Herbal medicinal products that affect the β2AR system are used to treat common cold and bronchitis accompanied with mucus covered and narrowed airways. The present work compares the influence of an ivy preparation and an ivy/thyme combination on the β2-adrenergic signal transduction. For receptor binding studies and characterization of the lateral mobility of β2AR we have used single molecule detection by fluorescence correlation spectroscopy and single particle tracking. For the determination of both the second messenger cAMP and the internalization of β2AR we have generated luciferase based reporter cell lines, which produce a cAMP-dependent luciferase in the cytosol and express β2AR with extracellular luciferase moiety in the plasma membrane. While both preparations increased the β2AR binding, a significant increase of the cAMP level was observed only for the ivy preparation, which can be explained by the inhibited internalization of HiBiT-tagged β2AR under stimulating conditions. In contrast, isoprenaline-mediated internalization of HiBiT-tagged β2AR of ivy/thyme combination pre-treated cells was not inhibited. Cells comparatively pre-treated with a thyme preparation did not show inhibition of ß2AR internalization either. Furthermore, SNAP-tagged β2AR of ivy preparation pre-treated cells, which were not internalized after isoprenaline stimulation, showed a redistribution from fast-to-slowly diffusing β2AR. A corresponding redistribution of these receptors was not observed after pre-treatment with both the ivy/thyme combination and the thyme preparation. Comparable to the ivy/thyme combination, no decrease in the intratrack transitioning probability ratio (p23/p32) for fast and slow diffusing β2AR was found for the thyme preparation, which, however, significantly decreased for control cells and for pre-treatment with the ivy preparation under stimulating conditions. It can therefore be concluded that the thyme fluid extract fraction in the ivy/thyme combination may have in part a negative effect on the β2-adrenergic signal transduction.The β 2-adrenergic receptor; Signal transduction; Fluorescence correlation spectroscopy, Single particle tracking; Optics; Dose-response relationship; Drug binding; Natural product; SpectroscopyPharmaceutical science toxicology; Biochemistry; Medicine; ivy; thyme; β Elevated β2-adrenergic signal transduction enhances cAMP levels in alveolar type 2 cells leading to an increased formation of surfactant, which is secreted onto the cell surface to lower the viscosity of sticky mucus ) ivy leaves dried extract (DER 5–7.5:1) on the binding behaviour of β22.1Initially, the content of α-hederin was determined by HPLC with the result of 7.7 mg/100 ml for the ivy preparation and 8.8 mg/100 ml for the ivy/thyme combination .Figure 12.22AR and the diffusion of β2AR-ligand complexes on the surface of A549 cells were investigated using FCS and Alexa-NA (norepinephrine coupled to the fluorescent dye Alexa532) as β2AR agonist. A549 cells pre-incubated with 5 nM Alexa-NA for 15 min revealed a total binding of 1.62 ± 0.27 nM (32.4 ± 10.2%), distributed in receptor-ligand complexes (RLC) with different diffusion time constants of τdiff2 = 0.32 ± 0.10 ms (1.33 ± 0.29 nM) for free diffusing RLC in the plasma membrane and τdiff3 = 23.90 ± 16,70 ms (0.29 ± 0.12 nM) for RLC with hindered lateral diffusion behaviour (n = 70) (The binding behaviour of β(n = 70) . DiffusiA549 cells pre-treated for 24 h with both the ivy preparation and the ivy/thyme combination each in a concentration corresponding to 1 μM α-hederin revealed changes in Alexa-NA binding . Pre-inc2.32AR-ligand binding on the intracellular cAMP level, HEK293 cells expressing a luciferase fused with a cAMP binding domain (HEK293 GloSensor™ cells) were investigated under stimulating conditions using 1 μM forskolin and 1 μM isoprenaline for 20 min. Compared to stimulated control cells, pre-incubation with the ivy preparation corresponding to 1 μM α-hederin  cells was investigated using SPT. In order to monitor trajectories of β2AR, the SNAP-tag was coupled to BG-CF640R  and D2 = 0.046 μm2/s (57%) for almost immobile (diffusion state S1) and slow diffusing (diffusion state S2) β2AR, respectively, while D3 = 0.162 μm2/s (26%) was found for fast diffusing (diffusion state S3) β2AR (n = 20)  , Table 22AR frequently switched diffusion states in sequential order from S1 to S3 and backwards. Switches between S1⇄S2 and S1⇄S3 were relatively rare, while the transition from S3→S2 clearly dominated   was applominated .Figure 72AR to change the diffusion state from S2 to S3 (p23) or vice versa from S3 to S2 (p32) can be described by the ratio p23/p32 = 0.41. Stimulation with 10 μM isoprenaline for 30 min induced internalization of β2AR, reducing the number of trajectories to 78 per 1000 frames per cell compared to 263 trajectories per 1000 frames per cell in control cells. For the β2AR remaining on the cell surface, stimulation did not affect the size of the diffusion coefficients, but significantly changed their proportions with D1 = 27%, D2 = 57%, and D3 = 16% . Accordingly, the intratrack transitioning probability ratio p23/p32 halved to a value of 0.25, compared to non-stimulated control cells. Thus, HEK293-β2AR-SNAP control cells respond to the binding of isoprenaline to β2AR and relocate desensitized β2AR in coated pits, which explains the large number of immobile receptors with diffusion state S1. Hegener et al. (2004) investigated the lateral mobility of β2AR on the surface of different cell types using FCS [2AR-Alexa-NA complex was detected immediately after addition of the ligand, whereas hindered mobility was observed after a delay of 15–20 min in A549 cells. The authors conclude that receptor regulation is required to form receptor-ligand complexes with slow lateral mobility. Comparable results were obtained from isoprenaline-stimulated HEK293-β2AR-SNAP cells pre-treated with ivy preparation showing a decreasing fraction of fast diffusing β2AR with D3 and an increasing fraction of immobile β2AR with D1 and a reduction in the intratrack transitioning probability ratio p23/p32  after pre-treatment with the ivy/thyme combination  per ml, ivy preparation containing 700 mg ivy leaves dried extract  per 100 ml and fixed fluid extract combination of thyme and ivy leaves  were purchased from local pharmacy store.5.22AR were reported by our group previously [Synthesis, identity and binding behaviour of Alexa532-norepinephrine (Alexa-NA) at βeviously .5.3Synthesis and identity of BG-CF640R were reported by Bosch et al. (2014) .5.42AR with N-terminal SNAPtag . Transfection was done by calcium phosphate transfection method. Cells were seeded in 12 well plates and allowed to attach for at least 24 h. 6.5 μl of 2 M CaCl2 and 50 μl sterile water were mixed with 1 μg plasmid DNA. The mixture was added dropwise to 2x HBS buffer . After 30 min the mixture was added to the cells. The medium was changed to fresh DMEM on the next day containing 750 mg/mL G418 for selection. Individual clones were selected in cloning rings and seeded in distinct wells of a 12 well plate. The clone with best β2AR-SNAP expression was used.Human embryonic kidney cells (HEK293) obtained from DSMZ  were cultivated in DMEM medium supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal calf serum. HEK293 cells were transfected with a commercially available plasmid coding for β5.56 HEK293 cells were diluted in 100 μl of a self-made electroporation buffer  with 2 μg GloSensor™ cAMP Plasmid DNA and transfected with the program Q-001. Afterwards they were transferred to a 10 cm plate with 10 ml fresh, fully supplemented DMEM medium. After 24 h the medium was changed to fully supplemented DMEM medium containing 100 μg/ml hygromycin for selection. When they were grown to confluency cells were diluted into a 96 well plate to generate a single cell dilution. Grown colonies were tested for functionality with the assay procedure described below.HEK293 cells were transfected by Amaxa electroporation technology using Nucleofector® II. For each transfection 2 × 105.62AR was performed using the Nano-Glo® HiBiT Extracellular Detection System from Promega. For this purpose, the human β2AR was provided with an N-terminal 11 amino acids long tag  using PCR technology. HEK293 cells were transfected with the HiBiT-β2AR DNA construct by polyethyleneimine (PEI) method. Briefly, cells were seeded in 12 well plates and allowed to grow until a confluency of ~80 % was reached. Before transfection medium was changed to 900 μl fresh medium. Two μg DNA were diluted in 100 μl 150 mM NaCl. 6.6 μl of a PEI stock solution (10 mg PEI in 10 ml ddH2O) were added. After an incubation of 10 min at room temperature, 100 μl of the mixture was added to the cells. Culture vessel was centrifuged for 5 min at 280 g. After 24 h, medium was changed to fresh DMEM containing antibiotic for selection of HEK-HiBiT-β2AR positive cell clones (150 μg/ml zeocin).Internalization of the β5.74 cells/cm2 on heat-sterilized glass coverslips  and maintained in 12x multi-well chambers  at 37 °C and 5% CO2. Cells were used for FCS experiments after three to four days in culture at 80–90 % confluency.The human lung carcinoma cell line A549 obtained from DSMZ was cultivated in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 % fetal calf serum . For FCS experiments cells were seeded in a density of 1.7–3.4 × 10Human embryonic kidney cells (HEK293) obtained from DSMZ (No. ACC 305) were cultivated in DMEM medium  supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum. Cells were maintained by five-to ten-fold dilutions with fresh medium every 3–4 days.2AR (HEK293-β2AR-SNAP) were grown on PDL coated 18 mm glass cover slips in 12-well plates. Cells were cultivated at 37 °C, 5% CO2 in DMEM medium (Gibco 31885-023) containing 10% fetal calf serum, 100 units/ml penicillin and 10 μg/ml streptomycin and used for SPT experiments at 80–90% confluency.For SPT measurements, HEK293 cells overexpressing SNAP-tagged β5.83PO4)) and B (acetonitrile) were used for the following linear gradient: 0–9 min 0% B (isocratic), 9–10 min to 6% B, 10–25 min to 15% B, 25–50 min to 40% B, 50–55 min to 60% B, and 55–65 min to 100% B. Flow rate was 1 ml/min. The ivy preparation and the ivy/thyme combination were diluted with methanol in a volume ratio of 1:2. 60 μl of these solutions were injected. 7.8 mg α-hederin reference substance were dissolved in 25.00 ml methanol, of which 40 μl were injected. Assignment of α-hederin in the HPLC chromatogram was performed through comparison of UV data and retention time of the corresponding reference substance.The following instrumentation was used: HPLC pump MSDS 600 E, diode array detector 996, WISP 712 and MILLENIUM V2.1 software were form Waters . For separation a LiChrospher® 100 RP18 column  and the eluents A (distilled water/acetonitrile = 88/4 (v/v), pH = 2 (H5.9p514nm = 2.4 kW/cm2). In order to separate the fluorescence light, a dichroic filter  and a bandpass filter  were integrated into the emission light path. After passing a variable pinhole (40 μm), the intensity fluctuations were detected by an avalanche single photon counting module . Using the MATLAB Software  autocorrelation curves were evaluated with the autocorrelation function . For excitation the 514 nm line of an argon laser  was separated by an appropriated filter  and focussed through a water immersion objective  into the sample (laser power: function :(1)G(τ)=withandNj is the average number of molecules of the species j in the volume element, τjD is the diffusion time constant of the species j, τ is the correlation time, ω0 is the radius of the observation volume in the focal plane, z0 is the radius of the observation volume in the z-direction, Dj is the translational diffusion coefficient of the species j, Qj is the quantum yield factor, σj is the absorption coefficient, ηj is the fluorescence quantum yield, gj is the fluorescence detection efficiency of the species j.where 5.102 dihydrate, 1.0 mM MgCl2 hexahydrate, 3.6 mM NaHCO3, 5.0 mM HEPES, 2.0 mM D-(+)-glucose monohydrate). The coverslips were mounted on a coverslip carrier above the microscope objective with an incubation volume of 300 μL. Cells were covered with either pure Locke's solution or Locke's solution containing the pre-incubation probes as listed above. After incubating the cells with 5 nM Alexa-NA for 15 min, the confocal volume element was positioned to the upper plasma membrane of a cell by motor aided cell scanning in z-direction. At position of half maximal fluorescence of the upper plasma membrane, simultaneous detection of fast diffusing free molecules and slow diffusing ligand bound to the receptor was possible. All measurements were performed for 60 s at 20 °C and cells were used for at best 60 min.A549 cells were pre-incubated for 24 h by adding 9.74 μl/ml of the ivy preparation (corresponds to 1 μM α-hederin), 8.55 μl/ml of the ivy/thyme combination (corresponds to 1 μM α-hederin) or 1 μM α-hederin to the culture medium. A maximum of 0.1 % ethanol was added as vehicle to control cells and treated cells. Prior to measurements cells were washed three times with Locke's solution pH 7.3 . Stimulation was done with 1 μM isoprenaline and 1μM forskolin simultaneously and cAMP increase was measured as heightened luminescence 20 min after stimulation.5.122AR-SNAP cells were incubated with 2.89 μl/ml thyme preparation (corresponds to the amount of thyme fluid extract that is present in the ivy/thyme combination), 9.74 μl/ml ivy preparation or 8.55 μl/ml ivy/thyme combination for 12 h. Subsequently, SNAPtag moiety was labeled using 10 nM BG-CF640R in phenol red free DMEM medium for 10 min at 37 °C. Unbound dye was removed by washing with PBS three times. The coverslip was mounted on a coverslip carrier, overlaid with PBS and treated with 10 μM isoprenaline for 20 min. Stimulated cells as well as non-treated control cells were imaged at 20 °C using an EMCCD camera , featuring 128 × 128 pixels of size 24 × 24 μm2. The camera is part of a custom-built setup, which uses an inverted wide-field epifluorescence microscope  equipped with a water immersion objective  and a 200-mm-focal length tube lens. Combined with 4-fold magnification lens , the setup has an effective 240-fold magnification, translating to an image pixel width of 100 nm. A continuous wave laser  was used for excitation of labeled receptors. Laser intensity was regulated using an acousto-optic tunable filter (AA Opto-Electronic) and set to 0.7 kW/cm2 in the object plane. Cells were imaged in phosphate buffered saline. In order to avoid photobleaching of emitted fluorescence before image acquisition, cells were searched and focused to the epical membrane, before opening the laser shutter and starting the recording. Image sequences were acquired with 20 fps (frames per second). Automated spot detection and track creation were executed by the Track-Mate plugin featured in Fiji software package (https://fiji.sc/). Further computing of diffusion coefficients, their distributions and intratrack transitioning probabilities was done by a Matlab based variational Bayes analysis (vbSPT), which detects diffusion states and can also differentiate between multiple diffusion regimes within one trajectory [HEK293-βajectory . Althougajectory ,16. This5.132AR cells were seeded in 96 well plates in a density of 2500 cells per well and allowed to grow another three days to a confluency of ~40–50 % in full-growth medium. Before incubation, the medium was changed to serum-free medium. Cells were incubated with 9.74 μl/ml of the ivy preparation, 8.55 μl/ml of the ivy/thyme combination, and 2.89 μl/ml of the thyme preparation for 16 h. Half of the cells were then incubated with 5 μM isoprenaline for 30 min. The luminescence of HiBiT-β2AR at the cell surface was determined using a Tecan Infinite 200 Pro microplate reader and the Nano-Glo® HiBiT Extracellular Detection System  according to the manufacturers' instructions. Briefly, the HiBiT tag consists of a small part of the luciferase NanoLuc, which is N-terminally coupled to the β2AR. After addition of the LargeBiT (LgBiT), the remaining part of the NanoLuc, a binding to the HiBiT moiety takes place, resulting in a active NanoLuc, which converts added furimazine to furimamide to generate chemiluminescence. The amount of receptors that remained on the cell surface after isoprenaline stimulation and internalization was calculated from the first ten luminescence measurements over a period of 20 min as follows (HEK293-HiBit-β follows :(5)RLU. The results were considered to be significant for p-values All data points from FCS experiments, cAMP determinations, and βH. Bussmann, J. Schulte-Michels, M. Bingel, F. Meurer, S. Aataz and F. Häberlein: Performed the experiments; Analyzed and interpreted the data.S. Franken and H. Häberlein: Conceived and designed the experiments; Wrote the paper.Engelhard Arzneimittel, Niederdorfelden, Germany.This work was supported by The authors declare the following conflict of interests: Sebastian Franken is an employee of Engelhard Arzneimittel, Niederdorfelden, Germany.No additional information is available for this paper.


Poison inhibitors of DNA topoisomerase II (TOP2) are clinically used drugs that cause cancer cell death by inducing DNA damage, which mechanism of action is also associated with serious side effects such as secondary malignancy and cardiotoxicity. In contrast, TOP2 catalytic inhibitors induce limited DNA damage, have low cytotoxicity, and are effective in suppressing cancer cell proliferation. They have been sought after to be prospective anticancer therapies. Herein the discovery of new TOP2 catalytic inhibitors is described. A new druggable pocket of TOP2 protein at its DNA binding domain was used as a docking site to virtually screen ~6 million molecules from the ZINC15 library. The lead compound, T60, was characterized to be a catalytic TOP2 inhibitor that binds TOP2 protein and disrupts TOP2 from interacting with DNA, resulting in no DNA cleavage. It has low cytotoxicity, but strongly inhibits cancer cell proliferation and xenograft growth. T60 also inhibits androgen receptor activity and prostate cancer cell growth. These results indicate that T60 is a promising candidate compound that can be further developed into new anticancer drugs. Drugs targeting DNA topoisomerase II TOP2) are widely used in clinics for a variety of hematological and solid tumors , 2. TOP2 are widee.g., etoposide) stabilize the covalent TOP2–DNA cleavage complex, resulting in DNA damage that can cause cell death  such as blocking TOP2 binding to DNA ; inhibite.g., mitoxantrone and doxorubicin) that indirectly block TOP2 enzyme activity. The resultant genotoxic and cytotoxic effects are inevitably not fully related to TOP2 inhibition. Some catalytic inhibitors  are TOP2 binders but have no accurate information of ligand–protein interactions, making it challenging to perform further medicinal chemistry  campaign to screen ~6 million molecules from the ZINC15 database  against https://www.rcsb.org). It was pre-processed using the Protein Preparation Wizard build in Maestro Schrödinger software  simulation by using the Desmond simulation package of Schrödinger . An SPC ZINC compounds were purchased from Enamine, Life Chemicals Ltd, Princeton Biomolecular Research, and Vitas. T60 and T60 derivatives were synthesized by Life Chemicals Ltd. with purity >95%.The human TOP2 assay kit  and TOP2B  were used for K-DNA decatenation assays. TOP2 enzymes were incubated with 250 ng of kDNA in a reaction buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM MgCl2, 2 mM ATP, 0.5 mM DTT, and 30 mg/ml BSA in the presence of candidate compounds or etoposide and ICRF193 as control inhibitors in a final volume of 20 µl. The reaction was incubated at 37C for 2 h after which 4 µl stop solution  was added. The reaction was resolved by electrophoresis on a 0.8% agarose gel at 60 V and stained with 0.5 µg/ml ethidium bromide for 15 min. DNA bands were visualized using a gel imager (Gel Doc™ EZ System) and quantified using the Image Studio™ Lite Quantification Software.The human TOP2A and TOP2B were used to perform DNA relaxation assays. TOP2 enzyme was incubated with the supercoiled pHOT-1 plasmid in a reaction buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM MgCl2, 2 mM ATP, 0.5 mM DTT, and 30 mg/ml BSA in the presence of candidate compounds or etoposide and ICRF193 as control inhibitors in a final volume of 20 µl. The reaction was incubated at 37C for 2 h and stopped by adding 2 µl of 10% SDS and 1 µl of 250 mM of EDTA, followed by the addition of 50 µg/ml of proteinase K at 45 C for 1 h. The reaction was then resolved by electrophoresis on a 1% agarose gel at 80 V and stained with 0.5 µg/ml ethidium bromide for 15 min. DNA bands were visualized using a gel imager (Gel Doc™ EZ System) and quantified using the Image Studio™ Lite Quantification Software.® F500.Ethidium bromide displacement assay was performed as previously described , 50 usinTOP2A or TOP2B was incubated with the supercoiled pHOT-1 plasmid in a reaction buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM MgCl2, 0.5 mM DTT, and 30 mg/ml BSA in the presence of candidate compounds or etoposide and ICRF193 as control inhibitors in a final volume of 20 µl. The reactions were incubated in 37C for 30 min and stopped by adding 2 µl of 10% SDS and 1 µl of 250 mM of EDTA, followed by the addition of 50 µg/ml of proteinase K at 45C for 30 min. The reaction was then resolved by electrophoresis on a 1% agarose gel containing 0.5mg/ml ethidium bromide in TAE buffer at 60 V. DNA bands were visualized using a gel imager (Gel Doc™ EZ System) and quantified by using the Image Studio™ Lite Quantification Software.in situ with AviTag technology. The AviTag sequence (GLNDIFEAQKIEWHE) followed by a six-residue glycine serine linker (GSGSGS) was incorporated with TOP2A (431–1193). Escherichia coli BL21 containing both biotin ligase and TOP2A (431–1193) vectors were induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and biotin at 16C overnight. The bacteria were then lysed by sonication, and the resulting lysate was purified by immobilized metal ion affinity chromatography (IMAC) with nickel nitrilotriacetic acid (NiNTA) resin and cation-exchange chromatography (HiTrap SP). Purified TOP2A (431–1193) (50 μg/ml) was bound to the super-streptavidin sensors over 50 min at room temperature. The sensor was kept in an assay buffer , when small molecules were added. After 200 s, a wash buffer was used to flush the sensor plate for 100 s. BLI signal was recorded throughout the 300 s following the manufacture’s instruction from OctetRED .BLI assays were performed as we previously reported . The dirLNCaP, PC3, DU145, K562, Jurkat, THP, HeLa, H-446, NCI-H82, NT2, NCCIT, U937, HEK293T, and NCI-H69 cell lines were purchased from the American Type Culture Collection . MR49F cell line was provided by Dr. Martin Gleave . BPH-1 cell line was provided by Dr. Simon Hayward . LNCaP95 was a generous gift from Dr. Alan Meeker . NT2 and NCCIT cells were generous gifts from Dr. Leendert Looijenga (3rd) . Jurkat, H-69, K562, H-446, THP, U937, H-82, NCCIT, LNCaP, BPH-1, and MR49F were cultured in RPMI-1640 with 10% Fetal Bovine Serum (FBS). Additionally, MR49F cells were maintained in media with 10 μM enzalutamide. LNCaP95 cells were cultured in phenol-free RPMI-1640 medium with 5% charcoal stripped serum (Hyclone). HeLa, NT2, 293T, PC3, and DU145 were cultured in DMEM supplemented with 10% FBS. All cell lines used in this study tested negative for mycoplasma contamination and were authenticated by short tandem repeat assays.Cell proliferation rates were measured by using the CellTitre 96 AqueousOne kit (Promega) and bromodeoxyuridine (BrdU) assay kit (Millipore) according to the manufacturer’s protocol as we have described . CytotoxIn the thermal shift assays, cells were treated with vehicle or T60 for 1 h and split into equal aliquots to be heated at 37 to 46°. Protein lyses were collected to perform immunoblotting assays with TOP2 antibodies . Our chromatin fractionation assays followed the protocol as previously described . Briefly, K562 cells 4 × 10 –53..53.EMSA followed the standard protocols as we and others described with minor modifications –56. The Cells were fixed with 4% paraformaldehyde, treated in 0.25% Triton X-100 for 15 min, incubated with F-actin conjugated to Phalloidin-iFluor 488 , and mounted with DAPI staining mount . Cell images were captured by confocal microscopy at 63× magnification  , 53.e.g. etoposide and T60) were incorporated as powders using a mortar and pestle to reach the targeted concentrations. The density of the paste was determined to be 1.2 g/ml ( ± 3%). One million K562 cells were inoculated subcutaneously in bilateral flanks of nude mice (Nu/Nu). Once tumors reached 100–200 mm3, mice were randomly selected for a single-dose treatment with 30 µl paste containing 0.3% etoposide, 1% T60 or T633, or an equivalent volume of vehicle. Tumor volumes were measured every three days and were calculated by length × width × depth × 0.5236. Mice were euthanized when tumor volumes reached 15% of body weight or weight loss >20% occurred. The remaining mice were euthanized after the control group was euthanized. All animal procedures were under the guidelines of the Canadian Council on Animal Care and were approved by the UBC Animal Care Committee (# A19-0256).The polymeric paste was prepared with 50% poly-DL-lactide-coglycolide (PLGA) and 50% PEG 300 (w/w) as we have reported . CompounStatistical analysis was performed using the GraphPad Prism 5.01 software . Differences between the two groups were compared by student t-test. One-way ANOVA followed t-test was used to compare differences among multiple groups. The levels of significance were set at p < 0.05 as *, p < 0.01 as **, and p < 0.001 as ***.Since we hypothesized that chemicals that interfere with TOP2 from binding their DNA substrates would likely be catalytic inhibitors, we focused on the potential docking sites at the TOP2–DNA interface. We analyze the TOP2A–DNA structure 4FM9  by usingin vitro TOP2A kinetoplast DNA (K-DNA) decatenation and supercoiled plasmid relaxation assays. We found that the compound T23 to be the most potent chemical to inhibit TOP2 activities.To screen for TOP2 binders, we initiated a CADD campaign. We first filtered the ZINC15 library down to ~6 million chemicals by two criteria: 1) are purchasable, and 2) have drug-like properties determined by Lipinski’s rule of 5  Figure. The topin silico screening was then initiated using T23 as a stating scaffold for compounds with better activity profiles  to test whether T60 blocks TOP2 from binding to DNA, because the T60 docking pocket is at the TOP2–DNA interaction interface. Human TOP2A protein (aa 431–1193) was expressed from BL21plyss bacteria  inactive. The ligand potency decreases with the increased polarity of Ring 1, which is observed in compounds in the following order: T609, T60, T635, and T636. The most active compound T609 lacks N atom on Ring 1; T60 with one N atom is less active, while T635 and T636 with two N atoms are nearly inactive. The two nitrogen atoms located on the linkers between the rings form crucial hydrogen bonds with the protein residues. Replacement of –NH groups with –NMe in T627 led to a complete abolishment of T60 activity. T630, T633, and T634 showed stronger activities than T60, which could be attributed to the presence of –OH and –Me fragments that could be accommodated by an adjacent protein cavity  assays. TOP2A (431–1193) was purified  to enable sustained local releases of the drugs wo weeks , while tvia an F877L mutation in its ligand-binding domain  pathway may repair the DNA damage and rescue the cells, it leaves genomic mutations, deletions, and gene rearrangements that are often oncogenic. Examples are etoposide and teniposide, which cause mutations of the MLL gene on chromosome 11q23 and therapy-related secondary leukemia . FurtherThe suppressive effects of T60 to AR may also have clinical benefit in treating PCa. While all AR inhibitors approve by the FDA target the ligand-binding domain (LBD) of AR to prevent AR from activation, tumor cells develop therapy-resistance by either overexpressing AR or synthesizing AR splice variants or drug-resistant AR mutants to resume AR signaling. Because TOP2B is essential for all AR isoforms to initiate transcription even after they are activated and recruited to targeted promoters under antiandrogen treatment conditions, blocking TOP2B serves as a new approach to inhibit both AR and AR-driven PCa growth. We showed that AR-positive PCa cell models are particularly sensitive to T60 treatment. T60 inhibits AR activity through multiple mechanisms including i) T60 mediated TOP2B inhibition prevents AR from initiating gene transcription .Conceptualization: XD and AC. Investigation and Discussion: VM-B, MR, YS, ZA, AL, NX, and JQ. Writing: XD, AC, and MG. Funding Acquisition: XD, and AC. Resources: VS, JL, FB, and NL. Supervision: XD, AC, and MG. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This finding opens the possibility to exploit the modulation of endothelial microvilli pattern to improve the uptake of anti-oxidant particles designed to counteract ROS-mediated cerebrovascular dysfunctions.Vascular oxidative stress is considered a worsening factor in the progression of Alzheimer’s disease (AD). Increased reactive oxygen species (ROS) levels promote the accumulation of amyloid-β peptide (Aβ), one of the main hallmarks of AD. In turn, Aβ is a potent inducer of oxidative stress. In early stages of AD, the concomitant action of oxidative stress and Aβ on brain capillary endothelial cells was observed to compromise the blood–brain barrier functionality. In this context, antioxidant compounds might provide therapeutic benefits. To this aim, we investigated the antioxidant activity of cerium oxide nanoparticles (CNP) in human cerebral microvascular endothelial cells (hCMEC/D3) exposed to Aβ oligomers. Treatment with CNP (13.9 ± 0.7 nm in diameter) restored basal ROS levels in hCMEC/D3 cells, both after acute or prolonged exposure to Aβ. Moreover, we found that the extent of CNP uptake by hCMEC/D3 was +43% higher in the presence of Aβ. Scanning electron microscopy and western blot analysis suggested that changes in microvilli structures on the cell surface, under pro-oxidant stimuli (Aβ or H Alzheimer’s disease (AD) is the most prevalent form of dementia, accounting for 50–75% of all 50 million people worldwide living with dementia in 2020 . The etiThese two factors, Aβ production and ROS increase, may interact and amplify each other in a vicious cycle of toxicity, promoting endothelial inflammatory response, dysregulation of cerebral blood flow, local hypoxia/ischemia and the parenchymal and perivascular deposition of Aβ .Therefore, reducing vascular oxidative stress could slow down AD progression. The use of antioxidant compounds for the management of neurodegenerative diseases is mainly limited by poor bioavailability, enzymatic degradation and scarce BBB crossing ,15. To o3+/Ce4+) on the surface of CNP, able to scavenge superoxide anions, H2O2 and peroxynitrite. By exploiting these properties, CNP act simultaneously like the endogenous antioxidant enzymes superoxide oxidase, catalase and oxidase [In this context, cerium oxide nanoparticles (CNP) have gained increasing relevance due to the promising results obtained as ROS scavenging agents . Most in oxidase ,20. Rece oxidase ,22,23,24However, CNP efficacy against vascular oxidative stress has not been evaluated so far. Thus, the present study aimed to investigate in vitro the potentiality of CNP in controlling the Aβ-induced oxidative stress on human cerebral microvascular endothelial cells (hCMEC/D3).2O2), and can be exploited to facilitate CNP cellular uptake. These discoveries may contribute to an increase of knowledge about the Aβ-induced changes on BBB and they open the possibility to exploit microvilli-like structures to enhance the antioxidant nanoparticle delivery to the brain.The most important significance and novelty of this research resides in the identification of microvilli-like protrusions on microvascular endothelial cells that undergo structural and functional modifications after exposure to pro-oxidant agents  oxide and 1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate (DiI) fluorescent dye was obtained from Sigma–Aldrich . Polyacrylic acid (PAA) was from Polysciences Inc. . 1,1,3,3,3-hexafluoro-2-propanol (HFIP), Aβ1-40 and Aβ1-42 peptides were purchased from Sigma–Aldrich . EBM-2 basal medium was purchased from Lonza . Fetal bovine serum (FBS) was from Eurobio . Penicillin–streptomycin (P/S) solution 100× was purchased from Euroclone . Chemically defined lipid concentrate (CDLC), Hepes, basal fibroblast growth factor (bFGF), rat tail collagen type I, trypsin-EDTA solution, NuPAGE Bis-Tris (4–12%) precast gels and 4–20% Tris-Glycine gels were all supplied by Invitrogen . Hydrocortisone and ascorbic acid were from Sigma–Aldrich . MTT was purchased from Sigma–Aldrich, LDH assay kit was from ThermoFisher Scientifics. Rabbit anti-ERM and rabbit anti-pERM antibodies were supplied by Cell Signaling Technology . Mouse anti-β-actin antibody, AlexaFluor 633 phalloidin and HOECHST were from Invitrogen. Mouse anti-Aβ 1–16 antibody (clone 6E10) was purchased from BioLegend . All other chemicals were of analytical grade and were obtained from either Sigma-Aldrich  or Merck .2 glass microscope slides, respectively, and left to dry. HRTEM imaging was performed using a FEI Titan 80–300 Cube transmission electron microscope , operating at an acceleration voltage of 300 kV, equipped with a S-Twin objective lens, a FEI XFEG Schottky electron source and a 2k × 2k US1000 Gatan CCD Camera.CNP were produced by direct precipitation from aqueous solution of cerium nitride and stabilized by PAA as described previously ,25. CNP The XRD diffractograms were acquired in θ–θ mode, with a step of 0.03° 2θ and acquisition time of 20 s per step using a Bruker D8 Advance diffractometer  with a Cu anticathode (λ-Cu-Kα = 1.541838 Å) operated at 40 kV and 40 mA.Hydrodynamic diameters were obtained by a Nano ZS90 DLS apparatus . Average sizes, distribution widths, polydispersion index, and associated standard deviations (SDs) were obtained for each sample from three measurements performed on diluted solutions (1 mg/mL).v/v). DiI, as a lipophilic cation, produces a non-covalent hydrophobic interaction with PAA. The CNP suspension was centrifuged at 17,000× g for 20 min to remove free DiI in solution, and the pellet recovered in deionized water. A 10 nm-shift in the maximum of the emission spectra between DiI in DMSO and CNP  and then added under stirring to the 6 mg/mL CNP suspension  were provided by Dr. S. Bourdoulous  and used as a model of the brain capillary endothelium . Cells a4 cells/cm2) for 48 h. To assess the biocompatibility of CNP, cells were incubated with different concentrations of CNP (ranging from 50 and 500 µg/mL) in complete cell culture medium, for 3 h and 24 h. At the end of treatment, cell viability was evaluated by MTT and LDH assays as previously described [hCMEC/D3 cells were cultured on collagen-coated 96-wells plates , according to the manufacturer’s instructions. The fluorescence intensity of dichlorodihydrofluorescein , which reflects the total level of ROS/RNS within the sample, was measured with Wallac 1420 Victor2 spectrofluorometer . A standard curve was obtained using scalar concentrations of DCF.hCMEC/D3 cells were seeded on collagen-coated 35 mm dishes at a density of 3.5 × 10proteins . Differe4 cells/cm2. After 2 days, cells were incubated with Aβ oligomers (1 µM) for 1 h in the complete culture medium without FBS in order to prevent the Aβ sequestering by serum proteins [v/v) in PBS for 15 min, followed by staining of actin cytoskeleton with Phalloidin AlexaFluor 633 (1:100 in PBS) for 1 h at room temperature (RT). After washes with PBS, nuclei were counterstained with Hoescht 33342 (1:5000 in PBS) for 15 min at RT. Cells were washed with PBS and images were acquired using the Operetta CLS High Content Analysis System (Perkin Elmer) equipped with 40× water objective and standard instrument filters. Ten different fields were imaged in each well. About 400 cells for each condition were analyzed and the fluorescence intensity of CNP was measured by the Harmony analysis software (PerkinElmer). For SEM analysis, fixed samples were coated with carbon film and analyzed with a field emission scanning electron microscope  equipped with an EDAX EDS microprobe and backscattered electron (BSE) detector. The SEM was operated at an accelerating voltage of 15kV.The internalization of CNP by hCMEC/D3 cells was evaluated under basal conditions and after exposure to Aβ oligomers. Cells were seeded on rat tail collagen I-coated 96-wells Cell Carrier Ultra plates (Perkin Elmer) at a density of 3 × 10proteins . Then, 5g for 15 min at RT. Supernatant (representing free Aβ) and pellet (representing Aβ-bound to CNP) were collected and the Aβ content was determined by the ELISA kit assay .To evaluate if CNP were able to bind Aβ, 1 uM of Aβ oligomers were added to 50 μg/mL of CNP in PBS, in order to prevent the Aβ sequestering by serum proteins . Samples4 cells/cm2) for 2 days, followed by incubation with 1 μM of Aβ oligomers in the complete medium for 24 h. Then, cells were washed with cacodylate 0.05 M and fixed with glutaraldehyde 2.5% (v/v) in cacodylate for 90 min. After washing with cacodylate 0.05 M, samples were dehydrated in an ethanol gradient , followed by chemical drying with hexamethyldisilazane (HMDS). Briefly, cells were incubated with HMDS:ethanol (1:2) for 20 min, then with HMDS:ethanol (2:1) for 20 min and finally with 100% HMDS for 20 min. Fresh HDMS was added to the dishes and evaporated in a chemical fume hood overnight.hCMEC/D3 cells were grown on collagen precoated 35 mm dishes  and backscattered electrons (BSE) detectors. Microvilli length was measured from SEM-SE images by using ImageJ software. At least 10 cells and 300–400 protrusions were analyzed for each condition. For each image, the microvilli length was obtained by using the “segmented line” tool of the program and the resulting values were exported in Excel. For each condition, microvilli were subdivided, based on the frequency analysis, in three classes in relation to their length  and expressed as a percentage.To evaluate CNP binding to microvilli-like structures, cells seeded on 35 mm dishes were treated with 1 μM of Aβ oligomers in complete medium without FBS for 24 h. Then, CNP (50 µg/mL) were added to the medium and cells were incubated for 15 min at 37 °C. Samples were fixed, dehydrated and dried as described for SEM analysis. To visualize cell-surface bound CNP, samples were imaged using BSE detectors and an accelerating voltage between 5 and 10 kV.4 cells/cm2 on the apical side of Transwell inserts  precoated with rat tail collagen type I (40 μg/cm2). The apical chamber (representing the blood) was filled with 500 μL of culture medium, while the basolateral chamber (representing the brain side) was filled with 1 mL of medium. hCMEC/D3 were grown for 3 days in complete cell culture medium. After 3 days, the medium was replaced with EBM-2 supplemented with 5% FBS, 1% CDLC, 1% P/S, 10 mM Hepes, 5 µg/mL ascorbic acid, 1.4 µM hydrocortisone and 10 mM LiCl. Cells were maintained at 37 °C, 5% CO2 and the medium was changed every 2 days.The in vitro BBB model was set up as previously described, with some modifications ,33,34. h2O2 or different concentrations of Aβ oligomers (0.1–10 µM) were added to the apical compartment in complete culture medium. Otherwise, to assess if the expression of ezrin–radixin–moesin (ERM) protein complex was related to cell polarization, Aβ oligomers (0.1–10 µM) were incubated in the basolateral compartment. After 24 h, cells were washed with PBS, detached from the filter using trypsin-EDTA and centrifuged at 130× g for 5 min. The pellet was washed in cold PBS, resuspended in cold lysis buffer ) and gently rotated for 30 min at 4 °C. After centrifugation at 13,000× g for 15 min at 4 °C, the supernatant was retained and total protein content was quantified by bicinchoninic acid assay . Of the total proteins 30 µg was separated by SDS-PAGE using 4–12% NuPAGE Bis-Tris gel and blotted onto nitrocellulose membrane. After blocking with TBS-Tween 0.1% + 5% bovine serum albumin (BSA) for 1 h at RT, membranes were immunodecorated alternatively with the primary antibodies: rabbit anti-ERM (1:1000), rabbit anti-pERM (1:1000) and mouse anti-β-actin (1:1500) in TBS-Tween 0.1% + 5% BSA overnight at 4 °C. After washes with TBS-Tween 0.1%, nitrocellulose membranes were incubated with the appropriate HRP-conjugated secondary antibody goat anti-rabbit  or goat anti-mouse  diluted in TBS-Tween 0.1% + 5% BSA for 90 min at RT. Protein bands were detected with an enhanced chemiluminescence system using Amersham Imager 600 and analyzed using NIH ImageJ software. All the data have been normalized to β-actin.Six days after hCMEC/D3 seeding on the Transwell system, 0.5 mM Ht-test. For all the other experiments, data were analyzed with one-way ANOVA. Experiments were performed at least in triplicate. The analysis was performed using GraphPad Prism software. A p-value < 0.05 was considered statistically significant.Data are expressed as the mean and standard deviation (SD). For CNP uptake quantification, data were analyzed by Student’s 2 crystal structure (PDF 98-002-8709) (The CNP used in this study were obtained by direct precipitation from aqueous solution and wrapped with a coating of PAA as the protective substance . XRD ana02-8709) A. HRTEM 02-8709) B,C. The Before testing the antioxidant properties of CNP, the experimental conditions were set up in order to preserve cell viability.4 CNP was selected for all the subsequent experiments. The viability of hCMEC/D3 cells was monitored after exposure to different doses of CNP, ranging from 50 to 500 μg/mL, using two techniques that are described in Then, we evaluated the effect of Aβ oligomers, the most toxic Aβ species ,38,39, oThe cytotoxicity of Aβ oligomers was checked by the MTT assay on cultured brain vascular endothelial cells and the results showed that after 24 h of incubation with Aβ1-42 oligomers, the cell viability was not affected even at the highest dose C. ConverBased on these results, 1 µM of Aβ oligomers was selected for all the subsequent experiments in order to maintain the cell viability >60%.To test if cell incubation with 1 μM of Aβ oligomers induces oxidative stress, the production of ROS/RNS by hCMEC/D3 was evaluated by the DCF assay, whose fluorescence is proportional to the level of ROS and RNS species produced. After 4 h of incubation with 1 μM Aβ oligomers, a 2-fold higher ROS/RNS production was observed compared to the basal condition of untreated cells.2O2 production [Prolonged exposure to Aβ up to 24 h did not further increase the DCF fluorescence, suggesting that Aβ-induced oxidative stress is time-independent. These results are comparable to previous published data showing that the treatment of HUVEC cells with Aβ25-35 induces a strong increase of Hoduction . HoweverIncubation with CNP for 3 h in the presence of Aβ oligomers  leads to a significant reduction (−36% ± 6.2%) of ROS/RNS production by hCMEC/D3 cells in the tested conditions , thus suTo obtain high specificity and high resolution in the imaging of the internalized CNP, fluorescent microscopy and SEM have been combined . Images These same observations were also confirmed by SEM analysis  where CNDespite the negative surface charge, due to the presence of PAA, the CNP cellular internalization observed suggests that the electrostatic interactions only partly contribute to nanoparticle/cell interaction, as already shown for other negatively charged nanoparticles ,46.Since CNP were not surface functionalized to target BBB endothelial cells and since human endothelial cells do not possess specific receptors for CNP internalization, to gain insight into the mechanisms involved in the uptake of CNP, we explore the possibility that Aβ could act as a carrier for CNP internalization, since it is known that Aβ can be exploited to permeate the BBB . Therefo2; Aβ1-42 = 0.48 microvilli/μm2; Aβ1-40 = 0.51 microvilli/μm2), interestingly, the pattern of microvilli length was different  was assessed to confirm endothelial microvilli formation under Aβ exposure. After 24 h of incubation, hCMEC/D3 cells were collected and the cell lysates were analyzed by WB to detect ERM proteins. The results showed that the apical exposure of hCMEC/D3 cells to Aβ induced a significantly increase of pERM/ERM ratio of 1.4-fold for Aβ1-42 A and of 2O2) was tested. The addition of a sublethal dose of H2O2 to culture medium showed a strong increase of pERM levels (+130%) compared to untreated cells, thus suggesting that a change in the pattern of microvilli protrusions correlates with the oxidative stress in endothelial cells . The results showed that abluminal exposure to Aβ did not induce any significant change of pERM/ERM levels , suggestCuriously, the presence of Aβ oligomers was sometimes observed in connection with membrane protrusions by SEM imaging . It willFinally, to demonstrate that CNP uptake is mediated by microvilli, hCMEC/D3 cells were exposed to 1 μM of Aβ1-42 oligomers for 24 h, followed by short-term incubation with CNP. Cells were then observed by SEM. Images showed that CNP co-localize with endothelial microvilli formed as a consequence of the stressor  . This reTaken together, our results suggest that Aβ induces oxidative stress on BBB endothelial cells that in turn promotes changes in the architecture of endothelial microvilli. This pathway supports particles recruitment, enhancing the uptake of CNP, which can exert their antioxidant effect.Oxidative stress induces reorganization of the microvilli pattern on polarized brain capillary endothelial cells. This event fosters the internalization of antioxidant NP, which efficiently reduce ROS/RNS levels in cerebral microvascular endothelial cells.Here we demonstrated that Aβ-induced oxidative stress promotes the formation of longer microvilli on endothelial cells, which favor the interaction of CNP with the cell surface and their internalization. The length of microvilli changed with the type of Aβ peptide (Aβ1-40 > Aβ1-42) and correlated with their cytotoxicity (Aβ1-40 > Aβ1-42). Internalized CNP efficiently reduces ROS/RNS levels in cerebral microvascular endothelial cells. It is important to highlight that the effect of CNP need to be evaluated in vivo because different issues could surface such as non-specific adhesion of CNP, long term toxicity, elimination, etc.Therefore, NP with tropism for endothelial microvilli could be exploited to deliver exogenous antioxidants in a way proportional to the level of oxidative stress, thus reducing the local production of ROS and restoring the functionality of brain endothelium.


Stachel sequence. However, the low affinity of p15 limits its utility for ADGRG2 characterization. In the current study, we used alanine scanning mutagenesis to examine the critical residues responsible for p15-induced ADGRG2 activity. We next designed systematic strategies to optimize the peptide agonist of ADGRG2, using natural and unnatural amino acid substitutions. We obtained an optimized ADGRG2 Stachel peptide T1V/F3Phe(4-Me) (VPM-p15) that activated ADGRG2 with significantly improved (>2 orders of magnitude) affinity. We then characterized the residues in ADGRG2 that were important for ADGRG2 activation in response to VPM-p15 engagement, finding that the toggle switch W6.53 and residues of the ECL2 region of ADGRG2 are key determinants for VPM-p15 interactions and VPM-p15-induced Gs or arrestin signaling. Our study not only provides a useful tool to investigate the function of ADGRG2 but also offers new insights to guide further optimization of Stachel peptides to activate adhesion GPCR members.The adhesion GPCR ADGRG2, also known as GPR64, is a critical regulator of male fertility that maintains ion/pH homeostasis and CFTR coupling. The molecular basis of ADGRG2 function is poorly understood, in part because no endogenous ligands for ADGRG2 have been reported, thus limiting the tools available to interrogate ADGRG2 activity. It has been shown that ADGRG2 can be activated by a peptide, termed p15, derived from its own N-terminal region known as the  The adhesion G protein–coupled receptors (aGPCRs) are a family of 33 receptors in humans that are involved in many diverse biological processes, including brain functions, immune responses, and fertility , 9, 10. Stachel (German for stinger), within the N-terminal region of an aGPCR member, which induces receptor activation through its interaction with the 7TM domain. The tethered Stachel sequence located at the CTF plays an important role in aGPCR activation in response to different physiological stimulations, such as ligand binding, mechanostress, or removing of the NTF  (Stachel peptide p15 (sequence: TSFGILLDLSRTSLP) derived from the Stachel sequence (A) (Adhesion G protein–coupled receptor G2(ADGRG2), also known as G protein–coupled receptor 64 (GPR64) or human epididymal gene product 6 (HE6), has attracted substantial attention for its specific expression and essential functions in the male reproductive system . Recent DGRG2-β) . In our sequence , to elicsequence A  . p15 doeStachel sequence–derived synthetic peptides have a significantly low affinity to the 7TM  (VPM-p15) that activated ADGRG2 with significantly improved affinity. Furthermore, we characterized the residues in ADGRG2 that are important for ADGRG2 activation in response to VPM-p15 engagement.In contrast to other peptidergic GPCRs,  the 7TM , 26. Ford ADGRG2 . The lowStachel peptide p15 on ADGRG2. We generated an ADGRG2 mutant in which the entire ECD (including the entire GPS motif) was removed (ADGRG2-ΔGPS-β). Then, we measured the concentration-dependent p15 activities toward ADGRG2-ΔGPS-β and full-length ADGRG2(ADGRG2-FL) receptor. Increasing the amount of p15 led to a significant increase in cAMP levels, which was determined by the GloSensor assay (A). p15 also elicited a rise in the intracellular calcium level, which was determined using a CalfluxVTN Ca2+ assay. This change could be inhibited by the Gq/11 selective inhibitor YM-254890, suggesting that p15 induced the calcium influx via a Gq-dependent pathway (B). Of interest, we found through the bioluminescence resonance energy transfer (BRET) assay that p15 promoted the recruitment of β-arrestin1 and β-arrestin2 to ADGRG2, respectively (C–D). It is notable that p15 induced significantly more recruitment of β-arrestin1 or β-arrestin2 through the truncated mutant compared with the wildtype ADGRG2. Moreover, the ADGRG2-β (CTF fragment) shows a significantly higher constitutive activity for arrestin recruitment compared with that of the wildtype ADGRG2 (E–F). Because the Stachel sequence of ADGRG2-β could have more chance to access to the 7TM domain compared with that of the Stachel sequence in the ADGRG2 full length, these results indicate that the arrestin recruitment to ADGRG2 might be strengthened by the activation of ADGRG2 via Stachel sequence interaction. Taken together, these data demonstrate that the Stachel peptide p15 activates multiple signaling pathways downstream of ADGRG2.First, it is necessary to confirm the activation of or assay A. p15 al pathway B. Of intectively , C–D. Ite ADGRG2 , E–F. BeStachel peptides of ADGRG2 , p15 induced the strongest ADGRG2 activation (B). To find out which amino acid (aa) residues of p15 are required for receptor activation, we performed a systematic alanine scanning. The results showed that mutations of T1A and T12A enhanced p15 activity. Conversely, mutations of any residues from the middle of LLDL at positions ranging from +6 to +9, F3A, or R11A all greatly impaired p15 activity .We next optimized p15 by amino acid substitution, according to the following strategies: (1) diversifying the noncritical residues identified in the alanine scanning to amino acids with different properties; (2) changing critical amino acids into natural or unnatural amino acids with different structures but similar properties ; (3) enhancing the hydrophobicity of the C terminus of the  peptide A.Figure B). Next, we changed the Ser(S) at +10 position to A, V, F, L; Thr(T) at +12 position to A, V, F, L to diversify the residue properties at these positions following strategy 2. We also changed +15 position to Tyr(Me) or P15, following strategy 3. However, these p15 mutants did not exhibit enhanced signal transduction capacities compared with the wildtype p15 (D). Finally, according to strategy 2, we mutated the Phe(F) at +3 position to unnatural amino acids Tyr(Me), Phe(4-CN), Phe(4F), Phe(4-Me), F3, F3, Phe(4-NO2). Interestingly, the F3Tyr(Me), F3Phe(4-Me), and F3 mutants showed higher signal transduction activities (C). According to the results of the above studies, concentration–response curves with p15 mutants T1V, F3Tyr(Me), F3Phe(4-Me), and F3 showed a lower EC50 value than wildtype p15 in the same second messenger assay (E). To further enhance the potency of the p15 mutants, we performed combined mutations of p15 for those who showed improvement at different p15 positions. The double mutants T1V/F3 and T1V/F3Phe(4-Me) (VPM-p15) showed a significant lower EC50 than other mutants, and the effect of the mutant VPM-p15 on EC50 was most profound, with an EC50 of 1.41 ± 0.16 μM and an approximately 170-fold increase compared with the wildtype p15 peptide  at +1 position to F, W, H, R, K, D, E, V, respectively. Following strategy 1, the substitution of the polar T to a hydrophobic V at +1 position in T1V peptide induced about three times more cAMP accumulation compared with the wildtype p15, as determined by the GloSensor assay B. Next, type p15 D. Finalltivities C. Accorder assay E. To fur2+ assay and to recruit β-arrestin by BRET assay. Concentration–response curves showed that VPM-p15 enhanced the abilities of ADGRG2 to activate both Gs and Gq , in both ADGRG2-FL, ADGRG2-ΔGPS-β overexpressed HEK293 cells (A–D). Subsequently, VPM-p15 promoted the recruitment of β-arrestin1 or β-arrestin2 to the ADGRG2 truncated mutant ADGRG2-ΔGPS-β, ADGRG2-β, and ADGRG2-FL . Therefore, the optimized VPM-p15 induced both G protein and arrestin activations downstream of ADGRG2.ADGRG2 has been shown to maintain the fluid reabsorption through coupling to both Gq and β-arrestin1 . We ther93 cells , A–D. Su-FL E–H, , A–B. ThStachel sequences of the region p15 are highly conserved among different species (A). Because adhesion GPCRs are most homologous to class B GPCRs, we therefore used the PTH1R (a class B GPCR) and a Swiss-model software to generate an ADGRG2 three-dimensional coordinate model (A). According to this model, we then selected ADGRG2 mutations in extracellular loops 1, 2, 3, and transmembrane helices and then mutated them to alanine by QuikChange (A–B and A). We transfected HEK293 cells with ADGRG2-ΔGPS-β WT or its corresponding mutants and detected the expression of receptors on the cell surface by cell-surface ELISA (B). We adjusted the amount of transfected plasmid and used the mutants without significant alterations of receptor-surface expression for further delineation of the ADGRG2-VPM-p15 engagement.To provide a foundation for further optimization of the ligand that activates ADGRG2, we then investigated the molecular mechanism of the engagement of VPM-p15 with ADGRG2. Through sequence alignment, we found that ADGRG2  species A. Becauste model A. Accord A–B and A. We trace ELISA B. We adjThe binding capacities of ADGRG2 WT or its mutants for VPM-p15 were determined by BRET assay, which has been used in previous studies , 28, 29.ECL2 and I758ECL2, as well as the mutations of W8246.53 in TM6 , almostD and ECL2, I758ECL2, and W8246.53) abolished intracellular cAMP accumulation, whereas F829 and F838 enhanced cAMP accumulation, and N759 ECL2, S760 ECL2, F8266.55, F8286.57, V836ECL3, I8447.53, and F8457.54 might reduce cAMP accumulation. The other residues, including F7645.35, Y7655.36, V7685.39, and F7725.43 may regulate receptor dynamics, which are important for transducing the ligand–binding signal to downstream coupled G proteins . These results indicate that G protein and arrestin activity are mediated by at least several common residues located in the ADGRG2 7TM region.Both p15 and VPM-p15 were able to induce β-arrestin recruitment Stachel peptides varies between 7 and 18 amino acids . Consistently, a recent study reported that ΔNTF and P622 mutants of ADGRG2 interacted with β-arrestin1 and β-arrestin2, which induced constitutive internalization of ADGRG2 in steady states . Similaf ADGRG2 , 24, 25.f ADGRG2 , 35, 36.f ADGRG2 , E–F. CoStachel peptides is that they require large amounts to elicit significant activation levels in vitro with EC50 values ranging from ∼80 to 400 μM in general  diversifying noncritical residues to natural or unnatural amino acids with different properties and (2) changing critical amino acids into natural or unnatural amino acids with similar properties with different structures  and Dual-Luciferase Reporter Assay System (E1960) were purchased from Promega. Tetramethylbenzidine was purchased from Thermo Fisher. Coelenterazine 400a (FP-BB839B) was purchased from Interchim. Gq/11 selective inhibitor YM-254890 (AG-CN2-0509-M001) was purchased from Adipogen. All other reagents or chemicals were purchased from Sigma-Aldrich unless otherwise specified.Wildtype ADGRG2 full-length (ADGRG2-FL) was cloned from mouse total cDNA libraries using the following primers: forward, ATTCTCGAGGATGCTTTTCTCTGGTGGG; reverse, ATTGAATTCCATTTGCTCGATAAAGTG, and then ADGRG2-FL, ADGRG2 C-terminal truncation mutant (ADGRG2-β), and ADGRG2-ΔGPS-β (the entire ECD of ADGRG2 was deleted) were subcloned into the pcDNA3.1 expression vector. The ADGRG2-ΔGPS-β mutants  were generated using a QuikChange Mutagenesis Kit (Stratagene). All mutations were verified by DNA sequencing. All primers are listed in Stachel peptides were synthesized using standard Fmoc-chemistry on an automated peptide synthesizer MultiPep (Intavis AG). Final side chain deprotection and cleavage from the solid support was achieved by using TFA, water, and thioanisole (95:2.5:2.5vol%). Peptides were subsequently purified to >95% purity by preparative RP-HPLC (Shimadzu LC-8). Peptides were suspended in dimethyl sulfoxide and diluted to ≤4% dimethyl sulfoxide (vol/vol) in experiments.ADGRG2 Human embryonic kidney 293 (HEK293) cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, penicillin (100 IU/ml), and streptomycin (100 μg/ml) as described , 38. For2-independent medium in each well for 2 h. The cAMP signal was examined using a luminescence counter (Mithras LB 940).The Glosensor cAMP assay was performed as described , 39, 40.2+ assays, HEK293 cells in 24-well dishes were cotransfected with plasmids encoding ADGRG2 or its mutants, CalfluxVTN constructs were used at a 6:1 ratio. Empty vector pcDNA3.1(+) was used to normalize the total amount of transfected plasmid DNA. The transfection and CalfluxVTN Ca2+ assays were performed as previously reported  under a Thermo plate reader. The BRET signal was calculated as the ratio of emission of YFP (527 nm) to Rluc (370–480 nm).The ADGRG2-YFP and β-arrestin1-Rluc or β-arrestin2-Rluc plasmids were transiently transfected into HEK293 cells. Forty-eight hours after transfection, the cells were washed with PBS, detached with PBS and 5 mM EDTA, and resuspended in PBS with 0.1% (w/v) glucose at room temperature. The cells were then distributed (80 μg of protein per well) in a 96-well microplate  and incubated in the presence or absence of ADGRG2 50 values of ADGRG2 WT and its mutants for binding VPM-p15 were calculated by GraphPad, and data were normalized by EC50 of WT.In the detection of binding capacities of ADGRG2 WT or its mutants for VPM-p15, HEK293 cells were transfected with Rluc-ADGRG2-ΔGPS-β or mutants. Transfected cells were stimulated by increasing the concentration of FITC-conjugated VPM-p15. Binding capacities were determined by BRET, ECAfter transfection for 24 h, HEK293 cells were cultured at 37 °C in 24-well plates pretreated with polylysine. After another 24-h incubation, the cells were washed with Tris Buffer Saline Tween20 (TBST) once and fixed with 4% paraformaldehyde at room temperature for 10 min. Then the cells were washed three times with TBST and sealed with 5% bovine serum albumin solution for 1 h at room temperature. After three washes with TBST, the cells were incubated with primary antibody overnight at 4 °C. After another three washes with TBST, secondary antibody was added, and the cells were incubated for 1 h at room temperature. Tetramethylbenzidine was added after washing three times with TBST. After 5 to 20 min, the reaction was stopped with 0.25 M HCl solution until the color of the solution changed appropriately to blue. Then the reaction solution was transferred to a 96-well plate, and the absorbance at OD450 was measured with a microplate reader.p < 0.05. The sequence alignments were performed using T-coffee.All the data are presented as the mean ± SEM from at least three independent experiments. Statistical comparisons were performed using an ANOVA with GraphPad Prism 6.0 . Significant differences were accepted at The structure presented in this paper has been deposited in the Protein Data Bank with the following code: 6NBI. All remaining data are contained within the article.The authors declare no conflicts of interest in regards to this manuscript.


Metabolomics analysis possibly identifies new therapeutic targets in treatment resistance by measuring changes in metabolites accompanying cancer progression. We previously conducted a global metabolomics (G-Met) study of renal cell carcinoma (RCC) and identified metabolites that may be involved in sunitinib resistance in RCC. Here, we aimed to elucidate possible mechanisms of sunitinib resistance in RCC through intracellular metabolites. We established sunitinib-resistant and control RCC cell lines from tumor tissues of RCC cell (786-O)-injected mice. We also quantified characteristic metabolites identified in our G-Met study to compare intracellular metabolism between the two cell lines using liquid chromatography-mass spectrometry. The established sunitinib-resistant RCC cell line demonstrated significantly desuppressed protein kinase B (Akt) and mesenchymal-to-epithelial transition (MET) phosphorylation compared with the control RCC cell line under sunitinib exposure. Among identified metabolites, glutamine, glutamic acid, and α-KG (involved in glutamine uptake into the tricarboxylic acid (TCA) cycle for energy metabolism); fructose 6-phosphate, D-sedoheptulose 7-phosphate, and glucose 1-phosphate (involved in increased glycolysis and its intermediate metabolites); and glutathione and myoinositol (antioxidant effects) were significantly increased in the sunitinib-resistant RCC cell line. Particularly, glutamine transporter (SLC1A5) expression was significantly increased in sunitinib-resistant RCC cells compared with control cells. In this study, we demonstrated energy metabolism with glutamine uptake and glycolysis upregulation, as well as antioxidant activity, was also associated with sunitinib resistance in RCC cells. Renal cell carcinoma (RCC) is a urological malignancy, and its incidence has steadily increased annually ,2. SeverMetabolomics is a comprehensive analysis that measures the wide range of metabolites, allowing for real-time quantification of changes in cellular metabolism . Cancer We previously investigated metabolites of clear cell RCC (ccRCC) by using our global metabolomics (G-Met) protocol in tissue samples ,17. PathIn the present study, we constructed a precise quantitative measurement system using liquid chromatography-mass spectrometry (LC-MS) to evaluate intracellular metabolites of sunitinib resistance among characteristic metabolites considered to contribute to sunitinib resistance.50). IC50 values were as follows: 786-O: 4.6 µM [95% confidence interval (CI) 1.1–18.4], ACHN: 1.9 µM (95% CI 0.75–5.9), and Caki-1: 2.8 µM (95% CI 0.6–12.7). 786-O had a higher IC50 value than the other two cell lines, and only 786-O could be stably subcultured using the previously reported sunitinib-resistant cell preparation protocol  at room temperature with shaking for 60 min. Immunoreactive bands were developed using Clarity™ Western ECL Substrate , detected using the ChemiDoc™ MP Imaging System , and quantified using Image Lab software version 4.1 . In a series of western blot analyses, the same experiment was performed at least three times.Cells were lysed in radioimmunoprecipitation lysis buffer  supplemented with phosphatase inhibitor cocktail . Protein amount in lysates was quantified by the Pierce BCA protein assay kit . Then, 20 µg of protein per sample were dissolved at 95 °C in 4 × Laemmli sample buffer  and β-mercaptoethanol . Separated proteins were then transferred to polyvinylidene difluoride (PVDF) membranes  using the TRANS-BlotForty-seven metabolites, which were increased in our ccRCC tissue study, were evaluated in this study . StandarAll LC-MS/MS analyses were performed using the LCMS-8050 triple quadrupole mass spectrometer coupled with the Nexera X2 UHPLC system  and Lab Solutions software . To analyze metabolites, analytes were separated into four groups and analyzed with each optimized method for improvement of the measurement sensitivity with each metabolite. The LC method used for each compound and optimized MS/MS conditions are summarized in 4 cells/well in 6-well plates and cultured at 37 °C with 5% CO2. Groups 3 and 4 were seeded at 2 × 105 cells/dish in a 10-cm dish and cultured at 37% with 5% CO2. After 24 h, culture supernatant was removed and medium containing DMSO or 5 µM sunitinib was added. After culturing for 48 h, cells were washed twice with D-PBS and recovered with a scraper. Cell pellets were stored at −80 °C until measurement.For intracellular metabolite concentration measurement, cells were divided into four groups. In groups 1 and 2, cells were seeded at 5 × 10g and 4 °C and 100 µL of supernatant was transferred to a 1.5-mL PP tube . Supernatants were then dried using a centrifugal evaporator for 1 h and redissolved in 50 µL water for group 1 samples or 50 µL 75% acetonitrile for group 2 samples. For group 3, 25 µL IS, 200 µL water, and 1 mL acetonitrile were added to cell pellets and vortexed. Cell disruption was subsequently performed using an ultrasonic vibrator. Samples were then centrifuged for 5 min at 15,000× g at 4 °C, and supernatants were transferred to a 1.5-mL PP tube. Samples were then dried using a centrifugal evaporator and reconstituted in 20 µL water. For group 4, 140 µL 50% acetonitrile, 10 µL IS, 175 µL water, and 175 µL acetonitrile were added to cell pellets. Samples were vortexed and cell disruption was performed using an ultrasonic vibrator. Samples were then centrifuged for 5 min at 15,000× g and 4 °C and supernatants were transferred to a 1.5-mL PP tube. Approximately 50 µL supernatant were used for measurement. The obtained intracellular metabolite concentration was counted for each cell, and the concentration per 10,000 cells was calculated. Cells were cultured in triplicate, and measurements were performed three times to calculate the mean metabolite concentration.In groups 1 and 2, 100 µL internal standard substance (IS) mix and 100 µL of 50% acetonitrile were added to cell pellets, then pellets were vortexed and disrupted using an ultrasonic vibrator. Samples were then centrifuged for 5 min at 15,000× RNA was extracted from cell lysates using the acid guanidinium-phenol-chloroform method, and total RNA was reverse transcribed into cDNA using the iScript™ cDNA Synthesis Kit  following the manufacturer’s protocol . Quantitwww.cBioPortal).Information for 512 patients with ccRCC (Pan-Cancer Atlas) in the Cancer Genome Atlas (TCGA) cohort database was used for analysis. Data utilized in this paper can be accessed via cBioPortal . Statistical significance was considered to exist at


Mutations in genes that encode components of tuberous sclerosis complex 2 (TSC2) are associated with tuberous sclerosis complex disease. TSC2 interacts with tuberous sclerosis complex 1 to form a complex that negatively regulates cell growth and proliferation via the inactivation of mechanistic target of rapamycin complex 1. The activity of TSC2 is mainly regulated via posttranslational modifications such as phosphorylation. However, the control of TSC2 activity is not entirely achieved by phosphorylation. In this study, we show that TSC2 is methylated at R1457 and R1459 by protein arginine methyltransferase 1 (PRMT1). Methylation of these two residues can affect the phosphorylation status through protein kinase B (Akt) of TSC2 at T1462 and is essential for TSC2 stability. Taken together, these findings indicate that novel posttranslational modifications are important for the regulation of TSC2 stability through PRMT1-mediated methylation. TSC2). In addition to TSC2 mutations, patients with TSC exhibit loss of TSC2 protein expression and concurrent increases in cell growth and proliferation2. TSC2 is expressed in lysosomes, where it interacts with tuberous sclerosis complex 1 (TSC1) to form a complex that has Rheb GTPase-activating protein (GAP) activity that negatively regulates cell growth through the inactivation of its mechanistic target, mechanistic target of rapamycin complex 1 (mTORC1)3. Our group and other groups have recently shown that TSC2 is a multifunctional protein that can also control other pathways, such as those related to Rab54 and Rac1 6.Tuberous sclerosis complex (TSC) is an autosomal dominant disorder that is characterized by benign tumours in the brain and other vital organs and is due to mutations in the tumour suppressor gene tuberous sclerosis complex 2  is a master regulator of survival, apoptosis, and cell growth 10 that is activated by insulin and various growth factors. Akt phosphorylates TSC2 at a common recognition motif, RxRxxS/T , that is located at multiple sites, including S939 and T1462, of human TSC2 12.TSC2 activity is regulated by insulin signalling 3. For methylation in particular, protein arginine methyltransferases (PRMTs) catalyse the addition of methyl residues from S-adenosyl methionine (SAM) to guanidino nitrogen atoms of arginine residues 13. In mammalian cells, 10 PRMTs are responsible for arginine methylation and have been classified into three groups, namely, monomethylarginine (MMA: type 1), asymmetric dimethylarginine (ADMA: type 2), or symmetric dimethylarginine (SDMA: type 3) 14. Protein arginine methyltransferase 1 (PRMT1) belongs to the type 1 enzyme group, which is ubiquitously expressed and preferentially recognizes glycine-arginine rich (GAR) motifs  on a target protein. PRMT1 regulates a wide variety of cellular processes, such as transcription, DNA repair, and transcriptional regulation 16 and is also a major methyltransferase 11 that is linked to diseases such as cancer and metabolic disorders 14.In addition to phosphorylation, in general, posttranslational modifications  can regulate protein activity In this study, we report that PRMT1 methylates TSC2 at R1457 and R1459. The methylation status of the TSC2 protein inhibits phosphorylation at T1462, leading to the regulation of its stability.https://research.bioinformatics.udel.edu/iptmnet/). This search identified R653, R751, R1122, R1355, and R1459 in TSC2 as candidate methylation sites  as a methyl donor in the presence of recombinant PRMT1. Obvious methylation was observed for the peptide containing R1459 [GST-TSC2 (aa 1432–1469)]  peptide, we replaced both R1457 and R1459 with alanine. In the following methylation assays using recombinant PRMT1, the methylation levels of these mutated peptides were decreased compared to that observed for the wild-type GST-TSC2 (aa 1432–1469) peptide  peptide, an RXR motif, which has been proposed as another PRMT1 target sequence, was present in the peptide 3H]-methionine in the presence or absence of the PRMT inhibitors 18 adenosine-2′,3′-dialdehyde (Adox) and 5′-deoxy-5′-(methylthio)adenosine (MTA) before the cells were harvested, and TSC2 was immunoprecipitated. The decrease in total protein methylation was first confirmed by Adox and MTA treatments -TSC2 co-expressing Myc-PRMT1 cells were prepared, and TSC2 was immunoprecipitated using an anti-HA antibody. As shown in Fig. Thus, we identified PRMT1 as a novel binding partner of TSC2, and together with the data shown in Fig. Homo sapiens, Mus musculus, and Rattus norvegicus , which is highly conserved in many species, including 19 and eosin Y disodium trihydrate  20 , and a TSC2 peptide that was methylated at R1457 and R1459 (methyl peptide) were synthesized and subjected to an in vitro kinase assay. Recombinant Akt phosphorylated the control TSC2 peptide at T1462, whereas phosphorylation of the methyl peptide was remarkably reduced Fig. A. Moreov 20 Fig. B,C. Thes22. To determine whether the inhibition of PRMT1 prevents the lysosomal localization of TSC2, we performed immunofluorescence analyses using an anti-TSC2 antibody and a marker antibody against the lysosomal protein lysosomal-associated membrane protein 2 (LAMP2). Extensive TSC2–LAMP2 colocalization was significantly reduced by treatment with insulin or the PRMT1 inhibitor AMI-5 . PRMT1 inhibitor-induced TSC2 degradation promoted S6K phosphorylation Fig. F. CollecTherefore, these data indicate that PRMT1-mediated methylation is required for the stability of TSC2 and further affects downstream signal transduction, i.e., mTORC1 activity.3.TSC2 is a tumour suppressor protein that is responsible for the autosomal dominant disorder TSC. The activity of TSC2 is mainly controlled through phosphorylation at various sites across the protein. In addition to phosphorylation, TSC2 activity is modulated by other posttranslational modifications, such as palmitoylation, acetylation and ubiquitination 23, which is present in TSC2 at R1457-R1459. Although we observed a weak band in the TSC2 R1457A mutant peptide  is the preferred target for PRMT1, it can also target the RXR motif 3. In particular, multiple sites of the Akt phosphorylation motif (RxRxxS/T) have been identified on TSC2; however, only two of these sites are conserved and phosphorylated in Drosophila at S939 and T1462. TSC2 lacking these two conserved sites can dominantly inhibit insulin-stimulated mTORC1 activity 24. The methylation sites at R1457 and R1459 overlapped with the Akt phosphorylation site, suggesting that TSC2 methylation could affect its phosphorylation status and in turn its activity. Indeed, methylation of FOXO1 at R248 and R250 by PRMT1 has been reported to interfere with phosphorylation at S253 and control its activity 25. Our finding that methylation at TSC2 R1457 and R1459 inhibited its phosphorylation led to the hypothesis that the methylation of TSC2 interferes with Akt-dependent phosphorylation at T1462.TSC2 activity is mainly regulated through phosphorylation via Akt and 5′ AMP-activated protein kinase (AMPK) at multiple sites 26. In our report, we also observed that hypomethylation decreased the protein level of TSC2, and further analysis revealed that TSC2 phosphorylation at the T1462 site was increased. Thus, we propose here that PRMT1 methylation blocked phosphorylation via Akt at T1462. In addition to T1462, Akt also phosphorylates TSC2 at S939 12 and S981 27. A study by Cai et al. showed that phosphorylation at these sites by Akt promotes the cytosolic localization of TSC2, where 14–3–3 is able to bind 21, but they did not examine phosphorylation at T1462. We did not observe methylation of R934 and R936 (data not shown), suggesting that the Akt motif at S939 could be protected by 14–3–3 to preclude methylation by PRMT1. The physiological implications of phosphorylation of T1462 are uncertain, but our findings suggest a novel PRMT1-mediated methylation modification mechanism.Plas et al. have shown that Akt results in a decrease in the total protein levels of TSC2 and FOXO3a. In this report, they showed that the Akt-stimulated decrease in TSC2 correlated with a decrease in the protein level of its binding partner TSC1 28. Furthermore, the TSC2 protein level in cells treated with AMI-5 in the absence of CHX was significantly decreased compared with that in untreated cells  30. These findings led to the conclusion that partial loss of TSC2 protein may cause metabolic stress and influence to the cause of TSC and giving ketogenic diet to these Eker rat have also increase the tumor development 31.Our findings suggest that hypomethylation leads to TSC2 degradation. We have identified several methylation sites that are not candidates for PRMT1 but may be methylated through other PRMTs, including the putative arginine methyltransferase NADH:ubiquinone oxidoreductase complex assembly factor 7 (NDUFAF7) . showed that TSC patients with mutations of arginine residues in TSC2 have low GAP activity 33, indicating the importance of arginine residues for both GAP functions 34 and TSC2 activity. Notably, the National Institutes of Health (NIH) mutation database  includes TSC patients who carry mutations at arginine residues, including in R1457 and R1459 of TSC2, suggesting that these sites do have physiological relevance for TSC pathology.Therefore, mutations in TSC2 may lead to unstable TSC2 and therefore led to the development of TSC tumours. Indeed, Guan et al35. In general, methionine restriction (MR), which reduces the methyl donor for PRMTs, has been well studied as inhibiting tumour development in various cancers. However, our data may suggest that PRMT inhibitors or MR may activate mTORC1, leading to tumour development in some patients. Therefore, treating TSC patients with PRMT1 inhibitors may have side effects, and specific activators may be useful alternative therapeutic treatments. However, the upstream signalling pathway that activates PRMT1 is still not clear. In addition, H2O2 and Ca2+ signals have been reported to enhance the activity of PRMT1 36. Additional studies are needed to define the mechanism by which methylation at these residues could affect TSC disease pathology and whether activation or inhibition of PRMT1 could be an effective strategy to mitigate the symptoms of TSC disease.In summary, we identified a novel posttranslational modification of TSC2 in the form of PRMT1-mediated methylation. This methylation promoted the stability of TSC2 by interfering with Akt-mediated phosphorylation at T1462. Inhibition of PRMT1 is often considered the target for treating cancer cells The following primary antibodies were used and diluted in 5% BSA/TBST: anti-PRMT1 polyclonal antibody , Cat. #2449), anti-TSC2 monoclonal antibody , anti-phospho-TSC2 (T1462) monoclonal antibody , anti-β-actin monoclonal antibody , anti-Akt polyclonal antibody , anti-phospho-Akt (S473) monoclonal antibody , anti-asymmetric di-methyl arginine motif (adme-R) MultiMab Rabbit mAb mix antibody , anti-HA HA-7 monoclonal antibody , anti-FLAG M2 monoclonal antibody , and anti-Myc My3 monoclonal antibody . Anti-rabbit IgG (Cat. #A0545) and anti-mouse IgG (Cat. #I5381) were purchased from Sigma. Dulbecco’s modified Eagle’s medium  was purchased from Wako. Foetal bovine serum (FBS) was purchased from Gibco (Cat. #10270-106).Methionine deprivation media or cysteine deprivation media was purchased from the Research Institute for the Functional Peptides Co., Ltd.The PRMT1 inhibitor AMI-5 was purchased from Santa Cruz (Cat. #133410U). The nonselective PRMT inhibitors Adox and MTA were purchased from Sigma-Aldrich . CHX (Cat. #C7698), chloramphenicol (Cat. #C0378), and CL (Cat. #A48105) were all purchased from Sigma-Aldrich. The proteasome inhibitor MG132 was obtained from Funakoshi (Cat. #133407-82-6).PRMT1, PRMT2, PRMT3, and PRMT6 were inserted into pRK7-N-Myc vectors. Human TSC2 was amplified from HeLa cDNA by PCR. The primer sets used in this experiment are listed in Table EV1.Human Human embryonic kidney (HEK) 293 T cells and HeLa cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin. 293 T cells were transfected with tagged Myc-PRMT1, Myc-PRMT2, Myc-PRMT3 or Myc-PRMT6 with HA-TSC2 using Lipofectamine LTX Reagent . Samples were analysed at 48 h post-transfection.PRMT1 expression, cells were transfected with a specific shRNA as reported previously, with minor modifications 22. For lentiviral infection, pLKO.1 shRNA vectors expressing shRNAs were transfected into HEK293T cells with the lentiviral packaging plasmids psPAX2 and pMD2. Viruses were collected 48 h after transfection and concentrated via ultracentrifugation at 4 °C for 1.5 h at 23,000 rpm. Cells were infected, selected for puromycin resistance, and analysed 7 days after infection. The primer sets used in this experiment are listed in Table EV2.To knock down PRMT1 expression, HEK293T cells were treated with siRNA as described previously using a scramble control (SC) siRNA, PRMT1 #1 siRNA (siPRMT1#1), or PRMT1 #2 siRNA (siPRMT1#2), which were purchased from Thermo Fisher Scientific . Each siRNA was transfected using RNAiMAX Lipofectamine  according to the manufacturer’s instructions. Cells were treated with 50 nM siRNA for 48 h.To knock down Escherichia coli cells were transfected with GST-labelled full-length proteins and mutant plasmids, grown to OD = 0.6, and induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 30 °C for 6 h. After centrifugation, cell pellets were lysed with sonication in buffer containing 50 mM Tris (pH 8.0), 5 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 0.5% NP-40, 50 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM dithiothreitol (DTT). Cell lysates were pulled down with glutathione Sepharose (GST-tagged proteins) at 4 °C overnight. Subsequently, beads were washed three times with wash buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM EDTA, 150 mM NaCl, and 0.5% Triton X-100, and bound proteins were eluted using elution buffer containing 50 mM Tris (pH 8.0) and 6.5 mM glutathione. The eluted proteins were then dialysed against a buffer containing 10 mM Tris (pH 7.4), 50 mM NaCl, 0.5 mM EDTA, and 0.05% 2-mercaptoethanol. The primer sets used in this experiment are listed in Table EV3.DH5α competent 18. Briefly, mock or TSC2-overexpressing HEK293T cells with cycloheximide (40 µg/µL) and chloramphenicol (40 µg/µL) cultured in medium containing [3H]-SAM with or without the PRMT1 inhibitor Adox/MTA (40 µg/µL) were pre-treated with methionine-free medium and incubated in the presence of l-[methyl-3H] methionine with/without the PRMT inhibitors Adox (40 µg/µL) and MTA (40 µg/µL). Immunoprecipitated TSC2 was detected by western blot analysis and autoradiography using a BAS scanner .Autoradiography and western blot analysis of TSC2 were performed as described previously, with minor changes 25. Briefly, 15 µg of purified recombinant GST-TSC2 (aa 1432–1470) was incubated with 1 ng GST-PRMT1  or immunoprecipitated with Myc-PRMT1 in the presence or absence of [3H]SAM  in a total volume of 40 µl with phosphate-buffered saline (PBS) containing 50 mM Tris-HCl (pH 7.4), 5 mM EDTA, 150 mM NaCl, and 0.5% Triton X-100 at 30 °C for 2 h. Reaction products were resolved using Coomassie brilliant blue (CBB) staining and detected by autoradiography using a BAS-2500 scanner.In vitro methylation of TSC2 was assessed as reported previously, with minor changes 37. Briefly, 500 µM TSC2 peptides were incubated in 500 µM MgCl2 before 100 ng of recombinant Akt was added (Thermo Fisher) together with 1 µCi of [γ-32P] ATP solution (Perkin Elmer) and 25 mM ATP (Sigma) to a final volume of 25 μl in H2O. Reaction mixtures were incubated in microcentrifuge tubes at 30 °C for 20 min. Subsequently, 15 μl aliquots of the mixture were loaded onto phosphor-cellulose paper discs , which were washed three times for 20 min each in 1% phosphoric acid . Relative activities were measured using a scintillation counter . The methyl-TSC2 peptide (methyl peptide) GL(mR)P(mR)GYTISDSAPS and the unmodified TSC2 peptide (control peptide) GLRPRGYTISDSAPS were synthesized by SCRUM, Inc.In vitro kinase assays with Akt were performed as reported previously, with minor changes Whole-cell lysates were resolved by 6–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Western blotting was carried out using a horseradish peroxidase (HRP)-conjugated anti-rabbit/mouse IgG antibody as the secondary antibody, and the GE Healthcare electrochemiluminescence (ECL) System (Cat. #RPN2236) was used for detection. Immune complexes were harvested from cells that were lysed in lysis buffer, followed by centrifugation at maximum speed for 10 min to remove cell debris. Protein G Sepharose 4 Fast Flow beads  were washed in 1 × PBS and incubated with each antibody overnight. The beads were then boiled in 1 × SDS sample buffer for 5 min before resolution on SDS-PAGE gels.38. Reactions were run on an Applied Biosystems StepOne Real-Time PCR system  using SYBR Green Master Mix . Expression levels were normalized to that of GAPDH mRNA. The primer sets used in this experiment are listed in Table EV4.Total RNA was extracted from cells for reverse transcriptase (RT)-PCR analyses, as described previously Cells were seeded onto microscope slides and incubated overnight before treatment. Cells were then washed in PBS, fixed in 4% paraformaldehyde in PBS for 10 min at room temperature and permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature. Cells were then washed twice in PBS and blocked for 1 h with 2% BSA in PBS. Cells were incubated with primary antibodies at 4 °C overnight, washed three times with 2% BSA in PBS, and incubated with secondary antibodies (goat anti-rabbit Alexa Fluor 488 or goat anti-rabbit Alexa Fluor 594) in PBS for 2 h at 4 °C. After three washes with PBS, coverslips were mounted, and images were taken using a confocal fluorescence microscope .t-test. Multiple group comparisons were analysed using Tukey–Kramer tests. Error bars represent standard deviations (SDs). Significance was set at *p < 0.05 and **p < 0.01.Significant differences between two groups were identified using the unpaired Student’s Supplementary Information.