phosphatidylinositol 5 phosphate  (Echelon Biosciences)

Journal: PLoS ONE

Article Title: A Homogeneous, High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel, Rapid Substrate Preparation

doi: 10.1371/journal.pone.0054127

Figure Lengend Snippet: Schematic representation of the 2∶1 DPPS:PI5P lipid preparation protocol.

Article Snippet: DPPS (1,2-dipalmitoyl- sn -glycero-3-phosphoserine) and PI5P (D-myo-phosphatidylinositol 5-phosphate diC16) were purchased from Echelon Biosciences (Salt Lake City, UT, USA).

Techniques:

Journal: PLoS ONE

Article Title: A Homogeneous, High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel, Rapid Substrate Preparation

doi: 10.1371/journal.pone.0054127

Figure Lengend Snippet: (A) The overnight (16 hour) stability of the assay reagents at 4°C when the enzyme and lipid were premixed, stored separately or made up fresh as compared to a no enzyme and 5 µM ADP (representing 0% and 100% conversion, respectively). The error bars represent the standard deviation (N = 2). (B) The PI5P lipid dependence of the PI5P4Kα enzyme reaction. The error bars represent the standard deviation (N = 2) and are not discernable on the plot. (C) and (D) Tyrphostin AG-82 (AG82) was identified as a weak inhibitor of PI5P4Kα (decreases the enzyme activity by 75%) by a radiometric assay that uses γ- 32 P-ATP and PI5P and measures the radiolabeled enzymatic product, PI(4,5)P 2 after the separation by thin layer chromatography. Five additional compounds were tested and found not to significantly inhibit PI5P4Kα (AG17 = tyrphostin AG-17, AG18 = tyrphostin AG-18, MP = mycophenolate, PVB = purvalanol B and SU6668). All compounds were tested at 100 µM, except for PVB, which was tested at 10 µM due to solubility limitations at higher concentrations. The raw image and the extracted data are shown in (C) and (D), respectively. The commercial PI5P substrate predominantly contains two palmitate groups with a very small amount of deacylated lipid lyso-PI5P that contains only one palmitate group. The intense top spots in (C) represent the PI(4,5)P 2 product with two palmitate groups, and the faint spots below represent the product with just one palmitate group.

Article Snippet: DPPS (1,2-dipalmitoyl- sn -glycero-3-phosphoserine) and PI5P (D-myo-phosphatidylinositol 5-phosphate diC16) were purchased from Echelon Biosciences (Salt Lake City, UT, USA).

Techniques: Standard Deviation, Activity Assay, Thin Layer Chromatography, Solubility

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Recruitment of Irgb6 to the membrane is a direct trigger for membrane deformation

doi: 10.3389/fcimb.2022.992198

Figure Lengend Snippet: Irgb6 deforms PS or PI5P-containing liposomes. Negative stain EM of 10% PI5P-, PS- or PC-containing liposomes (0.1 mg/ml) incubated with 1 μM Irgb6-WT or K275A/R371A (A–D) . Liposome alone were shown (E–G) . Note that remarkable membrane deformation of PI5P- or PS-containing liposomes (A, B) . Scale bar: 1000 nm (A–G) .

Article Snippet: Ten % (mol/mol) PI5P:phosphatidylinositol-5-phosphate (Cat#P-5016, Echelon Biosciences), 80% phosphatidylethanolamine (PE; Cat#840022C, Avanti Polar Lipids), 10% cholesterol (Chol; Cat#700000, Avanti Polar Lipids) were mixed in chloroform-methanol mixture (1:3 v/v).

Techniques: Staining, Incubation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Recruitment of Irgb6 to the membrane is a direct trigger for membrane deformation

doi: 10.3389/fcimb.2022.992198

Figure Lengend Snippet: Effect of nucleotide on membrane deformation of Irgb6-WT. Negative stain EM showing deformation of PI5P-containing liposomes by Irgb6-WT under nucleotide conditions as indicated. PI5P-containing liposomes (0.1 mg/ml) incubated 1 μM Irgb6-WT as in Figure 1 were absorbed on the grids. Then, buffer alone (A, E, I) , GMP-PNP at 0.5 mM (B, F, J) , GTP at 0.1 mM (C, G) or GDP at 0.1 mM (D, H) was added and incubated for 5 min as is in Supplemental Figure 1 . Scale bar: 1000 nm for upper panels (A–D) , 200 nm for middle panels (E–H) , 75 nm in bottom panels (I , J) .

Article Snippet: Ten % (mol/mol) PI5P:phosphatidylinositol-5-phosphate (Cat#P-5016, Echelon Biosciences), 80% phosphatidylethanolamine (PE; Cat#840022C, Avanti Polar Lipids), 10% cholesterol (Chol; Cat#700000, Avanti Polar Lipids) were mixed in chloroform-methanol mixture (1:3 v/v).

Techniques: Staining, Incubation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Recruitment of Irgb6 to the membrane is a direct trigger for membrane deformation

doi: 10.3389/fcimb.2022.992198

Figure Lengend Snippet: PI5P-containing liposomes stimulate Irgb6 GTPase activity. (A) GTPase activity of Irgb6-WT under low (15 mM NaCl) or high (100 mM NaCl) ionic strength conditions. Irgb6-WT at indicated concentrations was incubated in the buffer containing 15 mM or 100 mM NaCl at 37°C for 30 min. Data are means ± S.E.M. of three independent experiments. (*P < 0.05). (B) PI5P-containing liposomes stimulate GTPase activity of Irgb6-WT. Irgb6-WT at indicated concentrations were incubated with 0.1 mg/ml PI5P-containing liposomes in high ionic strength condition (100 mM NaCl) at 37°C for 30 min. Data are means ± S.E.M. of three independent experiments. (**P < 0.01).

Article Snippet: Ten % (mol/mol) PI5P:phosphatidylinositol-5-phosphate (Cat#P-5016, Echelon Biosciences), 80% phosphatidylethanolamine (PE; Cat#840022C, Avanti Polar Lipids), 10% cholesterol (Chol; Cat#700000, Avanti Polar Lipids) were mixed in chloroform-methanol mixture (1:3 v/v).

Techniques: Activity Assay, Incubation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Recruitment of Irgb6 to the membrane is a direct trigger for membrane deformation

doi: 10.3389/fcimb.2022.992198

Figure Lengend Snippet: Nucleotide-dependent binding of Irgb6 to PI5P-containing lipid monolayers. (A–J) Increased binding of Irgb6 to the lipid monolayers containing PI5P in the presence of GMP-PNP. Three μM Irgb6-WT was absorbed to the lipid monolayers without nucleotide (B, G) , or with 1 mM GMP-PNP (C, H) , GTP (D, I) or GDP (E, J) at room temperature for 3 h. TEM image of monolayer without Irgb6 is shown as a negative control (A, F) . TEM images taken at 300× (upper panels) or at 30000× (bottom panels) were shown. Note that binding of Irgb6-WT to monolayers was remarkedly increased in the presence of GMP-PNP (H) , and some appeared bulged (arrowheads in H ). Scale bar: 8.6 μm in upper panels (A–E) , 100 nm in bottom panels (F–J) . (K) Quantification of binding of Irgb6-WT to monolayers. Irgb6-WT on the lipid monolayers could be visible in negatively stained TEM images (512 x 512 pixel) taken as in (A–E) at low magnification. The area corresponding Irgb6-WT in TEM image was quantified using Image J from three to four independent grids. (***P < 0.0001). (L) Model for the molecular machinery of Irgb6 in PVM disruption. Recruitment of Irgb6 to the membrane leads to membrane deformation. The membrane binding is increased at GTP-binding state because of GTP-dependent polymerization of Irgb6. Dense packing of Irgb6 on lipid membrane results in the stimulation of GTPase activity, and upon GTP hydrolysis Irgb6 detaches from the membrane accompanying membrane damage.

Article Snippet: Ten % (mol/mol) PI5P:phosphatidylinositol-5-phosphate (Cat#P-5016, Echelon Biosciences), 80% phosphatidylethanolamine (PE; Cat#840022C, Avanti Polar Lipids), 10% cholesterol (Chol; Cat#700000, Avanti Polar Lipids) were mixed in chloroform-methanol mixture (1:3 v/v).

Techniques: Binding Assay, Negative Control, Staining, Activity Assay