S1868 and NasF5053) and reveal four critical residues  that control their regio- and stereo-selectivity to generate diverse dimeric DKP frameworks. Engineering these residues can alter the specificities of the enzymes to produce diverse frameworks. Determining the crystal structures (1.70–1.47 Å) of NasF5053 (ligand-free and substrate-bound NasF5053 and its Q65I-A86G and S284A-V288A mutants) and molecular dynamics simulation finally elucidate the specificity-conferring mechanism of these residues. Our results provide a clear molecular and mechanistic basis into this family of HTDKP-forming P450s, laying a solid foundation for rapid access to the molecular diversity of HTDKP frameworks through rational engineering of the P450s.Bacterial heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are a growing family of bioactive natural products. They are challenging to prepare by chemical routes due to the polycyclic and densely functionalized backbone. Through functional characterization and investigation, we herein identify a family of three related HTDKP-forming cytochrome P450s  are bioactive natural products that are difficult to access chemically. Here, the authors identify a family of three related HTDKP-forming cytochrome P450s and engineer key amino acid residues to produce distinct diketopiperazines frameworks. Among them, heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are particularly attractive for their unique structural architecture and fascinating bioactive properties, ranging from anticancer, anti-plasmodial, anti-HIV, and neuroprotective activities6. TDKPs are primarily produced by fungal systems, in which two pyrroloindoline units are predominantly fused together by a C3-C3’ bond8. Bacterially-sourced HTDKPs are much rarer, but their structural architectures are more versatile11. Based on their connectivities and stereochemistry, the dimeric DKP frameworks can be classified into five different types ; (II) C3-C7’, 2S-3R . (III) C3-C6’, 2S-3R ; (IV) C3-C6’, 2R-3S ; and (V) N1-C7’ .Natural products derived from tryptophan-containing diketopiperazine (TDKP) comprise a large class of secondary metabolites14. To develop efficient biocatalytic approaches, we recently investigated the biosynthesis of naseseazine C (NAS-C) and identified a key diketopiperazine (DKP) forming P450 enzyme (NascB)6. NascB catalyzes a radical cascade reaction to form intramolecular and intermolecular carbon–carbon bonds with both regio-specificity and stereo-specificity, which is very efficient in constructing the HTDKP frameworks and has been used to create 30 type I–IV NAS analogs employing different DKP substrates6. Very recently, Li and coworkers further identified two other P450 enzymes, AspB and NasB, which are responsible for the predominant formation of aspergilazine A (ASP-A) and NAS-B, respectively11 (Supplementary Table L-PL) into NAS-C (type III) and iso-NAS-B (type IV) accompanying the major product ASP-A (type V)11. This property of co-generation of different types of HTDKPs suggests these P450s have a regulatory mechanism in controlling different regio-specificities and stereo-specificities and presents a great potential for further improving catalytic efficiency, altering specificity and even creating diverse frameworks by rational protein engineering. However, such endeavors are reliant on understanding the molecular basis of HTDKPs-producing P450-catalyzed reactions, which currently remains elusive.The regio-specificity and stereo-specificity in the densely functionalized frameworks, especially at the quaternary stereocenter at the C3 position, renders chemical synthesis of bacterial HTDKPs very challengingS1868, and NasF5053), structurally characterize NasF5053 and its mutants by X-ray crystallography, and explore the further catalytic potential of the fourth pertinent P450 (NascB). Our results reveal that four key residues  are crucial for controlling the combination of the different regio-specificities and stereo-specificities. Based on our structural characterization, molecular dynamics and mutagenesis-validation of the residues involved, we elucidate how the regio-configuration and stereo-configuration in forming bonds is finely tuned in these P450s.To this end, we herein functionally characterize three HTDKP-forming P450s  and one adjacent P450. The P450 NascB (CYP nomenclature: CYP1190B2) encoded in locus-1 was responsible for the biosynthesis of NAS-C6. To verify the functionality of locus-2, its P450 NasbB was expressed in the Mycobacterium system. In the presence of spinach flavodoxin (Fd) and flavodoxin reductase (FdR), as well as an NADPH recycling system , NasbB was confirmed to efficiently dimerize the cWL-PL into NAS-B  in the , and 3 inascB or nasbB. We identified two previously uncharacterized P450 proteins: NasF5053 and NasS1868 from the Streptomyces strain sp. NRRL F-5053 and sp. NRRL S-1868, respectively. Soluble recombinant NasF5053 (CYP nomenclature: CYP1190B1) could be expressed in E. coli BL21 (DE3), while soluble NasS1868 (CYP nomenclature: CYP1190B1) could only be expressed in Mycobacterium smegmatis MC2 155.As the P450 enzymes with highly similar sequences can produce different products, we were interested in establishing the relationship between enzyme sequences and the corresponding products. Although NascB and NasbB share 68% sequence identity, it is difficult to extract the key residues responsible for the difference in product formation. In order to identify more P450s which could potentially generate some other kinds of C3-aryl pyrroloindolines, we used simple sequence searches to find genes homologous to S1868 can convert cWL-PL into Asp-A . Further, the in vitro assay confirmed that NasF5053 could produce NAS-C (47.4%), Asp-A (44.4%), as well as a minor product NAS-B (8.2%)  from spinach, Nasp-A Fig. ; a simil2%) Fig. , trace IscB Fig. . Except cal Fig. . This siF5053 and NascB, and the resultant mutant proteins were tested by enzyme assays. In the N-terminal part of NasF5053, we converted the following residues to their corresponding amino-acids in NasS1868: V44A-T49A-K52E (triple mutant), and P40H, Q65I, G84A, A86G, and I87V (point mutants). The in vitro enzyme assays of the NasF5053 mutants (P40H could not be tested as it was insoluble) using cWL-PL showed that the simultaneous mutation of V44A, T49A, and K52E and single mutation of G84A or I87V imposed a slight change to the ratio of NAS-C and ASP-A  almost abolished the production of NAS-C and generated ASP-A as the sole product  were introduced into NascB. The Q65I mutation in NascB also impacted on the production of NAS-C; it decreased the production of NAS-C and increased the production of ASP-A from none to reach the NAS-C to ASP-A ratio of 5:7  were chosen simultaneously for NNK-based saturation mutagenesis. The mutated plasmids were transferred into GBdir-T7 E. coli containing spinach Fd and FdR, a whole-cell biocatalysis system we developed previously6. A small library of four hundred colonies was selected and assayed using cWL-PL as a substrate. As expected, the production of NAS-B was significantly improved in some mutants. Among them, the mutant NasF5053-A86K-V288P not only yielded the highest ratio of NAS-B/ NAS-C  forms  and a small domain of four α-helices  and three β-sheets  .To further understand the molecular basis of product diversity of Nasrms Fig. , by X-raF5053 structures reveals binding of substrates only invokes minimal conformational changes, with a root-mean-square deviation (RMSD) of 0.362 Å (for 388 Cα atoms) between the two forms . Notably, Q65, D85, and E73 all reside in the long αB’-αC loop.Comparison between the substrate-free and substrate-bound NasF5053 structure, two cWL-PL molecules  are present in the binding site, with full occupancy. cWL-PL-E adopts an extended conformation and forms multiple contacts with the heme group, loop β3-1-β3-2, loop αB’-αC, and loop αK-β1-4. F387 and L77 form hydrophobic interactions with the proline portion of extended cWL-PL-E. Formation of hydrogen bonds is observed between S284 and the backbone amide nitrogen of G286, with N10’ and O19’ of the substrate, respectively. O18’ and N1’ are indirectly in contact with 7-propionate of the heme and E314, respectively, mediated via hydrogen bonding with water molecules. The hydrophobic side of V288 also protrudes towards 7-propionate of the heme and indole ring in cWL-PL-E , making the intramolecular cyclization between WL and PL in cWL-PL-U possible. Importantly, the indole ring of cWL-PL-U is positioned perpendicular to the heme group plane, with N1 forming a hydrogen bond with the heme-ligating water molecule  and NasF5053-S284A-V288A (PDB ID 6VZB). Comparisons among the three substrate-bound structures showed that all substrate-interacting residues, the heme, and the two substrates superimpose well  to high spin (HS) state16. This transition, however, is not complete because a small but significant fraction of LS signal still remains even in the saturating cWL-PL concentration  simulations with Amber -OH, until two indole rings of the two substrates are almost in a plane. The Q65I and A86G mutations result in a shift of the αB’-αC loop away from cWL-PL-U radical   Fig. . AccordiS-C Fig. . MD alsoble Fig. .L-PL-U radical and drives its rotation and shift towards cWL-PL-E. On the other side, the V288P mutation compresses the active site, slightly pushing and rotating cWL-PL-E  enzymes are among the most exquisite and versatile biocatalysts in nature to synthesize and modify natural products6. Although our previous DFT calculations and experiments preferred the N1-initiation over N10-initiation mechanism6, there was a lack of direct proof. Based on the crystal structures, we can now clearly see that the N1 of cWL-PL-U is indeed much closer than the N10 to the heme-ligating water molecule  that share high sequence similarities but generate unique overlapping product profiles across all the five types of bacterial dimeric DKP frameworks. Our systematic mutagenesis studies on the promiscuous NasF5053 and the versatile NascB identified four key resides, Q65, A86, S284, and V288, which play critical roles in controlling product regio-configurations and stereo-configurations. We demonstrate that the engineering of these residues is able to alter the product ratio and even generate an interesting framework, which has not previously been observed for the substrate cWL-PL. To obtain insights into the structural basis for regio-specificity, stereo-specificity, and chemical versatility, we further determined high-resolution crystal structures of wild-type NasF5053 in its substrate-free and substrate-bound form, and of two NasF5053 mutants (Q65I-A86G and S284A-V288A) in their substrate-bound forms. The binding mode of cWL-PL revealed by the complex structures supports the previous proposed intramolecular and intermolecular radical cascade addition mechanism. Molecular dynamics simulations were employed to uncover the specificity-conferring mechanism of these residues, based on the crystal structures. Therefore, our biochemical, structural, and computational characterizations across this representative group of HTDKP-forming P450s provide a clear mechanism of how these sophisticated catalytic mechanisms take place, which expands our knowledge on the chemical diversity of cytochrome P450s-catalyzed natural products and enables the rational engineering of this group of P450s and other homologs to obtain different HTDKP frameworks.In conclusion, through discovery, identification, and functional characterization, we have identified a suite of P450s  and pMS1 (nasbB and nasS1868), which were overexpressed in E. coli BL21 (DE3) and M. smegmatis mc2 155, respectively , 1 µM ferredoxin reductase (FdR), 2 mM NADP+, 2 mM glucose, and 2 mM glucose dehydrogenase (GDH) in 50 mM HEPES buffer, 100 mM NaCl, at pH 7.5. After incubating at 4 °C for 24 h, the reactions were quenched and extracted with ethyl acetate (2 × 200 µL). Then the combined organics were concentrated in vacuo, which were re-dissolved in HPLC-graded methanol and the resulting solutions were filtered through 0.45 µM membrane and finally analyed by UHPLC-MS. A Diamonsil  was used with a flow rate at 0.3 mL min−1 and a PDA detector over a 23 min gradient program with water (eluent A) and methanol (eluent B): T = 0 min, 40% B; T = 10 min, 40% B; T = 15 min, 70% B; T = 18 min, 40% B; T = 23 min, 40% B.P450 genes with codon optimized for 2, 20% (w/v) polyethylene glycol (PEG) 3350, pH 7.5 at 20 °C using the hanging drop vapor diffusion technique with the addition of 5% glycerol to the protein stock. The initial crystals were subsequently crushed for seeding by using the Seed Bead Kit (Hampton Research). Final crystals were obtained using the micro-seeding technique in 0.2 M CaCl2, and 22% (w/v) PEG 3350, pH 7.5 at 4 °C. Substrate-bound protein crystals were obtained by soaking the substrate-free crystals in the mother liquor containing 2.5 mM cWL-PL (diluting from 50 mM stock solution in DMSO) for 24 h or co-crystallization after mixing 0.13 mM protein with 2.5 mM cWL-PL. Both methods produce identical complex structures. The complex structure from soaking was chosen for structural analysis and presentation, due to a better overall quality, including resolution, of the diffraction data collected from the soaked crystals.Initial crystals were obtained in 0.2 M CaCl2, 22% (w/v) PEG 3350, pH 7.5, and 20% (v/v) glycerol prior to flash freezing in liquid. For the substrate-bound protein crystals, the cryoprotection solution also contained 2.5 mM cWL-PL. The X-ray diffraction data were collected at the Australian Synchrotron MX beamlines. The collected data were indexed and integrated using XDS25 and scaled and merged using Aimless26. A partial initial model of the holo-structure was obtained by the molecular replacement technique with Phaser in Phenix27 using the crystal structure of CYP121 from Mycobacterium tuberculosis (PDB accession code: 5WP2) as the search model. The initial model was improved by using the Morph Model tool in Phenix28 and manually modified in COOT29. The substrate-bound structure was solved by the molecular replacement technique using the holo-structure as the search model. The structures were refined using Phenix.Refine30 and manually modified in COOT iteratively. The graphic presentations of protein structures were prepared with Pymol.Crystals were mounted onto CryoLoops (Hampton Research) and soaked in a cryoprotection solution containing 0.2 M CaCl1H frequency of 400 MHz. Lyophilized samples (varying from 1 to 7 mg) were dissolved in 280 µL DMSO-d6 (Cambridge Isotope) and all spectra were recorded at 25 °C (298 K). 1H and 13C resonances were assigned through the analysis of 1D−1H, 1D 13C, 2D 1H–1H ROESY, 2D 1H–13C HSQC, and 2D 1H–13C HMBC (optimized for long-range heteronuclear couplings of 6 Hz). 1H and 13C chemical shifts were calibrated with reference to the DMSO solvent signal . NMR experiments were processed with Bruker Topspin program (version 3.57) and analyzed with MestReNova software.The NMR spectra were recorded on a Bruker Avance III spectrometer at a Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Although citrus leprosis disease has been known for more than a hundred years, one of its causal agents, citrus leprosis virus C2 (CiLV-C2), is poorly characterized. This study described the association of CiLV-C2 movement protein (MP) and capsid protein (p29) with biological membranes. Our findings obtained by computer predictions, chemical treatments after membrane fractionation, and biomolecular fluorescence complementation assays revealed that p29 is peripherally associated, while the MP is integrally bound to the cell membranes. Topological analyses revealed that both the p29 and MP expose their N- and C-termini to the cell cytoplasmic compartment. The implications of these results in the intracellular movement of the virus were discussed. Cilevirus and Dichorhavirus, has economic importance in the Americas, especially in citrus groves in Brazil. The infection is characterized by the formation of chlorotic and necrotic circular localized lesions in citrus leaves, fruits, and stems [Citrus leprosis disease, caused by viruses belonging to the genera nd stems .Cilevirus, family Kitaviridae, and a prevalent citrus leprosis-associated virus in South and Central America [Cilevirus.Citrus leprosis virus C (CiLV-C) is a member of genus  America , with th America ,3,4,5. CCileviruses have a bisegmented positive ssRNA genome carrying a 5′-cap structure and a 3′-poly (A) tail. For CiLV-C, its RNA1 codes for the protein precursor of the RNA-dependent RNA polymerase (RdRp) and the capsid protein p29) ,7,8, whi ,7,8, whMembrane-associated viral proteins can induce substantial cellular remodeling in the processes of viral replication and virion assembly. This intracellular disorder is usually associated with changes in organelles to form viral replication sites ,13. ViraTherefore, in the present study, we examined biochemical properties of the capsid (p29) and movement (MP) proteins encoded by CiLV-C2. Here, we reported the association of CiLV-C2 MP and p29 with biological membranes. Our findings obtained by computer predictions, chemical treatments after membrane fractionation, and biomolecular fluorescence complementation assay revealed that the p29 is peripherally associated, while the MP is integrally bound to the cell membranes. Topological analyses revealed that p29 and MP expose their N- and C-termini to the cell cytoplasmic compartment. These results allowed us to propose a topological model of the association of CiLV-C2 p29 and MP with cell membranes.p29 and MP genes [NcoI/NheI sites using the TaKaRa Taq DNA polymerase (TaKaRa Bio Inc) following the manufacturer’s specifications. All amplified genes were fused at their C-terminus with the human influenza hemagglutinin (HA) epitope. To do this, the genes were cloned into the vector pSK35-TSWVNSm:HA-PoPit [NSm gene. The correct in-frame insertions were confirmed by plasmid DNA sequencing. The expression cassettes corresponded to each CiLV-C2 isolated gene flanked by the 35S constitutive promoter from the cauliflower mosaic virus (CaMV) and the terminator from the potato proteinase inhibitor (PoPit) [HindIII restriction site.For the membrane association assay, the MP genes  were ampHA-PoPit , replaci (PoPit) . Next, tp29 and MP genes were amplified with specific primers, containing the NcoI/NheI restriction sites, and cloned into the pSK BiFC plasmids [For bimolecular fluorescence complementation (BiFC) assay, the plasmids ,22, whicplasmids .The pMOG vectors carrying the chrysanthemum stem necrosis orthotospovirus (CSNV) NSm, tobacco mosaic virus (TMV) 30K MP, and leader peptidase (Lep) proteins with the HA epitope fused at their C-terminus and the unfused green fluorescent protein (GFP) were described in previous works ,18.http://www.cbs.dtu.dk/services/TMHMM/), ΔG prediction server version 1.0 (http://dgpred.cbr.su.se/index.php?p=home), HMMTOP version 2.0 (http://www.enzim.hu/hmmtop/index.php), OCTOPUS (https://octopus.cbr.su.se), SOUSI (http://harrier.nagahama-i-bio.ac.jp/sosui/), and TMpred .Computer analysis from deduced amino acid sequences of the CiLV-C2 p29 (YP_009508071.1) and MP (YP_009508075.1), CiLV-C MP (ABC75825.1), CSNV NSm (AII20574.1), TMV 30K MP (AAD19281.1), and also for the Lep (WP_112485673.1) and GFP, were performed using computer tools MPEx , TMHMM sNicotiana benthamiana leaves were agroinfiltrated (OD600 = 0.5) with the pMOG800 construct carrying the CiLV-C2 p29 or MP ORFs fused to the HA epitope. As controls, we used N. benthamiana leaves agroinfiltrated with constructs expressing the HA-tagged Lep protein, unfused GFP, and HA-tagged NSm of CSNV. At 3 days post-infiltration (dpi), approximately 1.5 g of N. benthamiana leaves expressing the tested proteins were ground in lysis buffer . The homogenate was clarified by low centrifugation at 3000× g for 10 min at 4 °C, then the obtained supernatant was ultracentrifuged at 40,000× g for 40 min at 4 °C to yield the soluble (S30) and the crude (P30) microsomal fraction as reported previously [2CO3 (pH 11), 2, 4, or 8 M urea, and held for 30 min on ice. All fractions were analyzed by western blot in 12% SDS-PAGE gels as referred to previously [eviously ,24. Nexteviously  using ang for 20 min at 4 °C. The resultant supernatant was incubated at 37 °C for 10 min and centrifuged at 10,000× g for 10 min at room temperature to form the aqueous (AP) and organic phases (OP). Finally, the OP was resuspended in lysis buffer with the same volume obtained in the aqueous phase, and the fractions were analyzed by western blot in 12% SDS-PAGE gels, as above mentioned. Percentage values of relative protein accumulation was measured using Fiji ImageJ program Ver 2.0 with ISAC plugin. A reference value equivalent to 100% was given based on the pixel quantification of the controls treated with lysis buffer.For Triton X-114 analysis, the P30 fraction was resuspended in the lysis buffer containing 1% Triton X-114 and incubated on ice for 30 min. The mixture was clarified by centrifugation at 10,000× Agrobacterium tumefaciens cultures (C58) transformed with the constructs carrying the p29 or MP with the N-YFP or C-YFP fragments were co-infiltrated (OD600 = 0.4) with agrobacterium cultures carrying BiFC constructs containing the counterparts of the YFP addressed to cytosol/nucleus (N-YFPcyt and C-YFPcyt) or to the lumen of the endoplasmic reticulum (ER) (C-YFPer and N-YFPer) in N. benthamiana leaves. The plants were maintained in a FITOTRON growth chamber under conditions of 24 °C day/18 °C night at 16 h light/8 h dark and 70% humidity regime. The preparation for agroinfiltration was conducted as described previously [N. benthamiana were captured with the aid of a confocal laser scanning microscope Zeiss LSM 780 model. YFP was excited at 514 nm and emission was captured at 520–560 nm. The images were prepared using Fiji ImageJ program (version 2.0r).Fluorescence images of the leaf discs from Computer analysis from deduced amino acid sequences of the CiLV-C2 p29 and MP was performed using several computer tools. In the analysis, we also included the deduced amino acid sequences of the CSNV NSm and TMV 30K MP, which have been shown to be peripheral membrane proteins ,19, as wN. benthamiana leaves transiently expressing the CiLV-C2 p29 or MP ORFs fused to the HA epitope. As controls, we used N. benthamiana leaves agroinfiltrated with constructs expressing the HA-tagged Lep protein, unfused GFP, and HA-tagged NSm of CSNV, which corresponded to the transmembrane-, cytosolic-, and membrane-associated proteins, respectively. High-speed ultracentrifugation was performed to separate the plant leaf lysed extract, containing the abovementioned proteins, into pellet (P30) and supernatant (S30) fractions. Treatment with Na2CO3 is known to render microsomes into membrane sheets, releasing soluble luminal proteins [2CO3 treatment (2CO3) and the majority of the GFP protein remained in the soluble fraction (2CO3), as expected. After the urea treatments and specially after the more aggressive treatment with 8 M urea, the HA-tagged MP and Lep proteins remained in the pellet fraction (100% for both proteins), indicating that the CiLV-C2 MP is fully integrated to cell membranes. On the other hand, a considerable proportion of the HA-tagged p29 protein was observed to be associated with the soluble fraction (43%) after 8 M urea treatment. Similar behavior was observed for the CSNV NSm control  suggesting that p29 is peripherally associated with the membrane.In order to confirm these predictions and examine the membrane association of CiLV-C2 p29 and MP, we prepared subcellular microsomal fraction from proteins , while uproteins ,24. We oTo confirm the cell membrane association of the p29 and MP proteins, the P30 membrane-rich fraction was treated with Triton X-114. This treatment generated two phases, the aqueous (AP) and organic phases (OP), in which the integral membrane proteins should be portioned into the OP, meanwhile the AP should contain non-integral membrane proteins and soluble proteins . As expeA. tumefaciens cultures (C58) transformed with the constructs carrying the p29 or MP with the N-YFP or C-YFP fragments were co-infiltrated with agrobacterium cultures carrying BiFC constructs containing the counterparts of the YFP addressed to cytosol/nucleus (N-YFPcyt and C-YFPcyt) or to the lumen of the endoplasmic reticulum (C-YFPer and N-YFPer) in N. benthamiana leaves. All protein pair combinations are shown in N. benthamiana leaves  of CiLV-C2 is peripherally associated with cell membranes with the N- and C-termini exposed to the cytosol. The movement protein (MP) of CiLV-C2 is a transmembrane protein with the N- and C-termini exposed to the cytosol. The amino acid sequence analysis suggested that both CiLV-C2 and CiLV-C MPs could have a similar transmembrane topology. The cilevirus MPs are the first members of the 30K superfamily showing a transmembrane association pattern.


Escherichia coli reporter assay systems. As the tRNA chassis for engineering, I employed amber suppressor variants of allo-tRNAs having the 9/3 composition of the 12-base pair amino-acid acceptor branch as well as a long variable arm (V-arm). Although their V-arm is a strong binding site for seryl-tRNA synthetase (SerRS), insertion of a bulge nucleotide in the V-arm stem region prevented allo-tRNA molecules from being charged by SerRS with serine. The SerRS-rejecting allo-tRNA chassis were engineered to have another amino-acid identity of either alanine, tyrosine, or histidine. The tip of the V-arms was replaced with diverse hairpin RNA aptamers, which were recognized by their cognate proteins expressed in E. coli. A high-affinity interaction led to the sequestration of allo-tRNA molecules, while a moderate-affinity aptamer moiety recruited histidyl-tRNA synthetase variants fused with the cognate protein domain. The new design principle for tRNA-aptamer fusions will enhance radical and dynamic manipulation of the genetic code.Reprogramming of the genetic code system is limited by the difficulty in creating new tRNA structures. Here, I developed translationally active tRNA variants tagged with a small hairpin RNA aptamer, using  E. coli tRNA species . T. TSer vaof SerRS A,B. In aunstable . On the unstable C. Thus, unstable . Encouraunstable  26] in  in Ser vAG codon C.cat(Ser146TAG) . T. Tcat(Se E. coli C.cat(Ser146TAG) gene with Cm at a concentration of 34 µg/mL in the growth media  [C-terminal domain of archaeal O-phosphoseryl-tRNA kinase (PSTK-C) [Aquifex aeolicus (Aa) SelA-N or the Methanopyrus kandleri (Mk) PSTK-C were expressed at a moderate level by using the tac promoter on a pGEX vector and IPTG at a concentration of 10 µM  PSTK-C failed to sequester A005 tRNA molecules   and the (PSTK-C) . As expeof 10 µM C. In theof 10 µM C, which olecules C, possiblo-tRNAs . Similarlo-tRNAs ,11 and tECIS RNA , were trECIS RNA ,11 and tSer molecules was challenged. It is known that the allo-tRNAUTu1 variant (Ser as one of the E. coli tRNASer species in E. coli. S012 conferred at least 600 µg/mL Cm resistance to E. coli expressing the cat(Ser146TAG) gene (Mk PSTK-C (C-loop (U to C at the -5 position) [Mk PSTK-C domain, two V29I domains were fused with a long linker containing a SUMO domain, a 6xHis tag, and an Mk PSTK-C domain, in this order  gene (E. coli expressing the cat(Ser146TAG) gene and the Mk PSTK-C domain not fused with the MS2 coat protein  gene B and at k PSTK-C C,D. Thusosition) ,40 and tosition)  were useosition)  togetheris order B. This cAG) gene . HoweverAG) gene C, probabAG) gene A. One vaAG) gene A,C. Both protein D. Then,  protein E. Surpri protein E, while lo-tRNAs E. All fulo-tRNAs E. Thus, C domain E. These Tyr species of Methanocaldococcus jannaschii (Mj), or Nap3 [Candidatus Methanomethylophilus alvus Mx1201 (Ma) was used to be paired with these tRNAs. Three kinds of reporter genes were used for examining the orthogonality and the tRNATyr activity of these tRNA variants (bla) having an in-frame TAG codon at a permissive site (Ala182) [cat(Ser146TAG) reporter gene was cloned together with this bla(Ala182TAG) reporter gene to make a dual-reporter plasmid. The third one is a sfGFP gene in which the Tyr66 codon had been mutated to TAG, since Tyr66 is essential for the fluorophore formation [bla reporter gene, it was revealed that the Y072 and Y073 variants conferred only marginal carbenicillin (Car) resistance to cells, while the Y005 variant and the Mj tRNATyr Nap3 variant conferred up to 500 µg/mL Car resistance to cells (Tyr in E. coli [E. coli. Although the Y072-2 variant conferred up to 500 µg/mL Car resistance to cells (E. coli DH10B cells expressing the cat(Ser146TAG) reporter gene  (C1:G72 with A73) ,44,45 we or Nap3 , was usevariants B,C. One (Ala182)   reporter gene  reporter gene with tyrosine  (G-1:C73) [cat reporter genes, cat(Ser146TAG) and cat(His193TAG), were used for examining the orthogonality and the His-inserting activity of designed tRNA variants. In the cat(His193TAG) gene, the essential catalytic His193 was changed to TAG [E. coli expressing the cat(His193TAG) gene (E. coli cells expressing the cat(Ser146TAG) gene  tRNA molecules  were devG-1:C73) ,26,50 ind to TAG . It soonAG) gene B. HoweveAG) gene B. Ser inAG) gene A. Point AG) gene A; UUUUGAsuitable B,C, implis tRNAs F. Up to is tRNAs A,C. On tis tRNAs D,E. Thusposition .E. coli HisRS, several cognate pairs of RNA aptamers and protein domains [N-terminus of E. coli HisRS (E. coli expressing the cat(His193TAG) gene  too strong affinity between the cognate RNA-protein pair might have reduced the turnover rate of aminoacylation, and 3) a too short linker sequence SSGSNSNSGS between the N-terminal domain and HisRS might have kept the catalytic pocket of the tethered HisRS away from the CCA tail of tRNA. In contrast, the xRRM domain binds a 7SK SL4 RNA with a moderate affinity (Kd = 129 nM [C-terminus of the xRRM domain and the N-terminus of the E. coli HisRS may approach each other on the tRNA and can be fused by using the short linker (E. coli DH10B cells expressing the cat(His193TAG) reporter gene and xRRM-HisRS  gene C. The otAG) gene C. Three t linker E, accordRM-HisRS F, while RM-HisRS D, which RM-HisRS F. The U3RM-HisRS A variantRM-HisRS G, while RM-HisRS G. This mHis anticodon arm were used. It is well known that a large subgroup of α-Proteobacteria has a non-canonical tRNAHis lacking the G-1:C73 pair and has a non-canonical HisRS species that recognizes the non-canonical G1:U72 pair and A73 in addition to the GUG anticodon [Afifella pfennigii DSM 17,143 (Ap) [Consotaella salsifontis USBA 369 (Cs) [Caulobacter crescentus HisRS was not active enough in E. coli [His anticodon arm as their V-arms, to develop H072-αψHis-3 and αH005-αψHis-3/4/5 variants  be recruited to tRNA via the V-arm which mimics the anticodon arm that can be recognized by the ABD? In other words, can a pseudo-anticodon arm moiety ,56 on tRnticodon ,58,59,60369 (Cs) , becausevariants A. The H0d linker B. On thed linker B. The C- trimmed B. ApEc, ectively B.cat(His193TAG) reporter system, the His-inserting activities by combinations of the tRNA variants and HisRS variants were examined  which may partially impair anticodon recognition [ApAQEc and ApAQCs HisRS variants  and the H072-7SK-1 variant (having no pseudo-anticodon arm) were compared D. To parognition  were intvariants B. As expc failed D. H072-αc failed D. In conc failed G,7D. Bot72-7SK-1 D. Thus, eraction E, this Gancement D.C. crescentus tRNAHis variants having these anticodon changes are poor substrates of C. crescentus HisRS [Cs HisRS charged all pseudo-anticodon variants of αH005-αψHis-4 . The Ca. M. alvus TyrRS gene was cloned into pACYC184 by replacing the cat gene ORF. The HisRS ORFs were cloned immediately downstream of the tet gene of pACYC184-cat(193HisTAG). The bla variant gene was inserted downstream of the tet gene of pACYC184-cat(Ser146TAG) using Infusion. The other protein genes were cloned into the pGEX-6P-1 vector  and expressed under control of the tac promoter. Oligo DNAs and synthetic protein genes were purchased from Eurofin Genomics Co., Ltd. .The nucleotide and amino-acid sequences are provided in the E. coli DH10B and NEB 10-beta (NEB) cells were transformed with the indicated combinations of plasmids. Before spotting assays, transformed cells were pre-cultured in liquid LB media in the presence of 0.01% l-arabinose and 10 μM IPTG, if required, for a few hours [600 = 0.05 and then incubated for three hours. After spotting cell cultures (2 μL each) on LB agar plates, the plates were incubated at 37 °C overnight or for a longer time. For the induction of sfGFP genes, the final concentration of IPTG was 100 μM.ew hours . In typi


TriCEPS and bound to NK cells followed by mass spectrometric analyses. We could define NKG2A/CD94 as a cognate receptor of HLA-G. To verify the results, we used the reciprocal method by expressing recombinant soluble heterodimeric NKG2A/CD94 molecules and used them to target HLA-G*01:01 expressing cells. NKG2A/CD94 could be confirmed as an immune receptor of HLA-G*01:01. Despite HLA-G is marginal polymorphic, we could previously demonstrate that the most common allelic subtypes HLA-G*01:01/01:03 and 01:04 differ in peptide repertoire, their engagement to NK cells, their catalyzation of dNK cell proliferation and their impact on NK cell development. Continuing these studies with regard to NKG2A/CD94 engagement we engineered recombinant single antigen presenting K562 cells and targeted the surface expressed HLA-G*01:01, 01:03 or 01:04 molecules with NKG2A/CD94. Specificity and sensitivity of HLA-G*01:04/NKG2A/CD94 engagement could be significantly verified. The binding affinity decreases when using K562-G*01:03 or K562-G*01:01 cells as targets. These results demonstrate that the ligand-receptor assignment between HLA-G and NKG2A/CD94 is dependent of the amino acid composition in the HLA-G heavy chain. Understanding the biophysical basis of receptor-mediated events that lead to NK cell inhibition would help to remove non-tumor reactive cells and support personalized mild autologous NK cell therapies.Natural killer (NK) cell therapies are a tool to antagonize a dysfunctional immune system. NK cells recognize malignant cells, traffic to a tumor location, and infiltrate the solid tumor. The immune checkpoint molecule human leukocyte antigen (HLA)-G is upregulated on malignant cells but not on healthy surrounding cells, the requirement of understanding the basis of receptor mediated events at the HLA-G/NK cell interface becomes obvious. The NK cell receptors ILT2 and KIR2DL4 have been described to bind to HLA-G; however, their differential function and expression levels on NK cell subsets suggest the existence of an unreported receptor. Here, we performed a ligand-based receptor capture on living cells utilizing sHLA-G*01:01 molecules coupled to  HLA-G is expressed in pregnancies by cells of the trophoblast at the maternal–fetal interface ,2; in pronocytes ,11. Upreonocytes , cancer onocytes ,14, melaonocytes , breast onocytes ), and otonocytes ,18. Expronocytes , indicatThe HLA-G locus encodes for 19 distinct proteins . DespiteIn its function of mediating immune tolerance, HLA-G interacts with different immune effector cells such as natural killer (NK) cells, T and B cells, monocytes, and dendritic cells (DCs) . By bindILT2 (LILRB1) is expressed by NK cells, T cells, B cells, DCs, and decidual macrophages ,32,33 whThe NK cell receptor KIR2DL4 recognizes HLA-G , howeverWhile ligation between HLA-G and a TCR has never been detected , HLA-G e+ T cells are killed by apoptosis following interaction with HLA-G [It still remains to be ascertained how HLA-G has the ability to modulate the immune system by a subtle interplay between HLA expression, peptide presentation and immune receptor recognition. To date it is known for example that activated CD8th HLA-G . Specifith HLA-G  and matuth HLA-G . In vitrth HLA-G .VMAPRTLFL complex [VMAPRTLFL/NKG2A/CD94 interface and explain the inhibitory immune response as a result of pathogenic immune escape mechanism. Earlier studies on NKG2A/CD94 as a potential receptor for HLA-G molecules [VMAPRTLFL molecules on the surface of HLA-G expressing cells. NKG2A/CD94 could therefore not be implemented in the list of certain receptors for HLA-G molecules [Another non-classical HLA-Ib molecule, HLA-E, that is upregulated during pathogenic episodes and stabilized through binding to the HLA-G leader peptide VMAPRTLFL constitutes a ligand for the inhibitory NKG2A/CD94 receptor . Biophys complex  engaged  complex ,46. In 2olecules ,48 couldolecules .NK cell therapies are a tool to antagonize downregulation of HLA-Ia molecules during pathogenic episodes . NK cellTriCEPS ligand-based receptor capture technology to evaluate receptors for sHLA-G*01:01 on NK cells. The experiments are performed under almost physiological conditions on living cells allowing for the detection of receptors for a certain ligand. The TriCEPS molecule is comprised of three domains enabling (a) ligand binding, (b) receptor binding, and (c) detection and purification. The aim is to detect an unreported NK cell receptor for HLA-G and to analyze the magnitude of HLA-G allelic variants on receptor ligation.We used the + cells.In order to perform a ligand-based receptor capture several pretests had to be applied. Since transferrin was planned to be used as a positive control, the suitability of the cell line was verified by measuring the expression of the transferrin receptor TFR1 (CD71) on the cell surface of NKL cells. It could be shown that the transferrin receptor TFR1 (CD71) is highly expressed on NKL cells . The expNKL cells was ascertained. Following treatment of the target cells with 1.5 mM NaIO4 oxidation reagent no impact on the survival of NKL cells was observed  molecules and used them to target HLA-G*01:01, HLA-G*01:03 or HLA-G*01:04 expressing cells; recombinant soluble NKG2C/CD94 (sNKG2C/CD94) and non-transfected K562 cells served as negative control . In contrast to autologous NK cells, allogenic NK cell infusion bears the risk to develop unpredictable immune reactions. However, it remains questionable why not all tumor localizations are affected by these therapies. Due to the tumor microenvironment inhibiting immune effector functions, tumors become invisible for the immune system [NK cell-based immunotherapy against tumors has become an important field of research. NK cell function is regulated by an array of inhibitory and activating receptors of which NK cell-inhibitory receptors are specific for HLA class I molecules; alloreactive NK cells are reactive due to ing self ,72. Baserinciple , and monreatment ,75. The e system ,77. For e system ,79. In oe system . The rate system . Periphee system ,84,85. IIn summary, these experiments confirm and substantiate the assumption of NKG2A/CD94 to be a new receptor for HLA-G. Understanding the biophysical basis of receptor-mediated events that lead to NK cell inhibition would help to remove non-tumor reactive cells and support personalized mild autologous NK cell therapies.K562 was maintained in RPMI 1640  supplemented with 10% heat inactivated fetal calf serum , 2 mM L-glutamine , 100 U/mL penicillin and 100 µg/mL streptomycin .HLA class I negative cell line HEK293T cells, used for production of lentiviral particles, were cultured in DMEM  supplemented with 10% heat inactivated FCS, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin and 1 mg/mL geneticin .NKL cells were maintained in RPMI 1640 supplemented with 15% heat inactivated fetal calf serum, 1% Natriumpyruvate , 200 U/mL IL-2 , 100 U/mL penicillin and 100 µg/mL streptomycin.2.All cell lines were maintained at 37 °C and 5% COJEG-3 cDNA, subcloned into the lentiviral vector pRRL.PPT.SFFV.mcs.pre as previously described [site-directed mutagenesis by introducing single point mutation at position c.162A > T for HLA-G*01:03 or c.400C > A for HLA-G*01:04.The construct encoding for HLA-G*01:01 (exon 1–6) was generated from escribed . ConstrupRRL.PPT.SFFV.mcs.pre, as previously described [Constructs encoding for sHLA-G*01:0x were cloned into the lentiviral vector escribed . The resV5-His-tag were cloned into the lentiviral vector pRRL.PPT.SFFV.mcs.pre. The respective inserts were verified through sequencing.The method used for cloning of a vector encoding for sNKG2A/CD94 and sNKG2C/CD94 heterodimers is described by Pump et al. . ConstruHEK293T cells were transfected with the target plasmids (10 µg/5 × 106 cells) and the packaging and envelope vectors psPAX2 and pmD2.G (both 5 µg/5 × 106 cells) using ® 2000Lipofectamine . The lentiviral particles were utilized to transduce K562 cells.As described by Bade-Doeding et al. . HEK293TThe expression of sHLA-G was confirmed by ELISA  and detection antibody anti-β2m ) and western blot ). The presence of mHLA-G on the cell surface was verified by flow cytometry ). The presence of sNKG2/CD94 was confirmed by ELISA  or anti-hNKG2C  and detection antibody anti-V5-tag ).2 for 10 days, then the cells were pelleted  and the supernatant was filtered  prior to affinity chromatography. The sHLA-G molecules were purified at pH 8.0 using NHS-activated HiTrap columns coupled with w6/32. The sNKG2A and sNKG2C molecules were purified at pH 8.0 using NHS-activated HiTrap columns coupled with anti hNKG2A  or anti-hNKG2C antibody . For elution 100 mM glycine buffer at pH 2.7 (adjusted with HCl) was used. The samples were neutralized by addition of 1 M Tris-HCl (pH 8.5 at 4 °C). The success of the purification of HLA-G was confirmed quantitatively via an HLA class I-specific ELISA and qualitatively via western blot. The successful purification of sNKG2A/CD94 and sNKG2C/CD94 was confirmed quantitatively via ELISA and qualitatively via native PAGE  and allows under almost physiological conditions for recognition of receptors for an existing ligand on living cells [TriCEPS molecule to primary amines of the ligand. Aldehydes are introduced to carbohydrates of the receptor via mild oxidation, to facilitate binding of the TriCEPS molecule to the receptor. This enables the second domain of the TriCEPS molecule, a hydrazine group, to react with the aldehydes and thus to permanently bind to the receptor. The third domain is necessary to purify the TriCEPS molecule and all bound components before mass spectrometric analysis.In order to analyze HLA-G-receptor interactions, ligand-based receptor capturing was applied using purified sHLA-G and the NK-cell line ng cells ,63. ThisTriCEPS kit provided by Dualsystems Biotech AG. Pretests and the final binding reaction were conducted and then sent to Dualsystems Biotech AG for cell lysis, protein digestion, purification of the TriCEPS-molecule, peptide release, and the final mass spectrometric analysis. The receptor capturing during the final reaction was performed in the presence of a catalyzer.The experiment was performed using the K562 cells. Each experiment was performed using three technical replicates. K562 cells were lentivirally transduced with vectors encoding for HLA-G*01:0x variants and subsequently sorted for equal HLA-G expression as described by Celik et al. [K562 cells were analyzed for the expression of HLA-E molecules  for 30 min at 4 °C in the dark for detection.A flow cytometry-based assay was used to test whether recombinant sNKG2A/CD94 can bind to recombinant membrane bound HLA-G on k et al.  (Figure olecules . For all


C) of BoNT/B (HC/B) has been proposed to also contribute to toxin binding to neurons by interacting with lipid membranes (termed lipid-binding loop [LBL]). Analogous loops exist in the HCs of BoNT/C, D, G, and a chimeric toxin DC. However, it has been challenging to detect and characterize binding of LBLs to lipid membranes. Here, using the nanodisc system and biolayer interferometry assays, we find that HC/DC, C, and G, but not HC/B and HC/D, are capable of binding to receptor-free lipids directly, with HC/DC having the highest level of binding. Mutagenesis studies demonstrate the critical role of consecutive aromatic residues at the tip of the LBL for binding of HC/DC to lipid membranes. Taking advantage of this insight, we then create a “gain-of-function” mutant HC/B by replacing two nonaromatic residues at the tip of its LBL with tryptophan. Cocrystallization studies confirm that these two tryptophan residues do not alter the structure of HC/B or the interactions with its receptors. Such a mutated HC/B gains the ability to bind receptor-free lipid membranes and shows enhanced binding to cultured neurons. Finally, full-length BoNT/B containing two tryptophan mutations in its LBL, together with two additional mutations (E1191M/S1199Y) that increase binding to human receptors, is produced and evaluated in mice in vivo using Digit Abduction Score assays. This mutant toxin shows enhanced efficacy in paralyzing local muscles at the injection site and lower systemic diffusion, thus extending both safety range and duration of paralysis compared with the control BoNT/B. These findings establish a mechanistic understanding of LBL–lipid interactions and create a modified BoNT/B with improved therapeutic efficacy.Botulinum neurotoxins (BoNTs) are a family of bacterial toxins with seven major serotypes (BoNT/A–G). The ability of these toxins to target and bind to motor nerve terminals is a key factor determining their potency and efficacy. Among these toxins, BoNT/B is one of the two types approved for medical and cosmetic uses. Besides binding to well-established receptors, an extended loop in the C-terminal receptor-binding domain (H Botulinum neurotoxins are a family of bacterial toxins, some of which are approved for medical and cosmetic uses. This study shows that introducing aromatic residues to a lipid binding loop improved therapeutic efficacy of botulinum neurotoxin B by enhancing its ability to bind to lipid membranes at motor nerve terminals. Botulinum neurotoxins (BoNTs) are a family of protein toxins produced by various clostridial bacteria –3. They N, approximately 50 kDa) and the C-terminal receptor-binding domain . BoNTs target motor nerve terminals by binding to specific receptors and enter neurons via receptor-mediated endocytosis. Acidification of endosomes triggers conformational changes in the toxin, leading to translocation of the LC across endosomal membranes into the cytosol, where the disulfide bond is reduced. The released LC acts as a zinc-dependent protease cleaving specific neuronal proteins.BoNTs are approximately 150-kDa proteins composed of a light chain  and a heavy chain , connected via a single disulfide bond , 3, 5, 6N derived from BoNT/D and an HC most similar to BoNT/C-HC (HC/C) [C similar to BoNT/A1-HC [Clostridium botulinum strain [Enterococcus faecium strain [Paraclostridium bifermentans strains [BoNTs are traditionally classified into seven serotypes (BoNT/A–G) based on their distinct antigenicity. These serotypes may further contain a number of subtypes  . In addiC (HC/C) . Recent NT/A1-HC –13. Mostm strain , BoNT/Enm strain , and Par strains .BoNT/A, E, and C cleave the peripheral membrane protein synaptosomal nerve-associated protein 25 (SNAP-25) in neurons, and BoNT/B, D, F, and G cleave vesicle associated membrane protein (VAMP) 1, 2, and 3. BoNT/C can also cleave the plasma membrane protein Syntaxin 1. These three neuronal proteins belong to the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) protein family and form a complex that is the core machinery mediating fusion of synaptic vesicle membranes to neuronal membranes , 18. CleA “double-receptor” model has been well established for the majority of BoNTs , 20. As Cocrystal structures of BoNT/B-Syt II and BoNT/DC-Syt I/II reveal that the Syt-binding site is close to but separate from the GBS , 46–49. Among the seven BoNTs, BoNT/A and BoNT/B have been approved for clinical and cosmetic use, with BoNT/A as the dominant form in the market , 54. In MY) that can bind to human Syt II with high affinity. Whereas wild-type (WT) BoNT/B showed lower efficacy than BoNT/A in a humanized mouse model in which the Syt II luminal domain has been replaced with the human version, BoNT/BMY and BoNT/A showed similar efficacy in paralyzing skeletal muscles in this humanized mouse model [Enhancing toxin binding to nerve terminals facilitates their absorption into local neurons, thus reducing both diffusion from the injection site, which can lead to severe adverse effects , and these model .MY showed a higher potency than BoNT/A in paralyzing bladder muscles, which are smooth muscles [MY may offer a higher therapeutic efficacy than BoNT/A for treating smooth muscle–related disorders [Interestingly, BoNT/B and BoNT/B muscles . This is muscles , 63. Thuisorders .C/C, HC/D, and HC/DC to immobilized gangliosides and cells, leading to the suggestion that this loop is involved in ganglioside binding [C/DC binds directly to liposomes containing only phosphatidylcholine (PC), and mutations at the tip of this extended loop abolish lipid-binding capability, demonstrating that this extended loop, designated “lipid-binding loop” (LBL), mediates membrane binding independent of gangliosides in BoNT/DC [C/B, HC/G, and HC/DC with deletion mutations in their LBLs lost the ability to form stable binding to CGs or Syt II embedded in Triton X-100 micelles or in lipid environment in nanodiscs [When BoNT/B was modeled onto cell membranes via anchoring by gangliosides and Syt I/II, an extended loop between its GBS and Syt-binding site was recognized to be at a location ideal for membrane interactions , 47. Sim binding –69. Cons binding , 70. How BoNT/DC . A recen BoNT/DC . Furtheranodiscs .C/B, C, D, G, and DC using the nanodisc system. We found that HC/C, DC, and G can bind to receptor-free lipid nanodiscs directly, whereas HC/B and D showed no detectable binding. Mutagenesis studies demonstrated the key role of having consecutive aromatic residues at the tip of LBL for binding of HC/DC to lipid membranes. A “gain-of-function” mutant HC/B was then created by replacing the two nonaromatic residues at the tip of the HC/B-LBL with tryptophan. Crystal structural studies confirmed that the two introduced tryptophan residues were located at the tip of HC/B-LBL and did not alter the conformation of HC/B. Such an engineered HC/B showed robust binding to receptor-free lipid membranes and enhanced binding to cultured neurons. Finally, we produced a full-length active form of BoNT/B containing the same two tryptophan mutations, in combination with the E1191M/S1199Y mutations (BoNT/BMY-WW), and found that this mutant toxin showed improved efficacy in paralyzing local muscles and reduced systemic diffusion, offering expanded safety ranges and longer therapeutic duration compared with BoNT/BMY.Here, we carried out a systematic characterization of LBL–lipid interactions in HAmong BoNTs, five  contain LBLs of considerable length . To deteTo monitor toxin binding to nanodiscs, we utilized the biolayer interferometry (BLI) assay, a label-free technology that offers real-time analysis of binding kinetics. Immobilization of biotinylated nanodiscs onto SA biosensors showed robust binding and virtually no dissociation . As a coC/B, HC/C, HC/D, HC/DC, and HC/G to immobilized ND1, SA biosensors loaded with ND1 were first immersed in the buffer containing BSA until a stable baseline was reached and then soaked in the buffer containing HC for the association phase, followed by immersion in the buffer containing BSA again for the dissociation phase , whereas HC/B has only one (F1250). HC/D and HC/G both contain three aromatic residues, but they are not consecutive  of 4.8 × 10−5 M, whereas binding of HC/DC to ND-Syt showed an approximately 10-fold higher estimated KD of 4.7 × 10−6 M  and a double mutation . HC/BW showed a low level of binding to ND1, whereas HC/BWW showed a much higher binding than HC/BW , and it is impractical to draw a binding KD for comparison. Using a single–binding site model to fit the binding/dissociation curves suggests an estimated binding KD at approximately 10−9 M levels for HC/B and HC/BWW  [C/BWW showed overall stronger immunostaining signals than HC/B on both WT neurons and GD2 KO neurons  . Consist neurons . Binding neurons , indicat neurons . FurtherMY-WW) and control BoNT/B were produced recombinantly in Escherichia coli. Both toxins also contained the same set of additional mutations  that we previously developed for enhancing binding to human Syt II [MY-WW showed a slightly better ED50 than the control BoNT/BMY, although the difference was not significant . A series of toxin dilutions were injected into the hind legs of mice. Survival rates were monitored, and IM LD50s were estimated at 29.0 ± 4.3 pg for the control BoNT/B and 35.4 ± 1.0 pg for BoNT/BMY-WW . The structure was solved to a 2.4-Å resolution  protrude into the membrane bilayer  and biotin-labeled lipids to immobilize nanodiscs to the biosensors.The role of LBLs in BoNT/B, C, D, G, and DC for binding to lipid membranes has been hypothesized previously. Mutagenesis studies have demonstrated the importance of LBL for forming stable binding to membrane-embedded receptors and for the potency of these toxins –68, 71. C/DC contains three (YWF) and HC/C contains two (WY) aromatic residues at the tip of their LBLs, which appears to be the optimal design for mediating lipid interactions. The number and composition of aromatic residues both influence overall binding affinity. For instance, mutating any one of the three aromatic residues in HC/DC reduced its binding to lipids, whereas changing the F residue to W increased binding. The aromatic residues are separated in HC/D (WRFSF) and HC/G (YGYVW), which might be the reason for their loss of or low lipid-binding affinity. HC/B-LBL contains only a single aromatic residue (F), which is also away from the tip of LBL; thus, HC/B showed no detectable lipid-binding capability.Aromatic residues such as tyrosine, tryptophan, and phenylalanine have the capability to insert their bulky aromatic rings into the hydrophobic lipid bilayer while keeping their hydrophilic side chain groups in the aqueous environment . TherefoC/BWW with both gangliosides and Syt I peptide demonstrates conclusively that introducing two consecutive tryptophan residues does not alter the conformation of HC/B or affect the GBS and Syt-binding site. The only change is the extension of HC/B-LBL with two tryptophan residues at the tip. These data provided strong support for the role of consecutive aromatic residues at the tip of LBL in mediating lipid binding. The ternary structure also confirms that LBL is separate from the GBS and the Syt-binding site, suggesting that it contributes to overall binding affinity toward neuronal membranes independently.The ternary complex of HC/BWW to nanodiscs with different receptor components suggests that LBL–lipid interactions are more relevant to synergizing with ganglioside binding, with minimal impact on Syt binding for HC/B. This is likely because the HC/B-Syt interactions already have high affinity (approximately 10−7 M range), whereas HC/B–ganglioside interactions have low affinity (approximately 10−5 M range), making the impact of adding LBL–lipid interactions more obvious for ganglioside binding. Because binding of HC/DC to Syt II has a lower affinity (approximately 10−5 M range) than HC/B-Syt interactions, the synergistic effect with LBL–lipid interactions is obvious for HC/DC binding to membrane-embedded Syt II: WT HC/DC showed approximately 10-fold higher binding affinity toward ND-Syt compared with HC/DC containing mutations at the tip aromatic residues . These point mutations in HC/DC showed the same level of binding affinity toward ND-Syt as with isolated Syt II, indicating that mutations in LBL do not affect HC/DC binding to membrane-embedded Syt II. Thus, their lower binding affinity toward ND-Syt compared with WT HC/DC is due to loss of LBL–lipid interactions. It is interesting to note that HC/DC has the highest level of LBL–lipid interactions, which may compensate its lower binding affinity toward Syt I/II compared with HC/B.Examining binding of HC/BWW showed enhanced binding to neuronal surfaces than WT HC/B. The binding remains specific to nerve terminals rather than being diffused along neuronal membranes, and binding of HC/BWW to neurons was largely abolished by depleting CGs and by competition with a soluble Syt II fragment containing the toxin-binding site. This is likely because LBL–lipid interactions and HC/B–ganglioside interactions are both relatively weak, which allows BoNT/B to sample cell surfaces through fast-on/fast-off transient interactions. Only when Syt I/II are also present can the toxin be stably anchored to nerve terminals.HMY-WW over the control BoNT/BMY: (1) The safety ratio was improved: this is likely because BoNT/BMY-WW has less unwanted diffusion from the injection site. (2) There was longer therapeutic duration compared with the same dose of BoNT/BMY: this is likely because more BoNT/BMY-WW can enter the targeted local neurons than BoNT/BMY, which is also consistent with the suggestion that BoNT/BMY-WW has less diffusion and may thus remain near the injection site longer than BoNT/BMY. BoNT/B is currently utilized in the clinic as a therapeutic toxin and has the advantage of targeting smooth muscles more efficiently than the more widely used BoNT/A. Introducing lipid-binding capability to BoNT/B and potentially other BoNTs may present new ways to engineer toxins with improved therapeutic efficacy and reduced side effects.Finally, in vivo DAS assays revealed two major advantages of BoNT/BAll animal studies were approved by the Boston Children’s Hospital Institutional Animal Care and Use Committee (protocol number: 18-10-3794R). The Boston Children’s Hospital institutional animal care program holds an active animal welfare assurance on file with the Office of Laboratory Animal Welfare at National Institutes of Health, which describes Children’s adherence to Public Health Service’s Policy on Humane Care and Use of Laboratory Animals. It is also registered as a research facility with the US Department of Agriculture and follows the Animal Welfare Act and Regulations, when applicable. In addition, Children’s animal care and use program is fully accredited by AAALAC International.All procedures were approved by the Institute of Biosafety Committees at Boston Children’s Hospital (protocol number: IBC-P00000501). No part of the study has been deemed classified information. All active BoNTs are stored in a locked freezer. Used toxins and contaminated media/reagents/containers are exposed to 10% bleach solution for decontamination.C/B, HC/C, HC/D, HC/DC, and HC/G were subcloned into pET28a vector with an N-terminal HA tag. A modified version of ApoA1 was cloned into pNFXeX vector and expressed as an N-terminal His6-tagged protein [MY was subcloned into pET32a as previously described [E. coli BL21(DE3) strains. HC protein expression was induced using IPTG at 22°C for 16 hours. The full-length BoNT/B (BoNT/BMY and BoNT/BMY-WW) mutants were induced using IPTG at 16°C for 20 hours. For all other proteins, expression was induced using IPTG at 18°C for 16 hours. Cells were harvested at 4°C and lysed by sonication. Cell lysates were centrifuged for 60 minutes at 4°C and then filtered using syringe-driven filters . After centrifugation and filtration, harvested supernatants were loaded into Nickel-NTA columns (GE) using the AKTA Prime FPLC system (GE). Proteins were eluted using imidazole gradient elution and further desalted using the PD-10 column. For mouse Syt II 1–267, the cell lysis buffer contains 1% (v/v) Triton X-100, and the protein elution buffer contains 0.8% (W/V) n-octyl β-D-glucopyranoside (OG). For GST-tagged mSyt II 1–61, harvested supernatant was incubated with GST beads at 4°C for 1 hour. The beads were washed with phosphate-buffered saline (PBS), and then the protein was eluted with PBS containing 10 mM reduced L-Glutathione (GSH reduced). All the proteins used in the BLI assay were desalted in PBS.cDNAs encoding H protein , 79. cDNescribed . A stop MY and BoNT/BMY-WW), cells were harvested at 4°C and lysed by sonication in 50 mM Tris (pH 8), 200 mM NaCl, and treated with benzonase. Cell lysates were adjusted to 1.3 M (NH4)2SO4 and centrifuged for 60 minutes at 4,000g. The recovered supernatants were loaded onto a Butyl HP (GE) column using an AKTA Pure system (GE). Proteins were eluted with a 1.3–0 M (NH4)2SO4 in 50 mM Tris (pH 8) linear gradient. Fractions containing the target protein were pooled and desalted using a HiPrep Sephadex G-25 desalting column (GE). Further purification was performed by anion exchange chromatography with a Q HP HiTrap column (GE). Proteins were eluted with a 0–1 M NaCl in 50 mM Tris (pH 8) linear gradient. The fractions containing target protein were pooled and stored at −80°C.For full-length BoNT/B mutants . For ND1 and ND-Syt, the lipid mixture composition used was POPC/DOPS/cholesterol/biotin-PEG-DSPE (molar ratio: 74.5:5:20:0.5). For ND-CGs and ND-Syt-CGs, the lipid mixture composition used was POPC/DOPS/cholesterol/biotin-PEG-DSPE/GD1a/GT1b (molar ratio: 72.5:5:20:0.5:1:1). For nanodisc without biotin, the lipid mixture composition used was POPC/DOPS/cholesterol/ GD1a/GT1b (molar ratio: 73:5:20:1:1). For POPC ND, the lipid mixture composition used was POPC/biotin-PEG-DSPE (molar ratio: 99.5:0.5).The lipids dissolved in chloroform solution were first diluted and subsequently mixed together in glass tubes under a fume hood according to the aforementioned molar ratio. Then, the solvent in the mixtures was dried under nitrogen gas in a fume hood, and the remaining solvent was further removed at room temperature (22–24°C) overnight in a vacuum desiccator. The lipid films were resuspended in PBS with agitation. The final lipid stock concentration was 50 mM.For ND1 and ND-CGs, sodium cholate was used to solubilize lipids and then mixed with ApoA1 at a molar ratio of 60:1. For ND-Syt and ND-Syt-CGs, sodium cholate–solubilized lipids were mixed with ApoA1 and mouse Syt II 1–267 at a molar ratio of 60:1:1. Formation of nanodiscs was then triggered by mixing with methanol-activated SM2 Bio-Beads for 4 hours, followed by another round of mixing with new SM2 Bio-Beads for another 4 hours. Then, the nanodiscs were filtered using Costar Spin-X Plastic centrifuge tube filters (0.22-μm membrane pore size) and further purified using FPLC (Bio-Rad NGC chromatography systems) using a Superdex 200 GL 10/300 column . The formation of nanodiscs was confirmed and purity assessed using SDS-PAGE gel analysis and negative staining EM.EM grids  were first pretreated in a glow discharge unit for 2 minutes to allow adsorption of samples, after which 2 μl of the nanodisc samples was added to the grid surface. After 30 seconds, the sample droplets were adsorbed using filter paper on the edge of the grid. Then, the grids were soaked into 1.5% uranyl formate solution droplet for 30 seconds and dried using filter paper. The grids were further cleaned using 1.5% uranyl formate solution three times. Air-dried grids were then imaged on the Tecnai G2 Spirit BioTWIN transmission electron microscope and recorded with an AMT 2k CCD camera at the Harvard Medical School Electron Microscopy Facility.C for 40 seconds as the association step. The biosensors were then transferred to PBS for an 80-second dissociation step.For SA biosensor experiments, the Pall ForteBio's SA biosensors were prehydrated for 10 minutes. The nanodiscs containing biotin-lipids were loaded onto the biosensors, which were equilibrated in PBS for 400 seconds, and then exposed to solutions containing HFor anti-GST biosensor experiments, the Pall ForteBio's anti-GST biosensors were prehydrated for 10 minutes. M-Syt II 1–61 protein with a GST tag was first loaded onto the anti-GST biosensor, followed by equilibration in PBS for 400 seconds and then exposure to proteins for 40 seconds as the association step. The biosensors were then transferred to PBS for 80 seconds as the dissociation step.D values were calculated using the data analysis software BLItz Pro Software v8.1 following the BLItz system user guide .For both experiments, 0.5% BSA was added to all buffers to reduce nonspecific binding. Data were analyzed using the data analysis software BLItz Pro Software v8.1 . To calculate the total binding response during the association step, the last five signals (the last 500 milliseconds) of the association phase were averaged as the final response signal. The baseline value was subtracted from the final response signal to generate the 40-second response. The 40-second response curve was plotted using the GraphPad Prism software. In the response curve, the concentration was plotted along the x-axis, and the response was plotted along the y-axis. KC solutions for 800 seconds as the association step, followed by incubation in PBS for 800 seconds as the dissociation step. The experiments were carried out at 37°C with 1,000-rpm shaking. During all the experiments, all buffers contained 0.5% BSA. The data were collected using the data acquisition software  and analyzed using the data analysis HT11.0 software . During the data analysis, the data were aligned to the baseline step, and high-frequency noise was removed with Savitzky-Golay filtering. The 1:1 binding model was used for fitting. KD values were calculated using the data analysis HT11.0 software .For Biolayer Interferometry Octet RED 384 system assay, sample solutions and buffers were prepared in black 384-well plates. The program used was precompiled and run automatically during the experiments as follows: The Pall ForteBio's SA biosensors were prehydrated for 10 minutes. The nanodiscs with a biotin tag were loaded onto the biosensors for 400 seconds, equilibrated in PBS for another 500 seconds, and then exposed to HC/B proteins (1 μM) at room temperature for 30 minutes. The liposome–protein mixture was then added to sucrose solution to reach a final sucrose concentration of 30%, which served as the bottom layer in the centrifuge tube. The 200 μl of 25% sucrose was loaded on the top of the bottom layer, followed by 50 μl of Tris buffer. Loaded samples with sucrose gradient were centrifuged at 240,000g for 1 hour . After centrifugation, 50-μl solutions on the top of the gradient were collected and subjected to immunoblot analysis.POPC and gangliosides were purchased from Avanti Polar Lipids . POPC or POPC mixed with 1% ganglioside mixture was dried with nitrogen gas and further dried overnight at room temperature under vacuum to form the lipid films. The lipids were then resuspended with Tris buffer. The resuspended lipids were extruded through a polycarbonate filter  by 20 strokes to form liposomes. Liposomes (75 μl) were incubated with HMouse cortical neurons were prepared from newborn pups . Rat cortical neurons were prepared from E18–19 embryos dissected from pregnant rats . Dissected cortices were dissociated with papain according to the manufacturer’s instructions . Cells were plated on poly-D-lysine-coated coverslips in 24-well plates. Experiments were carried out using days in vitro (DIV) 11–13 for mouse neurons and DIV 13–15 for rat neurons.C/B in high-K+ buffer containing 87 mM NaCl, 56 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4, 0.5 mM MgCl2, and 1 mM CaCl2 for 5 minutes at 37°C. Cells were then washed three times with PBS. Binding of HC/B was examined with two complementary approaches: (1) For immunostaining, neurons were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 in PBS. Images were collected using a Zeiss 880 laser scanning confocal microscope with a 40× oil objective. At least three representative images were collected per condition. (2) For immunoblot analysis, the final concentration of proteins for neuron binding is 0.1 μM, and the total volume is 200 μl. Neurons in each well were lysed in a lysis buffer . The 20-μl 5× loading buffer was mixed with the supernatant of lysed cells. Samples were heated to 56°C for 5 minutes, lysates were centrifuged for 10 minutes at 4°C, and then 5-μl supernatants were subjected to SDS-PAGE and western blot analysis. Binding of HC/B was detected using a monoclonal human anti-HA antibody. The antibodies used were purchased from the indicated vendors: mouse monoclonal antibody for actin , mouse monoclonal anti-HA , and rabbit polyclonal antisynapsin (Millipore).Neurons were exposed to 100 nM HMY and BoNT/BMY-WW toxins both contain two point mutations E1191M/S1199Y [The control BoNT/BM/S1199Y . They weC/BWW construct used for crystallization was subcloned into a pET28a vector and expressed as an N-terminal His6-tagged protein. Cultures were grown at 37°C, and induction was carried out at 18°C using 0.5 mM IPTG. Bacterial pellets were lysed by sonication, and the protein was purified by affinity chromatography using a 5-ml HisTrap HP column  followed by gel filtration with a 16/60 Superdex 200 column . The final sample was stored at 11.3 mg/ml in 20 mM HEPES (pH 7.5), 300 mM NaCl, 10% glycerol, and 2 mM TCEP. A sample consisting of HC/BWW (10 mg/ml) mixed with 5 mM GD1a oligosaccharide  and 1 mM of the peptide corresponding to human Syt I (33–53) was prepared. Crystals of the complex grew within 3 days at 21°C using the sitting drop vapor-diffusion method, in which 200 nl of sample was mixed with 100 nl of reservoir solution composed of 20% v/v PEG6000, 0.1 M MES (pH 6.0), and 0.2 M sodium chloride. The crystals were transferred into a cryoprotectant solution supplemented with 15% v/v glycerol and then frozen in liquid nitrogen for data collection. Diffraction data were collected at station I04 of the Diamond Light Source  equipped with a PILATUS-6M detector . A complete data set to 2.4 Å was collected from a single crystal at 100 K. Raw data images were processed and scaled with DIALS [C/B dual receptor complex (PDB code 4BKZ) as a search model in PHASER [http://wwpdb.org).The Hth DIALS  and AIMLth DIALS  using thth DIALS . The strn PHASER . The worn PHASER  and manun PHASER . Water mn PHASER  at positn PHASER . CrystalS1 Fig(A) Representative FPLC elution profiles of nanodiscs (Superdex 200GL 10/300 column) and SDS-PAGE gel/Coomassie Blue staining analysis validated the presence of ApoA1 proteins in the collected nanodisc fraction. Lane 1 on the left represents the protein ladder. (B) Representative FPLC elution profiles of nanodiscs containing Syt II and SDS-PAGE gel/Coomassie Blue staining analysis validated the incorporation of Syt II in the collected nanodisc fraction. (C) Schematic drawing of nanodiscs  and negative staining EM showing the presence of nanodiscs. Scale bar represents 25 nm. (D) Nanodiscs were loaded to SA biosensors for BLI. All nanodiscs showed robust binding (association) with virtually no dissociation. (E) The presence of BSA (0.5%) further reduces background binding of nanodiscs lacking biotin-DSPE to SA biosensors. Top panel: ND-CGs without biotin-DSPE; bottom panel: ND-Syt-CGs without biotin-DSPE. ApoA1, Apolipoprotein A1; BLI, biolayer interferometry; BSA, bovine serum albumin; CG, complex ganglioside; DSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; EM, electron microscopy; FPLC, fast protein liquid size-exclusion chromatography; ND, nanodisc; SA, streptavidin; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide; Syt, synaptotagmin.(TIF)Click here for additional data file.S2 FigC/B (panel B), HC/C (panel C), HC/D (panel D), HC/DC (panel E), and HC/G (panel F) to POPC ND was measured by BLI assays. HC/B and HC/D showed no detectable binding to POPC ND. HC/DC showed the strongest binding, and HC/C showed modest levels, whereas HC/G showed low levels of binding to POPC ND. BLI, biolayer interferometry; HC, C-terminal receptor-binding domain; ND, nanodisc; POPC, 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine.(A) Schematic drawing of nanodiscs containing only POPC (POPC ND). (B–F) POPC ND was immobilized to biosensors. Binding of H(TIF)Click here for additional data file.S3 FigC/DC and the indicated HC/DC mutants to ND-Syt was analyzed by BLI assays. Binding kinetics were analyzed and are listed in C/DC and the indicated HC/DC mutants to immobilized GST-tagged Syt II (1–61) protein was analyzed by BLI assays. The binding kinetics were analyzed and listed in C/DC-LBL does not alter its binding to Syt II. BLI, biolayer interferometry; GST, glutathione S-transferase; HC, C-terminal receptor-binding domain; LBL, lipid-binding loop; ND, nanodisc; Syt, synaptotagmin; WT, wild-type.(A) Binding of WT H(TIF)Click here for additional data file.S4 FigC/DC and HC/B. (B) Sequence changes in HC/B. (C) The indicated HC/B mutants were examined for binding to ND1 using BLI at 10 μM concentrations. BLI, biolayer interferometry; HC, C-terminal receptor-binding domain; LBL, lipid-binding loop; ND1, receptor-free nanodisc.(A) Superposition of the LBLs of H(TIF)Click here for additional data file.S5 FigC/B mutants (10 μM) to ND1 were plotted in the bar graph. Error bars indicate means ± SD, n = 3. Numerical values for (B) are available in C, C-terminal receptor-binding domain; LBL, lipid-binding loop; ND1, receptor-free nanodisc.(A) Binding of the indicated mutants to ND1 was analyzed using BLI at 10 μM concentration. The specific mutations in LBL are marked in red. (B) The maximal binding signals of H(TIF)Click here for additional data file.S6 FigC/BWW complex. Main residues involved in binding are shown as sticks, with hydrogen bonds represented as dashed lines. (B) The Syt-binding site. Residues involved in binding are highlighted, illustrating the high similarity between the mutant and WT HC/B bound to hSyt I (red) and rSyt II (yellow), respectively. (C) The structures of HC/DC (cyan) in complex with hSytI (PDB 4isq) and the sialyl-T carbohydrate (PDB 5lr0) shows that the LBL loop (residues 1251–1253) interacts with the membrane. GD1a, disialoganglioside; HC, C-terminal receptor-binding domain; hSyt I, human Syt I; LBL, lipid-binding loop; PDB, Protein Data Bank; rSyt II, rat Syt II; Syt, synaptotagmin; WT, wild-type.(A) The ganglioside-binding site. GD1a in the WT costructure in pink, and blue in H(TIF)Click here for additional data file.S1 TableC, C-terminal receptor-binding domain; WT, wild-type.H(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 Raw Images(TIF)Click here for additional data file.


Recent findings have revealed that extracellular vesicles (EVs) are secreted from cells and circulate in the blood. EVs are classified as exosomes (40−100 nm), microvesicles  or apoptotic bodies . EVs contain mRNAs, microRNAs, and DNAs and have the ability to transfer them from cell to cell. Recently, especially in humans, the diagnostic accuracy of tumor cell type-specific EV-associated miRNAs as biomarkers has been found to be more than 90 %. In addition, microRNAs contained in EVs in blood are being identified as specific biomarkers of chemical-induced inflammation and organ damage.Therefore, microRNAs contained in the EVs released into the blood from tissues and organs in response to adverse events such as exposure to chemical substances and drugs are expected to be useful as novel biomarkers for toxicity assessment. In this study, C57BL/6 J male mice orally dosed with carbon tetrachloride (CCl4) were used as a hepatotoxicity animal model. Here, we report that not only the known hepatotoxicity biomarkers miR-122 and miR-192 but also 42 novel EV-associated biomarkers were upregulated in mice dosed with CCl4. Some of these novel biomarkers may be expected to be able to use for better understanding the mechanism of toxicity. These results suggest that our newly developed protocol using EV-associated miRNAs as a biomarker would accelerate the rapid evaluation of toxicity caused by chemical substances and/or drugs. To . To 6]. Recent reports demonstrate that EVs circulating in various fluids could be a diagnostic biomarker for various cancers  10] [11 [1110]  13]. EV. EV13]. Therefore, we focused on EV-associated miRNA in blood as a biomarker to establish a highly sensitive "next generation-type" toxicity test for chemical substances and drugs. The aim of this study was to screen EV-associated miRNAs as novel biomarkers of hepatotoxicity by comprehensive RNA-seq analyses of EV-associated miRNAs in C57BL/6 J male mice orally dosed with carbon tetrachloride (CCl4). First, we established more accurate protocol for evaluating rodent EVs. Then, we screened and evaluated differentially expressed EV-associated miRNAs in response to the administration of hepatic toxicant, carbon tetrachloride (CCl4). We used the known hepatotoxicity biomarkers, miR-122 and miR-192, as positive controls for the screening  21] [22 [2221]  18] [23 [2318] .22.1All animal studies were conducted in accordance with the guidelines of the animal care committee of the National Institute of Health Sciences (NIHS) (No. 664). C57BL6/J mice that were 12 weeks old were purchased from Charles River Laboratory Japan (Yokohama). The animals had access to a standard chow diet and water ad libitum and were housed in a pathogen-free barrier facility with a 12 L:12 D cycle.2.2To produce the hepatotoxicity models, carbon tetrachloride (CCl4)  (0 (corn oil), 7, or 70 mg/kg body weight) was orally administered to C57BL/6 J mice. Twenty-four hours after the administration of CCl4, blood and liver sample collections were performed under isoflurane anesthesia. Whole blood was collected by cardiac puncture, kept undisturbed at room temperature for 30 min and centrifuged at 3000 x g for 10 min at 4 ℃, and the serum was carefully separated. The separated serum was stored at -80 ℃ until use.2.3Liver samples were fixed with 10 % neutral buffered formalin. After conventional processing, paraffin-embedded sections were stained with hematoxylin and eosin (H&E) and examined histopathologically under a light microscope.2.4The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in each serum sample were determined using the automatic blood chemistry analyzer Dry-Chem NX 500 V .2.5The serum was centrifuged at 10,000 x g for 10 min to remove cellular debris and subsequently ultracentrifuged at 210,000 x g for 30 min (BECKMAN−COULTER Optima TLX). The pellet was resuspended in PBS and ultracentrifuged at 210,000 x g for 30 min again. The supernatant was carefully removed, and the EV pellets were resuspended in 30 μl PBS and stored at -80 ℃. Nanoparticle tracking analysis (NTA) of the EVs was performed with a Nanosight system (Nanosight) in EV diluted 1000-fold with PBS as previously described .2.6For EV-associated RNA, EVs were lysed with TRIzol, and EV-associated RNAs were extracted by a miRNeasy microelution kit (Qiagen) according to the manufacturer’s protocols. EV-associated RNA was evaluated by using a NanoDrop spectrophotometer , an Agilent 2100 Bioanalyzer and RNA pico chips .cDNA was prepared from the EV-associated RNAs with a SMARTer smRNA-Seq Kit for Illumina  according to the manufacturer’s protocol. Briefly, cDNA synthesis with the addition of both 5′ and 3′ adapters was followed by polyadenylation of EV-associated RNA (1 ng), first-strand synthesis and tailing with MMLV-derived PrimeScriptTM reverse transcriptase (RT), template switching with PrimeScript RT and the addition of full-length Illumina adaptors by PCR. The cDNA libraries were size-selected by using 3% agarose gel cassettes for the Blue Pippin System (Sage Science) with the parameters BP start = 148 and BP end = 185.The quality and quantity of the size-selected cDNA libraries were checked with KAPA Library Quantification Kit Illumina® Platforms  or Qubit dsDNA HS (High Sensitivity) Assay Kit in a Qubit® 2.0 Fluorometer . 2.0 pM of the cDNA library for each sample was sequenced on an Illumina Nextseq500 platform, and 75-bp reads were generated .2.7https://usegalaxy.org). The FASTQ quality filter was performed using the Filter FASTQ program, and both the 5′ and 3′ adapters were trimmed using the Trim FASTQ program. These processed sequence reads were aligned to the reference genome (mm10) using the TopHat program to generate BAM files.The raw data (raw reads) in the FASTQ format were first processed using the BCL2-FASTQ program . All the data analyses were performed on the Galaxy platform (http://mirbase.org) by the Cufflinks and Cuffnorm programs to generate gene expression data for each miRNA. The RNA-Seq data were analyzed based on the fold changes and Hierarchical clustering between samples, which were calculated with a two-tailed Student’s t-test (P < 0.01) using the Subio Platform and Subio Basic Plug-in .The BAM files were processed and normalized against the mouse miRNA database  was used to validate the RNA-Seq data. The cDNA libraries prepared for RNA-Seq were used to perform qRT-PCR. By using KAPA Library Quantification Kit Illumina® Platforms , real-time absolute quantification of each cDNA libraries was performed. By using 5′ adapter primer and a reverse primer for each specific miRNA, real-time absolute quantification of miRNAs was performed. The primer sequences used in the qRT-PCR assay are as follows; The 5′ adapter primer: AATGATACGGCGACCACCGA, miR-122−5p-specific primer : CAAACACCATTGTCACACTCCA, miR-192−5p-specific primer : GGCTGTCAATTCATAGGTCAG. Real-time quantitative PCR analyses were performed with a ABI7500HT instrument ; individual reactions were prepared with 0.2 pM of cDNA and Powerup SYBR Green Master Mix (Thermo Fisher Scientific) in a final volume of 20 μL. All reactions were performed in triplicate. Absolute quantification of miR-122a-5p and miR-192−5p was performed by using a standard curve method based on ten-fold serial dilution of synthesized oligo DNA miR-122−5p-Standard oligo: AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGCTGGAGTGTGACAATGGTGTTTG, and miR-192−5p- Standard oligo:AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGCCTGACCTATGAATTGACAGCC, raging from 5.0 × 102.9All data are presented as the means ± standard deviation (SD), and comparisons were performed by analysis of variance (ANOVA) (SAS). A probability of less than 0.01 was considered to be statistically significant.33.1Whole blood was collected by cardiac puncture from C57BL/6 J male mice (12 weeks). EVs were purified by ultracentrifugation of the serum extracted from whole blood. Microparticle analysis of the EVs was performed with a Nanosight a. The peIn general, these small RNAs are believed to be tRNA, microRNA (miRNA), snoRNA, and small RNA fragments. miRNAs, in particular, have attracted attention as biomarkers, as there have been several studies on the use of miRNAs as diagnostic biomarkers for tumors with high accuracy .3.2Each miRNA was first transcribed as a large precursor, which was termed the primary miRNA (pri-miRNA), by RNA polymerase II  30]. Pr. Pr30]. To quantify or sequence miRNAs, SMART (switching mechanism at the 5′ end of the RNA template) technology was used to generate cDNA libraries. Electropherogram results for cDNA library created from EV-associated small RNAs before size selection demonstrated that the region corresponding to the miRNA was a part of the whole library a 36].Fi.Fi36].FiWhether EV-associated RNAs were quantified by qRT-PCR or next generation sequencing (NGS), only the small RNA fractions of the cDNAs were further extracted by the size selector instrument to enrich the miRNA populations b.3.3C57BL/6 J male mice were orally dosed with carbon tetrachloride (CCl4) as hepatic toxicant because there are numerous reports that CCl4 induces hepatotoxicity in many experimental animals . In geneH&E staining showed CCl4 (70 mg/kg)-induced histopathological changes in the liver, with significant degeneration and necrosis of hepatocytes in the centrilobular region and the presence of perivenular inflammatory infiltrates 24 h after administration c, while 3.4The most commonly used diagnostic test for hepatotoxicity involves determining the activity of certain hepatocellular enzymes, including aspartate aminotransferase (AST or SGOT) and alanine aminotransferase (ALT or SGPT), in the blood . AST is AST d and ALTAs a result, serum biochemistry analysis and histology of the results of CCl4 administration demonstrate that only 70 mg/kg CCl4 administration results in apparent hepatotoxicity.3.5To identify differentially expressed EV-associated miRNAs, we performed RNA-Seq on size-selected, EV-associated small RNA libraries obtained from samples from mice treated with three dosages of CCl4, 0, 7, and 70 mg/kg. We then mapped the filtered clean reads to the reference mouse genome. We obtained an average of 68 million reads per sample that mapped to the reference genome. The library size-normalized counts for all samples were generated, and volcano plots of the differentially expressed EV-associated miRNAs are shown . We iden3.6To validate the RNA-Seq data, the known hepatotoxicity biomarkers, miR-122 and miR-192, were quantified in the same cDNA library as that used for RNA-seq by quantitative RT-PCR  21] [22 [2221]  18] [23 [2318] . Levels 3.7Liquid biopsy in medical examination is known as a very powerful tool. In fact, AST and ALT are known to be very good biomarkers of liver damage; however, it is difficult to distinguish the cause of liver damage or status of the liver based on only elevated levels of AST and ALT. And, further examination is needed for detailed lesion assessment in some cases. Recently, using EV-associated miRNAs or circulating free miRNAs in serum as biomarkers has made it possible to diagnose 13 types of cancer with an accuracy of 90 % or more  42] [43 [4342]  45] [46 [4645]  48]. Th. Th48]. In this study, we succeeded in isolating 44 miRNAs containing the known liver damage biomarkers miR-122 and miR-192. Using those novel biomarkers, it may be possible to elucidate the mechanism of hepatotoxicity by analyzing the behaviors of biomarkers when hepatotoxicity is caused by administration of drugs or chemical substances other than carbon tetrachloride. Since EVs in blood are secreted by a wide variety of cells, it is unclear whether the EV-associated miRNAs identified as novel biomarkers are secreted only by the liver. And effort to evaluate the origin of EVs should be necessary, however, to understand the systemic toxicity, no matter where EVs come from, whole biomarker analysis may be important. Because in addition to hepatotoxicity, it is also possible to comprehensively detect systemic toxicity by proceeding with analysis of EV-associated miRNAs as biomarkers by targeting various organs such as the kidney, lung and heart.Although this study analyzed the effects of carbon tetrachloride administrations at single time point, 24 h, clarifying the time courses of biomarkers in response to repeated administration of carbon tetrachloride may be applicable to shortening chronic toxicity tests and long-term carcinogenicity tests.in vivo.To date, circulating free miRNAs in serum have also been used in addition to EV-associated miRNAs as biomarkers of carcinogenesis. EV-associated miRNA is contained in serum RNA, but it is also known that some miRNAs have different expression profiles between EV-associated miRNA and circulating free miRNA in serum  50]. Th. Th50]. Finally, by quantification of EV-associated miR-122 and miR-192 using qRT-PCR and next-generation sequencing of the same serum sample, it was revealed that the miRNA expression profiles obtained from miRNA sequencing were almost identical to those obtained from qRT-PCR. Therefore, once useful biomarkers have been identified, systemic toxicity can be evaluated simply by performing qRT-PCR of each biomarker.R.O., T.O., S.K., and Y.H. conceived the study. R.O., Y.Y., and Y.F. participated in the experimental design and performed analyses. M.K. participated in histological analysis. R.O. and M.N. analyzed NGS data. R.O. wrote the manuscript. All authors read and approved the final manuscript.This work was supported in part by Research on the Regulatory Science of Pharmaceuticals and Medical Devices (20mk0101163j0202), Research on the Development of New Drugs (20ak0101093j003) from the Japan Agency for Medical Research and Development, the Health Sciences Research Grants from the Ministry of Health, Labor, and Welfare, Japan, (H30-KAGAKU-IPPAN-002), and JSPS KAKENHI (18K19315) to R.O. ; Grant-in-Aid from the Research Program on Hepatitis from Japan Agency for Medical Research and Development  and Grants-in -Aid from a Project for Cancer research and Therapeutic Evolution (P-CREATE) to T. O. ; JSPS KAKENHI (18K07053) to Y. H.The authors declare no competing interests.