Journal: Journal of Lipid Research

Article Title: Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2

doi: 10.1194/jlr.M073692

Figure Lengend Snippet: Enzymatic activity of AWAT2 toward geometric isomers of retinol. A: Western blot detection of mouse AWAT2 in microsomal fraction isolated from yeast cells transfected with YepM containing the enzyme’s cDNA. The control sample represents membranes from the cells transfected with empty vector. B: HPLC chromatograms show separation of retinols and products of their AWAT2-dependent esterification. Peaks 1 and 1’ represent 9-cis-retinol and 9-cis-retinyl myristyl, respectively; peaks 2, and 2’ represent 11-cis-retinol and the corresponding ester, respectively; peaks 3 and 3’ represent 13-cis-retinol and its ester, respectively; and peaks 4 and 4’ represent all-trans-retinol and all-trans-retinyl ester, respectively. The inset shows characteristic UV/Vis spectra for each of the isomers. C: Comparison of the time courses of retinyl ester formation from retinol and its geometric isomers (blue line, 9-cis-retinol isomer; red line, 11-cis-retinol isomer; green line, 13-cis-retinol isomer; black line, all-trans-retinol isomer). D: Determination of kinetic parameters for the enzymatic reaction in the presence of retinol and its isomers. The curves represent dependence of the initial velocity of retinyl myristate formation on the substrate concentrations. The kinetic parameters of the reaction were determined by fitting the experimental points to the Michaelis-Menten model.

Article Snippet: cDNA of mouse AWAT2 (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_177746","term_id":"594150398","term_text":"NM_177746"}} NM_177746 ) was purchased from OriGene Technologies.

Techniques: Activity Assay, Western Blot, Isolation, Transfection, Control, Plasmid Preparation, Comparison

Journal: Journal of Lipid Research

Article Title: Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2

doi: 10.1194/jlr.M073692

Figure Lengend Snippet: Kinetic parameters of AWAT2-dependent esterification of retinol isomers

Article Snippet: cDNA of mouse AWAT2 (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_177746","term_id":"594150398","term_text":"NM_177746"}} NM_177746 ) was purchased from OriGene Technologies.

Techniques:

Journal: Journal of Lipid Research

Article Title: Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2

doi: 10.1194/jlr.M073692

Figure Lengend Snippet: AWAT2 activity toward long-chain alcohol. A: LC-MS-based detection of myristate ester. The extracted ion chromatogram represents elution of product of the enzymatic reaction (myristyl myristate) detected at m/z 425.3 [MH]+. In addition to these ions, the MS spectrum averaged across this chromatography peak displays ions corresponding to palmityl myristate (m/z 453.3 [MH]+) that served as an internal standard for the purpose of quantification. B: Rate of myristyl myristate formation as a function of concentration of myristyl alcohol. By analogy to production of retinyl esters, the experimental points were used to determine the kinetic parameters for the enzymatic wax formation.

Article Snippet: cDNA of mouse AWAT2 (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_177746","term_id":"594150398","term_text":"NM_177746"}} NM_177746 ) was purchased from OriGene Technologies.

Techniques: Activity Assay, Liquid Chromatography with Mass Spectroscopy, Chromatography, Concentration Assay

Journal: Journal of Lipid Research

Article Title: Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2

doi: 10.1194/jlr.M073692

Figure Lengend Snippet: AWAT2-dependent esterification of 11-cis-retinol in the presence of holo-CRALBP. A: The apo-CRALBP expressed in E. coli cells was purified to homogeneity (inset) and preincubated with 11-cis-retinol to form holo-protein. After repurification, the efficiency of ligand binding was verified by recording UV/Vis absorbance spectra. The spectrum indicates two maxima at 278 nm corresponding to the protein scaffold and at 344 nm that is representative of bound 11-cis-retinol. B: The time course of 11-cis-retinol esterification in the presence of either free (open circle) or CRALBP-bound (filled circle) substrate. C, D: Verification of the isotopic composition of 11-cis-retinol (C) and its deuterated form (D). C: The MS spectrum of unlabeled retinoid reveals the main isotopic peak at m/z 269.3 that corresponds to ionization-driven loss of water from the parent ion. The conforming tandem spectrum (MS2) shows the fragmentation pattern of the retinoid moiety. D: The main peak for heavy atom-labeled 11-cis-retinol is at m/z 274.3 that indicates five deuterium atoms incorporated into the molecule. The ion at m/z 274.3 that corresponds to the molecules labeled with only four heavy atoms represents less than 20% of the mixture. Importantly, the unlabeled retinoid was not detectable. E: HPLC separation of 11-cis-retinol (blue peak) and its esterified form (green peak) after 2 min incubation of AWAT2 with holo-CRALBP and free 5D-11-cis-retinol. The isotopic composition of the substrates (blue) and products (green) are shown in (F), whereas (G) represents temporal changes in the ratio between labeled and unlabeled 11-cis-retinyl esters during the enzymatic reaction (filled circle) and the time course of ligand exchange between holo-CRALBP and free 11-cis-retinol (gray diamonds).

Article Snippet: cDNA of mouse AWAT2 (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_177746","term_id":"594150398","term_text":"NM_177746"}} NM_177746 ) was purchased from OriGene Technologies.

Techniques: Purification, Ligand Binding Assay, Labeling, Incubation

Journal: Journal of Lipid Research

Article Title: Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2

doi: 10.1194/jlr.M073692

Figure Lengend Snippet: Substrate selectivity of AWAT2 in the presence of a mixture of retinol isomers. A: Theoretical progress of the esterification reaction. Accumulation of 9-, 11-, and 13-cis-retinyl esters shown as dashed line, solid line, and dotted line, respectively, was simulated based on the kinetic parameters summarized in Table 1. The initial concentrations of the substrates resembled thermodynamic equilibrium between retinol isomers and were set below the Km values at 0.1, 1.0, and 2.3 μM for 11-, 9-, and 13-cis isomers, respectively. B: Representative chromatograms indicate changes in the composition of retinol isomers upon incubation with AWAT2. Trace (a) corresponds to separation of 9-cis-, 11-cis-, 13-cis-, and all-trans-retinol (peaks 1, 2, 3, and 4, respectively) from an equimolar mixture. Trace (b) shows the composition of retinoids extracted after 30 min of incubation in the standard condition for AWAT2 enzymatic assay. Disproportional (compared to other isomers) esterification of 11-cis-retinol is evident by diminishing of peak 2 and concomitant appearance of peak 2’ corresponding to 11-cis-retinyl myristate. C: The time course of the enzymatic reaction conducted in the presence of mixture of retinol isomers. Because formation of retinyl esters other than 11-cis was not sufficient for reliable quantification, the data are represented as ratios between substrates 11-cis- or 9-cis-retinol (filled circles) and 13-cis-retinol (open circles). The dashed line corresponds to the expected rate of 9-cis-retinyl myristate formation based on the kinetic parameters determined solely in the presence of 9-cis-retinol (Fig. 1A, C, D; Table 1). There is an apparent discrepancy between the efficiency of 9-cis-retinol esterification in the presence and absence of other retinol isomers.

Article Snippet: cDNA of mouse AWAT2 (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_177746","term_id":"594150398","term_text":"NM_177746"}} NM_177746 ) was purchased from OriGene Technologies.

Techniques: Incubation, Enzymatic Assay

Journal: Journal of Lipid Research

Article Title: Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2

doi: 10.1194/jlr.M073692

Figure Lengend Snippet: The effect of 11-cis-retinyl esters on AWAT2 activity. A: Inhibition of 9-cis-retinol esterification in the presence of 11-cis-retinyl palmitate. The enzymatic reaction was carried out in the presence of 6 μM 11-cis-retinyl ester and 100 μM 9-cis-retinol. After 20 min of incubation, the retinoid composition was examined by HPLC. The analysis indicated a lower amount of 9-cis-retinyl esters (peak 3’) produced in the presence of 11-cis-retinyl esters (gray trace) as compared with a control reaction (black trace). The chromatography peaks were identified based on the elution time and characteristic UV/Vis spectra (insets). The 11-cis-retinyl ester and the corresponding spectrum with maximum absorbance at 318 nm are marked with asterisks. Peak 3 and confirming UV/Vis spectra with maximum at 324 nm (inset) indicate 9-cis-retinol. B: The 11-cis-retinyl palmitate (11-cis-RP) inhibited esterification of 9-cis-, 13-cis-, or all-trans-retinol, but not 11-cis-retinol. Ten micromoles of selected retinyl esters were preincubated with AWAT2 containing microsomes prior to the addition of the alcohol substrate. C: The 9-cis-, 13-cis-, or all-trans-retinyl palmitates had no effect on esterification of retinol isomers. D: The inhibitory effect of 11-cis-retinyl esters was not dependent on the length of the acyl moiety. Results of the enzymatic assays performed in the presence of 11-cis-retinyl palmitate, laureate (LA), or acetate (AC) revealed a comparable level of inhibition for each of the tested compounds.

Article Snippet: cDNA of mouse AWAT2 (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_177746","term_id":"594150398","term_text":"NM_177746"}} NM_177746 ) was purchased from OriGene Technologies.

Techniques: Activity Assay, Inhibition, Incubation, Produced, Control, Chromatography

Journal: Journal of Lipid Research

Article Title: Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2

doi: 10.1194/jlr.M073692

Figure Lengend Snippet: Evaluation of the oligomeric state of AWAT2 by EM. A: A representative cryo-electron micrograph of purified GST-AWAT2. The red squares indicate fusion protein monomers, whereas the green circles indicate fusion protein homodimers. The 2D classes shown were produced using the 1,603 particle dataset. The top row of classes represents fusion protein monomers, whereas the second row corresponds to homodimers. Each row is marked with the percentage distribution of each oligomeric state calculated from the 12,846 particle dataset. B: Three views of the 3D reconstructed density adjusted to a reasonable volume using the volume/mass conversion of 0.81 Da/Å. The crystal structure of GST (purple) is docked into the density using the Chimera fit to map function.

Article Snippet: cDNA of mouse AWAT2 (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_177746","term_id":"594150398","term_text":"NM_177746"}} NM_177746 ) was purchased from OriGene Technologies.

Techniques: Purification, Produced

Journal: Journal of Lipid Research

Article Title: Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2

doi: 10.1194/jlr.M073692

Figure Lengend Snippet: A schematic representation of the modulation of AWAT2 enzymatic activity. A: In the absence of 11-cis-retinoids, AWAT2 reveals relaxed substrate selectivity allowing for esterification of a variety of geometric isomers of retinol. B: Interaction of the retinoid moiety in the 11-cis configuration with a putative allosteric site (marked with green boundary) leads to changes in the active site (blue and orange boundaries) that result in narrowing substrate specificity of the enzyme and selective production of 11-cis-retinyl esters.

Article Snippet: cDNA of mouse AWAT2 (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_177746","term_id":"594150398","term_text":"NM_177746"}} NM_177746 ) was purchased from OriGene Technologies.

Techniques: Activity Assay

Journal: The FASEB Journal

Article Title: Vascular Bed‐Specific Endothelial Dysfunction and Age‐Dependent Circadian Hypertension in Mice Lacking the Resolvin D2 Receptor GPR18

doi: 10.1096/fj.202503386R

Figure Lengend Snippet: Upper panels show In vivo MRI examinations of endothelial function in the femoral artery (A) and thoracic aorta (B) in response to acetylcholine (ACh, 16.6 mg/kg i.p.) administered to female wildtype (WT, open symbols, n = 6 for the femoral artery and n = 4 for the thoracic aorta) and GPR18 knockout (KO, green symbols, n = 5 for the femoral artery and n = 44 for the thoracic aorta) mice. The unaltered endothelium‐independent responses to infusion of the NO‐donor to sodium nitroprusside (SNP, 1 mg/kg i.v.; WT: N = 5 for the femoral artery and n = 3 for the thoracic aorta; KO: N = 5 for the femoral artery and n = 4 for the thoracic aorta) are shown as insets. Results are shown as changes in vessel volume. The horizontal line represents the mean. p ‐value from Student's t‐test vs. WT. (C) Ex vivo analysis of endothelium‐dependent relaxations in response to ACh administered to 10 μM PE‐precontracted arterial rings of the femoral artery derived from female mice (WT, n = 7; KO, n = 9) in the presence of 10 μM indomethacin. Inset shows endothelial‐independent relaxations in response to DEANO measured in 10 μM PE‐precontracted arterial rings of the femoral artery (WT, n = 8; KO, n = 8), in the presence of 10 μM indomethacin and 300 μM L‐NAME, to inhibit any possible confounding effects of endogenous NOS or COX activity, respectively. The overall P of significant two‐way ANOVA comparison of KO vs. WT is indicated in each concentration‐response graph. * p < 0.05 (Holm‐Sidak post hoc test vs. WT). (D) Representative images and analysis of immunofluorescent staining for endothelial nitric oxide synthase (eNOS) in femoral arteries derived from female GPR18 KO (green circles, n = 3) compared with female WT mice (open circles, n = 4). The horizontal line represents the mean; P‐value from Student's t‐test vs. WT. The micrographs show representative immunofluorescence stainings. Size bar represents 50 μm. (E) Concentration‐response relaxations to acetylcholine (ACh) in the absence (circles; WT, n = 7; KO, n = 10) and presence (squares; WT, n = 7; KO, n = 9) of 10 μM indomethacin in 10 μM PE‐precontracted isolated femoral arteries derived from female WT (left panel with open symbols) and GPR18 KO (left panel with green symbols). The overall p of two‐way ANOVA comparison of KO vs. WT is indicated in each concentration‐response graph. * p < 0.05 (Holm‐Sidak post hoc test vs. WT).

Article Snippet: The breeding and experimental protocols in GPR18 KO (MGI:107859; NCBI ID: 110168; C57BL/6J‐Gpr18 em1 cyagen) mice and WT littermates were approved by the local ethics committee and the Ministry of National Education, Higher Education, and Research and Innovation in France (APAFIS#22187‐2 019 093 011 424 973 v5).

Techniques: In Vivo, Knock-Out, Ex Vivo, Derivative Assay, Activity Assay, Comparison, Concentration Assay, Staining, Immunofluorescence, Isolation

Journal: The FASEB Journal

Article Title: Vascular Bed‐Specific Endothelial Dysfunction and Age‐Dependent Circadian Hypertension in Mice Lacking the Resolvin D2 Receptor GPR18

doi: 10.1096/fj.202503386R

Figure Lengend Snippet: Telemetry mean arterial blood pressures (MAP) in young (A–C) and old (D) WT (open symbols) and GPR18 KO (green symbols) mice. Each datapoint represents the MAP recorded during 1 h for all mice of each group during 3 days and nights. (A, B): The comparison between males (WT, n = 3; KO, n = 3) and females (WT, n = 4; KO, n = 3) in sex‐disaggregated analysis did not reveal any significant sex differences in either WT or GPR18 KO young mice. (C): In the sex aggregated analysis, with young KO ( n = 7; n = 3 males [4.64 ± 0.51 months] and n = 4 females [4.13 ± 0.19 months]) did not exhibit any significant differences compared with young WT ( n = 7; n = 3 males [4.49 ± 0.45 months] and n = 4 females [4.13 ± 0.08 months]) during either day or night. (D): Old KO ( n = 4 [3 females and 1 male; 20.7 ± 0.63 months]) exhibited a significantly increased MAP during daytime and a moderate increase during active night‐time compared with old WT mice ( n = 4 [2 females and 2 males; 21.3 ± 0.48 months]).

Article Snippet: The breeding and experimental protocols in GPR18 KO (MGI:107859; NCBI ID: 110168; C57BL/6J‐Gpr18 em1 cyagen) mice and WT littermates were approved by the local ethics committee and the Ministry of National Education, Higher Education, and Research and Innovation in France (APAFIS#22187‐2 019 093 011 424 973 v5).

Techniques: Comparison

Journal: Cell & Bioscience

Article Title: A minimal bile salt excretory pump promoter allows bile acid-driven physiological regulation of transgene expression from a gene therapy vector

doi: 10.1186/s13578-022-00803-9

Figure Lengend Snippet: AAV vectors expressing LucPEST downstream of BSEP minimal promoters. Schematic representation of AAV vectors containing a BSEP minimal promoter from human (ihPr) ( a ) and mouse (imPr) ( b ) origin. ITR: inverted terminal repeats; LucPEST: destabilized firefly luciferase sequence; pA: synthetic polyadenylation signal

Article Snippet: To generate pAAV-mBSEPpr-LucPEST, a 2488 nt synthetic sequence of full-length mouse BSEP promoter (NCBI AF190697.1) was also ordered from GenScript cloned into pUC57 flanked by Mlu I and Nhe I sites and used to substitute A1AT promoter in pAAV-A1AT-LucPEST, as described before (Additional file : Fig. S2a).

Techniques: Expressing, Luciferase, Sequencing

Journal: Cell & Bioscience

Article Title: A minimal bile salt excretory pump promoter allows bile acid-driven physiological regulation of transgene expression from a gene therapy vector

doi: 10.1186/s13578-022-00803-9

Figure Lengend Snippet: Bile acid induction of minimal BSEP promoters in wild-type (WT) mice. C57BL/6 male and female mice were administered 3 × 10 12 VG/kg of AAV8-imPr-LucPEST ( a ), AAV8-A1AT-LucPEST ( b ), or AAV8-ihPr-LucPEST ( d ) and received either normal diet (red open squares) or a diet supplemented with 0.2% cholic acid (CA) (blue closed circles) alternating with normal diet (blue open circles) as indicated in the figure. Luciferase expression was measured in live mice at the indicated times (n = 3 for each gender except in mice receiving AAV-ihPr-LucPEST where n = 2, only tested in females). c Representative bioluminiscence images of C57BL/6 male mice administered with AAV8-imPr-LucPEST and AAV8-A1AT-LucPEST (n = 3) that received alternating CA/normal diets. Images were taken at week 13 before the last CA cycle and at week 16 after 3 weeks of CA diet in the third cycle. Data are shown as mean + SEM of photon (Ph) units per second normalized with background signal. Statistical comparisons were calculated comparing the average luciferase expression during each CA cycle and its precedent normal diet cycle using a paired T test. *p < 0.05; **p < 0.01. Fold induction in each CA cycle was calculated by dividing the maximum expression under CA induction by basal expression prior to CA administration. imPr, AAV8-imPr-LucPEST; ihPr, AAV8-ihPr-LucPEST; A1AT, AAV-A1AT-LucPEST

Article Snippet: To generate pAAV-mBSEPpr-LucPEST, a 2488 nt synthetic sequence of full-length mouse BSEP promoter (NCBI AF190697.1) was also ordered from GenScript cloned into pUC57 flanked by Mlu I and Nhe I sites and used to substitute A1AT promoter in pAAV-A1AT-LucPEST, as described before (Additional file : Fig. S2a).

Techniques: Luciferase, Expressing

Journal: Cell & Bioscience

Article Title: A minimal bile salt excretory pump promoter allows bile acid-driven physiological regulation of transgene expression from a gene therapy vector

doi: 10.1186/s13578-022-00803-9

Figure Lengend Snippet: Luciferase expression in Abcb4 −/− mice. Abcb4 −/− mice exhibit high levels of bile acids in serum, making them a good model to test the physiological inducibility of minimal BSEP promoters. FVB Abcb4 −/− and WT male and female mice were administered 3 × 10 12 VG/kg of AAV8-imPr-LucPEST ( a ), AAV8-A1AT-LucPEST ( b ) or AAV8-ihPr-LucPEST ( c ). Luciferase was measured in live mice at the indicated times (n = 3 for each gender and vector, except in knock-out (KO) males inoculated with AAV-imPr-LucPEST where n = 4). The statistical comparison between Abcb4 −/− and WT mice was performed by using an unpaired T test at each time point. *p < 0.05; **p < 0.01; ***p < 0.001. Data are shown as mean ± SEM of photon (Ph) units per second normalized with background signal. imPr, AAV8-imPr-LucPEST; ihPr, AAV8-ihPr-LucPEST; A1AT, AAV8-A1AT-LucPEST

Article Snippet: To generate pAAV-mBSEPpr-LucPEST, a 2488 nt synthetic sequence of full-length mouse BSEP promoter (NCBI AF190697.1) was also ordered from GenScript cloned into pUC57 flanked by Mlu I and Nhe I sites and used to substitute A1AT promoter in pAAV-A1AT-LucPEST, as described before (Additional file : Fig. S2a).

Techniques: Luciferase, Expressing, Plasmid Preparation, Knock-Out, Comparison

Journal: Cell & Bioscience

Article Title: A minimal bile salt excretory pump promoter allows bile acid-driven physiological regulation of transgene expression from a gene therapy vector

doi: 10.1186/s13578-022-00803-9

Figure Lengend Snippet: BA induction of an optimized minimal BSEP promoter in wild type mice. C57BL/6 male and female mice were administered 3 × 10 12 VG/kg of AAV8-imPr-3xIR-LucPEST or AAV8-imPr-LucPEST and received either a diet supplemented with 0.2% CA (closed symbols) alternating with normal diet (open symbols) ( a ) or a continuous normal diet ( b ). Luciferase was measured in live mice at the indicated times (n = 4). Data are shown as mean + SEM of photon (Ph) units per second normalized to background signal. The statistical comparisons and fold induction were calculated as described in Fig. a (indicated in the upper part of the graph for imPr-3xIR and in the lower part for imPr). imPr, AAV8-imPr-LucPEST; imPr-3xIR, AAV8-imPr-3xIR-LucPEST

Article Snippet: To generate pAAV-mBSEPpr-LucPEST, a 2488 nt synthetic sequence of full-length mouse BSEP promoter (NCBI AF190697.1) was also ordered from GenScript cloned into pUC57 flanked by Mlu I and Nhe I sites and used to substitute A1AT promoter in pAAV-A1AT-LucPEST, as described before (Additional file : Fig. S2a).

Techniques: Luciferase