Reagents and antibodies
Dulbecco’s modified Eagle’s medium (DMEM), F-12 (1:1), and bovine serum (BS) were purchased from Gibco (Grand Island, NY). Fetal bovine serum (FBS) was purchased from Corning (Corning, NY). Melatonin, doxorubicin, H2DCF-DA, intracellular ATP assay kits, N-acetyl cysteine, and 4-P-PDOT (4-phenyl-2-propionamidotetralin) were purchased from Sigma-Aldrich (St. Louis, MO). MTT thiazolyl blue and D-luciferin were purchased from Duchefa Biochemie (Haarlem, the Netherlands). 4’,6-Diamidino-2-phenylindole (DAPI), enhanced chemiluminescence reagent, antibodies against GAPDH, E2F1, c-Myc, and voltage-dependent anion channel (VDAC), MitoTracker red, and control IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-AMPKα2 and anti-AMPKα1 antibodies were purchased from R&D Systems (Minneapolis, MN). MitoTracker green and anti-phospho-H2A.X (S139), anti-phospho-AMPKα (Thr172), anti-phospho-acetyl-CoA carboxylase (ACC) (S79), anti-ACC, anti-phospho-mitochondria fission factor (MFF) (S146), and anti-MFF antibodies were purchased from Cell Signaling Technology (Danvers, MA). MitoSOX Red was purchased from Invitrogen (Carlsbad, CA). The FITC-annexin V apoptosis kit was purchased from DB BioScience (San Diego, CA). The Total OXPHOS Rodent WB Antibody Cocktail was purchased from Abcam (Cambridge, MA).
H9c2 (rat cardiomyoblast) cells were purchased from American Type Culture Collection (ATCC CRL-1446) in 2012 and maintained in DMEM and F-12 (1:1) supplemented with 10% BS. H9c2 cells fuse to form multinucleated myotubes in response to low serum, and a recent characterization was performed in 2017. Microscopic analysis revealed that these cells formed multinucleated myotubes when cultured in media containing 1% horse serum, and RT-PCR analysis showed an increase in the expression of acetyl-CoA carboxylase β, which is a specific marker of myotube formation25. AMPK wild-type (WT) and AMPK knockout MEFs were generated by Benoit Viollet (INSERN, France) in 200626 and generously provided to us in 2008. Ampkα WT, Ampkα2−/−, Ampkα1−/−, and Ampkα double knockout (α1−/−α2−/−) MEFs were maintained in DMEM supplemented with 10% FBS. We characterized these cells according to the presence of AMPK isoforms and simian virus 40 large T antigen using Western blot and RT-PCR in 2018. All the cells were supplemented with 1% penicillin/streptomycin and cultured at 37 °C in a humidified environment with 95% air and 5% CO2.
Stable cell transfection
When the density of the H9c2 myocytes reached 50–60%, the cells were transfected with the expression vector pcDNA 3.1 encoding c-Myc-tagged wild-type (WT) or dominant negative (DN, K45A) forms of AMPKα2 using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). The cells were selected for 7 days in the presence of G418 (1 mg/mL), and the selected cells were used for the subsequent experiments.
The promoter region of the human AMPKα2 gene (−2.7 kb) was amplified by PCR from HEK293 genomic DNA and cloned into the pGL3-basic reporter vector. The [E2F]x4-Luc reporter construct was generously provided by Dr. Young-Chae Chang (The Catholic University, School of Medicine, Daegu, Korea). AMPKα2 wild-type (WT) and AMPKα2 dominant-negative (DN, D157A) forms were generated by PCR and cloned into the pCMV vector. The dsRed-mito plasmid was generously provided by Dr. Youngmi Kim Pak (Kyung hee University, School of Medicine, Seoul, Korea). Mito-ABKAR was a gift from Takanari Inoue and Jin Zhang (Addgene plasmid #61509). Tom20-mChF-AIP and mChF-AIP were gifts from Takanari Inoue (Addgene plasmids #61512 and #61527).
Cell viability assay
Cell viability was measured in 96-well plates using a quantitative colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which is an indicator of mitochondrial activity in live cells. Briefly, 30 μL MTT (final concentration, 0.5 mg/mL) was added to the medium at the indicated times after treatment and then incubated at 37 °C for 4 h. The MTT solution was removed, and 100 μL DMSO was added to each well. The absorbance of each well was measured at 540 nm using a microplate reader (BioTek, Winooski, VT).
Western blot analysis and immunoprecipitation
Cells were lysed in lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP40, 2 mM EDTA, 10 mM NaF, 2 mM Na3VO4). The protein concentrations in the total lysates were determined by the Bradford method (Bio–Rad, Hercules, CA, USA). Twenty micrograms of protein from the total lysates was subjected to SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Bedford, MA). Then, the membranes were incubated with primary antibodies in blocking solution (5% skim milk). The proteins were visualized using the ECL detection system. For the immunoprecipitation experiments, cell lysates were precleared with protein G/A beads and subsequently incubated for 1–2 h with protein G/A beads covalently coupled with anti-AMPKα2 or anti-c-Myc. The immune complexes were washed three times with cell lysis buffer. The eluted samples and whole lysates were resolved by SDS-PAGE, and the proteins were detected by western blot using the indicated antibodies. Quantification of western blots was performed by using ImageJ software.
H9c2 cells were cotransfected with 0.5 μg of the reporter vector. The empty pcDNA vector was used to adjust the total amount of DNA, and 0.5 μg of pCMV-EGFP expression plasmid was used as the internal control. Luciferase activity was determined using a luciferase assay kit (Promega) according to the manufacturer’s instructions. The relative luciferase activity was normalized against the GFP fluorescence intensity.
Fluorescence-activated cell sorting (FACS) analysis
Fluorescence was determined by flow cytometry (Beckman, Pasadena, CA). MitoTracker green, H2DCF-DA, and Annexin V-FITC fluorescence was measured by excitation at 488 nm and emission collection at 525 nm. Propidium iodide (PI), MitoTracker red, and MitoSOX Red fluorescence was measured by using excitation and emission wavelengths of 540 and 580 nm, respectively.
H9c2 cells were plated on glass coverslips and treated the following day with doxorubicin, with or without melatonin. The cells were fixed by incubating with 4% paraformaldehyde for 20 min and washed with phosphate-buffered saline (PBS). After permeabilization with 0.1% Triton X-100 in PBS for 3 min at room temperature, the cells were incubated with a blocking solution for 30 min at 37 °C. Solutions of anti-AMPKα2 (1:500) primary antibodies were added to the cells and incubated overnight at 4 °C. Thereafter, the cells were washed with PBS and then incubated with 1:500 dilutions of FITC- or Texas red-conjugated secondary antibodies for 2 h at 37 °C. MitoTracker was added before fixation, and DAPI was added after incubation with secondary antibodies. For the Förster resonance energy transfer (FRET) experiments, H9c2 cells were transfected with the mito-ABKAR plasmid and then treated the following day with doxorubicin and/or melatonin. Cyan fluorescent protein (CFP) was excited at 433 nm, and its emitted fluorescence was collected at 480 nm. FRET images were obtained by exciting yellow fluorescent protein (YFP) at 508 nm and collecting its emitted fluorescence at 527 nm. The lengths of mitochondria were calculated from each YFP image using ImageJ software (image.nih.gov/ij/), as previously described27, and the FRET fluorescence intensity was analyzed. Images of the cells were acquired using an LSM 510 confocal microscope (Carl Zeiss, Jena, Germany).
Total RNA was extracted from cells using TRIzol reagent (Invitrogen). cDNA was synthesized from 2 mg of total RNA using M-MLV reverse transcriptase (Fermentas, Hanover, MD, USA). The specific primers used for RT-PCR were as follows: AMPKα2, 5′-AGC TCG CAG TGG CTT ATC AT-3′ (sense) and 5′-GGG GCT GTC TGC TAT GAG AG-3′ (anti-sense); and GAPDH, 5′-AGA CAG CCG CAT CTT CTT GT-3′ (sense) and 5′-CTT GCC GTG GGT AGA GTC AT-3′ (anti-sense). The PCR products were analyzed by agarose gel electrophoresis and visualized using ethidium bromide. The signals were quantified using ImageJ software (National Institutes of Health (NIH), Bethesda, MD, USA).
Measurement of mitochondrial DNA content
Total genomic DNA was extracted from H9c2 cells and used as a template for the amplification of mitochondrially encoded cytochrome C oxidase II (MTCO2) and 18S rRNA by real-time quantitative polymerase chain reaction (qPCR). The following specific primer pairs were used: MTCO2, 5’-GCT TAC AAG ACG CCA CAT CA-3’ (sense) and 5′-GAA TTC GTA GGG AGG GAA GG-3′ (anti-sense); 18S rRNA, 5′-CGC GGT TCT ATT TTG TTG GT-3′ (sense) and 5′-AGT CGG CAT CGT TTA TGG TC-3′ (anti-sense). qPCR was performed using a 7500 Real-Time PCR System (Applied Biosystems, Branchburg, NJ, USA) with SYBRGreen PCR Master Mix (Applied Biosystems). A total of 40 thermal cycles were used for the PCR, and the expression of MTCO2 was analyzed using an absolute quantification method and normalized to the levels of 18S rRNA.
Measurement of intracellular ATP levels
Cells were seeded in 24-well plates and stimulated as indicated. The intracellular ATP content was measured using an ATP Determination Kit (Sigma-Aldrich) in accordance with the manufacturer’s instructions.
All the mice were purchased from Japan SLC (Shizuoka, Japan) and housed at 21 ± 2 °C and 50% ± 5% relative humidity with a 12-h light/12-h dark cycle. The mice were randomly divided into 4 groups (n = 5/group): sham, doxorubicin, melatonin, and doxorubicin + melatonin. Melatonin (1 mg/kg) and/or doxorubicin (2.5 mg/kg) were intraperitoneally injected into 4-week-old mice (Slic: ICR, male) every 2 days for 2 weeks. After 2 weeks of treatment, heart tissues were isolated, and five-micron-thick sections were subjected to H&E staining, Masson trichrome staining, immunohistochemical analysis (IHC) of AMPKα2, and colorimetric TUNEL assay (Promega Corp., Madison, WI). The thickness of the myofibrillum was measured as previously described28. The TUNEL-positive nuclei (dark brown) in the heart tissues were detected using a normal white light microscope (Olympus, Tokyo, Japan). Quantification was performed by counting the number of TUNEL-positive cells in at least five random fields. The animal protocol was approved by the Institutional Animal Care and Use Committee of Kyung Hee University (KHSASP-19-297).
Data and statistical analysis
The results are expressed as the means ± SEMs. The statistical significance of the data were analyzed by one-way analysis of variance (ANOVA) using the R program suite (version 3.2.4; http://www.r-project.org). By convention, a p-value < 0.05 was considered statistically significant. Individual p-values are indicated in the figures (*p < 0.05; **p < 0.01). Each experiment was repeated at least twice with three samples each to ensure statistical significance.
Structures of the glucocorticoid-bound adhesion receptor GPR97–Go complex
No statistical methods were used to predetermine sample size. The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessment.
HEK293 cells were obtained from the Cell Resource Center of Shanghai Institute for Biological Sciences (Chinese Academy of Sciences). Spodoptera frugiperda (Sf9) cells were purchased from Expression Systems (cat. 94-001S). Y-1 cells were originally obtained from the American Type Culture Collection (ATCC). The cells were grown in monolayer culture in RPMI 1640 with 10% FBS (Gibco) at 37 °C in a humidified atmosphere consisting of 5% CO2 and 95% air.
Constructs of GPR97 and miniGo heterotrimer
For protein production in insect cells, the human GPR97 (residues 21–549) with the autoproteolysis motif mutation (H248/A and T250/A) was sub-cloned into the pFastBac1 vector. The native signal peptide was replaced with the haemagglutinin signal peptide (HA) to enhance receptor expression, followed by a Flag tag DYKDDDK (China peptide) to facilitate complex purification. An engineered human Gαo1 with Gαo1 H domain deletion, named miniGαo1 was cloned into pFastBac1 according to published literature29. Human Gβ1 with the C-terminal hexa-histidine tag and human Gγ2 were subcloned into the pFastBacDual vector. scFv16 was cloned into pfastBac1 with the C-terminal hexa-histidine tag and the N-terminal GP67 signal peptide. To examine the activities of GPR97, the GPR97-FL-WT (wild-type full-length GPR97), GPR97-FL-AA (GPR97 GPS site mutation, H248/A and T250/A), GPR97β (GPR97 with the NTF removed, residues 250–549) and GPR97-β-T (GPR97β with the N-terminal tethered Stachel sequence removed, residues 265–549) were sub-cloned into the pcDNA3.1 plasmid. The GPR97 mutations E298A, R299A, F345A, F353A, H362A, L363A, Y364A, V370A, F371A, Y406A, W421A, W490A, A493G, I494A, L498A and N510A were generated using the Quikchange mutagenesis kit (Stratagene). The G protein BRET probes were constructed according to previous publications42,43. Human G protein subunits (Gαq, Gβ1 and Gγ2) were sub-cloned into the pcDNA3.1 expression vectors. The Gαq-RlucII subunit was generated by amplifying and inserting the coding sequence of RlucII into Gαq between residue L97 and K98. The Gqo probe, in which the six amino acids of the C-terminal of Gαq-RlucII were substituted with those from Gαo1, was constructed by PCR amplification using synthesized oligonucleotides encoding swapped C-terminal sequences. The GFP10–Gγ2 plasmid was generated by fusing the GFP10 coding sequence in frame at the N terminus to Gγ2. All of the constructs and mutations were verified by DNA sequencing.
High titre recombinant baculoviruses were generated using Bac-to-Bac Baculovirus Expression System. In brief, 2 μg of recombinant bacmid and 2 μl X-tremGENE HP transfection reagent (Roche) in 100 μl Opti-MEM medium (Gibco) were mixed and incubated for 20 min at room temperature. The transfection solution was added to 2.5 ml Sf9 cells with a density of 1 × 106 per ml in a 24-well plate. The infected cells were cultured in a shaker at 27 °C for 4 days. P0 virus was collected and then amplified to generate P1 virus. The viral titres were determined by flow cytometric analysis of cells stained with gp64-PE antibody (1:200 dilution; 12-6991-82, Thermo Fisher). Then, Sf9 cells were infected with viruses encoding GPR97-FL-AA, miniGαo, Gβγ, and with or without scFv16, respectively, at equal multiplicity of infection. The infected cells were cultured at 27 °C, 110 rpm for 48 h before collection. Cells were finally collected by centrifugation and the cell pellets were stored at −80 °C.
GPR97–Go complex formation and purification
Cell pellets transfected with virus encompassing the GPR97-FL-AA, miniGo trimer and scFv16 (only existed in cell pellets for purifying the cortisol–GPR97-FL-AA–Go–scFv16 complex) were resuspended in 20 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol, 10 mM MgCl2 and 5 mM CaCl2 supplemented with Protease Inhibitor Cocktail (B14001, Bimake) and 100 μM TCEP (Thermo Fisher Scientific). The complex was formed for 2 h at room temperature by adding 10 μM BCM (HY-B1540, MedChemExpress) or cortisol (HY-N0583, MedChemExpress), 25 mU/ml apyrase (Sigma), and then solubilized by 0.5% (w/v) lauryl maltose neopentylglycol (LMNG; Anatrace) and 0.1% (w/v) cholesteryl hemisuccinate TRIS salt (CHS; Anatrace) for 2 h at 4 °C. Supernatant was collected by centrifugation at 30,000 rpm for 40 min, and the solubilized complex was incubated with nickel resin for 2 h at 4 °C. The resin was collected and washed with 20 column volumes of 20 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol, 2 mM MgCl2, 25 mM imidazole, 0.01% (w/v) LMNG, 0.01% GDN (Anatrace), 0.004% (w/v) CHS, 10 μM BCM (or cortisol) and 100 μM TCEP. The complex was eluted with 20 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol, 2 mM MgCl2, 200 mM imidazole, 0.01% (w/v) LMNG, 0.01% GDN, 0.004% (w/v) CHS, 10 μM BCM (or cortisol) and 100 μM TCEP. The elution of nickel resin was applied to M1 anti-Flag resin (Sigma) for 2 h and washed with 20 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol, 2 mM MgCl2, 5 mM CaCl2, 0.01% (w/v) LMNG, 0.01% GDN, 0.004% (w/v) CHS, 10 μM BCM (or cortisol) and 100 μM TCEP. The GPR97–Go complex was eluted in buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol, 2 mM MgCl2, 0.01% (w/v) LMNG, 0.01% GDN, 0.004% (w/v) CHS, 10 μM BCM (or cortisol), 100 μM TCEP, 5 mM EGTA and 0.2 mg/ml Flag peptide. The complex was concentrated and then injected onto Superdex 200 increase 10/300 GL column equilibrated in the buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 0.00075% (w/v) LMNG, 0.00025% GDN, 0.0002% (w/v) CHS, 10 μM BCM (or cortisol) and 100 μM TCEP. The complex fractions were collected and concentrated individually for EM experiments.
Cryo-EM grid preparation and data collection
For the preparation of cryo-EM grids, 3 μl of purified BCM-bound and cortisol-bound GPR97–Go complex at approximately 20 mg/ml was applied onto a glow-discharged holey carbon grid (Quantifoil R1.2/1.3). Grids were plunge-frozen in liquid ethane cooled by liquid nitrogen using Vitrobot Mark IV (Thermo Fisher Scientific). Cryo-EM imaging was performed on a Titan Krios at 300 kV accelerating voltage in the Center of Cryo-Electron Microscopy, Zhejiang University. Micrographs were recorded using a Gatan K2 Summit direct electron detector in counting mode with a nominal magnification of ×29,000, which corresponds to a pixel size of 1.014 Å. Movies were obtained using serialEM at a dose rate of about 7.8 electrons per Å2 per second with a defocus ranging from −0.5 to −2.5 μm. The total exposure time was 8 s and intermediate frames were recorded in 0.2-s intervals, resulting in an accumulated dose of 62 electrons per Å2 and a total of 40 frames per micrograph. A total of 2,707 and 5,871 movies were collected for the BCM-bound and cortisol-bound GPR97–Go complex, respectively.
Cryo-EM data processing
Dose-fractionated image stacks for the BCM–GPR97–Go complex were subjected to beam-induced motion correction using MotionCor2.144. Contrast transfer function (CTF) parameters for each non-dose-weighted micrograph were determined by Gctf45. Particle selection, 2D and 3D classifications of the BCM–GPR97–Go complex were performed on a binned data set with a pixel size of 2.028 Å using RELION-3.0-beta246.
For the BCM–GPR97–Go complex, semi-automated particle selection yielded 2,026,926 particle projections. The projections were subjected to reference-free 2D classification to discard particles in poorly defined classes, producing 911,519 particle projections for further processing. The map of the 5-HT1BR–miniGo complex (EMDB-4358)47 low-pass filtered to 40 Å was used as a reference model for maximum-likelihood-based 3D classification, resulting in one well-defined subset with 307,700 projections. Further 3D classifications focusing the alignment on the complex produced two good subsets that accounted for 166,116 particles, which were subsequently subjected to 3D refinement, CTF refinement and Bayesian polishing. The final refinement generated a map with an indicated global resolution of 3.1 Å at a Fourier shell correlation of 0.143.
For the cortisol–GPR97–Go complex, particle selection yielded 4,323,518 particle projections for reference-free 2D classification. The well-defined classes with 2,201,933 particle projections were selected for a further two rounds of 3D classification using the map of the BCM-bound complex as reference. One good subset that accounted for 335,552 particle projections was selected for a further two rounds of 3D classifications that focused the alignment on the complex, and produced one high-quality subset with 75,814 particle projections. The final particle projections were subsequently subjected to 3D refinement, CTF refinement and Bayesian polishing, which generates a map with a global resolution of 2.9 Å. Local resolution for both density maps was determined using the Bsoft package with half maps as input maps48.
Model building and refinement
For the structure of the BCM–GPR97–Go complex, the initial template of GPR97 was generated using the module ‘map to model’ in PHENIX44. The coordinate of the 5-HT1BR–Go complex (PDB ID: 6G79) was used to generate the initial models for Go (ref. 44). Models were docked into the EM density map using UCSF Chimera49, followed by iterative manual rebuilding in COOT50 according to side-chain densities. BCM and lipid coordinates and geometry restraints were generated using phenix.elbow. BCM was built to the model using the ‘LigandFit’ module in PHENIX. The placement of BCM shows a correlation coefficient of 0.81, indicating a good ligand fit to the density. The model was further subjected to real-space refinement using Rosetta51 and PHENIX44.
For the structure of the cortisol–GPR97–Go complex, the coordinates of GPR97 and Go from the BCM-bound complex and scFv16 from the human NTSR1–Gi1 complex (PDB ID: 6OS9) were used as initial model. Models were docked into the density map and then were manual rebuilt in COOT. The agonist cortisol was built to the model using the ‘LigandFit’ module as described, showing a good density fit with a correlation coefficient of 0.80. The model was further refined using Rosetta51 and PHENIX44. The final refinement statistics for both structures were validated using the module ‘comprehensive validation (cryo-EM)’ in PHENIX44. The goodness of the fit of the model to the map was performed for both structures using a global model-versus-map FSC (Extended Data Fig. 2). The refinement statistics are provided in Extended Data Table 1. Figures of the structures were generated using UCSF Chimera, UCSF ChimeraX52 and PyMOL53.
Molecular dynamics simulation of the BCM–GPR97 and cortisol–GPR97 complexes
On the basis of the favour binding poses of BCM and cortisol with the receptor GPR97, which was calculated by the LigandFit program of PHENIX, the GPR97–agonist complexes were substrate from the two GPR97–agonist–mGo complexes for molecular dynamics simulation. The orientations of receptors were calculated by the Orientations of Proteins in Membranes (OPM) database. Following this, the whole systems were prepared by the CHARM-GUI and embedded in a bilayer that consisted of 200 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids by replacement methods. The membrane systems were then solvated into a periodic TIP3P water box supplemented with 0.15 M NaCl. The CHARMM36m Force Filed was used to model protein molecules, CHARMM36 Force Filed for lipids and salt with CHARMM General Force Field (CGenFF) for the agonist molecules BCM and cortisol.
Then, the system was subjected to minimization for 10,000 steps using the conjugated gradient algorithm and then heated and equilibrated at 310.13 K and 1 atm for 200 ps with 10.0 kcal mol−1 Å−2 harmonic restraints in the NAMD 2.13 software. Next followed five cycles of equilibration for 2 ns each at 310.13 K and 1 atm, for which the harmonic restraints were 5.0, 2.5, 1.0, 0.5 and 0.1 kcal mol−1 Å−2 in sequence.
Production simulations were run at 310.13 K and 1 atm in the NPT ensemble using the Langevin thermostat and Nose–Hoover method for 200 ns. Electrostatic interactions were calculated using the particle mesh Ewald (PME) method with a cut-off of 12 Å. Throughout the final stages of equilibration and production, 5.0 kcal mol−1 Å−2 harmonic restraints were placed on the residues of GPR97 that were within 5 Å of Go in the BCM (or cortisol)–GPR97–Go complex to ensure that the receptor remained in the active state in the absence of the G protein. Trajectories were visualized and analysed using Visual Molecular Dynamics (VMD, version 1.9.3)
cAMP ELISA detection in Y-1 cells
Y-1 cells were transfected with Gpr97 siRNA (si-97, GUGCAGGGAAUGUCUUUAA) or control siRNA (si-Con) for 48 h. After starvation for 12 h in serum-free medium, the cells were further stimulated with cortisone (8 nM), forskolin (5 μM) (Sigma-Aldrich) or control vehicle for 10 min. Then, cells were washed three times with pre-cooled PBS and resuspended in pre-cooled 0.1 N HCl containing 500 μM IBMX at a 1:5 ratio (w/v). The samples were neutralized with 1 N NaOH at a 1:10 ratio (v/v) after 10 min. The supernatants were collected after centrifugation of the samples at 600g for 10 min. The supernatants were then prepared for cAMP determination using the cAMP Parameter Assay Kit (R&D Systems) according to the manufacturer’s instruction. The Gpr97 expression level under various conditions were further confirmed using quantitative real-time PCR.
Mouse adrenocorticotoma cell line Y-1 cells were transfected with Gpr97 siRNA (si-97) or control siRNA (si-Con) for 48 h. Then, the cells were treated with serum-free medium for 12 h. After that, cortisone (16 nM) or ACTH (0.5 μM) were added to cells for 30 min. The supernatants of the cell culture medium were collected for measurements of corticosterone by ELISA according to the manufacturer’s instructions.
Quantitative real-time PCR
Total RNA of cells was extracted using a standard TRIzol RNA isolation method. The reverse transcription and PCR experiments were performed with the Revertra Ace qPCR RT Kit (TOYOBO FSQ-101) using 1.0 μg of each sample, according to the manufacturer’s protocols. The quantitative real-time PCR was conducted in the Light Cycler apparatus (Bio-Rad) using the FastStart Universal SYBR Green Master (Roche). The mRNA level was normalized to GAPDH in the same sample and then compared with the control. The forward and reverse primers for GPR97 used in the experiments were CAGTTTGGGACTGAGGGACC and GCCCACACTTGGTGAAACAC. The mRNA level of GAPDH was used as an internal control. The forward and reverse primers for GAPDH were GCCTTCCGTGTTCCTACC and GCCTGCTTCACCACCTTC.
cAMP inhibition assay
To measure the inhibitory effects on forskolin-induced cAMP accumulation of different GPR97 constructs or mutants in response to different ligands or constitutive activity, the GloSensor cAMP assay (Promega) was performed according to previous publications12,13. HEK293 cells were transiently co-transfected with the GloSensor and various versions of GPR97 or vehicle (pcDNA3.1) plasmids using PEI in six-well plates. After incubation at 37 °C for 24 h, transfected cells were seeded into 96-well plates with serum-free DMEM medium (Gibco) and incubated for another 24 h at 37 °C in a 5% CO2 atmosphere. Different ligands were dissolved in DMSO (Sigma) to a stock concentration of 10 mM and followed by serial dilution using PBS solution immediately before the ligand stimulation. The transfected cells were pre-incubated with 50 μl of serum-free DMEM medium containing GloSensor cAMP reagent (Promega). After incubation at 37 °C for 2 h, varying concentrations of ligands were added into each well and followed by the addition of forskolin to 1 μM. The luminescence intensity was examined on an EnVision multi-label microplate detector (Perkin Elmer).
The Gqo protein activation BRET assay
According to previous publications, the BCM dipropionate-induced GPR97 activity could be measured by chimeric Gqo protein assays25. The Gqo BRET probes were generated by replacing the six amino acids of the C-terminal of Gq-RlucII with those from GoA1, creating a chimeric Gqo-RlucII subunit47. GFP10 was connected to Gγ. The Gqo protein activation BRET assay was performed as previously described54. In brief, HEK293 cells were transiently co-transfected with control D2R and various GPR97 constructs, plasmids encoding the Gqo BRET probes, incubated at 37 °C in a 5% CO2 atmosphere for 48 h. Cells were washed twice with PBS, collected and resuspended in buffer containing 25 mM HEPES, pH 7.4, 140 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 12 mM NaHCO3, 5.6 mM d-glucose, 0.5 mM MgCl2 and 0.37 mM NaH2PO4. Cells that were dispensed into a 96-well microplate at a density of 5–8 × 104 cells per well were stimulated with different concentrations of ligands. BRET2 between RLucII and GFP10 was measured after the addition of the substrate coelenterazine 400a (5 μM, Interchim) (Cayman) using a Mithras LB940 multimode reader (Berthold Technologies). The BRET2 signal was calculated as the ratio of emission of GFP10 (510 nm) to RLucII (400 nm).
Measurement of receptor cell-surface expression by ELISA
To evaluate the expression level of wild-type GPR97 and its mutants, HEK293 cells were transiently transfected with wild-type and mutant GPR97 or vehicle (pcDNA3.1) using PEI regent at in six-well plates. After incubation at 37 °C for 18 h, transfected cells were plated into 24-well plates at a density of 105 cells per well and further incubated at 37 °C in a 5% CO2 atmosphere for 18 h. Cells were then fixed in 4% (w/v) paraformaldehyde and blocked with 5% (w/v) BSA at room temperature. Each well was incubated with 200 μl of monoclonal anti-FLAG (F1804, Sigma-Aldrich) primary antibody overnight at 4 °C and followed by incubation of a secondary goat anti-mouse antibody (A-21235, Thermo Fisher) conjugated to horseradish peroxide for 1 h at room temperature. After washing, 200 μl of 3,3′,5,5′-tetramethylbenzidine (TMB) solution was added. Reactions were quenched by adding an equal volume of 0.25 M HCl solution and the optical density at 450 nm was measured using the TECAN (Infinite M200 Pro NanoQuant) luminescence counter. For determination of the constitutive activities of different GPR97 constructs or mutants, varying concentrations of desired plasmids were transiently transfected into HEK293 cells and the absorbance at 450 nm was measured.
The FlAsH-BRET assay
HEK293 cells were seeded in six-well plates after transfection with GPR97-FlAsH with Nluc inserted in a specific N-terminal site. Before the BRET assay, HEK293 cells were starved with serum for 1 h. Then cells were digested, centrifuged and resuspended in 500 μl BRET buffer (25 mM HEPES, 1 mM CaCl2, 140 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 0.37 mM NaH2PO4, 5.5 mM d-glucose and 12 mM NaHCO3). The FlAsH-EDT2 was added at a final concentration of 2.5 μM and incubated at 37 °C for 60 min. Subsequently, HEK293 cells were washed with BRET buffer and then distributed into black-wall clear-bottom 96-well plates, with approximately 100,000 cells per well. The cells were treated with a final concentration of BCM and cortisol at 10−5 to 10−11 and then coelenterazinc H was added at a final concentration of 5 μM, followed by checking the luciferase (440–480 nm) and FlAsH (525–585 nm) emissions immediately. The BRET ratio (emission enhanced yellow fluorescent protein/emission Nluc) was calculated using a Berthold Technologies Tristar 3 LB 941 spectrofluorimeter. The procedure was modified from those described previously34,55,56.
A one-way ANOVA test was performed to evaluate the statistical significance between various versions of GPR97 and their mutant in terms of expression level, potency or efficacy using GraphPad Prism. For all experiments, the standard error of the mean of the values calculated based on the data sets from three independent experiments is shown in respective figure legends.
Further information on research design is available in the Nature Research Reporting Summary linked to this paper.
A ‘Build and Retrieve’ methodology to simultaneously solve cryo-EM structures of membrane proteins
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Glycoproteomics is coming of age, thanks to advances in instrumentation, experimental methodologies and computational search algorithms.
Glycosylation is one of the most common post-translational modifications, and glycoproteins play crucial roles in important biological processes like cell signaling, host–pathogen interaction, immune response and disease, including cancer and even the ongoing COVID-19 pandemic (Science 369, 330–333, 2020). Glycoproteomics aims to determine the positions and identities of the complete repertoire of glycans and glycosylated proteins in a given cell or tissue.
Glycans are everywhere. High-throughput glycoproteomics approaches offer insights. Credit: Katherine Vicari, Springer Nature
Mass spectrometry (MS)-based approaches allow large-scale global analysis; however, the structural diversity of glycans and the heterogeneous nature of glycosylation sites make comprehensive analysis particularly challenging. Glycans obstruct complete fragmentation of the protein backbone, and they were traditionally removed for simplicity at the cost of losing glycan information. The MS spectra tend to be complicated due to the presence of isomers, often requiring manual interpretation. Furthermore, database searching for spectral matches can quickly become a combinatorial problem and requires innovative bioinformatics solutions.
Recent developments in MS instrumentation, fragmentation strategies (J. Proteome Res. 19, 3286–3301, 2020) and high-throughput workflows have made analyzing intact glycoproteins a possibility. Several specific enrichment strategies have made even low-abundance glycans and glycopeptides detectable (Mol. Cell. Proteomics https://doi.org/10.1074/mcp.R120.002277, 2020). A variety of experimental workflows tailored for either N-linked glycans, which are found at consensus sites on the proteins, or O-linked glycans, which have no recognizable consensus sequence, have been developed (Nature 549, 538–542, 2017; Nat. Commun. 11, 5268, 2020; Nat. Methods 16, 902–910, 2019). New software packages based on fragment-ion indexing strategies offer substantial increases in speed for glycopeptide and site assignments (Nat. Methods 17, 1125–1132, 2020; Nat. Methods 17, 1133–1138, 2020).
With other -omics fields taking the lion’s share of attention in recent years, it is now time for glycoproteomics to shine. Comprehensive understanding of glycosylation at different levels of granularity is bound to serve both basic and translational research.
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Cite this article
Singh, A. Glycoproteomics. Nat Methods 18, 28 (2021). https://doi.org/10.1038/s41592-020-01028-9
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