free energy changes. X-axis limits were adjusted to match those in panel c. e. Percentage of mutations that significantly decrease (top) or increase (bottom) fitness in the binding assay (FDR=0.05) categorized by their biophysical mechanism. Pleiotropic mutations have significant changes in free energies of both folding and binding (FDR=0.05) and are classified as either synergistic or antagonistic depending on whether their effects are in the same or different direction respectively. See Figure S7 for the GB1 domain.

Article Snippet: To construct these plasmids, 7 gene blocks containing the mutated versions of GRB2-SH3 were synthesized with HindIII and NheI flanking restriction sites (gbGJJ002-8, Twist Bioscience, Table S2).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Global mapping of the energetic and allosteric landscapes of protein binding domains

doi: 10.1101/2021.09.14.460249

Figure Lengend Snippet: a. 3D structures of the GRB2-SH3 and PSD95-PDZ3 domains where residue atoms are colored by the position-wise average change in the free energy of folding. Ligands are shown as black sticks. b. Violin plots indicating distributions of confident changes in free energy of folding stratified by position in the structure (two-sided Mann-Whitney U test p-value<2.2e-16 comparing mutations in the core versus the remainder for both protein domains). c. Negative correlation between the position-wise average change in free energy of folding and the solvent exposure of the corresponding residue (RSASA). R = Pearson correlation. d. Percentage of core, surface or binding interface residues shown separately for de-stabilising residues (positions with ≥5 stabilizing mutations, folding ΔΔG<0, FDR=0.05) and the remainder. Inset numbers are total counts. See Figure S9 for the GB1 domain.

Article Snippet: To construct these plasmids, 7 gene blocks containing the mutated versions of GRB2-SH3 were synthesized with HindIII and NheI flanking restriction sites (gbGJJ002-8, Twist Bioscience, Table S2).

Techniques: MANN-WHITNEY, Binding Assay

Journal: bioRxiv

Article Title: Global mapping of the energetic and allosteric landscapes of protein binding domains

doi: 10.1101/2021.09.14.460249

Figure Lengend Snippet: a. 3D structures of the GRB2-SH3 and PSD95-PDZ3 domains where residue atoms are colored by the position-wise average absolute change in the free energy of binding. Ligands are shown as black sticks. b. Domain structures with orthosteric sites (ligand distance<5Å) highlighted in red spheres and major allosteric site residues highlighted in orange spheres. c. Relationship between the position-wise average absolute change in free energy of binding and the distance to the ligand (minimal side chain heavy atom distance). Major allosteric sites (orange) are defined as non-binding interface residues with weighted average absolute change in free energy of binding higher than the average of binding interface residue mutations (red). Class-switching residues in PSD95-PDZ3 are those that favour a change in specificity for a T-2F ligand defined in McLaughlin et al. 2012 . See Figure S12 for the GB1 domain.

Article Snippet: To construct these plasmids, 7 gene blocks containing the mutated versions of GRB2-SH3 were synthesized with HindIII and NheI flanking restriction sites (gbGJJ002-8, Twist Bioscience, Table S2).

Techniques: Binding Assay