pi3kγ (p110γ) recombinant human protein  (Thermo Fisher)

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: 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: 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