Journal: The Journal of Biological Chemistry

Article Title: Essential role of MptB in the biosynthesis of phosphatidylinositol mannosides, lipomannan and lipoarabinomannan in mycobacteria

doi: 10.1016/j.jbc.2026.113077

Figure Lengend Snippet: Effect of titrating the level of expression of mptB on the in vitro incorporation of GDP-[ 14 C]Man into mannolipids. A , effect of overexpressing mptBsm on the in vitro incorporation of GDP-[ 14 C]Man into mannolipids. Autoradiogram of the TLC of [ 14 C]-labeled lipids extracted from reaction mixtures containing membrane fractions prepared from Msmg carrying pSETetR or pSETetR- mptB sm . All reaction mixtures supplemented with purified PimA, PimB′, and PatA enzymes to ensure the quantity of PIM 2 substrates was not limiting. The TLC plate was developed in chloroform:methanol:ammonium hydroxide:water (65:25:0.5:4 by vol.) and radiolabeled lipids were visualized by phosphorimaging. The relative distribution of radioactivity incorporated into each PIM species was quantified using the ImageQuant software and the results are expressed as fold changes between the overexpressing and control strains. The individual data points and median values from four independent experiments are shown. Black dots correspond to the standard reaction mixture. Red , green , and blue dots correspond to reactions to which 0.2 mM GDP-Man, β-D-mannosyl farnesyl phosphate, or both GDP-Man and β-D-mannosyl farnesyl phosphate were added, respectively. B , effect of silencing mptBtb on the in vitro incorporation of GDP-[ 14 C]Man into mannolipids. Cell-free assays were conducted using cell envelope fractions prepared from Msmg WT and cKD- mptBsm (without added GDP-Man or β-D-mannosyl farnesyl phosphate). The amount of radioactivity incorporated into each PIM species in the ATc-treated WT mc 2 155 and cKD- mptBtb cultures are expressed relative to the corresponding values measured in the non-ATc–treated cultures arbitrarily set to 100%. See for a repeat experiment. C and D , LC-MS analyses of purified mannolipid X ( C ) and mannolipid Y ( D ) were conducted in the negative ion mode. The total ion chromatograms ( top ), extracted ion chromatograms (EIC) ( middle ), and corresponding mass spectra ( bottom ) for both compounds are shown. The EICs show single peaks eluting at retention times 3.25 min for mannolipid X and 3.26 min for mannolipid Y. The mass spectra of these peaks show a deprotonated molecular ion at m/z 1737.99 ([M-H] - ) for mannolipid X supporting the identity of this mannolipid as Ac 1 PIM 4 (acylated with two palmitoyl and one tuberculostearoyl chains) and a deprotonated molecular ion at m/z 1575.95 ([M-H] - ) for mannolipid Y, supporting the identity of this mannolipid as Ac 1 PIM 3 (acylated with two palmitoyl and one tuberculostearoyl chains). Other minor forms of Ac 1 PIM 3 and Ac 1 PIM 4 (not shown here) acylated with different combinations of palmitoyl, palmitoleyl, stearoyl, oleyl, and tuberculostearoyl chains were also detected. E and F , LC-MS/MS analysis in negative ion mode confirming the identity of mannolipid Y ( E ) and mannolipid X ( F ). The mass spectra reveal two related glycolipids that differ in their degree of mannosylation of a conserved phosphatidylinositol backbone. E , displays the fragmentation of the precursor ion at m/z 1575.95. Fragment ions at m/z 965.40, 983.41, 1295.67, and 1319.70 are consistent with a phosphatidylinositol core with three mannosyl residues and reflect neutral losses of acyl chains, including palmitate or tuberculostearate. The ion at m/z 1175.67 corresponds to the loss of an acylated mannosyl residue, while ions at m/z 689.51 and 433.27 further validate the presence of the phosphatidylinositol backbone. F , displays the fragmentation of the precursor ion at m/z 1738.01. A systematic 162.05 Da increase in fragment ions at m/z 1127.45, 1145.47, 1457.71, and 1481.75 relative to mannolipid Y in panel ( E ) indicates the presence of an additional mannosyl residue. Ions at m/z 689.51 and 433.27 further confirm the phosphatidylinositol core. MS/MS data therefore establish mannolipids Y and X as Ac 1 PIM 3 and Ac 1 PIM 4 , respectively.

Article Snippet: In some reactions, GDP-Man and/or β-D-mannosyl farnesyl phosphate (Avanti Research) were added at a final concentration of 0.2 mM.

Techniques: Expressing, In Vitro, Labeling, Membrane, Purification, Radioactivity, Software, Control, Liquid Chromatography with Mass Spectroscopy, Residue, Tandem Mass Spectroscopy

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: DNA-PKcs inhibits 3′3′-cGAMP- and agonist-associated STING activation. (A) ATP hydrolysis by DNA-PK was measured in vitro in the presence of increasing doses (0.8–2,500 µM) of 3′3′-cGAMP or c-di-AMP. Graphs present the mean of three independent experiments. Statistical significance was calculated by one-way ANOVA. (B) Recombinant DNA-PKcs was immunoprecipitated using either mock IgG or a DNA-PKcs–specific antibody prior to incubation with 3′3′-cGAMP or c-di-AMP and ELISA-based measurement of bound CDNs. Graph presents mean (±SEM) 3′3′-cGAMP and c-diAMP levels as measured in mock and DNA-PKcs–specific IP in three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (C) THP-1 cells were treated or not with 2 μM NU7441 in combination or not with 10 µg/ml fluorinated 3′3′-cGAMP for 6 h prior to WB analysis using the indicated antibodies. Representative WB of three independent experiments. (D) As in C, except that gene expression analyses were conducted. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (E) As in C, except that IFNβ, CXCL10, and CCL5 levels were measured by ELISA. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (F) Control and DNA-PKcs knockout THP-1 cells were treated with 3′3′-cGAMP for 6 h prior to gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (G) Human primary monocytes were isolated from buffy coats prior to treatment or not with 2 µM NU7441 for 1 h, followed by administration of 10 µg/ml fluorinated 3′3′-cGAMP for 6 h and gene expression analysis. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (H) ATP hydrolysis by DNA-PKcs was measured in vitro in presence of increasing doses (0.8–2,500 µM) of E7766 or ADU-S100. Statistical significance was calculated by one-way ANOVA. (I) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 1 µM of E7766 STING agonist for 3 h and gene expression analysis. Graphs present the mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (J) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 50 µM of ADU-S100 STING agonist for 3 h and gene expression analysis. Graphs present the mean (±SEM), n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (K) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 10 µM of diABZI for 3 h and gene expression analysis. Graphs present the mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Also see . IP, immunoprecipitation.

Article Snippet: The following drugs were used: NU7441 (Bio-Techne/Tocris, #3712, CID: 11327430); DMSO (Sigma-Aldrich, D2650, CID: 679); 2′3′-cGAMP (InvivoGen, tlrl-nacga23-02, CID: 137120248); 3′3′-cGAMP (InvivoGen, tlrl-nacga, CAS number: 849214-04-6); c-di-AMP (InvivoGen, tlrl-nacda, CAS number: 2734909-87-4/54447-84-6 [free acid]); c-di-GMP (InvivoGen, tlrl-nacdg, CAS number: 2222132-40-1/61093-23-0 [free acid]); diABZI (InvivoGen, tlrl-diabzi-2, CID: 137701219, CAS number: 2138299-34-8); E7766 (MedChemExpress, HY-111999A); ADU-S100 ammonium salt (MedChemExpress, HY-12885B); fluorinated 3′3′-cGAMP (InvivoGen, tlrl-nacgaf-05; CAS number: not available).

Techniques: Activation Assay, In Vitro, Recombinant, Immunoprecipitation, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Gene Expression, Control, Knock-Out, Isolation

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: DNA-PKcs selectively counteracts CDNs. (A) ATP hydrolysis by DNA-PKcs was measured in vitro in presence of increasing doses (0.8–2,500 µM) of c-di-GMP. Graph presents the mean (±SEM) performed in biological triplicates. Statistical significance was calculated by one-way ANOVA. ns, not significant. (B) T98G cells were treated or not with 2 μM NU7441 in combination or not with 10 µg/ml fluorinated 3′3′-cGAMP for 6 h prior to WB analysis using the indicated antibodies. Representative WB of three independent experiments. (C) As in B, except that gene expression analyses were conducted. Graphs present the mean (±SEM) performed in biological triplicates. Statistical significance was calculated by two-tailed Student's t test. ***: P < 0.001; **: P < 0.01; *: P < 0.05. (D) Experimental scheme for human primary monocyte isolation and treatment . Human primary monocytes were isolated from buffy coats prior to treatment or not with 2 µM NU7441 for 1 h, followed by administration of 10 µg/ml fluorinated 3′3′-cGAMP for 6 h and gene expression analysis. (E) Flow cytometry analysis of macrophages prepared as in . (F) Gene expression analyses were performed on human primary cells treated as described in . Graphs present the mean (±SEM) expression of IFIT1 in three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (G) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 1 µM of E7766 STING agonist for 3 h and analysis of gene expression. WB analyses were conducted using indicated antibodies and are representative of three independent experiments. (H) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 50 µM of ADU-S100 STING agonist for 3 h and analysis of gene expression. WB analyses were conducted using indicated antibodies and are representative of three independent experiments. (I) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 10 µM of diABZI for 3 h and analysis of gene expression. WB analyses were conducted using indicated antibodies and are representative of three independent experiments. (J) Control and DNA-PKcs knockout THP-1 cells were treated with 1 µM E7766 for 6 h prior to gene expression analysis. Graphs present mean (±SEM), n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (K) Control and DNA-PKcs knockout THP-1 cells were treated with 10 µM diABZI for 6 h prior to gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05. Related to . Source data are available for this figure: .

Article Snippet: The following drugs were used: NU7441 (Bio-Techne/Tocris, #3712, CID: 11327430); DMSO (Sigma-Aldrich, D2650, CID: 679); 2′3′-cGAMP (InvivoGen, tlrl-nacga23-02, CID: 137120248); 3′3′-cGAMP (InvivoGen, tlrl-nacga, CAS number: 849214-04-6); c-di-AMP (InvivoGen, tlrl-nacda, CAS number: 2734909-87-4/54447-84-6 [free acid]); c-di-GMP (InvivoGen, tlrl-nacdg, CAS number: 2222132-40-1/61093-23-0 [free acid]); diABZI (InvivoGen, tlrl-diabzi-2, CID: 137701219, CAS number: 2138299-34-8); E7766 (MedChemExpress, HY-111999A); ADU-S100 ammonium salt (MedChemExpress, HY-12885B); fluorinated 3′3′-cGAMP (InvivoGen, tlrl-nacgaf-05; CAS number: not available).

Techniques: In Vitro, Gene Expression, Two Tailed Test, Isolation, Flow Cytometry, Expressing, Control, Knock-Out