Journal: The Journal of Biological Chemistry

Article Title: A new functional assay reveals that membrane binding is critical for overactivation of the phosphoinositide 3-kinase H1047R mutant

doi: 10.1016/j.jbc.2026.111207

Figure Lengend Snippet: Establishment and titration of the optimal form of the substrate PIP 2 . A , the optimal form of substrate was determined by comparing two different ratios of lPIP 2 to total lipids, 1:10 and 1:2, both at a final lPIP 2 concentration of 7 μM. A water-soluble PIP 2 was also tested, at a concentration of 100 μM. Note that the most advantageous substrate preparation is a 1:2 mixture of lPIP 2 to total lipids. Two replicates from n = 10. B , different concentrations of lPIP 2 (1:2 mixture of lPIP 2 to total lipids) were tested. Note the dose-dependent effect. Two replicates from n = 4. C , schematic diagram of the experimental set up used in ( A ) and ( B ). The individual data points displayed above represent the means of replicates in each experiment. The statistical significance of difference between groups was examined by one-way ANOVA, followed by Tukey’s multiple comparisons test. ns : no significance, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Data were shown as mean ± SD. Specific activity values for PI3Kα, calculated as pmol PIP 3 produced per minute per nanogram of enzyme, are indicated in red fonts in the figure. lPIP 2 , lipid form of PIP 2 ; PIP 2 , phosphatidylinositol-4,5-biphospate; PIP 3 , phosphatidylinositol-3,4,5-triphosphate.

Article Snippet: Lipid PIP 2 [L-α-phosphatidylinositol-4,5 bisphosphate (Brain, Porcine) (ammonium salt)] and soluble PIP 2 [1,2-dioctanoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′,5-bisphosphate) (ammonium salt)] were from Avanti Polar Lipids.

Techniques: Titration, Concentration Assay, Activity Assay, Produced

Journal: The Journal of Biological Chemistry

Article Title: A new functional assay reveals that membrane binding is critical for overactivation of the phosphoinositide 3-kinase H1047R mutant

doi: 10.1016/j.jbc.2026.111207

Figure Lengend Snippet: Role of membranes in the overactivation of the mutants H1047R and E545K. We compare the PI3Kα enzymatic activity of H1047R, E545K, and WT, using as substrate 7 μM lPIP 2 ( A ), or 100 μM sPIP 2 ( B ). The activities of the mutants were normalized to WT, which was set to value “1.” Note that E545K shows higher activity than WT with either soluble or membranous PIP 2 , whereas H1047R exhibits increased activity only when membranes are used. Two replicates from n = 6 in ( A ) and n = 7 in ( B ). The individual data points displayed above represent the means of replicates in each experiment. The statistical significance of difference between groups in graphs ( A ) and ( B ) was examined by one-way ANOVA, followed by Tukey’s multiple comparisons test. ns : no significance, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Data were shown as mean ± SD. Specific activity values for PI3Kα, calculated as pmol PIP 3 produced per minute per nanogram of enzyme, are indicated in red fonts in the figure. lPIP 2 , lipid form of phosphatidylinositol-4,5-biphospate; SPR, surface plasmon resonance; sPIP 2 , soluble form of PIP 2 ; PIP 3 , phosphatidylinositol-3,4,5-triphosphate.

Article Snippet: Lipid PIP 2 [L-α-phosphatidylinositol-4,5 bisphosphate (Brain, Porcine) (ammonium salt)] and soluble PIP 2 [1,2-dioctanoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′,5-bisphosphate) (ammonium salt)] were from Avanti Polar Lipids.

Techniques: Activity Assay, Produced, SPR Assay

Journal: The Journal of Biological Chemistry

Article Title: Target validation uncouples mitochondrial translocator protein from 19-Atriol-mediated inhibition of steroidogenesis and identifies enzymatic targets

doi: 10.1016/j.jbc.2026.111191

Figure Lengend Snippet: 19-Atriol inhibits progesterone biosynthesis independently of TSPO and acts downstream of cholesterol import. A , schematic of the initial steps in the steroidogenic pathway: (i) Mitochondrial cholesterol import is mediated by the intermembrane space shuttle STAR. (ii) Cholesterol is converted to pregnenolone/P5 by CYP11A1 in the mitochondrial matrix. (iii) P5 is further converted to progesterone/P4 by 3β-hydroxysteroid dehydrogenase (HSD3B) at the endoplasmic reticulum. B , dose–response analysis of progesterone production in wild-type MA-10 and TSPO-null MA-10: Tspo Δ/Δ cells stimulated with Bt 2 cAMP. Increasing concentrations of 19-Atriol reduced P4 output in both genotypes (∗ p < 0.05) with comparable potency and maximal inhibition, indicating that its pharmacological action is independent of TSPO. C , immunoblot analysis of STAR and CYP11A1 expression following treatment with 19-Atriol (0,10 and 100 μM). TSPO was absent in MA-10: Tspo Δ/Δ cells, validating the knockout. Neither STAR nor CYP11A1 protein abundance was altered by 19-Atriol in either genotype (ACTB served as a loading control). D , Dose-response analysis of progesterone production in wild-type MA-10 and TSPO-null MA-10: Tspo Δ/Δ cells with supplementation of 22R-HC in the absence of stimulation. 19-Atriol continued to inhibit P4 production in both genotypes (∗ p < 0.05), indicating that the observed inhibitory effect occurs downstream of cholesterol import.

Article Snippet: Briefly, supernatants were incubated overnight at 4 °C with 125 I-labeled progesterone (MP Biomedicals) and an anti-progesterone antibody ( ) for competitive binding.

Techniques: Inhibition, Western Blot, Expressing, Knock-Out, Quantitative Proteomics, Control

Journal: The Journal of Biological Chemistry

Article Title: Target validation uncouples mitochondrial translocator protein from 19-Atriol-mediated inhibition of steroidogenesis and identifies enzymatic targets

doi: 10.1016/j.jbc.2026.111191

Figure Lengend Snippet: 19-Atriol is metabolized by HSD3B to 19-OHT, and 19-OHT also inhibits progesterone synthesis. A , reaction scheme showing 19-Atriol is converted by 3β-hydroxysteroid dehydrogenase (HSD3B) to 19-hydroxytestosterone (19-OHT). LC–MS/MS showed dose-dependent accumulation of 19-OHT with increasing 19-Atriol (1–100 μM) in MA-10 cells under basal conditions and with Bt 2 cAMP stimulation, indicating that 19-Atriol serves as an HSD3B substrate. B , similar to 19-Atriol, 19-OHT decreased P4 output in a dose-dependent manner in wild-type MA-10 and TSPO-null MA-10: Tspo Δ/Δ cells with comparable potency and maximal effect, with significant differences between the indicated concentrations (∗ p < 0.05). C , with 22R-HC supplementation, 19-OHT still reduced P4 production in both genotypes in a dose-dependent fashion similar to 19-Atriol, with significant differences between the indicated concentrations (∗ p < 0.05). D , in P5-supplemented assays, 19-OHT (0, 10, 100 μM) suppressed P4 formation at each P5 input (1, 5, and 10 μM). Distinct letters above bars indicate statistically significant differences within each P5 condition ( p < 0.05).

Article Snippet: Briefly, supernatants were incubated overnight at 4 °C with 125 I-labeled progesterone (MP Biomedicals) and an anti-progesterone antibody ( ) for competitive binding.

Techniques: Liquid Chromatography with Mass Spectroscopy