Journal: Biochemical Journal

Article Title: Nutrient transceptors physically interact with the yeast S6/protein kinase B homolog, Sch9, a TOR kinase target

doi: 10.1042/BCJ20200722

Figure Lengend Snippet: S. cerevisiae strains used in this study

Article Snippet: Using the Gap1 peptide and substitution peptide microarrays, synthesized by PEPperPRINT, we identified Gap1 peptides binding with Sch9 and also identified the most critical amino acid residues in the peptides for the interaction with Sch9.

Techniques:

Journal: Biochemical Journal

Article Title: Nutrient transceptors physically interact with the yeast S6/protein kinase B homolog, Sch9, a TOR kinase target

doi: 10.1042/BCJ20200722

Figure Lengend Snippet: Plasmids used in this study

Article Snippet: Using the Gap1 peptide and substitution peptide microarrays, synthesized by PEPperPRINT, we identified Gap1 peptides binding with Sch9 and also identified the most critical amino acid residues in the peptides for the interaction with Sch9.

Techniques: Expressing, Plasmid Preparation, Marker

Journal: Biochemical Journal

Article Title: Nutrient transceptors physically interact with the yeast S6/protein kinase B homolog, Sch9, a TOR kinase target

doi: 10.1042/BCJ20200722

Figure Lengend Snippet: Mep2, Sul1 or Pho84 and the Sch9 protein kinase. In vivo interaction between Sch9 and Gap1, Mep2, Sul1 or Pho84 was tested. Strains were constructed for the BiFC assay by tagging the transceptors with N-half of citrine fluorescent protein and Sch9 kinase with C-half of citrine. After substrate-specific starvation (see Materials and methods), samples were taken directly from the culture and imaged with a confocal microscope. Images of BiFC citrine fluorescent signal (left) and the DIC channel (right) are shown. ( A ) The Gap1–Sch9 BiFC strain showed in each cell a cytosolic stripe or arc, which was later found to locate at the nucleus–vacuole junction. ( B ) The Mep2–Sch9 BiFC strain showed a bright signal localized at the plasma membrane in each cell. ( C ) The Sul1–Sch9 BiFC strain showed a weak signal at the plasma membrane and a stronger signal in the vacuole. ( D ) The Pho84–Sch9 BiFC strain showed a similar but stronger plasma membrane signal and a weaker signal in the vacuole. All the scale bars indicate 10 µm.

Article Snippet: Using the Gap1 peptide and substitution peptide microarrays, synthesized by PEPperPRINT, we identified Gap1 peptides binding with Sch9 and also identified the most critical amino acid residues in the peptides for the interaction with Sch9.

Techniques: In Vivo, Construct, Bimolecular Fluorescence Complementation Assay, Microscopy, Clinical Proteomics, Membrane

Journal: Biochemical Journal

Article Title: Nutrient transceptors physically interact with the yeast S6/protein kinase B homolog, Sch9, a TOR kinase target

doi: 10.1042/BCJ20200722

Figure Lengend Snippet: ( A ) HA-tagged wild type Gap1 was pulled down by GST-tagged Pkh1 or Sch9, and detected with anti-HA-peroxidase antibody. Lane 1 : input from yeast cell extract directly loaded on the gel indicating the expression level of HA-Gap1 after 24 h nitrogen starvation. Lane 2 : negative control with only GST tag showing that no HA-Gap1 got pulled down unspecifically. Lane 3 : HA-Gap1 pulled down by GST-Pkh1. Lane 4 : HA-Gap1 pulled down by GST-Sch9. ( B ) HA-Gap1 mutant forms pulled down by GST-Sch9. Upper row input 1-6 : extract of yeast cells expressing HA-tagged Gap1 WT , Gap1 FVF-DKD , Gap1 F231D , Gap1 V232K , Gap1 F233D and Gap1 S234E was directly loaded on the gel for determination of their expression level with anti-HA-peroxidase antibody. Lower row pull-down 1–6 : HA-tagged Gap1 WT or mutant forms were pulled down by GST-Sch9. The quantification of band intensity below indicates the signal strength as a percentage of the signal with Gap1 WT , corrected with the expression level of the input and calculated as a mean of two independent experiments ± SD. There is no significant difference between the wild type and each mutant protein being observed.

Article Snippet: Using the Gap1 peptide and substitution peptide microarrays, synthesized by PEPperPRINT, we identified Gap1 peptides binding with Sch9 and also identified the most critical amino acid residues in the peptides for the interaction with Sch9.

Techniques: Expressing, Negative Control, Mutagenesis

Journal: Biochemical Journal

Article Title: Nutrient transceptors physically interact with the yeast S6/protein kinase B homolog, Sch9, a TOR kinase target

doi: 10.1042/BCJ20200722

Figure Lengend Snippet: A Gap1 peptide microarray was constructed and used for mapping the putative Sch9 binding site using 50 or 100 µg/ml GST-Sch9, or only GST as negative control (for description of peptide microarray, see ). ( A ) Microarray scan figures of GST or GST-Sch9 at concentration of 100 µg/ml. Upper figure : no interaction signal is observed on the microarray incubated with GST negative control. Lower figure : a range of positive signals (contiguous red spots) is observed on the microarray incubated with GST-Sch9. The HA peptides are synthesized on the edge of the peptide microarray, which are tested with anti-HA (12CA5) DyLight800 antibody after the mapping, and shown as green dots in the microarray scan figure. They are used as an internal control to confirm the assay quality and to facilitate grid alignment for data quantification. The clear and complete green dots of these control peptides validate the overall peptide microarray integrity and assay quality. ( B ) Quantification of the signals from the microarray scans is shown as intensity plots. For a better comparison of the response profiles with the two concentrations of GST and GST-Sch9, the intensity plots were shifted (the lines for GST-Sch9 at 50 µg/ml, GST at 100 µg/ml and GST-Sch9 at 100 µg/ml, were up-shifted with 500, 1000 and 1500 a.u., respectively). The control assays with pure GST did not reveal any background interaction at both protein concentrations. In contrast, many weak to moderate interactions were observed with GST-Sch9 at both protein concentrations, of which the strongest interactions were labeled on top of the peaks. ( C ) Location of the putative Sch9 binding domain in the predicted Gap1 topology (generated by Protter: http://wlab.ethz.ch/protter/start/ ) . Gap1 has 602 amino acid residues, arranged in 12 transmembrane domains (TMDs), with cytosolic N- and C-termini. The putative interaction motif ‘GVKGYGEAEFVFSFI’, indicated with circles with blue lining, is located in intracellular loop 2, with the 231 FVF 233 residues which are most critical for Sch9 binding and further investigated by mutagenesis, indicated with circles filled in red.

Article Snippet: Using the Gap1 peptide and substitution peptide microarrays, synthesized by PEPperPRINT, we identified Gap1 peptides binding with Sch9 and also identified the most critical amino acid residues in the peptides for the interaction with Sch9.

Techniques: Peptide Microarray, Construct, Binding Assay, Negative Control, Microarray, Concentration Assay, Incubation, Synthesized, Control, Comparison, Labeling, Generated, Mutagenesis

Journal: Biochemical Journal

Article Title: Nutrient transceptors physically interact with the yeast S6/protein kinase B homolog, Sch9, a TOR kinase target

doi: 10.1042/BCJ20200722

Figure Lengend Snippet: ( A ) Re-upstart of growth in NSM with 1 mM L-citrulline. OD 600nm was measured every 1 h for 90 h. ( B ) Re-upstart of growth in SC-URA medium. OD 600nm was measured every 1 h for 50 h. Standard deviation of the average of four replicates is shown with the error bars (for more details, see Materials and methods). ( C ) Transport of 1 mM [ 3 H]-labeled L-citrulline was measured for 1 min. Standard deviation of the average of three replicates is shown with the error bars. ( D ) As a read-out for Gap1-mediated PKA activation, short-term trehalase activation by the Gap1 mutant forms was measured. 5 mM L-citrulline was added after 24 h nitrogen starvation at time point 0. Trehalase activity was measured at different time points. Strains: gap1Δ strain transformed with empty plasmid (▽), plasmid expressing Gap1 WT (▪), Gap1 FVF-DKD (▴), Gap1 F231D (▾), Gap1 V232K (◆), Gap1 F233D (●) or Gap1 S234E (□).

Article Snippet: Using the Gap1 peptide and substitution peptide microarrays, synthesized by PEPperPRINT, we identified Gap1 peptides binding with Sch9 and also identified the most critical amino acid residues in the peptides for the interaction with Sch9.

Techniques: Standard Deviation, Labeling, Activation Assay, Mutagenesis, Activity Assay, Transformation Assay, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Crohn’s patients and healthy infants share immunodominant B cell response to commensal flagellin peptide epitopes

doi: 10.1101/2023.08.08.552496

Figure Lengend Snippet: (A) Schematic strategy of the flagellin peptide microarray. (B) Heatmap showing serum IgG reactivity for sequential, overlapping fifteen-residue flagellin peptides (108 peptides from each of the 19 different flagellins) in CD (n=135), HC (n=42), and UC (n=88) subjects. Sera were diluted at 1:100. Rows (subjects) were clustered using Euclidean distance and complete linkage applied to log-transformed data. Columns (peptides) are sorted by peptide position (and include peptides from up to 19 different flagellins per position). Cohort groups are indicated by colored dots on the right margin. Peptide starting position (relative to the N-terminus) is indicated on the bottom. For each 15-mer segment, per-residue information content is computed at each position across all flagellins (except L. CBir11 Fla and L. CBir66 Fla) and summated to indicate sequence conservation, shown as color. Although all peptides were used for the clustering, only peptides up to and including 121-136 are depicted in this heatmap. (See Supplementary Figure 1 for version showing all peptides). (C) 3D structure of L. A4 Fla2 as a representative flagellin showing the location of the hinge region. (D) Sequence alignment of flagellins included in the peptide microarray plus S. dublin FliC and E. coli FliC. Sequences were aligned in BioEdit using the ClustalW alignment feature. Flagellin D0 and D1 domains in the N-term are indicated by the color bars on top, whereas the hinge region is marked by the red box. (E) Motif discovery of the flagellin hinge region.

Article Snippet: In brief, biotinylated flagellin peptides were conjugated on to Streptavidin coated 4um polystyrene beads (Spherotech, Inc. Cat # PAK-4067-8K).

Techniques: Peptide Microarray, Transformation Assay, Sequencing

Journal: bioRxiv

Article Title: Crohn’s patients and healthy infants share immunodominant B cell response to commensal flagellin peptide epitopes

doi: 10.1101/2023.08.08.552496

Figure Lengend Snippet: (A) Schematic strategy of the flagellin peptide cytometric bead array. (B and C) Gating strategies of the flagellin peptide cytometric bead array. (D) Sera from CDhigh (n=39), CDlow (n=126), UC (n=181), and HC (n=95) subjects in the UAB cohort were probed against the flagellin peptide cytometric bead array. Representative flow plots of serum IgG reactivity to flagellin peptide epitopes in each group are shown. The red box highlights the hinge region peptides. (E) Histogram overlay of serum IgG reactivity to Lachnospiraceae D0-1N p25-59 in 3 representative CDhigh, CDlow, UC, and HC, respectively. (F-M) Epitope-specific IgG concentration in the serum of CDhigh, CDlow, UC, and HC subjects to indicated flagellin peptide epitopes. Data are presented as means ± SEM and analyzed with nonparametric Kruskal-Wallis test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: In brief, biotinylated flagellin peptides were conjugated on to Streptavidin coated 4um polystyrene beads (Spherotech, Inc. Cat # PAK-4067-8K).

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Crohn’s patients and healthy infants share immunodominant B cell response to commensal flagellin peptide epitopes

doi: 10.1101/2023.08.08.552496

Figure Lengend Snippet: (A) Sera of CD patients (grouped by disease behavior at 3-year follow-up using the Montreal classification, n=250 for B1 (inflammatory), n=142 for B2 (stricturing) or B3 (penetrating)) and non-IBD controls (n=72) from the RISK cohort were probed against the microbiota protein microarray. Mean fluorescent intensity (MFI) of serum IgG specific for indicated flagellin antigens is presented as means ± SEM and analyzed with nonparametric Kruskal-Wallis test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. (B) Heatmap of log 10 IgG response (color) on the microbiota protein microarray for 21 flagellins (rows) and 392 individuals with CD in the RISK cohort (columns). Rows and columns were hierarchically clustered using Euclidean distance on the log data, average linkage and optimal leaf ordering maximizing the sum of similarity between every leaf and all other leaves in the adjacent cluster. Montreal disease classification at follow-up (B2 and B3) is indicated below the heatmap, with unmarked individuals being B1. The bar graph at the bottom indicates the number of positive flagellin responses for each individual, where a positive response is two standard deviations above the mean (non-log transformed) of a non-IBD control cohort (n=72, not shown) per flagellin. (C and D) Summary of multi-flagellin reactivity in CD patients with B1 or B2/3 behavior at follow-up. (E-G) Sera of CD patients (grouped by disease behavior at 3-year follow-up, n=249 for B1, n=140 for B2/3) and non-IBD controls (n=72) from the RISK cohort were probed against the flagellin peptide cytometric bead array. Epitope-specific IgG concentration in different groups to indicated flagellin peptide epitopes is shown. Data are presented as means ± SEM and analyzed with nonparametric Kruskal-Wallis test. *P<0.05, **P<0.01, ****P<0.0001.

Article Snippet: In brief, biotinylated flagellin peptides were conjugated on to Streptavidin coated 4um polystyrene beads (Spherotech, Inc. Cat # PAK-4067-8K).

Techniques: Microarray, Transformation Assay, Concentration Assay

Journal: bioRxiv

Article Title: Crohn’s patients and healthy infants share immunodominant B cell response to commensal flagellin peptide epitopes

doi: 10.1101/2023.08.08.552496

Figure Lengend Snippet: (A) Heatmaps representing correlation coefficients between serum IgG anti-flagellin reactivity (log 10 MFI from microbiota protein microarray; rows) and serum IgG anti-peptide reactivity (hyperbolic arcsin MFI from flagellin peptide cytometric bead array; columns). Four separate heatmaps are shown for CD (n=148), HC (n=86), and UC (n=169) participants from the UAB cohort and CD (n=414) participants from the RISK cohort, respectively. Color corresponds to Pearson correlation coefficient indicated in the bar scale. Dots indicate correlations that were below Bonferroni-corrected p-value of 0.01 (based on 21 flagellins, 8 peptides, 4 groups = 672 tests) testing the hypothesis of no correlation against the alternative that there is a non-zero correlation. Rows (flagellins) are sorted based on the correlation for peptide D0-1N p25-59 in CD patients in the RISK cohort, as indicated by the triangle. (B) Hyperbolic arcsin MFI of serum IgG anti- Lachnospiraceae D0-1N p25-59 and D1C p391-407 of individual CD, HC, and UC participants from the UAB cohort and CD participants from the RISK cohort obtained from the flagellin peptide cytometric bead array was plotted against reactivity (log 10 MFI) to R. intestinalis Fla1 and E. coli FliC obtained from the microbiota protein microarray, respectively. Least squares regression was performed, with the correlation line in red and R value shown for each plot. (C and D) The value of hinge-specific IgG obtained from the flagellin peptide cytometric bead array is plotted on X axis, and the number of flagellins bound by sera in the same individual CD patients on the microbiota protein microarray on the Y axis. Data from the UAB cohort and the RISK cohort are presented as indicated.

Article Snippet: In brief, biotinylated flagellin peptides were conjugated on to Streptavidin coated 4um polystyrene beads (Spherotech, Inc. Cat # PAK-4067-8K).

Techniques: Microarray

Journal: bioRxiv

Article Title: Crohn’s patients and healthy infants share immunodominant B cell response to commensal flagellin peptide epitopes

doi: 10.1101/2023.08.08.552496

Figure Lengend Snippet: (A) Sera of healthy and non-IBD control infants at 0 month (cord blood, CB), 6 months, and 12 months of age (with all three timepoints) from the Uganda cohort (n=76) and the US (n=10) /Sweden (n=36) cohort were probed against the flagellin peptide cytometric bead array. Concentration of serum IgG specific to Lachnospiraceae flagellin D0-1N p25-59 in different groups is shown. Each dot represents an individual and responses of the same individual at different timepoints are linked with the grey lines. The means of serum IgG response to Lachnospiraceae flagellin D0-1N p25-59 of CD patients in the UAB (broken line) and RISK (solid line) cohort are plotted. Data are presented as means ± SEM and analyzed with repeated measures ANOVA and Friedman test. ****P<0.0001. (B and C) Sera of healthy and non-IBD control infants at indicated timepoints (including individuals not represented in all three timepoints) from the Uganda cohort (n=92) and the US (n=16) /Sweden (n=54) cohort were probed against the flagellin peptide cytometric bead array. IgG concentration specific to Lachnospiraceae D0N p25-44 and D1N p41-59 sub-epitopes in different groups is shown, respectively. Data are presented as means ± SEM and analyzed with two-way ANOVA and Tukey’s multiple comparisons test. *P<0.05, ***P<0.001, ****P<0.0001.

Article Snippet: In brief, biotinylated flagellin peptides were conjugated on to Streptavidin coated 4um polystyrene beads (Spherotech, Inc. Cat # PAK-4067-8K).

Techniques: Concentration Assay

Journal: Heart rhythm

Article Title: Calcium/Calmodulin-Dependent Protein Kinase II Regulation of I Ks during Sustained Beta-Adrenergic Receptor Stimulation

doi: 10.1016/j.hrthm.2018.01.024

Figure Lengend Snippet: (A) Phosphorylation status of KCNQ1 carboxy terminus in HEK 293 cells (co-expressing KCNQ1 and KCNE1) following treatment with 100 nM ISO for 3 minutes, 4 hours, and 24 hours via LCMS/MS analysis. (B) Peptide fragments corresponding to the intracellular regions of KCNQ1 were exposed to activated δCaMKII for 4 minutes and 30 seconds. Each peptide was 15 amino acids in length, tiled by two residues for 13 overlapping residues per consecutive peptide. Peptide fragments containing residues T482 and S484 (solid box at D5-D7) were the strongest substrates for CaMKII phosphorylation. The dashed box at D5-D7 is following 30 second exposure of activated δCaMKII. The solid box (F15-F19) contains a autocamtide-2 negative control (T→A mutation; F15), WT autocamtide-2 positive control (F17), and kemptide control (classical PKA substrate; F19). Full peptide sequences are in Supplemental Table 1. (C) Schematic of KCNQ1 and KCNE1 subunits showing carboxy terminal sites of potential CaMKII regulation investigated. *p<0.05 vs. control

Article Snippet: KCNQ1 Peptide Array Immobilized peptides were synthesized on a modified cellulose membrane using a robotic peptide synthesizer (Intavis MultiPep ® ) with routine Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry as previously described.

Techniques: Expressing, Negative Control, Mutagenesis, Positive Control

Journal: Heart rhythm

Article Title: Calcium/Calmodulin-Dependent Protein Kinase II Regulation of I Ks during Sustained Beta-Adrenergic Receptor Stimulation

doi: 10.1016/j.hrthm.2018.01.024

Figure Lengend Snippet: (A) Representative traces of IKs activation currents from WT KCNQ1/KCNE1 following 100 nM ISO or vehicle for 12-24 hours. (B) I-V plots, (C) activation curves (normalized to the voltage of maximum activation), and (D) rate constants of activation and (E) deactivation following treatment with ISO or vehicle. (F) Representative traces of IKs from KCNQ1 combination mimics of dephosphorylation (Triple-A) and phosphorylation (Triple-D) co-expressed with KCNE1. (G) I-V plots, (H) activation curves, and (I) rate constants of activation and (J) deactivation for Triple-A and Triple-D KCNQ1. (K) Peak current density at +60 mV for WT KCNQ1 and combination mimics following ISO or vehicle. *p<0.05 vs. WT+ISO, + p<0.05 for Triple-A vs. Triple-D, ǂp<0.05 for Triple-A+ISO vs. Triple-D+ISO, **p<0.05 for comparison indicated

Article Snippet: KCNQ1 Peptide Array Immobilized peptides were synthesized on a modified cellulose membrane using a robotic peptide synthesizer (Intavis MultiPep ® ) with routine Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry as previously described.

Techniques: Activation Assay, De-Phosphorylation Assay

Journal: Heart rhythm

Article Title: Calcium/Calmodulin-Dependent Protein Kinase II Regulation of I Ks during Sustained Beta-Adrenergic Receptor Stimulation

doi: 10.1016/j.hrthm.2018.01.024

Figure Lengend Snippet: (A) I-V plots, peak current density, and activation curves for WT, S457A, and S457D KCNQ1/KCNE1 (B) I-V plots, peak current density, and activation curves for WT, T482A, and T482D KCNQ1/KCNE1. (C) I-V plots, peak current density, and activation curves for WT, S484A, and S484D KCNQ1/KCNE1. *p<0.05

Article Snippet: KCNQ1 Peptide Array Immobilized peptides were synthesized on a modified cellulose membrane using a robotic peptide synthesizer (Intavis MultiPep ® ) with routine Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry as previously described.

Techniques: Activation Assay

Journal: Heart rhythm

Article Title: Calcium/Calmodulin-Dependent Protein Kinase II Regulation of I Ks during Sustained Beta-Adrenergic Receptor Stimulation

doi: 10.1016/j.hrthm.2018.01.024

Figure Lengend Snippet: (A) I-V plots and (B) peak current density for WT KCNQ1/KCNE1 following treatment with ISO (100 nM for 12-24 hours) and CN21, CN21-Ala, or myr-PKI. (C) Normalized activation curves following ISO with CN21, CN21-Ala, or myr-PKI. (D) Immunoblots and percent changes in CaMKII and CaMKII T287 phosphorylation following 100 nM ISO for 24 hours. *p<0.05

Article Snippet: KCNQ1 Peptide Array Immobilized peptides were synthesized on a modified cellulose membrane using a robotic peptide synthesizer (Intavis MultiPep ® ) with routine Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry as previously described.

Techniques: Activation Assay, Western Blot

Journal: Heart rhythm

Article Title: Calcium/Calmodulin-Dependent Protein Kinase II Regulation of I Ks during Sustained Beta-Adrenergic Receptor Stimulation

doi: 10.1016/j.hrthm.2018.01.024

Figure Lengend Snippet: (A) KCNQ1 carboxy peptides were exposed to activated δCaMKII. Each row contains peptides corresponding to the labeled KCNQ1 residue with columns for WT, A (phospho-acceptor site mutated to alanine), or KO (all serine and threonine mutated to alanine with the exception of T482 wherein S484 was not mutated; n=5 for each condition). (B) Quantification of phosphostimulated luminescence for WT, A, and KO peptides corresponding to KCNQ1 T482 and S484 during exposure to activated δCaMKII. *p<0.05, ns = not significant

Article Snippet: KCNQ1 Peptide Array Immobilized peptides were synthesized on a modified cellulose membrane using a robotic peptide synthesizer (Intavis MultiPep ® ) with routine Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry as previously described.

Techniques: Labeling

Journal: Heart rhythm

Article Title: Calcium/Calmodulin-Dependent Protein Kinase II Regulation of I Ks during Sustained Beta-Adrenergic Receptor Stimulation

doi: 10.1016/j.hrthm.2018.01.024

Figure Lengend Snippet: (A) I-V plots and (B) peak current density for WT or mutant KCNQ1 co-expressed with KCNE1 in cells overexpressing constitutively active δCaMKII or YFP control. (C) Normalized activation curves for WT KCNQ1 when expressed in CaMKII overexpression and control. (D) Rate constants of activation and (E) deactivation for WT KCNQ1 and S484A during CaMKII overexpression. + p<0.05 for Control, WT vs. CaMKII, WT, ǂp<0.05 for CaMKII, S484A vs. CaMKII, WT, **p<0.05 for comparison indicated, *p<0.05 for CaMKII, WT vs. CaMKII, S484A

Article Snippet: KCNQ1 Peptide Array Immobilized peptides were synthesized on a modified cellulose membrane using a robotic peptide synthesizer (Intavis MultiPep ® ) with routine Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry as previously described.

Techniques: Mutagenesis, Activation Assay, Over Expression