Journal: Biochemistry

Article Title: Inositol Hexaphosphate as an Inhibitor and Potential Regulator of p47 phox Membrane Anchoring

doi: 10.1021/acs.biochem.4c00117

Figure Lengend Snippet: General scheme of NOX2 activation. (A) Autoinhibition of p47 phox PX domain constrains the activation complex of NOX2, consisting of p40 phox , p67 phox , and p47 phox , to the cytosol resulting in deactivated NOX2. (B) Phosphorylation of p47 phox by PKC opens the p47 phox conformation and renders its PX domain accessible for lipid binding. Activation of p47 phox may also trigger trimerization of the activation complex. (C) Translocation of the activated trimeric complex is driven by p47 phox membrane anchoring through interactions between its PX domain and a PIP enriched phagosomal membrane, resulting in activation of NOX2 and production of ROS.

Article Snippet: His-tagged p47 phox -PX was purified on ice over a Ni-NTA column using wash buffer [0.1 M Tris (pH 7.4), 0.1 M NaCl, 50 mM imidazole, 1 mM DTT] and elution buffer [0.1 M Tris (pH 7.4), 0.1 M NaCl, 250 mM imidazole, 1 mM DTT] prior to an overnight dialysis at 4 °C with an in-house expressed and purified TEV protease, MBP-TEVcs-His6-uTEV2Δ-R5 [plasmid is a gift from Alice Ting, Addgene plasmid # 135456] to cleave the histidine tag.

Techniques: Activation Assay, Phospho-proteomics, Binding Assay, Translocation Assay, Membrane

Journal: Biochemistry

Article Title: Inositol Hexaphosphate as an Inhibitor and Potential Regulator of p47 phox Membrane Anchoring

doi: 10.1021/acs.biochem.4c00117

Figure Lengend Snippet: IP6 binds to the PX domain of p47 phox as observed by NMR. (A) 1 H– 15 N-HSQC overlay of 1:0 and 1:2 molar ratios of p47 phox -PX:IP6. Insets depict a titration series including 1:0.25, 1:0.5, 1:2, and 1:4 molar ratios of PX domain:IP6. High-affinity binding is implicated through line-broadening of resonances. (B) Mapping of CSPs upon addition of 2× IP6 to the PX domain (PDB: 1GD5 ). Residues depicting 1 and 2 standard deviations of a 20% trimmed mean are in pink and purple, respectively. Residues with unobservable or line-broadened peaks are depicted in black. (C) Comparison of CSPs upon 2× C 4 –PI(3,4)P 2 (purple) or IP6 (orange). Residues with unobservable or line-broadened peaks are shown as red circles offset from the x -axis.

Article Snippet: His-tagged p47 phox -PX was purified on ice over a Ni-NTA column using wash buffer [0.1 M Tris (pH 7.4), 0.1 M NaCl, 50 mM imidazole, 1 mM DTT] and elution buffer [0.1 M Tris (pH 7.4), 0.1 M NaCl, 250 mM imidazole, 1 mM DTT] prior to an overnight dialysis at 4 °C with an in-house expressed and purified TEV protease, MBP-TEVcs-His6-uTEV2Δ-R5 [plasmid is a gift from Alice Ting, Addgene plasmid # 135456] to cleave the histidine tag.

Techniques: Titration, Binding Assay, Comparison

Journal: Biochemistry

Article Title: Inositol Hexaphosphate as an Inhibitor and Potential Regulator of p47 phox Membrane Anchoring

doi: 10.1021/acs.biochem.4c00117

Figure Lengend Snippet: Docking simulation of IP6 against the PX domain of p47 phox (PDB: 1GD5 ). (A) Two highest scoring docking poses show IP6 bound either within the typical PIP binding pocket or the atypical shallow anionic lipid binding pocket. (B) IP6 bound within the PIP binding site is flanked by three residues known to be important to lipid binding (R43, W80, R90). (C) IP6 bound within the atypical binding site is flanked by four residues critical for lipid recognition (H51, K55, H74, and R70).

Article Snippet: His-tagged p47 phox -PX was purified on ice over a Ni-NTA column using wash buffer [0.1 M Tris (pH 7.4), 0.1 M NaCl, 50 mM imidazole, 1 mM DTT] and elution buffer [0.1 M Tris (pH 7.4), 0.1 M NaCl, 250 mM imidazole, 1 mM DTT] prior to an overnight dialysis at 4 °C with an in-house expressed and purified TEV protease, MBP-TEVcs-His6-uTEV2Δ-R5 [plasmid is a gift from Alice Ting, Addgene plasmid # 135456] to cleave the histidine tag.

Techniques: Binding Assay

Journal: Biochemistry

Article Title: Inositol Hexaphosphate as an Inhibitor and Potential Regulator of p47 phox Membrane Anchoring

doi: 10.1021/acs.biochem.4c00117

Figure Lengend Snippet: Apparent binding affinities of tested IPs as observed by NMR titration. (A) CSPs per residue of p47 phox -PX in the presence of IP6, IP5, IP4, and IP3. Comparison of CSPs induced by IPs display larger shifting and tighter binding to the PX domain with higher degrees of phosphorylation around the inositol ring. Residues that line-broadened and/or were unobservable are denoted as red circles offset from the x -axis. (B) Titration of the PX domain with IP6. Line-broadening prevented accurate K d fitting; curves are included here only as a guide for the eye. NMR titrations of the PX domain with IP5(1,3,4,5,6) (C), IP4(1,3,4,5) (D), and IP3(1,3,4) (E) were used to calculate K d values. Curves in panels (C)–(E) represent the fit of each residue to a global K d value, where all resonances with a CSP > 0.08 and R 2 > 0.95 when fit individually were included.

Article Snippet: His-tagged p47 phox -PX was purified on ice over a Ni-NTA column using wash buffer [0.1 M Tris (pH 7.4), 0.1 M NaCl, 50 mM imidazole, 1 mM DTT] and elution buffer [0.1 M Tris (pH 7.4), 0.1 M NaCl, 250 mM imidazole, 1 mM DTT] prior to an overnight dialysis at 4 °C with an in-house expressed and purified TEV protease, MBP-TEVcs-His6-uTEV2Δ-R5 [plasmid is a gift from Alice Ting, Addgene plasmid # 135456] to cleave the histidine tag.

Techniques: Binding Assay, Titration, Residue, Comparison, Phospho-proteomics

Journal: Biochemistry

Article Title: Inositol Hexaphosphate as an Inhibitor and Potential Regulator of p47 phox Membrane Anchoring

doi: 10.1021/acs.biochem.4c00117

Figure Lengend Snippet: Fluorescence polarization study to assess IP6 as a competitive inhibitor. (A) Binding of tracer BODIPY-PI(3,4)P 2 to p47 phox -PX and associated fit for K d determination. (B) Displacement of BODIPY-PI(3,4)P 2 tracer from the PX domain upon addition of IP6 and associated fit. Error bars represent standard deviation of experimental replicates; those not visible are smaller than the data points.

Article Snippet: His-tagged p47 phox -PX was purified on ice over a Ni-NTA column using wash buffer [0.1 M Tris (pH 7.4), 0.1 M NaCl, 50 mM imidazole, 1 mM DTT] and elution buffer [0.1 M Tris (pH 7.4), 0.1 M NaCl, 250 mM imidazole, 1 mM DTT] prior to an overnight dialysis at 4 °C with an in-house expressed and purified TEV protease, MBP-TEVcs-His6-uTEV2Δ-R5 [plasmid is a gift from Alice Ting, Addgene plasmid # 135456] to cleave the histidine tag.

Techniques: Fluorescence, Binding Assay, Standard Deviation

Journal: Biochemistry

Article Title: Inositol Hexaphosphate as an Inhibitor and Potential Regulator of p47 phox Membrane Anchoring

doi: 10.1021/acs.biochem.4c00117

Figure Lengend Snippet: IP6 acts as an inhibitor of membrane anchoring of p47 phox -PX with low micromolar potency as seen by a liposome FRET assay. Donor fluorophore is the PX domain intrinsic tryptophans, and acceptor fluorophore is Dansyl-PE incorporated into liposomes. Titrations of IP6 against p47 phox -PX and liposomes were completed in triplicate and normalized. Error bars represent the deviation of individual normalized titrations.

Article Snippet: His-tagged p47 phox -PX was purified on ice over a Ni-NTA column using wash buffer [0.1 M Tris (pH 7.4), 0.1 M NaCl, 50 mM imidazole, 1 mM DTT] and elution buffer [0.1 M Tris (pH 7.4), 0.1 M NaCl, 250 mM imidazole, 1 mM DTT] prior to an overnight dialysis at 4 °C with an in-house expressed and purified TEV protease, MBP-TEVcs-His6-uTEV2Δ-R5 [plasmid is a gift from Alice Ting, Addgene plasmid # 135456] to cleave the histidine tag.

Techniques: Membrane, Liposomes