Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: DRI-Pep #20 is a potent PI3Kγ/PKA disruptor peptide. A , chemical structure of DRI-Pep #20. The amino acid sequence of DRI-Pep #20 comprises the nonnatural D-peptide RHQGK, the D-retroinverso (DRI)-isoform of the cell penetrating peptide Penetratin 1 (P1) and a glycine (G) linker. B , schematic representation of the fluorescence spectroscopy assays for the characterization of the interaction between DRI-Pep #20 (or PI3Kγ MP) and the recombinant fluorescein 5-maleimide–labeled PKA-RIIα (PKA-F5M). C , steady-state emission spectra of PKA-F5M in the presence of increasing concentrations of DRI-Pep #20 (0–20 μM). K D : dissociation constant. Inset, nonlinear fitting of the fluorescence intensity maxima obtained at various concentrations of DRI-Pep #20 for the monitoring of bio-labeled PKA. K A : association constant. D , for kinetic analysis, fluorescence spectra of PKA-F5M in the presence of increasing concentrations of DRI-Pep #20 or PI3Kγ MP (inset) were analyzed and fitted to a single exponential function to obtain the observed rate constant ( k obs ). The binding of DRI-Pep #20 or PI3Kγ MP to biolabeled PKA was investigated under pseudo -first-order conditions, and the kinetic constants, k on and k off , were determined. E , schematic representation of the displacement assay between DRI-Pep #20 (or PI3Kγ MP) and the PI3Kγ/PKA-F5M complex. F , percentage displacement of the PI3Kγ/PKA-RIIα complex by DRI-Pep #20 or PI3Kγ MP, calculated from steady-state emission spectra of the PI3Kγ/PKA-F5M complex in the presence of increasing concentrations of the peptides (0–5 μM). The displacement efficiency was expressed as percentage of the binding between PI3Kγ and PKA-F5M relative to that in the absence of peptides. G , cAMP concentrations in peritoneal macrophages from WT (in green ) and PI3Kγ −/− mice (in gray ) treated with DRI-Pep #20 (1–25 μM) for 30 min. The amount of cAMP was expressed as percentage of cAMP accumulation observed in untreated PI3Kγ −/− cells. n ≥ 6 technical replicates from N > 3 independent experiments. ∗∗∗ p < 0.001 WT versus PI3Kγ −/− and # p < 0.05, ## p < 0.01, and ### p < 0.001 UT versus DRI-Pep #20 by one-way ANOVA, followed by Bonferroni’s post hoc test. Data are means ± SD. AU, arbitrary units; PKA, protein kinase A; PKA-RIIα, PKA regulatory subunit RIIα; PI3Kγ, phosphoinositide 3-kinase gamma; PKA-F5M, fluorescein 5-maleimide–labeled PKA-RIIα.

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: Sequencing, Fluorescence, Spectroscopy, Recombinant, Labeling, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: Binding kinetics of the interaction between DRI-Pep #20 or PI3Kγ MP and PKA-RIIα

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: Structural prediction of the binding between DRI-Pep #20 and PKA-RIIα. A , DRI-Pep #20 structure prediction by PEP-FOLD3.5. P1-G and RHQGK domains are shown as cartoons in gray and red , respectively. R-1, H-2, Q-3, and K-5 residues are indicated and shown as sticks . B , circular dichroism spectra of DRI-Pep #20 showing a peak at 190–240 nm. The percentage of α-helical and β-sheet secondary structures calculated by the K2D3 software are indicated. C , molecular docking simulation of the interaction between DRI-Pep #20 and the PKA-RIIα dimer by HADDOCK 2.4. The docked pose of DRI-Pep #20 in complex with residues 2 to 44 of PKA-RIIα (cartoon in green ) is shown. The key residues involved in the binding are indicated and shown as sticks , with DRI-Pep #20 residues in bold . Hydrogen bonds between DRI-Pep #20 and PKA-RIIα are indicated by yellow dashed lines . In ( A and C ), the structural models were developed using PyMOL. DRI, D-retroinverso; HADDOCK, high ambiguity driven biomolecular DOCKing; PI3Kγ, phosphoinositide 3-kinase gamma; PKA, protein kinase A; PKA-RIIα, PKA regulatory subunit RIIα.

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: Structural Proteomics, Binding Assay, Circular Dichroism, Software

Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: Structural prediction of the native binding between the N-terminal domain of PI3Kγ and PKA-RIIα. A , molecular docking simulation of the interaction between PI3Kγ and the PKA-RIIα dimer by HADDOCK 2.4. The docked pose of residues 109 to 159 of PI3Kγ in complex with residues 2 to 44 of the PKA-RIIα dimer ( green cartoon) is shown. The amino acids critical for the binding between the two proteins are shown and indicated as sticks , with the residues of PI3Kγ in bold . The putative PKA-binding motif of PI3Kγ (126–150) is shown in orange and blue . The sequence in orange indicates the region of PI3Kγ that was identified as being at the core of the interaction (KATHR). Hydrogen bonds between PI3Kγ and PKA-RIIα are indicated by yellow dashed lines . B , structural prediction of the KATHR sequence by PEP-FOLD3.5. KATHR and P1-G domains are shown as cartoons in orange and gray , respectively. K-18, H-21 and R-22 residues of the KATHR sequence (corresponding to K-126, H-129 and R-130 of native PI3Kγ) are indicated and shown as sticks . C , molecular docking simulation of the interaction between KATHR and the PKA-RIIα dimer by HADDOCK 2.4. The docked pose of KATHR in complex with residues 2 to 44 of PKA-RIIα (cartoon in green ) is shown. Yellow dashed lines indicate hydrogen bonds between KATHR and 2 to 44 PKA-RIIα. The amino acids critical for the binding are indicated and shown as sticks , with KATHR residues in bold . Throughout, the structural models were developed using PyMOL. HADDOCK, high ambiguity driven biomolecular DOCKing; PI3Kγ, phosphoinositide 3-kinase gamma; PKA, protein kinase A; PKA-RIIα, PKA regulatory subunit RIIα.

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: Structural Proteomics, Binding Assay, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: DRI-Pep #20 increases cAMP levels locally in vivo in the airway tract of mice. A , schematic representation of the treatment schedule. Mice received DRI-Pep #20 through intratracheal (i.t.) instillation. B – D , cAMP concentrations in tracheas ( B ), lungs ( C ) and hearts ( D ) from BALB/c mice 24 h after i.t. instillation of different doses of DRI-Pep #20 (0–750 mg/kg). Values in brackets indicate the dose of DRI-Pep #20 expressed as mg/kg. The number of mice (n) ranged from three to six per group. EC 50 , median effective concentration. E – G , cAMP concentrations in tracheas ( E ), lungs ( F ) and hearts ( G ) from WT and PI3Kγ −/− mice 24 h after i.t. instillation of 10 μg/Kg DRI-Pep #20 (in green ) or PBS (in gray ). The number of mice (n) ranged from three to four per group. In ( A and B ), ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001 by one-way ANOVA, followed by Bonferroni’s post hoc test. In ( E and F ) ∗ p < 0.05 and ∗∗ p < 0.01 PBS versus DRI-Pep #20 by two-way ANOVA test, followed by Bonferroni’s post hoc analysis. Throughout, data are means ± SD. DRI, D-retroinverso; PI3Kγ, phosphoinositide 3-kinase γ.

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: In Vivo, Concentration Assay

Journal: medRxiv

Article Title: Children develop robust and sustained cross-reactive spike-specific immune responses following SARS-CoV-2 infection

doi: 10.1101/2021.04.12.21255275

Figure Lengend Snippet: (A) Plasma from SARS-CoV-2 seropositive children (n=21) was assessed for binding to the spike protein of the four HCoV or the spike or nucleocapsid regions of SARS-CoV-2. Plasma was either applied neat (control) or following pre-absorption with either recombinant spike S1 domain (spike 1 Block) or spike S2 domain (spike 2 Block). S1 pre-absorption markedly reduced binding to SARS-CoV-2 spike whilst S2 pre-absorption reduced binding to OC43 and HKU-1. Repeated measure one-way ANOVA with Holm-Sidak’s multiple comparison test, or Friedman test with Dunn’s multiple comparisons test as appropriate

Article Snippet: Plasma samples were pre-diluted 1:10 with PBS then pre-absorbed by adding an equal volume of either recombinant spike S1 domain (10569-CV-100) or spike S2 (10594-CV-100) (R&D systems) at a concentration of 500ug/ml in PBS, or PBS alone (mock).

Techniques: Binding Assay, Recombinant, Blocking Assay, Comparison

Journal: Journal of Neuroscience

Article Title: Dynamic Changes in Presynaptic and Axonal Transport Proteins Combined with Striatal Neuroinflammation Precede Dopaminergic Neuronal Loss in a Rat Model of AAV -Synucleinopathy

doi: 10.1523/jneurosci.5427-08.2009

Figure Lengend Snippet: Figure3. Striatalchangesinthelevelsofproteinsinvolvedinsynaptictransmissionandaxonaltransportat8weekspostAAV -synuclein injection. Western blot was performed using membrane fraction (P2) of striatal lysate to measure levels of proteins involvedinsynaptictransmission.Amongtheseproteins,rabphilin3Aandsyntaxinlevelswerereduced(A,B).Westernblotwas performed using total lysate (T) of the striatum to measure levels of protein involved in axonal transport. Levels of anterograde transport motor proteins including KIF1A, KIF1B, KIF2A, KIF3A and myosin Va were markedly decreased, whereas levels of retrograde transport motor proteins including dynein, dynamitin and dynactin1 were dramatically increased (C, D). Levels of cytoskeletalproteinssuchasactin,neurofilaments,-tubulinand-tubulin,werealtered(C,D).Opticaldensitiesoftheindivid- ualbandswerequantifiedusingNIHImageJ.Opticaldensitiesof-synucleinoverexpressingconditionswerenormalizedbythe averagedvalueofGFPexpressingcondition.DataareshownasmeanSEM(AAVGFP,n46;AAVA53T-synuclein,n 46; *p 0.05 two tail t test).

Article Snippet: After at least 1 h of blocking in 5% nonfat dry milk, the membranes were incubated overnight at 4°C in various primary antibodies [ -synuclein (clone 42, BD Transduction Laboratories; 1:2000), GFP (Invitrogen; 1:5000) TH (Pel Freeze; 1:3000), dopamine transporter (DAT, Millipore Bioscience Research Reagents; 1:2000), Vesicular Monoamine Transporter 2 (VMAT2, Pel Freeze; 1:1000), SNAP-25 (Millipore Bioscience Research Reagents; 1:4000), Rabphilin3A (BD Transduction Laboratories; 1:2000), RAB3A (Affinity Bioreagent; 1:3000), syntaxin (Millipore Bioscience Research Reagents; 1:2000), synaptophysin (Santa Cruz; 1:500), synaptotagmin (BD Transduction Laboratories; 1:5000), synapsin (Millipore Bioscience Research Reagents; 1:5000), Munc-18 (Affinity Bioreagents; 1:3000), KIF1A (clone 16, BD Transduction Laboratories; 1:2000), KIF1B (Bethyl Laboratory; 1:2000), KIF2A (Abcam; 1:10,000), KIF3A (Abcam; 1:2000), KIF5 (Abcam; 1:1000), KIF17 (Abcam; 1:500), myosin Va (Sigma; 1:500), dynein (clone 74.1, Millipore Bioscience Research Reagents; 1:2000), dynamitin (Millipore Bioscience Research Reagents; 1:2000), dynactin1 (Novus,1:1000), -tubulin (clone DM1A, Upstate; 1:5000), -tubulin (Upstate; 1:5000), -tubulin (clone GTU-88, Sigma; 1:5000), Actin (clone C-2, Santa Cruz; 1:500), neurofilament 160 (clone NN18, Sigma; 1:1000), neurofilament 200 (clone N52, Sigma; 1:1000), and GAPDH (Millipore Bioscience Research Reagents; 1:5000)].

Techniques: Injection, Western Blot, Membrane