Journal: Bioscience Reports

Article Title: A conserved mechanism of sirtuin signalling through steroid hormone receptors

doi: 10.1042/BSR20193535

Figure Lengend Snippet: ( A ) A simplified modular structure of human SIRT1 showing locations of the STACs-activation domain (STACs-AD) and the Core Catalytic (deacetylation) Domain (CCD). NTD and CTD are the N-terminal and C-terminal domains respectively. A classical nuclear receptor box (NR-box) is located within the STACs-AD. Position of the partially conserved STACs activation residue E230 is shown for context . The alignment shows putative NR-boxes of SIRT1 (representing orthologues from different mammals) with orthologous sirtuins in the long-lived naked mole rat H. glaber , the short-lived killifish N. furzeri, S. cerevisiae, D. melanogaster and C. elegans . The NR-box core (LxxLL) of SIRT1 is homologous to those of prototypical coregulators GRIP-1 (Glucocorticoid Receptor-Interacting Protein-1), CBP (CREB-Binding Protein), TIF-1 (Transcriptional Intermediary Factor-1) and SRC-1/NCOA1 (Steroid Receptor Coactivator-1/ Nuclear Receptor Coactivator 1). ( B ) SIRT1 NR-box (see text) is a class III ERα-selective interface with a hydrophobic residue (Φ) at -1, and a hydrophilic residue (serine or threonine) at position -2 . In (A) and (B), positions of the aligned sequences within the coregulators are bracketed. ( C ) One-hybrid assay in HEK293 cells to determine activation functions in SIRT1 and its derivatives tethered to the Gal4 DNA-binding domain (Gal4DBD) as shown. Each construct or Gal4DBD (pM backbone) was cotransfected with the minimal promoter vector pFR-Luc (containing 5x Gal4-binding sites, hereafter labelled 5x Gal4-luc) and β-galactosidase (β-gal) as reporter gene and internal control respectively. The transcriptional activities of the indicated constructs were calculated from the activity of Gal4DBD that was set at 100% as reference. 5x Gal4-luc expression levels were normalized with β-gal and plotted in duplicates as means ± S.E/M; they are representative of three separate experiments. Note that wild-type Gal4DBD-STACs-AD Wt and its mutant Gal4DBD-STACs-ADΔNRB are identical except for mutations in the NR-box (LKDLL→ AKDAA), shown with a triangle within this construct. The activity of Gal4DBD-SIRT1 Wt was compared with its deacetylase-defective mutant Gal4DBD-SIRT1 H 363 Y; mutation is shown with a blue dot. Bracketed numbers besides each construct indicate cloned regions (residues) of SIRT1.

Article Snippet: VP16-ER alpha (# 11351) and VP16-ER beta long isoform (#11352), p413TEF-Sir2 (# 51742), 3xERRE/ERE-luciferase (# 37852), SIRT1.1 (# 13735), Flag-SIRT1 (# 1791), Flag-SIRT1 H 363 Y (# 1792) and Gal4-PGC-1 alpha (# 8892) were purchased from Addgene (Cambridge, MA, U.S.A.). pET28-Sir-2.1 was kindly provided by Leonard Guarente (MIT, Cambridge, MA, U.S.A.) while VP16-DAF-12 and its reporter gene lit -1K-TK-luc [ ] were kindly offered by David Mangelsdorf (UT Southwestern Medical Centre, Dallas, TX, U.S.A.).

Techniques: Activation Assay, Binding Assay, Hybrid Assay, Construct, Plasmid Preparation, Activity Assay, Expressing, Mutagenesis, Histone Deacetylase Assay, Clone Assay

Journal: Bioscience Reports

Article Title: A conserved mechanism of sirtuin signalling through steroid hormone receptors

doi: 10.1042/BSR20193535

Figure Lengend Snippet: ( A ) M2H assays were performed in HEK293 cells to determine interactions between VP16-ERα and SIRT1, Sir2 and Sir-2.1 tethered to Gal4DBD. ( B ) SIRT1-PGC-1α transcriptional complexes strongly interact with ERα and ERβ and overide Gal4 promoter repression by SIRT1 alone. M2H assay was repeated with Gal4DBD-SIRT1 and Gal4DBD-PGC-1α, and ERα or ERβ fused to VP16 activation domain. ( C ) M2H assay for interaction between the STACs-AD and VP16-ERα or VP16-ERβ. Gal4DBD-STACs-AD Wt or its NR-box mutant (ΔNRB) were cotransfected with the receptors as indicated. Schematic shows locations of Wt and mutant NR-boxes within the STACs-AD. Statistical significance of differences in interaction (B) or gene expression in response to DMSO or E2 (A–C) are shown with P values: * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001; differences are not significant where P values are not shown. 5x Gal4-luc and β-gal activities were determined as described and ER interaction with Gal4DBD was set as reference point. Data were plotted in duplicates as means ± S.E.M and are representative of three independent experiments.

Article Snippet: VP16-ER alpha (# 11351) and VP16-ER beta long isoform (#11352), p413TEF-Sir2 (# 51742), 3xERRE/ERE-luciferase (# 37852), SIRT1.1 (# 13735), Flag-SIRT1 (# 1791), Flag-SIRT1 H 363 Y (# 1792) and Gal4-PGC-1 alpha (# 8892) were purchased from Addgene (Cambridge, MA, U.S.A.). pET28-Sir-2.1 was kindly provided by Leonard Guarente (MIT, Cambridge, MA, U.S.A.) while VP16-DAF-12 and its reporter gene lit -1K-TK-luc [ ] were kindly offered by David Mangelsdorf (UT Southwestern Medical Centre, Dallas, TX, U.S.A.).

Techniques: Activation Assay, Mutagenesis, Expressing

Journal: Bioscience Reports

Article Title: A conserved mechanism of sirtuin signalling through steroid hormone receptors

doi: 10.1042/BSR20193535

Figure Lengend Snippet: Gal4DBD NR-box fusion peptides derived from C. elegans Sir-2.1 ( A,B ), Drosophila dSir2 ( C,D ), fish ( N. furzeri ) Sirt1, Nf Sirt1 ( E,F ), and S. cerevisiae Sir2 ( G,H ) were tested by the M2H assay for interaction with ERα (A, C, E, G) or ERβ (B, D, F, H). HEK293 cells were cotransfected with 5x Gal4-luc and increasing amounts of Gal4DBD NR-box (NRB) plasmids as indicated, or with Gal4DBD as negative control; cells were then treated with DMSO or 100 nM E2. In all cases, read-outs of ER interactions (fold activation) with the Gal4DBD were set as the reference point after normalizing luciferase activity against β-gal internal control. All data were plotted in duplicates as means ± S.E.M; data represent at least three separate experiments. Differences in gene expression between DMSO- and E2-treated cells were statistically significant if P ≤ 0.05.

Article Snippet: VP16-ER alpha (# 11351) and VP16-ER beta long isoform (#11352), p413TEF-Sir2 (# 51742), 3xERRE/ERE-luciferase (# 37852), SIRT1.1 (# 13735), Flag-SIRT1 (# 1791), Flag-SIRT1 H 363 Y (# 1792) and Gal4-PGC-1 alpha (# 8892) were purchased from Addgene (Cambridge, MA, U.S.A.). pET28-Sir-2.1 was kindly provided by Leonard Guarente (MIT, Cambridge, MA, U.S.A.) while VP16-DAF-12 and its reporter gene lit -1K-TK-luc [ ] were kindly offered by David Mangelsdorf (UT Southwestern Medical Centre, Dallas, TX, U.S.A.).

Techniques: Derivative Assay, Negative Control, Activation Assay, Luciferase, Activity Assay, Expressing, IF-P

Journal: Bioscience Reports

Article Title: A conserved mechanism of sirtuin signalling through steroid hormone receptors

doi: 10.1042/BSR20193535

Figure Lengend Snippet: Increasing doses (50, 100 and 200 ng) of Gal4DBD fusion peptides of wild-type (NRBWt) and mutant (NRBMt) SIRT1 NR-boxes were tested in M2H assays in HEK293 cells for interaction with ( A ) VP16-ERα and ( B ) VP16-ERβ; the reporter gene was 5x Gal4-luc. Luciferase expression was normalized to β-gal activity. Graphs were plotted by setting the activity of Gal4DBD interaction with the ERs as reference. P values show the significance of differences in ER interaction, comparing NRBWt with NRBMt; only P values ≤ 0.05 are significant. ( C–F ) Modified M2H assay of SIRT1 NR-box peptide binding specificity by competitive inhibition with wild-type (C,E) and mutant (D,F) NR-box peptides. Interactions were determined through dose-dependent repression of ERα (C,D) and ERβ (E,F) signalling; antagonism was observed with wild-type (C,E) but not with mutant (D,F) NR-box peptides. For panels (C–F), MCF-7 cells were cotransfected with VP16-ERα or VP16-ERβ, the ER reporter gene instead of 5x Gal4-luc, and increasing amounts (50, 100 and 200 ng) of Gal4DBD SIRT1 NR-box peptides. Cells were treated with DMSO or 100 nM E2 and ER reporter gene expression was normalized to β-gal internal control. Fold activation was determined from the activity of Gal4DBD (negative control) set at a value of 1. P values show significant ( P ≤ 0.05) differences between DMSO and E2-treated cells. Data are representative of three independent assays.

Article Snippet: VP16-ER alpha (# 11351) and VP16-ER beta long isoform (#11352), p413TEF-Sir2 (# 51742), 3xERRE/ERE-luciferase (# 37852), SIRT1.1 (# 13735), Flag-SIRT1 (# 1791), Flag-SIRT1 H 363 Y (# 1792) and Gal4-PGC-1 alpha (# 8892) were purchased from Addgene (Cambridge, MA, U.S.A.). pET28-Sir-2.1 was kindly provided by Leonard Guarente (MIT, Cambridge, MA, U.S.A.) while VP16-DAF-12 and its reporter gene lit -1K-TK-luc [ ] were kindly offered by David Mangelsdorf (UT Southwestern Medical Centre, Dallas, TX, U.S.A.).

Techniques: Mutagenesis, Luciferase, Expressing, Activity Assay, Modification, Binding Assay, Inhibition, Activation Assay, Negative Control

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Optimization of pGC-A expression in Sf9 cells. Western blot (anti-pGC-A) of total cell protein over different virus multiplicities of infection (MOI) and transfection time. MOI of 0 indicates no virus transfection. pGC-A (red arrow) is approximately 120 kDa.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Expressing, Western Blot, Infection, Transfection

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Determination of expressed full-length pGC-A functionality via whole-cell activity assay. The competitive ELISA assay measured the cGMP yield level in Sf9 cells expressing full-length pGC-A versus control Sf9 cells (n = 2). Both cell types were incubated with different concentrations of MANP ligand (0 to 10 7 pmol). Incubation with 0 pmol MANP served as a negative control.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Activity Assay, Competitive ELISA, Expressing, Incubation, Negative Control

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Purification of full-length pGC-A via affinity and size exclusion columns. ( A ) Western blot (anti-pGC-A) of samples from cell lysis to the affinity column. pGC-A (red arrow) is ~ 120 kDa. ( B ) Coomassie blue stain from cell lysis to the affinity column. ( C ) Coomassie blue stain of the eluted fraction from Superose 6 that was used for protein crystallization. M: marker; P: membrane pellet from ultracentrifugation after solubilization with n-dodecyl-β-D-maltoside (DDM) and cholesteryl hemisuccinate (CHS); FT: flowthrough from the affinity column; 50, 100, 500: imidazole (mM) at elution from the affinity column.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Purification, Western Blot, Lysis, Affinity Column, Staining, Crystallization Assay, Marker

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Presence of pGC-A in crystals was confirmed via western blot. The anti-pGC-A antibody was used in the western blot. The pGC-A monomer is 120 kDa. Lane 1: combined mix: combined crystallization drops collected in the PCR tube. Lane 2: supernatant: the supernatant collected from the centrifuged combined mix. Lane 3: washed pellet: the crystal pellet was washed with the precipitant solution and collected again via centrifugation. Lane 4: washed supernatant: the supernatant from washed crystal pellet. Lane 5: crystallization drop: one crystallization hanging drop directly mixed with SDS sample buffer. Lane 6: crystallization sample: purified pGC-A before crystallization, serving as a positive control.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Western Blot, Crystallization Assay, Centrifugation, Purification, Positive Control

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Indexed diffraction patterns from pGC-A microcrystals collected via serial crystallography at the Advanced Photon Source. Three indexable diffraction patterns with the highest resolution of 3 Å. ( A ) Blue/pink diffraction graphs show diffraction patterns analyzed using CrystFEL software. ( B ) The original diffraction patterns shown with white background. All diffraction dots were manually circled in red for better visualization.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Software

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Dynamic oligomeric states of pGC-A seen in replicate runs of Superose 6 size exclusion chromatography may be dependent on protein concentration. ( A ) The Superose 6 10/300GL column performance profile. Five standard proteins were used to generate relative molecule elution points based on different molecular sizes. Thyroglobulin (669 kDa) eluted at 14.19 mL, ferritin (440 kDa) eluted at 15.96 mL, aldolase (158 kDa) eluted at 17.58 mL, ovalbumin (44 kDa) eluted at 18.49 mL, and aprotinin (6.5 kDa) eluted at 21.60 mL. ( B-D ) Size exclusion chromatography of pGC-A. ( B ) The peak intensity at 17.3 mL corresponds to the pGC-A monomeric state (120 kDa). pGC-A monomer is the major peak determined by chromatography. Other ratios were faded out in the background and served as supplemental comparison. ( C ) pGC-A tetramer and monomer present similar ratios in the chromatographic separation. The peak intensity at 14.76 mL and 17.29 mL corresponds to pGC-A tetrameric (480 kDa) and monomeric states, respectively. ( D ) The pGC-A tetramer is the major peak. The peak intensity at 15.05 mL corresponds to the pGC-A tetrameric state.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Size-exclusion Chromatography, Protein Concentration, Chromatography

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Silver stain of high resolution clear-native PAGE from pGC-A Superose 6 fractions. Superose 6 column eluted tetramer (480 kDa) and monomer (120 kDa) size peaks were concentrated and analyzed in a 4–16% native gel. M, marker.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Silver Staining, Clear Native PAGE, Marker

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Silver stained clear-native PAGE of different oligomeric samples treated with or without dithiothreitol (DTT) overnight. Three peak samples, which represent the tetramer, dimer, and monomer of full-length pGC-A, were concentrated and split in half for treatment with or without DTT. S, concentrated sample only; S + DTT, concentrated sample incubated with 1 M DTT overnight.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Staining, Clear Native PAGE, Incubation

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Purified full-length pGC-A in vitro functional activity test. ( A ) Functional activity for two pGC-A oligomeric states with ATP incubation. The control group was GTP and ATP in sample buffer. For activity values in units of mg of purified pGC-A, the y-axis values of pmol/mL can be converted to nmol/mg protein by multiplying by 0.00873. ( B ) Functional activity for two pGC-A oligomeric states without ATP incubation. The control group was GTP only in the sample buffer. ( C ) Competitive cGMP ELISA standard fit in four parameters logistic (4PL) curve. The left Y-axis is the B/B0 (%) value and represents the percentage of bound cGMP. The right Y-axis represents the average net optical density (OD) reading at 405 nm. Both standard curves were generated with a 95% confidence interval. ( D ) The cGMP yield differences between pGC-A oligomer samples incubated with or without ATP were analyzed. All raw data points were analyzed via the ROUT method (Q = 1%) to remove significantly impossible outlier values before data analysis. One-way ANOVA was used to determine the statistical significance between samples and control in graphs ( A ) and ( B ). Two-way ANOVA was used to determine the statistical significance in graph ( D ). Each dot represented to the sample point and plotted as mean ± standard deviation (SD). * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001 and ns P ≥ 0.05, Not significant.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Purification, In Vitro, Functional Assay, Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay, Generated, Standard Deviation

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Proposed native state and three-step mechanism of full-length pGC-A. First, the full-length pGC-A forms a tetramer complex in the native state by non-covalent interactions (e.g., hydrogen bond and hydrophobic interactions). In each tetramer complex, there are two functional units, and each functional unit may represent a dimer. The narrowest part of the tetramer is the transmembrane domain. Second, the pGC-A signal transduction mechanism is not ATP-dependent. The current ATP-dependent two-step activation mechanism should instead be three-step. The first step is ligand (ANP) binding, which moderately activates the pGC-A; the second step is binding ATP, which partially boosts protein activity; the third step is the pGC-A phosphorylation, which fully activates the guanylyl cyclase.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Functional Assay, Transduction, Activation Assay, Binding Assay, Activity Assay