pi3kγ (p110γ  (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

Journal: bioRxiv

Article Title: Dual Targeting of STING and PI3Kγ Eliminates Regulatory B Cells to Overcome STING Resistance for Pancreatic Cancer Immunotherapy

doi: 10.1101/2024.02.14.580378

Figure Lengend Snippet: ( a ) PI3Kγ and STING expression at lymph node and pancreatic tumor from KPC mice compared to C57BL/6 mice. ( b ) PI3Kγ and STING expression at B cell, bone marrow derived dendritic cells and bone marrow derived macrophages from KPC mice and C57BL/6 mice. ( c ) Phosphorylation of IRF3 at splenic B cells, bone marrow derived dendritic cells (BMDCs), bone marrow derived macrophages (BMDMs) and CD4 T cells derived from KPC mice and THP-1 hSTING HAQ cells after treatments with or without MSA-2 or IPI-549. ( d ) Quantification and representative flow cytometry analysis of IL35 + and IL10 + Breg cells (from KPC spleen) after treatments with or without MSA-2 or IPI-549. ( e ) Quantification of mouse Interferon-β concentration of bone marrow derived dendritic cells (BMDCs) derived from C57BL/6 mice after treatments with or without MSA-2 or IPI-549. ( f, g ) Quantification of human Interferon-β concentration from THP-1 R232 hSTING R232 after treatments with or without MSA-2 or IPI-549. ( g ) Quantification of STING activation by reporter assay from THP1-Blue TM ISG by measuring optical density at 655 nm after treatments with or without MSA-2 or IPI-549. ( h ) Quantification of human Interferon-β concentration from THP-1 hSTING KO after treatments with or without MSA-2 or IPI-549. ( i, j ) Quantification of CD86 geometric mean fluorescent intensity of bone marrow derived dendritic cells (BMDCs) bone marrow derived macrophages (BMDMs) derived from C57BL/6 mice after treatments with or without MSA-2 or IPI-549. ( k ) Quantification of RAW264.7 M1 polarization ratios after treatments with or without MSA-2 or IPI-549. ( l ) Quantification of TNF-α concentration from RAW264.7 after treatments with or without MSA-2 or IPI-549. Data for quantification are shown as mean ± SD, n = 3. Statistical comparisons are based on one-way ANOVA, followed by post hoc Tukey’s pairwise comparisons or by Student’s unpaired T-test. The asterisks denote statistical significance at the level of **** p < 0.0001. ANOVA, analysis of variance; SD, standard deviation; n.s., no statistical significance. For ( d ) statistical comparisons are conducted between MSA-2 group with other groups. For ( e-l ), statistical comparisons are conducted between MSA-2 and MSA-2+IPI-549 groups with control group.

Article Snippet: PI3Kγ binding affinity was evaluated using PI3Kγ (p110γ/PIK3R5) assay kit from BPS Bioscience, following the provided protocol.

Techniques: Expressing, Derivative Assay, Flow Cytometry, Concentration Assay, Activation Assay, Reporter Assay, Standard Deviation, Control

Journal: JCI insight

Article Title: IRF5 governs macrophage adventitial infiltration to fuel abdominal aortic aneurysm formation.

doi: 10.1172/jci.insight.171488

Figure Lengend Snippet: Figure 4. IRF5 promotes macrophage migration via PI3Kγ. 845

Article Snippet: Antibodies aganist476 PI3Kγ (sc-166365), PCNA (Santa Cruz, sc-56) were purchased from Santa477 Cruz Biotechnology.

Techniques: Migration

Journal: JCI insight

Article Title: IRF5 governs macrophage adventitial infiltration to fuel abdominal aortic aneurysm formation.

doi: 10.1172/jci.insight.171488

Figure Lengend Snippet: Figure 7. IRF5 and PI3Kγ are induced in infiltrated macrophages in human AAA. 906

Article Snippet: Antibodies aganist476 PI3Kγ (sc-166365), PCNA (Santa Cruz, sc-56) were purchased from Santa477 Cruz Biotechnology.

Techniques:

Journal: JCI Insight

Article Title: IRF5 governs macrophage adventitial infiltration to fuel abdominal aortic aneurysm formation

doi: 10.1172/jci.insight.171488

Figure Lengend Snippet: ( A ) Venn diagram showing the overlapping genes among differentially expressed genes (DEGs) between WT and Irf5 –/– mice, migration-related genes, macrophage highly expressed genes, and genes with a potential IRF5 binding site. ( B ) Heatmap of the 15 overlapping genes in AAA tissues from WT and Irf5 –/– mice. ( C and D ) Macrophages from Irf5 fl/fl and Irf5 ΔMΦ mice were treated or not with TNF-α (50 ng/mL). The mRNA ( C , n = 7) and protein ( D , n = 4) levels of PI3Kγ were decreased in macrophages with IRF5 deletion. ( E ) Bone marrow–derived macrophages (BMDMs) stimulated with TNF-α (50 ng/mL) or not for 2 hours were collected, and ChIP assays were conducted ( n = 6). Chromatin was immunoprecipitated and assessed by PCR analysis to determine the binding site on the Pik3cg promoter. ( F ) Dual-luciferase assays indicated that the Irf5 expression plasmid transfected into HEK293T cells increased luciferase activity of Pik3cg promoter reporter plasmids, and luciferase activity was reduced when Pik3cg promoter reporter plasmids harbored the deletion of nt –1060 to –1047 ( n = 6). ( G and H ) Migratory abilities of macrophages from Irf5 fl/fl , Irf5 ΔMΦ , and Pik3cg MΦ+ Irf5 ΔMΦ mice were measured by wound healing ( G ) and Transwell ( H ) assays ( n = 9). Excessive PI3Kγ expression promoted macrophage migration. Scale bar: 50 μm. Data are presented as mean ± SD, and the significance was determined by 2-way ANOVA followed by Bonferroni’s test ( C – E ) or 1-way ANOVA followed by Bonferroni’s test ( F – H ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against PI3Kγ (catalog sc-166365) and PCNA (catalog sc-56) were purchased from Santa Cruz Biotechnology.

Techniques: Migration, Binding Assay, Derivative Assay, Immunoprecipitation, Luciferase, Expressing, Plasmid Preparation, Transfection, Activity Assay

Journal: JCI Insight

Article Title: IRF5 governs macrophage adventitial infiltration to fuel abdominal aortic aneurysm formation

doi: 10.1172/jci.insight.171488

Figure Lengend Snippet: ( A ) Representative images of immunofluorescent staining of PI3Kγ and CD68 in elastase-induced (E-induced) versus inactive elastase–induced (IE-induced) AAA from WT mice. Asterisks indicate aortic lumen. Scale bar: 100 μm. ( B ) Quantification of PI3Kγ in mice treated with IE ( n = 7) or E ( n = 7). ( C ) Western blot analysis suggested that PI3Kγ expression in E-induced AAA tissues was significantly increased compared with the IE group. ( D ) Representative photographs of WT mice and Pik3cg –/– mice subjected to IE or E treatment. Scale bar: 2 mm. ( E ) Pik3cg –/– mice showed a reduced AAA expansion compared with that of WT mice ( n = 7 WT mice with IE, n = 7 Pik3cg –/– mice with IE, n = 7 WT with E, n = 7 Pik3cg –/– with E). ( F ) CD68 immunostaining in AAA tissues from WT mice and Pik3cg –/– mice after E treatment for 2 weeks. Quantification is shown on the right. Asterisks indicate aortic lumen. Scale bar: 100 μm. Data are presented as mean ± SD, and the significance was determined by unpaired, 2-tailed Student’s t test ( B and C ) or 2-way ANOVA followed by Bonferroni’s test ( E and F ). * P < 0.05, *** P < 0.001.

Article Snippet: Antibodies against PI3Kγ (catalog sc-166365) and PCNA (catalog sc-56) were purchased from Santa Cruz Biotechnology.

Techniques: Staining, Western Blot, Expressing, Immunostaining

Journal: JCI Insight

Article Title: IRF5 governs macrophage adventitial infiltration to fuel abdominal aortic aneurysm formation

doi: 10.1172/jci.insight.171488

Figure Lengend Snippet: ( A ) Representative images of immunofluorescent staining of IRF5 and CD68 in human AAA tissues. In AAA tissues, IRF5 was mostly located in the aortas and present in infiltrated CD68-positive cells. ( B and C ) Quantification of IRF5 in normal aortas ( B , n = 3) and AAA samples ( C , n = 5). ( D ) Representative images of immunofluorescent staining of PI3Kγ and CD68 in human AAA tissues. PI3Kγ was highly expressed in macrophages of human AAA samples. ( E and F ) Quantification of PI3Kγ in normal aortas ( E , n = 3) and AAA samples ( F , n = 5). Scale bars: 100 μm. Data in B , C , E , and F are presented as mean ± SD, and significance was determined by unpaired, 2-tailed Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against PI3Kγ (catalog sc-166365) and PCNA (catalog sc-56) were purchased from Santa Cruz Biotechnology.

Techniques: Staining