Shigella effector OspI targets Ubc13 and deamidates Gln100 of Ubc13 to a glutamic acid residue, leading to the inhibition of host inflammatory responses. Here we report the crystal structure of the OspI-Ubc13 complex at 2.3 Å resolution. The structure reveals that OspI uses two differently charged regions to extensively interact with the α1 helix, L1 loop and L2 loop of Ubc13. The Gln100 residue is bound within the hydrophilic catalytic pocket of OspI. A comparison between Ubc13-bound and wild-type free OspI structures revealed that Ubc13 binding induces notable structural reassembly of the catalytic pocket, suggesting that substrate binding might be involved in the catalysis of OspI. The OspI-binding sites in Ubc13 largely overlap with the binding residues for host ubiquitin E3 ligases and a deubiquitinating enzyme, which suggests that the bacterial effector and host proteins exploit the same surface on Ubc13 for specific recognition. Biochemical results indicate that both of the differently charged regions in OspI are important for the interaction with Ubc13, and the specificity determinants in Ubc13 for OspI recognition reside in the distinct residues in the α1 helix and L2 region. Our study reveals the molecular basis of Ubc13 deamidation by OspI, as well as a convergence of E2 recognition by bacterial and host proteins.Ubc13 is an important ubiquitin-conjugating (E2) enzyme in the NF-κB signaling pathway. The  Shigella infects human intestinal epithelium cells and causes severe inflammatory colitis (bacillary dysentery). Shigella harbors an approximately 220-kb virulence plasmid that encodes a type III secretion system (T3SS) protein secretion apparatus and many effector proteins. Using the T3SS, Shigella delivers the effector proteins into the host cells, targeting key signal molecules and manipulating the host physiological processes and thereby promoting infection and multiplication. OspI, a newly identified Shigella effector, targets the host Ubc13 protein, a critical ubiquitin-conjugating enzyme in the NF-κB signaling pathway. OspI deamidates Gln100 of Ubc13 to a glutamic acid residue, thereby disrupting TRAF6-catalyzed polyubiquitination and dampening host inflammatory responses. However, the structural mechanism of this specific deamidation is unclear. Through crystallography, we have determined the structure of the OspI-Ubc13 complex. The structure illustrates how OspI interacts with Ubc13 and how Ubc13 induces conformational changes in OspI. Combining structural analysis and biochemical assays, we revealed how OspI distinguishes Ubc13 from other ubiquitin conjugating enzymes and found that OspI binds to the same surface region on Ubc13 as host TRAF6, CHIP and OTUB1. Our study sheds light on the molecular mechanism of Ubc13 deamidation by OspI and provides new insights into E2 recognition by bacterial and host proteins.The Gram-negative pathogenic bacterium  Ubiquitination is a post-translational protein modification involved in many important cellular processes, such as signal transduction, protein degradation and vesicle trafficking Shigella, a Gram-negative pathogenic bacterium, causes human shigellosis by invading the intestinal epithelium cells after ingestion. Shigella delivers a subset of effectors into host cells via a specifically evolved type III secretion system, modulating the cellular processes and promoting infection and multiplication Shigella OspF effector exhibits phosphothreonine lyase activity and irreversibly dephosphorylates MAPKs, inhibiting the MAPK signaling pathway Shigella VirA effector inactivates Rab1 with TBC-like GAP activity, inhibiting the host autophagy-mediated defense Shigella effector, OspI, targets the host Ubc13 and deamidates Gln100 to a glutamic acid residue, leading to the disruption of TRAF6-catalyzed polyubiquitination A recent study revealed that a newly identified To generate the OspI-Ubc13 complex, we mutated the catalytic residue Cys62 of OspI 10-helix (α2). We designated the β3–β4 linking loop as L1 and the loop between α2 and α3 as L2  Similarly to the non-complexed wild-type alone structure 2, which covers 11% of the total accessible surface of OspI  and a short helix (α4) into a long, integrated helix (α4′ in the Ubc13-bound OspI)  . A compand OspI) . The actnd OspI)  and coulnd OspI) . Upon Ubnd OspI)  and S5. nd OspI) , which sin vitro pull-down assays  into host cells, specifically targets RhoA GTPase to regulate the host cytoskeleton Burkholderia pseudomallei, deamidates the host translation factor elF4A and abolishes its helicase activity, causing host translational inhibition Vibrio parahaemolyticus, acts on Rac1 and Cdc42 for mediating pathogen invasion Pasteurella multocida, causes the constitutive activation of G proteins by targeting Gαi2 and GαqB. pseudomallei, and Cif, from EPEC, deamidate ubiquitin/Nedd8 and induce host cell cycle arrest Five additional glutamine deamidases have been identified from various bacterial pathogens. CNF1, secreted by uropathogenic Interestingly, although OspI, PMT, CHBP and Cif are largely different in overall structure , each ofAn emerging concept is that bacterial pathogens employ effector proteins to modulate the host ubiquitination pathway for promoting infection and multiplication Given that a large number of papain-like proteins exist in eukaryotic cells, it is possible that some host papain-like proteins might function as glutamine deamidases to inactivate proteins in the ubiquitination pathway. A recent study revealed that the OUT-type deubiquitinating enzyme A20 regulates Ubc13 and UbcH5c to inhibit the E3 ligase activities of TRAF6, TRAF2 and cIAP1 Shigella flexneri 2a strain 301 and cloned into the pGEX-6p-2 vector. The mutation C62A of OspI was generated by using the standard PCR method. Human Ubc13 was cloned to the pET14b vector with an N-terminal 6×His tag. The human E2 enzymes, UBE2T and UBE2L3, and their mutant were cloned into an adapted pGEX-4T-1 vector with a TEV cut site and a C-terminal 10×His tag. The OspI mutations for the pull-down assays were generated from the C62A construct. The OspI mutations for the deamidation assays were generated from the wild-type OspI construct. All Mutagenesis were performed with the QuickChange Site-Directed Mutagenesis Kit (Stratagene) and all plasmids were verified by DNA sequencing.DNA for OspI was amplified from E. coli BL21 (DE3) strain (Novagen) at 22°C for 12 h after 0.4 mM IPTG induction when OD600 reached 0.6–0.8. The C62A mutant of OspI was purified with glutathione-sepharose resin followed by the PreScission protease digestion. Further purification was performed with the Hitrap Q HP ion exchange and subsequent gel-filtration chromatography . Tag-free OspI proteins including wild type and its mutants used in the deamidation assays were purified as C62A. GST-fused C62A and its mutant proteins used in His-tag pull-down assays were purified with the glutathione-sepharose resin and gel filtration chromatography. GST tags of wild-type UBE2T and UBE2L3, and their mutant in which the α1 helix is replaced by the Ubc13 sequence were removed by the TEV protease digestion after GST affinity purification. Ubc13 proteins (wild type and mutants) were purified with Ni-NTA resin and gel filtration chromatography. All purification processes were performed at 4°C.All recombinant proteins were expressed in the To prepare OspI-Ubc13 complex, purified C62A was incubated with wild-type Ubc13 at the molar ratio of 1∶1 for 12 h at 4°C in the buffer containing 50 mM Tris-HCl pH 8.0 and 150 mM NaCl. The final concentration of the mixture was 20 mg/ml. In initial crystal screening, crystals of OspI-Ubc13 complex appeared after 7 days. After optimization, diffraction-qualified crystals were obtained after 12 h in the well condition containing 0.2 M Sodium thiocyanate, 20% PEG3350 and 0.1 M HEPES pH 7.0. Crystals of OspI-Ubc13 complex were transferred into the well solution supplemented with 15% glycerol as the cryoprotectant, and then flash-cooled into the liquid nitrogen. All crystallization experiments were carried out with the hanging-drop vapor diffusion method at 16°C. The diffraction data of OspI-Ubc13 complex was collected on the BL17U1 beamline at Shanghai Synchrotron Radiation Facility (SSRF). The diffraction data was processed by using the HKL-2000 package.http://www.delanoscientific.com/). Statistics of data collection and refinement are listed in The OspI-Ubc13 complex structure was solved with the molecular replacement method by using the wild-type OspI alone structure (pdb ID: 3B21) and the Ubc13 model from TRAF-UBC13 complex (pdb ID: 3HCT) as the searching modes and the Phaser program in CCP4 1QRAS5, R9 and M13) in the α1 helix replaced with those of Ubc13 , wild-type UBE2L3, and UBE2L3 with the exposed residues (M1AAS4 and E13) in the α1 helix replaced with those of Ubc13 (M1AGLP5 and R14) were performed similarly. For the deamidation assays, 10 µg Ubc13 proteins (wild type or mutants) were incubated with 7.16 ng (0.03 µM) wild-type OspI or its mutants at 30°C for the indicated time in the reaction buffer containing 20 mM Tris-HCl pH 7.4, 100 mM NaCl and 0.5 mM DTT. The samples were separated in native PAGE, stained with Coomassie Brilliant Blue and then quantified with the program ImageJ.10 µg His-Ubc13 (wild type or mutants) was preloaded onto 5 µl Ni-NTA resin and then incubated with GST-fused C62A or its mutants at 4°C for 2.5 hours in the binding buffer containing 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 20 mM imidazole, 0.5% NP-40 and 10% Glycerol. After extensive washing, the pull-down samples were loaded into SDS-PAGE and stained with Coomassie Brilliant Blue. The pull-down assays for wild-type UBE2T, UBE2T with the surface residues  at 30°C for 1 h. Samples were analyzed by immunoblotting and using anti-IκBα antibody.Luciferase activities were determined in HEK293 cells at 12 h after co-transfection with the NF-κB reporter plasmid (10 ng), Renilla construct (5 ng), TRAF6 (100 ng) and OspI variants (50 ng) by using the dual luciferase assay kit (Promega) according to the manufacturer's instructions. For cell-free assay for NF-κB signaling, U937 cell lysate were centrifuged at 100,000 g for 1 h to get the supernatant for preparing the S100 cell extract. 70 ng TRAF6 were incubated with 12 ng OspI mutants in the U937 S100 cell extract in the reaction buffer . 200 ng E1, 500 ng Uev2, 500 ng Ubc13 and 70 ng TRAF6 were incubated with the indicated amount of C62A at 30°C for 1 h. Reactions were terminated with the SDS-PAGE sample buffer and analyzed by immunoblotting with anti-ubiquitin antibody.Ubiquitination assays were carried out in 10 µl reaction buffer  and cDNA was generated with M-MLV reverse transcriptase (Promega). Real-time PCR was performed on Applied Biosystems 7500 Fast Real-Time PCR System using the SYBR Green system (TaKaRa). The primers used for quantitative RT–PCR analysis of human IL8 and human GAPDH have been verified in previous paper OspI mutant genes were cloned into the rescue plasmid PME6032. The secretion assays were carried out by strictly following the previously described procedure OspI Shigella strains complemented with the Flag-tagged OspI mutant genes were cultured in BHI broth at 37°C. Aliquots of 2.5 ml of the Shigella cultures were washed with ice-cold PBS and resuspended in 1 ml PBS. After incubation at 37°C for 5 min, 3 µl 1% Congo red were added to the bacterial suspension, which was incubated for 10 min at 37°C and centrifuged at 14 000 g for 5 min at 4°C. The supernatant was passed through a 0.45 µm pore size filter, and trichloroacetic acid was added to the resultant supernatant (0.5 ml) at a final concentration of 6%. The secreted OspI mutant proteins present in PBS containing 0.003% Congo red were pelleted down at 14,000 g for 5 min at 4°C. Each pellet from the same number of bacteria was separated by 12% SDS–PAGE and immunoblotted with the anti-Flag antibody.To test the secretion level of OspI mutant proteins, the C-terminal Flag-tagged Figure S1OspI and Ubc13 Form a Heterodimeric Complex in Crystals. The obtained crystals of the OspI-Ubc13 mixture were washed for more than 3 times by using the well solution and denatured in SDS sample loading buffer at 95°C for 10 minutes. The sample was loaded into SDS-PAGE and stained by Cossmassie Brilliant Blue.(TIF)Click here for additional data file.Figure S2The Conformational Changes of Ubc13 upon OspI Binding. (A) Two views of the conformational changes of Ubc13 upon OspI binding. The Ubc13 structures from Ubc13 alone (pdb ID: 1JBB), Ubc13-MMS2 (pdb ID: 1J7D), TRAF6-Ubc13 (pdb ID: 3HCT) and CHIP-Ubc13 complexes (pdb ID: 2C2V) are superimposed with the Ubc13 structure in the complex with OspI. The structures are colored and labeled as indicated. The conformational changes of the α1 helix were marked as the black arrows. (B) The Gln142 wedging into the cleft between Leu4 and Arg6 in the α1 helix of Ubc13 induces the redirection of Pro5, which is highlighted with a black arrow.(TIF)Click here for additional data file.Figure S3Comparison of OspI with Structural Homologues. (A) Crystal structure of Ubc13-bound OspI. (B–I) Crystal structures of OspI homologues, AvrPphB (B), CHBP (C), Cif (D), CYLD (E), USP7 (F), USP14 (G), USP8 (H) and USP21 (I). All structures represented as cartoon are labeled and colored as indicated. The core secondary structural elements of papain-fold in each structure are highlighted in dark purple. The OspI-like structural regions in the deubiquitinating enzymes, CYLD, USP7, USP14, USP8 and USP21, are indicated in black circles (E–I). (TIF)Click here for additional data file.Figure S4Similar Expression and Secretion Levels of OspI Mutant Proteins. The C-terminal Flag-tagged OspI mutant proteins secreted from the indicated OspI-complemented Shigella strains by addition of Congo red  were analyzed by immunoblotting with the anti-Flag antibody.(TIF)Click here for additional data file.Figure S5Conformational Changes of OspI upon Ubc13 Binding and Structural Comparison of the Catalytic Residues of OspI and AvrPphB. (A) Structural superimposition between Ubc13-bound (green) and wild-type free OspI . The residues and the secondary structures undergoing conformational changes are labeled as indicated. (B) Structural superimposition of the catalytic residues of wild-type OspI with that of AvrPphB (pdb ID: 1UKF). The catalytic residues of OspI and AvrPphB are shown as sticks, colored in blue and grey, respectively. Besides the catalytic triads, Gln162 of OspI and Asn93 of AvrPphB participating in formation of the oxyanion holes for catalysis (TIF)Click here for additional data file.Figure S6The residues, Gly3, Pro5, Arg14, Arg102 and T103 of Ubc13 are Required for the Recognition and Deamidation by OspI. (A) Effects of the indicated Ubc13 mutations on OspI interactions. The interactions of GST-C62A with the indicated Ubc13 variants were examined in His-tag pull-down assays. (B) Quantitative analysis of the deamidation activities of wild-type OspI on the mutant Ubc13 proteins. The percent product (Ubc13-E100) of the mutant Ubc13 proteins formed as a function of time by wild-type OspI is plotted. All assays were repeated more than 2 times.(TIF)Click here for additional data file.Figure S7OspI, TRAF6, CHIP and OTUB1 Bind to the Same Surface Regions of Ubc13. (A–D) Complex structures of OspI-Ubc13 (A), TRAF6-Ubc13 , CHIP-Ubc13  and OTUB1-Ubc13  are represented in the same orientation of Ubc13. Ubc13 in all complex structures are colored in purple. OspI, TRAF6, CHIP and OTUB1 are colored in green, blue, red and orange, respectively, and labeled as indicated.(TIF)Click here for additional data file.Figure S8Inhibition of TRAF6-catalyzed Polyubiquitination and IκBα Phosphorylation by the OspI C62A Mutant. (A) Inhibition of TRAF6-catalyzed polyubiquitination by the C62A mutant. Ubiquitination assays were carried out in 10 µl reaction buffer . 200 ng E1, 500 ng Uev2, 500 ng Ubc13 and 70 ng TRAF6 were incubated with C62A at 30°C for 1 h. Reactions were terminated with SDS-PAGE sample buffer and analyzed by immunoblotting with anti-ubiquitin antibody. The recombinant C62A protein was titrated with four concentrations ) in the ubiquitination assays. (B) Inhibition of TRAF6-induced phosphorylation of IκBα by C62A in U937 S100 cell extract. The cell-free assays were performed as (TIF)Click here for additional data file.Figure S9Two Types of Secreted Glutamine Deamidases from Various Bacterial Pathogens. (A–B) The CNF-C type of glutamine deamidases, CNF1  and BFL1 . The catalytic residues of CNF1 and BFL1 are shown as sticks in purple and labeled as indicated. (C–F) The papain-like type of glutamine deamidases, wild-type OspI (pdb ID: 3B21), CHBP (pdb ID: 3EIR), Cif (pdb ID: 3EFY) and PMT (pdb ID: 2EBF). Surface structure representations of these glutamine deamidases are placed at the right panels. The catalytic cysteine residues of the deamidases are highlighted in purple in the left cartoon structures and their positions in the surface structures (right) are marked with black arrows. The catalytic residue Cys62 of OspI harboring three conformations is buried in the glutamine-binding pocket (C) and covalently bound by a disulfide bond formed with Cys65 (TIF)Click here for additional data file.


Background: Biological roles for intracellular dipeptidyl peptidases 8 and 9 are unknown.Results: By degradomics, 29 new in vivo substrates were identified  for DP8/DP9, including adenylate kinase 2 and calreticulin.Conclusion: These substrates indicate roles for DP8 and DP9 in metabolism and energy homeostasis.Significance: Being the first proteomics screen for DP8/DP9 substrates, unexpected new cellular roles were revealed. in vivo DP8 and DP9 substrates. In vivo roles for DP8 and DP9 in cellular metabolism and homeostasis were revealed via the identification of more than 29 candidate natural substrates and pathways affected by DP8/DP9 overexpression. Cleavage of 14 substrates was investigated in vitro; 9/14 substrates for both DP8 and DP9 were confirmed by MALDI-TOF MS, including two of high confidence, calreticulin and adenylate kinase 2. Adenylate kinase 2 plays key roles in cellular energy and nucleotide homeostasis. These results demonstrate remarkable in vivo substrate overlap between DP8/DP9, suggesting compensatory roles for these enzymes. This work provides the first global investigation into DP8 and DP9 substrates, providing a number of leads for future investigations into the biological roles and significance of DP8 and DP9 in human health and disease.Dipeptidyl peptidases (DP) 8 and 9 are homologous, cytoplasmic N-terminal post-proline-cleaving enzymes that are anti-targets for the development of DP4 (DPPIV/CD26) inhibitors for treating type II diabetes. To date, DP8 and DP9 have been implicated in immune responses and cancer biology, but their pathophysiological functions and substrate repertoire remain unknown. This study utilizes terminal amine isotopic labeling of substrates (TAILS), an N-terminal positional proteomic approach, for the discovery of  In vitro studies have demonstrated nonenzymatic roles for DP8 and DP9 in cell migration, proliferation, and apoptosis ) and DP9 (DPP9/DPRP-2) are highly conserved and ubiquitously expressed intracellular exopeptidases of the serine protease SC clan S9b subfamily (which includes (DPP4/DPIV/DPPIV/CD26 EC 3.4.14.5)) that share 61% identity at the amino acid level in humans . In vitrpoptosis . In cancpoptosis  and DP9 poptosis . Ubiquitpoptosis . Despiterocesses , 17.In vitro, both DP8 and DP9 cleave the well known DP4 substrates neuropeptide Y(1–36), glucagon-like peptide-1(7–36), glucagon-like peptide-2(1–33), and peptide YY(1–36) -α/β, interferon-γ-inducible protein, and interferon-inducible T cell α-chemoattractant, also known DP4 substrates, has been demonstrated  with DP9 proteolysis preventing major histocompatibility complex class I cell surface antigen presentation  , 19. In nstrated . Howeverrs cells . To dateentation . Besidesin vivo in inflamed mouse skin , to identify the substrate degradome of DP8 and DP9. TAILS is focused around the isolation of protein and peptide N termini for proteomic identification of neo-N termini resulting from proteolytic events . This meuse skin . Stable All chemicals were purchased from Sigma unless stated otherwise.(882 aa) and DP9 (where aa is amino acid) or catalytically inactive mutants DP8(S739A) and DP9(S729A) (SKOV3 cells were maintained  with G419(S729A) . Clonal 7) pooled from three flasks were pelleted by centrifugation , resuspended in homogenization buffer  containing protease inhibitors , and then gently lysed by nitrogen cavitation  using a Parr Cell Disruptor (Parr Instrument Co.). Lysates were clarified by centrifugation , and then supernatants were subjected to ultracentrifugation  for separation of membrane and cytosolic fractions. Cytosolic fractions were further clarified by additional ultracentrifugation . Protein concentrations were determined by micro-Bradford assay (Bio-Rad). Aliquots were made and stored at −70 °C until TAILS proteome analysis.Cells were grown to confluence in three T175 flasks, washed with PBS  to remove serum proteins, and then incubated for 3 h in 15 ml of phenol red-free, serum-free DMEM (Invitrogen). Cells were washed with ice-cold PBS, detached with 0.2% (w/v) EDTA/PBS, and then resuspended in ice-cold PBS. Cells  from enzyme-active and catalytically inactive cell lines (both DP8 and DP9) were concentrated and purified by 12%  TCA precipitation. Protein denaturation, reduction, and alkylation of cysteine residues was performed as described previously . Residual formaldehyde was quenched; then heavy and light isotopically labeled samples were combined, concentrated, and purified by acetone precipitation and tryptically digested, and then labeled peptides were enriched by a negative selection step using a dendritic polyglycerol aldehyde polymer as described . Three biological replicates for both DP8 and DP9 were subjected to the TAILS process in three independent experiments.TAILS was performed using dimethylation labeling of primary amines of protein N termini and ϵ-amino acids of lysine side chains as described . In brieeviously . Sampleseviously   were desalted using reverse phase-solid phase extraction via Sep-Pak® C18 column  according to the manufacturer's recommendations.Desalting and pre-fractionation of samples (∼100 μg) by strong cation exchange-HPLC, prior to LC-MS/MS analysis, was performed as described . N-termi−1) coupled to a quadrupole time-of-flight QStar XL hybrid electrospray ionization mass spectrometer  or a QStar Pulsar mass spectrometer . Samples were loaded, eluted, and separated on the C18 column, and MS data were acquired automatically using Analyst QS version 1.1 software  as described )  against the human International Protein Index (IPI) database . MASCOT searches of MS data were performed separately for heavy- and light-labeled peptides. Searches were performed using the following modifications: fixed carbamidomethylation of cysteines (+57.021 Da (Cys)), fixed heavy lysine (+34.0631 Da (Lys)), or light lysine (+28.0311 Da (Lys)); variable methionine oxidation (+15.995 Da (Met)), and fixed and variable modifications of N termini with heavy formaldehyde (+34.0641 Da (N termini)), light formaldehyde (+28.0311 Da (N termini)), and acetylation (+42.011 Da (N termini)). The additional search criteria used were as follows: semi-ArgC cleavage specificity with up to three missed cleavages; a monoisotopic mass error window for the parent ion of 0.4 to 0.6 Da; peptide mass tolerance of 0.4 Da for MS/MS fragment ions; and the scoring scheme ESI-QUAD-TOF. Allowed peptide charge states were 1(MinGW))  with Pep(MinGW))  sensitiv(MinGW)) .2-P2-P1-P1′-P2′-). Because of this strict canonical specificity, datasets were also manually parsed to identify all peptides with a cleaved or noncleaved Pro residue in what would be the P1 position of a DP8/DP9 substrate. Pairs of peptides that differed in length by two amino acids at their N termini, but did not contain a Pro in P1, were also selected as candidate substrates.DP8 and DP9 are exopeptidases with a strong preference for cleavage of N-terminal dipeptides mainly after a Pro residue in the P1 position  via DP8/DP9-affected pathways were identified from quantitative analysis of heavy/light (protease/control) isotope-labeled peptide abundance ratios. Only peptides identified with ≥95% confidence (PeptideProphet) were used for quantitative analysis. ASAPRatios were normalized by recentering the raw datasets around a median of one and then applying a natural logarithm transformation. This enabled the exclusion of obvious outliers and established a normal range for determining significance by using means and S.D. of the normalized data to determine 90% confidence intervals of the nontransformed data. This was achieved using the R statistical package. High and low ratio peptides were considered to be either substrates of DP8/DP9 or derived from proteins that were either differentially expressed or processed by an alternative protease to DP8/DP9.database for annotation, visualization and integrated discovery (DAVID), version 6.7 (UniProt Knowledge Base (UniProtKB) accession numbers were mapped to protein IPI numbers. Functional annotation and biological pathway information were obtained from UniProtKB entries or by using the sion 6.7   as described previously  and DP9(892aa) were expressed and purified as described previously (m) were incubated with 1.7 milliunits of recombinant DP8 or DP9 in assay buffer: 50 mm Tris, 100 mm NaCl, pH 8.0, at 37 °C for up to 24 h. Calreticulin and adenylate kinase 2 were also incubated in assay buffer alone or with DP8/DP9 in the presence of 10 μm of the DP inhibitor, Val-Boro-Pro  with approval from DARA BioSciences Inc., Raleigh, NC). At time points of 0, 1, 4, and 24 h, 5 μl of each reaction was removed and stopped by the addition of 1% TFA. Peptides were desalted and cleaned by OMIX® C18 tip . Eluates were mixed 1:1 with α-cyano-3-hydroxycinnamic acid matrix solution (1% (w/v) α-cyano-3-hydroxycinnamic acid, 49.5% acetonitrile, 49.5% ethanol, 0.001% TFA) and spotted onto a standard stainless steel MALDI sample plate, and then masses of intact peptides and DP8/DP9 cleavage products were obtained by MALDI-TOF MS analysis performed on a Waters Micromass® M@ALDI  or a Bruker Autoflex III MALDI MS/MS . All cleavage experiments with MALDI-TOF analysis were performed in triplicate. MS spectra were processed using MassLynx 4.0 (Waters) software package.Recombinant human DP8eviously , 32. N-teviously  were synAll LC-MS/MS data (.wiff files) associated with this study may be downloaded from ProteomeCommons.org Tranche using the following hash: YRRnIo2jM9SRJ7vSBk7OG65e/3cVbIQ7ksECUIT97Doxwfg1YAGWJ5eHBqzMXog/IQFcGWzlS08gVKP/RqN+E1KLoVwAAAAAAAAYRg.A). DP8- and DP9-EGFP proteins were visualized by immunoblot (∼118 kDa in mobility) confirming the heterologous expression of active and inactive proteases (B). All cell lines were found to express basal levels of endogenous DP8 and DP9 protein (∼98 kDa in mobility) (B) that may contribute to background proteolysis and affect the discovery of substrates by TAILS. DP activity increased in the wild-type DP8- and DP9-transfected SKOV3 cells when compared with the catalytically inactive DP8(S739A), DP9(S729A), and vector-transfected controls (C). Enzymatic assays of fractionated clonal cell lines confirmed that this increase in DP activity is due to the overexpression of cytosolic DP8/DP9 (D and E).SKOV3 cells were stably transfected with constructs encoding enzymes DP8 and DP9, with a size of 882 and 863 residues, respectively, including the N-terminal β-propeller and C-terminal α/β-hydrolase domains. Clonal cell lines for both DP8 and DP9 were selected for equal expression of the active and inactive version of each enzyme in fusion with the fluorescent EGFP protein A. DP8- aroteases B. All ceobility) B that macontrols C. Enzyma DP8/DP9 , D and EA and B). Each TAILS experiment yielded 200–400 unique proteins that were identified from more than one N-terminal peptide occurrence, i.e. peptide/spectra (A and B). The union of each DP8 TAILS experiment yielded 59 unique proteins (A), whereas the union of each DP9 TAILS experiment yielded 92 unique proteins (B). From all three experiments, a total of 543 unique proteins were identified for DP8, and 597 were identified for DP9, of which 394 proteins were common to both DP8 and DP9 (C).TAILS was performed on three isolations of cytoplasmic proteomes from both wild-type and catalytically inactive DP8- and DP9-stable SKOV3 cell lines. The total number of unique peptides and proteins identified within each experiment was determined , A and B/spectra , A and Bproteins A, whereaproteins B. From a and DP9 C.supplemental Figs. S2 and S3). Functional annotation and pathway mapping revealed the involvement of candidate substrates in biochemical pathways relating to lysosomal processes, carbohydrate metabolism, and nucleotide metabolism and synthesis .A total of 22 and 21 proteins were identified by parsing for potential DP8 and DP9 substrates, respectively. Of these, 8/22 and 7/21 candidate substrates were uniquely identified for DP8 and DP9, respectively . Most casupplemental Table S10). Of these, 17/37 and 10/55 proteins were identified as being candidate substrates of DP8 and DP9, respectively (e.g. peptides located at mature N termini. All other proteins identified (20 for DP8 and 45 for DP9) are listed in supplemental Table S10. Evidence of non-DP8/DP9 N- and C-terminal truncation of proteins was found , demonstrating that non-DP proteolytic pathways are altered by increasing DP8/DP9 activity. Interestingly, approximately half of the proteins identified for DP8 are mitochondrial, potentially occurring during mitochondrial turnover . Functional annotation and pathway mapping revealed that many of the identified proteins play key roles in carbohydrate, nucleotide, and protein metabolism .Quantitative analysis was only performed on datasets derived from the first two TAILS experiments for both DP8 and DP9 due to the poor recovery of labeled peptides from experiment 3 (data not shown). In total, 37 proteins were identified as potential DP8 substrates or DP8 pathway-affected proteins, and 55 proteins were identified as potential DP9 substrates or DP9 pathway-affected proteins; only 10 of these proteins were common to both DP8 and DP9  followed by an Ala in the P2 position (C and D). The frequency of residues in the P1 and P2 position for the remaining candidate substrates was variable for both DP8 and DP9 . In total, cleavage of 9/14 substrates by both DP8 and DP9 was confirmed. No unique cleavage was identified for DP8 or DP9 demonstrating similar enzyme specificity and large substrate overlap between these enzymes.DP8 and DP9 cleave A) demonstrating a probable higher affinity of recombinant DP9 for this substrate in vitro. Recombinant DP8 cleaved N-terminal peptides of adenylate kinase 2 and calreticulin with similar kinetics, whereas DP9 displayed a faster rate of cleavage for the calreticulin N-terminal peptide compared with adenylate kinase 2 (A and 6A). Specificity of DP8/DP9 cleavage was confirmed by performing peptide catalysis reactions in the presence of the nonselective DP inhibitor, Val-Boro-Pro  (B and 6B). Co-localization of DP8- and DP9-EGFP with both adenylate kinase 2 and calreticulin was demonstrated by confocal microscopy (A) confirming physical proximity between protease and substrate. No difference in the localization of either adenylate kinase 2 or calreticulin was detected between the active DP8- and DP9-EGFP cell lines compared with the catalytically inactive DP8(S739A)-EGFP and DP9(S729A)-EGFP cells (data not shown). Immunoblots demonstrated that full-length adenylate kinase 2 and calreticulin are not altered in their expression levels in the DP8- or DP9-EGFP SKOV3 cells lines (B) indicating that DP8/DP9 proteolysis is unlikely to alter the stability of these two proteins.In comparison with DP8, DP9 more readily cleaved the N-terminal peptide of adenylate kinase 2 A demonstkinase 2 A and 6A.abostat) B and 6B.croscopy A confirmls lines B indicatin vitro assays  and DP9 (45 proteins) enzyme activity. Many of the proteins identified by quantitative analysis and those confirmed as DP8/DP9 substrates are involved in regulating cellular metabolism and energy homeostasis. Thus, this study for the first time reveals potential roles for DP8 and DP9 in cellular metabolic pathways, including glycolysis, gluconeogenesis, fatty acid metabolism, and nucleotide metabolism/biosynthesis. In addition, the involvement of calreticulin in the antigen processing and presentation is consistent with a role for DP9 proteolysis in preventing cell surface presentation of the major histocompatibility complex class I antigen  or has been removed. These findings suggest that in the DP8 cell lines, there may be increased mitochondrial turnover/degradation resulting in leakage of proteins from autophagosomes  suggestin vivo of a protease with its substrate is required for cleavage. By demonstrating this by co-localization studies, it supports the in vivo relevance of adenylate kinase 2 and calreticulin as natural substrates of DP8 and DP9. Although determination of the biological importance of the cleavage of adenylate kinase 2 and calreticulin by DP8/DP9 is difficult due to the pleiotropic effects of both adenylate kinase 2 and calreticulin .The N-end rule specifies that the stability of a protein can be affected by the identity of its N-terminal residue . AlthougFig. 7in vivo substrates for both DP8 and DP9, both previously enigmatic proteases with only one in vivo substrate known. Furthermore, this work has highlighted roles for both of these proteases in cellular energy metabolism and homeostasis. Importantly, adenylate kinase 2 and calreticulin were identified and validated as substrates of both DP8 and DP9. Adenylate kinase 2 plays an important role in maintaining cellular energy homeostasis, and thus DP8/DP9 proteolysis may contribute to regulating cellular energy homeostasis through adenylate kinase 2. TAILS is a powerful proteomic approach for the discovery of protease substrates. The recent use of TAILS in in vivo analysis of tissue samples opens the possibility for similar analysis of DPs in the future. For the first time, we have applied TAILS to a cytoplasmic proteome and found the approach to be particularly well suited to the discovery of N-terminal substrates of the exopeptidases DP8 and DP9. This proteomic investigation has identified some unique but largely overlapping roles and substrates for DP8 and DP9, thus paving the way for ongoing investigations into the fundamental roles of DP8 and DP9 in cellular metabolism and homeostasis.In conclusion, this study has identified and validated a number of biologically important candidate


P = 0.001) with abnormal metaphases (P < 0.001) were observed and a significantly increased rate of apoptosis was found (P = 0.006) compared to full-length RASSF1A. Although the association with microtubules and their stabilization was unaffected, mitotic spindle formation was altered by deletion of the SARAH domain of RASSF1A. In summary, our results suggest that the SARAH domain plays an important role in regulating the function of RASSF1A.The Ras association domain family 1A (RASSF1A) tumor suppressor encodes a Sav-RASSF-Hpo domain (SARAH), which is an interaction domain characterized by hWW45 (dSAV) and MST1/2 (dHpo). In our study, the interaction between RASSF1A and RASSF1C with MST1 and MST2 was demonstrated and it was shown that this interaction depends on the SARAH domain. SARAH domain-deleted RASSF1A had a similar growth-reducing effect as full-length RASSF1A and inhibited anchorage independent growth of the lung cancer cell lines A549 significantly. In cancer cells expressing the SARAH deleted form of RASSF1A, reduced mitotic rates ( RASSF1) was identified on chromosome 3p21.3, a region frequently deleted in cancer [RASSF1, termed RASSF1A and RASSF1C, which are transcribed from different CpG island promoters [RASSF1A is often hypermethylated in cancer, whereas the promoter region of RASSF1C is never methylated [RASSF1A harbors a diacyl glycerol binding domain [RASSF1A encodes a tumor suppressor gene, which reduces tumor growth in vivo and in vitro [Rassf1a in mice significantly increased spontaneous and induced tumorigenesis [The Ras association domain family 1 gene  and mediates proapoptotic signals through binding of the mammalian sterile 20-like kinase 1 and 2 (MST1 and MST2) –19. MoreDrosophila leads to cell cycle arrest and apoptosis [In the C-terminal part of RASSF1A and RASSF1C, a protein-protein interaction domain called SARAH (Sav/RASSF/Hpo) has been determined . The SARpoptosis –30.Drosophila orthologue of the human RASSF proteins restricts Hpo activity by competing with Sav for binding to Hpo [It was shown that the single g to Hpo . Praskovg to Hpo  and moreg to Hpo . MST1 hag to Hpo . The cleg to Hpo , 35. H2Bg to Hpo . Followig to Hpo . The cleg to Hpo  by chromg to Hpo .Both RASSF1A and WW45 activate MST2 by promoting its autophosphorylation . MoreoveTo gain new insights into the tumor suppressor function of RASSF1A, we deleted its SARAH domain and analyzed its altered function. Deletion of the SARAH domain resulted in a decreased colony formation of tumor cells. During mitosis, abnormal spindle formation was observed. We demonstrate that the interaction of RASSF1A and RASSF1C with MST1 and MST2 depends on the SARAH domain. Deregulation of the SARAH domain may contribute to altered proapoptotic and mitotic signaling of RASSF1A.The localization experiments and the protein expression experiments were performed in HEK293 and COS7 cells . For stable transfection, the lung cancer cell line A549 (ATCC) was used. A549 cells harbor epigenetic silenced RASSF1A, but express RASSF1C [μg of each plasmid using the PEG/LiAc method. The interaction analysis was carried out on SD minimal medium plates without adenine and histidine and the transformation efficiency was controlled on SD plates with adenine and histidine. The strength of interaction was investigated by quantification of the expression of the β-galactosidase reporter gene with o-nitrophenyl-β-D-galactopyranoside (ONPG) as substrate at 420 nm.The Matchmaker Two-hybrid system  was utilized. cDNAs of RASSF1A and RASSF1C were described previously , 45. MutMST1 and MST2 were cloned into the vector pCMV-Tag1  and/or in the vector pEBG. To investigate the interaction of specific RASSF1 forms, MST1 and MST2, cotransfections  were performed in HEK293 cells. Plasmids (pEBG and pCMV-Tag 1) were used, that express GST-Flag-RASSF1A, GST-Flag-RASSF1C, GST-Flag-RASSF1AΔSARAH, GST-Flag-RASSF1CΔSARAH, and GST or Flag-MST1, Flag-MST2, and Flag-WW45 [μM staurosporine for 3 h or transfected with 10 μg of constructs with Turbofect for 36 h . Total protein was isolated using Flag-lysis buffer, samples were denatured with Laemmli-buffer, separated in 10% SDS-PAGE, and blotted onto PVDF membranes. First antibodies are: anti-GAPDH , anti-P-MST1 (Thr183)/MST2 (Thr180) , anti-Flag , and secondary antibodies are HRP coupled . ECL  was used for detection with Versadoc .A549 were treated with 3 RASSF1A, RASSF1AΔSARAH, and RASSF1C were cloned into the vector pCMV-Tag1 . The lung cancer cell line A549 was transfected using Lipofectamine 2000 . Colonies were selected under 1 mg/mL Geneticin  in DMEM and clones were picked after 4 weeks. Expression of Flag-RASSF1 was confirmed by RT-PCR using the FLAG-specific primer (5′-TGGATTACAAGGATGACGACG) and RASSF1-specific primer L27111 (5′-TCCTGCAAGGAGGGTGGCTTC). PCR products were analyzed on a 2% Tris-borate EDTA agarose gel.μL of 5 mg/mL INT and the size of 25 colonies was determined with MetaVue . Growth curves of stable transfected clones were analyzed by seeding 150,000 cells in triplicates in 6-well plates. Every 24 hours, cells were counted using a Neubauer counting chamber. In order to investigate the proliferation in soft agar, stable transfected cells were seeded in 0.3% agarose. Experiments were performed in duplicates with 5,000 cells per plate under selection with 1 mg/mL Geneticin. Colony size was measured after 4 weeks with a microscope . Therefore, colonies were stained with 400 α-tubulin  antibodies and Alexa Fluor goat anti-mouse  antibodies to show the colocalization with the microtubules and spindle poles, respectively. Nuclei of the cells were visualized by staining with DAPI . Cells in mitoses were scored by microscopy (ZEISS Axioplan 2). Cells with highly condensed chromosomes and spindle structures were classified as mitotic cells. For microtubule stability analysis, the cells were treated one day after transfection for one hour with 20 μM nocodazole, fixed, and stained with DAPI and anti-α-tubulin antibodies. YFP constructs are shown in green color.RASSF1A and RASSF1C were cloned into the fluorescence vector pEYFP-C2 . The deletion of the SARAH domain of RASSF1A and RASSF1C was accomplished by the QuickChange XL Site-Directed Mutagenesis Kit  with the upper SARAH deletion primer 5′-AGGAAAATGACTCTGGGCCCCTTGGGTGACCTCT and the complementary lower primer. After transient transfection into HEK293 cells with Lipofectamine 2000 , the localization of YFP-RASSF1A, YFP-RASSF1AΔS, YFP-RASSF1C, and the vector control were investigated with a fluorescence microscope. Cells were co-stained with anti-In Situ Cell Death Detection Kit, TMR red . The nuclei were costained with DAPI  solution. Slides were quantified with a fluorescence microscope (ZEISS Axioplan 2). Yellow fluorescence expressing cells (500) were counted and the rate of apoptotic cells (red fluorescence) was calculated. All experiments were done in triplicates.The lung cancer cell line A549 was transiently transfected with different RASSF1A constructs tagged with yellow fluorescence (pEYFP-C2) using Lipofectamine 2000 . After two days, the transfected cells were harvested and centrifuged on a slide. After fixation with formaldehyde, a TUNEL staining was performed with the P < 0.05 was considered as significant.All statistical evaluations were performed using the SPSS 12.0 Software . A Probability of In silico analysis of RASSF1A (340 aa) and RASSF1C (270 aa) by PROSITE (www.expasy.org) revealed the presence of a Sav-RASSF-Hpo (SARAH) domain at their C-terminus   assay  . To gain, resp.) . Both RAG) assay . The intG) assay . There wRASSF1-specific forms was confirmed by RT-PCR . Two RASSF1AΔS expressing clones showed a similar growth to RASSF1A expressing cells. RASSF1AΔS clone1 and clone2 had a significant reduction of growth at 96 h compared to RASSF1C  and controls . Subsequently, the anchorage independent growth was determined in soft agar experiments  compared to RASSF1A  and control .Growth effects of the SARAH domain of RASSF1 were investigated in stable transfected lung cancer cells. The lung cancer cell line A549 was transfected with RASSF1A, RASSF1C, and RASSF1AΔS, and the control vector (pCMV-Tag1) and the growth of these cells were evaluated . A549 hay RT-PCR . Subsequ Figures . In thesP = 0.001) reduced to 3.4% compared to 8.6% in RASSF1A transfected HEK293 cells (P < 0.001) of mitoses in RASSF1AΔS expressing cells were abnormal (multi- or monopolar) compared to RASSF1A transfected cells, where only 0.6% of abnormal mitosis were counted (We transfected yellow fluorescent protein-tagged RASSF1A and RASSF1AΔSARAH (RASSF1AΔS) into HEK293 and COS7 cells and the localization was determined by fluorescence microscopy . RASSF1A93 cells . The mit counted .μM nocodazole for one hour  higher, when the cells expressed RASSF1A (28%) in comparison to RASSF1C (16%) and the YFP control  and RASSF1A  , 32. RASIt was demonstrated that RASSF1A colocalizes with the microtubule network during interphase and is found at the spindles and centrosomes during mitosis , 23. RASRASSF1A was also reported to interact with MAP1B (microtubule-associated protein 1B) and C19ORF5 (chromosome 19 open reading frames 5), both microtubule-associated proteins , 49, 53.In summary, our data indicate that RASSF1A is important for several signals, which are frequently altered in tumorigenesis, including apoptosis, mitotic spindle organization, and proliferation. Our data suggest that other domains  than SARAH also significantly contribute to the proapoptotic and antiproliferative function of RASSF1A. Specific interaction of RASSF1A with MST/LATS and other binding partners  might be important in the regulation of proliferation and apoptosis and in the formation of normal mitotic spindles and processes of dividing chromosomes by RASSF1A.


CDKN2A and CDK4 and one low-penetrance gene, MC1R, are well-defined genetic risk factors for melanoma. MITF has been recently identified as a novel intermediate risk melanoma-predisposing gene.Concomitant primary cutaneous melanoma in monozygotic twins has been reported in only two pairs but in neither of them genetic analysis was performed. Two high-penetrance susceptibility genes, CDKN2A, exon 2 of CDK4, the entire open reading frame of MC1R and the recently described MITF c.952 G > A (p.Glu318Lys) variant were investigated by direct sequencing. Sequencing analysis of the high-penetrance susceptibility genes showed no changes in CDKN2A and in exon 2 of the CDK4 gene. Both patients were heterozygous for the same CDKN2A UTR c.*29C > G variant. Interestingly, the same two heterozygous variants of the MC1R were identified in both twins: the c.451C > T (p.Arg151Cys) and the c.456C > A (p.Tyr152*) variants. Neither patient showed the c.952 G > A (p.Glu318Lys) substitution in the MITF gene.We describe the extraordinary occurrence of a primary cutaneous invasive melanoma in two 44-year-old identical, female twins, on the same body site within 30 days of each other and report for the first time the genetic analysis of melanoma susceptibility genes in both twins. Data on characteristics of the twins were collected through a standardized questionnaire and skin examination. Exons 1α, 1β, 2 and 3 of MC1R variants in our identical twins in the absence of CDKN2A and CDK4 mutations highlights the contribution of low penetrance genes, such as MC1R, in melanoma susceptibility.Identification of two high-risk  CDKN2A  and CDK4  and one low-penetrance gene, MC1R , have been identified so far as genetic risk factors for melanoma [CDKN2A is the major melanoma susceptibility gene, mutated in approximately 40 % of melanoma-prone families with at least three affected members. Predisposing mutations in CDK4 have been identified in a limited number of melanoma families worldwide. Allelic variants in the MC1R gene, important in the pigmentation process, have been associated with increased melanoma risk, with the strongest effect observed for red hair color (RHC) variants . Recently, whole genome sequencing lead to the identification of MITF  as a novel melanoma-predisposing gene with the p.Glu318Lys variant defined as an intermediate risk variant [Genetic, phenotypic and environmental factors are known to affect melanoma risk. Two high-penetrance susceptibility genes, We describe the extraordinary occurrence of a primary cutaneous invasive melanoma in two 44-year-old female, identical twins, on the same body site within 30 days of each other and report for the first time genetic analysis of melanoma susceptibility genes in both twins.A pair of female monozygotic twins who were included in a surveillance program at our Pigmented Lesion Clinic for the presence of multiple atypical nevi and at risk phenotype Figure A had beeCDKN2A (NCBI accession NM_000077.3), exon 2 of CDK4 (NCBI accession NM_000075.3) and the entire open reading frame of MC1R (NCBI accession NM_002386.3) were amplified using PCR and directly sequenced on ABI Prism® 310 Genetic Analyzer  as described previously [MITF c.952 G > A (p.Glu318Lys) variant (NCBI accession NM_000248.3) was screened by direct sequencing of a PCR-amplified 589 amplicon using the following primers: forward, 5'-TACATTCCCTCTGGTATTGT-3'; reverse, 5'-AGCAGTTTGTGCGAATGCA-3'. Primers for PCR amplification were designed from the MITF genomic nucleotide sequence (NCBI accession NG_011631.1) and were the followings: forward, 5'-TACATTCCCTCTGGTATTGT-3'; reverse 5'-AGCAGTTTGTGCGAATGCA-3'. An amplicon of 589 bp was obtained by standard PCR using 1.25 U of AmpliTaq Gold® 360 (Applied Biosystems) in a 50-μl volume, containing the 1X reaction buffer provided by the manufacturer, 1.6 mM of MgCl2, 200 μM of each deoxynucleoside triphosphate, 0.2 μM of each primer and 100 ng genomic DNA template. Five per cent dimethyl sulfoxide was added to the reaction solution. PCR amplification conditions were as follows: 95°C for 7 min, 35 cycles of 94°C for 1 min, 52.2°C for 1 min, and 72°C for 1 min, followed by a final extension step at 72°C for 7 min. Written informed consent was obtained from our patients under an Institutional Review Board-approved protocol.Genomic DNA was isolated from whole blood of both patients using QIAamp® DNA Blood kit . Exons 1α, 1β, 2 and 3, including the exon-intron boundaries of CDKN2A, although both patients were heterozygous for the same UTR c.*29C > G variant. No mutations were detected in exon 2 of the CDK4 gene. Interestingly, sequencing of the MC1R identified the same two heterozygous variants in both twins: the c.451C > T (p.Arg151Cys) and the c.456C > A (p.Tyr152*) variants  and the c.456C > A (p.Tyr152*), was detected in our twins. The p.Arg151Cys has been shown to confer the highest melanoma risk with summary estimates ranging from 1.78 (95%CI 1.45–2.20) to 1.93 (95%CI 1.54–2.41) in two recent meta-analyses [MC1R-associated melanoma risk has been shown to increase with the number of variants in the genotype supporting the MC1R-associated susceptibility in our patients carrying two nonfunctional MC1R variants. Finally, carriers of MC1R RHC variants have been recently suggested to develop melanomas lacking significant pigmentation [variants -12. A doanalyses ,11. We panalyses . The nonanalyses ,15. Inteentation ,17 as alMC1R with melanoma, other low-penetrance melanoma risk alleles have been described in genes related to pigmentation, nevus count, immune responses, DNA repair, metabolism and the vitamin D receptor [In addition to the well-defined association of receptor . In thisMC1R variants in our identical twins in the absence of CDKN2A and CDK4 mutations highlights the contribution of low penetrance genes, such as MC1R, in melanoma susceptibility, although additional known or as yet unknown genetic risk factors might be involved.The concordance for cutaneous melanoma as well as the congruence for its location and identity of the age of onset in monozygotic twins support the role of genetic rather than environmental factors in the development of melanoma. Detection of two high-risk Written informed consent was obtained from both patients for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.CDKN2A: Cyclin-dependent kinase inhibitor 2A; CDK4: Cyclin-dependent kinase 4; MC1R: Melanocortin-1 receptor; MITF: Microphtalmia-associated transcription factor; RHC: Red hair color; SSM: Superficial spreading melanoma.The authors declare that they have no competing interests.All authors have read and approved the final manuscript. CP carried out the molecular genetic studies, participated in the analysis and interpretation of data and drafted the manuscript; MCF was involved in conception and design of the study, supervised the genetic analysis and participated in the interpretation of data and drafting the manuscript; MS collected the clinical data and participated in drafting the manuscript; KP diagnosed the patients, was involved in conception and design of the study and revised the manuscript critically.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/13/81/prepub


Guanine quadruplex (G4 DNA) is a four-stranded structure that contributes to genome instability and site-specific recombination. G4 DNA folds from sequences containing tandemly repetitive guanines, sequence motifs that are found throughout prokaryote and eukaryote genomes. While some cellular activities have been identified with binding or processing G4 DNA, the factors and pathways governing G4 DNA metabolism are largely undefined. Highly conserved mismatch repair factors have emerged as potential G4-responding complexes because, in addition to initiating heteroduplex correction, the human homologs bind non-B form DNA with high affinity. Moreover, the MutS homologs across species have the capacity to recognize a diverse range of DNA pairing variations and damage, suggesting a conserved ability to bind non-B form DNA.E. coli MutS and a heteroduplex recognition mutant, MutS F36A, were capable of recognizing and responding to G4 DNA structures. We find by mobility shift assay that E. coli MutS binds to G4 DNA with high affinity better than binding to G-T heteroduplexes. In the same assay, MutS F36A failed to recognize G-T mismatched oligonucleotides, as expected, but retained an ability to bind to G4 DNA. Association with G4 DNA by MutS is not likely to activate the mismatch repair pathway because nucleotide binding did not promote release of MutS or MutS F36A from G4 DNA as it does for heteroduplexes. G4 recognition activities occur under physiological conditions, and we find that M13 phage harboring G4-capable DNA poorly infected a MutS deficient strain of E. coli compared to M13mp18, suggesting functional roles for mismatch repair factors in the cellular response to unstable genomic elements.Here, we asked if E. coli MutS has a binding activity specific for non-B form G4 DNA, but such binding appears independent of canonical heteroduplex repair activation.Taken together, our findings demonstrate that  E. coli MutS and the eukaryotic MutS homologs, MSH2/MSH6 (MutSα), have the extraordinary capacity to recognize and initiate repair of unrelated types of DNA damage, such as UV photoproducts , hum, humNeisbination , and probination  genomes.bination .c-MYC gene and the immunoglobulin switch regions occur at G4 sequences , DNA briewed in ,28).E. coli MutS recognizes G4 DNA structures. Further, we ask if such binding is affiliated with canonical mismatch repair. With mobility shift assays, we identify G4 DNA as a high-affinity substrate for E. coli MutS. Interestingly, we also find that G4 DNA and heteroduplex binding constitute separable MutS functions. Our results argue that the genome instability and recombination associated with G4 DNA may involve repair-independent functions of highly conserved mismatch repair proteins.Evidence has emerged that connects the highly conserved mismatch repair proteins with cellular responses to G4 DNA. Human MSH2/MSH6 (MutSα) has been directly visualized by electron microscopy bound to the G-rich and G4 capable strand of expressed immunoglobulin switch regions, enriched in chromatin IPs of the transcriptionally activated switch regions, and shown to specifically recognize G4 oligonucleotide DNA via mobility shift assays . Also inE. coli[+ ion but not Li+ ion  established this highly conserved residue as critical for heteroduplex recognition and mismatch correction . We ther Winkler ) with alD values for G4 binding were essentially unchanged in the presence and absence of up to 1 mM ATPγS . Addition of either ATP or slowly hydrolyzing analogs to binding assays interferes with specific complex formation between MutS and heteroduplexes -46 refle4 Figure B which mE. coli MutS will respond to non-B form DNA structures, independent of repair activation, in vivo. While the genome of E. coli is mostly coding and generally non-repetitive compared to higher eukaryotes, the genomes of bacteriophage are characteristically non-B form. In particular, filamentous phages, such as M13, infect E. coli and replicate by rolling-circle replication using a circular duplex intermediate. By nature of a single-stranded circular genome, these viruses present a DNA molecule that is already non-B form, although we have increased the structure forming potential of M13 even further by introducing the G-rich Sγ3 fragment , or JW1 expressing MutS F36A (JW1-MutS F36A) to ask if MutS and mismatch repair are needed for infection efficiency when M13 harbors this additional non-viral DNA sequence. Phage infection success for MutS defective strain was measured by counting plaques normalizing to an isogenic MutS proficient strain.To ask if there are repair-independent and structure-associated roles for MutS in the cell, we infected several Successful M13 phage infection of bacteria results in the formation of a plaque on LB agar plates. We first titered phage stocks of M13-G and M13mp18 using NM522 and defined the volume required to generate ~100 plaques/plate for each stock. Using identical volumes and conditions, M13mp18 infection showed nearly equal plaques/plate for both NM522 and JW1 . RegardNeisseria recombination [Genomic regions rich in repetitive guanine are common in higher eukaryotes, but rarer in prokaryotes. G-rich introns located at the 5’ end of expressed loci have been correlated with transcriptional pausing, providing a gene regulation rational for why genetically unstable G-rich DNA may be retained in mammals . Howeverbination , few recbination . In otheE. coli MutS has such a robust G4 binding ability. We consider it unlikely that the high affinity G4-binding activity of E. coli MutS . ATPγS was purchased from MP biomedicals. The mutS:Tn10 allele from FC1124  was transferred to NM522 (NEB) by P1 transduction to create JW1 then tested for mutator phenotype by nalidixic acid screening. Both PCR and DNA sequencing confirmed MutS interruption and the absence of full length MutS gene in JW1.All oligonucleotides . The identical Sγ3 sequence was transferred from pCR 2.1-G and pCR2.1-C to the multiple cloning site (XbaI/HindIII fragment) of M13mp18 for phage infections assays.Phagemids containing G4 capable sequences were created using a 564 bp sequence PCR amplified from genomic template (Ramos B cells) with primers specific for the intronic human Sγ3 sequence  as described previously [Over expression and purification of MutS proteins followed previously published protocols using an original MutS expression clone pTX412 created by Malcom Winkler  and proveviously . Purifie32P end-labeled G4 or G-T mismatched radiolabeled oligonucleotide in the presence of 200 fmols of homoduplex competitor DNA. G-T heteroduplex and homoduplex DNA were created by annealing HPLC purified oligonucleotides (FisherOperon), which were DNA substrates previously designed by the Hays laboratory for MutS homolog mobility shift assays [2, 1 mM DTT, 50 μg/ml BSA, 10 mM KCl and the relevant 32P 5’ end labeled substrate. Sucrose was added to 15% and reactions resolved by 6% native PAGE in 0.5X TBE with 5 mM MgCl2 and 10 mM KCl. Gels were then transferred to Whatman paper, dried, imaged by phosphorimager Storm 840  and bands quantified with ImageQuant software . All mobility shifts followed the above procedure, with the only exception being the addition of 1 mM ATPγS , as indicated. Apparent KD was determined as previously described [2D. TP-G4 characteristically shows a more diffuse band compared to heteroduplex oligonucleotide [Binding assays used purified MutS and 10-20 fmols of 5’ t assays  at an OD of 0.5 with a single plaque, followed by culturing in 25 ml of LB overnight. Phage were concentrated by standard 2.5 M NaCl/20% PEG precipitation protocol (NEB) and then resuspended in 800 μl of TE buffer. Titers were determined by serial dilution and counting plaques. Appropriate volumes of either M13mp18 or M13-G were added to experimental NM522 to correspond to approximately 100 plaques for each plate. Plaque-forming efficiency for mutS::Tn10 NM522 (JW1) for M13mp18 or M13-G is presented relative to plaque-forming efficiency for NM522 (isogenic and MutS proficient). JW1 cells were transformed with MutS F36A under control of the pTrc promoter in pTrcHIS2B or with pTrcHIS2B empty vector, and plaque-forming efficiency determined relative to NM522 infection. In both empty vector and MutS F36A, expression from the pTcr promoter was induced by addition of 1 mM IPTG for 20 minutes prior to phage infection and plated on LB agar containing 1 mM IPTG.Identical Ig Sγ3 sequence, described in Figure Bp, Base pair; IP, Immunoprecipitation; Ig, Immunoglobulin; CD, Circular Dichroism; G4, Guanine quadruplex; MSH, MutS homolog.The authors declare that they have no competing interests.EAE, experimental design, experimentation, and manuscript preparation. BJ, experimental design and experimentation. JDW, experimental design, and experimentation. GMB, experimental design, experimentation, and manuscript preparation. EDL, research design and manuscript preparation. All authors read and approved the final manuscript.Oligonucleotide master list. All oligonucleotides were synthesized commercially .Click here for fileMutS binds G4 DNA with high affinity. Table and graph depicting the percent of G-T mismatch and G4 bound by MutS. Data represent the mean of three independent experiments with standard error. The protein concentration where 50% of the labeled substrate is bound (indicated) was used as the value for apparent KD. Click here for fileF36 is not required for MutS binding G4. Table and graph depicting the percent of G4 bound by MutS and MutS F36A. Data represent the mean of three independent experiments with standard error. The protein concentration where 50% of the labeled substrate is bound (indicated) was used as the value for apparent KD. Click here for fileMutS binds G4 in presence of ATPγS. Table and graph depicting the percent of G4 bound by MutS in the presence or absence of ATPγS. Data represent the mean of three independent experiments with standard error. The protein concentration where 50% of the labeled substrate is bound (indicated) was used as the value for apparent KD. Click here for fileMutS immunoprecipitation. Representative immunoprecipitations performed on E. coli cell lysates. Amplicons were verified by sequencing. Input and IgG controls are indicated. G4 DNA refers to a transformed plasmid containing a portion of the human Sγ3 G4 sequence. Ctl DNA refers to a plasmid identical to the G4 DNA plasmid except the G4 motif is inverted disallowing G4 formation upon IPTG induction. NEB Turbo (Cat# C2986) bacterial cells were transformed with G4 DNA plasmid or control, grown to mid log phase, induced with IPTG, immunoprecipitations performed using anti-MutS or IgG control, and MutS association examined by plasmid-specific PCR. Click here for fileNeither MutS nor MutS F36A specifically bind homoduplex DNA. Mobility shift assay using purified MutS and MutS F36A and labeled homoduplex oligonucleotide. Lane 1, far left, is a negative control with radiolabeled homoduplex DNA but no protein. Lanes 2–4 contain increasing amounts of MutS . Lanes 6–8 contain increasing amounts of MutS F36A . Click here for file


The 2009 influenza pandemic affected people in almost all countries in the world, especially in younger age groups. During this time, the debate over whether to use corticosteroid treatment in severe influenza H1N1 infections patients resurfaced and was disputed by clinicians. There is an urgent need for a susceptible animal model of 2009 H1N1 infection that can be used to evaluate the pathogenesis and the therapeutic effect of corticosteroid treatment during infection.We intranasally inoculated two groups of C57BL/6 and BALB/c mice (using 4- or 6-to 8-week-old mice) to compare the pathogenesis of several different H1N1 strains in mice of different ages. Based on the results, a very susceptible 4-week-old C57BL/6 mouse model of Beijing 501 strain of 2009 H1N1 virus infection was established, showing significantly elevated lung edema and cytokine levels compared to controls. Using our established animal model, the cytokine production profile and lung histology were assessed at different times post-infection, revealing increased lung lesions in a time-dependent manner. In additional,the mice were also treated with dexamethasone, which significantly improved survival rate and lung lesions in infected mice compared to those in control mice. Our data showed that corticosteroid treatment ameliorated acute lung injury induced by the 2009 A/H1N1 virus in mice and suggested that corticosteroids are valid drugs for treating 2009 A/H1N1 infection.Using the established, very susceptible 2009 Pandemic Influenza A (H1N1) mouse model, our studies indicate that corticosteroids are a potential therapeutic remedy that may address the increasing concerns over future 2009 A/H1N1pandemics. During the spring of 2009, a novel swine origin influenza A (H1N1) virus (S-OIV) emerged in Mexico and affected almost all countries in the world, bringing about the first influenza pandemic since 1968 et al. have explored the potential of corticosteroid treatment against lethal H5N1 influenza infection in mice It has been reported for decades that severe viral pneumonia can cause acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) Wild-type 4- and 6- to 8-week-old BALB/c and C57BL/6 mice were purchased from Vital River, Beijing, China. Mice were kept under specific-pathogen-free conditions and all mouse research were approved by the Institutional Animal Care and Use Committee of the Academy of Military Medical Sciences (ID: SYXK 2007-005). Operations including intranasal inoculation of live virus or AF and bronchoalveolar lavage (BAL) were performed under sodium pentobarbital anesthesia (60–80 mg/kg). The trachea was cannulated for BAL after anesthesia and tissues were handled gently. After BAL, the mice were euthanasized by cervical dislocation and then carefully observed and palpated to confirm that there was no respiration and heart beat. Mice showing >30% of body weight loss were considered to have reached the experimental end point that was approved by the Institutional Animal Care and Use Committee of the Academy of Military Medical Sciences and were humanely euthanized.The influenza viruses used in this study were A/Beijing/05/2006 seasonal H1N1 strain , influenza A (H1N1) virus strain A/Beijing/501/2009 (BJ501) which is a pandemic H1N1 isolate, influenza A (H1N1) virus strain A/California/07/2009 (CA07), and influenza A (H1N1) virus strain A/PR/8/1934 (PR8). Live virus experiments using BJ501, CA07 and their mock-infected control, AF(P3), were performed in Biosafety Level 3 (P3) laboratory and live virus experiments using seasonal H1N1 and PR8 and their mock-infected control, AF(P2), were performed in Biosafety level 2 (P2) laboratory, under governmental and institutional guidelines. Viruses were propagated by inoculation into 9 to 10-day-old SPF embryonated eggs via the allantoic route. For BJ501, virus from earlier 3 generations was used. For UV inactivation, a 6-well plate containing 0.8 ml/well virus dilution of BJ501 was placed near the UV light (about 5 cm in distance) with the light on for 3 h in a biosafety cabinet.5.5 TCID50 of virus or virus dilution. The survival in each group of ten mice was monitored daily for 10 days. Survival data were analyzed using Kaplan-Meier survival analysis.4-week-old C57BL/6 mice were inoculated intranasally with 105.5 TCID50 of virus or virus dilution buffer and then sacrificed at various days post-infection (DPI). The lungs of each group of three mice were fixed in formalin and then embedded in paraffin. 4 µm sections were obtained and stained with hematoxylin-eosin.After being anesthetized with pentobarbital sodium, 4-week-old C57BL/6 mice were inoculated intranasally with 105.5 TCID50 virus or virus dilution buffer. At 5 days post-infection (DPI), the wet weight of the lungs from the mice was measured. The lungs were then heated to 68°C for 24 hours and the dry weight of the lungs was recorded, after which the wet to dry ratios were calculated.To assess the extent of acute pulmonary edema, the lung wet to dry weight ratios were calculated. 4-and 6-to 8-week-old BALB/c and C57BL/6 mice were used in this study. Six mice per group were anesthetized with pentobarbital sodium and intranasally inoculated with 10For cytokine measurement, the bronchoalveolar lavage fluid (BALF) of mice was collected after the mice were inoculated with virus or virus dilution buffer for 24 hours, and the BALF was processed with the IL-6 and TNF-α ELISA MAX™ Deluxe Sets .5.5 TCID50 of BJ501 after being anaesthetized with pentobarbital sodium. Dexamethasone phosphate (1 mg/kg or 10 mg/kg)  or placebo were administered intraperitoneally 6 h before BJ501 infection and continued daily until the end of each experiment. Survival rate, histology, acute pulmonary edema and BALF cytokine levels were measured as described in the Viral Pathogenesis in Mice section.4-week-old C57BL/6 mice were infected intranasally with 10P<0.05 indicates statistical significance.All data are shown as the means ± S.E.M. Measurements at single time points were analyzed by ANOVA, and if they demonstrated significance, these points were further analyzed by a two-tailed t-test. Time courses were analyzed by repeated measurement (mixed model) ANOVA with Bonferroni post-t-tests. Survival data were analyzed using Kaplan-Meier survival analysis. All statistical tests were conducted using the GraphPad Prism 5 software. Four H1N1 strains, including the seasonal H1N1, BJ501, CA07 and the commonly used mouse-adapted PR8 strain were used to infect 4- and 6- to 8-week-old C57BL/6 and BALB/c mice. The lung edema reflected by the wet/dry ratio of the lung tissue were investigated in our study. As a result, PR8 induced the most severe lung edema; BJ501 infection induced the next highest wet/dry ratios for most infection groups; whereas seasonal H1N1 infection did not induce any changes. CA07 infection had an intermediate virulence phenotype when compared to BJ501 and seasonal virus infection, causing relatively mild damage to the lung .We further compared the degree of lung injury in weanling and young mice. The wet/dry ratio changes for 4-week-old mice were significantly increased over strain-matched mice . In partWe infected weanling C57BL/6 mice with the BJ501 isolate to determine the pathological and cytokine changes that occur in the mice over multiple DPI. Pathological analysis of the mice showed progressive inflammatory reactions and lung lesions with bronchiolitis and alveolitis. Increased alveolar interstitial space, infiltrating cells, bronchitis with occasional necrosis of epithelial cells and alveolar hemorrhage can be easily recognized in the mice at 4 d, 5 d, 6 d after BJ501 infection  and alsoIn summary, these studies demonstrated that BJ501 virus strain induced high levels of virulence, causing ALI and a high degree of hypercytokinemia in infected weanling C57BL/6 mice. Thus, we hypothesized that in the cases of 2009 pandemic influenza infection, the host produced an over-exaggerated immune response and that the cytokine storm might play a critical pathological role in the disease.in vivo.Corticosteroid administration is among the most powerful treatments used to curb inflammation. Considering the unusual high cytokine levels assessed in the above referenced data and the clinical hypercytokinemia reported The 2009 influenza A (2009 H1N1) pandemic caused illness with severe complications, including fatality, worldwide. Many clinical reports have indicated that, in comparison with seasonal influenza, pandemic 2009 influenza A (H1N1) disproportionately affects younger individuals and that infants and children infected with influenza are more likely to develop complications compared to adults Mice infected with the typical 2009 H1N1 virus were reported to exhibit mild lung lesions and inflammation 5.5 TCID50 of BJ501 virus showed obvious respiratory symptoms, most importantly causing lethal disease. The lungs of virally infected mice were highly edematous, which was also demonstrated by a dramatically increased lung wet/dry weight ratio. Pathological changes rendered a progressive pattern with diffuse alveolar damage, hemorrhage, interstitial edema and extensive inflammatory accumulation from 4 to 6 DPI (Mice that were infected intranasally with 10to 6 DPI . It is rAmerican Journal of Respiratory and Critical Care Medicine also presented the arguments, including both pros and cons, on the concept of corticosteroid treatment for ALI associated with H1N1 viral pneumonia within different groups Corticosteroids, the immune-modulators, have strong anti-inflammatory activities and are widely prescribed for many immune and inflammatory diseases In conclusion, weanling C57BL/6 mice infected with the BJ501 isolate proved to be an ideal animal model for the severe cases of 2009 pandemic influenza infection in humans. Our experimental results from the mouse model of 2009 A/H1N1 infection suggested that corticosteroid treatment could be useful in patients infected with the 2009 pandemic influenza virus and help us to be better prepared for the next influenza pandemic.


Plum pox virus (PPV) causes the most economically-devastating viral disease in Prunus species. Unfortunately, few natural resistance genes are available for the control of PPV. Recessive resistance to some potyviruses is associated with mutations of eukaryotic translation initiation factor 4E (eIF4E) or its isoform eIF(iso)4E. In this study, we used an RNA silencing approach to manipulate the expression of eIF4E and eIF(iso)4E towards the development of PPV resistance in Prunus species. The eIF4E and eIF(iso)4E genes were cloned from plum (Prunus domestica L.). The sequence identity between plum eIF4E and eIF(iso)4E coding sequences is 60.4% at the nucleotide level and 52.1% at the amino acid level. Quantitative real-time RT-PCR analysis showed that these two genes have a similar expression pattern in different tissues. Transgenes allowing the production of hairpin RNAs of plum eIF4E or eIF(iso)4E were introduced into plum via Agrobacterium-mediated transformation. Gene expression analysis confirmed specific reduced expression of eIF4E or eIF(iso)4E in the transgenic lines and this was associated with the accumulation of siRNAs. Transgenic plants were challenged with PPV-D strain and resistance was evaluated by measuring the concentration of viral RNA. Eighty-two percent of the eIF(iso)4E silenced transgenic plants were resistant to PPV, while eIF4E silenced transgenic plants did not show PPV resistance. Physical interaction between PPV-VPg and plum eIF(iso)4E was confirmed. In contrast, no PPV-VPg/eIF4E interaction was observed. These results indicate that eIF(iso)4E is involved in PPV infection in plum, and that silencing of eIF(iso)4E expression can lead to PPV resistance in Prunus species. Plum pox virus (PPV) causes disease in Prunus trees, including peaches, plums, apricots, cherries and ornamental species. Plum pox, also known as Sharka, is the most devastating Prunus viral disease in terms of economic and agronomic importance worldwide Potyvirus, family Potyviridae. The PPV genome consists of a positive-sense single-stranded RNA molecule approximately 9.8 kb in length and has a 5′-terminal VPg 4E showed resistance to PPV Viruses encode a limited number of proteins and depend on the recruitment of host factors to complete their life cycle. These host factors are potential targets for alternative antiviral strategies. Many antiviral recessive resistance genes encode components of the translation initiation complex, including the eukaryotic translation initiation factor 4E (eIF4E) and eIF4G and their isoforms P. domestica) by specifically silencing either eIF4E or eIF(iso)4E. The results indicated that silencing of eIF(iso)4E but not eIF4E provided effective resistance to PPV in Prunus species. This is the first report confirming the involvement of eIF(iso)4E in PPV infection in its natural woody host.In this study, the involvement of plum eIF4E and eIF(iso)4E in PPV infection was investigated in its natural host plum (eIF4E and eIF(iso)4E genes from plum, eIF4E sequences for peach (AJ823667), apple (CV149907), apricot (CO370600) and Arabidopsis (Y10548) and eIF(iso)4E sequences for peach (DY638147), lettuce (AAP86603) and Arabidopsis (Y10547) were aligned and compared (data not shown). Conserved sequences were chosen for primer pairs Pd4E-F1 and Pd4E-R1, and Pdiso-F5 and Pdiso-R1 (eIF4E and eIF(iso)4E, respectively. Reverse transcription (RT)-PCR was conducted using total RNA isolated from plum leaves. Amplified PCR products of the expected length were cloned and sequenced. The sequences were used to design new primers  and 3′-UTR (Pd4E-R3)  of 702 bp, a 5′-UTR of 85 bp and a 3′-UTR of 173 bp. The predicted protein has 233 amino acid residues with a calculated molecular mass of 26.4 kDa. The sequence similarity of the cloned gene to other eIF4Es was confirmed using BLAST. The closest matches with the predicted translation product were eIF4E from pea , soybean  and muskmelon .To design primers for cloning of Pdiso-R1  to ampli primers  to obtaiPd4E-R3) . The asseIF(iso)4E was obtained by assembling the sequences of 5′-RACE and 3′-RACE and verified by RT-PCR using primers of Pdiso-F2 and Pdiso-R3 , eIF4E protein from castor bean  and eIF(iso)4Es from lettuce  and cucumber , suggesting that the cloned gene is eIF(iso)4E. The cloned plum eIF4E and eIF(iso)4E genes were designated PdeIF4E and PdeIF(iso)4E. The sequence identity between PdeIF4E and PdeIF(iso)4E coding sequences is 60.4% at the nucleotide level and 52.1% at the amino acid level. Both PdeIF4E and PdeIF(iso)4E sequence data have been deposited into GeneBank (accession numbers are JX137116 for PdeIF4E and JX137117 for PdeIF(iso)4E).Similarly, the cDNA sequence of plum Pdiso-R3 . The cDNTo compare the relationships of eIF4E superfamily members from different plant species, full length protein sequences of eIF4E and eIF(iso)4E were aligned and a phylogenetic analysis was conducted. Two distinct branches were formed in the phylogenetic tree  with allGene specific primer pairs Pd4E-Fm3/Pd4E-Rm3, and Pdiso4E-Fm3/Pdiso-Rm3  were usePdeIF4E and PdeIF(iso)4E were examined in different plum tissues using quantitative real-time PCR. Total RNA was isolated from roots, stems, leaves, petals, green immature fruit, flower buds, anthers and leaf buds of wild type plum trees. Expression levels of PdeIF4E and PdeIF(iso)4E were normalized to the expression level of an internal control gene. The Ps-actin gene was chosen as a control as its expression is consistent under different conditions in different tissues PdeIF4E and PdeIF(iso)4E were very similar in different tissues 4E mRNA were found in leaf buds, leaves and flower buds (Expression profiles for  tissues . When co tissues . The hig anthers . Similarwer buds . Lowest wer buds .eIF4E and eIF(iso)4E genes are silenced, plants exhibit a semi-dwarf phenotype eIF4E and eIF(iso)4E were selected to design the gene silencing constructs (nts 1–300 for PdeIF4E and nts 1–272 for PdeIF(iso)4E). Each resulting construct contains the target gene sequences in forward and reverse orientations separated by an intron (To study the involvement of PdeIF4E and PdeIF(iso)4E in PPV infection, we used a gene silencing strategy. DNA constructs containing self-complementary hairpin structure of target genes were developed. It has been previously reported that when both n intron . The conPutative transgenic shoots formed on the selection medium after four weeks. The developed shoots were excised from explants and cultivated on the shoot regeneration medium supplemented with selective agent for shoot propagation. Induced shoots showed vigorous and continuous growth on the selection medium . At leasPdeIF4E and Pdiso-siR for PdeIF(iso)4E, respectively. The presence of the Neomycin Phosphotransferase (NPTII) gene in transgenic plants was also analysed using primers nptII-F and nptII-R (data not shown).Tissue from putative transformants were analysed by PCR using primer 35S-F, located in the CaMV35S promoter region, and gene specific primers Pd4E-siR for NPTII. DNA samples from all selected transgenic plants showed hybridization with DIG-labelled NPTII probe. One to seven hybridization signals were detected in the different plants analysed (PdeIF(iso)4E transgenic lines and 6 PdeIF4E transgenic lines were obtained. These lines were designated Pdiso4E-ISH and Pd4E-ISH, respectively.Southern blot was used to confirm transgene integration into the genome of putative transformants using a probe specific for analysed , suggestPdeIF4E or PdeIF(iso)4E transgene-specific siRNAs was determined in representative transgenic lines via Northern blot using 32P labelled probes specific for each transgene. Previous reports showed that two classes of siRNAs, 21–22 bp and 24–26 bp are usually present in transgenic plants The presence of PdeIF4E and PdeIF(iso)4E genes in representative transgenic plum lines was investigated by qRT-PCR. In Pd4E-ISH lines, PdeIF4E mRNA transcript levels decreased significantly (50%–90% reduction compared to wild-type plants), and in contrast, PdeIF(iso)4E levels increased (122% to 164% relative to the wild type plants expression levels) (PdeIF(iso)4E gene expression level decreased by 76–88% compared to wild-type plants and the PdeIF4E expression level increased to 141% to 230% of the wild-type level (Expression of  levels) . Similarpe level .eIF4E did not result in a decrease of expression of the other isoform. Pd4E-ISH and Pdiso4E-ISH transgenic plants did not exhibit any morphology changes when compared to wild type plants (data not shown).The results indicated that the pPd4E-ISH and pPdiso4E-ISH constructs were effectively silencing the expression of the respective target gene. The silencing was specific and silencing of one isoform of Transgenic plum plants were inoculated with the PPV-D strain by chip-budding To test the stability of PPV resistance, the plants went through a second cycle of cold treatment in which the plants were placed in the cold room for three months. After the cold treatment, the plants were brought back to the greenhouse with normal growth conditions. The presence of PPV was tested 6 and 16 weeks after the second cold treatment. All Pd4E-ISH plants that tested positive in the first test remained PPV positive. All control plants tested PPV positive and exhibited stunted growth. As for Pdiso4E-ISH lines, only 3 of the 26 plants that tested PPV negative after the first cold treatment became PPV positive after the second cold treatment . In totaPPV symptoms were visually monitored after cold treatments. Only two wild type plants exhibited typical PPV symptoms, e.g. chlorotic spots and ring patterns, on leaves , four weADE, HIS, MEL1) was only observed when VPg and eIF(iso)4E were cloned into the bait and prey vectors and expressed in the same cell. This was evidenced by the ability of these cells to grow in absence of adenine and histidine and the development of the blue color (To determine if there is an interaction between PPV-VPg and eIF4E/eIF(iso)4E, the GAL4 based yeast two hybrid (Y2H) system was used. Reporter gene activation  assay. The PPV-VPg protein was fused to the C-terminal fragment of the yellow fluorescent protein (YFP) and the PdeIF(iso)4E protein was fused to the N-terminal fragment of YFP. Following transient co-expression of PPV-VPg-YC and PdeIF(iso)4E-YN in Nicotiana benthamiana, interaction of these proteins allowed reconstitution of fluorescence (PdeIF(iso)4E in plum can confer resistance to PPV, while silencing of PdeIF4E did not. Thus, the VPg-eIF(iso)4E interaction is probably important for PPV infection in plum.To test if the positive interaction between PPV-VPg and PdeIF(iso)4E can be observed rescence . This inrescence  and nucleIF(iso)4E expression using an RNA silencing-based strategy. The resistance was stable over an extended period of time and over two growth and dormancy cycles.In this study, we have shown that PPV resistance can be efficiently achieved in a PPV natural host plant by silencing eIF4E and eIF(iso)4E in PPV nature woody host plant (plum). We did not find significant difference in the expression profiles of the two genes in different tissues. Both genes were transcribed abundantly in leaves, leaf buds and flower buds, and were expressed at a relatively lower level in petals 4E are part of the eIF4F complex and function in recruiting mRNAs to ribosomes for translation initiation n petals . This suArabidopsis, an eIF4E mutant line confers resistance to Clover yellow vein virus (ClYVV), while eIF(iso)4E mutant lines are resistant to Turnip mosaic virus (TuMV) and Tobacco etch virus (TEV) ArabidopsisPepper veinal mottle virus (PVMV) ArabidopsiseIF(iso)4E were resistant to PPV-D over an extended period of time involving two growth and dormancy cycles (eIF(iso)4E gene is an effective approach for generating host factor based resistance to PPV.Mutant plants lacking either eIF4E or eIF(iso)4E do not show phenotype change compared to wild type plants, suggesting some degree of functional redundancy between the two isoforms y cycles . In conty cycles . The reseIF4E gene was effectively silenced 4E gene expression can effectively confer PPV resistance to a Prunus plant. To our knowledge, this is the first demonstration that manipulation of the expression of a host factor can provide an effective resistance source for the control of PPV in woody trees. It is likely that the transgenic plants are also resistant to other strains of PPV or to other viruses recruiting the same host factor for infection. Additional tests will answer this question.In this study, we have shown that eIF(iso)4E is involved in PPV infection in its natural host woody plant and that silencing of Prunus domestica L.) were collected from the orchard of the University of Guelph, Vineland Station, Ontario, Canada. Unless stated otherwise, young leaves were collected and used for RNA and DNA extractions. Four to five leaf discs (approximately 50∼100 mg) from each plant were collected and immediately frozen in liquid nitrogen. Leaf tissues were homogenized with a Mixer Mill MM 300 . A Plant RNA Isolation Kit  was used for RNA extraction. The genomic DNA was removed using DNase I  before RNA was eluted according to the manufacturer instructions. siRNAs were isolated from young leaves using the MicroRNA Purification Kit (Norgen Biotek Corp.). One microgram of purified total RNA was used for cDNA synthesis using the QuantiTect® Reverse Transcription kit . The RT reactions were incubated at 42°C for 30 min, followed by 3 min at 95°C to inactivate the reverse transcriptase. This was followed by standard PCR.Tissues from plum (eIF4E and eIF(iso)4E genes, 5′-RACE and 3′-RACE were carried out using the First Choice RLM-RACE kit  following the manufacturer's instructions. The eIF4E gene specific 5′-RACE and 3′-RACE primers, Pd4E-5′-RACE-OP, Pd4E-5′-RACE-IP, Pd4E-3′-RACE-OP and Pd4E-3′-RACE-IP 4E gene-specific 5′-RACE and 3′-RACE primers, Pdiso4E-5′-RACE-OP, Pdiso4E-5′-RACE-IP, Pdiso4E-3′-RACE-OP and Pdiso4E-3′-RACE-IP (eIF(iso)4E amplification. Purified PCR fragments were cloned into pGEM T-easy vector  and sequenced.To obtain the 5′- and 3′- sequences of plum -RACE-IP , were us-RACE-IP , were usTotal RNA samples were extracted from root, stem, leaf, petal, immature green fruit, flower bud, anther and leaf bud tissues, and reverse transcribed to cDNAs. Tissues used for RNA extraction were pooled from three plum plants. For mRNA transcript level analysis, three RNA samples were isolated for each type of tissues and used as template for biological repeats. For transgenic plants, three RNA samples were isolated for each plant using leaf tissues pooled from the same plant.PdeIF4E and PdeIF(iso)4E amplification, respectively. The ACTIN gene from Prunus salicina L. was used as the internal reference gene using primers Ps-actin-F and Ps-actin-R PdeIF4E, PdeIF(iso)4E or plum ACTIN genes. Each standard curve composed of 15 datapoints representing 3 replicates for 5 different DNA concentrations of 2× dilution series starting from 12.5 ng. The reaction efficiency for all standard curves was greater than 95%. The level of each mRNA was calculated using the mean Ct normalized to the corresponding reference gene for gene expression profiles in different tissues. All results are shown as means of the three biological replicates with corresponding standard errors.qRT-PCR reaction preparations were conducted by using the QuantiTect® SYBR® Green PCR Kit  on a LightCycler® 480 real-time PCR system . Primer sets Pd4E-Fm3 and Pd4E-Rm3, and Pdiso-Fm3 and Pdiso-Rm3 were used for attB1 and attB2 sites, Pd4E-attB1 and Pd4E-attB2 for Pd4E and Pdiso-attB-1 and Pdiso-attB-2 for PdeIF(iso)4E (Agrobacterium tumefaciens strain EHA105 Plasmid constructs containing ihp-RNAs of target genes were cloned using the bacteriophage lambda site-specific recombination based Gateway® technology F(iso)4E . PurifiePlum hypocotyls of mature seeds were used as explants for transformation and the procedure was as described previously et al., (2009), except that genomic DNA samples were extracted from transgenic plum plants using the Plant DNA Isolation kit  and each DNA sample was digested with BamHI .DNAs isolated from putative transgenic plants were used as templates and the PCR was conducted with Taq DNA polymerase (Genscript) reaction with primers CTAP and Pd4E-siR and CTAP and Pdiso-siR respectively in 20 µl PCR reaction. CTAP primer is located at the 35S promoter and the reverse primers Pd4E-siR and Pdiso-siR are sequence specific. Southern blots were conducted using DIG High Prime DNA Labelling and Detection Starter Kit II  as described by Tian et al., 2006) with the following modifications. Approximately 8 µg of small RNA samples were mixed with 2× RNA loading buffer  and separated on a 7 M urea denatured 20% polyacrylamide gel in 0.5× TBE buffer ran for approximately 90 min at 180 V in a BIORAD Mini-Protein Cell . The RNAs were transferred to Hybond-N+ nylon membrane  using semi-dry transfer cell  at 180 mA for approximately 2 h. The RNAs were fixed to the membrane by UV cross-linking. DNA fragments corresponding to the entire length of the transgenes were amplified from plasmids containing PdeIF4E and PdeIF(iso)4E coding sequences, respectively, by PCR using primers Pd4E-F8 and Pd4E-siR, and Pdiso-F1 and Pdiso-siR .Detection of small interfering RNAs was performed as described (Zhang diso-siR . The ampPdeIF4E and PdeIF(iso)4E coding sequences were amplified using primers Pd4E-attB1/Pd4E-attB2-Y2H and Pdiso-attB1/Pdiso-attB2-Y2H (PPV-VPg and PdeIF4E and PdeIF(iso)4E cDNA sequences were cloned into both bait and prey vectors, but only results using PPV-VPg as a bait and PdeIF4E or PdeIF(iso)4E as prey are shown. Reciprocal assay using PPV-VPg as a prey and PdeIF4E or PdeIF(iso)4E as bait gave the same results. For co-transformation of bait and prey constructs, yeast strain Y2HGold was used according the Yeastmaker Yeast Transformation System protocol . After transformation, cells were spread on SD double drop out (DDO), SD/-Trp/-Leu, agar plates and incubated at 28°C for 3–5 days. Colonies growing on DDO plates were suspended in DDO liquid medium. A 10× dilution series of 5 µl aliquots of co-transformed Y2HGold were spotted onto DDO/X  and QDO/X/A  agar plates. Plates were incubated at 28°C for 3–5 days. Colonies growing on the DDO plates were analyzed following the manufacturer's protocol (Clontech).Protein interactions were tested using the Matchmaker Gold Yeast Two-Hybrid System . ttB2-Y2H  and the ttB2-Y2H . All seqPdeIF4E and PdeIF(iso)4E cDNA sequences were amplified using the primer pairs listed PPV-VPg cDNA sequences were cloned to be expressed as fusion to the C-terminal fragment of YFP (construct VPg-YC). All YFP fusion constructs were transformed into A. tumefaciens strain GV3101. To infiltrate epidermal N. benthamiana cells, Agrobacterium cultures were grown to an OD600 of 1.0. Equal volumes of each culture were mixed and infiltrated in N. benthamiana leaves as described The Gateway compatible BiFC vectors pEarleygate201-YN and pEarleygate202-YC Plum plants were inoculated with PPV strain D virus by chip-budding http://blast.ncbi.nlm.nih.gov). cDNA sequences assembling, genomic DNA alignments and translation of nucleotide to protein sequences were performed with SeqMan, MegaLign and EditSeq programs of Lasergene 6. Full length amino acid sequences from select plant species andhuman were aligned (DNAman Version 6) using an optimal sequence alignment and a PAM protein weight matrix eIF4E and eIF(iso)4E protein sequences were identified by BLASTP searches at NCBI (


HIV infected individuals face an increased risk of serious illness from influenza. Current guidelines recommend vaccination of all HIV infected adults. Several studies have analysed the immunogenicity of adjuvant H1N1 vaccines in these individuals. However, no such analysis exists for Celvapan (Baxter), an inactivated H1N1 vaccine without adjuvants, derived from verocells and based on whole virions. Celvapan contains A/California/07/2009 (H1N1)v virus strain. Vaccination was provided in two i.m. doses, at least three weeks apart. This vaccine has been used widely in Austria during the recent H1N1 pandemic.3 and the median CD4 nadir 209 cell/mm3. 38 % of the patients seroconverted after receiving both doses of the vaccine. Only 7 patients had HAI titers <1:40 before vaccination; all of them showed post-vaccination titers of >1:40. There was no significant difference with regard to age, the recent CD4 cell count or the CD4 nadir between seroconverters and non-responders and the share of patients with a recent VL BLQ or a VL BLQ for at least 12 months was roughly equal in both groups.Adult HIV-1 infected individuals scheduled for H1N1 vaccination where included in this study. Serum samples were taken before the first vaccination (baseline) and after the second vaccination. Antibody titers were determined by hemagglutination inhibition (HAI) assay using chicken red blood cells and the Influenza A/H1N1pdm virus A/California/7/2009 (NIBSC 09/146). Clinical and HIV-related data were taken from patient charts. Seroconversion was defined as a ≥4-fold increase in antibody titer between the pre- and post vaccination serum, and a post-vaccination titer of ≥1:40 was considered protective. 78 patients were included in the study. 42 patients provided serum samples after the second vaccination. 74 % of the patients were male. Median age was 38.5 years. 21 patients had a VL below the limit of quantification (BLQ) since more than 12 months, 27 patients received currently HAART therapy with a VL BLQ. The median CD4 cell count was 442 cell/mmSeroconversion rates in HIV-infected individuals were significantly lower compared to those in otherwise healthy individuals in spite of a high percentage of individuals with well-controlled VL BLQ and high CD4 cell counts. In addition, seroconversion rate for HIV-infected individuals in this observational trial during 2009 pandemic flu season were lower than those recently published in studies using adjuvant H1N1 vaccines.