Initiation factor IF3 is an essential protein that enhances the fidelity and speed of bacterial mRNA translation initiation. Here, we describe the dynamic interplay between IF3 domains and their alternative binding sites using pre-steady state kinetics combined with molecular modelling of available structures of initiation complexes. Our results show that IF3 accommodates its domains at velocities ranging over two orders of magnitude, responding to the binding of each 30S ligand. IF1 and IF2 promote IF3 compaction and the movement of the C-terminal domain (IF3C) towards the P site. Concomitantly, the N-terminal domain (IF3N) creates a pocket ready to accept the initiator tRNA. Selection of the initiator tRNA is accompanied by a transient accommodation of IF3N towards the 30S platform. Decoding of the mRNA start codon displaces IF3C away from the P site and rate limits translation initiation. 70S initiation complex formation brings IF3 domains in close proximity to each other prior to dissociation and recycling of the factor for a new round of translation initiation. Altogether, our results describe the kinetic spectrum of IF3 movements and highlight functional transitions of the factor that ensure accurate mRNA translation initiation. IF3 is the mRNA ,8,9  and pre-steady state kinetics techniques combined with molecular modelling to monitor IF3 movements on the ribosome and to construct a comprehensive model depicting the dynamics of IF3 domains along the cycle of translation initiation, from the initial binding until the recycling of the factor.InfA, InfB, InfC and InfC-E166C were acquired commercially . Competent E. coli BL21 (DE3) cells were CaCl2 transformed  with expression vectors coding for IF1, IF2 with an NTD His-tag, IF3 or IF3E166C. Typically, 2 L of Luria Bertani (LB) medium were used to grow the BL21 (DE3) strains to an OD600 nm of 0.5. Protein expression was induced by adding 1 mM Isopropyl β-d-1-thiogalactopyranoside . Cells were allowed to express the factors for 3 hours prior to harvesting by centrifugation at 5000 × g at 4°C. Cells were lysed by sonication (20 cycles of 10 s followed by 30 s without sonication) in Buffer A (50 mM HEPES (pH 7.1), 100 mM NH4Cl, 10 mM MgCl2, 10% glycerol, 6 mM 2-mercaptoethanol) for IF1 and IF3; or Buffer B (50 mM sodium phosphate buffer (pH 7.7), 300 mM NaCl, 30 mM imidazole) for IF2. Debris and supernatant were separated by centrifugation at 15 000 × g for 30 min a 4°C.Expression vectors pET24c containing 4Cl gradient (0.05–1 M) in Buffer A using a HPLC system . IF3 and IF1 eluted at 700 mM and 400 mM NH4Cl, respectively. Best separation conditions were found at 1 ml/min flow rate and 20 CVs long gradient, collecting fractions of 1 ml each. Protein elution was followed by absorbance at 290 nm and SDS-Polyacrylamide Gel Electrophoresis . While IF3 eluted with an elevated degree of purity, IF1 fractions contained high molecular weight contaminants. Protein contaminants were eliminated by subjecting the combined IF1 fractions to Amicon® Ultra 30K Da centrifugal filters  followed by concentration on a HiTrap™ SP HP , single step eluted with 1 M NH4Cl in Buffer A. Eluted proteins were dialyzed against Storage buffer (50 mM Tris–HCl (pH 7.1), 200 mM NH4Cl, 10% Glycerol, 6 mM 2-mercaptoethanol) and 50 μl aliquots were stored at –80°C. Purity was assayed by 15% SDS-PAGE and total protein staining using blue Coomassie. Initiation factor IF2 was purified by affinity chromatography on a Histrap FF crude column (1 ml) . Supernatants were manually loaded to the column, followed by 5 ml of wash buffer (50 mM Imidazole in Buffer B) and to 2.5 ml of elution buffer (250 mM Imidazole in Buffer B). The first 1.5 ml were collected and subjected to dialysis in Buffer A. Finally, IF2 was further purified by Cation exchange chromatography as explained above for IF1 and IF3.Initiation factors IF1 and IF3 were purified by Cation exchange chromatography on a HiTrap™ SP HP column . Cleared lysates were loaded onto the column  and subsequently subjected to a linear NHE166C was subjected to extensive dialysis in Labelling buffer (50 mM HEPES (pH 7.1), 100 mM NH4Cl, 10% glycerol and 0.5 mM TCEP) in a D-Tube™ Dialyzer Maxi  to remove traces of 2-mercaptoethanol as the reducing agent strongly inhibits the coupling to maleimide linked dyes to cysteines. First, the C-terminal was labelled at the recombinant cysteine (C166) as it is exposed and efficiently reacts with maleimide derivatives. IF3E166C with a 10-fold excess of Atto-540Q maleimide  was incubated in Labelling buffer for 20 min. The reaction was stopped by the addition of 6 mM 2-mercaptoethanol. The modified IF3, termed IF3C540Q, was purified from unreacted dyes on a HiTrap SP HP column  as described above. After 10 CV washes with Buffer A containing 100 mM NH4Cl, a single step elution was applied using 3 ml of 1 M NH4Cl in Buffer A. Typically, elution of the labelled protein is readily visible in the first 0.5 ml fraction. IF3C540Q was subsequently dialyzed as mentioned above in labelling buffer containing 2 M UREA. The partial denaturation of IF3 results in the exposure of the otherwise buried cysteine at position 65 of the NTD. The unfolded protein was incubated with a 20-fold molar excess of Alexa488 maleimide  for 1 h at RT, under mild shacking. IF3C540Q–N488 (IF3DL) was purified from the unreacted dye as described above using HiTrap SP HP column . Eluted proteins were dialyzed against Storage buffer and 20 μl aliquots were stored at –80°C. Purity and efficiency of labelling was assayed by 15% SDS-PAGE where fluorescence was observed under a UV trans-illuminator and total protein was detected by Blue Coomassie staining.IF32), essentially as described , 70mM NH4Cl, 30 mM KCl, 10 mM MgCl2, 6 mM 2-Mercaptoethanol) at 25°C. Unless otherwise stated, 30S complexes were prepared by mixing 0.1 μM 30S, 0.08 μM IF3DL, 0.3 μM IF1, 0.3 μM IF2, 100 μM GTP, 0.5 μM mRNA and/or fMet-tRNAfMet. Mixtures were incubated at 37°C for 30 min and centrifuged at 14 000 × g for 5 min prior to the measurement. Fluorescence time courses were baseline removed, and to highlight the overall conservation of the signal along the full cycle of IF3, an amplitude factor corresponding to the previous reaction was added. All graphical representations have the same Y axis range and represent changes of Volts as measured by the Instrument Photomultiplier. Time courses were analysed by non-linear regression with one or two exponential terms  or a SX20  by rapidly mixing equal volumes of each reacting solution. The excitation wavelength for Alexa 488 was 470 nm. The emission signal was measured after a long-pass optical filter with a 515 nm cut-off. 1000 points were acquired in each measurement. 7 to 10 replicates were recorded for each reaction and subsequently averaged. All stopped flow reactions were performed in TAKMi; With DL slower than their respective bimolecular reactions \docuWith Equation ; \documefMet (Flu) (7 buffer (50 mM Tris (pH 7.5), 70 mM NH4Cl, 30 mM KCl, 7 mM MgCl2, 6 mM 2-mercaptoethanol) using the BioSuite 450HR size exclusion column  for macromolecular complexes. tRNA fluorescence was excited at 460 nm and measured at 540 nm. 70S absorbance was measured at 260 nm. Coelution of fMet-tRNAfMet (Flu) and 70S ribosomes was assigned to 70S IC formation. The efficiency of complex formation was calculated from the areas under the peaks corresponding to 70S ICs and unbound tRNA as follows:fMet were formed in the absence or presence of IF3 or IF3DL. Then, a mixture of ternary complex EF-Tu·GTP·[14C]-Val-tRNAVal (0.2 μM) was added to the initiation complexes. The experiments were performed in TAKM7 buffer and 1 mM GTP at 25°C and incubated for 1 min. Reactions were quenched with 1/10 volume of 5 M KOH and hydrolysed for 30 min at 37°C. Samples were neutralized with 1/10 volume of glacial acetic acid and analysed by reversed-phase HPLC and liquid scintillation counting as described before   binding E166C was then modelled with the Swiss Modeler server using each IF3 PDB file as template  of each fluorophore on the 30S ICs structures was determined with FRET-restrained Positioning and Screening software  . IF3 wastemplate . Finallysoftware  29). Th. ThE166CE. coli wt IF3 was modelled using Modeller v.9.25 , and to build the IF3 model with Alexa 488 and Cy5 fluorophores following the procedure described by Schepers and Gohlke.  involved minimal mutagenesis of the factor, which was modified at a single amino acid position (E166C). The cysteine in IF3E166C is solvent exposed for the free or 30S-bound IF3, does not disturb any secondary structure of IF3C, and was used previously for FRET-based kinetics . Th. Th36). kinetics ,13,25,37ch other .DL showed that the fluorescent derivative promoted 70S IC formation similarly to wt IF3 . In. InDL onDL to the 30S ribosomal subunit resulted in a biphasic increase of fluorescence over time with both apparent rates saturating with 30S concentration , IF3 st1 Figure , Table 11 Figure , Table 11 Figure ,38. Thusd to 30S . To furtsolution .DL complex, we compared our FRET efficiencies to all combinations of IF3C and IF3N sites retrieved from available structures. The structural modelling of IF3DL combined with FRET changes suggest that in the absence of any other ligand, IF3 acquires the most extended layout with IF3C likely occupying the C1 site and IF3N moving into the NR site with an interdomain distance of about 60 Å , yet accounting for a smaller amplitude , positin ≈30 ms  and rearn ≈30 ms ,9, and bn ≈30 ms   of IF3 from the 30S subunit  and IF3 accommodation during 30S pre-IC formation shall take around 50 ms.Thus, the initial interaction of fMet-tRNAS pre-IC . Indeed,I states . AltogetDL FRET signal as a function of fMet-tRNAfMet recruitment depended on the presence of the mRNA. In contrast to the absence of mRNA, mRNA-programmed 30S complex appeared biphasic in time, indicating a further accommodation of IF3DL following the initial binding of fMet-tRNAfMet  followed by the closure of IF3 caused by the ejection of the factor from the resulting 70S IC (decrease from 1.0 to 0.0)  Figure . The ratt Figure . Consiste Figure . In the n Figure  in agreeC Figure . AltogetfMet and the initiation triplet of the mRNA (IF3 enhances the fidelity of translation initiation with three defined functions. IF3 prevents the premature joining of the large 50S subunit by impairing inter-subunit bridges B2a and B2b (reviewed in ). The fathe mRNA . In latethe mRNA . This wothe mRNA , assignithe mRNA .fMet binding site architecture. In this context, IF3C moving to the C2 site would be coupled to the dissociation of IF3N from the NR site to adopt a dynamic layout that is ready to interact with the initiator tRNA . The former would ensemble in less than 30 ms and rearrange in around 1 s, being able to reject the 50S by maximal occupancy of the C2 site by IF3C Figure , and IF2In summary, we describe the functional conformations of IF3 defining with high accuracy the temporal framework covered by both domains of the factor in correlation with all events taking place during the translation initiation process. Altogether, this work allows to accurately assign the speed and nature of elemental movements of IF3 behind its essential fidelity function.gkab522_Supplemental_FilesClick here for additional data file.


Background: Cachexia is a multifactorial disorder characterized by weight loss and muscle wasting, making up for about 20% of cancer-related death. However, there are no effective drugs to combat cachexia at present.Methods: In this study, the effect of CT26 exosomes on C2C12 myotubes was observed. We compared serum HMGB1 level in cachexia and non-cachexia colon cancer patients. We further explored HMGB1 expression level in CT26 exosome. We added recombinant HMGB1 to C2C12 myotubes to observe the effects of HMGB1 on C2C12 myotubes and detected the expression level of the muscle atrophy-related proteins. Then, we used the HMGB1 inhibitor glycyrrhizin to reverse the effects of HMGB1 on C2C12 myotubes. Finally, HMGB1 inhibitor glycyrrhizin was utilized to relieve cachexia in CT26 cachexia mouse model.Results: Exosomes containing HMGB1 led to muscle atrophy with significantly decreased myotube diameter and increased expression of muscle atrophy-related proteins Atrogin1 and MuRF1. Further, we detected that HMGB1 induced the muscle atrophy mainly via TLR4/NF-κB pathway. Administration of the HMGB1 inhibitor glycyrrhizin could relieve muscle wasting in vitro and attenuate the progression of cachexia in vivo.Conclusion: These findings demonstrate the cachectic role of HMGB1, whether it is soluble form of HMGB1 or secreted from tumor cells as part of exosomes. HMGB1 inhibitor glycyrrhizin might be a promising drug in colon cancer cachexia. Cancer cachexia is a multifactorial symptom with the feature of weight loss and muscle wasting, which is caused by a combination of various reasons, including reduced food intake, metabolic changes, excess catabolism, and inflammation . It is avia plasma membrane fusion and participant in cell communication (Exosomes (EVs), deriving from endosomal compartment of numerous cells, are released outside the cells nication . EVs connication . Emerginnication . Esophagnication .Systemic inflammation is one of the main causes of cachexia muscle wasting. Pro-inflammatory cytokines such as IL-6, TNF α, and IL-1β promote the transcriptional activation of atrophy-related genes Atrogin1 and MuRF1, which play a major role in the occurrence and development of muscle wasting. Activation of inflammatory pathways such as NF-κB signaling pathway could cause muscle wasting both in cachexia mouse models and muscles of cancer cachexia patients . InflammIn vivo study confirmed the cachexia effects of HMGB1 and anti-cachexia effects of glycyrrhizin on CT26 tumor-bearing mice.In this article, we explored the effects of EVs containing HMGB1 on C2C12 myotubes. Then, we used the HMGB1 inhibitor glycyrrhizin to confirm the cachectic role and tried to find out the possible pathways. Mouse CT26 colon cancer cells (CT26 cells) and mouse myoblasts (C2C12 cells) were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. CT26 cells and C2C12 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin G-streptomycin  in 37°C incubators with 5% CO2. When C2C12 myoblasts reached 90%, the proliferation medium was discarded and replaced with DMEM medium containing 2% horse serum . The differentiation medium was replaced every day. Glycyrrhizin was obtained from SelleckChem  and dissolved in dimethyl sulfoxide (DMSO). TAK-242, BAY 11-7082, PD169316 were purchased from MedChem Express .When the cell density of CT26 reaches 80%, discard the complete medium and wash with sterile PBS gently for at least 3 times. Then continue to incubate for 48 h with serum-free DMEM. The CT26 cell supernatant was collected and centrifuged at 1,200 rpm for 10 min to remove the cell debris. Filter the centrifuged CT26 supernatant through a 0.2 μm sterile syringe filter and the medium was used as a cachectic factor. CT26 conditioned medium (CCM) were prepared by CT26 supernatant and DMEM at a ratio of 1:1 with 2% horse serum.g for 15 min at 4°C, 3,000 × g for 15 min, and 12,000 × g for 30 min to remove tumor cells and fragments. The exosomes were precipitated by an ultracentrifuge  at 120,000 × g for 90 min. The supernatant was collected as exosome-depleted CCM. The pallet was resuspended in PBS and the exosomes were purified by centrifugation at 120,000 × g for 90 min. The resulting exosome pellet was resuspended in PBS for cell experiments. The exosome pellet obtained after ultracentrifugation was lysed with radioimmunoprecipitation assay (RIPA) for 30 min, and then centrifuged at 12,000 g for 30 min at 4°C. The supernatants containing total proteins were transferred to a new centrifuge tube, and proteins in exosomes were quantified by bicinchoninic acid (BCA) assay (Beyotime Biotechnology). Murine serum exosomes were obtained by ultracentrifugation using the above method after dilution with PBS at a ratio of 1:1.The CT26 supernatant was centrifuged at 500 × The exosomes derived from CT26 were fixed with 1% glutaraldehyde in PBS (pH 7.4) and then washed. Take 30 μl of exosome suspension on the formvar/carbon coated grid, and then negatively stain with 3% (W/V) aqueous phosphotungstic acid for 90 s, and observe with a transmission electron microscope (Hitachi H-7000FA).The exosomes from CT26 were labeled with 2 μM PKH26 (Sigma-Aldrich) for 4 min. Complete medium was used to neutralize with PKH26, then the excess dye was removed by ultracentrifugation for an hour. The precipitation was re-suspended in PBS and incubated with C2C12 myotube cells for 8 h. The cells were then counterstained with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) and observed by fluorescence microscope .We analyzed the fraction of exosomes on a ZETAVIEW instrument . Use nanoparticle tracking analysis software to count and analyze EV particles.The cross-sectional study collected patients with advanced colon cancer in Tongji hospital  from May 2020 to September 2020. Ethical approval was granted by the Committee on the Ethics of Tongji hospital. The inclusion criteria: the patients were pathologically diagnosed as advanced colon cancer, no history of other malignancies, clear consciousness, more than 18 years old, blood samples available, available abdominal CT scan within 1 month, no previous diabetes, no thyroid disease and no other factors that may cause weight loss. Cancer cachexia was diagnosed according to the international consensus .Human serum HMGB1 were quantified by the HMGB1 ELISA Kit . After anesthesia, mouse blood samples were obtained through the retro-orbital sinus. Serums were collected by centrifugation at 2,500 rpm at 4°C for 15 min for ELISA test. IL-6 , TNF-α , HMGB1  were quantitatively detected according to the instructions.After treating C2C12 myotubes in a 24-well plate with different concentrations of HMGB1 for 72 h, they were washed 3 times with PBS, fixed with 4% paraformaldehyde for 20 min, and permeabilized with 0.5% Triton X-100 at room temperature for 30 min. The primary antibody MyHC antibody was incubated overnight at 4°C, and then incubated with Alexa Fluor 488 goat anti-mouse IgG antibody  at room temperature for 1 h, and the cells were stained with DAPI. Images of myotubes were taken by a fluorescence microscope. The diameters of more than 100 myotubes in at least 10 fields were measured by ImageJ software .Proteins were extracted in RIPA lysis buffer with 1% phenylmethylsulphonyl fluoride (PMSF) and 1% phosphorylase inhibitor cocktail  for 30 min at 4°C. Proteins were quantified by BCA assay. Each sample of 30 μg protein was electrophoresed on 10% SDS-PAGE gel. Then, electro-transfer proteins to polyvinylidene difluoride (PVDF) membranes. Membrane were incubated with primary antibodies overnight at 4°C after being blocked in 5% bovine serum albumin at room temperature for 1 h. Wash the membranes in Tris-buffered saline with 0.1% Tween-20 (TBST) for 3 times. The membranes were incubated with the corresponding secondary antibody conjugated with horseradish peroxidase . Finally, membranes were detected using ECL solution  following TBST washed for 3 times. The antibodies used were listed as follows: NF-κB p65 , phosphorylated NF-κB p65 , IκBα , phosphorylated IκBα , MuRF1 , Atrogin1 , α-tubulin , Anti-myosin heavy chain . The HRP-conjugated anti-mouse and anti-rabbit antibodies were obtained from Invitrogen. Replicates of western blotting were shown in For HMGB1 knockdown vector, the sequences used were: ATGCAGCTTATACGAAGATAA. The sequences of scramble shRNA (shCON) were: TTCTCCGAACGTGTCACGT. When CT26 cells reached 50% confluence, we replaced the complete medium with a serum-free medium containing 5 μg/ml polybrene and lentiviruses  at a multiplicity of infection of 50. Then, change to complete medium after 12 h. The empty vector lentivirus transduction was performed in the same way. When GFP was detected under the fluorescence microscope, we added puromycin to the complete medium at a final concentration of 3 μg/ml for 1 week to select stable transfected cells.All experimental protocols were approved by the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Six weeks old healthy male BALB/c mice were raised in a regular 12 h light-dark cycle and specific pathogen free environment.in vivo, mice were randomly divided into five groups (n = 8 per group): control group, CT26 group, shCON group, shHMGB1 group, shHMGB1+EVs group. The control group was normal mice without CT26 injection, and the CT26 group mice were injected subcutaneously with untreated CT26. The mice in the shCON group were injected with CT26 cells transduced with empty lentiviruses. The mice in the shHMGB1 group were injected with HMGB1-lowexpressed CT26 cells. The mice in shHMGB1+EVs group were injected with HMGB1-lowexpressed CT26 cells and 20 μg CT26 exosomes were injected three times a week. The remaining mice were injected with 100ul of PBS. The weight of mice and the size of tumors were recorded every 3 days.To explore the role of HMGB1 6) into the right flank and healthy mice were injected with equal volume of PBS. Treatment started 7 days after tumor implantation when the tumors were palpable. Mice in the control group were injected with equivalent drug vehicle. Mice in glycyrrhizin group were treated with glycyrrhizin every day for 2 weeks . The body weights of the mice, food intake and tumor volumes were measured every 3 days. Two weeks after treatment, mice were euthanized. Tumors, gastrocnemius muscles and epididymal adipose tissues were removed, weighed, and quickly frozen in liquid nitrogen for subsequent analysis.For glycyrrhizin treatment in CT26 cachexia model, mice were randomly divided into four groups (n = 8 per group): healthy mice without tumor, CT26 tumor-bearing mice without glycyrrhizin treatment, CT26 tumor-bearing mice with glycyrrhizin (5 mg/kg/day) treatment, CT26 tumor-bearing mice with glycyrrhizin (20 mg/kg/day) treatment. Tumor-bearing mice were injected subcutaneously with CT26 cells  under applicable conditions  and western blot. We found double-layered vesicles, with the diameters of most particles were around 110 nm . WB analIncubating C2C12 myotubes with different concentrations of CT26-EVs for 24 h, we detected higher expression of muscle atrophy-related proteins Atrogin1 and MuRF1, indicating that a certain level of CT26-EVs could induce muscle wasting in C2C12 myotubes . Furtherin vitro. Different concentrations of recombinant HMGB1 were added to C2C12 myotubes. The expression of atrophy-related genes Atrogin1 and MuRF1 were elevated  pretreated with myotubes followed by HMGB1. As shown in Glycyrrhizin is reported to bind with HMGB1 directly and inhibit HMGB1 . To furtIn vitro, we found that HMGB1 was significantly down-regulated after transduction , which may indicate that different tumor types have different cachexins. To further confirm the role of HMGB1 in muscle wasting, we knocked down HMGB1 in CT26 cells and HMGB1-lowexpressed CT26 tumor-bearing mice showed alleviated cachexia symptoms.In a systematic literature review of 18 studies involving 11 cancers, high expression of HMGB1 was associated with poor prognosis . ElevateGlycyrrhizin is the most important active ingredient extracted from licorice root. It has a broad spectrum of anti-cancer and anti-inflammatory properties. It can also reduce the level of alanine aminotransferase and protect the liver . It can In conclusion, we found that exosomes containing HMGB1 could induce muscle wasting and serum HMGB1 was elevated in cachexia patients. Glycyrrhizin, HMGB1 inhibitor, may be a potent drug in cancer cachexia.


In vitro assays confirmed that Pgrmc1 KO livers exhibit reduced cytochrome P450 activity consistent with reduced enzyme levels. Mechanistic studies in cultured cells demonstrated that PGRMC1 stabilizes cytochromes P450 and that binding and stabilization do not require PGRMC1 binding to heme. Importantly, Pgrmc1-dependent stabilization of cytochromes P450 is physiologically relevant, as Pgrmc1 deletion protected mice from acetaminophen-induced liver injury. Finally, evaluation of Y113F mutant Pgrmc1, which lacks the axial heme iron-coordinating hydroxyl group, revealed that proper iron coordination is not required for heme binding, but is required for binding to ferrochelatase, the final enzyme in heme biosynthesis. PGRMC1 was recently identified as the causative mutation in X-linked isolated pediatric cataract formation. Together, these results demonstrate a heme-independent function for PGRMC1 in cytochrome P450 stability that may underlie clinical phenotypes.Progesterone receptor membrane component 1 (PGRMC1) is a heme-binding protein implicated in a wide range of cellular functions. We previously showed that PGRMC1 binds to cytochromes P450 in yeast and mammalian cells and supports their activity. Recently, the paralog PGRMC2 was shown to function as a heme chaperone. The extent of PGRMC1 function in cytochrome P450 biology and whether PGRMC1 is also a heme chaperone are unknown. Here, we examined the function of Pgrmc1 in mouse liver using a knockout model and found that Pgrmc1 binds and stabilizes a broad range of cytochromes P450 in a heme-independent manner. Proteomic and transcriptomic studies demonstrated that Pgrmc1 binds more than 13 cytochromes P450 and supports maintenance of cytochrome P450 protein levels posttranscriptionally.  Cytochromes P450 are an essential superfamily of heme-containing monooxygenase enzymes that catalyze biosynthetic reactions, detoxify xenobiotic compounds, and metabolize pharmaceutical drugs . These eSchizosaccharomyces pombe and human cells (Progesterone receptor membrane component 1 (PGRMC1) participates in protein–protein interactions with cytochromes P450 in the fission yeast an cells . PGRMC1 an cells . In addian cells , 12, 13.an cells , 7, 8. Ian cells . Additioan cells , 12, 13.an cells . HoweverPGRMC1 is a heme-binding membrane protein, and despite its name, PGRMC1 binding to heme is better understood than its binding to progesterone . As a tyPGRMC1 has a paralog in mammals, named progesterone receptor membrane component 2 (PGRMC2), with which it shares 60% amino acid identity. In a landmark study, the Saez lab demonstrated that PGRMC2 functions as a heme chaperone and plays a critical role in mitochondrial homeostasis in mouse brown adipose tissue . Heme chin vivo, we generated mice with a conditionally targeted Pgrmc1 allele. The conditional allele contains loxP sites flanking exons 1 and 2 of Pgrmc1 with a neomycin resistance cassette (A). Mice carrying the conditionally targeted allele were crossed to mice expressing Sox2-Cre recombinase to produce whole body Pgrmc1 knockout (KO) mice. Male and female whole body Pgrmc1 KO mice are viable. Because Pgrmc1 is X-linked  (A and B) (B and C)  , A and BA and B) , B and CB and C) , and plaB and C) . Liver mB and C) , except Pgrmc1 KO mice were infected with AAV8 GFP or AAV8 Flag-Pgrmc1 to express the protein in the liver for 8 days (A and B). A Flag affinity purification was performed on detergent-solubilized liver membrane fractions from these mice (C), and bound proteins were identified by mass spectrometry. Flag-Pgrmc1 binding partners (p = 4.35 × 10−14), which reflects the cytochromes P450 (C). The second most enriched GO term was “oxidation-reduction process” (p = 6.70 × 10−13), which reflects the cytochromes P450 and additional proteins involved in electron transfer reactions, such as lathosterol oxidase and retinol dehydrogenase. Binding of Flag-Pgrmc1 to Cyp1a2, Cyp2e1, Cyp3a, and Cyp51a in the liver membrane fraction was validated by western blotting; binding to Cyp2f2 was not confirmed (D). In each confirmed instance, Flag-Pgrmc1 bound to 0.2% to 2% of cytochrome P450 protein in the liver membrane fraction (D). This coimmunoprecipitation experiment demonstrated that Flag-Pgrmc1 binds cytochromes P450 and, more broadly, may bind enzymes involved in redox processes in mouse liver.Our previous studies demonstrated that PGRMC1 binds cytochromes P450 in yeast and human cells . To testr 8 days , A and Bese mice C, and bopartners  and S2.2mes P450 C. The seonfirmed D. In eacfraction D. This cD and S2E). To determine if Pgrmc1 affects cytochrome P450 protein levels globally, we performed quantitative mass-spectrometry proteomics with isobaric tagging on liver membrane samples from WT and Pgrmc1 KO mice and detected a total of 936 proteins in both of two biological replicates , with its detection in the Pgrmc1 KO samples likely an artifact of coisolation interference during the mass spectrometry run . Protein expression of Cyp1a2, Cyp2f2, Cyp7b1, Cyp51a, Cyp2e1, and Cyp3a was confirmed to be decreased in Pgrmc1 KO mouse liver membranes by western blotting (B and C). Quantitative proteomics revealed a 14 to 38% reduction in protein across these specific cytochromes P450, and western blotting revealed a 22 to 70% reduction. Together, these complementary methods show that Pgrmc1 functions to maintain protein levels of these cytochromes P450.We consistently noted an increase in levels of cytochromes P450 expression in Flag-Pgrmc1-expressing KO liver as compared with GFP-expressing KO liver D and S2Eplicates . We deteFig. 2A, , a compaFig. 2A, . DiffereFig. 2A,  and S3.3etry run . Among t. To investigate this, we performed RNA-seq on WT and Pgrmc1 KO liver. Expression of 16,318 genes was measured, including 83 cytochromes P450  and “oxidation-reduction process” (p = 2.07 × 10−3) . Notably, Pgrmc1 binding partners were enriched for these same GO terms (C). For the subset of the 83 cytochromes P450 detected in the RNA-seq data . When the PGRMC1 KO cells were transfected with Flag-PGRMC1, overexpressed PGRMC1 was twofold higher than endogenous PGRMC1 (B). Next, we sought to validate the PGRMC1-CYP1A2 binding observed in mouse liver using human cells. Specific binding between Flag-PGRMC1 and CYP1A2-1XMyc was confirmed by Flag coimmunoprecipitation in PGRMC1 KO cells (C). To test the effect of PGRMC1 on CYP1A2 stability, PGRMC1 KO cells were transfected with CYP1A2-1XMyc with or without Flag-PGRMC1. At 48 h posttransfection, cells were exposed to the irreversible translation inhibitor emetine, and protein levels were measured over time. PGRMC1 stabilized CYP1A2-1XMyc protein, increasing its half-life by 67% (F and G). This experiment demonstrated that PGRMC1 stabilizes CYP1A2 posttranslationally.Our obiotics . We generoblasts . Knockoublotting A. When ts PGRMC1 B. Next, KO cells C. To tese by 67% , F and GPgrmc1 KO mice. Our previous work showed that PGRMC1 binds CYP51A1 and supports its activity in cholesterol synthesis in HEK293 cells  using membrane samples from WT and Pgrmc1 KO mice. ECOD is mediated by CYP1A1 and CYP1A2 in humans and rodents with additional cytochromes P450 contributing to metabolism in humans  (A). Product formation was reduced at all substrate concentrations tested in the Pgrmc1 KO samples, and the difference was statistically significant between 0.125 mM and 2 mM. The Km of the reaction did not differ in the KO (B). However, the Vmax of the reaction was 42% lower in the KO (B), consistent with reduced levels of Cyp1a2 protein in Pgrmc1 KO livers (B and C).ECOD product formation (7-hydroxycoumarin) was monitored over a range of substrate (7-ethoxycoumarin) concentrations, and the data exhibited Michaelis–Menten kinetics (WT R= 0.934) A. Producn the KO B. Howeven the KO B, consisO livers , B and CPgrmc1 KO mice (C). In the KO samples, paraxanthine formation was reduced by 30% at 50 μM caffeine. Taken together, these two enzyme assays demonstrate that loss of Pgrmc1 reduces Cyp1a2 activity, consistent with measured decreases in Cyp1a2 protein (B and C).Caffeine is another well-established Cyp1a2 substrate . We assa KO mice C. In the protein , B and CPgrmc1 KO mice (D). In the KO samples, p-nitrocatechol formation was reduced by 62% at 100 μM p-nitrophenol, consistent with reduced levels of Cyp2e1 protein in Pgrmc1 KO livers (B and C). These activity assays show that loss of Pgrmc1 reduces cytochrome P450 activity, consistent with measured decreases in cytochrome P450 protein.We also tested the effect of loss of Pgrmc1 on Cyp2e1 activity. Cyp2e1 is a cytochrome P450 involved in the metabolism of ethanol, pharmaceutical drugs, and low-molecular-weight carcinogens, and p-nitrophenol is a known CYP2E1 substrate . We assa KO mice D. In theO livers , B and Cin vitro, we asked whether Pgrmc1 supports liver Cyp2e1 cytochrome P450 activity in vivo. Overdosing on the common over-the-counter analgesic acetaminophen (APAP) is the leading cause of acute liver failure in patients (N-acetyl-p-benzoquinone imine (NAPQI), which is subsequently glutathionylated for excretion .Since Pgrmc1 was required for maximal Cyp2e1 activity in liver membranes patients , 35. In xcretion . When noxcretion . Howeverxcretion . Cyp2e1 toxicity , 37. FlaFig. 1D,  and S2.2KO liver , B and CPgrmc1 KO protected against APAP-induced liver injury. Mice were injected with 600 mg APAP per kg body weight and euthanized 24 h later. All mice survived the study. Liver damage was surveyed by measuring serum ALT and AST and by histologic analysis of hematoxylin and eosin-stained liver sections. In WT animals treated with APAP, liver damage was apparent. ALT was elevated 37-fold, and AST was elevated 20-fold compared with vehicle-treated controls (A and B). APAP-treated WT mice had centrilobular hepatocellular necrosis characteristic of APAP hepatotoxicity and cytoplasmic microvesicular vacuolation (C). Notably, Pgrmc1 KO mice treated with APAP had the same ALT and AST levels as vehicle-treated mice (A and B). While the APAP-treated KO mice exhibited cytoplasmic microvesicular vacuolation, they did not have the extensive centrilobular necrosis observed in APAP-treated WT mice (C). Thus, lack of Pgrmc1 expression was protective against the damage of APAP-induced liver injury, which is mediated by Cyp2e1. These data show that Pgrmc1-dependent stabilization of cytochromes P450 can have a clinically significant impact in a model of drug-induced liver injury.To investigate a functional role for Pgrmc1 in Cyp2e1 activity, we tested whether controls , A and Buolation C. Notablted mice , A and B WT mice C. Thus, A) . Cytochromes P450 had no significant differential expression between Flag-Pgrmc1 and Y113F Flag-Pgrmc1 samples in the proteomics dataset . Expression of Cyp1a2, Cyp2e1, Cyp3a, and Cyp51a was the same in the Y113F Flag-Pgrmc1 sample as the Flag-Pgrmc1 sample . Thus, the axial iron-coordinating residue is not required in mouse liver for Pgrmc1 to maintain cytochrome P450 levels.The most notable biochemical property of PGRMC1 is its ability to bind heme. Residue Y113 was identified in the PGRMC1 crystal structure (PDB 4X8Y) as the axial iron-coordinating residue in the heme-binding pocket A 18). K. KA) . Consistent with the result for Flag-Pgrmc1, binding of Y113F Flag-Pgrmc1 to Cyp2f2 was not confirmed (B). The top enriched GO terms among the candidate binding partners were “exogenous drug catabolic process” (p= 9.96 × 10−18) and “oxidation-reduction process” (p = 5.45 × 10−16) and reflect the cytochromes P450 and additional proteins that are involved in electron transfer reactions (C). Flag-Pgrmc1 candidate binding partners were also enriched for these GO terms (C). Like Flag-Pgrmc1, Y113F Flag-Pgrmc1 binds cytochromes P450 and, more broadly, may bind enzymes involved in redox processes in the liver.We next tested whether Y113F Flag-Pgrmc1 binds cytochromes P450 in mouse liver by Flag coimmunoprecipitation coupled to mass spectrometry. Y113F Flag-Pgrmc1 bound 75 proteins  , which Figs. 5onfirmed B. The toeactions C. Flag-PGO terms C. Like FC). Y113F Flag-PGRMC1 also stabilized CYP1A2-1XMyc to the same extent as Flag-PGRMC1 (D and E). This experiment showed that the axial iron-coordinating residue of PGRMC1 is not required to stabilize CYP1A2.Since Y113F Flag-Pgrmc1 bound and affected steady-state protein levels of cytochromes P450 in the liver similar to Flag-Pgrmc1, we asked if Y113F Flag-PGRMC1 stabilized CYP1A2 in cell culture. We confirmed that CYP1A2-1XMyc bound Y113F Flag-PGRMC1 C. Y113F g-PGRMC1 , D and EPgrmc1 KO mice infected with AAV GFP, AAV Flag-Pgrmc1, or AAV Y113F Flag-Pgrmc1, we performed the ECOD metabolism assay at a 2 mM saturating substrate concentration (F). As expected, the AAV GFP sample generated the same amount of product as an uninfected Pgrmc1 KO sample, and Flag-Pgrmc1 restored product formation to the level of the uninfected WT control (F). Notably, Y113F Flag-Pgrmc1 also restored product formation to the level of the uninfected WT control. Thus, the axial iron-coordinating residue Y113 of Pgrmc1 is not required for Pgrmc1 to support Cyp1a2 activity. Altogether, Y113F PGRMC1 behaved like wild-type PGRMC1 in all mouse liver and cell culture assays.Because Y113F Flag-PGRMC1 stabilized CYP1A2-1XMyc in human cells, and Y113F Flag-Pgrmc1 stabilized and bound Cyp1a2 in liver, we tested whether Y113F Pgrmc1 can also support Cyp1A2 activity in the liver. Using liver membrane fractions from ntration F. As exp control F. Notablin vitro. The crystal structure of human PGRMC1 revealed that four residues  coordinate heme in the binding pocket (A)  as well as the mutants Y113F PGRMC1 and Y113F, K163A, Y164F (3X MUT) PGRMC1, and the affinity tag was removed by thrombin cleavage (A). After incubation with a 100-fold molar excess of hemin, recombinant PGRMC1 (rPGRMC1) had the deep reddish-brown color characteristic of a heme-binding protein (B). Y113F rPGRMC1 was a similar reddish-brown color (B), suggesting that it retained the ability to bind heme. Unlike rPGRMC1 and Y113F rPGRMC1, 3X MUT rPGRMC1 was much lighter in color, suggesting it has a lower binding affinity for heme than either rPGRMC1 or Y113F rPGRMC1 (B). To measure the heme-binding affinity of each rPGRMC1 protein, 10 μM of each protein was incubated with 0 to 30 μM hemin for 16 h. The amount of hemin bound was measured spectrophotometrically at A394. The data were fit to the Hill equation or a linear model as appropriate. rPGRMC1 bound heme with a Kd of 4.9 ± 0.33 μM (B), which is similar to the value reported for PGRMC2 (1.4 μM) (d = 4.7 ± 0.42 μM) (B). In contrast, 3X MUT rPGRMC1 bound heme nonspecifically (B). Taken together, these results indicate that mutation of the iron-coordinating residue Y113F in PGRMC1 does not prevent heme binding and that additional residues in the heme-binding pocket of PGRMC1 must be mutated to prevent heme binding.Since Y113F Flag-Pgrmc1 binds and stabilizes cytochromes P450 like Flag-Pgrmc1, we investigated the ability of Y113F Pgrmc1 to bind heme g pocket A 18). F. Fin vitcleavage A. After  protein B. Y113F wn color B, sugges rPGRMC1 B. To mea 0.33 μM B, which C1 aa 43–5 as well0.42 μM) B. In conifically B. Taken PGRMC1 KO cells, we found that 3X MUT Flag-PGRMC1 binds CYP1A2-1XMyc (C). Additionally, 3X MUT Flag-PGRMC1 stabilized CYP1A2-1XMyc in an emetine chase more effectively than Flag-PGRMC1 or Y113F Flag-PGRMC1 (C and D). These results indicate that PGRMC1 binds and stabilizes CYP1A2 in a heme-independent fashion.Having identified a heme-binding mutant of PGRMC1, we next tested whether heme binding by PGRMC1 is required for PGRMC1 to bind and stabilize CYP1A2. Using A2-1XMyc C. Additig-PGRMC1 , C and DE and F). Fech is responsible for the final step in heme synthesis (E and F). Taken together, these results suggest that Y113F PGRMC1 is capable of binding heme with the affinity of the wild-type protein, but the axial iron-coordinating residue of Pgrmc1 is critical for Pgrmc1 binding to the heme biosynthetic enzyme ferrochelatase.Despite the shared ability of PGRMC1 and Y113F PGRMC1 to bind and stabilize cytochromes P450 and their affinity for heme, we discovered one notable difference between Flag-Pgrmc1 and Y113F Flag-Pgrmc1. We performed a stringent quantitative analysis of the proteins that bound Flag-Pgrmc1 and Y113F Flag-Pgrmc1 in the Flag pull-down from liver membrane fractions . As expeynthesis , and binynthesis . Bindingynthesis , E and Fin vivo. Using a whole-body Pgrmc1 KO mouse, we observed (1) PGRMC1 binds many cytochromes P450 from diverse families in the liver; (2) PGRMC1 stabilizes cytochromes P450 posttranslationally in a heme-independent manner; (3) PGRMC1 is required for maximal activity in the liver of cytochrome P450 enzymes that it binds; (4) PGRMC1 alters cytochrome P450 activity at a physiologically significant level that may have a clinical impact; and (5) PGRMC1 binds the terminal heme synthesis enzyme Ferrochelatase (FECH) in the liver. Our observations expand knowledge on in vivo cytochrome P450 biology and implicate PGRMC1 in heme metabolism.PGRMC1 is a membrane-bound, heme-binding protein implicated in a plethora of biological processes . PreviouPGRMC1 binds many cytochromes P450 from diverse families in the liver. Pgrmc1 bound to 56% of the 23 cytochromes P450 assayed by mass spectrometry. We validated the binding interaction with Pgrmc1 for five cytochromes P450  by western blotting, and all interactions were confirmed, except Cyp2f2 for reasons that are unclear. PGRMC1 bound cytochromes P450 from families involved in different functions, including xenobiotic, pharmaceutical drug, cholesterol, and arachidonic acid metabolism  were less abundant in Pgrmc1 KO liver. The decrease in protein expression cannot be attributed to a decrease in mRNA level as gene expression was either unchanged  or elevated  in Pgrmc1 KO liver. The elevation of cytochrome P450 transcripts in Pgrmc1 KO liver, like Cyp3a11, may be a compensation mechanism to restore cytochrome P450 protein levels. Indeed, such a mechanism may actually mask the effect that loss of Pgrmc1 has on cytochrome P450 protein levels. For the three cytochromes P450 that were among the least abundant proteins in Pgrmc1 KO mice , all three were shown to bind Pgrmc1 by at least one method (mass spectrometry or western blotting), and likewise their mRNAs were either unchanged  or elevated . Cyp1a2 bound Pgrmc1 and was less abundant in Pgrmc1 KO liver. Using cultured cells, we confirmed that Flag-PGRMC1 bound CYP1A2 and stabilized CYP1A2 in a chase assay with a translation inhibitor. Collectively, these in vivo and in vitro data demonstrate that PGRMC1 binds and posttranslationally stabilizes cytochrome P450 enzymes in mouse liver.PGRMC1 stabilizes cytochromes P450 posttranslationally. Cytochrome P450 proteins that bound PGRMC1 tended to be less abundant in Vmax) in Pgrmc1 KO microsomes for the Cyp1a2-dependent ECOD reaction was reduced by 40%, but there was no change in enzyme affinity (Km) , Pgrmc1 may affect either of these parameters to increase cytochrome P450 activity. Our data demonstrate that a decrease in Cyp1a2 and Cyp2e1 protein underlies the decrease in cytochrome P450 activity in Pgrmc1 KO liver. Both Cyp1a2 and Cyp2e1 proteins bind Pgrmc1, and the proteins are expressed at lower levels in Pgrmc1 KO liver. PGRMC1 most likely controls enzyme activity by stabilizing cytochromes P450 rather than affecting the catalytic cycle of the enzyme, but a concomitant effect on the kcat of cytochromes P450 upon PGRMC1 binding cannot be ruled out as protein–protein interactions are also known to alter cytochrome P450 activity  . Likewisr injury  and 4 . Contrary to this result, our work shows that full-length Y113F PGRMC1 binds CYP1A2 in both cultured cells and mouse liver and that Y113F PGRMC1 promotes CYP1A2 stability as well as wild-type PGRMC1. In fact, Y113F Pgrmc1 binds all 13 cytochromes P450 that Pgrmc1 binds in mouse liver plus an additional five cytochromes P450. These data suggest that the N-terminal transmembrane domain of PGRMC1 may contribute to cytochrome P450 binding.The axial heme coordinating residue Y113 of PGRMC1 is not required for PGRMC1 to bind heme or to bind and stabilize cytochromes P450  and 6. Tet al.  solved te et al.  reportedTo render PGRMC1 unable to bind heme, Y113 and two additional residues that coordinate the protoporphyrin ring  must be mutated. This 3X MUT PGRMC1 bound CYP1A2 and stabilized it in cultured cells  and S6, et al. previously reported that PGRMC1 binds to FECH in human embryonic kidney and leukemia cells (PGRMC1 of which 96 (5.5%) are missense mutations. No missense mutations occur in the Y113 codon or those of the other three residues that compose the heme-binding site  in mouse liver. As FECH is the terminal enzyme in mitochondrial heme synthesis , this obia cells . Here, wia cells . In the ia cells . The Y11ia cells , a more ia cells , 21. To ing site , consistThe binding of PGRMC1 to FECH, which is associated with the inner membrane in the mitochondrial matrix, and ER-localized cytochrome P450 enzymes raises the question of how PGRMC1 localizes to these different subcellular compartments and whether its localization is dynamic. The binding of PGRMC1 and FECH places PGRMC1 in a key position to transfer heme throughout the cell. It is difficult to explain how an ER-resident Type 1 membrane protein like PGRMC1 can interact with FECH, which resides in the mitochondrial matrix associated with the inner membrane, such that heme from FECH could be transferred to the cytochrome-b5 domain of PGRMC1. While studies have described PGRMC1 as localized to many different subcellular compartments, no model of the PGRMC1-FECH interaction has yet provided a satisfactory explanation that respects the rules of protein trafficking and membrane topology , 21, 47.et al. recently showed that the PGRMC1 paralog, PGRMC2, is a heme chaperone, which suggests that PGRMC1 may also be a heme chaperone  with a single N-terminal Flag tag in the vector pcDNA3.1 was previously described  from Invitrogen Ultimate ORF IOH52560 (Johns Hopkins University High Throughput Biology HiT Center) to pcDNA3.1. The sequence contains a point mutation (C1200T) that is nonsynonymous . CYP1A2 is tagged at the C-terminus with a single Myc tag. The GP78-5X Myc plasmid was a gift of Dr Russell DeBose-Boyd  with Flag-Pgrmc1 or Y113F Flag-Pgrmc1 subcloned from the mammalian expression vectors described here: The mammalian expression vector, Flag-Pgrmc1, encoding murine Pgrmc1 was generated by subcloning the Pgrmc1 CDS (NM_016783.4 nucleotides 95–682) to pcDNA3.1. Pgrmc1 was tagged at the N-terminus with a single Flag tag. The mammalian expression vector Y113F Flag-Pgrmc1 was generated from the Flag-Pgrmc1 mammalian expression vector by site-directed mutagenesis (NM_016783.4 nucleotide 432 A->T). AAV8 EGFP, AAV8 Flag-Pgrmc1, and AAV8 Y113F Flag-Pgrmc1 viral particles were produced by the University of Pennsylvania Vector Core.The plasmids for the production of AAV8 viral particles encoding murine Pgrmc1 were generated by replacing the The bacterial expression vectors for truncated human PGRMC1 and Y113F PGRMC1 were generated by subcloning the sequence for amino acids 43 to 195 (NP_006658.1) from the Flag-PGRMC1 and Y113F Flag-PGRMC1 mammalian expression vectors into pET28a (EMD BioScience). The bacterial expression vector for human Y113F, K163A, K164A (3X MUT) PGRMC1 was generated by PCR mutagenesis (NM_006667.5 nucleotides 1585–1590 AAGTAT->GCGTTT) of the Y113F PGRMC1 bacterial expression vector.PGRMC1 KO cell line from the human fibroblast cell line SV589 (gift of Drs Michael Brown and Joseph Goldstein) was generated by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated genome editing ; 10% (v/v) FBS; and penicillin-streptomycin  at 37 °C with 5% CO2. Human PGRMC1 (NM_006667.5) contains three exons and is translated into a 195-aa protein. A CRISPR guide RNA (gRNA) to target sequence nucleotides 114 to 133 (5′-GCTCTCCAGATCGCTTGGGT-3′) located in exon 1 was cloned into the Cas9-gRNA vector PX459 (Addgene #48139) . Transfected SV589 cells were selected for growth in the presence of 1 μg/ml puromycin  for 3 days. Single clones were isolated by dilution cloning. Genomic DNA flanking the gRNA target site was amplified by standard PCR, then sequenced by Sanger sequencing. Primer sequences were human PGRMC1 forward: 5′-CTCCCAGGTAGAACTGAG-3′ and reverse 5′-CACATC GAACACCTTGCC-3′. One isolated clone contained a 10 bp deletion and an 11 bp deletion in both PGRMC1 alleles. Knockout of PGRMC1 was further confirmed by immunoblotting.A  editing . SV589 a #48139) . To genePGRMC1 KO cell line, 1 × 106 SV589 or 2 × 106PGRMC1 KO cells were plated in 10-cm plates. The next day, the cells were transfected with 15 μg plasmid DNA (15 μg pcDNA 3.1 or 10 μg Flag-PGRMC1 plus 5 μg pcDNA3.1), with 50 μl Polyfect (Qiagen) according to manufacturer’s instructions for HeLa cells. The cells were harvested 24 h after transfection. For chase experiments, 1.6 × 106PGRMC1 KO cells were seeded in a 10-cm plate. Two days after seeding, cells were transfected with a total of 15 μg plasmid DNA , with 50 μl Polyfect (Qiagen) according to manufacturer’s instructions for HeLa cells. After 24 h, the cells were split at 1:6 to a 6-well plate. At 48 h posttransfection, the cells were treated with 100 μg/ml emetine (Sigma) for 4 h. For Flag coimmunoprecipitation, 1.5 × 106PGRMC1 KO cells were seeded in a 10-cm plate. The next day the cells were transfected with 15.05 μg plasmid DNA  with 50 μl Polyfect (Qiagen) according to manufacturer’s instructions for HeLa cells. Cells were harvested 24 h after transfection and used immediately.For validation of the Mycoplasma pulmonis, fur mites, and pinworms. Mice were housed in social groups (2–5 mice) of the same sex in individually ventilated cages (Allentown Caging Inc) with autoclaved corncob bedding  and nesting material . Cages were changed every 14 days. Autoclaved feed  was provided ad libitum, and water was provided via in-cage automated watering systems . The room was maintained at 22 ± 1 °C on a 14:10 light:dark cycle at 40 to 70% humidity. Euthanasia was performed under isoflurane anesthesia by cervical dislocation. Whole blood was collected via cardiocentesis and placed in a heparin-coated green-top tube for plasma separation or a yellow-top tube with serum separator gel for serum separation (BD Biosciences). Clinical chemistry was performed with a Vet AceTM analyzer (Alfa Wassermann). Complete Blood Count (CBC) was performed with a Procyte Dx (Idexx Laboratories Inc). After harvest, liver tissue was flash frozen in liquid nitrogen for molecular analysis or stored in 10% neutral buffered formalin (Sigma) for histology.The Johns Hopkins animal care and use program is accredited by AAALAC international, and the Johns Hopkins Institutional Animal Care and Use Committee (IACUC) reviewed and approved all procedures. Routine health surveillance using dirty bedding sentinel serology indicated that the mice were free of the following organisms: mouse hepatitis virus, minute virus of mice, mouse parvovirus, epizootic diarrhea of infant mice (rotavirus), Theilers murine encephalomyelitis virus, murine norovirus, Sendai virus, pneumonia virus of mice, reovirus, lymphocytic choriomeningitis virus, ectromelia virus, mouse adenovirus (FL & K87), mouse cytomegalovirus, Pgrmc1 floxed mice (C57BL/6N) were generated by inGenious Targeting Laboratory (iTL). The long homology arm of the targeting construct was 889 to 6921 bp upstream of exon 1 (Ensembl GRCm38 X chromosome), and the short homology arm was 296 to 2102 bp downstream of exon 2. The targeting construct inserted loxP sites 568 bp upstream of exon 1 and 1906 bp downstream of exon 2. The targeting construct also contained a neomycin cassette flanked by FRT sites within the loxP sites. Targeted iTL IC1 (C57BL/6N) male embryonic stem cells were microinjected into Balb/c blastocysts. Resulting chimeras with a high percentage black coat color were mated to C57BL/6N mice to generate F1 heterozygous offspring. F1 female heterozygotes were crossed to male Sox2-Cre mice (B6.Cg-Tg(Sox2-cre)1Amc/J, Jackson Laboratories) at Johns Hopkins animal facilities. The resulting whole-body Pgrmc1 knockout (KO) mice lacking exons 1 and 2 were bred for this study. Male mice aged 8 to 12 weeks were used for this study, except for the acetaminophen-induced liver injury study where male mice aged 13 to 15 weeks were used.E. coli using Ni2+-NTA agarose (Qiagen). PGRMC1 antiserum (5944) was generated by Covance using a standard protocol.Hexahistidine-tagged recombinant human PGRMC1 (amino acids 43–195) antigen was purified from g for 10 to 15 min at 4 °C, and the supernatant was processed as the sample.To make liver lysates, liver tissue was disrupted with a Tissue Lyser (Qiagen) and steel beads in the presence of RIPA buffer , 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS) plus 1X protease inhibitors  at 4 °C. Cell lysates were made by resuspending cell pellets in RIPA buffer (approximately five pellet volumes) with protease inhibitors  on ice. Lysates were cleared by spinning in a microfuge at 21,130g for 30 min at 4 °C. The supernatant was spun at 100,000g for 90 min at 4 °C. The pellet was washed with Resuspension Buffer  and spun at 100,000g for 60 min at 4 °C. The pellet was defined as the enriched membrane fraction. It was resuspended in Storage Buffer  glycerol) with 0.1 mM DTT to 10 to 20 mg protein/ml. DTT was omitted for samples intended for coimmunoprecipitation. All buffers contained protease inhibitors  at 1X or at 2X for samples intended for coimmunoprecipitation.Liver membrane fractions were prepared as described previously  with somProtein concentration in lysates and membrane fractions was measured using the BCA kit (Pierce), and samples were mixed with 5X SDS loading buffer  SDS, 25% (v/v) glycerol, 0.2% (w/v) bromophenol blue) with or without 12.5% (v/v) β-mercaptoethanol (BME). After heating at 65 °C for 10 to 15 min, proteins were subjected to SDS-PAGE and transferred to nitrocellulose membranes (BioRad). The membranes were blocked with 5% (w/v) dried milk in PBS-T [PBS +0.05% (v/v) Tween 20], then incubated with primary antibodies. Primary antibodies and working concentrations included: rabbit anti-PGRMC1 5944 (1:500–1:1000), mouse anti-Cyp1a2 (D15) , rabbit anti-Cyp2e1 , rabbit anti-CYP3A4 , mouse anti-β-Actin (C4) , mouse anti-c-Myc (9E10) , rabbit anti-GFP , rabbit anti-calnexin , rabbit anti-Cyp2F2  , rabbit 11 particles of AAV8 GFP, Flag-Pgrmc1, or Y113F Flag-Pgrmc1. All buffers contained protease inhibitors . One milligram of liver membrane protein in 150 μl microsome Storage Buffer (6.67 mg/ml) was diluted to a final concentration of 2 mg/ml in 1 mM MgCl2. The sample was treated with ten units of benzonase (EMD Millipore) on ice for 30 min. Next, an equal volume of 2X TAP lysis buffer  was added to dilute the sample to 1 mg/ml. Insoluble material was removed by centrifugation at 20,000g for 10 to 15 min at 4 °C. Flag-M2 agarose (Sigma) was washed once in three bead volumes of 1X TAP lysis buffer containing 0.715 mM MgCl2. Then, beads were blocked in 20 bead volumes of wild-type liver lysate at 1 mg/ml prepared from a 12-week-old, wild-type male mouse in 1X TAP lysis buffer. The beads were blocked for 1 h at 4 °C and then washed three times in ten bead volumes of 1X TAP lysis buffer containing 0.715 mM MgCl2. Each sample was incubated with 25 μl Flag M2 beads for 1 h at 4 °C while rotating. The beads were washed 3X in 20 bead volumes of 1X TAP lysis buffer. The fourth and final wash was done in 20 bead volumes of 1X TAP lysis buffer containing 0.1% (w/v) DDM, and the samples transferred to a fresh tube. The bound fraction was eluted by incubating the beads at 65 °C for 10 min in 50 μl of elution buffer  SDS). For mass spectrometry analysis of Pgrmc1-binding proteins, the Flag coimmunoprecipitation was performed in technical triplicate for each biological replicate . Equal volumes of eluate from each technical replicate were combined to form a pooled sample for each biological replicate that was subsequently analyzed by mass spectrometry. For mass spectrometry of the membrane proteome of AAV8 GFP, Flag-Pgrmc1, or Y113F Flag-Pgrmc1 infected Pgrmc1 KO liver, the samples were prepared as described above with benzonase treatment and diluted to 1 mg/ml before sonication.Flag pull-downs from mouse liver were performed on samples from male mice sacrificed 8 days after tail vein infection with 5 × 10g for 10 to 15 min at 4 °C. Flag-M2 agarose (Sigma) was washed once in six bead volumes of 1X TAP lysis buffer. Then, the beads were blocked in six bead volumes of 3% BSA  in 1X TAP lysis buffer. The beads were blocked for 45 to 60 min at room temperature and then washed twice in six bead volumes of 1X TAP lysis buffer. Each sample (300 μg protein) was incubated with 10 μl Flag M2 agarose (Sigma) at a protein concentration of 1 mg/ml for 1 h at 4 °C while rotating. The beads were washed 3X in 30 to 50 bead volumes of 1X TAP lysis buffer. After the second wash, the samples were transferred to a fresh tube. The bound fraction was eluted by incubating the beads at 65 °C for 10 min in 30 μl of 1X SDS loading buffer without BME.For Flag pull-downs from transfected cultured cells, cells were lysed in 1X TAP lysis buffer with protease inhibitors . Lysates were solubilized by rotating at 4 °C for 1 h and cleared by spinning in a microfuge at 20,000Tissues fixed in 10% neutral buffered formalin (Sigma) were processed, paraffin embedded, sectioned (4 μm), and stained with hematoxylin and eosin according to standard protocols by Oncology Tissue Services (Johns Hopkins University). Slides were viewed on a Nikon Eclipse Ci microscope and images captured using Nikon DS-Fi2 camera. Images were edited for white balance across the whole image using Adobe Photoshop.g at 4 °C for 15 min. The supernatant was dried in a speed vac overnight, and pellets were saved for protein quantification. Dried samples were constituted with 20 mM phosphate buffer containing 0.1 mM trimethylsilylpropionic acid (TMSP) and 0.1 mM NaN3. Proton NMR spectra were acquired, analyzed, and quantified as previously described (Mice were fasted for 4 h before sacrifice around 1:00 PM (ZT-7). Liver tissue (approximately 300 mg) was snap frozen in liquid nitrogen and processed the same day. Briefly, the tissue was homogenized in two tissue volumes of 20 mM phosphate buffer, pH 7.4. To the supernatant, four tissue volumes of methanol were added, and the samples vortexed before incubation at −20 °C for 30 min. The samples were spun at 13,000escribed .Pgrmc1 KO livers, quantitative proteomics was employed. Equal masses of liver membrane protein from four WT and four KO mice (Replicate #1) or five WT and five KO mice (Replicate #2) were pooled by genotype to produce two biological replicates per genotype. In each MS run, a WT and a KO pool were labeled with three different isobaric tags, resulting in three technical replicates per genotype per experiment. The samples were digested with the protease trypsin, which specifically cleaves at the carboxyl side of the amino acids lysine and arginine. Next, the samples were labeled with unique Tandem Mass Tag (TMT) 10-plex reagent (Thermo Fisher) according to the manufacturer’s protocol. The combined labeled sample was cleaned up from excess TMT tag with a detergent removal spin column (Pierce).To quantify protein levels in Mus musculus RefSeq v.69  using the Mascot (Matrix Science) search engine v.2.5  running through Proteome Discoverer v.1.4 (DBVersion: 79) (Thermo Fisher) with one or two missed cleavages allowed, respectively; fixed modifications: methylthio (C), TMT6plex (N-term); variable modifications: oxidation (M), deamidation (NQ), and TMT6plex (K); and a tolerance of 10 ppm MS and 0.018 or 0.03 Da MS2, respectively.The labeled sample was fractionated with five or four basic, reversed-phase fractions of 5%, 15%, 20%, 30%, and 75% or 5%, 15%, 25%, and 75% (v/v) acetonitrile in 10 mM triethylammonium bicarbonate buffer (TEAB). Each fraction was analyzed by liquid chromatography interfaced with tandem mass spectrometry (MS/MS) using a Nano-Acquity HPLC system interfaced with a QExactive HF (Thermo Fisher). A stepped collision energy 32 s/30 s was used for fragmentation. Precursor and fragment ions were analyzed at resolutions 60,000 and 120,000, respectively, and automatic gain control target values at 1e5 with 200 ms maximum injection time and 3e5 with 50 ms maximum injection time, respectively. Data was searched against t test statistics  less than 1% (based on a concatenated decoy database) in which all reporter ions were detected were included for downstream analyses. Spectra with isolation interference greater than 30% were excluded as well. Within each TMT experiment, relative protein abundances were quantified by a robust median sweep algorithm , 58. Briatistics . For mulatistics  were calvia Oasis HLB cartridges (Waters). Briefly, cartridges were conditioned with 100% (v/v) methanol and equilibrated with 100% (v/v) water. Samples were loaded and then washed twice with 5% (v/v) methanol in water. Peptides were then eluted with 100% (v/v) methanol and dried via vacufuge.For proteomics on AAV-infected liver, the samples were digested with the protease trypsin, which has specificity for the amino acids lysine and arginine at the C-terminus of a cleaved peptide. Next, the samples were labeled with unique TMT 10-plex reagent (Thermo Fisher) according to the manufacturer’s protocol. The combined labeled samples were cleaned up from excess TMT tag with a detergent removal spin column (Pierce). The labeled samples were resuspended in an aqueous 5% (v/v) methanol and 0.5% (v/v) formic acid solution and then desalted via Agilent 3100 OFFGEL isoelectric focusing gel in a 12-well setup according to the manufacturer’s protocol. Upon completion, fractions were separately desalted with Pierce C18 spin columns (Thermo Fisher) following the provided protocol. Column resin was activated with 50% (v/v) acetonitrile and equilibrated with 5% (v/v) acetonitrile +0.5% (v/v) trifluoroacetic acid solutions. Fractions were loaded onto columns and washed with 5% (v/v) acetonitrile +0.5% (v/v) trifluoroacetic acid solution. Samples were eluted via two cycles of addition of 70% (v/v) acetonitrile solution to the columns. All sample fractions were dried separately via vacufuge.Peptides were then fractionated via full scan  and a data-dependent MS2 top 10 scan , respectively.Each fraction was then analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) using an nLC-1200 nano-flow liquid chromatography system (Thermo Fisher) interfaced with a Q-Exactive mass spectrometer (Thermo Fisher). Precursor and fragment ions were analyzed Pgrmc1 KO liver were searched together against the M. musculus 10090 Uniprot reference proteome  using the Sequest HT search engine running through Proteome Discoverer v.2.1 (Thermo Fisher) with one or two missed cleavages allowed and a precursor mass tolerance of 8 ppm and fragment mass tolerance of 0.02 Da. The trypsin protease cleavage sites permitted were only on the C-terminal sides of lysine and arginine. Dynamic modifications of methionine oxidation and deamidation of asparagine and glutamine, along with static modification of carbamidomethylation of cysteine, were permitted. Peptide assignments were validated using the Target Decoy Peptide Spectral Match Validator node with a relaxed FDR of <0.05 and a strict FDR of <0.01. Peptides from the LC-MS/MS run for the quantification of Flag-Pgrmc1 and Y113F Flag-Pgrmc1 binding partners had a Spectrum File beginning with “03,” while peptides from the LC-MS/MS run for the quantification of the membrane proteome of AAV8-infected Pgrmc1 KO liver had a Spectrum File beginning with “04.” Included peptides had (1) a Master Accession number, (2) were marked “Unique,” (3) had an isolation interference that was ≤30%, and (4) the Peptide Quan Info was used.Data from both LC-MS/MS runs for the quantification of Flag-Pgrmc1 and Y113F Flag-Pgrmc1 binding partners and the quantification of the membrane proteome of AAV8 infected For candidate Flag-Pgrmc1 and Y113F Flag-Pgrmc1 binding partner identification, reporter ion abundances for each spectrum of a protein were summed, then the protein abundance medians for each condition were taken. For Flag-Pgrmc1 and Y113F Flag-Pgrmc1, the ratio of protein abundance to GFP was compared for enrichment. Candidate Flag-Pgrmc1 or Y113F Flag-Pgrmc1 binding partners were those proteins with a fold change (Flag-Pgrmc1/GFP or Y113F Flag-Pgrmc1/GFP) ≥20%. For stringent quantitative analysis of Flag-Pgrmc1 and Y113F Flag-Pgrmc1 binding partners, only peptides present in at least two biological replicates were used to calculate the protein abundance. Peptides present only in Y113F Flag-Pgrmc1 samples were also eliminated.Pgrmc1 KO liver, all reporter ion intensities that belong to the same protein were used as the measure (sum) of that protein abundance in the sample. Relative protein abundances were quantified by a robust median sweep algorithm as described above except the measure of a protein’s abundance in a sample was the sum of the peptides. For statistical analysis, conditions were analyzed in R by linear modeling with limma and p-values adjusted by the Benjamini and Hochberg FDR method  . For RNA, up- and downregulated genes were considered to be those genes with fold change ≥40% and a probability of differential expression ≥0.95. Genes were searched by Ref-seq gene name. For protein, proteins were searched by Uniprot ID. In the experiment comparing protein expression in WT and Pgrmc1 KO liver, differentially expressed proteins were considered to be those proteins with fold change ≥20% and the absolute value of the signal-to-noise ratio ≥2. The differentially expressed proteins were compared with the reference list of all proteins measured in the experiment. In the experiment comparing protein expression in AAV8 GFP, Flag-Pgrmc1, or Y113F Flag-Pgrmc1 infected Pgrmc1 KO livers, differentially expressed proteins were those with unadjusted p-values ≤ 0.05. The differentially expressed proteins were compared with the reference list of all proteins measured in the experiment.GO Term Analysis was conducted using PANTHER version 14  . A FisheM. musculus mm10 reference genome. The “rsem-run-ebseq” and “rsem-control-fdr” scripts provided by Rsem were used to run EBSeq to perform differential expression analysis. All default parameters were used, except “FDR_rate” was set to 0.05.Total RNA was prepared from ∼30 mg of snap frozen liver with RNA STAT-60 (Amsbio) reagent according to the manufacturer’s instructions. DNase digestion was performed on-column with an RNAeasy Kit (Qiagen) according to manufacturer’s instructions. RNA concentration was estimated with a NanoDrop (Thermo Fisher). For RNA-seq, equal masses of RNA (2.5 μg) from mice of the same genotype were pooled to create one sample per genotype. Library preparation was completed with an Illumina TruSeq Stranded Total RNA kit. Samples were analyzed on a HiSeq 2500 (Illumina) machine in Rapid Run mode with paired-end 100 bp × 100 bp sequencing. CASAVA 1.8.2 (Illumina) was used to convert BCL files to FASTQ files. Default parameters were used. Rsem-1.2.09 was used for running the alignments as well as generating gene and transcript expression levels. The “rsem-calculate-expression” module was used with the following options: “bowtie-chunkmbs 200,” “calc-ci,” “output-genome-bam,” “paired-end,” and “forward-prob.” The data were aligned to the Km and Vmax determined using GraphPad Prism 7.05.The 7-ethoxycoumarin O-deethylation (ECOD) assay was conducted as described previously . For eacvia the addition of caffeine (Sigma) to 50 μM and incubated for 60 min at 37 °C while shaking. Reactions were quenched via protein precipitation by direct addition of 50 μl of ice-cold acetonitrile (to 50% v/v) and incubated on ice for 10 min. Precipitate was pelleted by centrifugation for 10 min at 10,000g at 4 °C. The supernatant was transferred and dried in a vacuum centrifuge. Samples were reconstituted in 25 μl water prior to mass spectrometry.Pooled membrane protein as described for the ECOD assay was preincubated at 2 mg/ml in 100 mM potassium phosphate, pH 7.4 with an NADPH regenerating system (Corning) at 37 °C for 5 min. Reactions were initiated 18 column  was performed using a mobile-phase system of water with 0.1% (v/v) formic acid (mobile phase A) and acetonitrile with 0.1% (v/v) formic acid (mobile phase B) at a flow rate of 400 μl/min. The gradient used was as follows: 0% B from 0 to 4 min, 0 to 5% B from 4 to 15 min, 5 to 100% B from 15 to 16 min, 100% B from 16 to 21 min, 100 to 0% B from 21 to 22 min, and 0% B from 22 to 25 min. Paraxanthine detection was performed in positive ion mode using a transition of m/z 181.0720 > 124.0509. Comparisons of relative amounts of paraxanthine formed were performed using the integrated peak area from the paraxanthine chromatograms.Reconstituted samples were resolved using a Dionex Ultimate 3000 uHPLC system and analytes were detected using a coupled Q-Exactive benchtop Orbitrap mass spectrometer (Thermo Fisher). Separation of analytes on an Agilent Polaris Cet al.  visible spectrophotometer. Peak absorption at 515 nm was used to determine the Cyp2e1 activity using a p-nitrocatechol  standard curve as described in Chang et al.  per kg body weight in 50% saline/DMSO (v/v). All mice were given DietGel Recovery (Clear H2O) in the cage. Mice were then euthanized 24 h postinjection. Whole blood was collected via cardiocentesis, and serum was separated as described above. The liver was harvested, and the median and left lateral lobes were individually separated. A horizontal, transverse section  was taken from the median lobe. A diagonal, transverse section to include the hilus was taken from the left lateral lobe. All sections were fixed in 10% neutral buffered formalin (Sigma). Liver sections were processed for histologic analysis as described above.Wild-type and 2, 2 mM ATP, 20 mM Imidazole, and 1x protease inhibitors  at room temperature for 15 min. The cell lysates were clarified by centrifugation at 80,000g at 4 °C for 45 min and purified over a 1 ml HisTrap HP column  using a 25 mM-500 mM imidazole gradient in 20 mM HEPES-KOH, pH 7.4; 150 mM NaCl buffer. The purified proteins were desalted using PD10 columns  equilibrated with 50 mM HEPES-KOH, pH 7.4; 150 mM NaCl; 2 mM MgCl2; 5% (v/v) glycerol and concentrated using 10,000 MW cutoff filters (Amicon). The N-terminal histidine tag was removed by incubating the concentrated proteins with 50 U/ml Thrombin  at room temperature for 16 h, followed by desalting by PD10 column. The recombinant proteins had a residual tetra peptide (Gly-Ser-His-Ser) tag at the N-terminus after thrombin cleavage.Recombinant proteins were expressed in BL21 CodonPlus(DE3)RIPL cells (Agilent) using 200 μM IPTG and overnight shaking at 20 °C. Cells were lysed in B-PER (Thermo Fisher) reagent supplemented with 25 U/ml Benzonase (EMD Biosciences), 2 mM MgClThe majority of the bacterially expressed, tag-removed PGRMC1 lacked heme (apo PGRMC1). To test the heme-binding ability of the recombinant PGRMC1 (rPGRMC1), Y113F rPGRMC1, and 3X MUT rPGRMC1, proteins (1 μM) were incubated with 100 μM Hemin  in PBS for 15 min at 30 °C. Hemin was diluted from a 10 mM stock in DMSO. Thereafter, the proteins were desalted using PBS equilibrated PD10 columns and concentrated using 10,000 MW filters (Amicon). Hemin without any added protein was subjected to the same procedure as a negative control.403 = 170 mM−1 cm−1 in DMSO  were performed in GraphPad Prism versions 7.05 to 8.2.0. Analysis of large datasets was performed using R.Sample sizes and numbers of biological and technical replicates are noted for each experiment. Statistical tests (https://www.ncbi.nlm.nih.gov/geo/), accession GSE174375. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (http://www.ebi.ac.uk/pride) with the dataset identifiers PXD028238, PXD028284, and PXD028288. All remaining data are contained within the article and RNA-Seq data are deposited at NCBI GEO (he PRIDE  partner This article contains


However, there has been no study concerning the continued use of blonanserin.Unlike other antipsychotics, our previous positron emission tomography (PET) study demonstrated that a single dose of blonanserin occupied dopamine D2 and D3 receptor occupancies in patients with schizophrenia who had been treated with blonanserin.We examined D2 and D3 receptor occupancies were evaluated by [11C]-(+)-PHNO. We used nondisplaceable binding potential (BPND) of 6 healthy subjects which we previously reported as baseline. To consider the effect of upregulation of D3 receptor by continued use of antipsychotics, D3 receptor occupancy by blonanserin in seven subjects who completed 2 PET scans were re-analyzed by using BPND of olanzapine condition as baseline.Thirteen patients with schizophrenia participated. PET examinations were performed on patients treated with clinical dosage of blonanserin or olanzapine alone. A crossover design was used in which seven patients switched drugs after the first scan, and PET examinations were conducted again. D50 was 0.30 ng/mL for D2 receptor for caudate and putamen  and 0.70 ng/mL for D3 receptor for globus pallidus and substantia nigra . EC50 for D3 receptor of blonanserin changed to 0.22 ng/mL  when we used BPND of olanzapine condition as baseline.Average occupancy by olanzapine (10.8 ± 6.0 mg/day) was as follows: caudate 32.8 ± 18.3%, putamen 26.3 ± 18.2%, globus pallidus − 33.7 ± 34.9%, substantia nigra − 112.8 ± 90.7%. Average occupancy by blonanserin (12.8 ± 5.6 mg/day) was as follows: caudate 61.0 ± 8.3%, putamen 55.5 ± 9.5%, globus pallidus 48.9 ± 12.4%, substantia nigra 34.0 ± 20.6%. EC2 and D3 receptors in patients with schizophrenia.Our study confirmed that blonanserin occupied both D In terms of the treatment of schizophrenia, D2 receptor has been thought to be strongly associated with the pathology of schizophrenia and be a major target for its treatment. Dopamine D3 receptor has similarities to the other members of the D2-like receptor family, but D3 receptor has very high affinity for dopamine and modulates dopamine release as an autoreceptor -PHNO indicated that 100% of the signal in the substantia nigra (SN), 67% in the globus pallidus (GP), and 26% in the ventral striatum represent D3 receptor sites  study with [11C]-(+)-PHNO that several antipsychotics  did not decrease, or even increased the in vivo nondisplaceable binding potential (BPND) of D3 receptors in human brain -PHNO gives a mixed D2/3 signal composed of differing D2 and D3 proportions, and therefore previous studies measured BPND of D3 receptors in D3-receptor rich-regions. Another study also reported that chronically administered antipsychotics  showed lower selectivity for D3 compared with D2 receptors ex vivo than in vitro in rat brain  similarly to a D2-rich region  in rat brain, while risperidone, olanzapine, and aripiprazole did not -PHNO and PET, we demonstrated that a single dose of 12 mg of blonanserin occupied D3 receptors to the same degree as D2 receptors  -IV for schizophrenia. Inclusion criteria were as follows: (1) schizophrenia patients treated with blonanserin or olanzapine alone for 4 weeks or more, and those who did not change their dose for at least 2 weeks; (2) subjects who agreed to change from one drug to the other, (3) subjects who scored less than 120 on the Positive and Negative Syndrome Scale (PANSS15) at screening. Exclusion criteria were as follows: (1) subjects with past or current serious medical illness and/or organic brain diseases, (2) subjects with contraindication for the use of magnetic resonance imaging (MRI), (3) subjects with contraindication for blonanserin and olanzapine, (4) subjects treated with electroconvulsive therapy within 3 months before the screening, (5) subjects taking tandospirone at the time of screening, as there was a report that buspirone, which is in the same drug family, occupies DThis study was designed as an open-label protocol. PET examination was performed on subjects who had been treated with blonanserin or olanzapine. A crossover design was then used in which patients switched drugs after the first scan, and PET examination was performed again after 2 weeks or longer. The doses of both drugs were within their clinical dose range. The mean dose of olanzapine was 10.8 ± 6.0 (range: 2.5–20) mg/day, and that of blonanserin was 12.8 ± 5.6 (8–24) mg/day. After complete explanation of the study, written informed consent was obtained from all participants. This study was approved by the institutional review board of Nippon Medical School Hospital, Japan.Thirteen subjects participated in this study. The patients’ characteristics are listed in Table 137Cs source. Dynamic PET scan was performed for 90 min  after i.v. bolus injection of [11C]-(+)-PHNO. Injected radioactivity was 139.1 to 386.4 MBq (309.8 ± 79.7 (mean ± SD MBq) for olanzapine-condition; 351.2 ± 34.3 MBq for blonanserin-condition). The injected mass of [11C]-(+)-PHNO was 0.5–2.5 μg . Molar radioactivity was 55.3–141.0 GBq/μmol  at the time of injection.PET scans were performed with Eminence SET-3000GCT-X  to measure regional brain radioactivity. This scanner provides 99 sections with an axial field of view of 26.0 cm. Spatial resolution was 3.45 mm in-plane and 3.72 mm axially full-width at half-maximum. A head fixation device was used during the scans. A 15-min transmission scan was done to correct for attenuation using a MRI of the brain was acquired with 1.5 T MR imaging, Intera 1.5 T Achieva Nova  as proton density image . These images were used for analysis of the PET scans.Venous blood samples were taken just before the PET scans, collected in tubes containing EDTA-2Na, and centrifuged at 3000 rpm for 10 min at 4 °C. Separated plasma samples were stored at − 80 °C until analysis. The plasma concentration of blonanserin was measured by validated method using high-performance liquid chromatography-tandem mass spectrometry with a target lower quantification limit of 0.001 ng/mL . The plasma concentration of olanzapine was measured by validated method using high-performance liquid chromatography-tandem mass spectrometry with a target lower quantification limit of 0.0001 ng/mL .2-rich regions and the substantia nigra and globus pallidus as D3-rich regions, based on Searle’s study with [11C]-(+)-PHNO . Regions of interest (ROIs) were defined for the caudate, putamen, globus pallidus, substantia nigra, and cerebellum in accordance with Tziortzi’s study -PHNO was performed using a simplified reference tissue model (Lammertsma and Hume 11C]-(+)-PHNO binding. This model has been validated to reliably estimate BPND, which compares the concentration of radioligand in the receptor-rich region with the receptor-free region -PHNO  , which we reported in a previous study  , putamen , globus pallidus , and substantia nigra .BPC is the plasma concentration of drug, Emax is the maximum occupancy, and EC50 is the plasma concentration required to achieve 50% occupancy  was as follows: caudate nucleus 32.8 ± 18.3%, putamen 26.3 ± 18.2%, globus pallidus − 33.7 ± 34.9%, substantia nigra − 112.8 ± 90.7%. The average level of occupancy by blonanserin (12.8 ± 5.6 mg/day) was as follows: caudate nucleus 61.0 ± 8.3%, putamen 55.5 ± 9.5%, globus pallidus 48.9 ± 12.4%, substantia nigra 34.0 ± 20.6%. Correlations between the plasma concentration of blonanserin and receptor occupancy in D2-rich and D3-rich regions are shown in Fig. 50 of D2 receptor was 0.30 ng/mL , while EC50 of D3 receptor was 0.70 ng/mL .We analyzed D3 receptor occupancy by blonanserin using individual BPND of olanzapine condition as baseline. The results are shown in Table 3 was higher than when using the baseline BPND in healthy volunteers (67.9 ± 11.8% versus 44.7 ± 16.6%) . EC50 of D3 receptor occupancy was 0.22 ng/mL , which was close to that of D2 receptor occupancy  was slightly lower than the D2 receptor occupancy (55.5 to 61.0%) using HV as baseline. This result seemed to be due to the influence of upregulation. Second, schizophrenia patients showed increased [11C]-(+)-PHNO binding compared to healthy subjects even if they were untreated  for blonanserin in the same 7 subjects by using HV as baseline. Thus, our results confirmed that blonanserin occupied D3 receptor as well as D2 receptor in patients with schizophrenia. Blonanserin might be an important target for further studies regarding the therapeutic efficacy of D3 receptor blockade by antipsychotic drugs.The occupancy of D3 receptor occupancy in the substantia nigra by olanzapine in 8 patients with schizophrenia was − 124.1 ± 87.6%. We thought that the negative occupancy might reflect upregulation. Previous studies did not measure the occupancy of the substantia nigra (Graff-Guerrero et al. 3 receptor by olanzapine; however, the evaluation of a large number of subjects and/or using same subjects both before and after its administration will be needed for clarification.In this study, the degree of DND of D2 receptor was negatively correlated with age in the caudate, while that of D3 receptor was not correlated with age in the globus pallidus and substantia nigra (Nakajima et al. 2 receptor using younger-aged controls for baseline might be influenced by an age effect if controls would be older. Third, we used all-male HVs, whereas 46% of the patients were female. In this regard, a previous study indicated that D3 receptor differed between male and female rhesus monkeys (Martelle et al. 3 upregulation exactly as there was no drug-free baseline condition. Fifth, 7 of the 13 participants in this study were smokers while all HVs were non-smokers. We could not rule out the effects of smoking, as it has been shown to have an effect on the dopamine system (Le Foll et al. We should acknowledge several limitations to this study. First, our sample size was small. Furthermore, 6 of the 13 subjects underwent only one PET scan. Therefore, additional studies including larger numbers of subjects and longitudinal designs are essential for the generalization of our findings. Second, we used a younger-aged control group compared to the patients. BP3 receptors to the same degree as D2 receptors in the brains of schizophrenia patients. By more discussions on the therapeutic effects of blonanserin, which is now known to clearly possess in vivo D3 receptor antagonism, we may be able to consider the relevance of anti-dopamine D3 receptor acivities as well as the therapeutic effects on cognitive impairments and negative symptoms of mental disorders.In conclusion, our study confirmed that continuous usage of blonanserin occupied dopamine D