The alkaloid camptothecin analog SN38 is a potent antineoplastic agent, but cannot be used directly for clinical application due to its poor water solubility. Currently, the prodrug approach on SN38 has resulted in 3 FDA-approved cancer therapeutics, irinotecan, ONIVYDE, and Trodelvy. However, only 2–8% of irinotecan can be transformed enzymatically in vivo into the active metabolite SN38, which severely limits the drug’s efficacy. While numerous drug delivery systems have been attempted to achieve effective SN38 delivery, none have produced drug products with antitumor efficacy better than irinotecan in clinical trials. Therefore, novel approaches are urgently needed for effectively delivering SN38 to cancer cells with better efficacy and lower toxicity.Based on the unique properties of human serum albumin (HSA), we have developed a novel single protein encapsulation (SPE) technology to formulate cancer therapeutics for improving their pharmacokinetics (PK) and antitumor efficacy and reducing their side effects. Previous application of SPE technology to doxorubicin (DOX) formulation has led to a promising drug candidate SPEDOX-6 , which will undergo a human phase I clinical trial. Using the same SPE platform on SN38, we have now produced two SPESN38 complexes, SPESN38-5 and SPESN38-8. We conducted their pharmacological evaluations with respect to maximum tolerated dose, PK, and in vivo efficacy against colorectal cancer (CRC) and soft tissue sarcoma (STS) in mouse models.0-∞ of 0.05 and 4.5 nmol × h/mL for SN38 and SN38 glucuronidate (SN38G), respectively, with a surprisingly high molar ratio of SN38G:SN38 = 90:1. However, PK of SPESN38-5 by IV at 55 mg/kg yielded much higher mouse plasma AUC0-∞ of 19 and 28 nmol × h/mL for SN38 and SN38G, producing a much lower molar ratio of SN38G:SN38 = 1.5:1. Antitumor efficacy of SPESN38-5 and irinotecan (control) was evaluated against HCT-116 CRC xenograft tumors. The data indicates that SPESN38-5 by IV at 55 mg/kg is more effective in suppressing HCT-116 tumor growth with lower systemic toxicity compared to irinotecan at 50 mg/kg. Additionally, SPESN38-8 and DOX (control) by IV were evaluated in the SK-LMS-1 STS mouse model. The results show that SPESN38-8 at 33 mg/kg is highly effective for inhibiting SK-LMS-1 tumor growth with low toxicity, in contrast to DOX’s insensitivity to SK-LMS-1 with high toxicity.The lyophilized SPESN38 complexes can dissolve in aqueous media to form clear and stable solutions. Maximum tolerated dose (MTD) of SPESN38-5 is 250 mg/kg by oral route (PO) and 55 mg/kg by intravenous route (IV) in CD-1 mice. SPESN38-8 has the MTD of 45 mg/kg by IV in the same mouse model. PK of SPESN38-5 by PO at 250 mg/kg gave mouse plasma AUCSPESN38 complexes provide a water soluble SN38 formulation. SPESN38-5 and SPESN38-8 demonstrate better PK values, lower toxicity, and superior antitumor efficacy in mouse models, compared with irinotecan and DOX.The online version contains supplementary material available at 10.1186/s12967-023-04778-0. As a topoisomerase I Top1) inhibitor, the alkaloid SN38 (7-ethyl-10-hydroxy camptothecin) is one of the most potent cytotoxic camptothecins (CPTs) against cancer cells  inhibito. AlthougTo overcome these problems of using SN38 as an anticancer drug, numerous drug delivery systems, such as prodrugs, polymeric micelles, and liposome-based formulations, have been studied extensively . These aIt is well-documented that human serum albumin (HSA) is a desired drug delivery carrier –30 due t0-∞. SPESN38 also has a higher antitumor efficacy than that of irinotecan without increased toxicity. These results demonstrate SPESN38 complexes as novel effective anticancer agents with great potential for clinical applications, thereby warranting further studies to develop them into cancer therapeutics.In this study, by adopting the SPE technology, we have successfully encapsulated SN38 to create two SPESN38 complexes, SPESN38-5 (5 SN38 molecules per HSA) and SPESN38-8 (8 SN38 molecules per HSA as the maximum capacity). Preclinical evaluations using CRC and STS mouse models show that SPESN38 complexes have better PK values than those of irinotecan, resulting in 1.8-fold higher SN38 AUCHSA (25% solution) and SN38 were purchased from Octapharma USA and GLPbio Technology, respectively. Methanol, ethanol, other chemicals and suppliers were purchased from VWR. UV spectrum measurement and quantitation were conducted on a UV-1600 PC spectrometer (VWR). Both SPESN38-5 and SPESN38-5 were prepared following similar protocols for making SPEDOX-6 .Igh-1b/IcrTac-Prkdcscid, 5 to 7 weeks old were from Roswell internal breeding.All in vivo studies were performed at Roswell Park Comprehensive Cancer Center Animal Facility following the animal protocol approved by the Institutional Animal Care and Use Committee (IACUC). Male and female CD-1(ICR) mice (haired) (5 to 7 weeks old) were purchased from Charles River Lab. Severe combined immunodeficiency (SCID) mice  at doses of 300, 250 and 200 mg/kg on Day 1. For IV route, the SPESN38-5 solution was intravenously injected to mice at doses of 80 , 55 , 45  and 35 mg/kg . The percent body weight change of all mice was recorded vs days. For SPESN38-8, the same procedures were used for preparation and the resulting SPESN38-8 solution was intravenously injected to mice at doses of 45 and 35 mg/kg .2 are at the timepoints of 1, 2, 4, 8, 12, and 24 h (triplicate blood samples at each time point). Serum from each blood sample was obtained by 2,500 rpm centrifugation for 3 min, and the serum on the top layer was collected using pipet and transferred into 1.5 mL Eppendorf tubes and then frozen immediately in liquid nitrogen until PK analysis. After IV administration of SPESN38-5 (tail vein injection) was performed at the dose of 55 mg/kg to six groups , the same procedure was used for sample preparation.After PO administration of SPESN38-5 at the dose of 250 mg/kg to six groups , blood samples were collected into 1.5 mL Li-Heparin LH/1.3 tubes after anesthetizing mouse with CO10  and camptothecin , respectively] for a protein precipitation extraction, followed by centrifugation and injection of the supernatant for analysis. Chromatographic separation was achieved using a Waters CORTECS C18+ LC column  maintained at 50 ℃ and sample elution carried out at flow rate of 300 µL/min with a biphasic gradient (water with 0.1% acetic acid and acetonitrile with 0.1% acetic acid). SN-38 and SN-38G were detected by multiple reaction monitoring (MRM) using an AB SCIEX 5500 mass spectrometer with an electrospray ionization source in positive ion mode controlled by AB SCIEX Analyst® software, version 1.6.2. All sample results were obtained within one analytical run. Samples above the calibration range were diluted to be below the point of saturation of the detector.Mouse plasma samples were analyzed for SN-38 and SN-38G by LC–MS/MS using a previously described method  over themax), Area Under the plasma Concentration–time curve (AUC), elimination half-life (t1/2), apparent clearance (CL/F), and clearance (CL). AUC values were calculated using the linear-up log-down method.Non-compartmental analysis (NCA) was performed utilizing mouse plasma concentrations of SN38 and SN38-G that were obtained by LC–MS/MS. Plasma samples that were included in the NCA were collected at t = 0, 1, 2, 4, 8, 12, and 24 h post-dose for PO route and at t = 0, 0.083, 1, 2, 4, 8, and 24 h post-dose for IV route. The PK parameters were calculated using Phoenix WinNonlin software: maximum plasma concentration (C6 per injection) were suspended in 200 µL of a 1:1 solution of ice-cold PBS and Matrigel  solution. HCT-116 cancer xenograft tumors were first generated by injecting 2 × 106 cancer cells into the flank area of SCID mice. After the tumors grew to 800–1200 mm3, they were isolated, and approximately 50 mg of non-necrotic tumor masses were subcutaneously implanted into the flank area of individual female SCID mice. When the implanted xenograft tumors grew to 250 to 350 mm3 on Day 7 after tumor transplantation, mice were randomly divided into 4 groups for intravenous injection: (1) vehicle , (2) SPESN38-5 , (3) irinotecan , (4) SPESN38-5 . The intended dose for irinotecan  at 100 mg/kg was attempted on 2 SCID mice bearing HCT-116. Surprisingly, both died immediately. Other doses at 75 and 50 mg/kg were tried on healthy CD-1 mice. Both mice from 75 mg/kg IV died instantly, but 2 mice from 50 mg/kg IV were safe, which is consistent with the literature report. Therefore, irinotecan treatment group had only 6 female mice for this study. Mice in group 1 and group 2 on Day 10 were sacrificed due to the large tumor size with diameter ≥ 20 mm. One mouse from group 4 showed health issues early on and was euphanized. Tumor volume (TV) and BW were measured three times per week or daily depending on the condition of the mouse. TV was calculated using the formula: v = 0.5 (L x W2). Progression at the endpoint was a tumor size with diameter ≥ 20 mm or a moribund condition.HCT-1116 cell line (CCL-247) was purchased from ATCC. After growing in Eagle's Minimum Essential, HCT-116 cells were harvested by trypsinization and washed twice with PBS. HCT-116 cells (2 × 106 per injection) were suspended in 200 µL of a 1:1 solution of ice-cold PBS and Matrigel  solution. SK-LMS-1 cancer xenograft tumors were first generated by injecting 1 × 106 cancer cells into the flank area of SCID mice. After the tumors grew to 800–1200 mm3, they were isolated, and approximately 50 mg of non-necrotic tumor masses were subcutaneously implanted into the flank area of individual mice. Equal number (12) of female and male mice were used in this experiment. When the implanted xenograft tumors grew to 250 to 350 mm3 on Day 7 after tumor transplantation, mice were randomly divided into 6 groups for intravenous injection: (1) vehicle , (2) DOX , (3) SPESN38-8 , (4) vehicle , (5) DOX , (6) SPESN38-8 . The intended schedule for drug or vehicle treatment was weekly for 3 doses. However, mice in groups 2 and 5 with DOX at 5 mg/kg after 2 doses lost > 20% BW, indicating severe toxicity. Mice in group 1, 2, 4 and 5 on Day 9 were sacrificed due to the large tumor size with diameter ≥ 20 mm and severe BW loss (> 20%). One male mouse from group 6 had some health issues early on and was sacrificed on Day 14. Tumor volume (TV) and BW were measured three times per week or daily depending on the condition of the mouse. TV was calculated using the formula: v = 0.5 (L x W2). Progression at the endpoint was a tumor size with diameter ≥ 20 mm or a moribund condition.SK-LMS-1 cell line (HTB-88) was purchased from ATCC. After growing in Eagle's Minimum Essential, SK-LMS-1 cells were harvested by trypsinization and washed twice with PBS. SK-LMS-1 cells  for 20 min using a DAKO PT Link. With an Autostainer, the following steps and reagents were used for IHC analysis:2O2 for 15 min; (2) Incubation with 10% normal goat serum 10 min (Thermo Fisher #50062Z) 10 min; (3) Incubation with Avidin/Biotin block (Vector Labs Cat#SP-2001); (4) Incubation with primary KI67 antibody (Abcam #ab15580 or Cleaved Caspase-3 (Asp175) antibody  diluted in 1% BSA for 30 min; (5) Incubation with secondary Goat anti Rabbit (Vector labs #BA-1000) for 15 min; (6) Incubation with ABC reagent (Vector Labs Cat #PK 6100) for 30 min; (7) Incubation with DAB substrate (Dako Cat #K3467) for 5 min; (8) Counterstained with DAKO Hematoxylin for 20 s; (9) Coversliped slides.(1) Incubation in 3% HStatistic analyses of tumor volume and tumor weight change are described in Additional file.Following the successful preparation of SPEDOX-6 by the SPE technology , we madeo) of − 9.5 to − 7.3 kcal/mol, corresponding to Kd values of 0.12–4.8 µM. These binding sites show well-defined SN38 binding pockets that involve multiple non-covalent interactions  and IV routes and SPESN38-8 in IV route. In the PO route, the first dosing at 300 mg/kg was fed to two female CD-1 mice, but one mouse died in less than 72 h, indicative of exceeding MTD. The second and third doses at 250 and 200 mg/kg were evaluated , Area Under the plasma Concentration–time curve (AUC), elimination half-life (t1/2), apparent clearance (CL/F), and clearance (CL) are listed in Table 0-∞ for SN38 and SN38G are 22 and 2559 ng × h/mL, respectively. When converted into nmol, AUC0-∞ for SN38 and SN38G are 0.05 and 4.5 nmol × h/mL, with the molar ratio of SN38:SN38G = 1:90 in mouse plasma. It is surprising that most SN38 was glucuronidated to SN38G, an inactive form from SN38 metabolism. The oral bioavailability of SPESN38-5 was estimated to be only ~ 3%. However, the IV route at a dose of 55 mg/kg gave much better AUC0-∞ of 7378/15795 ng × h/mL and 19/28 nmol × h/mL for SN38 and SN38G, with SN38:SN38G = 1:1.5. Therefore, it is expected that antitumor efficacy of SPESN38-5 would be higher by IV over the PO route.A single dose of 250 mg/kg in PO route and 55 mg/kg in IV route were administered into CD-1 mice in triplicate. The total amount of SN38 and its metabolite SN38G in collected blood samples were extracted under acidic conditions and were analyzed by LC–MS/MS. Under acidic conditions, the carboxylate forms of both SN38 and SN38G would be converted into their respective lactone forms. The total mouse serum SN38 and SN38G concentration–time profiles are shown in Fig. Irinotecan, a prodrug of SN38, is the standard treatment of CRC. Therefore, we wanted to test the antitumor efficacy of SPESN38-5 in a CRC mouse model. The HCT-116 CRC xenograft model was chosen for the following reasons: (i) its low FcRn level  , (ii) it. In agreement with TV measurement, the average tumor weight in the SPESN38-5 at 200 mg/kg PO group on Day 10, irinotecan at 50 mg/kg IV group and SPESN38-5 at the 55 mg/kg IV group on Day 21 was significantly less than that in the control group on Day10. In particular, SPESN38-5 IV group significantly reduced tumor growth relative to the irinotecan group on Day 21, resulting in the average tumor weight ratio of irinotecan:SPESN38-5 = 8:1, indicating superior SPESN38-5 efficacy over irinotecan in the CRC mouse model. Furthermore, photographic images of the tumors removed at the end of experiments for each treatment group . On Day 21, 6 out of 7 mice in the SPESN38-8 treatment reached tumor-free status . On Day 9, TV change was 281, 142, and − 40% for the control group, the DOX treatment group, and the SPESN38-8 treatment group  dose is very effective in suppressing SK-LMS-1 STS tumor growth with low systemic toxicity, eliminating the implanted tumor from 6 out of 7 mice while significantly reducing the tumor size of the other Fig. . Therefo1/2 for the ring opening reaction and reach the equilibrium state with ~ 20/80% L/C forms in PBS buffer at 37 ℃. However, the presence of HSA significantly changes the kinetics and thermodynamics of the ring opening reaction, due to differential HSA binding to L/C forms of different CPTs. While HSA binds the L/C forms of CPT with a 157-fold higher affinity for the C form, SN38 binding to HSA is reversed, with 4.3-fold stronger binding for the L form. As a result, HSA increases the ring opening of CPT with decreasing t1/2 and < 0.5% L form at the equilibrium. On the contrary, SN38’s t1/2 and the L form at the equilibrium both increase substantially in the presence of HSA, from 20 to 35 min and 13 to 38%, respectively [CPTs belong to the class of TOP1 inhibitors , 47–53. ectively , 16–20. ectively –13 have ectively , 22.The current study represents the first example of developing unmodified SN38 in soluble and stable forms for in vivo antitumor evaluation. Based on the similar preparation procedure except for different SN38/HSA ratios, SPESN38-5 and SPESN38-8 are expected to display similar pharmacological properties such as MTD, PK, and antitumor efficacy. Toxicity study in mice indicated SPESN38-5 and SPESN38-8 with respective 55 and 45 mg/kg MTD, confirming their similar but not identical properties. We thus proceeded to conduct PK and different tumor model studies with either SPESN38-5 or SPESN38-8.0-∞ of SPESN38-5 at a single IV dose of 55 mg/kg . Furthermore, drug molecules are often linked to the carrier through covalent conjugation. In contrast, the SPE technology has the following unique properties: (1) The formulation process involves no chemical steps. HSA encapsulation of drug molecules are achieved through multiple non-covalent interactions between HSA and drug molecules under a specific set of conditions; (2) The binary system contains a single native HSA molecule that encapsulates a predefined number of a specific drug molecule in its unmodified form; (3) The resulting SPEDRUG complex is uniform in size (monodisperse) and has the same size of a native HSA molecule; (4) The number of drug molecules per HSA molecule may be adjusted according to specific application; (5) The HSA-drug binding strength is tunable by adjusting formulation conditions to effect PK and antitumor efficacy. The successful development of SPEDOX-6 , SPESN38Using the newly developed SPE technology, we prepared SPESN38-5 and SPESN38-8, demonstrating the first examples of unmodified SN38 in clear, stable, and injectable solution. Compared with irinotecan and DOX in animal models, SPESN38-5 and SPESN38-8 showed favorable pharmacokinetic values, superior antitumor efficacy against CRC and STS, and lower systemic toxicity. The successful development of SPEDOX-6, SPESN38-5, and SPESN38-8 has validated the SPE platform in drug formulation. These SPEDRUG complexes represent a new uniform macromolecular nanodrug that may be used to target low FcRn expressing cancer cells, further improving their antitumor efficacy while reducing side effect toxicities. These promising preclinical results have prompted these SPEDRUG complexes to be aggressively pursued for their clinical applications.Additional file 1: Fig. S1. Tumor volume of different treatment groups on Day 10. SPESN38-5 by IV was more efficacious than clinical drug irinotecan, while PO route was not effective. Fig. S2. Tumor volume comparison on Day 21 (ending day of experiments). SPESN38-5 by IV was significantly efficacious than irinotecan. Fig. S3. Tumor weigh comparison of different treatment groups on ending days. The mice from the control and PO groups had to been sacrificed on Day 10 due to the large tumor size. Table S1. Summary of antitumor efficacy of irinotecan and SPESN38-5 Against HCT-116. Table S2. Summary of antitumor efficacy of DOX and SPESN38-8 against SK-LMS-1. Table S3. Summary of PK parameters for irinotecan at 200 mg/kg from literature. Table S4. Summary of tumor volume change for all groups on Day 10. Table S5. Summary of tumor volume change for all groups on Day 21. Table S6. Summary of tumor wight for all groups on the ending days. Table S7. Summary of tumor volume by day and group. Table S8. Summary of tumor volume change from Day 0 by different groups and days. Table S9. Result summary of Tukey-Kramer adjusted pairwise tests. Table S10. Tumor weight summary on their ending days


Gene deletion is one of the standard approaches in genetics to investigate the roles and functions of target genes. However, the influence of gene deletion on cellular phenotypes is usually analyzed sometime after the gene deletion was introduced. Such lags from gene deletion to phenotype evaluation could select only the fittest fraction of gene-deleted cells and hinder the detection of potentially diverse phenotypic consequences. Therefore, dynamic aspects of gene deletion, such as real-time propagation and compensation of deletion effects on cellular phenotypes, still need to be explored. To resolve this issue, we have recently introduced a new method that combines a photoactivatable Cre recombination system and microfluidic single-cell observation. This method enables us to induce gene deletion at desired timings in single bacterial cells and to monitor their dynamics for prolonged periods. Here, we detail the protocol for estimating the fractions of gene-deleted cells based on a batch-culture assay. The duration of blue light exposure significantly affects the fractions of gene-deleted cells. Therefore, gene-deleted and non-deleted cells can coexist in a cellular population by adjusting the duration of blue light exposure. Single-cell observations under such illumination conditions allow the comparison of temporal dynamics between gene-deleted and non-deleted cells and unravel phenotypic dynamics provoked by gene deletion. Escherichia coli that has been used to infer the roles of genes in various biological phenomena, such as cellular growth and morphogenesis  system allows us to induce gene deletion at desired timings by blue light exposure . The PA-nce gene . We obseThe duration of blue light illumination significantly influences the fraction of gene-deleted cells in the population. Therefore, by adjusting the illumination duration, we can simultaneously observe the dynamics of gene-deleted cells and non-deleted cells in a cell population. Since the target chloramphenicol resistance gene is tagged with the gene for the fluorescent protein mCherry, the fractions of gene-deleted cells are measurable simply by fluorescent microscopy. Here, we describe the protocol for measuring the fraction of gene-deleted cells based on batch-culture assay.Glass test tube 16.5φ × 165L Molton cap 1.5 mL microtube ® syringe filter MinisartSterilized petri dish φ90 × 15 mm Aluminum foilE. coli strain, carrying a fluorescently tagged target gene flanked with two loxP sequences and photoactivatable Cre (PA-Cre) system. Here, we used YK0083, which has a chloramphenicol resistance gene, cat, as a target gene for demonstration. The mCherry-tagged cat gene sandwiched with two loxP sequences was introduced into the intC locus on the genome. The genes for the PA-Cre system, creN-nmag and pmag-creC, are placed downstream of PLlacO1 promoter on a plasmid. These genes are inducible by IPTG. This strain is available by contacting the authors.TM LB broth, Miller (Luria-Bertani) DifcoAgar TM M9 minimal salts, 5× DifcoD(+)-glucose 4·7H2O MgSO2·2H2O CaClMEM amino acids solution (50×) Ampicillin sodium Isopropyl-β-D(-)-thiogalactopyranoside (IPTG) ® water produced by Milli-Q® Integral 3 Elix® water produced by Milli-Q® Integral 3 Milli-QLB broth (see Recipes)LB agar containing 100 μg/mL of ampicillin (Amp) (see Recipes)M9 medium with 0.2% glucose and amino acids (see Recipes)1 M IPTG (see Recipes)50 mg/mL Amp (see Recipes)Centrifuge Spectrometer Blue light illuminator BioShaker Incubator Stereomicroscope Portable photodiode-based laser power meter Sample preparationE. coli cells in LB broth with 50 μg/mL of Amp at 37 °C with shaking overnight.Culture g for 1 min at room temperature.Centrifuge 100 μL of the overnight culture at 21,500× Remove the supernatant.Resuspend the cellular pellet in 1 mL of M9 medium with 0.2% glucose and amino acids.600 of the resuspended cells.Measure the OD600 to 0.001 in 2 mL of M9 medium with 0.2% glucose and amino acids containing 50 μg/mL of Amp and 0.1 mM IPTG.Adjust the ODCover the test tube with the aluminum foil.Cultivate the cells at 37 °C with shaking for 3 h.Gene deletion inductionRemove the aluminum foil after the cultivation.Expose the test tube to blue light using blue-light illuminator (light intensity: 6.8 mW) for the desired duration 25 g of Difco® water1 L of ElixAutoclave at 121 °C for 15 min.Store at room temperature.At the time of use, add 50 mg/mL Amp on a clean bench as appropriate, if needed.LB agar containing 100 μg/mL of AmpMix the following:TM LB broth, Miller (Luria-Bertani)25 g of Difco15 g of agar® water1 L of ElixAutoclave at 121 °C for 15 min.Add 2 mL of 50 mg/mL Amp on a clean bench.Dispense 25 mL in each 90 mm dish on a clean bench.Store at 4 °C.M9 medium with 0.2% glucose and amino acidsPrepare the following reagents:5× M9TM M9 minimal salts, 5× in 1 L of Milli-Q® water.1) Dissolve 56.4 g of Difco2) Autoclave at 121 °C for 15 min.3) Store at room temperature.20% glucose® water to 50 mL.1) Dissolve 10 g of D(+)-glucose in Milli-Q2) Sterilize using a 0.2 μm pore filter.3) Store at 4 °C.41 M MgSO4·7H2O in Milli-Q® water to 50 mL.1) Dissolve 12.3 g of MgSO2) Sterilize using a 0.2 μm pore filter.3) Store at 4 °C.21 M CaCl2·2H2O in Milli-Q® water to 50 mL.1) Dissolve 7.4 g of CaCl2) Sterilize using a 0.2 μm pore filter.3) Store at 4 °C.Sterilized water® water at 121 °C for 15 min.1) Autoclave Milli-Q2) Store at room temperature.Mix the following on a clean bench:200 mL of 5× M910 mL of 20% glucose10 mL of MEM amino acids solution (50×)42 mL of 1 M MgSO2100 μL of 1 M CaCl778 mL of sterilized waterStore at 4 °C.At the time of use, add antibiotics or IPTG on a clean bench as appropriate, if needed.1 M IPTG® water to 10 mL.Dissolve 2.383 g of IPTG in Milli-QSterilize using a 0.2 μm pore filter.Dispense in microtubes on a clean bench.Store dispensed tubes at -20 °C.50 mg/mL Amp® water.Dissolve 500 mg of ampicillin sodium in 10 mL of Milli-QSterilize using a 0.2 μm pore filter.Dispense in microtubes on a clean bench.Store dispensed tubes at -20 °C.


Pedersen, Snoberger et al. measure the force sensitivity of the yeast endocytic myosin-1 called Myo5 and find that it is more likely to generate power than to serve as a force-sensitive anchor in cells. Implications for Myo5’s role in clathrin-mediated endocytosis are discussed. Saccharomyces cerevisiae endocytic type I myosin called Myo5, a motor whose role in clathrin-mediated endocytosis has been meticulously studied in vivo. We report that Myo5 is a low-duty-ratio motor that is activated ∼10-fold by phosphorylation and that its working stroke and actin-detachment kinetics are relatively force-insensitive. Strikingly, the in vitro mechanochemistry of Myo5 is more like that of cardiac myosin than that of slow anchoring myosin-1s found on endosomal membranes. We, therefore, propose that Myo5 generates power to augment actin assembly-based forces during endocytosis in cells.Myosins are required for clathrin-mediated endocytosis, but their precise molecular roles in this process are not known. This is, in part, because the biophysical properties of the relevant motors have not been investigated. Myosins have diverse mechanochemical activities, ranging from powerful contractility against mechanical loads to force-sensitive anchoring. To better understand the essential molecular contribution of myosin to endocytosis, we studied the in vitro force-dependent kinetics of the  Saccharomyces cerevisiae and Schizosaccharomyces pombe, turgor pressure opposes plasma membrane invagination, so actin is required at every CME site (During clathrin-mediated endocytosis (CME), the plasma membrane invaginates and undergoes scission to become a cytoplasmic vesicle. Coat proteins like clathrin can deform membranes under low tension , but wheCME site .S. cerevisiae . Myo5 is insensitive to resistive force compared to related myosins. We, therefore, propose that Myo5 actively powers CME. Because actin and myosin collaborate in a variety of membrane remodeling processes, we expect that these results will be instructive beyond CME.S. cerevisiae  was well fit by:We measured the steady-state actin-activated ATPase activities of phosphorylated and unphosphorylated Myo5 using the NADH-coupled assay  in the pM actin. . PhosphoM actin. . The actKATPase) was determined to be 5.1 ± 0.88 µM, and the maximum ATPase rate (Vmax) was found to be 3.3 ± 0.15 s–1  and ATP binding to (K1′ and k+2′) actoMyo5.Resistive force impacts the rate of myosin detachment from actin and two biochemical transitions, ADP release and subsequent ATP binding, determine the detachment rate. Therefore, we used stopped-flow kinetics to measure ADP release from , k+5′ anWe found that yeast Myo5 does not quench the fluorescence of actin labeled at cys-374 with pyrene iodoacetamide, which is the probe most used to measure myosin binding . Thus, wTo determine the rate constant for ATP binding, we mixed nucleotide-free actoMyo5 (100 nM) with varying concentrations of ATP and monitored 90° light scattering. Time courses followed single exponential functions . For phoK1′ is a rapid equilibrium binding step, k2′ is a rate-limiting isomerization to the AM.ATP state, and kdiss is the rapid actin dissociation step. The apparent second-order rate constant for ATP binding to phosphorylated actoMyo5 was determined by a linear fit to the data (K1′k2′ = 0.39 ± 0.017 µm–1 s–1). The unphosphorylated actoMyo5 data were fit byk2′ = 290 ± 24 s–1) and ATP affinity (K1′ = 0.006 ± 0.0016 µM–1) were determined. The apparent second-order rate constant for ATP binding (K1′k2′) was determined from a linear fit of the observed rates below 100 µM ATP to be 1.1 ± 0.28 µM–1 s–1 (The mechanism was modeled as in µM–1 s–1 .k+5′) was measured by preincubating 100 µM ADP with 200 nM actoMyo5 and then rapidly mixing with 2.5 mM ATP, as shown in The rate constant for ADP dissociation (k+5′ = 74 ± 2.0 s–1) and for unphosphorylated actoMyo5  and since we have measured the overall ATPase rate (Vmax = 3.3 ± 0.15 s–1), we can estimate the duty ratio as follows:Our results suggest that both phosphorylated and unphosphorylated Myo5 have low duty ratios . Since ADP release limits the rate of phosphorylated Myo5 detachment from actin at saturating ATP .6 antibody. Coverslips were incubated with a range of concentrations of phosphorylated and unphosphorylated Myo5, creating a titration series of surface densities. At low Myo5 surface densities , actin filaments failed to bind the coverslip  of the time-forward average was limited by the covariance smoothing window, but at 10 and 1,000 µM ATP . In this condition, an initial substep of 4.8 nm was followed by a second substep of 0.2 nm . We deteectively , which a−1 at 1 and 10 µM ATP, respectively (−1) was limited by the covariance smoothing window  is the detachment rate at force F, k0 is the detachment rate in the absence of load, d is the distance parameter (the distance to the force-dependent transition state and a measure of force sensitivity), kB is Boltzmann’s constant, and T is the temperature. Best fit parameters for k0 and d were determined by MLE of the unaveraged data from −1, in agreement with the measured detachment rate under low load conditions at 1,000 µM ATP  is most like that of β-cardiac (muscle) myosin (d = 1.3 nm), suggesting that it is well-suited for generating power. The difference between Myo5 and acutely force-sensitive Myo1b, a tension-sensitive anchor myosin (d = 15 nm), is dramatic. From 0 to 2 pN of resistance, Myo1b attachment lifetimes slow from ∼600 ms to ∼45 s, resulting in negligible power generation . Steady-state, transient, and single molecule experiments were performed at 20°C in KMg25 buffer . Apyrase VII was obtained from Sigma-Aldrich. The purity and concentration of purified proteins were determined by comparing in-gel Coomassie blue staining to staining of known amounts of bovine serum albumin (Pierce).ATP concentrations were determined spectrophotometrically after each experiment by absorbance at 259 nm, ∈filtered . Actin cS. cerevisiae calmodulin gene CMD1 was cloned from genomic DNA into a bacterial expression plasmid with a sequence encoding His6-TEV situated at the 5′ end to generate pDD2743. pDD2743 was transformed into Rosetta E. coli, optimized for expression (Novagen). A saturated overnight culture in LB  was used to inoculate a 1-L culture in LB to OD600 = 0.1. Cells were grown to OD600 = 0.6–1, induced with 0.5 mM IPTG for 5 h at 37°C, pelleted at 4,225 × g for 20 min at 4°C in a Sorvall SLA-3000 (fixed angle) rotor, washed with cold 20 mM HEPES pH 7.5, and re-pelleted at 2,250 × g for 10 min at 4°C in a Jouan CR3i (swinging bucket) centrifuge. Cell pellets were flash-frozen in 45 ml lysis buffer . Upon thawing, cells were lysed by sonication, 2 mg DNase I (Roche) and Triton X-100 to 1% were added, and the resulting lysate was incubated on ice for 30 min, then spun at 92,000× g for 25 min in a Beckman Type 70 Ti rotor. The supernatant was loaded onto a 1-ml HisTrap HP column  pre-equilibrated with binding buffer . The column was washed with 20 ml binding buffer, and Cmd1 was eluted using a 30 ml linear gradient from 0–100% elution buffer . Fractions containing Cmd1 were pooled, Cmd1 was cleaved from His6 with TEV protease, and dialyzed overnight at 4°C into a low salt buffer . Following dialysis, purified, cleaved Cmd1 was bound to a MonoQ column and eluted using a 10 ml linear gradient from 0–70% high salt buffer . Fractions containing Cmd1 were pooled, dialyzed into KMg50 buffer , and stored at −80°C.The S. cerevisiae. The MYO5 open reading frame (ORF) from S. cerevisiae was cloned from genomic DNA and truncated at Gly763, generating a construct containing the motor domain and both Cmd1-binding IQ motifs of the lever arm. The SHE4 ORF was cloned in its entirety from S. cerevisiae genomic DNA. Both ORFs were ligated into a 2-µm expression plasmid with a partially defective LEU2 gene (leu2d) to ensure a high copy number, creating plasmid pDD2744  were used to inoculate 1.5 liters of cultures in the same media with raffinose substituted for glucose to OD600 = 0.1. After 18 h of growth at 30°C, cultures were induced with 2% galactose, Bacto yeast extract was added to 10 g/L, and Bacto peptone to 20 g/L. After 8 h of expression, the cells were harvested at 4,225 × g for 20 min at 4°C in a Sorvall SLA-3000 rotor, washed with 25 ml cold Milli-Q water, re-pelleted at 2,250 × g for 10 min at 4°C in a Jouan CR3i centrifuge, resuspended in 0.2 vol of cold Milli-Q water, and drop frozen into liquid nitrogen. Lysis was achieved through cryomilling (10 cycles of 3 min grinding with one min cooldown) in the large vials of a 6870 freezer/mill (SPEX Sample Prep).pDD2744 was transformed into D1074 yeast . Saturat2, 2 mM ATP, 20 mM imidazole, 5 mM DTT) supplemented with 1 mM PMSF, 1 × cOmplete protease inhibitor cocktail without EDTA (Roche), and 1 µM Cmd1. For purification of phosphorylated Myo5, 1 μg Pak1 , and Myo5 was eluted using a 20 ml linear gradient from 0-100% elution buffer (wash buffer with 1 M imidazole).Cell powders were thawed in binding buffer  overnight at 4°C and again into 500 ml of the same buffer for 2 h at 4°C and then stored at −20°C.Fractions containing Myo5 were pooled and supplemented with Cmd1 to 1 µM. For unphosphorylated Myo5 purification, a further 20,000 units of lambda phosphatase were added along with MnCl2, 2 mM ATP in KMg25) and the other syringe contained the mixture of actin (0–80 µM) and myosin (100 nM) in KMg25. The concentrations above are after mixing. After mixing, the concentration of NADH loss due to ATP hydrolysis was monitored by absorbance at 340 nm , and the linear regions of the curve were fitted to a straight line to determine ATPase activity.Steady-state actin-activated ATPase activity was measured using the NADH enzyme-linked assay in an Applied Photophysics SX.18 MV stopped-flow apparatus . In one ATP-induced dissociation of actoMyo5 was measured and analyzed as described . Briefly6 (Sigma-Aldrich), made with 0.2 mg/ml in motility buffer , was first added to the flow chamber and incubated there for 5 min to coat the nitrocellulose-coated coverslip with the antibody. The flow chamber was then blocked for 2 min with 2 mg/ml casein. Blocking coverslips with bovine serum albumin (BSA) led to inferior gliding. Phosphorylated or unphosphorylated Myo5 in motility buffer with 2 mg/ml casein, diluted to a range of concentrations as indicated in Motility assays were carried out essentially as in Flow chambers for optical trapping were constructed with double-sided tape and vacuum grease as previously described . Briefly−1; Sigma-Aldrich) at 15,000 × g for 1 min and adding 2 μl of catalase supernatant to 20 μl of 19.1 U/μl glucose oxidase (Sigma-Aldrich).Trapping buffer (KMg25 with 1 mM DTT freshly added) was used for all trapping assays. A 100× stock of glucose oxidase + catalase (GOC) was freshly prepared by centrifuging catalase  was flowed in the chamber and incubated between 30 s and 3 min and then immediately blocked with 2, 3-min incubations of 1–2 mg/ml BSA. Stocks of phosphorylated His9-tagged Myo5 were diluted to 1 nM in trapping buffer with 300 mM added KCl and incubated in the flow cell for 2 min. The number of myosins bound to the surface was limited by the surface concentration of anti-His6 antibody and the incubation time of anti-His6 antibody was adjusted daily between 30 s and 3 min such that one of the three to five pedestals tested showed clear myosin interactions with the actin dumbbell.0.01 mg/ml anti-HisFollowing incubation with Myo5, a second blocking step with two 3-min incubations of 1–2 mg/ml BSA was performed. The final buffer added to the flow cell contained trapping buffer with the indicated amount of ATP, 1 μl of GOC added immediately prior to addition to the chamber, and 0.1–0.25 nM rabbit skeletal muscle actin polymerized with 15% biotinylated actin (Cytoskeleton) stabilized by rhodamine-phalloidin (Sigma-Aldrich) at a 1.1–1.2 molar ratio with G-actin concentration. Neutravidin-coated beads were prepared by incubating 0.4 ng of 0.8 μm diameter polystyrene beads (Polysciences) and coated with 5 mg/ml neutravidin (Thermo Fisher Scientific). 3 μl of neutravidin-coated beads were added to one side of the chamber prior to sealing. All trapping data were acquired within 90 min of the addition of the final buffer to the chamber.Optical trapping experiments were performed at room temperature (20 ± 1°C) using a dual beam 1,064-nm trapping laser as previously described , 2018. AIndividual 0.8-μm-diameter neutravidin-coated beads were caught in the two traps and held ∼5 μm apart. Trap stiffnesses were adjusted to 0.05–0.1 pN/nm for each trap. A biotinylated actin filament visualized by rhodamine-phalloidin was bound to the two trapped beads, creating a bead-actin-bead dumbbell. The dumbbell was pretensioned (3–5 pN) by steering one beam using a piezo-controlled mirror conjugate to the back focal plane of the objective, and the surface of the pedestal beads was probed for myosins. Putative myosin interactions were detected via drops in the variance of the two beads, and the three-dimensional position of the dumbbell relative to the myosin was refined further by maximizing the rate and size of the observed power stroke deflections. Every 30–60 s, the dumbbell was moved axially along the actin filament in ∼6-nm steps between trace acquisition to ensure even accessibility of actin-attachment target zones. Stage drift was corrected via a feedback system using a nano-positioning stage and imaging the position of the pedestal bead with nm precision . In expeIsometric optical clamping experiments were performed as previously described  using a Actomyosin interactions for non-isometric optical clamping experiments were detected using the single-molecule computational tool SPASM (Software for Precise Analysis of Single Molecules, Events detected in isometric optical clamping experiments were detected as described in  using a Review HistoryClick here for additional data file.SourceData F2is the source file for Fig. 2.Click here for additional data file.


BRAF non-V600 mutation occupies a relatively small but critical subset in colorectal cancer (CRC). However, little is known about the biological functions and impacts of BRAF class III mutation in CRC. Here, we aim to explore how D594A mutation impacts on biological behaviors and immune related signatures in murine CRC cells.BRAF V600E (class I), G469V (class II) and D594A (class III) mutant cell lines were established based on MC38 cells. The biological behaviors of cells were evaluated in respect of cell growth, cell proliferation, cell apoptosis, cell migration and invasion by the methods of colony-forming assay, CCK-8 assay, Annexin V/PI staining and transwell assay. The concentrations of soluble cytokines were detected by ELISA. The membrane expression of immuno-modulatory molecules and the pattern of tumor infiltrating lymphocyte were evaluated by flow cytometry. The molecular mechanism was explored by RNA sequencing. Immunohistochemistry (IHC) staining was used for the detection of CD8α in tumor tissues. qRT-PCR and western blot were performed to assess the mRNA and protein expression. Anti-PD-L1 treatment and cytokines neutralization experiments were conducted in in vivo models.+ T cells was significantly high within D594A mutant tumors, which may provide potential response to anti-PD-L1 therapy. RNA sequencing analysis showed that D594A mutation led to enhanced expression of ATF3 and THBS1, which thus facilitated CXCL9 and CXCL10 production upon IFN-γ treatment. In addition, CXCL9 or CXCL10 neutralization reduced the infiltration of CD8+ T cells into THBS1-overexpressing tumors.D594A mutant cells displayed lower grade malignancy characteristics than V600E (class I) and G469V (class II) mutant cells. Meanwhile, D594A mutation led to evident immuno-modulatory features including upregulation of MHC Class I and PD-L1. In vivo experiments displayed that the frequency of infiltrated CD8+ T cells infiltration into the microenvironment of D594A mutant CRC. Our present study is helpful to define this mutation in CRC and provide important insights in designing effective immunotherapeutic strategies in clinic.D594A mutant CRC exhibited lower aggressiveness and immune-activated phenotype. ATF3-THBS1-CXCL9/CXCL10 axis mediated functional CD8The online version contains supplementary material available at 10.1186/s12967-023-04606-5. Mitogen-activated protein kinase (MAPK) cascade is involved in the progression of most human cancers, and mutations of RAS-RAF-MEK-ERK are frequently occurred. BRAF is an oncogenic kinase and contains diverse mutations in approximately 10% colorectal cancer (CRC) .Among the distinct BRAF mutations, the proportion of non-V600 (class II and class III) was approximately 22–30%, and V600 mutation is predominantly possessed in BRAF mutant CRC , 3. Furt+ T cells, the main player of anti-tumor immunity and producers of interferon gamma (IFN-γ), is highly relevant for predicting the therapeutic response to anti-PD-1/PD-L1 therapy [Recently, immunotherapy emerged to represent the promising treatment modality for advanced CRC patients, including immune checkpoint inhibitors (ICIs), which acts largely within the tumor immune microenvironment (TIME) , 6. Inte therapy , 9.It is becoming increasingly clear that V600E mutation possessed multiple malignancy properties  and poor+ T cells recruitment and anti-PD-L1 sensitivity. Our work expands the understanding of the biological role and immune related signatures of BRAF D594A mutation in CRC and provides important insights in designing effective immunotherapeutic strategies in clinic.In this study, we established three mutant MC38 cell lines possessed V600E (class I), G469V (class II) and D594A (class III) mutation, respectively. We showed that D594A mutant cells were less aggressive and displayed increased expression of immuno-modulatory molecules. Moreover, D594A mutation enhanced the secretion of CXCL9 and CXCL10 via ATF3-THBS1 axis, which was associated with CD8BRAF gene in MC38 cells was knockout by lenti-CRISPR Cas9 technique. The BRAF WT and mutant plasmids were purchased from Shanghai Jikai Gene Chemical Technology Co., LTD. Briefly, the WT BRAF (NM_004333) gene sequence was synthesized by whole gene synthesis technique. Sequences of V600E (T1799A), G469V (G1406T) and D594A (A1781C) were obtained by point mutation technique. The recombinant plasmids were transfected into BRAF knockout MC38 cell lines respectively according to the instructions . Neomycin was added into medium to obtain positive clones .Endogenous 2.MC38 cell line was purchased from ATCC (American Type Culture Collection). The cell line was verified by Short Tandem Repeat (STR) profiling . Mycoplasma were detected routinely. MC38 cells were cultured in DMEM  medium contained 10% fetal bovine serum (FBS) . The medium also supplemented with 100 U/mL penicillin  and 100 μg/mL streptomycin . Cells were maintained in a humidified atmosphere at 37 °C with 5% CO−△Ct.Cells were digested with trypsin  and washed using PBS. RNA was extracted with TRIzol reagent , and reversed transcription into cDNA according to manufacturer's instructions . Primers were designed using Primer-BLAST (PubMed) and synthesized from Genewiz Corporation (China). qRT-PCR reactions were performed in triplicate . The expression was normalized using β-actin . The relative expression of genes was calculated by 2The protein extraction, concentration measurement and imaging were performed referring to the method described before . Rabbit Exponential growth phase of BRAF WT and mutant MC38 cells were collected. 1,000 cells were seeded in 35 mm dish. The culture medium was changed every 3–4 days. After 10–12 days, clones were washed by PBS gently and fixed using 4% paraformaldehyde for 15 min at room temperature. After that, clones were washed again and stained by 0.1% crystal violet reagent for 20 min. The dishes were washed gently with PBS until the background was clean. Inverted microscope was used to count and photograph.Cell viability was evaluated by CCK-8 assay . Cells (2000 cells/well) were seeded into 96-well plate and incubated at 37 °C for 24, 48 and 72 h, respectively. Four replicates were performed. 10 µL CCK-8 reagent was added to each well. After incubation at 37 °C for 2 h, the OD values at 450 nm of each well was measured. The data was analyzed by GraphPad Prism 8.0.Cells  were seeded into 6-well plate and cultured for 24 h. The trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) reagent  was added to induce cell apoptosis for 24 h. All the cells were collected and stained by the APC-Annexin V Binding Apoptosis assay kit  according to the manufacturer's protocol. The proportion of apoptotic cells was analyzed by flow cytometry.Transwell chamber  was used in the migration and invasion assay. Before the invasion assay, 100 µL diluted matrigel was added into the upper chamber. Cells were resuspended in DMEM medium (without FBS) at the concentration of 100,000 cells/100 µL or 300,000 cells/100 µL and added to the upper chamber. DMEM medium (600 µL) supplemented with 15% FBS was added in the lower chamber of each well. After incubation for 24 h, cells on the upper chamber were fixed using 4% paraformaldehyde for 15 min. The upper chamber was washed twice with PBS, and stained with 0.1% crystal violet for 20 min. Cells on the upper surface of the filter were wiped by cotton swabs. Cells passing through the transwell membrane were observed and photographed under an inverted microscope.Tumor cells in exponential growth stage were collected. 4,000 cells were seeded into 96-well plate and incubated overnight. On the second day, Sorafenib, Trametinib and SCH772984  were serially diluted to the concentrations as 100 µM, 80 µM, 60 µM, 40 µM, 20 µM, respectively, and were added into the wells of each group. Three replicates were performed. The cells were treated with the inhibitors for 24 h, 48 h and 72 h, respectively. The OD values at 450 nm were obtained using CCK-8 method. IC50 values were calculated and the sensitivity of different mutant cell models to MAPK inhibitors were analyzed.Cells  were incubated in 6-well plate overnight. The culture medium was discarded. The plate was washed gently by pre-warmed PBS. The cells were starved by culturing in FBS-free DMEM medium. After 36 h, the medium in the wells were replaced by 2 mL DMEM medium containing 100 ng/mL EGF  for 15 min at 37 °C. The whole protein was extracted immediately using RIPA. The control group was avoided contacting with FBS throughout the operation.Cells  were incubated in 6-well plate. 20 ng/mL IFN-γ was added in the culture medium on the second day and treated for 48 h. The supernatant of tumor cells was collected and centrifuged at 4˚C, 4,000 rpm for 10 min to discard the cell debris. The concentrations of CXCL9  and CXCL10  were detected using ELISA method follow the manufacturer's instructions. The OD values at 450 nm and 570 nm were detected. The standard curve was drawn and the concentrations of CXCL9 and CXCL10 were calculated.Tumor cells  were seeded in 6-well plate and incubated overnight. The culture medium was replaced by fresh complete medium containing 20 ng/mL IFN-γ. The tumor cells were harvested 48 h later and washed by PBS. Anti-mouse-PD-L1 antibody  and anti-mouse-MHC Class I antibody  were diluted using FACS buffer (PBS containing 10% FBS) at the ratio of 1:200. The cells were resuspended by the dye solution and stained for 25 min on ice. After washing with FACS buffer, cells were resuspended by FACS buffer and filtered by nylon membrane. The single cell suspension was analyzed by BD LSRFortessa™ Cell Analyzer . The data was processed using flowjo Vx 10.0 software.+ T cells, TNF-α, IFN-γ, PD-L1 and TIM3 were detected by flow cytometry.The supernatant of tumor cells was collected as above. Lymph nodes from OT-1 mice were grinded, and T cells were collected to be activated with IL-2 and N4-peptide for three days. Live T cells were collected by density gradient centrifugation. 500,000 T cells were incubated in medium containing equal volume of T cells culture medium and tumor supernatant. Three replicates were performed. After co-culture for 48 h, the expression of functional markers of CD8PD-1  and TIM3  were detected as above. For the detection of secretory TNF-α and IFN-γ, T cells were firstly incubated in culture medium containing PMA (5 ng/mL), Ionomycin (500 ng/mL), Brefeldin A  and Monensin  for 4 h at 37 °C. T cells were collected and resuspended by FACS buffer. Fixable viability dye efluor 506  was added for staining for 25 min on ice. Then T cells were washed and resuspended in 1 mL freshly prepared Fix/Perm solution (BD Biosciences) for 20 min at 4 °C. After being washed with Perm/Wash buffer (BD Biosciences), T cells were stained with anti-TNF-α  and anti-IFN-γ  for 25 min, washed and filtered. All samples run on a flow cytometer and analyzed using flowjo V× 10.0 software.2)/2. All animal procedures were conducted in accordance with the guidelines of the Animal Care and Welfare Committee of Nanjing medical university.C57BL/6 and BALB/C Nude female mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and Cavens Biogle Model Animal Research Co.,Ltd. (China), respectively. BRAF mutant and WT cell lines at exponential growth stage were prepared and resuspended by PBS. 1,000,000 cells were injected subcutaneously on the right flank of the mice. Tumor volumes were measured every 2–3 days. The long diameter (L) and short diameter (W) of the tumors were measured, and tumor volume was calculated as: volume =  in a wet box at 4 °C overnight. The slices were washed with PBS for 4 times and incubated with secondary antibody at 37 °C for 15 min. The color reaction was performed using DAB Substrate kit . The slices were observed and photographed under an inverted microscope. The positive cells were counted and analyzed by GraphPad Prism 8.0.BRAF WT and D594A mutant MC38 cells were harvested at exponential growth stage. Total RNA was isolated using Trizol reagent. RNA quality and integrity were confirmed via an Agilent Bioanalyzer 2100. Then, mRNA libraries were established and sequenced at Genewiz Corporation (China).THBS1 mRNA expression using the following primers: forward primer, 5′- GGGGAGATAACGGTGTGTTTG-′3, and reverse primer, 5′- CGGGGATCAGGTTGGCATT-3′. Western blot was used to detect the THBS1 protein expression in cells.THBS1-overexpressing plasmid (pMSCV-THBS1) was constructed based on pMSCV-PIG  by homologous recombination technique . Briefly, the sequence of THBS1 was obtained by cloning the MC38 cDNA template using the following primers; forward primer, 5′-gccggaattagatctctcgagATGGGGCTGGCCTGGGGACTA-′3 and reverse primer, 5′- gtagaattcgttaacctcgagTTAATGGTGATGGTGATGATGGGGATCTCTACATTCGTATTTCAGGT-′3, and recombined to linearized pMSCV-PIG at XhoI restriction site. THBS1-overexpressing (hTHBS1) and control (hctrl) cell lines were established by transfecting either pMSCV-THBS1 plasmid or pMSCV-PIG into MC38 cells using jetPRIME reagents . qRT-PCR was used to determine The shRNA sequences targeting THBS1 were as following; sense: CCGGTGAAACCGATTTCC GACAATTCTCGAGAATTGTCGGAAATCGGTTTCATTTTTGA; anti-sense: ATTCAAAAATGAAACCGATTTCCGACAATTCTCGAGAATTGTCGGAAATCGGTTTCA. THBS1-knockdown plasmid (pLKO.1-shTHBS1) was constructed based on pLKO.1  by homologous recombination technique. THBS1-knockdown (siTHBS1) and control (sictrl) cell lines were established by transfecting either pLKO.1-shTHBS1 or pLKO.1 plasmid into D594A mutant MC38 cells using jetPRIME reagents . Cells were maintained in a culture medium containing 400 μg/mL neomycin  for two weeks. qRT-PCR and western blot were used to determine the mRNA and protein levels of THBS1.+ T cells were resuspended in DMEM medium at the concentration of 500,000 cells/120 µL and added into the upper chamber. Culture supernatant (600 µL) from tumor cells was added to the lower chamber of each well. 0.1 µg/mL CXCL9 and CXCL10 neutralizing antibody were used. After incubation for 3 h, cells that migrated through the filter was counted and photographed under an inverted microscope. Three replicates were performed.Transwell chamber  was used in the T cell migration assay. CD8hTHBS1 and hctrl cells were injected subcutaneously into C57BL/6 mice respectively. The mice injected hTHBS1 cells were divided into three groups randomly. Two experimental groups were given 5 µg/100 µL CXCL9 and CXCL10 neutralizing antibody by injecting intraperitoneally, respectively. The remaining group was given 100 µL PBS. The injection was performed on day 8, 11 and 14 after tumor transplantation. The tumor growth curve was drawn and the tumors were weighed. TILs were analyzed by flow cytometry.t-test. P < 0.05 was considered to indicate a statistically difference. All statistical analyses were two-sided and performed by GraphPad Prism 8.0. Figures were drawn using GraphPad Prism 8.0.All experiments were repeated at least three times. Data were expressed as the mean ± standard deviation. The differences between two groups were analyzed by non-parametric To investigate the potential biological impacts of distinct BRAF mutations on CRC cells, BRAF V600E (class I), G469V (class II), D594A (class III) mutant and WT MC38 stable cell lines were established by virus infection. As shown by western blot, no significant differences in protein expression of BRAF were observed among the four established cells Fig. A. The muWe further examined the impacts of different BRAF mutations on cell growth. Compared to WT cells, cells harboring V600E mutation exhibited stronger growth ability, but the growth rate was not altered with D594A mutation, suggesting that D594A mutation may have little effects on tumor proliferation or progression Fig. C. Next, In short, CRC cells with distinct classes of BRAF mutation present biological behaviors with different malignancy degree. Overall, the D594A mutation was demonstrated to display relatively weaker expansion and metastasis capacity than V600E and G469V mutations.PD-L1 was also the highest in D594A among the four classified cells upon IFN-γ treatment  class I and II, PD-L1 and PD-L2. MHC class II and PD-L2 were not expressed in MC38 cells in our previous and other’s studies . We thusPsmb8  between D594A and WT cells were analyzed by RNA sequencing (RNA-seq). Compared to WT, 540 genes were upregulated, and 658 genes downregulated in D594A  in its promoter. To further explore the potential transcriptional factors mediating the upregulation of THBS1, we made a prediction using an online tool (http://jaspar.genereg.net), which showed that THBS1 can be regulated by numerous transcriptional factors, among which, qRT-PCR verified that ATF3 expressed excessively higher in D594A -expressing CD8To further investigate the role of THBS1 in the secretion of CXCL9/CXCL10, we established THBS1-overexpressing cell line  mutation had hyper-activated BRAF kinase, which was dramatically reduced in D594A (class III) mutation. It is well-known that MAPK signaling directly participates into cell proliferation  and apopWe found D594A was relatively sensitive to BRAFi and MEKi, established distinct biological performance from BRAF V600E and WT CRC. BRAF inhibitor (BRAFi) alone display limited clinical benefit in CRC patients with BRAF V600E mutation . A combiIn this study, we observed that IFN-γ had dual effects on MC38 cells . On the + T cells exert the tumor eradication through the production of cytotoxic molecules, including perforin, granzyme, IFN-γ and TNF-α [+ T cells retained better effector function when co-cultured with supernatant from D594A mutant cells. In vivo experiments displayed a higher frequency of CD8+ T cells in D594A tumors, with lower expression of TIM3 and PD-1, which are the co-inhibitory molecules and indicating terminally exhaustion of CD8+ T cells. These findings suggest that effective suppression of D594A mutant tumor rely largely on the infiltration and function of CD8+ T cells in the TIME.CD8nd TNF-α . We foun+ T cells, we performed RNA-seq and identified THBS1 as an important candidate related to chemotaxis. THBS1 (thrombospondins 1) is a multifunctional matricellular protein and was first described as an angiogenesis inhibitor in 1990. The role of THBS1 in CRC is multifaceted and controversial. Maeda et al. thought that THBS1 was a negative-regulator of tumor angiogenesis and correlated with good prognosis [. proved that THBS1 was significantly correlated with liver metastasis and poor prognosis of CRC [+ T cell adhesion and chemotaxis by binding to α4β1 integrin [+ T cells had been reported rarely. But THBS1 knockdown indirectly increased CD8+ TILs by increasing tumor vascularization in triple-negative breast cancer [To further explore the mechanism driving the infiltration of CD8rognosis , while Ls of CRC . In the integrin . THBS1 eintegrin . THBS1 aintegrin . Howevert cancer .THBS1 by binding to its promoter. ATF3 has also been proved to drive the transcription of PD-L1 and can be used as a biomarker for predicting the efficacy of PD-1 checkpoint blockade therapy [ATF3 and PD-L1 were the highest in D594A in our study. However, the correlation of ATF3 with the expression of THBS1 haven’t been explored further.We found that THBS1 is highly expressed in cells with D594A mutation. Dual-luciferase reporter system validated that ATF3 could regulate the expression of  therapy . Consist+ T cells was largely driven by the CXCL9/CXCL10-CXCR3 axis [+ T cells via CXCL9 and CXCL10. CXCL10 not only recruits CD8+ T cells, but also regulates its differentiation during chronic infection [+ T cells in this study. However, the molecular mechanism underlying the improved CD8+ T cell function in the TIME of D594A tumor needs further in-depth investigations.It is well recognized that the accumulation of CD8CR3 axis . We founCR3 axis . We furtnfection , while C+ T cells, D594A mutant CRC responded to anti-PD-L1 treatment efficiently. We surprisingly found that the infiltrated CD8+ T cells in D594A tumor expressing lower PD-1 and TIM3 after anti-PD-1 treatment, which predicted a better prognosis to some extent. Collectively, the effector function of CD8+ T cells may be more essential compared to its abundance, but the infiltration of CD8+ T cells was indeed more abundant in D594A mutant CRC anyway. D594A mutant CRC may be classified as “immunological tolerance type” in the CMS system, which is the ideal type to respond to immune therapy [. proposed that PD-L1 expression was associated with CD8+ T cell infiltration in Melanoma. Blocking PD-L1/PD-1 pathway may benefit patients with PD-L1+ tumors [PD-L1 is a promising drug target in cancer therapy. PD-L1 inhibitors including atezolizumab, avelumab and durvalumab were recently approved by United States Food and Drug Administration (FDA) for treating diverse types of cancer , 37. MC3 therapy . Taube e+ tumors .However, MC38 is an MSI-H model and may not accurately reflect the impacts of BRAF D594A mutation in MSS CRC . In addi+ T cells was, at least in part, mediated by ATF3-THBS1-CXCL9/CXCL10 axis in the TIME of D594A.In total, D594A mutation exhibits lower aggressiveness among the three mutant types. Tumors with D594A mutation can be defined as “hot tumor” , and theAdditional file 1: Fig. S1.A Validation of mutation sites in MC38 cells with different BRAF mutations by sequencing. V600E, T1799A; G469V, G1406T; and D594A, A1781C. B Quantification of the bands intensity towards the blots in Fig. C Detection of the sensitivity of BRAF WT and mutant cells to MAPK inhibitors. Sorafenib, BRAF inhibitor; Trametinib, MEK1/2 inhibitor; SCH772984, ERK1/2 inhibitor. Three independent replicates were performed for above experiments.Additional file 2: Fig. S2. Effects of different BRAF mutations on MC38 tumor growth in BALB/C nude mice (n = 8 mice per group). The tumor sizes, growth curves and weights of the tumors were shown as (A–C), respectively. Actin serves as an internal reference. *P < 0.05; **P < 0.01.Additional file 3: Fig. S3. The mRNA levels of representative chemotaxis genes, THBS1, B2M, CCL9, IFLH1, C3, CPL3A1, HFE and IL4RA in different mutant cells analyzed by qRT-PCR. Three independent replicates were performed. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.Additional file 4: Fig. S4. Establishment of THBS1 overexpressing and knockdown cells. A, B The validation of THBS1-overexpression on mRNA and protein levels by qRT-PCR and western blot, respectively. hTHBS1 and hctrl cells were established based on MC38 cells. C, D The validation of THBS1-knockdown on mRNA and protein levels by qRT-PCR and western blot, respectively. siTHBS1 and sictrl cells were established based on BRAF D594A mutant MC38 cells. *P < 0.05; ****P < 0.0001.


Although many reports have elucidated the function of HCAR2 and its potential as a therapeutic target for treating not only dyslipidemia but also neuroimmune disorders such as multiple sclerosis and Parkinson’s disease, the structural basis of ligand recognition and ligand-induced Gi-coupling remains unclear. Here we report three cryo-EM structures of the human HCAR2–Gi signaling complex, each bound with different ligands: niacin, acipimox or GSK256073. All three agonists are held in a deep pocket lined by residues that are not conserved in HCAR1 and HCAR3. A distinct hairpin loop at the HCAR2 N-terminus and extra-cellular loop 2 (ECL2) completely enclose the ligand. These structures also reveal the agonist-induced conformational changes propagated to the G-protein-coupling interface during activation. Collectively, the structures presented here are expected to help in the design of ligands specific for HCAR2, leading to new drugs for the treatment of various diseases such as dyslipidemia and inflammation.Hydroxycarboxylic acid receptors  transduce G Niacin is used to treat cardiovascular disease through its activation of the GPCR HCAR2. Here, the authors present cryo-EM structures of HCAR2 bound to niacin and other drug-like small molecules, which explain the basis of HCAR2 ligand recognition. HCAR2 and HCAR3 share 95% sequence identity, differing mainly in the ligand pocket and intracellular C-terminal tail. HCAR2 responds to niacin3  as well as other ketone body-containing metabolites such as beta-hydroxybutyrate (BHB)4. In contrast, HCAR1 and HCAR3 respond to different metabolites such as lactic acid or 3-hydroxyoctanoic acid, respectively6. Niacin has been used for decades as an antiatherogenic drug that is also able to decrease plasma levels of low-density lipoprotein (LDL). Its ability to reduce the production of certain proinflammatory cytokines and macrophage chemotaxis is mediated by HCAR27. Since HCAR2 was believed for many years to help lower the plasma levels of LDL, drug development campaigns focused on the receptor to develop new treatments for dyslipidemia9. More recently, HCAR2 was shown to mediate neuroprotective effects and inflammatory response regulation, as well as playing a role in the suppression of colorectal cancer13. HCAR2-knockout mice show a greater susceptibility to colonic inflammation and colon cancer12. Although conceived initially as a drug target for the control of lipid metabolism, HCAR2 therefore has potential as a route to novel treatments of a wide range of inflammatory diseases, as well as atherosclerosis, multiple sclerosis and Parkinson’s disease, and as a result it is of growing interest for pharmaceutical applications.Humans and other higher primates possess three highly similar G-protein-coupled receptors (GPCRs) in the hydroxy-carboxylic acid (HCA) receptor family, which consists of HCAR1, HCAR2, and HCAR3, also known as GPR81, GPR109A, and GPR109B, respectively15. Several synthetic agonists of HCAR2 have been developed with a view to lowering plasma LDL concentrations, including the niacin derivatives acipimox16, acifran17 and MK-035418, as well as novel compounds such as GSK2560738 and MK-190319. In 2003, acipimox was approved by the Food and Drug Administration for the treatment of hyperlipoproteinemia. Although acipimox showed higher efficacy against dyslipidemia than niacin, it still caused the side-effect of flushing. Potent HCAR2 agonists were developed after the commercialization of acipimox, but only acipimox and acifran proved able to deliver the desired lipid profile. Although many studies have indicated that HCAR2 is involved in various inflammatory disorders and dyslipidemia, the signaling mechanism of HCAR2 is still unknown at the molecular level. To assist in the understanding of HCAR2 activation and the development of novel HCAR2 agonists, we have determined the structure of the niacin-bound signaling complex by single particle cryogenic electron microscopy (cryo-EM).Despite the usefulness of niacin in the treatment of atherosclerosis, patient compliance is greatly weakened by cutaneous flushing, caused by HCAR2 triggering the release of vasodilatory prostaglandins from Langerhans cells and keratinocytesi heterotrimer  and recruitment of β-arrestin1 . The NanoBiT assays confirmed that the BRIL-HCAR2 construct fully retained the Gi and β-arrestin1 responses to all three ligands when compared with the expression-matched wild-type HCAR2  in the presence of agonists   and C177ECL2. The presence of P168ECL2 and a beta hairpin loop further reduce the flexibility of ECL2  Fig. . Three dts) Fig. . Cys18N-CL2 Fig. . These sCL2 Fig. . As withCL2 Fig. . Within  21 Fig. . The modECL1 presses against the ligands, helping to hold them in a stable pose  F2777.36 form stacking interactions with the ligands  reduced Gi activity and β-arrestin1 recruitment, probably by steric hindrance to ligand binding  reduces Gi activity and β-arrestin1 recruitment, possibly by allowing greater ligand mobility, so that the W91ECL1S mutation has a smaller impact on signaling by GSK256073 than by the smaller ligands  of niacin for the L1073.32F mutant is similar to that of the wild-type, but the maximum response (Emax) of this mutant is less than that of the wild-type, both for dissociation of the Gi heterotrimer and also recruitment of β-arrestin1. These results imply that L1073.32 plays a more crucial role in HCAR2 activation rather than niacin binding . Molecular docking of MK-1903 provides an insight into its interactions with HCAR2 , and W91ECL1 is replaced with serine in HCAR3, it is possible that some selectivity is achieved through the replacement of S17845.51 with isoleucine. Active-state HCAR3 modeling with AlphaFold222 suggests that isoleucine at this position in HCAR3 leads to a conformational change in ECL2 that causes a clash with MK-1903 27 and the P2Y purinoceptor 1 (P2Y1) bound to an antagonist (PDB:4XNV)28. P2Y1 has the highest sequence homology to HCAR2 of all proteins in the PDB. In comparison to the models of inactive HCAR2 and P2Y1, the Gi-coupled HCAR2 structures adopt an active form, with a significant shift of the extracellular end of TM2, and the marked outward movement of TM6 typical of class A GPCRs  in HCAR2. Agonist binding causes rotamer conformational changes that are relatively modest at the toggle switch, but which gradually increase in the surrounding residues including the PIF, N/DPxxY and DRY motifs . This aspartate residue, together with D732.50, S1143.39, N2867.45 and S2877.46, form the putative sodium ion binding site which is generally important for activation by agonists34. Structural comparison shows rearrangements of N2867.45, S2877.46 and D2907.49 residues which may involve displacements of water molecules and the sodium ion during the activation of typical class A GPCRs ifs Fig. . The outTM3 Fig. . In the .50 Fig. , which iCRs Fig. . The sid.50 Fig. , and hasues Fig. . HCAR2 uund Fig. . This coi coupled class A GPCRs have now been reported, allowing their general features to be studied36. To allow binding to the Gα protein C-terminal α5 helix, a cytoplasmic cavity  is opened by movement of TM6 towards the cell interior. The HCAR2-Gi complex structures show very similar Gi interfaces to other class A GPCRs. The HCAR2-Gi complex is stabilized by hydrophobic interactions between TM3 (V1293.54), TM5 and TM6  and apolar side-chains of Gαi, such as I344G.H5.16 (Gα numbering is shown as superscripts), L348G.H5.20, L353G.H5.25 and Phe354G.H5.26  has recently been reported to play a ‘toggle switch’ role, allowing signaling biased towards either the G-protein or β-arrestin1 downstream pathway, and the size of the ligand binding to CCR1 affects signal bias39. Biased signaling of GPCRs can occur not only through orthosteric agonists but also by allosteric modulators41. In a recently published study, the allosteric modulator of HCAR2 Compound 9n42 showed a Gi protein-biased allosteric effect, and specific residues involved were identified43.In this work, we determined the structures of niacin, acipimox and GSK256073-bound HCAR2 coupled to the GECL2 of HCAR2, for example, is replaced by leucine in HCAR1 and HCAR3. The three human proteins in this family each possess some unique residues forming the orthosteric binding site, and these residues, such as W91ECL1 and S17845.51, could be exploited to design ligands specific for particular HCAR subtypes  cells and synthesized by Integrated DNA Technologies. The gene was cloned into a modified pFastBac HT-B vector containing N-terminal haemagglutinin (HA) signal sequence, Flag-tag (DYKDDDD), 10 × His tag and HRC3V protease cleavage site. HCAR2 was expressed in Sf9 insect cells using a Bac-to-bac expression system. Cells were infected at a density of 2 × 106 cells per ml and incubated at 27°C for 60–72 h till harvest. To purify membrane fractions, harvested cells were disrupted in a hypotonic buffer  and subsequently in a high osmotic buffer  in the presence of protease inhibitor cocktail (Roche). Purified membranes were solubilized at 4°C for 2 h in a buffer containing 50 mM HEPES pH 8.0, 500 mM NaCl, 10% glycerol, 1% (w/v) n-dodecyl-beta-D-maltopyranoside , 0.2% (w/v) cholesterol hemisuccinate  with 100 μM of ligands. Insoluble debris was removed by ultracentrifugation in 264,902 g at 4°C for 40 min, and the supernatant was incubated with TALON® metal affinity resin (Clontech) overnight at 4°C. The resin was washed three times with 10 column volumes washing buffer I containing 50 mM HEPES pH 8.0, 500 mM NaCl, 10% glycerol, 20 mM IMD, 0.05% DDM, 0.01% CHS, 100 μM ligand and then washing buffer II containing 50 mM HEPES pH 8.0, 500 mM NaCl, 10% Glycerol, 20 mM IMD, 0.05% LMNG, 0.005% CHS, 0.01% DDM, 0.002% CHS, 100 μM ligand and then another 10 column volumes of washing buffer III containing 50 mM HEPES pH 8.0, 500 mM NaCl, 10% Glycerol, 20 mM IMD, 0.05% LMNG, 0.005% CHS, 100 μM ligand. The protein was then eluted using the washing buffer III supplemented with 350 mM imidazole. Eluted protein was concentrated and subjected to PD-10 Desalting Column (Cytiva). To cleave the Flag-tag and 10 × His tag, HRV3C protease was treated to purified protein. After reverse column work, fractions containing HCAR2 were pooled and subjected to size-exclusion chromatography on a Superdex 200 Increase 10/300 GL column (Cytiva) pre-equilibrated in 50 mM HEPES pH 8.0, 100 mM NaCl, 1 mM MgCl2, 0.5 mM TCEP, 0.05% LMNG, 0.005% CHS in presence 100 μM ligand. The monomeric fractions were collected and concentrated for receptor-G protein complex formation.A coding sequence was designed to express full-length human HCAR2 (UniProtKB-Q8TDS4) carrying thermostabilized apocytochrome b562RIL (BRIL) at the N-terminus. The DNA sequence was codon optimized for expression in i1, Gβ1 and Gγ2 was designed and purified as the same scheme as the previous report32. Briefly, Gγ2-(GSA)3-Gαi1 was inserted in downstream of P10 promoter and Gβ1 was inserted downstream of AcMNPV polyhedrin promoter of pFastbac-dual vector. The scFv16 gene was cloned into a modified pFastBac1 vector with N-terminal GP67 signal sequence and C-terminal HRV3C protease cleavage site followed by 6×His-tag and purified as previously reported32 with simple modification. In brief, media containing secreted scFv16 was separated by centrifugation at 72–96 h after infection. The pH of the medium was adjusted to 7.5–8.0 by adding Tris-base powder. Chelating agents were quenched by addition of 1 mM nickel chloride and 5 mM calcium chloride and stirring at room temperature for 1 h. After centrifugation, the supernatant was mixed and incubated with 5 ml of Ni-EXCEL resin (Cytiva). After 2 h, the resin was collected and washed with 20 column volumes of buffer containing 20 mM HEPES pH 7.5, 500 mM NaCl and 20 mM imidazole. The scFv16 was eluted with 20 mM HEPES pH 7.5, 100 mM NaCl and 350 mM imidazole. After HRV3C protease treatment, sample was further purified by size-exclusion chromatography using a Superdex 200 16/600 pg column (Cytiva). The peak fraction was collected and concentrated to 5 mg per ml for future use.The construct for heterotrimeric Gαi heterotrimer were mixed in a 1:1.2 molar ratio with 2.5 µl of Apyrase (NEB) and incubated at 25 °C for 30 min. Purified scFv16 was added to a 1:1.3, Gi heterotrimer:scFv16 molar ratio. The mixture was incubated on ice for 1 h. Then the mixture was subjected to size-exclusion chromatography on a Superdex 200 Increase 10/300 GL column (Cytiva) pre-equilibrated with 20 mM HEPES pH 8.0, 100 mM NaCl, 1 mM MgCl2, 0.5 mM TCEP, 0.001% LMNG, 0.0001% CHS and 100 μM ligand. Peak fractions containing HCAR2–Gi heterotrimer-scFv16 complex were collected and concentrated to 5 mg per ml.HCAR2 and G2, exposure time of 4.7 s, and dose on camera of 7.5 e−1/px/s. Defocus range was −0.8 to −1.8 µm. All data were collected automatically using EPU software.Three µL of the sample was applied to a glow-discharged holey carbon grid . The grid was blotted with blotting force of 10 for 5 s at 4 °C, 100% humidity and flash-frozen into liquid ethane using Vitrobot Mark IV instrument (Thermo Fisher Scientific). After being plunge-frozen in liquid ethane, grids were stored in liquid nitrogen and subjected to cryo-EM data collection. Cryo-EM imaging was performed on a Titan Krios G4 (Thermo Fischer Scientific) operated at 300 kV, equipped with a Gatan Quantum-LS Energy Filter (slit width 15 eV) and a Gatan K3 direct electron detector at a nominal magnification of 105,000× in Correlated Double Sampling (CDS) mode, corresponding to a pixel size of 0.83 Å per pixel for 49 frames with total dose of 51.7 e/Å44. Motion correction was performed by Patch motion correction. CTF estimation for micrographs was performed by Patch CTF estimation. Micrographs under 5 or 10 Å CTF resolution were cut off by Curate Exposures.The processing of the collected data HCAR2 complex with each compound was carried out by cryoSPARC (v.3.3.1 and v.4.0.3)45 were conducted using extracted particles to obtain a 2.96 Å map. To improve further the quality of density for the receptor, a soft mask was applied to the receptor for Local Refinement. The final 3.43 Å map was used for modeling the structure.For the HCAR2 complex with niacin, 5001 movies were collected. The 2,135,798 particles were automatically picked by blob picker and extracted using binning state (3.35 Å/pixel). Extracted particles were classified by 2D classification, and particles included in the classes showing good alignment were subjected to Ab-Initio Reconstruction. Hetero Refinement was conducted using all volumes from Ab-Initio Reconstruction. One class having a clear volume was selected and particles included in that class were extracted in 1.07 Å/pix. Using re-extracted particles, NU-refinement was conducted and 3.2 Å map was obtained. Three rounds of Hereto Refinement were conducted using the 3.2 Å map and four of junk volume. The particles included in one class having a well resolved volume were extracted in 1.245 Å/pix, then NU-refinement and DeepEMhanceri. 3D classification was performed with Relion, and two classes were chosen with a clear density for the receptor. Finally, 228,127 particles were selected, and 3D reconstruction was performed with non-uniform refinement by cryoSPARC, and a 2.77 Å map was obtained. To improve further the quality of density maps of the receptor and Ligand (Acipimox), a soft mask was applied to the receptor for Local Refinement. The final 3.13 Å map was used for modeling the structure.For the HCAR2 complex with acipimox, 4301 movies were collected. Particles were automatically picked by blob picker from 500 micrographs, and extracted in binning state (3.11 Å/pixel). 2D classification was conducted to make template for Template picker. The 4,581,295 particles were picked by Template picker from all micrographs and were extracted in binning state (3.11 Å/pixel). Extracted particles were classified by 2D classification, and particles included in the classes showing good alignment were subjected to Ab-Initio Reconstruction. Two rounds of Hetero Refinement were conducted using all of volumes from Ab-Initio Reconstruction. One class having a clear volume was selected and particles included in that class were extracted in 1.245 Å/pix. Using re-extracted particles, NU-refinement was conducted and a 2.63 Å map was obtained. Subsequently, new masks were made to cover the entire complex. Relion (v.4.0.0) was then used for multibody refinement focusing on the receptor and a part of GαFor the HCAR2 complex with GSK256073, 4957 movies were collected. Particles were automatically picked by blob picker from 500 micrographs, and extracted in binning state (3.11 Å/pixel). 2D classification was conducted to make template for Template picker. The 5434,103 particles were picked by Template picker from all micrographs and were extracted in binning state (3.11 Å/pixel). Extracted particles were classified by 2D classification, and particles included in the classes showing good alignment were subjected to Ab-Initio Reconstruction. Two rounds of Hetero Refinement were conducted using all of volumes from Ab-Initio Reconstruction. One class having a clear volume was selected and particles included in that class were extracted in 1.245 Å/pix. Using re-extracted particles, NU-refinement was conducted and 3.22 Å map was obtained. Subsequently, new masks were made to cover the receptor and the complex of G-protein. Relion (v.4.0.0) was then used for multibody refinement focusing on the receptor, 3D classification was performed with Relion, and one class was chosen with clear density for the receptor. Then Hetero Refinement was conducted using the complex model from previous NU-refinement model and volumes from junk 2D images. Finally, 153,633 particles were selected, and 3D reconstruction was performed with non-uniform refinement by cryoSPARC, and a 3.39 Å map was obtained. To improve further the quality of the density map, a soft mask was applied to the receptor for Local Refinement. The final 3.74 Å map was used for modeling the structure.46. At first, the initial models were roughly fitted to each cryo-EM map using ChimeraX47. Fitted models were subjected to real-space refinement in PHENIX48. The refined model was manually adjusted using COOT49 and then further refined with the Real-space refinement procedure in PHENIX48. ChimeraX47, LigPlot+50 and CASTp 3.051 were used to prepare figures illustrating structural information in the paper.The predicted model of HCAR2 from AlphaFold Protein Structure Database was used as the initial template for modeling the receptor. Model of G-proteins and scFv16 complexes was used from the structure of Type 2 bradykinin receptor-Gq complex (PDB:7F2O)i activation was measured by the NanoBiT-based G-protein dissociation assay20, in which the interaction between a Gα subunit and a Gβγ subunit was monitored by the NanoBiT system (Promega). Specifically, a NanoBiT-Gi1 protein consisting of Gαi1 subunit fused with a large fragment (LgBiT) at the α-helical domain (between the residues 91 and 92 of Gαi1) and an N-terminally small fragment (SmBiT)-fused Gγ2 subunit with a C68S mutation was expressed along with untagged Gβ1 subunit and HCAR2 . The mutants of HCAR2 were generated using primers listed in Supplementary Table 5 cells ml−1 (4 ml per well in DMEM (Nissui) supplemented with 10% fetal bovine serum (Gibco), glutamine, penicillin and streptomycin), 1 day before transfection. Transfection solution was prepared by combining 12 µL (per dish hereafter) of polyethylenimine (PEI) Max solution , 400 µL of Opti-MEM (Thermo Fisher Scientific) and a plasmid mixture consisting of 400 ng ssHA-FLAG-HCAR2 (or an empty plasmid for mock transfection), 200 ng LgBiT-containing Gαi1 subunit, 1 µg Gβ1 subunit and 1 µg SmBiT-fused Gγ2 subunit (C68S). After incubation for 1 day, the transfected cells were harvested with 0.5 mM EDTA-containing Dulbecco’s PBS, centrifuged and suspended in 2 ml of HBSS containing 0.01% bovine serum albumin  and 5 mM HEPES (pH 7.4) (assay buffer). The cell suspension was dispensed in a white 96-well plate at a volume of 80 µL per well and loaded with 20 µL of 50 µM coelenterazine (Carbosynth) diluted in the assay buffer. After a 2 h incubation at room temperature, the plate was measured for baseline luminescence  and titrated concentrations of niacin  were manually added. The plate was immediately read at room temperature for the following 10 min, at measurement intervals of 20 s. The luminescence counts from 8 min to 10 min after ligand addition were averaged and normalized to the initial count. The fold-change values were further normalized to those of vesicle-treated samples and used to plot the G-protein dissociation response. Using the Prism 9 software (GraphPad Prism), the G-protein dissociation signals were fitted to a four-parameter sigmoidal concentration-response curve with a constrain of the HillSlope to absolute values <2. For each replicate experiment, the parameters Span (= Top – Bottom) and pEC50  of individual HCAR2 mutants were normalized to those of WT HCAR2 performed in parallel and the resulting Emax values were used to calculate ligand response activity of the mutants.HCAR2 ligand-induced GHCAR2 ligand-induced β-arrestin1 recruitment to the receptor was measured by the NanoBiT-based β-arrestin recruitment assay. Briefly, the N-terminally LgBiT-fused human β-arrestin1 (200 ng plasmid per 6-cm dish) was expressed with the C-terminally SmBiT-fused ssHA-FLAG-HCAR2  in HEK293A cells in a 6-cm culture dish. As a negative control, ssHA-FLAG-V2R-SmBiT (vasopressin V2 receptor) was used. The transfected cells were dispensed into a 96-well plate, and ligand-induced luminescent changes were measured by following the same procedures as described for the NanoBiT-G-protein dissociation assay, except that luminescence counts were taken from 13 min to 15 min after compound addition.Transfection was performed according to the same procedure as described in the “NanoBiT-G-protein dissociation assay” section. One day after transfection, the cells were collected by adding 200 μl of 0.53 mM EDTA-containing Dulbecco’s PBS (D-PBS), followed by 200 μl of 5 mM HEPES (pH 7.4)-containing Hank’s Balanced Salt Solution (HBSS). The cell suspension was transferred to a 96-well V-bottom plate in duplicate and fluorescently labeled with an anti-FLAG epitope (DYKDDDDK) tag monoclonal antibody ) and a goat anti-mouse IgG secondary antibody conjugated with Alexa Fluor 488 . After washing with D-PBS, the cells were resuspended in 200 μl of 2 mM EDTA-containing-D-PBS and filtered through a 40-μm filter. The fluorescent intensity of single cells was quantified by an EC800 flow cytometer equipped with a 488 nm laser (Sony). The fluorescent signal derived from Alexa Fluor 488 was recorded in an FL1 channel, and the flow cytometry data were analyzed with the FlowJo software (FlowJo). Live cells were gated with a forward scatter (FS-Peak-Lin) cutoff at the 390 setting, with a gain value of 1.7. Values of mean fluorescence intensity (MFI) from ~20,000 cells per sample were used for analysis. For each experiment, we normalized an MFI value of the mutants by that of WT performed in parallel and denoted relative levels.54. Missing hydrogen atoms were added. The protonation states of the protein, charged residues and histidine residues at pH 7 were set according to the PROPKA estimation implemented in PDB2PQR:55 D73 and E37 were set to a protonated aspartic and glutamic acid, respectively. H134 was set to the neutral histidine with only the protonated Nε atom (HSE in the CHARMM force field), while other histidine residues were set to the neutral histidine with only the protonated Nδ atom (HSD in the CHARMM force field). The N- and C-termini were set to NH3+ and COO−, respectively. The protonation state of ligands was assigned using the Epik module in Maestro  simulations were conducted using HCAR2 (residues 9–316) and the ligand. A membrane-water system was prepared using the Membrane Builder implemented in CHARMM-GUI56 with PLUMED ver. 2.7.157. Simulations were performed under periodic boundary conditions. The CHARMM36m force field60 with WYF parameter61 for cation-pi interactions was used for all components except the ligand. The CHARMM General Force Field (CgenFF)62 was used for niacin. An energy minimization and six equilibration runs were performed according to the default setting in the Membrane Builder: Simulation setups were the same as those of our previous simulations63. After equilibration runs, production runs were performed. Due to the periodic boundary condition, the ligand at the dissociated position sometimes moved across the periodic boundary of the z-direction and reached to the intracellular side . Here, the restraint does not bias the specific binding pathway, and the ligand spontaneously approached the extracellular surface of HCAR2 during production runs. In each ligand, three 1-μs production runs were performed independently. The ensemble was NPT ensemble, and the temperature and pressure were set at 300 K and 1 atm, respectively. The thermostat was using the Nose-Hoover scheme. The barostat was using the semi-isotropic Parrinello-Rahman approach. The electrostatic interactions were handled by the smooth particle mesh Ewald method. The van der Waals interactions were smoothly truncated using the switching function within a range of 10–12 Å. Bond lengths involving hydrogen atoms were constrained by the P-LINKS algorithm, and the time step was set to 2 fs. From each 1-μs production run, snapshots were extracted every 1 ns and used for trajectory analysis. The contact was defined as <4 Å distance between nonhydrogen atoms on the protein residues and the ligand. In addition to the wild-type, MD simulations for the R221.27W mutant were performed. The R221.27W substitution was done using the setup procedure of the Membrane Builder implemented in CHRARMM-GUI54, and other setups and conditions of MD simulations were performed as the same for the wild- type as described above.All-atom conventional MD simulations were conducted using the MD program package GROMACS ver. 2019.664. The receptor structure was prepared by the experimental structure of the acipimox bound HCAR2 complex solved by cryo-EM. Hydrogen atoms were added by the protein preparation wizard in Maestro (Schrödinger Release 2020-4)65. After an optimization and an energy minimization of the receptor structure, a cubic grid was determined with the Acipimox bound at the orthosteric site as the center. The grid size was the default size, which contained enough residues to interact with the ligand, and the grid potential parameter of the receptor was calculated without the ligand. The structure of MK-1903 was obtained from the PubChem database (CID 49763030)56. The protonation state of the ligand was determined using the LigPrep wizard in Maestro (Schrödinger Release 2020-4). Then, the prepared MK-1903 was docked into the grid in HCAR2 using Glide with the standard precision mode. A docking pose of MK-1903 was selected by the best docking score and shown in Supplementary Fig. A docking simulation of MK-1903 to the HCAR2 structure was performed using Glide (Schrödinger Release 2020-4)Each NanoBiT-based assay was performed with at least three independent replicates, and the data were analyzed using GraphPad Prism 9 (GraphPad Software). The data are presented as means ± standard error of the mean.Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting SummarySource Data