Journal: International Journal of Molecular Sciences

Article Title: Structural Insights Uncover the Specific Phosphoinositide Recognition by the PH1 Domain of Arap3

doi: 10.3390/ijms24021125

Figure Lengend Snippet: Phospholipid-binding abilities of Arap3-PH1 domain analyzed using liposome pull-down assay and SPR measurements. ( A ) Schematic representation of human Arap3 protein. The first PH domain of Arap3 is marked in cyan. ( B ) Sequence alignment of Arap3-PH1 orthologs in vertebrates and human Arap1-PH1, Arap2-PH1. Sequence accession number in the Uniprot database are: human, Q8WWN8; bovine, E1BBA0; mouse, Q8R5G7; zebrafish, A0A140LH27; human Arap1, Q96P48; human Arap2, Q8WZ64. Alignment was performed using Clustal X and illustrated with ESPript 3.0. Strictly conserved (white letters filled with red color) and conservatively substituted (red letters with blue box) residues are denoted. The secondary structure element for human Arap3-PH1 is labeled on the top. The KXnQXR motif are marked by black dots. ( C ) Arap3-PH1 (20 μg) mixed with liposomes (640 μg) composed of 98% PC as the fixed component and 2% of specific phospholipids, respectively. Proteins in the absence of liposome were used as a control. After centrifugation, the pellet (P) and supernatant (S) were analyzed by SDS/PAGE and Coomassie. ( D ) SPR measurements of the binding affinities of the Arap3-PH1 domain for diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2. The upper panel shows representative sensorgrams of diC4-PI(3,4,5)P3 (left) and diC4-PI(4,5)P2 (right) when mixed with Arap3-PH1. Data were collected by injecting increasing concentrations of diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2 samples over Arap3-PH1 proteins immobilized on the surface of a CM5 biochip. The lower panel shows representative binding curves fitting for diC4-PI(3,4,5)P3 (left) and diC4-PI(4,5)P2 (right) during their interaction with Arap3-PH1. A one-site binding model was utilized to fit the curves. The experiment was carried out in triplicate. The KD value is presented as mean ± SD, n = 3.

Article Snippet: DiC4-PI(3,4,5)P3 (Echelon, Salt Lake City, UT, USA) were diluted from 75 μM to 1.172 μM as 1:2 dilution series and diC4-PI(4,5)P2 (Echelon, USA) were diluted from 600 μM to 4.688 μM.

Techniques: Binding Assay, Pull Down Assay, Sequencing, Labeling, Centrifugation, SDS Page

Journal: International Journal of Molecular Sciences

Article Title: Structural Insights Uncover the Specific Phosphoinositide Recognition by the PH1 Domain of Arap3

doi: 10.3390/ijms24021125

Figure Lengend Snippet: Crystallographic data collection and refinement statistics.

Article Snippet: DiC4-PI(3,4,5)P3 (Echelon, Salt Lake City, UT, USA) were diluted from 75 μM to 1.172 μM as 1:2 dilution series and diC4-PI(4,5)P2 (Echelon, USA) were diluted from 600 μM to 4.688 μM.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Structural Insights Uncover the Specific Phosphoinositide Recognition by the PH1 Domain of Arap3

doi: 10.3390/ijms24021125

Figure Lengend Snippet: Structure of the Arap3-PH1 domain in complex with diC4-PI(3,4,5)P3. ( A ) Cartoon diagram of Arap3-PH1 complexed with diC4-PI(3,4,5)P3. Arap3-PH1 is colored turquoise, with secondary structures labeled. The loops of β1/β2 and β6/β7 that interact directly with diC4-PI(3,4,5)P3 are colored blue. The diC4-PI(3,4,5)P3 (gold) is shown in stick mode. ( B ) Surface electrostatic potential of Arap3-PH1 complexed with diC4-PI(3,4,5)P3. Blue areas, positive; red areas, negative. ( C ) Detailed interactions of the diC4-PI(3,4,5)P3 with Arap3-PH1 domain. The side chains of crucial residues are shown in stick mode and labeled, respectively. The phosphate groups on diC4-PI(3,4,5)P3 are also labeled. Selected hydrogen bonds or salt bridges are shown as dotted lines.

Article Snippet: DiC4-PI(3,4,5)P3 (Echelon, Salt Lake City, UT, USA) were diluted from 75 μM to 1.172 μM as 1:2 dilution series and diC4-PI(4,5)P2 (Echelon, USA) were diluted from 600 μM to 4.688 μM.

Techniques: Labeling

Journal: International Journal of Molecular Sciences

Article Title: Structural Insights Uncover the Specific Phosphoinositide Recognition by the PH1 Domain of Arap3

doi: 10.3390/ijms24021125

Figure Lengend Snippet: The binding interfaces of Arap3-PH1 for diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2 revealed by NMR titration. ( A ) Overlay of 1 H- 15 N HSQC spectra of Arap3-PH1 in the absence (black) and in the increasing amounts of diC4-PI(3,4,5)P3. The molar ratios of the protein to diC4-PI(3,4,5)P3 are shown in the inset: 1:0 (black), 1:0.25 (turquoise), 1:0.5 (lime green), 1:0.75 (orange), 1:1 (pink) and 1:1.25 (red). ( B ) Overlay of 1 H- 15 N HSQC spectra of Arap3-PH1 in the absence (black) and in increasing amounts of diC4-PI(4,5)P2. The molar ratios of the protein to diC4-PI(4,5)P2 are shown in the inset: 1:0 (black), 1:0.5 (royal blue), 1:1 (turquoise), 1:2 (lime green), 1:4 (orange), 1:6 (pink) and 1:8 (red). ( C ) The chemical shift perturbations (CSPs) of each residue during NMR titrations (up, diC4-PI(3,4,5)P3 titration; down, diC4-PI(4,5)P2 titration) are calculated and shown with the secondary elements on top. White dots indicate pro residues. Black dots indicate residues with no data. The mean value and the mean value plus one standard deviation are indicated by dash and solid lines, respectively. Residues with CSPs between mean value and mean value plus one standard deviation are colored gold, and above mean value plus one standard deviations are colored red. ( D , E ) Surface representations of the Arap3-PH1 structure with the perturbed residues upon binding to diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2 are colored and labeled.

Article Snippet: DiC4-PI(3,4,5)P3 (Echelon, Salt Lake City, UT, USA) were diluted from 75 μM to 1.172 μM as 1:2 dilution series and diC4-PI(4,5)P2 (Echelon, USA) were diluted from 600 μM to 4.688 μM.

Techniques: Binding Assay, Titration, Standard Deviation, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Structural Insights Uncover the Specific Phosphoinositide Recognition by the PH1 Domain of Arap3

doi: 10.3390/ijms24021125

Figure Lengend Snippet: The R308H mutation within the Arap3-PH1 domain abolishes its binding to PI(3,4,5)P3 lipid, and impairs the capacity of Arap3 to inhibit breast cancer cell invasion. ( A ) Liposome binding assays of the Arap3-PH1 R308H mutant with liposomes composed of 99% PC and 1% PI(3,4,5)P3. Arap3-PH1 WT was used as a positive control. ( B ) Transwell migration assays were performed to measure the cell invasion activities of MDA-MB-231 cells transfected with GFP-Arap3 WT , GFP-Arap3 R308H and the GFP control. ( C ) Quantification of cell invasion activities of cells transfected with GFP-Arap3 WT , GFP-Arap3 R308H and the GFP control from the experiment described in ( B ). Data are expressed as mean ± SEM for each group from three independent experiments. * p < 0.05, p values were calculated by Student’s t test.

Article Snippet: DiC4-PI(3,4,5)P3 (Echelon, Salt Lake City, UT, USA) were diluted from 75 μM to 1.172 μM as 1:2 dilution series and diC4-PI(4,5)P2 (Echelon, USA) were diluted from 600 μM to 4.688 μM.

Techniques: Mutagenesis, Binding Assay, Positive Control, Migration, Transfection

Journal: Cell Research

Article Title: Endothelial CDS2 deficiency causes VEGFA-mediated vascular regression and tumor inhibition

doi: 10.1038/s41422-019-0229-5

Figure Lengend Snippet: PI-PIPs metabolism is involved in CDS2-deficient vessel regression. a Schematic diagram of CDS2-controlled phosphoinositide recycling. DAG, diacylglycerol; PA, phosphatidic acid; PIS1, phosphatidylinositol synthase 1; PI, phosphoinositol; PIK, phosphoinositol 3/4/5-kinase; PIPK, phosphatidylinositol 4/5-phosphate 5/4-kinase; PTEN, phosphatase and tensin homolog; PI3K, phosphoinositide 3 kinase; PLC, phospholipase c; PG, phosphatidylglycerol; CL, cardiolipin. b Model of timing for phospholipid-carrier mixture microinjection, heatshock induction and confocal imaging analysis in ( c ) and ( d ). c Quantification of rescue effects of different phospholipids (PI, PG, PIP2 and PIP3) on vegfa OE-induced ISV regression in cds2 mutant embryos. Bars show percentages of vessel deficiency. The representative images of classified vascular phenotype are shown on the right. The counted ISV numbers were shown on the top, 10 ISV per embryo. d Representative images of trunk vessels from cds2 mutants at 76–80 hpf with or w/o vegfa OE and with microinjection of lipid–carrier complex, including PG, PI, PIP2 or PIP3 at 28–30 hpf. Scale bars, 50 μm ( c ) and 100 μm ( d )

Article Snippet: For phospholipid delivery, phosphatidylinositol (PI) (Echelon Biosciences, P-0004; 1 mg ml −1 ), phosphatidylglycerol (PG) (Sigma, P8318; 2.5 mg ml −1 ), phosphatidylinositol 3, 4, 5-trisphosphate (PIP3) (Echelon Biosciences, P-3904; 1 mg ml −1 ) and phosphatidylinositol 4, 5-bisphosphate (PIP2) (Echelon Biosciences, P-9045; 1 mg ml −1 ) were dissolved in water and incubated with equal molar concentration of histone H1 carrier (Echelon Biosciences, P-9C2) for 10 min at room temperature before injection.

Techniques: Imaging, Mutagenesis

Journal: Cell Research

Article Title: Endothelial CDS2 deficiency causes VEGFA-mediated vascular regression and tumor inhibition

doi: 10.1038/s41422-019-0229-5

Figure Lengend Snippet: PIP3 exhaustion governs VEGFA-induced regression of CDS2-deficient endothelium. Confocal images ( a ) and quantitative analysis ( b ) of vessel-deficient phenotype in WT zebrafish embryos or cds2 mutants with or w/o vegfa OE, with control MO or pten MO (combination of ptena and ptenb MO) injection. The counted ISV number shown on the top ( b ) is from 15–20 embryos per group. c Relative vegfa mRNA level of cds2 mutants with vegfa OE injected with ctrl or pten MO. The expression was normalized to WT embryos. n = 3 samples per group, 15–20 embryos pooled for each sample. d pten knockdown partially restored PIP3, but not PIP2 level in cds2 -deficient endothelium with vegfa OE. Western blotting analysis on a-Tubulin serves as the internal control for cell amounts in each sample collected during lipid quantitation analysis. n = 4 samples each group. Confocal images ( e and f ) and quantitative analysis ( g ) of P7 retinal vessels stained by IB4 ( e ) and IB4/COL4 ( f ) from control or VEGFA-injected Cds2 iΔEC mice treated with bpV (PTEN inhibitor) or vehicle (saline). bpV (2 mg kg −1 ) or vehicle was given three times at P2, P4 and P6. Arrows show regressed vessels. Angiogenic sprouts, COL4 + /IB4 − empty sleeves, endothelial area and branch points were quantified in ( g ), n = 6–10 mice per group. Scale bars, 100 μm ( a and f ) and 200 μm ( e ). Error bars, mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant ( P ≥ 0.05). See also Supplementary information, Fig.

Article Snippet: For phospholipid delivery, phosphatidylinositol (PI) (Echelon Biosciences, P-0004; 1 mg ml −1 ), phosphatidylglycerol (PG) (Sigma, P8318; 2.5 mg ml −1 ), phosphatidylinositol 3, 4, 5-trisphosphate (PIP3) (Echelon Biosciences, P-3904; 1 mg ml −1 ) and phosphatidylinositol 4, 5-bisphosphate (PIP2) (Echelon Biosciences, P-9045; 1 mg ml −1 ) were dissolved in water and incubated with equal molar concentration of histone H1 carrier (Echelon Biosciences, P-9C2) for 10 min at room temperature before injection.

Techniques: Injection, Expressing, Western Blot, Quantitation Assay, Staining

Journal: Cell Research

Article Title: Endothelial CDS2 deficiency causes VEGFA-mediated vascular regression and tumor inhibition

doi: 10.1038/s41422-019-0229-5

Figure Lengend Snippet: PIP3 reduction is mainly caused by PLCγ mediated PIP2 hydrolysis. Representative confocal images ( a ) and quantitative analysis ( b ) of trunk vessel phenotypes of WT or cds2 mutant embryos with vegfa OE and with or w/o plcg1 MO. The ISV number counted from 20–22 embryos per group is shown on the top ( b ). c Relative vegfa mRNA level of cds2 mutants with vegfa OE injected with ctrl or plcg1 MO. vegfa expression level was determined at 2 h post heatshock induction and normalized to WT embryos without vegfa OE. n = 3 samples, 15–20 embryos pooled for each sample. d plcg1 knockdown partially restored PIP2 and PIP3 level in cds2 -deficient endothelium with vegfa OE. Western blotting analysis on a-Tubulin serves as the internal control for cell amounts in each sample collected during lipid quantitation analysis. n = 4 samples per group. e Working model of VEGFA-triggered vessel regression on CDS2-deficient endothelium. The outcome of VEGFA signaling can be reversed from angiogenesis to vessel regression, which is dependent on CDS2-controlled PIP2 and PIP3 availability and FOXO1 signaling activation. Scale bar, 100 μm. Error bars, mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant ( P ≥ 0.05). See also Supplementary information, Fig.

Article Snippet: For phospholipid delivery, phosphatidylinositol (PI) (Echelon Biosciences, P-0004; 1 mg ml −1 ), phosphatidylglycerol (PG) (Sigma, P8318; 2.5 mg ml −1 ), phosphatidylinositol 3, 4, 5-trisphosphate (PIP3) (Echelon Biosciences, P-3904; 1 mg ml −1 ) and phosphatidylinositol 4, 5-bisphosphate (PIP2) (Echelon Biosciences, P-9045; 1 mg ml −1 ) were dissolved in water and incubated with equal molar concentration of histone H1 carrier (Echelon Biosciences, P-9C2) for 10 min at room temperature before injection.

Techniques: Mutagenesis, Injection, Expressing, Western Blot, Quantitation Assay, Activation Assay