Shigella T3SS needle complex. The inner membrane (IM) region of the basal body adopts 24-fold rotational symmetry and forms a channel system that connects the bacterial periplasm with the export apparatus cage. The secretin oligomer adopts a heterogeneous architecture with 16- and 15-fold cyclic symmetry in the periplasmic N-terminal connector and C-terminal outer membrane ring, respectively. Two out of three IM subunits bind the secretin connector via a β-sheet augmentation. The cryo-EM map also reveals the helical architecture of the export apparatus core, the inner rod, the needle and their intervening interfaces.The Type III Secretion Systems (T3SS) needle complex is a conserved syringe-shaped protein translocation nanomachine with a mass of about 3.5 MDa essential for the survival and virulence of many Gram-negative bacterial pathogens. This system is composed of a membrane-embedded basal body and an extracellular needle that deliver effector proteins into host cells. High-resolution structures of the T3SS from different organisms and infection stages are needed to understand the underlying molecular mechanisms of effector translocation. Here, we present the cryo-electron microscopy structure of the isolated  Shigella causing dysentery utilizes the type 3-secretion system (T3SS) to inject virulence factors into the gut cells. The T3SS needle complex is a syringe-shaped nanomachine consisting of two membrane-embedded ring systems that sheath a central export apparatus and a hollow needle-like structure through which the virulence factors are transported. We present here the structure of the Shigella T3SS needle complex obtained by high-end electron microscopy. The outer membrane (OM) ring system adopts a mixed 15- and 16-fold cyclic symmetry and the near-atomic structure shows the connection of the inner membrane (IM) and OM rings. Conserved channels in the IM ring connect the bacterial periplasm with the central export apparatus. Similar to the Salmonella flagellar system, the export apparatus and its connected needle-like structure assemble in a helical manner. This study advances our understanding of the role of essential structural elements in the T3SS assembly and function.Diarrheal diseases evoke about 2.2. million dead people annually and are the second leading cause of postneonatal child mortality worldwide.  Shigella causes dysentery, a disease that accounts for approximately half a million infant deaths annually [Shigella colonize the gastrointestinal tract by delivering virulence factors called effectors in a time-dependent fashion to the target cell [Salmonella spp and Yersinia spp, Shigella strains belong to the phylogenetic family of Salmonella pathogenic island– 1 (SPI-1) bacteria [Bacterial diarrheal diseases cause million deaths in children under age five worldwide . Shigellannually . Common get cell . Togethebacteria , which eShigella [Shigella) and outer-  membrane proteins to form their respective rings in the corresponding membranes and periplasm [Shigella [Shigella needle complex and dimension, however, is still unclear. The export apparatus is thought to serve as entry gate for unfolded substrates in the cytoplasm and an anchor for the inner rod in the periplasm [Salmonella consists of five membrane associated proteins [Shigella needle complex orthologues are SpaPQRS  and MxiA. On the cytoplasmic side of the needle complex, a set of different proteins assemble together to form the sorting platform, a complex that recruits, unfolds and delivers T3SS substrates to the cytoplasmic components of the export apparatus [The T3SS structural core is a membrane-embedded transporter called needle complex, which is an evolutionary conserved syringe-like multiprotein assembly with a molecular weight of about 3.5 MDa. This complex is made of a basal body and a hollow needle that extends from the bacterial surface. The needle is a homo-oligomeric structure of different length depending on the species, formed by a helical arrangement of a single small protein of 9 kDa, MxiH in Shigella . The baseriplasm . These rShigella , functioeriplasm . The strproteins ,11. Its pparatus ,13. Effepparatus . The sorpparatus . Severalpparatus –19, but Shigella T3SS IM periplasmic ring and of the N-terminal domains of the secretin, revealing their interface and a network of channels in the IM ring, based on high-resolution cryo-EM symmetrized maps obtained by single-particle analysis . Further, the unsymmetrized map at 5.1 Å resolution of the basal body reveals a mixed C15/C16 symmetry of the secretin oligomer and provides a model for the architecture of the export apparatus core, the inner rod and the start of the needle filament. Altogether, our obtained maps and molecular models provide new insights into the structure and assembly of the Shigella needle complex and allow a comparative analysis with the well-studied orthologous complex from Salmonella.Here, we report the near-atomic structure of the Shigella T3SS needle complex by single particle cryo-EM analysis. For this, we imaged vitrified needle complexes with a 300 kV FEI Titan Krios microscope in combination with a Falcon II direct electron detector. 104,272 particle images obtained after 2D classification were subjected to 3D classification in three classes. The main class included 69% of the particles, which we used for high-resolution refinement  yielded a map with an overall resolution of 5.1 Å (Shigella needle complex measures approximately 31 nm in length and is tapering from 25 nm diameter in the IM ring down to 15 nm in the outer membrane (OM) ring  ring . In our OM) ring . The mapOM) ring . The IM nt paper .Consequently, we performed focused refinements for different regions of the needle complex to exploit the symmetry and obtain higher resolution maps . We applThe periplasmic IM ring map shows clear backbone and side chain densities that allowed us to build and refine the first high-resolution experimental structures of the periplasmic domains of the IM proteins MxiG and MxiJ . Local rThe periplasmic IM ring is composed of 24 copies of the inner membrane proteins MxiG and MxiJ forming two concentric rings . MxiJ foThe MxiG N-terminal cytoplasmic domain (residues M1 to H125) is connected by a predicted transmembrane helix from residue S126 to L143 to the periplasmic domains D2 (E152 to L200), D3 V201 to L272), D4 (S273 to I340) ,26 and a2, D4 (S2The domains D2 to D4 of MxiG and both MxiJ domains share a similar fold known as the ring-building motif (RBM), where two α-helices fold against a mixed three-stranded β-sheet . The Mxi2, which corresponds to 20% of the monomer surface, whereas MxiG subunits share a relatively small homo-interface area of ~875 Å2 (8% of the monomer surface). The two MxiG-MxiJ interfaces have area of ~800 Å2 and ~650 Å2.Several homo- and hetero-interactions between MxiG and MxiJ stabilize the IM ring structure. Each protein molecule in the ring interacts with two identical subunits and two of the other type. The most extensive interface is provided by MxiJ dimerization with a surface area of ~2450 ÅSalmonella [In MxiG, the D2 and D4 domains are mainly responsible for hetero-interactions. The helix α2 and the strand β3 of D2 are in contact with the C-terminal stretch (P188 –V197) of one MxiJ subunit and with the loop connecting the helix α1 and the strand β2 of the adjacent MxiJ subunit . In D4, lmonella ,30.MxiG homo-interfaces mostly involve the D3 and D4 domains, as a cleft separates the domain D2 of adjacent molecules, although long side chains can bridge the gap. For instance, K160 is hydrogen-bonded to Y172 of the neighboring MxiG subunit. In domain D4, the C-terminal end of the helix α5 of one MxiG subunit is packed against the sheet β7-β9 of the neighboring MxiG subunit. In addition, the C-termini of the helices α6 and α3 of two adjacent subunits are in contact .Pseudomonas T3SS [Salmonella [Salmonella orthologue of SpaP has been observed also in crosslinking experiments [MxiJ dimerization involves both domains. A network of hydrogen bonds, salt bridges and hydrophobic contacts connects helices α1, α2 and α4, most of the β-strands, the loop connecting α3 and α4, and the C-terminal stretch that folds back into the direction of the membrane . Severalnas T3SS . Hydrophlmonella . The hyderiments .Salmonella basal body . The N0-N1 interface is stabilized by three hydrogen bonds and hydrophobic interactions involving several conserved residues  is moderately hydrophobic (2) and is stabilized by five hydrogen bonds  includes a single intermolecular hydrogen bond between S107 and I60.The N0-N0 interaction completes the interfaces between adjacent MxiD subunits. It involves the end of the two helices, the loops connecting them with the three-stranded β-sheet and the loop linking N0 and N1. The small interface  supports the relevance of N3 domain in driving the oligomerization process [The proximal edge of the β-barrel is in contact with the ring formed by the N3 domain . This do process .The C1 map of the needle complex shows distinct tubular densities at the resolution of 5–6 Å in both the export apparatus and the inner rod and needle , suggestSalmonella flagellar system [Shigella proteins SpaP, SpaQ and SpaR. These proteins are mostly composed of helices, which allow the fit into the low-resolution map. Random search followed by rigid body and flexible fitting allowed us to place five subunits of SpaP and SpaQ, respectively, and one of SpaR into our C1 map .In structures obtained from isolated secretin oligomers the connector N0 domain appears to have a high degree of flexibility ,35. The Shigella needle complex shows C16 and C15 symmetries for the connector  and the OM ring , respectively. Likely, this asymmetry is responsible for the tilt between connector and OM ring observed in the reconstruction  is present across the bacterial inner membrane ,19,48,49Yersinia ,17 and flmonella ,48,50,51 complex ,53. The pparatus ,13,54. Ipparatus , it is pThe IM ring and the export apparatus interact through a structurally conserved loop comprising residues P91 to P99 in MxiJ. The hydrophobic residues of this loop might promote the close contact between these two needle complex components despite their different symmetries and allowing some rotational freedom relative to each other for functionality , as a hyShigella T3SS. This molecular arrangement differs from the 5:4:1 stoichiometry found in the reconstruction of the isolated export apparatus of Shigella [Salmonella. The difference could be consequence of the isolation process of the complex or its heterologous overexpression. Native MS reveals the presence of the 5:5:1 complex when purified with a mild detergent in Pseudomonas savastatoi [Salmonella cross-linking data suggest that the additional subunit SpaS is also bound close to the location that we assigned to the 5th SpaQ [We found evidence in our map for a 5:5:1 stoichiometry of the export apparatus proteins SpaP:SpaQ:SpaR of the Shigella  and Salmvastatoi . However5th SpaQ . TherefoIn vivo cross-linking studies in Salmonella have shown that PrgJ  interacts with SpaP and SpaR [Salmonella needle complex revealed the presence of only few copies of PrgJ [Comparison of our structure with the isolated export apparatus showed different conformation at the inner rod interface, where the N-terminal helix of a SpaP subunit occludes the opening in the isolated export apparatus, while it is bent and moved aside in the needle complex. The helix movement provides additional space for the passage of transported molecules . Our C1 and SpaR ,57. A st of PrgJ , suggest of PrgJ ,57, whic of PrgJ  and the Shigella needle complex described in this study contributes to a better understanding of the molecular mechanisms underlying protein secretion, with the ultimate aim of supporting the development of novel therapeutics to fight against multi-drug resistant Gram-negative bacteria that employ the T3SS.The structure of the Shigella mutant M90T ΔIpaDΔMxiH was generated by λ Red recombination [ΔIpaDΔMxiH was complemented with N-terminal Strep-tagged MxiH encoded on an anhydrotetracycline hydrochloride (AHT) inducible plasmid pASK-IBA5plus. M90T ΔMxiG was complemented with an N-terminal Strep-tagged MxiG encoded on an AHT inducible plasmid pASK-IBA5plus. For co-localization of the T3SS needle with the bacterial membrane, N-terminal Strep-tagged MxiH encoded on pASK-IBA3C was co-transformed with Strep-MxiG on pASK-IBA5plus in M90T ΔMxiG. Shigella were kept on Tryptic Soy Broth (TSB) agar plate supplemented with 0.01% (w/v) Congo red and antibiotics as selection markers. E.coli DH5α were kept on lysogeny broth (LB) agar plates with antibiotics as selection markers.The knockout bination . M90T ΔI600 0.4–0.6. Cells were washed with phosphate buffered saline (PBS) and osmotically shocked by resuspension in buffer containing 18% (w/v) sucrose, 1 mg/ml Lysozyme, 100 mM Tris pH 8.0, 100 mM NaCl and 1.25 mM EDTA at 37°C, until vast majority of bacteria formed spheroplasts. Cell lysis was performed with 2% (v/v) Triton-X-100, in the presence of DNAse 1 and 10 mM MgSO4 and Complete Ultra Protease Inhibitor Cocktail  at room temperature for 15 min. Cell debris was removed by centrifugation in a JA25.50 rotor at 15000 rpm at 4°C for 20 min. The resulting supernatant was centrifuged at 40,000 rpm with rotor 45Ti at 4°C for 4h, the pellet was solubilized in 50 mMTris pH 8.0, 5 mM EDTA, 100 mM NaCl, 0.02% w/v n-Dodecyl β-D-Maltopyranoside containing PIC at 4°C overnight. Needle complexes were isolated using Strep-tactin sepharose resin (IBA Lifescience) and used immediately for further structural analysis.Bacteria were grown in TSB at 37°C, MxiH expression was induced by AHT to final concentration of 0.2 μg/ml and bacterial cells were harvested at OD-/Å2. Pixel size was 1.38 Å, corresponding to a magnification of about 100,000 fold. Nominal underfocus value was set randomly in the interval 1.5–4 μm.400-mesh Quantifoil R2/1 holey grids with additional ~4–5 nm continuous thin carbon layer were incubated with 5 μl of sample and vitrified with FEI Vitrobot Mark IV. Different glow-discharging, incubation and blotting times were tested and the grids were inspected either at the electron microscope FEI Tecnai Spirit at the Max Planck Institute for Molecular Genetics  or FEI Tecnai G2 F20 in CEITEC . 5,238 micrographs were collected with a FEI Titan Krios microscope in CEITEC  at 300 kV equipped with a FEI Falcon II detector from a grid vitrified after 15 sec glow-discharging, 5 min sample incubation on ice and 2 sec blotting time. Each micrograph is composed of 7 frames, with a total exposure time of 1.5 sec per micrograph and total dose of 25 eShigella mutants have been collected with a FEI Talos L120C microscope at the cryo-EM facility of the Centre for Structural System Biology . Samples were stained with 1% uranyl acetate.Negative-stained micrographs of needle complex from Individual frames were aligned with Motioncorr 2.1  and the Focused refinement of the IM ring was performed imposing C24 symmetry with a solvent mask obtained from the C1 reconstruction. Angular sampling started from 1.8° and decreased stepwise to 0.2° during the process. The resulting map had resolution 3.9 Å and post-processing yielded a map with resolution 3.6 Å sharpened with B-factor -128. A second focused map was obtained including in the mask the IM ring and the connector regions and imposing C8 symmetry, which is common between the C24 fold symmetry of the IM ring and the C16 of the secretin, with angular sampling 0.9° and 0.5°. The map at 4.5 Å resolution was post-refined yielding 3.9 Å and sharpened with B-factor -120.The high-resolution maps of the periplasmic IM ring and connector obtained applying C24 and C8 symmetry, respectively, allowed us to choose the correct handedness by modeling the structures of MxiG, MxiJ and the N-terminal domains of MxiD into them. The correct handedness of the other maps was established by comparing them with the former two maps.Salmonella homologues PrgH (PDB entry 3GR0) [22-189 was split into two domains, MxiJ22-80 and MxiJ98-189. MxiG172-340, MxiJ22-80 and MxiJ98-189 were fitted as rigid body in the periplasmic ring of the focused C24 map low-pass filtered at 3.2 Å IM reconstruction with the software Chimera version 1.11.2 [21-197 and MxiG152-340. Phenix CC_mask is 0.83, the EM ringer score [The map for periplasmic IM ring shows backbone and side chain features which allowed modeling of the atomic structure for MxiJ and the periplasmic domains of MxiG. We used Phyre2  to generry 3GR0)  and an Ery 3GR0)  due to tn 1.11.2  and refin 1.11.2  and phenn 1.11.2  applyinger score  is 3.02.37-171 was built using the Escherichia coli EscC structure (PDB entry 3GR5) [de novo with Coot 0.8.9.1 using the bulky aromatic side chains as sequence markers. The entire connector model was refined with Coot and phenix.real_space_refine 3357 imposing secondary structure restraints and NCS-like constraints corresponding to C8 symmetry. Phenix CC_mask is 0.85 and EMringer score is 3.50 [The connector region is formed by the two N-terminal domains of MxiD and the C-terminal domain of MxiG. An initial model of the MxiDry 3GR5)  as templ is 3.50 .Salmonella (EMDB entry 4173) [Shigella were generated with SWISS-MODEL [Salmonella flagellar export apparatus as templates (PDB entry 6F2D) [Shigella needle filament (PDB entry 2MME) [To build a model of the export apparatus core, the cryo-EM map of the flagellar export apparatus from ry 4173)  was initry 4173)  are 1.88SS-MODEL  using thry 6F2D) . We fittry 2MME)  as searcry 2MME)  includinThe atomic models of the IM ring, connector, export apparatus core and inner rod were validated as described  by compaSalmonella open secretin pore [The OM ring map does not show distinct secondary structure features, therefore we did not attempt flexible fitting. We built a homology models for the N3 and secretin domains of MxiD using SWISS-MODEL and the recently published structure of the tin pore . We usedth 0.3261) and adjusted p value 3.5e-12 [Salmonella. Each secretin domain includes residues 301–521.For the secretin domain homology model, 500,000 global searches with Chimera fitmap yielded 270,138 unique fits. The top 15 fits have correlation in the range 0.4453–0.3849 . The small dimension of this domain and low quality of the C1 map on the tilted side of this region prevents the global search from finding the correct poses in the top hits. Thus, we modelled the remaining N3 domains by superposing one of the secretin-N3 subunits found by Chimera to the lonely secretin domains. Each N3 domain includes residues 179–230 and 259–300. The C-terminal S domain has not been modelled as explained in the main text.For the multiple sequence alignment, a structural alignment between MxiG/PrgH and MxiJ/PrgK was generated with PDBeFold  and orthLocal resolution maps were calculated with Relion 2.1. Electrostatic analysis was carried out with APBS  using twMaps were segmented and edited with Segger inside Chimera  and maskThe Channel analysis was performed using CHEXVIS  with a pCalculation of helical parameters of the export apparatus core is based on the centre of mass of SpaP subunits and has been performed with HELFIT . SpaP haHydrophobicity of the protein surfaces was depicted following the Eisenberg scale .http://www.ebi.ac.uk/msd-srv/ssm) [RMSD of structural alignments has been calculated with the protein structure comparison service PDBeFold version 2.59 at the European Bioinformatics Institute (srv/ssm)  with defAll molecular structure figures were generated with PyMol  or UCSF E. coli DH5α for selection and amplification. Sanger sequencing was used to verify mutagenesis and plasmids harboring the mutation were transformed in Shigella M90T ΔMxiG.To generate the MxiG mutants, the following primers and their reverse complements were used: 5´CTGTCAGAGAAGAACTGACAAAGGAAAAGCTTGAGCTC3´ for D311K, 5’GGTGTCAAACAAAGAAATAAATGAGATTCAACAATATATCAATC3’ for R208E, 5’GGTGTCAAACAAAGAAATAAATGCAATTCAACAATATATCAATC3’ for R208A, 5’ CTGGTGTCAAACAAAAGAATAAATAGAATTCAACAATATATC3’ for E205R, 5’ CTGGTGTCAAACAAAGCAATAAATAGAATTCAACAATATATCAATC3’ for E205A, 5’ GTTGAATTTCCGTATTTCAAAAATATTAAA3’ for Y263F. 5’ GATGATGATTTTTAAGGTAAATCATATC3’ for 347-Stop. The polymerase chain reaction was performed on MxiG encoded on the pASK-IBA5plus vector with a Phusion High-Fidelity DNA Polymerase. The PCR product was Dpn1 digested, and transformed in Shigella M90T ΔMxiG strains, encoded on a pASK-IBA5plus plasmid. For the secretion assay and the expression test, the sample cell density was normalized, lysed and analyzed by a SDS-PAGE gel or Western Blot. The Western Blot was performed using a polyvinylidene fluorid membrane with anti-IpaB, anti-IpaC and anti-DnaK (Enzo) antibodies. For the expression test, a western blot with a polyvinylidene fluorid membrane was performed using anti-DnaK (StressGene Biotechnologies Corp.) and anti–MxiG antibodies.The secretion assay was performed as published  inducingShigella M90T ΔMxiG strains were co-transformed with MxiG mutants encoded on a pASK-IBA5plus plasmid and Strep-MxiH on a pASK-IBA3C plasmid. For co-localization studies of the MxiG mutants in the bacterial membrane, Shigella M90T ΔMxiG was transformed with MxiG mutants encoded on a pASK-IBA5plus plasmid only and treated with 0.1% Triton X-100 in Dubelcco´s—Phosphate buffered Saline (DPBS) and 10% w/v Lysozyme, 5 mM EDTA in DPBS at room temperature to permeabilize the membrane. Bacterial cells were fixed for 15’ at RT with 4% paraformaldehyde, followed by membrane permeabilization, if required. Staining was performed with a monoclonal primary mouse anti-Strep-tag II antibody  in combination with an Alexa Flour 647 secondary antibody (Invitrogen). The bacterial membrane was stained with a DiO lipophilic tracer (Invitrogen). Bacteria were mounted in Mowiol 4–88 medium on 33 mm μ-dishes (ibidi) and visualized on a Leica SP8 confocal microscope equipped with a 63x, N.A. 1.40 oil immersion objective using excitation at 471 nm and 653 nm and operated in HyVolution mode. Deconvolution was performed with Huygens Essential (Huygens Compute Engine 18.04.0p7 64b) software using express deconvolution and standard profile. Experiments were performed multiple times on biological duplicates.To analyze the co-localization of the T3SS needle with the bacterial membrane, S1 FigShigella needle complexes (left) and class averages obtained in the final round of 2D classification (right).Representative micrograph of purified (TIF)Click here for additional data file.S2 FigA) Full reconstruction without symmetry, resolution 5.1 Å. (B) Focused reconstruction of the IM ring and connector with C8 symmetry, resolution 3.9 Å. (C) Focused reconstruction of the IM ring with C24 symmetry, resolution 3.6 Å.FSC curves from gold-standard refinement and post-refinement. The vertical line indicates the resolution of the maps at 0.143 FSC. ((TIF)Click here for additional data file.S3 FigA) Full reconstruction of the needle complex without symmetry. (B) Focused reconstruction of the IM ring and connector with C8 symmetry. (C) Focused reconstruction of the IM ring with C24 symmetry.Cross-sections of the maps obtained in this study, locally low-pass-filtered and colored according to the local resolution (Å) as shown in the color scales. ((TIF)Click here for additional data file.S4 FigA and B) Detail of the C24 density map of the IM ring with the atomic model represented as sticks. Selected residues close the channel exit or involved in MxiG-MxiJ interaction and subjected to mutational analysis are labeled (C) Model-map FSC curves of the IM ring. Model versus the full map used for building and refinement (black line), model refined against the first of the two independent half maps versus the same map  and versus the second half map . The line at FSC 0.5 marks the approximate resolution of ~3.5 Å of the model.( labeled . (C) Mod(TIF)Click here for additional data file.S5 FigA and B) Interface between two adjacent MxiJ (shades of orange) and one MxiG subunit (blue). Interface MxiJ D2 –MxiG D4 (A). Interface MxiJ–MxiG D2 (B). (C) Interface between two adjacent MxiG subunits, involving the D3 and D4 domains.Residues involved in polar interactions are depicted as sticks and labeled; dotted lines represent hydrogen bonds and salt bridges. ((TIF)Click here for additional data file.S6 FigShigella proteins. Blue boxes indicate conserved residues with regards to their physicochemical properties. Fully conserved residues are depicted in white letters on red background. Residues involved in intermolecular interactions are marked with +, x or O. The green boxes indicate the MxiJ loops pointing in the direction of the export apparatus. MxiJ orthologues of the following species were aligned (UniProt code in brackets): Shigella flexneri (Q06081), Salmonella typhimurium (P41786), Candidatus regiella insecticola (E0WTJ1), Sodalis glossinidius (Q2NVJ4), Burkholderia pseudomallei (Q3JL03), Chromobacterium violaceum (Q7NUV9), Yersinia enterocolitica (Q01251) and Pseudomonas aeruginosa (Q9I314). All proteins mentioned belong to the SPI-1 family, except for Pseudomonas PscJ, which is part of the Ysc family. MxiG orthologues of the following species are aligned: Shigella flexneri (P0A221), Salmonella typhimurium (P41783), Candidatus regiella insecticola (G2H2F2), Sodalis glossinidius (Q2NR71), Burkholderia pseudomallei (Q63K19), Chromobacterium violaceum (Q7NVC0).MxiG and MxiJ protein sequences were aligned with orthologues from other Gram negative bacteria expressing T3SS. The highlighted secondary structure elements correspond to the (TIF)Click here for additional data file.S7 FigA) The MxiG D311K mutant does not secrete effector proteins. Secreted proteins of wild type M90T strain (WT) and mutant strain were precipitated and visualized by coomassie stained SDS-PAGE. Protein secretion was induced adding Congo red (CR). SepA serves as a T3S-independent loading control. IpaA, IpaB, IpaH and IpaC are T3SS effectors. (B) Western blot of Shigella total cell lysates stained with antibodies against MxiG and DnaK for wild type M90T and MxiG mutants. (C) Secretion assay of the MxiG Y263F, Y263F_E205R and 347-Stop mutants. Secreted proteins and total cell lysates of wild type M90T strain (WT) and mutant strains were separated via SDS-PAGE and effector proteins (IpaB and IpaC) visualized by Western Blot; DnaK served as loading control. The effector proteins IpaB and IpaC are produced but not secreted in the MxiG mutant strains 347-Stop, Y263F and Y263F_E205R. (D) Representative immunofluorescence images of Shigella M90T ΔMxiG strains complemented with the Strep-MxiG mutants E205R and Y263F_E205R. The bacterial membrane was permeabilized prior to antibody staining. Strep-MxiG localizes at the bacterial membrane in both mutants. Red: anti-Strep antibody; green: lipophilic membrane dye. The scale bar corresponds to 2 μm.((TIF)Click here for additional data file.S8 Fig(A and B) Interface between adjacent MxiJ subunits in cartoon representation; residues involved in polar interactions are depicted as sticks and are labeled; dotted lines represent hydrogen bonds and salt bridges. (A) Polar interactions involving residues of the D1 domains. (B) Polar contacts at the MxiJ–MxiJ interface in the D2 domain. Only conserved or conservatively substituted residues involved in hydrogen bonds or salt bridges are depicted. (C) MxiJ surface involved in homo-interaction colored according to the Eisenberg hydrophobicity scale (white residues being most hydrophobic). Hydrophobic residues at the D2-D2 domain interface are labeled.(TIF)Click here for additional data file.S9 Fig(A) Side view of the outer surface of our model of the export apparatus core colored according to the electrostatic potential . Two MxiJ and two MxiG subunits  located on opposite sides of the IM ring are shown as cartoon. Two MxiJ loops are in close proximity to the export apparatus. The tip residues of the lower loop  are conserved and interact mostly with the hydrophobic surface of SpaQ, while the residues of the upper loop  interact mostly with the more hydrophilic surface of SpaP. (B) Vertical cutaway of the export apparatus core showing the inner surface colored according to the electrostatic potential . The inner chamber is mostly positively charged. The bottom opening is closed, preventing substrate access, while the upper opening provides connection to the channel of the inner rod.(TIF)Click here for additional data file.S10 Fig(A) Top view of a quarter of the IM ring with MxiG and MxiJ models in sky blue and orange cartoon representation, respectively. The IM channel surface is depicted in yellow. The radial channels connecting the periplasm with the export cage are interconnected by a circular channel. (B) Radial channel in the IM ring of Salmonella (PDB ID 5TCP) with PrgH and PrgK subunits in purple and beige cartoon representation, respectively. The channel is depicted in yellow, the exits A and B are labeled. (C) Radius and electrostatic potential along the central line of the Salmonella IM ring radial channel.(TIF)Click here for additional data file.S11 Fig(A) Model-map FSC curves of the connector (MxiD34-171 and MxiG338-367). Model versus the full map used for building and refinement (black line), model refined against the first of the two independent half maps versus the same map  and versus the second half map . The line at FSC 0.5 marks the approximate model resolution of ~ 3.7 Å. (B) Detail of the β-sheet augmentation of MxiD69-74 with MxiG362-371 (left) and of the density map for MxiG363-371 (right). MxiD and MxiG carbon atoms are green and blue, respectively. Hydrogen bonds are depicted as dotted lines, the donor-acceptor distance indicated in Angstrom. (C) Slice through the focused C8 map and the cartoon models of IM ring and connector (right) and detail of the region between them (left). MxiG is depicted in blue, MxiJ in orange, MxiD in green. The MxiG C-terminal stretch connects IM ring and connector; the unassigned density between them could be occupied by the C-terminus of an adjacent MxiG subunit, which does not participate in the β-sheet augmentation.(TIF)Click here for additional data file.S12 FigShigella flexneri (Q04641), Salmonella typhimurium (P35672), E.coli EPEC (B7UMB3), Yersinia enterocolitica (Q7BRZ9), and Pseudomonas aeruginosa (P95431) were aligned.Multiple sequence alignment of MxiD with orthologous from different T3SS expressing Gram-negative bacteria. Numbering and secondary structure elements of MxiD are indicated. Blue boxes highlight conserved residues with regards to their physicochemical properties. Identical residues are depicted in white letters on red background. MxiD orthologues of (UniProt code in brackets) (TIF)Click here for additional data file.S13 Fig(A) N0 and N1 domains of MxiD colored from N- to C-terminus from blue to yellow (left) and close-up of the polar interaction (right). Residues forming hydrogen bonds are depicted as sticks and labeled. The side chains of Y96, Y105 and D136 bind backbone atoms of L134, A157 and K97, respectively (B) Tilted top view of the N0 and N1 domains of MxiD colored according to Eisenberg hydrophobicity scale (white residues being most hydrophobic). Residues involved in hydrophobic N0-N1 interactions are labeled and depicted as sticks .(TIF)Click here for additional data file.S14 Fig(A) Two neighboring MxiD connector subunits (left). N1 residues involved in polar interaction are represented as sticks and labeled in the close-up (right). Black dotted lines represent N1-N1 interface hydrogen bonds , yellow dotted lines hydrogen bonds between the N0 and N1 domains  (B) Two neighboring MxiD N1 domains colored according to the Eisenberg hydrophobicity scale (white residues being more hydrophobic) (left). Residues at the hydrophobic interface are depicted as sticks and labeled in the close-up (right) . (C) N0 and N1 domains of two neighboring InvG subunits of the Salmonella connector (PDB ID 6DV3) (left); residues involved in polar intermolecular interactions represented as sticks and labeled in the close-up (right).(TIF)Click here for additional data file.S15 Fig(A) Side view of the Shigella T3SS needle surface, colored according to its electrostatic potential . (B) Vertical cutaway of the OM ring, the surface colored as in (A). (C) Tilted top view of the surface of the open OM pore, colored according to the Eisenberg hydrophobicity scale (white representing hydrophobic areas). A ring of hydrophobic residues is visible on the membrane-facing side of the MA subdomain.(TIF)Click here for additional data file.S16 Fig(A) Vertical cross section of the semitransparent C1 map of the export apparatus, inner rod and start of the needle, with the protein subunits represented as cartoons (same color code as in (B) Model-map FSC curves. Model versus the full map used for building and refinement (black line), model refined against the first of the two independent half maps versus the same map  and versus the second half map . The line at FSC 0.5 estimates the model resolution ~8 Å.(TIF)Click here for additional data file.S17 Fig(A) Subunits of the first inner rod turn (dark magenta and purple) and distal SpaP (light blue) of the Shigella needle complex superposed with the distal SpaP subunit of the isolated Salmonella flagellar system (dark blue). The N-terminal helix of the SpaP subunits occludes the opening between the export apparatus and the inner rod in the isolated flagellar system, while it is kinked and leaves the passage open in the Shigella needle complex. (B) Secondary structure prediction of MxiI generated with the PSIPRED server (http://bioinf.cs.ucl.ac.uk/psipred). Blue bars represent the confidence value. Pink tubes represent helices. C = coil, H = helix.(TIF)Click here for additional data file.S18 FigSalmonella domains has been obtained from the models with PDB ID 5TCP  or 6DV3 (InvG). Fraction of matched residues are relative to the Shigella domains.The structure of the (PDF)Click here for additional data file.S19 FigReconstruction obtained from a preliminary dataset composed of ~3000 particle images collected on a FEI Tecnai Spirit microscope at 120 kV. The map was low-pass filtered at 60 Å before using it as reference for initial classification of the high-resolution dataset collected at the FEI Titan Krios.(TIF)Click here for additional data file.


Pannexin 1 (Panx1) is a ubiquitously expressed hexameric integral membrane protein known to function as an adenosine triphosphate (ATP) release channel. Panx1 proteins exist in unglycosylated core form (Gly0). They undergo critical post-translational modifications forming the high mannose glycosylation state (Gly1) in the endoplasmic reticulum (ER) and the complex glycosylation state (Gly2) in the Golgi apparatus. The regulation of transition from the ER to the cell membrane is not fully understood. Using site-specific mutagenesis, dye uptake assays, and interaction testing, we identified two conserved aromatic residues, Trp123 and Tyr205, in the transmembrane domains 2 and 3 of the zebrafish panx1a protein. Results suggest that both residues primarily govern the assembly of panx1a subunits into channels, with mutant proteins failing to interact. The results provide insight into a mechanism enabling regulation of Panx1 oligomerization, glycosylation, and trafficking. N254Q demonstrated limited membrane localization, with only a subpopulation reaching the cell surface [Pannexin 1 (Panx1) is an integral membrane glycoprotein forming a large pore membrane channel ubiquitously in the mammalian central nervous system (CNS) ,3. The p surface ,11. Desp surface . A singlDanio rerio) Panx1 ortholog (panx1a) has been described and found to form functional membrane channels in the Neuroblastoma 2a (Neuro 2a) cell line [Previously, a zebrafish  open reading frame (amino acids 1-416) was cloned into the enhanced yellow fluorescent protein plasmid (pEFYP-N1) expression vector  as described [panx1a WT and mutants were cloned into a pdTomato-His expression vector. For localization studies, ER and Golgi organelle markers tagged with DsRed2 were generated as described [The full-length escribed . Mutagen2. For Western blots, ~50,000 cells were seeded in 24-well plates. For imaging of fixed cells and live-cell imaging experiments, ~25,000 cells were seeded in 24-well plates or 35mm glass-bottom dishes . For Ni-NTA His pull-down assays, a combined total of ~3,000,000 cells were seeded in two 100mm plates. For the cell surface biotinylation assay, ~800,000 cells were seeded in 60mm plates.Neuroblastoma 2a (Neuro 2a) cells were cultured in DMEM containing 2mM glutamine, 1% nonessential amino acids, 1% penicillin and streptomycin, and 10% fetal bovine serum. Cells were maintained at 37 °C in a humidified atmosphere with 5% CONeuro 2a cells were transiently transfected using the Effectene™ Transfection Reagent  according to the manufacturer’s protocol. For 24-well plates and 35mm glass-bottom dishes, cells were transfected with 200ng DNA for single transfections and 400ng DNA for double transfections. For each 100mm plate, 2000ng of DNA was used for single transfections, and 4000ng DNA was used for double transfections. For 60mm plates, 600ng of DNA was used. Expression and functional studies were performed 48 h post-transfection. For live microscopy experiments, the medium was changed for DMEM lacking phenyl red at least 30min prior to imaging.® CLx Infrared Imaging System (LI-COR Biosciences).Proteins were separated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 100-150V for 1.5 h. The gel was placed onto a nitrocellulose membrane, and proteins were transferred using the Trans-Blot Turbo Transfer  system. Transfer conditions were 1.3A and 2.5V for 7 min. The membrane was blocked with Odyssey blocking buffer  for 1h and incubated overnight with specific antibody mixtures. Primary antibodies used included rabbit anti-green fluorescent protein (GFP) (1:500) , mouse anti-GFP (1:500) , rabbit anti-His (1:1000) , and mouse anti-beta-actin (1:15000) . Secondary antibodies were donkey anti-rabbit IRDye680LT (1:15000) (LI-COR Biosciences) and goat anti-mouse IRDye800CW (1:15000) (LI-COR Biosciences). Membranes were imaged using an OdysseyTM (Sigma-Aldrich).For immunofluorescence, transfected cells were fixed with 100% methanol for 10 min at room temperature (RT). Cells were blocked in Phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA) for 1h at RT. Next, the primary antibody dilution containing rabbit anti-His (1:1000) (Bethyl Laboratories Inc.) in PBS with 0.1% BSA was applied for 1h at RT. Cells were then incubated with 2ug/mL of secondary antibody dilution containing Alexa Fluor 568 goat anti-mouse  in PBS with 0.1% BSA for 1h at RT. Lastly, cells were washed in PBS and mounted with FluoroshieldTM (Sigma-Aldrich). Images were taken with a Zeiss LSM 700 confocal microscope using a Plan-Apochromat 63x/1.4 Oil DIC M27 objective. Zeiss ZEN imaging software was used to control all imaging parameters.For imaging of fluorescently tagged proteins, transfected cells were fixed with 4% paraformaldehyde for 30 min at RT and mounted with FluoroshieldTM Buffer provided with the His60 Ni Gravity Column Purification kit according to the manufacturer’s protocol (Clontech Laboratories Inc.). The lysates were loaded onto equilibrated columns and rocked at 4 °C for 1h. The columns were washed, and proteins were eluted according to the manufacturer’s protocol. All samples were recovered in 1X Laemmli sample buffer, heated at 95 °C for 5–10 min, and analyzed by Western blotting.Neuro 2a cells were double transfected with either WT panx1a-dTomato-His and WT panx1a-EYFP, Y205A-dTomato-His and WT panx1a-EYFP, or dTomato-His with WT panx1a-EYFP. The cells were lysed under native conditions using His60 Ni xTractorTM Sulfo-NHS-Biotin (Thermo Scientific) per plate for 30 min at room temperature. The reaction was quenched with three washes of 50mM glycine buffer for a total of 15 min at RT. Cells were collected in PBS lacking calcium and magnesium, washed, and lysed in NP40 lysis buffer for 30 min on ice with periodic vortexing. Cells were then incubated with 90uL of prewashed DynabeadsTM MyONETM Streptavidin C1 (Invitrogen) on a shaker at 4 °C overnight. Bead complexes were washed with the following buffers: buffer 1 (2% SDS in dH20), buffer 2 , buffer 3 , and buffer 4 . Bead complexes were then resuspended in 60uL of 1X Laemmli buffer and boiled for 5 min to elute the proteins. Eluted proteins were analyzed by Western blot analysis using rabbit anti-GFP (1:500) (Santa Cruise Biotechnologies) to detect wild type and mutant panx1a-EYFP. A mouse anti-beta-actin antibody (1:15000) (Sigma-Aldrich) was used to as reference protein.Neuro 2a cells transfected with fluorescently tagged WT panx1a or mutants were subject to cell surface biotinylation assay 48 h post-transfection. Cells were grown on 60mm plates and washed once with warm PBS containing calcium and magnesium. Cells were then labeled with 0.25mg of membrane-impermeable EZ-linkTransfected cells were treated with 5ug/mL Brefeldin A (BFA) (Sigma-Aldrich) for 19h before localization studies and Western blot analysis. BFA has been used as an inhibitor for the transport of Panx1 between the ER and Golgi compartments [2 using a Zeiss 700 confocal microscope. Ethidium bromide (EtBr) was added to a final concentration of 10uM immediately prior to imaging. Images were taken every 1 min for a duration of 10 min at a 512 × 512 pixel resolution with a scan speed of 4. Fluorescent values after 10 min of EtBr uptake were subject to statistical analysis.Neuro 2a cells transfected with fluorescently tagged WT panx1a and panx1a mutants were tested for dye uptake 48 h post-transfection using a protocol described previously ,22. CellFt is the fluorescence intensity at t seconds, F0 is the fluorescence intensity upon bleaching, and Fi is the fluorescence intensity immediately prior to bleaching.Neuro 2a cells were grown on 35mm glass-bottom dishes and transfected with fluorescently tagged WT or mutant panx1a to assess cell surface dynamics . Cells wDpost is the average intensity after the bleach, and Dpre is the average intensity before the bleach. The threshold value of 10 nm distance was converted into FRET efficiency and was calculated to be 1.4% for DsRed and EYFP pair, based on the reference distance between the two fluorescent tags (4.9 nm) [oR is the distance between two fluorescent tags, E is the FRET efficiency, and R is the FRET distance.Neuro 2a cells were transfected with combinations of EYFP and DsRed-tagged WT and mutant panx1a using a previously established protocol . Cells w(4.9 nm) . FRET diTotal RNA was extracted 48h post-transfection using RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s protocol from Neuro 2a cells, with or without 5ug/mL BFA treatment for 19h. A total of 1ug of RNA was used to synthesize cDNA using the ReadyScript cDNA Synthesis Kit (Sigma-Aldrich). qPCR was performed using the SsoFast EvaGreen Supermix (Bio-Rad) with the oligonucleotide pairs described . QuantifStatistical analysis was performed using MatLab R2018b, GraphPad Prism 8, or the Relative Expression Software Tool (REST) . Data weSequence alignment of multiple pannexin orthologs and paralogs, as well as the distant invertebrate relative INX-6, revealed that aromatic amino acids W123 and Y205 of panx1a were conserved A. W123 aIn Neuro 2A cells, the membrane localization of EYFP-tagged panx1a was compromised when mutating either Y205 or W123, thus identifying them as amino acids required for cell surface localization C. SubstiThe efficient membrane trafficking of panx1a requires N-glycosylation. A Western blot analysis determined the state of glycosylation for the aromatic mutants. The Y205A, R204Y:Y205R, I203Y:R204I:Y205R, W123A, and Y205A:W123A mutants showed a loss of the complex glycosylation (Gly2) form of the protein. All mutants still expressed the high mannose glycosylation form (Gly1) D.The possibility of Y205 being a site for tyrosine phosphorylation in panx1a was tested by replacement with the aromatic amino acid phenylalanine, which cannot be phosphorylated. Unlike Y205A, the EYFP-tagged Y205F mutant localization was similar to wild type panx1a A. Western = 17) and both Y205A and W123A mutants was detected  , which was considered to represent baseline uptake of EtBr. The Y205F mutant showed no significant difference in total dye uptake when compared to wild type panx1a . The general trend of the dye uptake over the course of 10 min was similar for wild type and the Y205F mutant (A dye uptake assay and Fluorescent Recovery After Photobleaching (FRAP) analysis were used to assess the channel function and the cell surface dynamics of panx1a and mutants. As expected, both Y205A and W123A mutants were unable to uptake EtBr due to the lack of transport to the cell membrane. Fluorescent images showed the dye uptake at 0 (start) and 10 min (endpoint) post-treatment with EtBr A. Only t 0.0001) B. Both mF mutant C.n = 35; Y205F: 52.28 ± 11.96, n = 33; p = 0.43)  E. The ge = 0.43) F. These First, we confirmed that the complex glycosylation of zebrafish panx1a was added in the Golgi apparatus by treating panx1a-EYFP transfected Neuro 2a cells with Brefeldin A (BFA). Previous studies have shown that 21 h treatment of 5ug/mL BFA disrupts the trafficking of rodent pannexin-1 between the ER and Golgi . This wan = 34; Cav-1: 0.71 ± 0.12, n = 36; p = 0.018), and with ER than Golgi biomarkers  , and with ER than Golgi markers , suggesting that trafficking of Y205A out of the ER was impaired , Cav-1-His (Caveolin-1 marker), calreticulin-DsRed (ER marker), or galactosyltransferase-DsRed (Golgi apparatus marker) C. The sa 0.0003) E. In conimpaired F.p < 0.001, CHOP: p < 0.000, EDEM: p < 0.000, BiP: p < 0.000, sXBPI: p < 0.000, usXBPI: p = 0.019). Except for one upregulated gene, expression of Y205A did not affect cell stress markers (BiP: p = 0.005). Overexpression of both wild type and mutant panx1a caused no significant difference in the expression levels of stress genes when compared to EYFP-expressing cells.Next, ER stress was ruled out by quantitative real-time PCR of established biomarkers E. The mRDouble transfection of WT panx1a-DsRed with Y205A-EYFP did not rescue the observed localization phenotype. The intracellular retention of the mutant remained A. Howeven = 103) . No significant difference in FRET efficiency was detected between the intracellular panx1a-DsRed and panx1a-EYFP pair when compared to panx1a-DsRed paired with Y205A-EYFP mutant , or pairs of mutants Y205A-DsRed and Y205A-EYFP , suggesting neither combination had the ability to reach a compact state. To complement these results, we also calculated the FRET distance based on the FRET efficiencies (Förster Resonance Energy Transfer (FRET) experiments determined the proximity between panx1a WT and the Y205A mutant. Panx1a-DsRed and panx1a-EYFP had a FRET efficiency above the threshold of 0.014 at the cell membrane. We concluded that the channels had assembled in a compact state after insertion into the cell membrane  B. As expciencies .The FRET experiment did not allow us to conclude the ability of wild type and mutant proteins to interact. This question was addressed using a His60 Ni column pull-down assay. The panx1a-dTomato-His protein pulled down panx1a-EYFP efficiently C. The Y2The highly conserved aromatic amino acid residues located on the intracellular side of transmembrane domain 2 (TM2) and 3 (TM3) were identified by sequence alignment with human pannexin proteins and the INX-6 channel. Presently, only the high-resolution crystal structure of INX6 has been reported. The mutation of either the Y205 or the W123 residues resulted in the retention of the protein. Since the two residues are in close proximity to each other, as seen with the INX-6 channel, it is possible that they interact by aromatic–aromatic interactions similar to those reported previously [+ channel [Xenopus laevis oocyte model [Many integral membrane proteins contain transmembrane domains made up of bundles of lipid-bilayer-spanning alpha helices. The TM alpha-helices are generally composed of a core of mostly hydrophobic amino acids, with basic and aromatic amino acids at each end of the helix. This arrangement is suggested to play a unique role in membrane protein interactions with water/lipid interfaces, anchoring the proteins into the membrane through an interaction of their aromatic rings with the lipid head groups ,29,30. P channel . The stu channel . Howeverte model  has showHere, we present results that provide insight into the post-translational processing of panx1a and the relevance for trafficking. The presence of the high mannose glycosylation form (Gly1) on Western blots indicated the ability to undergo limited post-translational modification. Both W123A and Y205A mutant proteins still underwent the first modification step towards complex glycosylation (Gly2). N-glycosylation of rodent or human pannexin-1 at amino acid position N254 and the corresponding position N246 in zebrafish panx1a is considered necessary for trafficking to the cell membrane ,10,14. WThe co-localization analysis revealed that the Y205A mutant was more abundant in the ER than the Golgi. Previous research has shown that the transport of Panx1 between the ER and Golgi is mediated via COP II vesicles . When weThe accumulation of integral membrane proteins like panx1a can occur upon transient overexpression, causing ER stress when misfolded or when otherwise altered proteins trigger a cellular stress response. The RT qPCR analysis tested whether ER stress was a confounding factor in this investigation. Transcriptional regulation of Atf-4, CHOP, EDEM, BiP, and XBP1 is known as an indicator of ER stress and the unfolded protein response (UPR) . BrefeldFörster resonance energy transfer (FRET) tests and pull-down experiments tested the interaction between Y205A-EYFP and WT panx1a-DsRed protein pairs. In contrast to wild type:wild type pairs, the low FRET efficiency detected between wild type:Y205A, as well as Y205A:Y205A pairs, suggested that the mutant protein failed to form higher-order complexes. Consistently, the pull-down experiment demonstrated that the interaction between wild type and mutant proteins was low or even nonexistent.This study identified two critical aromatic amino acids, W123 and Y205, that together help panx1a proteins to oligomerize and efficiently traffic to the cell membrane. Based on the high-resolution structure of the INX-6 channel , it is r


The role of PI3K-mTOR pathway in regulating NK cell development has been widely reported. However, it remains unclear whether NK cell development depends on the protein kinase B (PKB), which links PI3K and mTOR, perhaps due to the potential redundancy of PKB. PKB has two phosphorylation sites, threonine 308 (T308) and serine 473 (S473), which can be phosphorylated by phosphoinositide-dependent protein kinase-1 (PDK1) and mTORC2, respectively. In this study, we established a mouse model in which PKB was inactivated through the deletion of PDK1 and Rictor, a key component of mTORC2, respectively. We found that the single deletion of PDK1 or Rictor could lead to a significant defect in NK cell development, while combined deletion of PDK1 and Rictor severely hindered NK cell development at the early stage. Notably, ectopic expression of myristoylated PKB significantly rescued this defect. In terms of mechanism, in PDK1/Rictor-deficient NK cells, E4BP4, a transcription factor for NK cell development, was less expressed, and the exogenous supply of E4BP4 could alleviate the developmental defect of NK cell in these mice. Besides, overexpression of Bcl-2 also helped the survival of PDK1/Rictor-deficient NK cells, suggesting an anti-apoptotic role of PKB in NK cells. In summary, complete phosphorylation of PKB at T308 and S473 by PDK1 and mTORC2 is necessary for optimal NK cell development, and PKB regulates NK cell development by promoting E4BP4 expression and preventing cell apoptosis. NK cfunction , 7.We prfunction . Ablatiofunction . mTOR bifunction , 10. Usifunction , 11.As a central regulator, the serine/threonine kinase PKB/Akt links the upstream PI3K with the downstream mTOR signaling, and converts the environmental signals into cellular response signals. To date, three PKB family members have been identified in mammals, designated PKB1, PKB2 and PKB3, which share similar domain structure and function redundantly. The germline deletion of PKB leads lethal disorder. The contributions of PKB to immune cells such as T cells , B cell The activity of PKB is delicately modulated by the phosphorylation of two sites, Thr308 and Ser473. The first step for PKB activation is the phosphorylation of Thr308 by PDK1. This process is mediated by the tethering of PKB and PDK1 to the plasma membrane . The lipCre/+CD122 mice to established a mouse model in which PKB was inactivated through the deletion of PDK1 and Rictor, a key component of mTORC2, respectively. In Cre/+CD122 mice, Cre begins to be expressed during and after NK cell commitment. We demonstrated that complete phosphorylation of PKB at T308 and S473 by PDK1 and mTORC2 is necessary for optimal NK cell development. Mechanistically, PKB regulates NK cell development by promoting E4BP4 expression and preventing cell apoptosis.To investigate the role of PKB in early NK cell development, we used newly generated flox/floxPDK1 mice were a gift from Dario Alessi (University of Dundee). CD122-Cre mice was described previously  were used in our experiment. All mice were bred and maintained in specific pathogen-free animal facilities of Tsinghua University. All animal procedures were approved by the Animal Ethics Committee of Tsinghua University.eviously . Rictorf308 (D25E6) and anti-phospho-AKT S473 (D9E) were obtained from Cell Signaling Technology. For the detection of phosphorylated signaling proteins, NK cell was fixed with Phosflow Lyse/Fix buffer, permeabilized with Phosflow Perm buffer III (eBioscience), and then stained with antibodies. Data were acquired on an LSRII (Becton Dickinson) flow cytometer and analyzed using FlowJo software (Treestar). The expression level was presented as percentage or net MFI, which was determined by subtracting the mean fluorescence intensity of isotype control.Monoclonal antibodies against mouse CD3 (17A2), NKp46 (29A1.4), NK1.1 (PK136), CD127 (SB/199), Ly49A (A1), Ly49H (3D10), Ly49G2 (4D11), Ly49D (4E5), NKG2A (20D5), NKG2D (CX5), KLRG1 (2F1), CD11b (M1/70), CD27 (LG.7F9), Bcl-2 (10C4), E4BP4 (S2M-E19), Eomes (Dan11mag), T-bet (4B10), Ki67 (B56) and isotype controls were purchased from eBioscience. Anti-Ly49C/I (YLI-90) and anti-phospho-STAT5 (pY694) were purchased from BD Biosciences. Anti-phospho-S6 (D57.2.2E). Anti-phospho-AKT T6 cells in 500 µL 1640 medium) with the stimulation of rmIL-15/IL-15Rα complex (10 ng/ml). Twenty-four hours later, cells were collected for flow cytometry analysis. Meanwhile, spleen lymphocytes without stimulation were treated with the same staining, then used as control.To detect NK cell responsiveness to IL-15, splenocytes were incubated in 24-well plates (2×10NK cells were purified from splenocytes using a positive selection kit (StemCell Technologies) and then cultured in RPMI 1640 medium containing 20% fetal bovine serum, β-Mercapto alcohol, and 1,000 U/ml IL-2. Seven days later, NK cells were further stimulated with or without rmIL-15/IL-15Rα complex (10 ng/ml) for 24 h. Cells were collected and the protein was extracted as previously described. Equal amounts of protein were fractionated by 12% SDS-PAGE and transferred to polyvinylidene difluoride membrane. Membranes were blocked with TBS/Tween 20 (TBST) containing 5% bovine serum albumin (BSA) for 1 h. After blocking, the membranes were incubated with the appropriate specific primary antibodies at 4°C overnight, followed by incubation with HRP-conjugated secondary antibodies . Detection was performed using an enhanced chemiluminescence kit  according to the manufacturer’s instructions. Densitometry analysis was performed using ImageJ software.NK cell Caspase activity was measured with fluorescein isothiocyanate (FITC)- conjugated z-VAD-fmk according to the manufacturer’s instruction (eBioscience).Mice were treated with 5-FU for 4 days, and bone marrow cells were collected for spin-infection with MSCV retrovirus encoding target genes. The infected BM cells were then transferred into recipient mice that were sub-lethally irradiated. After eight weeks, the reconstruction of recipients was assessed by flow cytometry.β2m-deficient mice were depleted of red blood cells by Ficoll-Hypaque density gradient centrifugation and then labeled with 5µM CFSE (Molecular Probes). At the same time, splenocytes from C57BL/6 mice were labeled with 0.5µM CFSE (10-fold less than β2m cells). Two types of CFSE-labeled splenocytes were mixed at a 1:1 ratio. A mixture of 2×106 splenocytes was intravenously injected into mice pre-treated with 200 µg Poly (I:C). Eighteen hours later, CFSE-positive cells in the spleen and lymph nodes were determined by flow cytometry.Splenocytes from 6), and NK-non-sensitive cells RMA expressing DsRed (106). Tumor cells were mixed at a 1:1 ratio. Eighteen hours later, the mice were sacrificed, and cells in the peritoneal cavity were collected by repeated washing with PBS containing 2 µM EDTA. The relative percentage of residual RMA-S-GFP and RMA-DsRed cells were measured by flow cytometry.Mice treated with 200 µg Poly (I:C) for 18 h were intraperitoneally injected with a mixture of tumor cells, NK-sensitive cells RMA-S transfected with GFP (105 cells/mouse). Fourteen days later, the mice were sacrificed. The lung was weighed and the number of lung surface nodules was counted under a dissecting microscope.B16F10 melanoma cells in the log phase were resuspended in 1×PBS and intravenously injected into the mice  across multiple groups were used to determine significance. A fl/fl/CD122Cre/+ (PDK1KO) mice. However, there were still residual NK cells in the spleen and bone marrow (We previously showed that PDK1 deletion at the NK progenitor (NKp) stage caused a reduction of NK cell number in PDK1e marrow . To inve). Therefore, combined with the published data, we suggest that the inactivation of PDK1 leads to the increased activation of PKB S473, which may be a compensating effect.Our group had reported that the deletion of PDK1 on NK progenitor diminished mTORC1 activation in NK cells . Yang etfl/fl/CD122Cre/+Rictor mice (RictorKO) to disrupt Rictor in NKp cells. Rictor deletion nearly abolished PKB S473 phosphorylation . As expected, the deletion of Rictor in PDK1KO mice could notably diminish the enhanced phosphorylation level of PKB S473 in total and NK cell subsets . Interestingly, the additional deletion of Rictor also decreased the amount of phosphorylated form of S6 and STAT5 in total and different NK cell subsets , CD122+NK1.1+CD11b– immature NK (iNK) and CD122+NK1.1+CD11b+ mature NK (mNK)  , 21. We To prove the above point, we examined the repertoire of NK cell receptors, which is sequentially acquired by NK cells during their development. CD127 was usually expressed only on a fraction of early NK cells in Control mice. However, the proportion of CD127 positive cells was increased in PDK1 and Rictor DKO mice. Ly49 family members  and KLRG1, which represent NK cell mature and senescence respectively, were hardly expressed in PDK1 and Rictor DKO NK cells , National Key Research & Developmental Program of China (2018YFC1003900), Natural Science Foundation of Guangdong Province , Guangzhou Science and Technology Project  and the China Postdoctoral Science Foundation . The Dong laboratory was also supported by Tsinghua-Peking Center for Life Sciences.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.


Moreover, reactive oxygen species (ROS) production, apoptosis, and neurodegeneration were significantly lower in the CYP2E1(−/−) I/R group (p < 0.001). The BBB damage was significantly lower in CYP2E1(−/−) mice compared to wild-type (WT) (p < 0.001), while 20-HETE production was increased by 41%. Besides, inflammatory cytokines expression and the number of activated microglia were significantly lower in CYP2E1(−/−) mice following I/R. CYP2E1 suppression ameliorates I/R injury and protects BBB integrity by reducing both oxidative stress and inflammation.Despite existing strong evidence on oxidative markers overproduction following ischemia/reperfusion (I/R), the mechanism by which oxidative enzyme Cytochrome P450-2E1 (CYP2E1) contributes to I/R outcomes is not clear. In this study, we sought to evaluate the functional significance of CYP2E1 in I/R. CYP2E1 KO mice and controls were subjected to middle cerebral artery occlusion (MCAo-90 min) followed by 24 h of reperfusion to induce focal I/R injury as an acute stage model. Then, histological and chemical analyses were conducted to investigate the role of CYP2E1 in lesion volume, oxidative stress, and inflammation exacerbation. Furthermore, the role of CYP2E1 on the blood-brain barrier (BBB) integrity was investigated by measuring 20-hydroxyecosatetraenoic acid (20-HETE) activity, as well as, in vivo BBB transfer rate. Following I/R, the CYP2E1 KO mice exhibited a significantly lower lesion volume, and neurological deficits compared to controls ( Clinically effective treatments to reduce ischemia-reperfusion (I/R) injury are still a major unfulfilled medical need. Several barriers and limitations exist in the wide use of favorite reperfusion methods. For example, reperfusion of ischemic cells results in a reactive oxygen species (ROS) burst that contributes to lipid peroxidation, as well as DNA oxidation and irreversible tissue injury. The most important sources of ROS are mitochondrial dysfunction, NADPH oxidases, cellular enzymes, as well as metabolic enzymes such as the cytochrome P450 family . CytochrWhile CYP2E1 is predominantly expressed in the liver, significant levels of CYP2E1 are expressed and induced in many extrahepatic tissues  ,7,8. ComBrain indigenous substrates of CYP2E1 are arachidonic acids, linoleic acids, and oleic acids gluconeogenic precursors and estrogenic metabolites . MetabolCerebral ischemia is shown to induce CYP2E1 expression . IncreasCYP2E1(−/−) mice (CYP2E1-null) on the C57BL/6 background selected from our breeding colony established at The University of South Florida  . Experimn = 12F + 12M), WT (n = 12F + 12M), and Sham groups (n = 5M + 5F), unless otherwise mentioned, for both sexes were studied for (1) The role of CYP2E1 on ROS activity, (2) Stroke complication because of CYP2E1 expression (permanent vs. transient), and (3) The role of CYP2E1 in neurovascular inflammation, 20-HETE expression, and the BBB health. Animals were randomly assigned to the experimental groups.Three experimental groups CYP2E1 KO  to induce permanent MCAo or followed by 24 h reperfusion to induce focal cerebral I/R injury or transient MCAo (tMCAo). The filament occlusion was omitted to induce sham-operated models. To induce MCAo, mice surgically manipulated to expose the common carotid arteries. A silicon-coated filament inserted into the left common carotid artery, up through the internal carotid artery to block the MCA. For the permanent MCAo model, the filament remained in place permanently. But for tMCAo the filament remained in place for 90 min (occlusion) and then retracted to allow for cerebral region reperfusion. During the surgery, 2.0% isoflurane was used for induction and 1.0–2.0% for maintenance. As for post-surgery treatments, mice were given the same amount of analgesic agents and normal saline 2 h after the operation. Rules for inclusions and exclusions of the models are presented in Materials and Methods in the Data Supplement.The neurological deficits were assessed and scored on a 5-point scale based on the report of Longa et al. . Mouse wAfter the reperfusion, five mice were randomly selected as samples from each group, the brains were removed and sectioned into five coronal sections, 2 mm thick for TTC staining. Details on TTC staining and infarct volume measurement are presented in Materials and Methods in the Data Supplement.Total cell membranes were prepared because it has been shown that brain CYPs are present more in multiple membrane fractions, including microsomal, mitochondrial, and nuclear membranes ,26,27. TCYP2E1 activity was assessed by measuring the rate of p-nitrophenol oxidation to p-nitrocatechol with samples of brain membranes as described previously ,29,30,31TUNEL assay was used to assess neuronal apoptosis in the ischemic hemisphere. Coronal brain slices that contain the ischemic region and the corresponding brain regions in controls were used for the TUNEL assay. Immunohistochemistry protocols and antibodies are presented in Materials and Methods in the Data Supplement. Tissue samples were taken from the ischemic region of the brain for microglial activity and astrogliosis analysis. Cerebral ionized calcium-binding adaptor protein-1 (Iba-1), glial fibrillary acidic protein (GFAP), and β-actin protein levels were determined at 24 h after I/R because Iba-1 and GFAP protein levels are known to be significantly increased at that time post-ischemia . ImmunohProtein levels of inflammatory markers; tumor necrosis factor (TNF)-α, interleukin (IL)-6, and monocyte chemoattractant protein 1 (MCP-1) in the brain were measured using an ELISA kit  according to the manufacturer’s protocol. Tissue samples were taken from ischemic regions and corresponding regions in control brains. Samples were measured in duplicates. Readings from each sample were normalized for protein concentration. Moreover, tissue 20-HETE levels were assessed using 20-HETE ELISA kits  according to the manufacturer’s instructions, and results were normalized to dry weight.ROS generation was measured using the CM-H2DCFDA  a peroxide-sensitive fluorescent probe as described previously with minor modifications . The homMagnetic resonance imaging was performed with 7T BioSpec MR Scanner  equipped with 500 mT/m gradient (rise time 80–120 μs), and a cryogenic quadrature RF surface coil (Bruker Biospin) as the RF transmitter/receiver  at 24 h t-test or Mann–Whitney test was applied based on parametric or non-parametric distribution, respectively. One-way analysis of variance (ANOVA) with Dunnett’s post-hoc was used for comparison of more than two data sets. Differences were considered to be significant at p < 0.05.We conducted all statistical analyses in the “R” environment (R core team 2017). Study groups were blinded to the person (S.T.) who conducted the statistical analysis. Data are expressed as mean ± standard deviation (SD), and n refers to the number of animals used. All data sets were tested for normality using the Shapiro–Wilk test, and a subsequent unpaired p < 0.005), and tMCAo (* p < 0.01) as shown in a representative TTC stains and quantitative analysis of total brain infarct volume ratio. Though the lesion volume was higher in tMCAo (both genders) compared to the MCAo group, we did not find any statistically significant differences . As expected, no significant CYP2E1 activity found in CYP2E1(−/−) group  following I/R in WT mice compared with age- and sex-matched controls (** −) group A.p < 0.001) in CYP2E1 KO. The analysis of fluorescence intensity illustrates the significant reduction in ROS production in CYP2E1 KO mice after the insult of I/R in comparison to age-matched WT mice. No fluorescence was detected in wells without the incubation of MC-H2DCFDA. Data are shown as mean + SD. n = 7 M per group, and *** p < 0.001  mice following tMCAo. Apoptosis of mice MCAo brain tissue in each group measured by TUNEL  in the middle cerebral artery territory of the cortex. TUNEL-positive cells (brown staining) significantly decreased in CYP2E1 compared with those in the WT group, see p < 0.005 and *** p < 0.001, respectively). Representative Western blots of Iba-1 and GFAP protein levels in the ischemic brain are shown at the bottom of the panels respectively. β-actin was used as a control for loading.The quantified level of protein expression of Iba-1 (Right) and GFAP (Left) in the peri-infarct region of CYP2E1(−/−), CYP2E1(+/+), and WT control mice brains subject to 90 min I/R are shown in the top panel of p < 0.05), (B) MCP-1 (p < 0.05), and (C) TNF-α (p < 0.0001) in cerebral ischemic tissues at 24 h of I/R, as measured by ELISA. Data are presented as mean ± SD in We observed more than two-fold reduction in protein concentration of (A) IL-6 (p < 0.01) difference between the mean of BBB transfer rate of CYP2E1(−/−) I/R and WT I/R. (We have observed that CYP2E1 (−/−) I/R models have a lower area with abnormal BBB transfer rate in comparison to WT in I/R models. Interestingly, there was no significant damage to the BBB of CYP2E1 (−/−) I/R models as measured by the rate of leakage of the contrast agent (Gd-DTPA) from the blood into cerebral tissues through the BBB. The BBB transfer rate maps which represent a pixel-wise calculated BBB transfer rate are depicted for three consecutive slices that cover most of the ischemic lesion volume are shown in panels A and B of  WT I/R. C. p < 0.05) in CYP2E1(−/−) mice following the insult of I/R . Interestingly we observe that the I/R insult did not significantly increase 20-HETE synthesis in WT controls. We measured the 20-HETE levels in WT controls and CYP2E1(−/−) mice brain with and without I/R insult. The 20-HETE level in WT controls n was 37.1 ± 2.5 ng/mL D. HoweveHere, we report results from an experimental study designed to evaluate the potential of CYP2E1 inhibition as a target for neuroprotection in I/R. Our results suggest that cerebral I/R insult activates CYP2E1 and increases ROS production. Using genetic approaches, we provide evidence that oxidative enzyme CYP2E1 critically defines I/R outcome. Indeed, mice in which CYP2E1 was deleted had the best stroke outcome . More spCYP2E1 is significantly expressed in different brain cell compartments, including the endoplasmic reticulum, the plasma membrane, the Golgi apparatus, as well as mitochondria ,39,40,41Various stimulants such as CNS inflammation , chronicIn this study, we confirmed that CYP2E1 protein is expressed in brain tissues of WT controls when we compare to KO mice brain tissues. Furthermore, we showed that in the MCAo mouse models, I/R insult increases CYP2E1 protein activity by (61% increase) compared to WT controls. Ischemic insult, as well as traumatic brain injury (TBI), increases the activity of phospholipase A2 (PLA2), which hydrolyzes membrane phospholipids to generate arachidonic acid (AA) . An incrFree radicals surge exacerbates cerebral damage following I/R insult. However, little is known about the CYP2E1 role in free radicals production following I/R. In this study, a marked decrease was observed in cerebral I/R damage following CYP2E1 suppression. In these models, a marked reduction in lesion volume, as well as ROS activity following I/R insult, supports the notion that CYP2E1 activity plays a crucial role in oxidative stress following I/R. Of note is that these observations were not sex-specific. Although there is a sex-specific expression of some hepatic CYPs , there iIncreased ROS activity is observed in cerebral reperfusion . ConsideOf note is that along with CYP2E1 there are other cellular oxidase scavenger systems such as lipoxygenase (LOX) , cyclooxInflammation contributes significantly to the pathogenesis of post-ischemic insult ,58,59. OThese lines of evidence indicate that CYP2E1 not only has a crucial role in oxidative stress but also contributes to proinflammatory markers activation following I/R. The connection between CYP2E1 and inflammatory markers is complicated. For example, studies have shown that IL-6 has a critical role in enabling lipopolysaccharides (LPS) to increase CYP2E1 activity by using IL-6 KO mice . Therefo. However, the debate remains on how a 20-HETE surge impacts I/R injury. A study connects 20-HETE vasoconstrictors property to reduced I/R injury [Here, we further explored the role of CYP2E1 activity on damaging BBB integrity following I/R insult. Our data indicate that CYP2E1 suppression alleviates BBB damage following I/R insult. To explore the underlying mechanism by which CYP2E1 suppression impacts BBB health following I/R, here we measured 20-HETE expression. Furthermore, 20-HETE is a cytochrome P450-derived metabolite of AA . 20-HETER injury . AnotherR injury . We prop2e1 inhibition is a crucial factor for the expression of EETs as an endothelium-derived hyperpolarizing factor (EDHF). Regarding hepatic CYP2E1 expression, it will merit investigating if CYP2E1 plays a role in liver-brain communications. Moreover, it will be interesting to investigate the morphology and 3D architecture of brain vasculature in the CYP2E1 KO mouse. In this study, histochemical experiments were designed to enable us to draw conclusions based on group statistics. On the other hand, MRI data enabled us to draw individual longitudinal analysis, as well as group analysis. We admit that employing a histological group analysis method limits the acquired knowledge of the specific CYP2E1 dynamics in each animal. Another limitation is that knocking CYP2E1 out may interfere with other detoxification mechanisms that may reduce the toxicity of ischemia. Therefore, it may confound with the goals of this study, i.e., the reduction of oxidative stress of ischemia. To avoid this confounding, we monitored animals for their health and also examined serum inflammatory markers at least twice before I/R. We planned to exclude animals if a complication happens, which was not the case in this study. As a future direction of this study, we recommend considering the conditional KO model, which is now available, to study the specific role of CYP2E1 in the brain. However, this study using CYP2E1(−/−) mice enabled us to identify whether cypIn conclusion, I/R insult activates oxidative enzyme CYP2E1 that induces oxidative stress, increases inflammatory markers, and exacerbates I/R damages. Genetic inhibition of this enzyme significantly reduces oxidative stress, specifically on the BBB tight junctions following I/R. Following I/R insult, the cascade of inflammatory/oxidative stress, that CYP2E1 plays a major role, destroys the BBB tight junctions. Understanding the specific roles of the CYP on exacerbation of oxidative stress and inflammation following I/R may lead to more refined therapeutic strategies.


G-quadruplexes (G4) are considered new drug targets for human diseases such as cancer. More than 10,000 G4s have been discovered in human chromatin, posing challenges for assessing the selectivity of a G4-interactive ligand. 3,6-bis(1-Methyl-4-vinylpyridinium) carbazole diiodide (BMVC) is the first fluorescent small molecule for G4 detection in vivo. Our previous structural study shows that BMVC binds to the MYC promoter G4 (MycG4) with high specificity. Here, we utilize high-throughput, large-scale custom DNA G4 microarrays to analyze the G4-binding selectivity of BMVC. BMVC preferentially binds to the parallel MycG4 and selectively recognizes flanking sequences of parallel G4s, especially the 3′-flanking thymine. Importantly, the microarray results are confirmed by orthogonal NMR and fluorescence binding analyses. Our study demonstrates the potential of custom G4 microarrays as a platform to broadly and unbiasedly assess the binding selectivity of G4-interactive ligands, and to help understand the properties that govern molecular recognition. The bui+ ,4,5. Thes are fou+ ,7,8.G4 structures are involved in many cellular processes of DNA, including gene transcription ,10, DNA ex,max = 435, λem,max = 580) to detect G4 structures in human cells [3,6-bis(1-Methyl-4-vinylpyridinium) carbazole diiodide BMVC, a is a G4an cells ,23,24. Ban cells ,26. Wheran cells . We haveMicroarray glass slides with hundred thousands of DNA sequences are a fast, straightforward, and high-throughput platform that has been employed to screen, profile, and quantify ligand and protein interactions with DNA and RNA molecules ,29,30. Wex,max = 647, λem,max = 665) labeled small molecule pyridostatin [Herein, we report a binding-selectivity analysis of BMVC to the MycG4 and other G4 structures using custom G4 microarrays and competition experiments between Cy5-fluorophore  using the competition microarray experiments. The differential reduction of Cy5-PDS binding to variants in the flanking sequences induced by BMVC addition reveals the binding selectivity for specific MYC G4 flanking sequences a. In theWe further analyzed the effects of BMVC on the Cy5-PDS-binding to MYC G4 loop and single flanking-base sequence variants (5′-NGGGNGGGNNGGGNGGGN-3′), which include all possible loop and flanking variants c. ConsisThe sequence selectivity shown by the flanking variants explains the markedly weaker binding of BMVC to Bcl-2_P1G4 . Bcl2_55+), and hybrid type human telomeric G4 (Tel26 in K+)  . BMVC bi6 in K+) . Our pre6 in K+) . Mutatio6 in K+) . In cont6 in K+) . In addi6 in K+) . These r6 in K+) .While BMVC binds the MYC_14/23T G4 with the highest affinity , BMVC caWe have established a high-throughput, large-scale custom G4 DNA microarray to assess the binding selectivities of proteins and small molecules across ~20,000 potential G4 structures simultaneously. Competition binding experiments of the Cy5 labeled PDS and the unlabeled G4-interactive small molecule BMVC demonstrate that the custom G4 microarray platform can assess the binding selectivity of BMVC to various G4 structures and flanking sequences, as well as differential G4 binding selectivity between BMVC and PDS. Our results reveal that BMVC selectively binds parallel G4s, in particular the MYC_14/23T G4. Moreover, the G4 microarray data shows BMVC selectively recognizes the flanking sequences of parallel G4s, especially the 3′-flanking T. Importantly, the binding and sequence selectivity revealed by the large-scale DNA microarray data is in good agreement with the individual binding data by NMR and fluorescence. Our study demonstrates that the G4 DNA microarray provides a high-throughput and unbiased platform to assess the binding selectivity of G4-targeting molecules on a large scale and can help understand the properties that govern molecular recognition.We designed a custom microarray that contains four identical sectors that contain ~177,440 ssDNA 60-mers to examine G4 binding selectivity (NCBI GEO Platform GPL28372). The microarray contains different sets of G4 variants designed to examine several sequence parameters that affect G4 formation and binding selectivity such as loop length, loop sequence, flanking tail sequence, and single nucleotide variants of known G4s . BrieflyDNA microarray experiments were performed and analyzed as described previously . MicroarMolecule-bound microarrays were scanned with an Agilent G5761A SureScan Dx Microarray Scanner System to detect Cy5 signal at two laser settings (30 and 100 PMT). Spot intensities from microarray images were extracted using Agilent Feature Extraction Software and are reported as raw fluorescence intensities. All binding assays were performed twice with high agreement between replicates (R > 0.8). Microarrays with the fewest number of saturated spots were used for further analysis. Median intensity was then computed for probes containing identical sequence on each microarray. Sequence logos were generated from a position frequency matrix generated from selected sequences using ggseqlogo .G4 DNA oligonucleotides were synthesized using β-cyanoethylphosphoramidite solid-phase chemistry (Applied Biosystem Expedite 8909), as described previously . NMR exp


Human respiratory syncytial virus (RSV) and parainfluenza virus type 3 (HPIV3) are among the most common viral causes of childhood bronchiolitis and pneumonia worldwide, and lack effective antiviral drugs or vaccines. Recombinant (r) HPIV3 was modified to express the RSV fusion (F) glycoprotein, the major RSV neutralization and protective antigen, providing a live intranasal bivalent HPIV3/RSV vaccine candidate. This extends previous studies using a chimeric bovine-human PIV3 vector (rB/HPIV3). One advantage is that rHPIV3 expresses all of the HPIV3 antigens compared to only two for rB/HPIV3. In addition, the use of rHPIV3 as vector should avoid excessive attenuation following addition of the modified RSV F gene, which may occur with rB/HPIV3. To enhance its immunogenicity, RSV F was modified (i) to increase the stability of the prefusion (pre-F) conformation and (ii) by replacement of its transmembrane (TM) and cytoplasmic tail (CT) domains with those of HPIV3 F (H3TMCT) to increase incorporation in the vector virion. RSV F (+/- H3TMCT) was expressed from the first (F/preN) or the second (F/N-P) gene position of rHPIV3. The H3TMCT modification dramatically increased packaging of RSV F into the vector virion and, in hamsters, resulted in significant increases in the titer of high-quality serum RSV-neutralizing antibodies, in addition to the increase conferred by pre-F stabilization. Only F-H3TMCT/preN replication was significantly attenuated in the nasal turbinates by the RSV F insert. F-H3TMCT/preN, F/N-P, and F-H3TMCT/N-P provided complete protection against wt RSV challenge. F-H3TMCT/N-P exhibited the most stable and highest expression of RSV F, providing impetus for its further development. Orthopneumovirus and family Pneumoviridae 16]. In tAfrican green monkey kidney epithelial Vero cells (ATCC CCL-81), rhesus macaque kidney LLC-MK2 epithelial cells (ATCC CCL-7), and baby hamster kidney BHK BSR-T7/5 cells that constitutively express the T7 RNA polymerase were maintained as previously described . RecombiBlpI (pre-N) or a unique AscI (N-P) site was introduced in the antigenome cDNA at nt position 103–109 (TTTCAAA mutated to GCTTAGC) or 1675–1682 (ATCTAAAT mutated to GGCGCGCC), respectively, by site directed mutagenesis without affecting the viral genome length  or second (N-P) gene position of rHPIV3 , two mode length . The Blps system  were corBlpI or AscI sites, as shown in BlpI sites  gene position of rHPIV3  gene position of HPIV3 to yield the constructs F/N-P and F-H3TMCT/N-P. All constructs were designed to maintain a hexameric genome length and preserve hexamer phasing of the vector genes. The RSV F inserts at the pre-N and N-P position had the hexamer phasing of the N and P genes, respectively. The vector constructs were recovered in duplicate by co-transfecting BHK BSR T7/5 cells with the anti-genome plasmids and three expression plasmids expressing the HPIV3 N, P, and L proteins (see cDNA encoding the RSV F protein of strain A2 (GenBank accession number KT992094) was designed to be codon-optimized  (GenBank accession # MN746317) for human expression. Two amino acid substitutions K66E and Q101P  were included to make the amino acid sequence identical to that of an early passage of strain A2, and which also increase expression . In addipI sites  or AscI cI sites . These ff rHPIV3 , to yieleins see .As described previously , Vero ce50 per cell. After virus adsorption, the inoculum was removed, cells were washed, and 3 ml of medium was added to each well followed by incubation at 32C for a total of 7 days. At 24 h intervals, 0.5 ml of culture medium was collected and flash-frozen, and 0.5 ml of fresh medium was added to each well. The collected samples were titrated together in LLC-MK2 cells in 96-well plates by the TCID50 limiting dilution method using hemadsorption with guinea pig erythrocytes to identify the infected wells.Vero cells in 6-well plates were infected in triplicate with rHPIV3 vectors at a multiplicity of infection (MOI) of 0.01 TCID50 per cell or wt RSV at an MOI of 3 PFU per cell. After incubation at 32C for 48 h, cells were washed once with cold PBS and lysed with 300 ul 1X LDS lysis buffer (Thermo Fisher Scientific) containing 1X reducing reagent (Thermo Fisher Scientific). Cell lysates were passed through a QIAshredder column , heated for 5 min at 95°C, and 20 μl of each sample was loaded onto a 4–12% Bis Tris NuPAGE gel (Thermo Fisher Scientific). Proteins were electrophoresed in the presence of antioxidant (Thermo Fisher Scientific) and transferred to PVDF membranes using the iBlot system (Thermo Fisher Scientific). Membranes were blocked with blocking buffer  for 1 h and probed with primary antibodies in blocking buffer overnight at 4°C, followed by incubation with blocking buffer containing infrared dye-labeled secondary antibodies (LiCor). Blot images were acquired and analyzed using Image Studio software (LiCor). Primary antibodies included (i) a murine anti-RSV F MAb ; (ii) rabbit polyclonal hyperimmune serum against sucrose gradient-purified HPIV3 virions, or E. coli-expressed rHPIV3 F ectodomain, or an HPIV3 HN peptide (YWKHTNHGKDAGNELETC); and (iii) mouse anti-tubulin monoclonal antibody (MAb) . Secondary antibodies included donkey anti-rabbit IRDye 800CW and donkey anti-mouse IRDye 680LT (LiCor) that were used as per manufacturer’s instructions.LLC-MK2 or Vero cells in 6-well plates were infected with rHPIV3 or rHPIV3-RSV F at an MOI of 3 TCID5 TCID50 of rHPIV3, or 106 PFU of wt RSV. At day 5 post-inoculation, nasal turbinates and lungs were collected and virus replication was assessed by titration of homogenized samples on LLC-MK2 cells using limiting dilution and hemadsorption assay for rHPIV3 or on Vero cells by immunoplaque assay for RSV.Groups (n = 6) of 6-week old female Golden Syrian hamsters , pre-screened to be RSV- and HPIV3-seronegative, were inoculated IN with 100 μl of Leibovitz’s L15 medium (Thermo Fisher Scientific) containing 106 PFUs of wt RSV A2 strain. Nasal turbinates and lungs were collected on day 3 post-challenge to assess protection against RSV replication by immunoplaque titration of tissue homogenates on Vero cells. Serum titers of RSV F- or HPIV3-specific neutralizing antibodies were determined by a 60% plaque reduction neutralization test (PRNT60) on Vero cells in 24-well plates using rRSV or rHPIV3 expressing green fluorescent protein. Sera were heated at 56°C for 30 min to inactivate serum complement, and 4-fold serial dilutions of each serum in duplicate were mixed with an equal volume of diluted virus either with or without added guinea pig complement  [http://imagej.nih.gov/ij/]), and PRNT60 titers were calculated by linear regression analysis.In a separate study, hamsters in groups (n = 6) were inoculated as described above and sera were collected on day 28 to measure serum RSV- and HPIV3-neutralizing antibody titers. On day 30, hamsters were challenged IN with 10lle, MD) . After iin vitro passages in Vero cells, the cell type used for preparing clinical trial material. Briefly, Vero cells in T-25 flasks were infected with the P4 stock of F-H3TMCT/N-P, the P2 stocks of the other three constructs, or the P2 stock of wt rHPIV3, four replicates of each, at an MOI of 0.01 TCID50/cell. Following incubation at 32C for 3 days, culture supernatant was collected and was used to infect fresh Vero cells  under the animal study protocol number LID 34E. The National Research Council’s Guide for the care and use of laboratory animals and the Public Health Service Policy on humane care and use of laboratory animals served as the guidelines for the care and use of hamsters in this study.S1 Checklist(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Autophagy, a self-degradative physiological process, is critical for homeostasis maintenance and energy source balancing in response to various stresses, including nutrient deprivation. It is a highly conserved catabolic process in eukaryotes and is indispensable for cell survival as it involves degradation of unessential or excessive components and their subsequent recycling as building blocks for the synthesis of necessary molecules. Although the dysregulation of autophagy has been reported to broadly contribute to various diseases, including cancers and neurodegenerative diseases, the molecular mechanisms underlying the epigenetic regulation of autophagy are poorly elucidated. Here, we report that the level of lysine demethylase 3B (KDM3B) increases in nutrient-deprived HCT116 cells, a colorectal carcinoma cell line, resulting in transcriptional activation of the autophagy-inducing genes. KDM3B was found to enhance the transcription by demethylating H3K9me2 on the promoter of these genes. Furthermore, we observed that the depletion of KDM3B inhibited the autophagic flux in HCT116 cells. Collectively, these data suggested the critical role of KDM3B in the regulation of autophagy-related genes via H3K9me2 demethylation and induction of autophagy in nutrient-starved HCT116 cells. Autophagy is highly evolutionarily conserved and involved in several pathological pathways, including those related to cancers and neurodegenerative diseases . AutophaAutophagy has context-dependent roles, which either promote or inhibit the cell death in cancers , 8. For Although autophagy is a cytosolic event, several studies have revealed the importance of epigenetic regulation occurred in nucleus for proper autophagic flux –17. Sustlmo2 by reducing H3K9me2 on the promoter in HL-60, which are acute promyelocytic leukemia cells . Se. Se20]. 16 cells . ATG5, A16 cells . Consistarvation . In shKD reduced . HoweverATG5 and ATG7 in stable shKDM3B HCT116 , we selected the ATPase VCP/p97 as a putative interacting partner. VCP can interact with KDM3B and has various roles, including its control over the ubiquitin-dependent protein degradation [VCP HCT116 cells using shRNAs targeting VCP and found that KDM3B was induced with VCP depletion  ATG5 was induced by starvation in HCT116 cells. (B) Stable sh(TIF)Click here for additional data file.S2 Fig(A-B) plenti-Flag-VCP-overexpressing HCT116 cells were harvested and the cell extracts were immunoprecipitated with anti-KDM3B and anti-Flag antibodies. The interactions between KDM3B and VCP were detected by immunoblots. * indicates non-specific bands. (C) HCT116 cells were treated with starvation media for 4, 6, and 8 hours. The mRNA levels of VCP in HCT116 cells were confirmed by qPCR. The results represent at least three independent experiments (± SEMs).(TIF)Click here for additional data file.S3 FigKDM3B knockdown HCT116 cells. The result is expressed as means ± SEM. (n = 3). * P < 0.05.Cell proliferation was accessed through MTT assays in stable (TIF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.