Here, we study in mice the role of the intracellular Ca2+ channel Ryanodine Receptor 2 (RyR2) in synaptic plasticity and memory formation. We demonstrate that loss of RyR2 in pyramidal neurons of the hippocampus impairs maintenance and activity-evoked structural plasticity of dendritic spines during memory acquisition. Furthermore, post-developmental deletion of RyR2 causes loss of excitatory synapses, dendritic sparsification, overcompensatory excitability, network hyperactivity and disruption of spatially tuned place cells. Altogether, our data underpin RyR2 as a link between spine remodeling, circuitry dysfunction and memory acquisition, which closely resemble pathological mechanisms observed in neurodegenerative disorders.Dendritic spines are postsynaptic domains that shape structural and functional properties of neurons. Upon neuronal activity, Ca The membrane was incubated with the radioactively labeled probe ON at 65 °C. Fragment size was visualized using HyperfilmTM MP : labeled membranes were incubated in an autoradiography chamber at −80 °C for 3 days and films developed using a CURIX 60 (AGFA) automated developing machine.Genetic deletion of Ryr1 F 5′-AGCTGACAATAAGAGCAAAATGG-3′; Ryr1 R 5′-TGATCTGAGCCACCTGACTG-3′; Ryr2 F 5′-CTACCCGAACCTCCAGCGATACT-3′; Ryr2 R 5′-GCAAAAGAAGGAGATGATGGTGTG-3′; Ryr3 F 5′-CAGTGGGTATGCTTCCCATAA-3′; Ryr3 R 5′-GGCCAGCTTGCAGAATAGG-3′; Ip3r1 F 5′-CCACAGAGCAGGAGCTTGAA-3′; Ip3r1 R 5′-TTGCCAAAGCTGGTAAGGCT-3′; Ip3r2 F 5′-ACATCGTGTCCCTGTACGC-3′; Ip3r2 R 5′-TGGCTGCAAAGAGGTCAACT-3′; Ip3r3 F 5′-CTACCCGAACCTCCAGCGATACT-3′; Ip3r3 5′-CAGGAACTTCTCCTGCTCCG-3′; Serca2 F 5′-GATGGGGCTCCAACGAATTG-3′; Serca2 R 5′-TCTTCCCCTTCCTCGAACCA-3′; Serca3 F 5′-GACGCTCACCACCAATCAGA-3′; Serca3 R 5′-CTCCCCTTGCCTCACTTCG-3′; CaV1.2 F 5′-CCTGGCCATGCAGCACTAT-3′; CaV1.2 R 5′-GCTCCCAATGACGATGAGGA-3′; CaV1.3 F 5′-TGGCCATGCAGCACTATGAG-3′; CaV1.3 R 5′- CGTGTTCCAGGCGTCACTAA-3′; ΔΔCt values were normilized to β-actin and represented as fold change compared to control mice.RT-PCR was used to quantify gene expression levels. RNA was extracted from snap frozen brains using RNeasy kit (Qiagen) and tested for purify (A260/280) and integrity. RNA extracted (100 ng) was retrotranscribed with qScript cDNA SuperMix (Quanta Biosciences). Gene expression analysis was conducted with Fast SYBR Green Master Mix (Applied Biosystems) on a Step One Plus Real-Time PCR System (Applied Biosystems). The following primers were used: Samples were prepared homogenizing frozen tissues in RIPA buffer (Sigma Aldrich) supplemented with proteases  and phosphatases inhibitor  using an IKA Ultra-Turrax homogenizer in ice. Proteins extracted were subjected to SDS-page and immunoblot on nitrocellulose membranes (Amersham). Between 20 and 40 μg of protein were loaded and resolved on 10–12% acrylamide gel and transferred onto nitrocellulose membranes (Bio-Rad). Due to the high molecular weight of RyR2, 7% acrylamide gels were used and stacking gels were kept intact. Overnight transferring on nitrocellulose membranes were run at 20 V for 15 h at 4 °C. Membranes were blocked in 5% milk 0.05% TBS-Tween−20 for 1 h, incubated with the primary antibodies overnight at 4 °C and finally incubated with secondary antibodies for 1 h at room temperature. Immunoblots were developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo-Scientific) and imaged with the chemiluminescent analyzer Chemidoc imaging system (Bio-Rad). Bands were quantified by densitometry .t test, one-way ANOVA, RM one-way ANOVA or Pearson correlation. Non-normal distributed data was statistically compared with Mann–Whitney U-Test, Friedmann-Test, or Spearman Correlation, respectively. Statistical significance was defined as ∗p < 0.05.GraphPad Prism Software (GraphPad Software) was used for statistical analyses. Data are expressed as mean ± SEM or median [25th and 75th percentile]. Number of biological replicates is indicated in the figure legends. Data was tested for normality with the D’Agostino & Pearson normality test. Statistical comparison of normal distributed data was performed with two-tailed Student’s Supplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Figure S4Supplementary Figure S5Supplementary Figure S6Table S1Table S2.Supplementary Figure Legends


P21/n. The packing is directed mainly by H⋯H van der Waals interactions and C—H⋯Br hydrogen bonds, as revealed by Hirshfeld surface analysis.The reaction of 1,5-dibromopentane with urotropine results in crystals of the title molecular salt, 5-bromourotropinium bromide, crystallizing in space group  3,7]decane bromide], C11H22BrN4+·Br− (1), crystallizing in space group P21/n. The packing in compound 1 is directed mainly by H⋯H van der Waals interactions and C—H⋯Br hydrogen bonds, as revealed by Hirshfeld surface analysis. Comparison with literature examples of alkylurotropinium halides shows that the interactions in 1 are consistent with those in other bromides and simple chloride and iodide species.The reaction of 1,5-dibromopentane with urotropine results in crystals of the title molecular salt, 5-bromourotropinium bromide [systematic name: 1-(5-bromopentyl)-3,5,7-triaza-1-azoniatricyclo[3.3.1.1 The pentyl chain is in the all-trans configuration, unlike its hexyl relative , H8B  and H15A. C—H⋯Br—C bonds are also seen from H5B to Br1 at the end of the pentyl chain, linking neighbouring cations into an inversion dimer with an H⋯Br distance of 3.00 Å , white surface areas represent contacts at the sum of van der Waals radii, red is shorter (close contact) and blue is longer (long contact).The Hirshfeld surface analysis  with the next major contributor being H⋯Br  with a small N⋯H component . The interaction of H and the alkylbromine residue accounts for 12.6% of the surface , leaving the remaining 18.4% as H⋯Br− hydrogen bonds to the bromide ions , as detailed above.The Hirshfeld surface primarily consists of H⋯H van der Waals interactions . However, the interactions between hydrogen and bromide account for a similar percentage of the overall Hirshfeld surface (16.6% in YOYWIS versus 18.4% in compound 1).A close relative to compound et al., 19831 can be seen where chloride is the halide anion: BIDBIZ 4(hmta)]2Br2·2H2O  and NaI  were dissolved in ethanol and 1,5-dibromopentane  was added. Clear block-like crystals appeared after 8 days, which were found to be a mixture of compound Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989020015601/hb7951sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020015601/hb7951Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020015601/hb7951Isup3.cmlSupporting information file. DOI: 2046576CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


However, the detailed molecular mechanisms by which S1P induces COX-2 expression in human cardiac fibroblasts (HCFs) remain unknown. HCFs were incubated with S1P and analyzed by Western blotting, real time-Polymerase chain reaction (RT-PCR), and immunofluorescent staining. Our results indicated that S1P activated S1PR1/3-dependent transcriptional activity to induce COX-2 expression and PGE2 production. S1P recruited and activated PTX-sensitive Gi or -insensitive Gq protein-coupled S1PR and then stimulated PKCα-dependent phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK1/2, leading to activating transcription factor NF-κB. Moreover, S1P-activated NF-κB was translocated into the nucleus and bound to its corresponding binding sites on COX-2 promoters determined by chromatin immunoprecipitation (ChIP) and promoter-reporter assays, thereby turning on COX-2 gene transcription associated with PGE2 production in HCFs. These results concluded that in HCFs, activation of NF-κB by PKCα-mediated MAPK cascades was essential for S1P-induced up-regulation of the COX-2/PGE2 system. Understanding the mechanisms of COX-2 expression and PGE2 production regulated by the S1P/S1PRs system on cardiac fibroblasts may provide rationally therapeutic interventions for heart injury or inflammatory diseases.In the regions of tissue injuries and inflammatory diseases, sphingosine 1-phosphate (S1P), a proinflammatory mediator, is increased. S1P may induce the upregulation of cyclooxygenase-2 (COX-2)/prostaglandin E Large adiseases . Furthernd human  detectednd human . Therefo2 leading to the release of proinflammatory PGs, such as PGE2 in various types of cells and tissues. Previously, several studies have demonstrated that S1P could induce COX-2 expression accompanied by the release of PGE2, and leading to inflammatory responses in various models of cells and organs (Cyclooxygenase (COX) catalyzes the rate-limiting reaction in the synthesis of prostaglandins (PGs) in various tissues, which consists of two isoforms of COX, in terms as COX-1 and COX-2. COX-1 is constitutively expressed and maintains the homeostasis of physiological functions . In contd organs . In addid organs . Moreoved organs . In the d organs . Therefo3 coupling Gq proteins to activate phospholipase C (PLC)-β, hydrolyze phosphoinositide (PI), and result in the formation of inositol trisphosphate (IP3) and diacylglycerol, ultimately leading to Ca2+ increase and protein kinase C (PKC) activation, respectively , Gαi-3 (sc-262), PKCα (sc-208), p42 (sc-154), p38 (sc-33688), JNK1 (sc-474), and p65 NF-κB (sc-7151) antibodies were from Santa Cruz . Sphingosine 1-phosphate (S1P), W123, JTE-013, and CAY10444 were from Cayman . GPA2A, pertussis toxin (PTX), Gö6976, PD98059, SB202190, SP600125, and helenalin were from Biomol . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was purchased from Biogenesis . Bicinchoninic acid (BCA) protein assay kit was purchased from Pierce . Enzymes, siRNAs for S1PR1 , S1PR3 , Gq , Gi , PKCα , p42 , JNK1 , and p65 NF-κB  and other chemicals were from Sigma . SDS-PAGE supplies were from MDBio Inc .DMEM/F-12 medium, fetal bovine serum (FBS), TRIzol reagent and PLUS-Lipofectamine, and p38 siRNA (MAPK14HSS102352) were purchased from Invitrogen . Hybond C membrane, enhanced chemiluminescence (ECL) and Western blotting detection system were purchased from GE Healthcare Biosciences . COX-2 antibody was from Abcam . PhosphoPlus p42/p44 MAPK (#9101), p38 MAPK (#9211), JNK1/2 (#4668), p65 NF-κB (#3031), and I-κB (#2859) antibodies were from Cell Signaling . S1PR1 (sc-48356), S1PR3 (sc-30024), G2 atmosphere. When these cells reached confluence, cells were treated with 0.05% (w/v) trypsin/0.53 mM EDTA for 1 min at 37°C. The cell suspension was diluted with DMEM/F-12 containing 10% FBS and 2 mM glutamine to a concentration of 2 cells/ml × 105 cells/ml. The cells were plated onto 12-well culture plates and made quiescent at confluence by incubation in serum-free DMEM/F-12 containing 0.1% BSA and 2 mM glutamine for 24 h, and then incubated with S1P at 37°C for the indicated time intervals as the experimental protocol. When the inhibitors were used, cells were pretreated with the inhibitor for 1 h, and then incubated with S1P. Experiments were performed using cells from passages 4 to 7.HCFs were isolated from the human heart obtained from ScienCell Research Laboratories . DMEM/F-12 containing 10% (v/v) FBS, 2 mM glutamine and antibiotics  was used for cell culture, and these cells were grown at 37°C with a humidified 5% COq, Gi, PKCα, p38α, JNK1, p42, p65, or GAPDH antibody overnight. TTBS washed the membranes with four times for 5 min each, then the membranes were incubated with an anti-rabbit horseradish peroxidase antibody of a 1:2000 dilution for 1 h. ECL reagents were used to detect the immunoreactive bands, quantified by UN-SCAN-IT gel version 6.1 . The densitometry of the specific band was normalized to GAPDH. Each experiment was performed in three individual experiments.Growth-arrested HCFs were incubated with S1P at 37°C for the indicated time intervals. To yield the whole-cell extract, the cells were washed with ice-cold PBS, scraped, and collected by centrifugation at 45,000 × g for 1 h at 4°C, as previously described . Samplesβ-actin. The following primers were used for amplification reactionAccording to the protocol of the manufacturer, total RNA was isolated from HCFs treated with S1P for the indicated time in 10 cm culture dishes using TRIzol. To produce a template for PCR amplification, the cDNA obtained from 0.5 μg total RNA was prepared, as previously described . Oligonu5′-AAAACCGTGGGGAATGTATGAGC-3′ (forward primer);5′-GATGGGTGAAGTGCTGGGGAAAG-3′ (reverse primer);5′-GTGTAGACCCAGAGTCCTGCG-3′ (forward primer);5′-AGCTTTTCCTTGGCTGGAGAG-3′ (reverse primer);5′-GGCCTAGCCAGTGCTCAGC-3′ (forward primer);5′-CCTTGGTGTAATTGTAGTGTTCCAGA-3′ (reverse primer);5′-GGAGCCCCTAGACGGGAGT-3′ (forward primer);5′-CCGACTGCGGGAAGAGTGT-3′ (reverse primer);5′-GAACCCTAAGGCCAACCGTG-3′ (forward primer);5′-TGGCATAGAGGTCTTTACGG-3′ (forward primer).β-actin was performed in parallel as a relatively invariant internal reference RNA, and cDNA amounts were standardized to equivalent β-actin mRNA levels. These primer sets recognized only the genes of interest as indicated by amplification of a specific single band of the expected size  and direct sequence analysis of the PCR product.PCR mixes contain 1.25 mM of each dNTP, 10 μl of 5× PCR buffer, 100 pmol of each forward and reverse primer, and 2.5 units of Taq polymerase . Fifty microliter was the final reaction volume. The program for COX-2 amplification was performed in 25 cycles as follows: at 94°C for 20 s, 60°C for 40 s, and 72°C for 40 s. After the last cycle, all samples were incubated for an additional 10 min at 72°C. 2% agarose 1× TAE gel containing ethidium bromide was used to analyze the PCR fragments and their sizes were compared to a molecular weight marker. Amplification of Real time-PCR was performed with the TaqMan gene expression assay system, using primers and probe mixes for COX-2 and endogenous GAPDH control genes as previously described . PCRs weβ-galactosidase activity standardized firefly luciferase activities.For the construction of the κB-Luc and COX-2-Luc plasmids, a human COX-2 promoter located within a region spanning from −483 to +37 bp was cloned into the pGL3-basic vector . A series of point mutations were introduced into the NF-κB binding site of COX-2 promoter by mismatched primer mutation PCR as previously described . The plaq, Gi, PKCα, p42, p38α, JNK1, and p65 were from Sigma . HCFs cells were plated at 3 cells/ml × 105 cells/ml (1 ml per well) in 12-well culture plates for 24 h, reaching about 80% confluence. Cells were incubated with 0.4 ml of serum-free DMEM/F-12 medium each well after washed once with PBS and once with serum-free DMEM/F-12. The siRNA was prepared and transiently transfected using Lipofectamine 2000 transfection reagent according to the instructions of the manufacturer.The SMARTpool-siRNAs of scramble, S1PR1, S1PR3, GAfter treatment, harvested cells were sonicated for 5 s at output 1.5 with a sonicator . Then, the cell lysates were centrifuged at 8,000 rpm for 15 min at 4°C. Both the pellet and supernatant were collected, respectively. The pellet was saved as the nuclear fraction. The supernatant was saved for further centrifugation at 14,000 rpm for 60 min at 4°C. The pellet and the supernatant were respectively collected as membrane fraction and cytosolic fraction.HCFs were plated on 6-well culture plates with coverslips. Cells were shifted to a serum-free DMEM/F-12 for 24 h and treated with 15 μM S1P. The cells were washed twice with ice-cold PBS, then fixed with 4% (w/v) paraformaldehyde in PBS for 30 min and permeabilized with 0.3% Triton X-100 in PBS for 15 min. Next, the staining procedures were performed by incubating with 10% normal goat serum in PBS for 30 min, followed by incubating with a primary anti-p65 NF-κB polyclonal antibody (1:200 dilution) in PBS with 1% BSA for 1 h. After washing thrice with PBS, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody (1:200 dilution) in PBS with 1% BSA for 1 h. Finally, after washing thrice with PBS, the cells were mounted with aqueous mounting medium. The images were taken under a fluorescence microscope .in vivo association of nuclear proteins with human COX-2 promoter, chromatin immunoprecipitation (ChIP) analysis was conducted as previously described .The cells in 12-well plates were grown to confluence. Then, cells were incubated with serum-free DMEM/F-12 medium for 24 h. After that, the growth-rested cells were treated with S1P as indicated time intervals. The conditioned medium was collected to analyze PGEp values of 0.05 were considered to be statistically significant. Post hoc tests were run only if F achieved p < 0.05 and there was no significant in homogeneity of variance. All the data were expressed as the mean ± SEM, at least three individual experiments (n = number of independent cell culture preparations). The n values are provided in the figure legends. Error bars were omitted when they fell within the dimensions of the symbols.Statistical analysis was performed by using GraphPad Prizm Program 6.0 software . We used one-way ANOVA followed by Dunnett’s post hoc test when comparing more than two groups of data and one-way ANOVA, nonparametric Kruskal–Wallis test, followed by Dunnett’s post hoc test when comparing multiple independent groups. via turning on COX-2 gene expression determined by a COX-2-promoter luciferase report assay. As shown in 2, the cultured media were collected to measure PGE2 levels using an ELISA kit. The data showed that S1P enhanced PGE2 generation in a time-dependent manner  was used. As shown in i in the response, a pertussis toxin (PTX) was used. As shown in q and Gi in S1P-induced COX-2 expression, HCFs were transfected with Gq or Gi siRNA. As shown in q or Gi siRNA knocked down the level of Gq or Gi protein expression, respectively, and attenuated S1P-induced COX-2 expression. These results indicated that S1P induces COX-2 expression via S1PR1 and S1PR3 coupling to either Gq protein or PTX-sensitive Gi protein in HCFs.Extracellular S1P interacts with a family of specific G protein-coupled receptors (GPCRs) to evoke multiple cellular responses. First, to determine whether Gq/Gi protein-coupled S1PR1/3 signaling pathway. As shown in q/Gi protein-coupled S1PR1/3-mediated activation of PKCα in HCFs.The classic PKC isoforms such as PKCα have been demonstrated to participate in the regulation of several proteins expression in various cell types . Hence, q/Gi) protein-coupled S1PR1/3-mediated PKCα pathway leading to COX-2 expression in HCFs.Activation of MAPKs by S1P could modulate cellular functions of vascular smooth muscle cells . To invevia the G (Gq/Gi) protein-coupled S1PR1/3-mediated PKCα pathway leading to COX-2 expression in HCFs.To investigate the effect of p38 MAPK on S1P-induced COX-2 expression in HCFs, a p38 MAPK inhibitor SB202190 was used. As shown in q/Gi) protein-coupled S1PR1/3-mediated PKCα pathway in HCFs.To determine the role of JNK1/2 in S1P-induced COX-2 expression, a JNK1/2 inhibitor SP600125 was used. As shown in q/Gi, PKCα, p38 MAPK, and JNK1/2, but not p42/p44 MAPK, leading to COX-2 induction in HCFs.S1P could activate NF-κB signaling to regulate cellular functions in various cell types . To provWe have found that S1P stimulates translocation of p65 NF-κB leading to COX-2 expression . Next, w2 production  synthesized by COX-2 are involved. Moreover, S1P is elevated in the regions of tissue injuries and inflammation , and NF-κB signaling pathway is essential for S1P-induced COX-2 gene expression and PGE2 production , a heterotrimeric GPCR, which stimulates multiple signaling pathways and regulates diverse cellular functions  is involved in S1P-induced COX-2 expression in HCFs. Moreover, S1P-stimulated p65 NF-κB translocation, binding to COX-2 promoter region, and NF-κB transcriptional activity was significantly inhibited by helenalin and the MAPK inhibitors including SB202190 (p38 MAPK) or SP600125 (JNK1/2), but not PD98059 (MEK1/2), suggesting that NF-κB activated by S1P is mediated through p38 MAPK- and JNK1/2-dependent mechanisms in HCFs. Interestingly, in HCFs, the p42/p44 MAPK is not involved in activation of NF-κB by S1P, consistent with our previous report indicating that activation of NF-κB by S1P independent of p42/p44 MAPK in rat VSMCs , and NF-κB signaling pathways in HCFs. These results implicate a role for cardiac fibroblasts, in addition to their organized and preservative functions, as inflammatory cells involved in the production of chemical mediators, which may contribute to the inflammatory responses in heart diseases. Although the limitation of the present study is the lack of in vivo evidence to support our findings, it is worth further conducting experiments using animal models. Overall these findings imply that S1P might play a crucial role in the pathogenesis of heart inflammatory diseases and provide useful insights for developing effective therapeutic targets in heart diseases.In conclusion, we reported here that the S1P/S1PR system exerted its stimulatory effects on COX-2 gene expression in HCFs. The GThe raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.C-CY, L-DH, M-HS, and C-MY substantially contributed to the conception or design of the work, the acquisition, analysis, and interpretation of data for the work. C-CY, L-DH, M-HS, and C-MY drafted the work and revised it critically for important intellectual content. C-CY, L-DH, M-HS, and C-MY finally approved the version to be published. C-CY, L-DH, M-HS, and C-MY agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.This work was supported by the Ministry of Science and Technology, Taiwan ; China Medical University, Taiwan [Grant numbers: CMU109-MF-08]; Chang Gung Medical Research Foundation, Taiwan .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sphingobium sp. strain SYK-6 is an alphaproteobacterial degrader of lignin-derived aromatic compounds, which can degrade all the stereoisomers of β-aryl ether-type compounds. SYK-6 cells convert four stereoisomers of guaiacylglycerol-β-guaiacyl ether (GGE) into two enantiomers of α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV) through GGE α-carbon atom oxidation by stereoselective Cα-dehydrogenases encoded by ligD, ligL, and ligN. The ether linkages of the resulting MPHPV enantiomers are cleaved by stereoselective glutathione (GSH) S-transferases (GSTs) encoded by ligF, ligE, and ligP, generating (βR/βS)-α-glutathionyl-β-hydroxypropiovanillone (GS-HPV) and guaiacol. To date, it has been shown that the gene products of ligG and SLG_04120 (ligQ), both encoding GST, catalyze GSH removal from (βR/βS)-GS-HPV, forming achiral β-hydroxypropiovanillone. In this study, we verified the enzyme properties of LigG and LigQ and elucidated their roles in β-aryl ether catabolism. Purified LigG showed an approximately 300-fold higher specific activity for (βR)-GS-HPV than that for (βS)-GS-HPV, whereas purified LigQ showed an approximately six-fold higher specific activity for (βS)-GS-HPV than that for (βR)-GS-HPV. Analyses of mutants of ligG, ligQ, and both genes revealed that SYK-6 converted (βR)-GS-HPV using both LigG and LigQ, whereas only LigQ was involved in converting (βS)-GS-HPV. Furthermore, the disruption of both ligG and ligQ was observed to lead to the loss of the capability of SYK-6 to convert MPHPV. This suggests that GSH removal from GS-HPV catalyzed by LigG and LigQ, is essential for cellular GSH recycling during β-aryl ether catabolism. The industrial production of useful substances from lignin is desired, since it is the most abundant aromatic resource and the second most abundant bioresource on Earth after cellulose. In recent years, microbial conversion processes known as “biological funneling” have attracted attention for their ability to upgrade heterogeneous mixtures of low-molecular-weight aromatic compounds obtained through chemical lignin depolymerization into platform chemicals5. Based on these facts, elucidating the bacterial lignin catabolic system has become more critical.Lignin is one of the major components of plant cell walls, accounting for 15–40%7. Although lignin has asymmetric carbons, it is considered optically inactive because of its equal amounts of optical isomers8. Among the intermonomer linkages in lignin, β-aryl ether is the most abundant, comprising 45%–50% of all linkages in softwood lignin and 60–62% in hardwood lignin9. Accordingly, cleavage of the β-aryl ether linkage is considered a crucial step in lignin biodegradation. Furthermore, β-aryl ether-type biaryls have two distinct isomeric forms, erythro and threo, and each form has enantiomeric forms11.Lignin is formed through the dehydrogenative polymerization of hydroxycinnamic alcohols, resulting in various C–C and C–O–C bonds between the phenylpropane unitsSphingobium sp. strain SYK-6 is an alphaproteobacterium that exhibits the best-characterized catabolic system for lignin-derived aromatic compounds12. SYK-6 can utilize lignin-derived biaryls, including β-aryl ether, phenylcoumaran, biphenyl, and diarylpropane, and monoaryls, including ferulate, vanillin, and syringaldehyde, as its sole sources of carbon and energy. In SYK-6 cells, four stereoisomers of a model β-aryl ether compound, guaiacylglycerol-β-guaiacyl ether (GGE), are converted into two enantiomers of α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV) through the oxidation of the α-carbon, which is catalyzed by Cα-dehydrogenases, LigD, LigL, and LigN -GGE and -GGE into (βS)-MPHPV and (βR)-MPHPV, respectively, whereas LigL/LigN converts -GGE and -GGE into (βR)-MPHPV and (βS)-MPHPV, respectively. The ether linkage in the resulting MPHPV is cleaved by enantioselective glutathione (GSH) S-transferases (GSTs), LigF, LigE, and LigP (β-etherases), to produce α-glutathionyl-β-hydroxypropiovanillone (GS-HPV) and guaiacol through the nucleophilic attack of GSH on the MPHPV β-carbon atom -MPHPV and (βR)-MPHPV, subsequently producing (βR)-GS-HPV and (βS)-GS-HPV, respectively18. Recently, X-ray crystal structures of LigE and LigF were determined, suggesting that LigE was most similar to the GSTFuA fungal class, and LigF was classified in a new structural class closely related to GSTFuAs or fungal Ure2p-like GST19. LigF and LigE cleave the β-aryl ether linkage through an SN2 nucleophilic attack of GSH on the β-carbon of the substrates19. Another GST, LigG, catalyzes the cleavage of the thioether linkage in (βR)-GS-HPV by transferring the (βR)-GS-HPV GSH moiety to another GSH molecule, producing achiral β-hydroxypropiovanillone (HPV) and oxidized glutathione (GSSG)16. Meux et al. and Pereira et al. independently solved the X-ray crystal structure of LigG21. LigG belongs to the omega-class GST, and Cys15 is a catalytic residue20. LigG had little to no activity for (βS)-GS-HPV, suggesting the involvement of an alternative GST in (βS)-GS-HPV conversion14. A recent study reported that a Nu-class GST, NmGST3 of Novosphingobium sp. strain MBES04, exhibited GSH-removing activity toward both GS-HPV isomers23. Kontur et al. reported that NaGSTNu, belonging to the Nu-class GST, showed activity toward both GS-HPV isomers, and it was the only enzyme essential for converting both GS-HPV isomers in Novosphingobium aromaticivorans DSM 1244424. Furthermore, they showed that the gene product of SLG_04120, a putative Nu-class GST in SYK-6, had GSH-removing activity for both GS-HPV isomers.In this study, we identified the GS-HPV-converting GST genes in SYK-6 and uncovered the roles of these genes in β-aryl ether catabolism, since the genes involved in GS-HPV catabolism have not been clarified in SYK-6 to date.14. High-performance liquid chromatography (HPLC) analysis of the reaction mixtures showed that MPHPV was converted into GS-HPV by purified LigF and LigE at 60 min . The conversion of MPHPV by LigF and LigE stopped at approximately 50%, and approximately equimolar GS-HPV to the converted MPHPV was generated . Chiral HPLC analysis was conducted to confirm whether LigF and LigE enantioselectively convert MPHPV. The MPHPV preparation that was used as the substrate was an equimolar mixture of (βR)-MPHPV and (βS)-MPHPV (53:47) . LigF and LigE completely converted (βS)-MPHPV and (βR)-MPHPV, respectively, indicating (βR)-GS-HPV and (βS)-GS-HPV generation in each reaction . We attempted to remove the remaining MPHPV and guaiacol from the reaction mixtures by ethyl acetate extraction; however, we found that the prepared GS-HPV isomers racemized after incubation in buffer A at 30 °C for 24 h (data not shown). Hence, we prepared each GS-HPV isomer by incubating 400 µM MPHPV with purified LigF or LigE and 5 mM GSH in buffer A before usage.Racemic MPHPV (400 µM) was reacted with purified LigF and LigE  with 5 mM GSH in 50 mM Tris–HCl buffer  to obtain enantiopure GS-HPV isomers25 containing 10 mM sucrose, 10 mM glutamate, 0.13 mM methionine, and 10 mM proline (Wx-SEMP) were incubated with 100 µM (βR)-GS-HPV or (βS)-GS-HPV and 2.4 mM GSH in buffer A. HPLC analysis indicated that SYK-6 converted both GS-HPV isomers into HPV after incubation for 5 min . The activities of the extracts of SYK-6 cells grown in a Wx-SEMP with or without 5 mM GGE supplementation were measured to evaluate the inducibility of GS-HPV conversion. The specific activity of the extracts from the cells grown in Wx-SEMP for (βS)-GS-HPV (142 ± 3 nmol·min−1·mg−1) was approximately six-fold higher than that for (βR)-GS-HPV (24.5 ± 0.2 nmol·min−1·mg−1). The specific activities for (βS)-GS-HPV (150 ± 19 nmol·min−1·mg−1) and (βR)-GS-HPV (31.4 ± 1.1 nmol·min−1·mg−1) of the extracts from the cells grown in Wx-SEMP supplemented with 5 mM GGE were not significantly higher than those of the extracts from the cells grown in Wx-SEMP. These results suggest that GSH-removing enzyme genes are constitutively expressed.In order to verify the ability of SYK-6 to convert GS-HPV isomers, the extracts of SYK-6 cells grown in Wx minimal mediumNovosphingobium sp. strain MBES04, N. aromaticivorans DSM 12444, Novosphingobium sp. strain PP1Y, and Thiobacillus denitrificans ATCC 25259. This phylogenetic tree showed that SLG_00360 is relatively close to a clade, including ligG (19% identity), and SLG_04120 belongs to a clade, including NmGST3 of MBES04 (29% identity) and NaGSTNu of DSM 12444 (58% identity), which can convert both GS-HPV isomers. Moreover, Cys15 and Pro16, shown to be the active center of SYK-6 LigG, were conserved in SLG_29340 (Cys10 and Pro11) . On the basis of these findings, we selected ligG, SLG_00360, SLG_04120, and SLG_29340 as candidates for GS-HPV-converting GST genes. To examine whether the gene products of ligG, SLG_00360, SLG_04120, and SLG_29340 exhibit activities toward GS-HPV, these genes fused with a His-tag at the 5ʹ terminus were expressed in E. coli. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) showed the protein production of 33, 28, 36, and 33 kDa, appearing as products of ligG, SLG_00360, SLG_04120, and SLG_29340 . The cell extracts of these E. coli transformants were incubated with 100 µM (βR)-GS-HPV or (βS)-GS-HPV and 2.4 mM GSH in buffer A. HPLC analysis showed that ligG gene product completely converted (βR)-GS-HPV into HPV after incubation for 30 min, whereas it only slightly converted (βS)-GS-HPV -GS-HPV, which is similar to that of previous reports26. By contrast, the SLG_04120 gene product completely converted both GS-HPV isomers into HPV, verifying its specificity, as previously reported , ligQ mutant (ΔligQ), and ligG ligQ double mutant (ΔligG ligQ)  to determine whether ligG and ligQ are indeed involved in converting GS-HPV in SYK-6. The cell extract of each mutant was reacted with 100 µM (βR)-GS-HPV or (βS)-GS-HPV and 2.4 mM GSH in buffer A, and their ability to convert GS-HPV isomers was compared with SYK-6. ΔligG and ΔligQ both showed a decreased conversion of (βR)-GS-HPV, but only ΔligQ exhibited the loss of (βS)-GS-HPV conversion  and ligQ (pQFligQ) to confirm whether this activity defect was due to the disruption of ligG and ligQ. A cell extract of ΔligG ligQ harboring pQFligG showed higher specific activity for (βR)-GS-HPV than that of the wild type -GS-HPV as the wild type -GS-HPV than the wild type -GS-HPV in SYK-6 cells, whereas only ligQ is involved in (βS)-GS-HPV conversion.We then created E. coli expressing His-tagged ligG and ligQ, respectively . Purified LigG and LigQ were incubated with 100 µM (βR)-GS-HPV or (βS)-GS-HPV and 2.4 mM GSH in buffer A. LigG showed approximately 300-fold higher specific activity for (βR)-GS-HPV (33 ± 1 µmol·min−1·mg−1) than for (βS)-GS-HPV (0.11 ± 0.01 µmol·min−1·mg−1). By contrast, LigQ showed approximately six-fold higher specific activity for (βS)-GS-HPV (110 ± 14 µmol·min−1·mg−1) than for (βR)-GS-HPV (17 ± 2 µmol·min−1·mg−1).The specific activity of LigG for (βR)-GS-HPV was approximately two-fold higher than that of LigQ. These results indicated that LigG is highly specific for (βR)-GS-HPV, and LigQ prefers (βS)-GS-HPV more than (βR)-GS-HPV. The Km values of LigG and LigQ for GSH were determined to be 1.19 ± 0.22 and 0.34 ± 0.10 mM, respectively .LigG and LigQ were purified to near-homogeneity through nickel affinity chromatography from the cell extracts of ligG and ligQ in SYK-6 affects MPHPV conversion. Here ΔligG and ΔligQ cells converted MPHPV similar to the wild type, whereas ΔligG ligQ cells lost the ability to convert MPHPV . The introduction of pQFligG or pQFligQ into ΔligG ligQ cells restored their MPHPV-converting ability, indicating that the disruption of both ligG and ligQ led to the loss of the ability to convert MPHPV in vivo -GS-HPV, and LigQ converts both GS-HPV isomers. Our study further elucidated that both ligG and ligQ participate in converting (βR)-GS-HPV in SYK-6 cells, whereas only ligQ is involved in converting (βS)-GS-HPV -GS-HPV conversion by ΔligQ for 60 min was lower than that by ΔligG, ligQ appears to more involved in (βR)-GS-HPV conversion than ligG -GS-HPV was approximately two-fold lower than that of LigG in the presence of 2.4 mM GSH. The reason why the disruption of ligQ had more influence on (βR)-GS-HPV conversion than the disruption of ligG may be the higher GSH affinity of LigQ than that of LigG . Alternatively, ligQ expression level in SYK-6 may be higher than that of ligG.We confirmed that LigG is highly specific for  and NmGST6 (MBENS4_2530) that showed 29% identity with LigQ and 62% identity with LigG, respectively. NmGST3 had activities for both isomers, and NmGST6 was highly specific for (βR)-GS-HPV23; however, the involvement of these genes in GS-HPV catabolism has not been investigated. We found another GST gene in the MBES04 genome, MBENS4_4395 (accession no. GAM07399), exhibiting 59% and 63% identity with LigQ and NaGSTNu, respectively. GAM07399 may be involved in GS-HPV isomer conversion besides the NmGST3 and NmGST6 genes. In Novosphingobium sp. strain PP1Y, NsLigG (PP1Y_AT11674), exhibiting 63% identity with LigG, was highly specific for (βR)-GS-HPV26. Furthermore, we found PP1Y_AT11650 (accession no. CCA92086) in the genome of this strain, which showed 80% identity with NmGST3; and PP1Y_AT20084 (accession no. CCA92884) that showed 57% and 66% identity with LigQ and NaGSTNu, respectively. These genes may also be involved in GS-HPV isomer conversion.In ligD, ligF, ligE, and ligG, constitute an operon, whereas ligQ locates at a different locus 14. In the case of MBES04, the NmGST4–NmGST5–NmGST6 genes (MBENS4_2528–2530) corresponding to ligFEG are adjacent, and the NmGST3 gene  locates just upstream of MBENS4_2528 with a different transcription direction 22. By contrast, another Nu-class GST gene MBENS4_4395 (accession no. GAM07399) locates at a different locus. Almost the same gene organization was also observed in the PP1Y genome 22. Based on the conservation of these gene arrangements, the conversion of both GS-HPV isomers may become possible by acquiring a Nu-class GST gene in strains carrying ligFEG or by adding ligFEG to a Nu-class GST gene-carrying strain. Unlike the aforementioned strains, the genes corresponding to ligDFE are scattered through the DSM 12444 genome.In the SYK-6 genome, the β-aryl ether catabolic genes, S)-GS-HPV (0.11 ± 0.01 µmol·min−1·mg−1) was only 0.33% of that for (βR)-GS-HPV (33 ± 1 μmol·min−1·mg−1). This result is similar to previous reports demonstrating that LigG had little to no activity for (βS)-GS-HPV derivatives26. By contrast, Rosini et al. reported that 500 µM racemic GGE was fully converted into HPV and guaiacol by purified LigD, LigL, LigF, LigE, and LigG in 2 h27, suggesting that LigG catalyzed the thioether cleavage of both (βS)- and (βR)-GS-HPV. However, the conversion of (βS)-GS-HPV by LigG was likely to have proceeded because a large amount of purified LigG (200 µg/ml) was used for this conversion.The specific activity of LigG for (βS)-GS-HPV than for (βR)-GS-HPV in this study. Kontur et al. reported that LigQ showed approximately 11-fold higher kcat/Km value for (βS)-GS-HPV than for (βR)-GS-HPV (Table S2)24. Also, NaGSTNu of DSM 12444 showed approximately four-fold higher kcat/Km value for (βS)-GS-HPV than for (βR)-GS-HPV24, and NmGST3 of MBES04 had approximately two-fold higher specific activity for (βS)-GS-HPV than for (βR)-GS-HPV (Table S2)23. Therefore, all these Nu-class GSTs have higher specificity for (βS)-GS-HPV than for (βR)-GS-HPV.Conversely, LigQ showed approximately six-fold higher specific activity for . These values were considerably higher than Km of LigG for (βR)-GS-HPV (0.016 ± 0.001 mM)21 and Km of LigQ for (βR)-GS-HPV (0.055 ± 0.007 mM) and (βS)-GS-HPV (0.011 ± 0.002 mM)24. The Km value of LigG for GSH is supported by the observation of Picart et al. that the specific activity of LigG was halved when reacted in the presence of 0.4 mM GSH compared to 1 mM GSH26. Based on the quantum mechanics/molecular mechanics simulations, Prates et al. suggested that HPV leaves the active site after GS– of GS-HPV is transferred to Cys15 of LigG, giving enough space to accommodate a second GSH for a subsequent thiol-disulfide reaction, which forms GSH disulfide and restores the catalytic ability of LigG18. The lack of specific interactions to bind the GSH in the active site of LigG was suggested to be associated with high Km value for GSH estimated by the results of Picart et al18. In addition, the Km values for GSH of E. coli Nu-class GSTs (YghU and YfcG) have been reported to be on the order of mM when using 2-hydroxyethyl disulfide as a substrate 29.The Nu was proposed based on X-ray crystal structure, modeling of substrate binding, and mutant analysis. Thr51 and Asn53 of NaGSTNu provide hydrogen bonds that stabilize a reactive GSH thiolate anion, which attacks the GS− moiety of GS-HPV to form GS–SG disulfide. The rupture of the thioether bond is facilitated by the formation of a transient enolate intermediate. This intermediate is stabilized by the interactions between GS-HPV and Tyr166 and Tyr224 hydroxy groups. The capture of a solvent-derived proton by the carbanion collapses the enolate to form HPV24. All these residues are conserved in LigQ, GAM07399, and CCA92884, suggesting that these GSTs catalyze GSH removal from GS-HPV using the same reaction mechanism as that of NaGSTNu.The mechanism for the cleavage of the thioether bond in GS-HPV by NaGSTligG ligQ lost the ability to convert MPHPV in vivo  could not proceed , Wx-SEMP, and Wx-SEMP containing 5 mM GGE at 30 °C. When necessary, 50 mg kanamycin/liter, 100 mg streptomycin/liter, or 12.5 mg tetracycline/liter were added to the cultures. E. coli strains were grown in LB at 37 °C. The media for E. coli transformants were supplemented with 100 mg ampicillin/liter, 25 mg kanamycin/liter, or 12.5 mg tetracycline/liter.Table S3 lists the strains and plasmids that were used in this study. 31. In preparing (βR)-GS-HPV and (βS)-GS-HPV, purified LigF (40 µg protein/ml) and LigE (150 µg protein/ml) were incubated in 1 ml reaction mixtures containing 400 µM racemic MPHPV, 5 mM GSH, and buffer A, respectively, for 60 min at 30 °C14. The resulting mixtures were used as preparations of 200 µM GS-HPV isomers, including 4.8 mM GSH. GGE and guaiacol were purchased from Tokyo Chemical Industry, Co., Ltd.MPHPV and HPV were prepared as described previouslyHPLC analysis was conducted using the ACQUITY UPLC system (Waters). The MPHPV and GS-HPV reaction products were analyzed using a TSKgel ODS-140HTP column . Additionally, a CHIRALPAK IE column  was used to analyze MPHPV reaction products. All analyses were conducted at a flow rate of 0.5 ml/min. The mobile phase was a mixture of solution A (acetonitrile containing 0.1% formic acid) and B (water containing 0.1% formic acid) under the following conditions: detection of MPHPV reaction products using TSKgel ODS-140HTP: 0–2.0 min, 10% A; 2.0–4.0 min, linear gradient from 10 to 60% A; 4.0–4.5 min, linear gradient from 60 to 90% A; 4.5–5.0 min, 90% A. Detection of MPHPV reaction products using CHIRALPAK IE: 0–25.0 min, 30% A. Detection of GS-HPV reaction products using TSKgel ODS-140HTP: 0–5.0 min, 10% A. MPHPV, GS-HPV, guaiacol, and HPV were detected at 280, 286, 276, and 276 nm, respectively.ligG, ΔligQ, and ΔligG ligQ) grown in LB were washed with Wx medium, resuspended in Wx-SEMP medium to an optical density at 600 nm (OD600) of 0.2, and incubated for 16 h. The resultant cells were washed twice with buffer A. The cells that were resuspended in the same buffer were then broken by an ultrasonic disintegrator , and the supernatants were obtained as cell extracts after centrifugation . Protein concentration was determined using the Bradford method, using bovine serum albumin as the standard (Bio-Rad Laboratories).GS-HPV-transforming activities of the cell extracts of SYK-6 and its mutants were determined by measuring HPV production using HPLC. The MPHPV-transforming activities of the cell extracts of SYK-6 and its mutants were determined by measuring the decrease in MPHPV using HPLC. The reaction products were also detected using HPLC analysis. SYK-6 and its mutant cells (ΔR)-GS-HPV, (βS)-GS-HPV, or 200 µM racemic MPHPV in the presence of 2.4 mM (for GS-HPV) or 5.0 mM GSH (for MPHPV) in buffer A at 30 °C. The reactions were stopped by adding acetonitrile  after 3 min (measurement of the specific activities for GS-HPV), 5 min (identification of GS-HPV metabolites), 20 min (measurement of the specific activities for GS-HPV in mutant-complemented strains), or 60 min (for MPHPV). Precipitated proteins were removed by centrifugation at 19,000 × g for 15 min, and the resulting supernatants were diluted with water to a final acetonitrile concentration of 12.5%, filtered, and analyzed using HPLC. The specific activity of GS-HPV conversion was expressed in moles of HPV produced per min per milligram of protein. The cells of SYK-6 grown in LB were washed with Wx medium, resuspended in Wx-SEMP medium to an OD600 of 0.2, and incubated at 30 °C to examine the effects of GGE on enzyme induction. When the cultures reached an OD600 of 0.4–0.5, we added 5 mM GGE to them. After 12 h of further incubation, cell extracts were prepared and used for the enzyme assay. For complementing ΔligG ligQ, pQFligG and pQFligQ were constructed by In-Fusion cloning (Takara Bio) of ligG and ligQ fragments amplified by PCR using SYK-6 total DNA and their corresponding primer pairs (Table S4) into pQF. After confirming the nucleotide sequences of the inserts, each plasmid was introduced into ΔligG ligQ cells through electroporation. The transformed cells were grown in Wx-SEMP containing 100 µM cumate and tetracycline for 16 h, and the cell extracts were prepared and used for the enzyme assay.Cell extracts (5–1000 µg protein/ml) were incubated with 100 µM , applying 1,000 bootstrap replicates.Nucleotide sequences were determined through Eurofins Genomics. Sequence analysis was conducted using the MacVector program . Sequence similarity searches, pairwise alignments, and multiple alignments were conducted using the BLASTP programligQ (SLG_04120), ligF (SLG_08650), ligE (SLG_08660), ligG (SLG_08670), and SLG_29340 were amplified through PCR using SYK-6 total DNA and the primer pairs listed in Table S4, and each amplified fragment was cloned into pET-16b by In-Fusion Cloning. The nucleotide sequences of the inserts were confirmed by sequencing. Each expression plasmid was introduced into E. coli BL21(DE3), and the transformed cells were grown in LB. Gene expression was induced for 4 h at 30 °C by adding 1 mM isopropyl-β-D-thiogalactopyranoside when the OD600 of the cultures reached 0.5. The cells were then harvested by centrifugation at 5000×g for 5 min at 4 °C and washed with buffer A. The cells were then resuspended in the same buffer and broken using an ultrasonic disintegrator. The supernatants were obtained as cell extracts after centrifugation at 19,000×g for 15 min at 4 °C. For purification of LigF, LigE, LigG, and LigQ, the cell extracts of E. coli BL21(DE3) harboring pET08650, pET08660, pET08670, and pET04120, respectively (Table S3), were transferred onto a His SpinTrap column . The purified fractions were subjected to desalting and concentration using an Amicon Ultra centrifugal filter unit , and the enzyme preparations were stored at − 80 °C. Gene expressions and the purity of the preparations were examined using SDS-12% PAGE. The protein bands in gels were stained with Coomassie Brilliant Blue.DNA fragments carrying SLG_00360, R)- and (βS)-GS-HPV, respectively; LigQ, 0.2 and 1.0 µg protein/ml for the conversion of (βS)- and (βR)-GS-HPV, respectively) were incubated in the 100 µl reaction mixtures containing 100 µM (βR)-GS-HPV or (βS)-GS-HPV, 2.4 mM GSH, and buffer A at 30 °C. The reactions were stopped by adding acetonitrile or methanol to a final concentration of 50% after 30 min of incubation (for cell extracts) and 15 s (for purified enzymes). Protein precipitates were removed through centrifugation at 19,000×g for 15 min. The resulting supernatants were diluted with water to a final concentration of acetonitrile or methanol of 12.5%, filtered, and analyzed using HPLC. Specific activities for GS-HPV conversion of purified enzymes were calculated after 15 s incubation and expressed in moles of HPV produced per min per milligram of protein. The Km values of LigG and LigQ for GSH were determined using 100 µM (βR)-GS-HPV under the following protein and GSH concentrations: LigG, 1.25 µg/ml protein and 0.25–4.9 mM GSH; LigQ, 5.0 µg/ml protein and 0.1–1.9 mM GSH. The Km values were calculated through non-linear regression analysis using the GraphPad Prism 7 software  fitted to the Michaelis–Menten equation and expressed as means ± standard deviations of three independent experiments.GS-HPV-transforming activities were determined by measuring HPV production using HPLC. The reaction products were also detected through HPLC analysis. The cell extracts (100 µg protein/ml) and purified enzymes  were incubated at 30 °C with shaking for 24 h. Portions of the cultures were periodically collected, and the amounts of the MPHPV were measured using HPLC. For complementation analysis of ΔligG ligQ, the resting cells (OD600 of 5.0) of SYK-6 harboring pQF, ΔligG ligQ harboring pQF, ΔligG ligQ harboring pQFligG, or ΔligG ligQ harboring pQFligQ, grown in Wx-SEMP containing cumate (100 µM) and tetracycline, were incubated with 200 µM MPHPV at 30 °C with shaking for 24 h.The cells of SYK-6, ΔSupplementary information.


Therapeutic antibodies targeting programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis induce potent and durable anti-tumor responses in multiple types of cancers. However, only a subset of patients benefits from anti-PD-1/PD-L1 therapies. As a negative regulator of anti-tumor immunity, TGF-β impairs the efficacy of anti-PD-1/PD-L1 and induces drug resistance. Developing a novel treatment strategy to simultaneously block PD-1/PD-L1 and TGF-β would be valuable to enhance the effect of anti-PD-1/PD-L1 and relieve drug resistance.Based on the Check-BODY™ technology platform, we developed an anti-TGF-β/PD-L1 bispecific antibody YM101. The bioactivity of the anti-TGF-β moiety was determined by Smad-luciferase reporter assay, transwell assay, western blotting, CCK-8, and flow cytometry. The bioactivity of the anti-PD-L1 moiety was measured by T cell activation assays. EMT-6, CT26, and 3LL tumor models were used to investigate the anti-tumor activity of YM101 in vivo. RNA-seq, immunohistochemical staining, and flow cytometry were utilized to analyze the effect of YM101 on the tumor microenvironment.YM101 could bind to TGF-β and PD-L1 specifically. In vitro experiments showed that YM101 effectively counteracted the biological effects of TGF-β and PD-1/PD-L1 pathway, including activating Smad signaling, inducing epithelial-mesenchymal transition, and immunosuppression. Besides, in vivo experiments indicated the anti-tumor activity of YM101 was superior to anti-TGF-β and anti-PD-L1 monotherapies. Mechanistically, YM101 promoted the formation of ‘hot tumor’: increasing the numbers of tumor infiltrating lymphocytes and dendritic cells, elevating the ratio of M1/M2, and enhancing cytokine production in T cells. This normalized tumor immune microenvironment and enhanced anti-tumor immune response might contribute to the robust anti-tumor effect of YM101.Our results demonstrated that YM101 could simultaneously block TGF-β and PD-L1 pathways and had a superior anti-tumor effect compared to the monotherapies. It has been well-established that cancer cells could escape from immune surveillance by activating some immune checkpoint pathways . Among aA series of clinical studies showed that anti-PD-1/PD-L1 antibodies had robust and durable anti-cancer activities across several solid and hematologic cancers, such as lung cancer –9, renalTransforming growth factor-beta (TGF-β) has three isoforms: TGF-β1, TGF-β2, and TGF-β3. As a versatile cytokine, TGF-β is usually overexpressed in advanced tumors and related to poor prognoses . The rolTGFB1 gene expression is higher in the non-responder’s tumor tissues [In the TME with hyperactive TGF-β signaling, the effect of anti-PD-1/PD-L1 therapy is limited . After a tissues . Corresp tissues , 43. Giv tissues . To optiCheck-BODY™ platform is designed by Wuhan YZY Biopharma Co., Ltd for the development of symmetric tetravalency bispecific antibodies. Check-BODY™ platform is characterized by high production yield, easy purification, and high structural stability. YM101 is constructed based on the Check-BODY™ technology platform. In the present study, we explored the biochemistry characteristics of YM101 in vitro and assessed its anti-tumor activity in vivo.CT26 (murine colon cancer cell), EMT-6 (murine breast cancer cell), 4T1 (murine breast cancer cell), A549 (human lung cancer cell), and NCI-H358 (human lung cancer cell) were cultured in RPMI-1640 (Gibco) containing 10% fetal bovine serum (FBS) . HT-2 (murine T cell) and CTLL-2 (murine T cell) were cultured in RPMI-1640   with 10% FBS and 200 IU/ml interleukin-2 . Primary murine T cells were isolated from C57BL/6 mouse-derived splenocytes and cultured in RPMI-1640 containing 10% FBS. NF639 (murine breast cancer cell) and 3LL (murine lung cancer cell) were cultured in DMEM (Gibco) with 10% FBS.The therapeutic antibodies and isotype control antibody used in the present study included YM101, human IgG, anti-TGF-β, and anti-PD-L1. The anti-TGF-β antibody was constructed based on GC1008 . The antThe prepared YM101 was analyzed using SDS-PAGE and Coomassie Brilliant Blue staining. To verify the purity and molecular weight of YM101, reduced and non-reduced SDS-PAGE were conducted as previously described . After CCapillary electrophoresis with sodium dodecylsulfate (CE-SDS) assay was performed following the standard protocol . For theThe molecular weight of the intact antibody was measured with 1 μg/μl YM101, and the molecular weights of the short chain and long chain were measured using reduced samples with Dithiothreitol (50 mM). Liquid chromatograph , chromatographic column , and mass spectrometer  were used in this assay.96 well flat-bottom plates  were coated with TGF-β1 , TGF-β2 , TGF-β3 , and PD-L1  (200 ng per well) at 4 °C overnight. On the next day, the plates were washed 3 times using PBS containing 0.05% Tween-20  (PBST). The assay plates were blocked with PBS containing 3% bovine serum albumin  for 2 h. Then, serially diluted YM101 or controls were added into the plates and incubated for 1 h. Afterwards, the plates were washed and incubated with anti-hIgG-HRP  for 1 h. ELISA substrates (100 μl per well)  were added into plates, and the HRP reaction was terminated using 2 N HCl. The absorbance values were read at 450 nm (Molecular Devices) .For the double-antigen sandwich ELISA assay, 96 well plates  were coated with TGF-β1, TGF-β2, and TGF-β3 (200 ng per well) at 4 °C overnight. After washing and blocking, serially diluted YM101 or controls were added into the plates and incubated at 37 °C for 1 h. Then, the plates were washed and incubated with PD-L1-Biotin  for 1 h. Subsequently, the assay plates were washed and incubated with peroxidase-conjugated streptavidin . According to the standard protocol of ELISA, the simultaneous binding of YM101 to TGF-β and PD-L1 was quantified by absorbance value.4 viable NF639 or 4T1 cells were seeded in 96 well flat-bottom plates  and incubated at 37 ℃ overnight. NF639 or 4T1 cells were transiently transfected with Smad luciferase reporter plasmid (0.1 μg per well) by Effectene® Transfection Reagent . After transfection, NF639 or 4T1 cells were incubated with TGF-β1 (10 ng/ml) and serially diluted YM101 or controls for 24 h. Luminescence was detected using Bright-Glo™ Luciferase Assay System .3 × 105 pM antibodies for 96 h. Untreated cells were employed as the negative control. All cells were cultured with DMEM or RPMI-1640 containing 1% FBS during treatment. Then, 5 × 104 NF639 and 4T1 cells were suspended in 100 µl DMEM or RPMI-1640 containing 1% FBS and seeded in the upper chambers. The lower chambers were filled with 600 µl DMEM or RPMI-1640 containing 10% FBS. Migratory and invasive cells were stained with crystal violet solution after incubating for 12 h.Transwell migration or invasion assays were performed using 8.0 µm pore size inserts  without or with Matrigel . NF639 and 4T1 cells were treated with 10 ng/ml TGF-β1 plus 10Protein was extracted by Mammalian Total Protein Extraction Kit . 25 µg protein from each sample was separated by SDS-PAGE gel and then transferred to a polyvinylidene fluoride membrane (Millipore). The primary antibodies were anti-E-cadherin , anti-N-cadherin , anti-Vimentin , anti-Snail , anti-β-Actin , anti-phospho-Erk1/2 , anti-Erk1/2 , and anti-GAPDH . The secondary antibodies were anti-rabbit-IgG-HRP . Signal detection was performed as previously described .96 well flat-bottom plates  were pre-coated with 5 μg/ml anti-CD3  at 4 °C overnight. After lysing red blood cells , murine splenocytes were washed and cultured in RPMI-1640 containing 10% FBS, 2 μg/ml anti-CD28 , 10 ng/ml TGF-β1, 100 IU/ml IL-2 for 6 days. Fixable Viability Stain 700 , anti-CD4 , anti-CD25 , anti-Foxp3 , eBioscience™ FOXP3/Transcription Factor Staining Buffer Set  were used to detect the ratio of Treg.3 per well) were seeded in 96-well plates . Then, TGF-β1 and antibodies (105 pM) were added into plates. Within one week after treatment, cell viability was continuously monitored by CCK-8 reagent .TGF-β could inhibit the IL-2 dependent proliferation of CTLL-2 and HT-2. We performed CCK-8 assays to measure the capability of YM101 antagonizing the effect of TGF-β. CTLL-2 and HT-2 cells (1 × 105 pM) for 4 days. Then, cells were harvested for cell cycle analysis. After treatment with 75% cold ethanol at 4 °C for 30 min, cells were dyed by the staining buffer containing 50 μg/ml Propidium Iodide (PI)  and 200 μg/ml RNase  for 30 min. The ratios of cells in different phases were detected by flow cytometry and analyzed by Flowjo v10 .CTLL-2 and HT-2 were pretreated with TGF-β1 (10 ng/ml) plus antibodies (105 pM) for 5 days. Cell apoptosis was evaluated according to the standard protocol of manufacturers  [5 cells were suspended with 200 μl Annexin V binding buffer, 5 μl Annexin V, and 10 μl PI. After incubation for 20 min at room temperature, the ratio of apoptotic cells was measured by flow cytometry.CTLL-2 and HT-2 were pretreated with TGF-β1 (10 ng/ml) plus antibodies (10oLegend) . For eac6/ml) were mixed with TGF-β1 (10 ng/ml) and antibodies (105 pM) and cultured at 37 ℃ for 4 days. Then, the cellular supernatants were harvested, and the concentrations of cytokines were measured by Multi-Analyte Flow Assay Kit .TGF-β regulates the differentiation of naïve T cells and affects the levels of multiple cytokines during T cell activation . In the 6/ml) were mixed with PD-L1 and antibodies (105 pM). The T cells were cultured at 37 °C for 4 days. Then, the cellular supernatants were harvested, and the IL-2 concentration was measured by Multi-Analyte Flow Assay Kit. Besides, we detected the T cell proliferation by CFSE  dilution assays. Murine T cells were activated by precoated anti-CD3 (2 μg/ml) and cultured with exogenous PD-L1 (2 μg/ml) as well as antibodies (105 pM) for 4 days. The ratio of daughter cells was measured by flow cytometry.Additionally, to assess the activity of the anti-PD-L1 moiety of YM101, we performed a T cell activation assay  in the presence of exogenous PD-L1 (2 μg/ml). Viable T cells  = length × width2 × 0.5. Mice were euthanized when tumor volume exceeded 2500 mm3 or when the study ended.To explore the optimal dose of YM101, BALB/c mice were inoculated with 5 × 104 EMT-6 cells. Therapy was initiated on 11 to 12 days after inoculation, when the tumor volumes reached ≈ 250 mm3. Mice were randomly assigned to 4 groups (8 mice per group): vehicle , anti-PD-L1, anti-TGF-β, and YM101. Mice received equivalent mole antibodies every two days by intraperitoneal injection. The mice received treatments for 6 times. Tumor size was measured with a digital caliper every two days.To investigate the effect of YM101 on the TME, BALB/c mice were inoculated in the right mammary fat pad with 5 × 106 CT26 cells were inoculated subcutaneously in BALB/c mice. Treatment was started on 9 to 10 days after inoculation, when the tumor volumes reached 100–200 mm3. Mice received treatment every two days by intraperitoneal injection. Tumor size was measured with a digital caliper every 2 days.1 × 106 3LL cells in the right flank. Therapy was initiated on 8 days after inoculation when the tumor volumes reached ≈ 100 mm3. Mice received treatment every two days. On the 10 days after the final YM101 treatment, YM101-cured or treatment-naive C57BL/6 mice were rechallenged by subcutaneously inoculating 1 × 106 3LL cells. Additionally, we repeated the 3LL challenging assay to explore the effect of YM101 on survival. 3LL tumor-bearing mice received 6 doses of antibodies. Then, the mice were followed up for survival for 2 weeks.C57BL/6 mice were inoculated with 1 × 10Mice were sacrificed, and tumor tissues were harvested. Single-cell suspensions were prepared using Collagenase B  and Hyaluronidase . After digestion, the suspensions were filtered by 40 μm Nylon cell strainers . Before staining, cells were suspended in PBS and dyed by Fixable Viability Stain 700. Fluorescent staining was performed according to the manufacturer’s recommendations. Fluorescent antibodies recognizing murine CD45 , CD3 , CD8a , CD107a , granzyme B , CD11b , I-A/I-E , CD11c , CD206 , F4/80  were used in this assay. Flow cytometry buffers utilized in the experiment included Brilliant Stain Buffer  and eBioscience™ FOXP3/Transcription Factor Staining Buffer Set . Flow cytometry was performed using Beckman CytoFLEX S or Beckman CytoFLEX LX. Flow cytometry data were analyzed by Flowjo v10 .Isolated tumors were fixed using 4% paraformaldehyde for 48 h, dehydrated, and embedded with paraffin wax. Tumor tissues were sectioned and transferred to slides. Picrosirius red staining was conducted using Direct Red 80 . Anti-TGF-β1 , anti-p-Smad3 , anti-α-SMA , anti-E-cadherin , anti-Vimentin , anti-Ki67 , anti-PCNA , anti-cleaved-Caspase 3 , anti-CD3 , anti-CD4 , and anti-CD8  immunohistochemistry staining assays were performed according to the two-step protocol . Bright-+ cell was assessed using the proportions of positive pixels in ROIs. For picrosirius red, anti-α-SMA, anti-E-cadherin, anti-Vimentin, anti-Ki67, anti-PCNA, and anti-cleaved-Caspase 3 staining, the expression abundances were measured by integral optical density (IOD) values. For any ROI, the infiltration depth of T cell was calculated by the mean nearest distance of all CD3+ cells to the tumor border. The mean nearest distance was scaled by the distance between the corresponding tumor border to tumor center [The digital quantitation of immunohistochemistry staining was conducted with ImageJ software . The ratio of CD3r center .p-value less than 0.05. The comparisons were performed as following: YM101 vs. vehicle, YM101 vs. anti-TGF-β, and YM101 vs. anti-PD-L1. The expression profile of DEGs was visualized using pheatmap package. The immune signatures were designed based on public lists [The total RNA of EMT-6 tumors was extracted by Trizol, as previously described . For eacic lists , which wic lists .t-test or t-test with Welch’s correction was used for the statistical comparison between two groups. For data not meeting normal distribution, Mann–Whitney test was used for the statistical analysis between two groups. All tests were two-sided, and the significance level was 0.05.Statistical analyses were conducted using Prism 8 (GraphPad Software Inc.). For data following a normal distribution, Student’s YM101 is a recombinant IgG1/IgG2 hybrid antibody. The short chain of YM101 consists of three domains: VLa, CL, and VHb  . However, for TGF-β3, the affinity of YM101 was weaker than anti-TGF-β    and 4T1 (IC50 = 417 pM)  Fig. a, b.Fig.TGF-β enhances the movement capability and promotes the EMT in cancer cells . Consistp < 0.0001)   had a modest anti-tumor effect , anti-TGF-β (p < 0.0001), anti-PD-L1 (p < 0.05) , anti-TGF-β (p < 0.001), and anti-PD-L1 (p < 0.01) groups , anti-TGF-β (p < 0.0001), and anti-PD-L1 (p < 0.05) , anti-TGF-β (p < 0.001), and anti-PD-L1 (p = 0.09) with or on the verge of statistical significance    Fig. i, j. In 29) Fig. i. The tu29) Fig. k. In the+ and CD8+ T cells in the peritumoral stroma , anti-TGF-β (p < 0.05), and anti-PD-L1 (p < 0.05) , anti-TGF-β (p < 0.05), and anti-PD-L1 (p = 0.09) with or on the verge of statistical significance , anti-TGF-β , anti-PD-L1  , anti-TGF-β (p < 0.01), anti-PD-L1 (p < 0.05)  of tumor infiltrating lymphocytes (TILs) compared to vehicle (05) Fig. a. In addnce Fig. b. Apart ly) Fig. c, d. Mor05) Fig. e.Fig. 8Fp < 0.001), anti-TGF-β-treated (p < 0.01), and anti-PD-L1-treated tumors (p < 0.05) , anti-TGF-β (p < 0.05), and anti-PD-L1 (p < 0.05)  Fig. f. Furthe05) Fig. g.Prf1 (perforin), Ifng (interferon), Gzma (granzyme A), and Gzmb (granzyme B) were upregulated in YM101-treated group  and a mesenchymal marker (Vimentin) to assess EMT phenotype. The results of IHC staining showed that YM101 upregulated E-cadherin but downregulated Vimentin  had been developed. The results of preclinical studies of M7824 showed that the dual blockade of TGF-β and PD-L1 was feasible in cancer treatment . MoreoveIn conclusion, we developed a novel bispecific antibody YM101, which simultaneously blocked TGF-β and PD-1/PD-L1 pathways. YM101 exhibited a potent anti-tumor activity, even in the murine models in which single anti-TGF-β or anti-PD-L1 treatment did not trigger substantial tumor regression. Further investigations on the TME revealed that YM101 promoted the formation of immune inflamed tumor, normalized the dysregulated anti-tumor immunity, and provided an immunosupportive TME. Based on the encouraging results of this pilot experiment, it is promising to further develop the anti-TGF-β/human PD-L1 bispecific antibody. As increased TGF-β is a dominantly immune inhibitory pathway in multiple types of cancer, an anti-TGF-β/PD-L1 bispecific antibody might provide a choice for cancer patients resistant to immune checkpoint inhibitors.Additional file 1. Supplementary tables and figures.


Moreover, the reduction of mitochondrial membrane potential (MMP) and the impairment of mitochondrial oxygen consumption triggered by Aβ42O were also prevented by PL171. Further experiments demonstrated that PL171 reduced the acetylation of mitochondrial proteins, and particularly the acetylation of manganese superoxide dismutase (MnSOD) and oligomycin-sensitivity-conferring protein (OSCP), two mitochondrial SIRT3 substrates, was suppressed by PL171. Mechanism studies revealed that PL171 upregulated SIRT3 and its upstream peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) under basal and Aβ42O-treated conditions. The inhibition of SIRT3 activity could eliminate the protective effects of PL171. Further, long-term treatment with Aβ42O increased the number of senescent neuronal cell, which was also alleviated by PL171 in a SIRT3-dependent manner. Taken together, our results indicated that PL171 rescued Aβ42O-induced oxidative stress, mitochondrial dysfunction, and cell senescence via upregulating SIRT3 and might be a potential drug candidate against AD.Oxidative stress-induced mitochondrial dysfunction and cell senescence are considered critical contributors to Alzheimer's disease (AD), and oxidant/antioxidant imbalance has been a therapeutic target in AD. SIRT3 is a mitochondrial protein regulating metabolic enzyme activity by deacetylation and its downregulation is associated with AD pathology. In the present study, we showed that a newly synthesized rhamnoside derivative PL171 inhibited the generation of reactive oxidant species (ROS) induced by amyloid- Mitod fibril . The redd fibril . In receory loss , 8. Aβ dal (MMP) , 10. Andoduction .β-induced neuron dysfunction is unknown. The present study demonstrated that a newly synthesized rhamnoside derivative PL171 attenuated Aβ42 oligomer- (Aβ42O-) induced oxidative stress, mitochondrial dysfunction, and cell senescence by upregulating SIRT3-mediated antioxidant effects, indicating PL171 can counteract Aβ42O defects via SIRT3.Protein acetylation is a posttranslational process regulating global mitochondrial functions . SIRT3 bO-tert-butyldimethylsilyl) ferulic chloride [2Cl2 5.0 mL was added to the solution of 2,3,4-tri-O-acetyl-α-L-rhamnopyranosylamine [2Cl2 10 mL and pyridine 0.1 mL at 0°C. The resulting mixture was stirred for 2 hours at rt, then diluted with CH2Cl2 20 mL. The mixture was washed with brine and the combined organic layers were dried over MgSO4 and concentrated in vacuum. The residue was purified by chromatography on silica gel with eluent , and the β-L-rhamnopyranoside product 820 mg (75%) and α-L-rhamnopyranoside product 120 mg (11%) were obtained, respectively.The  was dissolved in 8.0 mL of dry THF, and TBAF  was added dropwise to the solution at rt. The reaction mixture was stirred for 2 hours, after which it was diluted with THF 20 mL. The mixture was washed with brine and the combined organic layers were dried over MgSO4, and concentrated in vacuum. The residue was purified by chromatography on silica gel with eluent , and the phenol product 340 mg  was obtained.Then, the 2Cl2. 33% CH3NH2 in CH3OH 0.5 mL was added to the solution at 0°C during 5 min. The resulting mixture was stirred for 1 hour at 0°C. The mixture was concentrated in vacuum and the resulting residue was purified by chromatography on silica gel with eluent , and 140 mg  of PL171 was obtained. ESI (+)-MS: 340.3 (M+1)+; H-NMR  δ 7.56  7.19 , 7.07 , 6.82 , 6.62 , 5.28 , 3.92 , 3.85 , 3.54-3.52 , 3.38-3.34 , 1.32 . 13C NMR  δ 168.58, 149.92, 149.10, 143.64, 127.92, 123.51, 118.04, 116.32, 111.55, 79.06, 75.29, 75.22, 73.41, 72.30, 56.28, 17.92.The obtained phenol product 300 mg (0.65 mmol) was dissolved in 3.0 mL of CH1H-NMR and 1H-1H COSY spectra are provided in Figs. A flowchart to summarize the synthesis of PL171 is provided in Fig. β42O were prepared according to the previous publications with some modifications [β42 peptides were purchased from . Briefly, the hexafluoroisopropanol- (HFIP-) treated Aβ42 peptides were resuspended in dimethyl sulfoxide (DMSO) and then diluted in DMEM/F12 phenol-red free medium to achieve a 100 μM concentration. The diluted Aβ42 peptides were then vortexed for 15 s followed by incubation for 24 h at 4°C. The formation of Aβ42O were previously validated in our laboratory by dot blots, atomic force electromicroscopy, and western blot assays [The Aications –25. Aβ42t assays , 26.v/v) at 37°C.SK-N-SH cells were purchased from ATCC. The cell line was cultured in Modified Eagle's Medium (MEM) with 10% fetal bovine serum (FBS) and 100 U/mL penicillin and 0.1 mg/mL streptomycin in a humidified incubator with 5% CO2/95% air , following the manufacturer's guidelines. The values were measured by BioTek SynergyNEO .SK-N-SH cells were seeded in 96-well plate at 1 × 106) were seeded into 60 mm dishes. After the required treatments, cells were washed once with PBS, dissociated with trypsin-EDTA solution, and collected by centrifugation at 200 g for 10 min. The cell pellets were gently resuspended in PBS precooled in an ice bath followed by centrifugation at 600 g for 5 min at 4°C. The pellets were gently resuspended with 1 mL mitochondrial separation reagent supplemented with (100 μM) PMSF and then incubated on ice for 10 minutes. Cell suspensions were then homogenized on ice with a 1 cc insulin syringe 28G1/2, drawing through the needle 10 times. After centrifugation at 600 g for 10 min at 4°C, the supernatants were collected and recentrifuged at 11,000 g for 10 min at 4°C to get the mitochondria. The mitochondria lysates were then used for western blot analysis.The cellular mitochondria were extracted following the manufacturer's instructions with some modifications . SK-N-SH cells (1.5 × 104 cells/well density. Cells were treated with Aβ42O or PL171 at indicated concentrations followed by staining with 10 μM DCFH-DA in the serum and phenol-red free medium for 30 min at 37°C in a humidified incubator with 5% CO2/95% air (v/v) at 37°C. The cells were washed with PBS twice and then observed under a laser-scanning confocal microscope . Alternatively, the cells in the 96-well black plate were extracted with 1% Triton X-100 for 10 min at 37°C and the fluorescence was measured using BioTek SynergyNEO  at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA)  was used as a probe to detect intracellular ROS levels. Briefly, SK-N-SH cells were seeded in a 96-well plate at 1 × 10μM Aβ42O for 24 h following preincubation with or without 30 μM PL171 for 4 h. At the end of treatment, cells were costained with 2.5 μM MitoSOX Red mitochondrial superoxide indicator  and 3 μg/mL the nuclear staining dye Hoechst  for 20 min at 37°C. The fluorescent signals were recorded using BioTek SynergyNEO at 510/580 nm (excitation/emission) for MitoSOX and 350/461 nm for Hoechst. The MitoSOX fluorescence signal was normalized to the Hoechst.Cells were treated with 10 4 cells/well. Cells were challenged of Aβ42O or Aβ42-1O at indicated concentrations or pretreated with PL171 for 4 or 24 h followed by incubation with 10 μM Aβ42O for another 24 h. JC-1 kits  were used to assess the MMP level of cells according to the manufacture's protocols. Briefly, cells were loaded with mixed JC-1 staining solution for 30 minutes at 37°C and then washed twice with the staining buffer. The cells were observed under a Zeiss Observer Z1 microscope. The fluorescence intensity was detected using the BioTek SynergyNEO  at 490/530 nm (green) for monomers and 525/590 nm for aggregates (red), and the membrane potential was represented as the ratio of red/green fluorescence intensity.SK-N-SH cells were seeded into a 96-well plate  at the density of 1 × 104 cells/well. The cells were pretreated with or without 30 μM of PL171 for 4 h followed by stimulation with 10 μM Aβ42O for 24 h. The cells were then incubated in a nonbuffered bicarbonate-free DMEM (Sigma) containing 25 mM glucose, 2 mM glutamax, and 1 mM sodium pyruvate at 37°C in an incubator without CO2 for 45 min. OCR was measured under basal condition and also after the injection of oligomycin (1 μM), FCCP (1 μM), rotenone (1 μM), and antimycin A (1 μM). Data were analyzed using Seahorse XF-24 software.The cellular oxygen consumption rate (OCR) of SK-N-SH cells was examined using SeahorseXFe24 following the manufacturer's guidance (Seahorse Bioscience). SK-N-SH cells were seeded in XF24-well microplates at 3 × 10β-gal staining using a commercial kit . SK-N-SH cells (5 × 104 cells/well) were cultured in the medium with 5% FBS in 24-well plates. After 72 h treatments with Aβ42O in the absence or presence of PL171, cells were prepared for SA-β-gal staining following the manufacturer's guidelines. The cells were imaged by a Zeiss Observer Z1 microscope. The blue-stained cells from at least 10 different fields (60-100 cells/field) were counted under each experiment.Cell senescence was measured by SA-5 cells/well density were extracted by 1 mL TRI Reagent  to obtain total RNA according to the manufacturer's instructions. Reverse transcription was conducted using PrimeScript RT master mix  under the following conditions: 37°C, 15 min and 85°C, 15 sec. Then, the reaction consists of 4 μL of prediluted cDNA in a total volume of 25 μL reaction containing 0.25 μM each primer. And gene transcripts were analyzed by quantitative real-time PCR conducted with 2x HotStart SYBR Green qPCR master mix  on a Stratagene Mx3000P (Agilent Technologies). The reaction parameters were as follows: 95°C for 10 min; 95°C for 30 s, 40 cycles; 60°C for 30 s; 72°C for 30 s. An additional cycle was performed for evaluation of primer's dissociation curve: 95°C for 1 min, 60°C for 30 s and 95°C for 30 s. Each cDNA sample was amplified twice. HPRT was used as an internal control. Primers used were as follows:After treatment with PL171 at indicated concentrations, cells at 2 × 10α, forward 5′TCTGAGTCTGTATGGAGTGACAT-3′, reverse 5′-CCAAGTCGTTCACATCTAGTTCA-3′; HPRT, forward 5′-CCTGGCGTCGTGATTAGTGAT-3′, reverse 5′-AGACGTTCAGTCCTGTCCATAA-3′.SIRT1, forward 5′-AAGTTGACTGTGAAGCTGTACG-3′, reverse 5′-TGCTACTGGTCTTACTTTGAGGG-3′; SIRT3, forward 5′-CCCCAAGCCCTTTTTCACTTT-3′, reverse 5′-CGACACTCTCTCAAGCCCA-3′; PGC-15 cells/well) were treated with PL171 for 24 h or pretreated with PL171 for 4 h followed by Aβ42O treatment for another 24 h. For mitochondria lysate preparation, cells at 1.5 × 106 cells/well density were seeded and mitochondria were isolated as previously described. Total cell lysates or mitochondria lysates were separated by 10 or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes . Membranes were blocked with 5% nonfat milk in TBS containing 0.1% Tween-20 for 1 h at room temperature (RT). Membranes were subsequently incubated with relevant antibodies: SIRT3 , ATP5A , SIRT1 , OSCP , ATP5Ok139 , SOD2 , SOD2k68 , PGC-1α , AMPKα Rabbit Monoclonal Antibody , Phospho-AMPKα (Thr172) Antibody , and actin , at 4°C overnight followed by horseradish peroxidase- (HRP-) conjugated secondary antibody. Membranes were then incubated with an ECL substrate and visualized by Mini Chemiluminescent Imaging and Analysis System.Cells (1 × 10t-test (two-tailed) was applied for the comparisons of two datasets, and one-way or two-way analysis of variance (ANOVA) with Bonferroni's posttest was used where more than two datasets were compared. Statistical significance was accepted at p < 0.05.The data were analyzed by Prism 6.0 . Unpaired Student's β42O could induce the generation of ROS, thus causing oxidative stress of neurons. Here, we treated human neuronal cells SK-N-SH with different concentrations of Aβ42O for 24 h and measured the cellular ROS level by staining with DCFH-DA, which has no fluorescence and can pass freely through plasma membrane, and produce fluorescent DCF when oxidized by ROS. Data showed that treatment with Aβ42O dose dependently increased DCF fluorescence and Aβ42O at 10 μM significantly promoted the signal by about 30% to the control  consistently induced the increase of ROS which however was dose dependently reduced by the pretreatment with PL171  stimulated mitochondrial ROS by about 26% which was significantly suppressed by the preincubation with PL171   . These rβ42O can induce the loss of MMP. In the present study, JC-1 probe was used to evaluate MMP in SK-N-SH cells. Red fluorescence and green fluorescence represented high and low mitochondrial membrane permeability, respectively, and the ratio could represent the change of MMP. Compared with the control group, treatment with Aβ42O largely enhanced green fluorescence intensity  decreased MMP by about 24%, 32%, and 36% for 8 h, 16 h, and 24 h, respectively  induced the reduction of MMP by 34% which was attenuated to around 10% by preincubation with 30 μM of PL171 for 4 h. This protective effect of PL171 was even more profound when extending the period of PL171 preincubation to 24 h  impaired OCR, and however, the presence of PL171  inhibited Aβ42O-induced mitochondrial impairment  for 4 h restored the ATP level to the level similar as the control  and OSCP (ATP5O/OSCP) by about 20% and 36%, respectively  significantly blocked the effect of PL171  increased the acetylation level of MnSOD which was significantly downregulated by preincubation with 30 μM of PL171 for 4 h  signal pathway [α mRNA and protein expressions by PL171 was detected  abolished PL171-mediated AMPK activation and SIRT3 upregulation  reduced SIRT3 and PGC-1α expressions compared to the control group  reduced MMP by 32% to the control group, which was successfully prevented by PL171 . In SIRT3 inhibitor-pretreated cells , Aβ42O decreased MMP by 28% which was not changed by PL171  increased the number of SA-β-gal-positive cells by more than twofold  resulted in a similar increase in the number of SA-β-gal-positive cells as in the cells without 3-TYP  mitochondrial dynamic or morphology, (2) bioenergetics (ATP and oxidative stress), and (3) transport . Regardifunction , 36. Altfunction , 38, curfunction , and idefunction , 41. In β-mediated mitochondrial dysfunction is not exactly clear yet. In recent years, some groups have explored relevant mechanisms to impact mitochondrial function in AD. SIRT3 is the main mitochondrial sirtuin involved in protecting stress-induced mitochondrial integrity and energy metabolism and is highly associated with the pathogenesis of AD [β, both the mRNA and protein levels of SIRT3 are declined [β level in AD patients [β42O induced the reduction of SIRT3 expression and its activity, further proving that SIRT3 is involved in Aβ-mediated mitochondrial dysfunction. The prevention of SIRT3 reduction by PL171 attenuated Aβ42O-induced neuronal defects, which were abolished by the SIRT3 inhibitor, suggesting that SIRT3 could be a therapeutic target for AD treatment.The mechanism of Ais of AD . In the declined  and litepatients . Thus, Spatients , 44. Herα, a transcriptional coactivator for the peroxisome proliferator-activated receptor-γ (PPARγ) and for other transcription factors is involved in the regulation of oxidative phosphorylation, lipid metabolism, and mitochondrial biogenesis [α has protective effects against AD pathology. For example, PGC-1α has been reported to downregulate the transcription and expression of BACE1, which results in reduced Aβ generation and increased nonamyloidogenic sAPPα levels [α was decreased in the brain of AD patients and the content of PGC-1α protein was negatively correlated with Aβ levels [in vitro studies demonstrated that Aβ reduced PGC-1α expression and PGC-1α could restore Aβ neurotoxicity [α and prevented Aβ-induced decline of protein. It has been reported that AMPK activation can stimulate CREB-mediated PGC-1α expression which regulates ERRα binding to the motif in SIRT3 promoter and promotes SIRT3 gene level [α/SIRT3 axis.PGC-1ogenesis . PGC-1α α levels . Notablyβ levels . Furthertoxicity , 48. Herne level , 50. We β42O facilitate the number of senescent neuronal cells properly by stimulating ROS generation and mitochondrial dysfunction. Selective elimination of senescent cells or inhibition of cell senescence process is now considered a promising strategy for the treatment of age-associated disorders [β42O-induced neuronal senescence is unknown. Here, as a designed rhamnoside derivative, PL171 can not only prevent Aβ42O-induced oxidative stress and mitochondrial impairment but also inhibited Aβ42O-mediated cell senescence. All these effects were absent when the activity of SIRT3 was blocked, indicating that PL171 has antiaging or anti-AD effects via targeting SIRT3. Notably, all these effects of PL171 were examined in a cell line which is deficient for in vivo interoperation. In the future, more relevant in vivo models should be applied to further investigate the therapeutic potential of PL171 on aging or AD intervention.Cell senescence is a biological process that involves several key elements including mitochondrial dysfunction, ROS production, inflammation, and DNA damage and plays a key role in promoting aging and age-related diseases, such as AD , 52. Togisorders . Rhamnosisorders , 20, whiβ-induced oxidative stress-mediated mitochondrial dysfunction and cell senescence via promoting SIRT3 function in human neuronal cells.PL171 can counteract A


In the title pyridazinone derivative, the chlorophenyl and pyridazinone rings being almost perpendicular, while the phenyl ring of the styryl group is coplanar with the pyridazinone ring. In the crystal, N—H⋯O hydrogen bonds form inversion dimers with an  19H14Cl2N2O, an important pharmacophore with a wide variety of biological applications is not planar, the chlorophenyl and pyridazinone rings being almost perpendicular, subtending a dihedral angle of 85.73 (11)°. The phenyl ring of the styryl group is coplanar with the pyridazinone ring [1.47 (12)°]. In the crystal, N—H⋯O hydrogen bonds form inversion dimers with an R22(8) ring motif and C—H⋯Cl hydrogen bonds also occur. The roles of the intermolecular interactions in the crystal packing were clarified using Hirshfeld surface analysis, and two-dimensional fingerprint plots indicate that the most important contributions to the crystal packing are from H⋯H (37.9%), C⋯H/H⋯C (18.7%), Cl⋯H/ H⋯Cl (16.4%) and Cl⋯C/C⋯Cl (6.7%) contacts.The title pyridazinone derivative, C Pyridazinone is an important pharmacophore possessing a wide range of biological activities including antitumor  of 3.9107 (15) Å where Cg1 and Cg3 are the centroids of the N1/N2/C8–C11 and C14–C19 rings, respectively pyridazine -one -yl}acetate -one -one . Other interactions and their respective contributions are C⋯H/H⋯C (18.7%), Cl⋯H/H⋯Cl (16.4%), Cl⋯C/C⋯Cl (6.7%), O⋯H/H⋯O (6.5%), N⋯H/H⋯N (4.8%), C⋯O/O⋯C (3.3%) and C⋯N/N⋯C (2.5%). The acceptor and donor atoms participating in the hydrogen bond appear as blue (donors) and red regions (acceptors) corresponding to positive and negative potential, respectively, in the HS mapped over the electrostatic potential, in the range −0.099–0.165 a.u., as shown in Fig. 6A Hirshfeld surface (HS) study of the title compound was undertaken using E)-6-styryl-4,5-dihydropyridazin-3(2H)-one  and 2,6-dichlorobenzaldehyde  in 30 ml of ethanol, sodium ethanoate  was added. The mixture was refluxed for 3 h. The reaction mixture was cooled, diluted with cold water and acidified with concentrated hydrochloric acid. The precipitate was filtered, washed with water, dried and recrystallized from ethanol. Colourless single-crystals were obtained by slow evaporation at room temperature.To a solution of (Uiso(H) = 1.2Ueq(C). The N-bound H atom was located in a difference-Fourier map and refined with N—H = 0.86 Å. Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698902001573X/dx2034sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S205698902001573X/dx2034Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698902001573X/dx2034Isup3.cmlSupporting information file. DOI: 2047452CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


N,N′-bisarylamidine family is reported herein: the N,N′-bis-4-pyridylamidine is a symmetrically N,N′-disubstituted arylamidine containing a 4-pyridyl substituent on the carbon atom of the N–C–N linkage and bulky 2,6-diisopropylphenyl groups on the nitrogen atoms.A new member of the bulky  30H39N3·0.5C7H8, is a symmetrically N,N′-disubstituted arylamidine containing a 4-pyridyl substituent on the carbon atom of the N–C–N linkage and bulky 2,6-diisopropylphenyl groups on the nitrogen atoms. It crystallizes in the Z-anti configuration and its amidine C—N bonds present amine [1.368 (1) Å] and imine [1.286 (1) Å] features. Intramolecular hydrogen bonds are present in the structure together with intermolecular N—H⋯N and C—H⋯N interactions linking the molecules in chains along the a- and c-axis directions.The title compound, C It is important to note that for the compounds crystallized in mixtures of Z-anti and E-syn tautomeric forms, the value of ΔCN is, as expected, significantly lower . The second type of intermolecular hydrogen bond is a much weaker Csp2—H ⋯N interaction between the para proton H10 of aryl ring 1 and the N2 amidine bridge atom of an adjacent molecule .In the crystal structure of s Table 1 (Desirajs Table 1 can be ia axis) alternate with layers of co-crystallized toluene molecules, but no real attractive interactions were identified between the main amidine and the toluene.In the crystal packing, the chains of main amidine moieties ] and Csp3—H ⋯π type [C15—H15 ⋯π (pyridyl ring)] arylamidines -2,4,6-trimethylbenzamidine -2-oxybenzamidinato ligand also exist phenyl]-4-pyridinecarboximidamide (1)1 was obtained from N--4-pyridinecarboxamide phenyl]-4-pyridinecarboxamide , SOCl2 , dry Et3N , 2,6-diipropylaniline , and dry toluene (50 mL) were combined following the general procedure for benzamidine synthesis reported in the above-mentioned reference. A beige precipitate was obtained directly from the reaction mixture, which was recrystallized in hot EtOH, to yield the desired product as a beige solid. X-ray quality crystals (colourless blocks) were obtained in EtOH/water (1:1) at 263 K. Yield 7.5 g, 66%. 1H NMR  δ, ppm: 8.58–8.49 , 8.45 , 7.57–7.49 , 7.41–7.33 , 7.33–7.25 , 7.23 , 6.87 , 6.83–6.76 , 3.43 , 2.99 , 1.30 .Compound k × Ueq(parent C-atom), where k = 1.2 for the aromatic H atoms and 1.5 for the methyl H atoms. The NH proton (H1) was located in the difference-Fourier map and refined freely.Crystal data, data collection and structure refinement details are summarized in Table 4Co-crystallized disordered solvent  present on a symmetry position was modelled as two component disorder using PART −1 and PART −2 instructions. The occupancy factor was fixed at 0.25. The following constraints and restraints were also used: DFIX, FLAT and SADI (on position), ISOR and SIMU . The model was refined anisotropically.10.1107/S2056989021000141/dj2016sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021000141/dj2016Isup2.hklStructure factors: contains datablock(s) I. DOI: 2054114CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Data 10.1038/s41597-020-00600-4, published online 14 August 2020Correction to: Programa de Pós-graduação em Agroecologia, Universidade Estadual do Maranhão (UEMA), São Luís, Brazil was spelled incorrectly. This has now been corrected in both the HTML and PDF versions.Following publication of this Data Descriptor it was found that the affiliation,


Charge-assisted N—H⋯O and O—H⋯O hydrogen bonds along with π–π interactions stabilize the crystalline state. Intermolecular interactions are quantified by Hirshfeld surface analysis. 2H4N3+·C2HO4− (I), comprises one 1,2,4-triazolium cation and one hydrogen oxalate anion. In the crystal, the hydrogen oxalate anions are linked by O—H⋯O hydrogen bonds into chains running parallel to [100]. In turn, the anionic chains are linked through the 1,2,4-triazolium cations by charge-assisted +N—H⋯O− hydrogen bonds into sheets aligned parallel to (01The asymmetric unit of the title 1:1 salt 1,2,4-triazolium hydrogen oxalate, C C—H⋯N) between the 1,2,4-triazolium cations play a minor role in the stabilization of the crystal packing. The differences between the individual fingerprints of cations and anions result from different distributions of the C⋯N/N⋯C contacts (i.e. π–π stacking). It was found that the H⋯H contacts have a relatively small contribution of only 7.7% to the entire Hirshfeld surfaces of the title salt.In order to quantify the nature of the intermolecular interactions present in the crystal structure, Hirshfeld surfaces  under constant stirring for one h. The resulting solution was filtered to remove any undissolved solid. The filtrate was allowed to slowly evaporate at room temperature. After two weeks, colourless block-shaped crystals of the title salt (I) suitable for X-ray analysis were obtained.An aqueous solution (5 ml) of oxalic acid  and 1,2,4-triazole  was added dropwise to an aqueous solution (5 ml) of TbClUiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019017304/wm5529sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019017304/wm5529Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019017304/wm5529Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019017304/wm5529Isup4.cmlSupporting information file. DOI: 1974526CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The tumour suppressor protein p53 regulates various genes involved in cell-cycle arrest, apoptosis and DNA repair in response to cellular stress, and apparently functions as a pioneer transcription factor. The pioneer transcription factors can bind nucleosomal DNA, where many transcription factors are largely restricted. However, the mechanisms by which p53 recognizes the nucleosomal DNA are poorly understood. In the present study, we found that p53 requires linker DNAs for the efficient formation of p53-nucleosome complexes. p53 forms an additional specific complex with the nucleosome, when the p53 binding sequence is located around the entry/exit region of the nucleosomal DNA. We also showed that p53 directly binds to the histone H3-H4 complex via its N-terminal 1–93 amino acid region. These results shed light on the mechanism of nucleosome recognition by p53.  In genomes, transcription factors bind to specific DNA sequences on regulatory elements to control their target gene expression. In eukaryotes, genomic DNA is tightly compacted as chromatin, in which the repeating unit is the nucleosome . The nucThe tumour suppressor p53 is a pioneer transcription factor , and regGenomic studies have suggested that the p53 binding sites are located near the entry/exit sites of nucleosomes in cells , 16. In To reveal the mechanisms by which p53 binds to the nucleosomes with and without its target sequence, p53BS, we prepared the recombinant human p53 protein and performed biochemical analyses with reconstituted nucleosomes.Escherichia coli BL21(DE3) cells with the p53 expression plasmids were grown at 37°C until the optical density reached 1.2 (OD600). The expression of the GST-fused p53 proteins was then induced by the addition of 0.5 mM isopropyl β-d-1-thiogalactopyranoside, and the cells were further cultured at 18°C overnight. The cells were harvested by centrifugation and resuspended in buffer A (50 mM Tris–HCl (pH 7.5), 0.5 M NaCl, 10% glycerol, 1 mM EDTA and 2 mM 2-mercaptoethanol). The cells were then disrupted by sonication, and the lysates were centrifuged. The supernatants were gently mixed with glutathione sepharose 4B beads  at 4°C for 1 h. The beads were then loaded into an Econo-column (Bio-Rad), and were washed with 20 column volumes (CVs) of buffer B (50 mM Tris–HCl (pH 7.5), 1 M NaCl, 1 mM EDTA and 2 mM 2-mercaptoethanol), followed by washing with 20 CVs of buffer C (30 mM Tris–HCl (pH 7.5), 0.15 M NaCl and 2 mM 2-mercaptoethanol). For the electrophoretic mobility shift assay (EMSA) experiments, the GST-tags on the p53 proteins were removed by on-column GST-tagged human rhinovirus 3C protease cleavage, at 4°C overnight. The eluted proteins were further purified by Heparin Sepharose  chromatography, using stepwise elution with buffer D (30 mM Tris–HCl (pH 7.5), 1 M NaCl and 2 mM 2-mercaptoethanol), and finally purified by chromatography on a HiLoad 16/600 Superdex200 pg  or HiLoad 26/600 Superdex75 pg  column with buffer E (30 mM Tris–HCl (pH 7.5), 0.5 M NaCl and 2 mM 2-mercaptoethanol). For the pull-down assay, the GST-fused p53 proteins on the Glutathione Sepharose 4B beads were eluted with buffer F (30 mM Tris–HCl (pH 9.0), 0.15 M NaCl, 2 mM 2-mercaptethanol, 20 mM glutathione), and were finally purified by chromatography on a HiLoad 16/600 Superdex200 pg column , eluted with buffer E.The DNA fragments encoding the human p53 full-length (p53FL), p53 N-terminal region  and N-terminus deleted p53  proteins were inserted into the pGEX-6P-1 vector . The 193 bp DNA fragments derived from the Widom 601 sequence were prepared as described , 23. Accet al.  were mixed with p53 or a p53 mutant (0–0.8 µM) in reaction solution (22 mM Tris–HCl (pH 7.5), 100 mM NaCl, 0.4 mM 2-mercaptoethanol and 0.8 mM DTT), and the samples were incubated at 25°C for 30 min. The p53 binding was visualized by non-denaturing 5% PAGE in 0.5 × TBE buffer , followed by ethidium bromide staining.For the preparation of the p53-nucleosome complex, p53 (270 pmol) and the nucleosome (60 pmol) were incubated in reaction solution (25 mM Tris–HCl (pH 7.5), 250 mM NaCl, 1 mM 2-mercaptoethanol and 0.5 mM DTT) at 25°C for 30 min. The p53-nucleosome complex was fractionated on a non-denaturing 5–12% SuperSep™ Ace gel  in 1 × TBE buffer , followed by ethidium bromide staining (first dimension). The gel was soaked in denaturing buffer  and 100 mM DTT), and was gently agitated at room temperature for 1 h. The gel was then analysed by 18% SDS-PAGE, followed by Oriole (Bio-Rad) staining (second dimension).p53 (0.8 µM) was incubated with the nucleosome (0.1 µM) in 10 µl of reaction solution (20 mM Tris–HCl (pH 7.5), 100 mM NaCl, 0.4 mM 2-mercaptoethanol and 0.8 mM DTT), at 25°C for 30 min. The 24 bp DNA (0–1.6 µM) was then added to the reaction solution, and incubated at 25°C for 30 min. The incubated samples were fractionated by non-denaturing biphasic PAGE, using a lower 12% acrylamide gel and an upper 5% acrylamide gel. The PAGE was performed in 0.5 × TBE, followed by ethidium bromide staining.The nucleosome acidic patch antibody, PL2-6, was prepared as described previously (The H3-H4 complex (15 µM as dimer) was mixed with increasing amounts (0–18 µM) of p53 in 10 µl of reaction solution (30 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.1% NP-40 and 1 mM 2-mercaptoethanol), and incubated at 25°C for 30 min. The bands were visualized by non-denaturing 5% PAGE in 0.5 × TBE buffer, followed by Coomassie Brilliant Blue (CBB) staining as described .The H3-H4 complex (2.5 µM as dimer) was mixed with 2.5 µM of GST, GST-p53, GST-p53NTD or GST-p53ΔN in 20 µl of reaction solution (20 mM Tris–HCl (pH 8.0), 150–164 mM NaCl and 0.1% NP-40), and incubated at 25°C for 30 min. Glutathione Sepharose 4B beads  and 500 µl of binding solution (20 mM Tris–HCl (pH 8.0), 150 mM NaCl, 10 µg/ml BSA and 0.1% NP-40) were added to the reaction mixture, and the samples were rotated at 4°C for 30 min. The beads were collected by centrifugation, and washed three times with 1 ml of wash solution (20 mM Tris–HCl (pH 8.0), 150 mM NaCl and 0.1% NP-40). The washed beads were mixed with 10 µl of sample buffer (100 mM Tris–HCl (pH 6.8), 4% SDS, 20% glycerol, 200 mM 2-mercaptoethanol and 0.2% bromophenol blue), and were heated at 95°C for 15 min. The samples were analysed by 18% SDS-PAGE, followed by CBB staining.We first tested the nucleosome-binding activity of purified p53 . To do sTo eliminate the possibility that p53 binds to the 24 bp linker DNA regions, we next performed a competitive nucleosome-binding assay. In this assay, the nucleosome containing the 193 bp DNA was used. The competitor was a 24 bp double-stranded oligonucleotide containing the same DNA sequence as the linker DNA of the nucleosome. As shown in Previous single molecule studies reported that the sequence independent DNA binding of p53 may facilitate the efficient search for its target sequence in genomes . The seqin vivo (We next tested if the p53 target sequence, p53BS, affects the p53-nucleosome complex formation by EMSA. It has been reported that p53 stably binds to the p53BS located at the entry/exit site of the nucleosome in its natural target locus in vivo . Yu et ain vivo  also shoin vivo . As showin vivo , which cin vivo . These rin vivo . TherefoThe p53 binding to its target sequence in the nucleosome is probably mediated by its central DNA-binding region. However, the C-terminal region of p53 also possesses DNA-binding activity . TherefoWe next tested the histone-binding activity of p53 by a pull-down assay. As shown in Finally, we tested whether the N-terminal region of p53 affects the nucleosome binding. To do so, we performed an EMSA with p53ΔN and the nucleosomes containing the 193 bp DNA. Although p53ΔN bound to the nucleosome, the bands corresponding to the specific p53-nucleosome complexes were barely detected . TherefoThe p53 N-terminal region contains TAD1 and TAD2 domains, which are required for p53 target gene induction . A histo