Journal: Ecotoxicology and environmental safety

Article Title: PRKAA1 induces aberrant mitophagy in a PINK1/Parkin-dependent manner, contributing to fluoride-induced developmental neurotoxicity.

doi: 10.1016/j.ecoenv.2023.114772

Figure Lengend Snippet: Fig. 5. NaF exposure induces elevated phos phorylation of PRKAA1 expression in vivo and in vitro. (A) The hierarchical cluster heatmap of phosphorylated proteins in rat hippocampus. (B) Statistics of different phosphorylation sites and proteins in rat hippocampus. (C) Volcano plot of differentially phosphorylated proteins in rat hippocampus. The criteria for significance were set at logarithmic fold change (log2 FC) of > 1.2 or < 0.8 (P < 0.05). (D) Phosphorylation of PRKAA1 protein in rat hippocampus. (E-F) Representative western blot and relative quan tifications of PRKAA1 and p-PRKAA1 in rat hippocampus. (G-H) Representative western blot and relative quantifications of PRKAA1 and p-PRKAA1 in SH-SY5Y cells. All experiments were performed independently and repeated 3 times. The data were presented as the means ± SD. *P < 0.05 compare with the control group.

Article Snippet: PRKAA1, p-PRKAA1 (Thr172), Parkin, and Cyt C antibodies were obtained from Boster Biotech (China).

Techniques: Expressing, In Vivo, In Vitro, Phospho-proteomics, Western Blot, Control

Journal: Ecotoxicology and environmental safety

Article Title: PRKAA1 induces aberrant mitophagy in a PINK1/Parkin-dependent manner, contributing to fluoride-induced developmental neurotoxicity.

doi: 10.1016/j.ecoenv.2023.114772

Figure Lengend Snippet: Fig. 6. DM inhibits aberrant mitophagy by pre venting the phosphorylation of PRKAA1 in vitro. (A-B) Representative western blot and relative quantifications of PRKAA1and p-PRKAA1 in SH- SY5Y cells. (C-D) Representative western blot and relative quantifications of PINK1 and Parkin in SH-SY5Y cells. (E-F) Representative images and relative quantifications of immunofluores cence staining of PINK1 and Mito-tracker, after DM intervention. The scale bar represents 50 µm. (G-H) Representative western blot and relative quantifications of TOMM-20 in SH-SY5Y cells. All experiments were performed independently and repeated 3 times. The data were presented as the means ± SD. *P < 0.05 compare with the control group, @P < 0.05 compare with the NaF group.

Article Snippet: PRKAA1, p-PRKAA1 (Thr172), Parkin, and Cyt C antibodies were obtained from Boster Biotech (China).

Techniques: Phospho-proteomics, In Vitro, Western Blot, Staining, Control

Journal: Arthritis Research & Therapy

Article Title: Aberrant mechanical loading induces annulus fibrosus cells apoptosis in intervertebral disc degeneration via mechanosensitive ion channel Piezo1

doi: 10.1186/s13075-023-03093-9

Figure Lengend Snippet: Piezo1 promotes AFCs apoptosis via Ca2 + /Calpain2/Caspase3 pathway. a The activity of Calpains in the Ctrl group, CMS group, CMS + Lv-Ctrl group, and CMS + Lv-Piezo1 group as determined by Calpain activity detection kit ( n = 3). b Western blotting analysis showing the Calpain1, Calpain2, Bax, Cleaved-Caspase3 expression in the Ctrl group, CMS group, CMS + Lv-Ctrl group, and CMS + Lv-Piezo1 group ( n = 3). c Representative flow cytometry scatter plots detecting AFCs apoptosis in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group. d Representative Tunel staining fluorescence pictures detecting AFCs apoptosis in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group (scale bar = 50 μm). Apoptotic AFCs appeared red and nuclei were counterstained with Hoechst 33,358 (blue). e Representative MMP staining fluorescence pictures detecting AFCs apoptosis in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group (scale bar = 50 μm). MMP appeared red and nuclei were counterstained with Hoechst 33,358 (blue). f Statistic data of apoptotic AFCs in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group determined by flow cytometry ( n = 3). g Statistic data of Tunel-positive apoptotic AFCs in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group determined by Tunel staining ( n = 3). h Statistic data of mean MMP fluorescent intensity in AFC in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group determined by MMP staining ( n = 3). i Western blotting analysis showing the Bax, Cleaved-Caspase3 expression in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group ( n = 3). ** P < 0. 01, *** P < 0.001, **** P < 0.0001

Article Snippet: Primary antibodies contain calpain1 (10,538–1-AP, Proteintech), calpain2 (11,472–1-AP, Proteintech), Bax (50,599–2-Ig, Proteintech), cleaved-Caspase 3 (9664, Cell Signaling Technology, MA, USA), Piezo1 (5939–1-AP, Proteintech), and Gapdh (60,004–1-Ig, Proteintech).

Techniques: Activity Assay, Western Blot, Expressing, Flow Cytometry, TUNEL Assay, Staining, Fluorescence

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: International Journal of Molecular Medicine

Article Title: lncRNA MEG3 promotes hepatic insulin resistance by serving as a competing endogenous RNA of miR-214 to regulate ATF4 expression

doi: 10.3892/ijmm.2018.3975

Figure Lengend Snippet: miR-214 inhibition and ATF4 overexpression reverse MEG3 knockdown-mediated decreases in FoxO1, G6pc and Pepck protein expression. Cells were transfected with the indicated plasmids, followed by treatment with 0.5 mM palmitate for 4 h. (A–D) Western blot analysis revealed that MEG3 knockdown significantly suppressed the palmitate-mediated increase in (B) FoxO1, (C) Pepck, and (D) G6pc protein expression. (E–H) By contrast, the miR-214 inhibitor additionally induced the palmitate-mediated increase in (F) FoxO1, (G) Pepck and (H) G6pc protein expression. (I–L) ATF4 overexpression also induced the palmitate-mediated increase in (J) FoxO1, (K) Pepck and (L) G6pc protein expression. β-tubulin served as the loading control in the western blot analyses. * P<0.05 vs . group 1; # P<0.05 vs. group 3; $ P<0.05 vs . group 4. si, small interfering RNA; ctrl, control; MEG3, maternally expressed gene 3; miR, microRNA; FoxO1, forkhead box protein O1; Pepck, phosphoenolpyruvate carboxykinase; G6pc, glucose-6-phosphatase catalytic subunit; ATF4, activating transcription factor 4.

Article Snippet: The PVDF membranes were then blocked with 5% fat-free milk for 1 h at room temperature and sequentially incubated at 4°C overnight with the corresponding primary antibodies against ATF4 (1:1,000; cat. no. ab23760; Abcam, Cambridge, MA, USA), FoxO1 (1:1,000; cat. no. ab39670; Abcam), Pepck (1:200; cat. no. sc-377027; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and G6pc (1:1,000; cat. no. TA334651; OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Inhibition, Over Expression, Expressing, Transfection, Western Blot, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: lncRNA MEG3 promotes hepatic insulin resistance by serving as a competing endogenous RNA of miR-214 to regulate ATF4 expression

doi: 10.3892/ijmm.2018.3975

Figure Lengend Snippet: miR-214 inhibition and ATF4 overexpression reverse the MEG3 knockdown-mediated decrease in FoxO1, G6pc and Pepck mRNA expression. Cells were transfected with the indicated plasmids, followed by treatment with 0.5 mM palmitate for 4 h. (A–C) RT-qPCR results revealed that MEG3 knockdown significantly suppressed the palmitate-mediated increase in (A) FoxO1, (B) Pepck, and (C) G6pc mRNA expression. (D–F) By contrast, the miR-214 inhibitor induced the palmitate-mediated increase in (D) FoxO1, (E) Pepck, and (F) G6pc mRNA expression. (G–I) ATF4 overexpression also induced the palmitate-mediated increase in (G) FoxO1, (H) Pepck, and (I) G6pc mRNA expression. * P<0.05 vs . group 1; # P<0.05 vs. group 3; $ P<0.05 vs . group 4. miR, microRNA; ATF, activating transcription factor 4; MEG3, maternally expressed gene 3; FoxO1, forkhead box protein O1; Pepck, phosphoenolpyruvate carboxykinase; G6pc, glucose-6-phosphatase catalytic subunit; si, small interfering RNA; ctrl, control; MEG3.

Article Snippet: The PVDF membranes were then blocked with 5% fat-free milk for 1 h at room temperature and sequentially incubated at 4°C overnight with the corresponding primary antibodies against ATF4 (1:1,000; cat. no. ab23760; Abcam, Cambridge, MA, USA), FoxO1 (1:1,000; cat. no. ab39670; Abcam), Pepck (1:200; cat. no. sc-377027; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and G6pc (1:1,000; cat. no. TA334651; OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Inhibition, Over Expression, Expressing, Transfection, Quantitative RT-PCR, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: lncRNA MEG3 promotes hepatic insulin resistance by serving as a competing endogenous RNA of miR-214 to regulate ATF4 expression

doi: 10.3892/ijmm.2018.3975

Figure Lengend Snippet: Proposed associations between target genes. Palmitate time-dependently increased MEG3 and ATF4 expression, but decreased miR-214 expression. MEG3 functioned as a competing endogenous RNA of miR-214 to upregulate ATF4 expression, leading to the promotion of FoxO1 and its downstream gluconeogenic enzymes G6pc and Pepck. This thereby increases gluconeogenesis and promotes insulin resistance. MEG3, maternally expressed gene 3; miR, microRNA; ATF4, activating transcription factor 4; FoxO1, Forkhead box protein O1; O1; Pepck, phosphoenolpyruvate carboxykinase; G6pc, glucose-6-phosphatase catalytic subunit.

Article Snippet: The PVDF membranes were then blocked with 5% fat-free milk for 1 h at room temperature and sequentially incubated at 4°C overnight with the corresponding primary antibodies against ATF4 (1:1,000; cat. no. ab23760; Abcam, Cambridge, MA, USA), FoxO1 (1:1,000; cat. no. ab39670; Abcam), Pepck (1:200; cat. no. sc-377027; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and G6pc (1:1,000; cat. no. TA334651; OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Expressing