Our results suggest that (1) three distinct lineages of EV-D68 were co-circulating in 2013 and 2014; (2) isolates associated with AFM/AFP belong to a single phylogenetic subclade – B1; (3) the majority of isolates from the B1 subclade have 21 unique substitutions that distinguish them from other isolates, including amino acid substitutions in the VP1, VP2, and VP3 capsid proteins and the 3D RNA-dependent RNA polymerase, and nucleotide substitutions in the internal ribosome entry sequence (IRES); (4) at 12 of these positions, B1 isolates carry the same residues observed at equivalent positions in paralysis-causing enteroviruses, including poliovirus, EV-D70 and EV-A71. Based on these results, we hypothesize that unique B1 substitutions may be responsible for the apparent increased incidence of neuropathology associated with the 2014 outbreak.Enterovirus D68 (EV-D68) caused a severe respiratory illness outbreak in the United States in 2014. Reports of acute flaccid myelitis (AFM)/paralysis (AFP) in several independent epidemiological clusters of children with detectable EV-D68 have raised concerns that genetic changes in EV-D68 could be causing increased disease severity and neurological symptoms. To explore the potential link between EV-D68 genetic variations and symptom changes, we performed a series of comparative genomic analyses of EV-D68 2014 outbreak isolate sequences using data and analytical tools in the Virus Pathogen Resource (ViPR;  Picornaviridae family is composed of non-enveloped, single-stranded, positive-sense RNA viruses. The genomes of picornaviruses encode a single open-reading frame that is translated into a long polyprotein, which is subsequently processed into mature peptides by virus-encoded proteases , as the most common viral agents of the common cold, polioviruses , causing poliomyelitis within the Enterovirus C species, enterovirus A71 (EV-A71), causing a variety of neurological diseases, and enterovirus D68 (EV-D68) (The roteases . The Ent(EV-D68) . Before (EV-D68) . From Au(EV-D68) . This ou(EV-D68) . Statist(EV-D68)  and Cali(EV-D68)  both sug(EV-D68) . For exa(EV-D68) , and sev(EV-D68) . Outside(EV-D68) , France (EV-D68) , Norway (EV-D68) , and Aus(EV-D68) , each reTo explore the evolutionary source of the EV-D68 outbreak viruses and the potential link between a possible novel lineage and the apparent changes in symptomatology associated with the 2014 EV-D68 outbreak, we performed a series of comparative genomics analyses of the EV-D68 2014 outbreak isolates to identify amino acid and nucleotide substitutions in 2014 outbreak isolates that could be contributing to increased disease severity and neurological symptoms.Picornaviridae family (http://www.viprbrc.org/brc/home.spg?decorator=picorna) on 9 February 2016. Sequences from the same genomic regions were aligned using the MUSCLE algorithm (All sequences were retrieved from the Virus Pathogen Resource (ViPR)  – Picornlgorithm  implemenTo compare EV-D68 sequences with those of other enteroviruses that are associated with neurological illnesses, the full-length EV-D70 isolate (J670/71), two AFP-associated EV-A71 isolates (AFP2001064/EV71/GX/CHN/2001 and AFP2001071/EV71/GX/CHN/2001), and representative neurovirulent poliovirus isolates  were used.Phylogenetic relationships between the nucleotide sequences of complete VP1 coding regions were inferred using RAxML  with 1,0Each EV-D68 isolate was assigned a neurovirulence trait for phylogeny–phenotype association analysis. Isolates associated with either AFM/AFP or encephalitis case reports were classified as neurovirulent. EV-D68 isolates that were not explicitly associated with an AFM/AFP or encephalitis case report were classified as non-neurovirulent, based on the fact that historically EV-D68 had been predominantly linked to respiratory symptoms only, with the exception of two cases for which no genome sequence data is available. In order to determine if the neurovirulent isolates demonstrated a non-random distribution during viral evolution, the same VP1 nucleotide dataset used in phylogenetic analysis was first input into the Bayesian Evolutionary Analysis Utility (BEAUTi) and then the Bayesian Evolutionary Analysis Sampling Trees (BEAST). The BEAST tree was subsequently input into the Bayesian Tip-association Significance (BaTS) program  to calcuz-test to determine whether the difference between two proportions is significant assumes that the samples in each group are independent. The second statistical test used in this study extends the z-test by allowing the samples (i.e. isolates) to be dependent. For each position, the test statistic is constructed based on the comparison of mutation proportions between the B1 and non-B1 isolates. The approach assumes a common evolutionary correlation matrix among the isolates across all positions, where the correlation matrix is a Bayesian estimate based on the sample correlation matrix. By incorporating the correlation matrix in the test statistic, the test can account for the dependency among the isolates due to shared evolutionary relatedness.Genetic differences between EV-D68 B1 and non-B1 isolates were identified by two separate statistical tests, using protein sequences for mature peptides and nucleotide sequences for 5’UTR. The first statistical test is a Chi-squared statistical test implemented in the ViPR meta-CATS tool . The secTP is the number of isolates containing the B1-representative residue/nucleotide in the subclade B1 group, FN is the number of isolates containing non-representative residue(s)/nucleotide(s) in the B1 group, TN is the number of isolates containing non-representative residue(s)/nucleotide(s) in the non-B1 group, and FP is the number of isolates containing the B1-representative residue/nucleotide in the non-B1 group.For each significant position identified by these statistical tests, sensitivity and specificity were calculated based on the following formulae:P value < 0.05, P value corrected for evolutionary relatedness among isolates < 0.05, specificity > 0.80, and sensitivity > 0.75 in the subclade B1 group were considered to be significant.Positions with a Chi-squared To compare the B1 isolates with paralysis-causing poliovirus, EV-D70, and EV-A71 enteroviruses, EV-D68 sequences were aligned with the corresponding sequences of representative poliovirus, EV-D70, and EV-A71 viruses using the MUSCLE algorithm  on the VTo explore the evolutionary origin of the recent EV-D68 isolates since its initial detection in 1962 , a phyloSince all EV-D68 isolates associated with neurological illnesses belong to the B1 subclade, we tested the association between neurovirulence and phylogeny using the BaTS program . Each EVTo determine if there are any consistent substitutions that characterize subclade B1, which contains the AFM/AFP-associated isolates, we first used the ViPR meta-CATS statistical comparative analysis tool  to analyIt is well established that other enteroviruses, especially PV, EV-D70, and EV-A71, are associated with paralysis symptoms . To explTable 2 andThe manifestation of clinical symptoms following virus infection results from the combined effects of both virus and host factors. Most enterovirus infections are asymptomatic. However, when enterovirus infections are symptomatic, they can cause a spectrum of clinically distinct syndromes . As an eThe phylogenetic and phenotypic association analysis results obtained using BaTS clearly indicates that the neuropathology phenotype is correlated with phylogenetic clustering. Since all neurological illness-associated isolates belong to the monophyletic subclade B1, we hypothesize that this subclade has acquired genetic changes enhancing neurotropism of EV-D68. Comparative genomic analyses reveal that 12 B1 diagnostic positions carry the same residues as those observed at equivalent positions in other enteroviruses including poliovirus, EV-D70, and/or EV-A71. Since these enteroviruses are known to cause paralysis symptoms more frequently, one or more of these substitutions found in the B1 subclade could be linked to a more neurovirulent phenotype, perhaps by enabling EV-D68 to replicate more efficiently in neuronal cells. This hypothesis is currently being tested in a variety of different cell culture model systems.The mechanisms of pathogenicity and cellular tropism of EV-D68 are not fully understood. Capsid proteins, non-structural proteins, and UTR regions may all affect EV-D68 infectivity, replication efficiency, and pathogenicity in different host tissues and cell types . Our stuPicornavirus entry into cells begins with the attachment of viral proteins to host receptors. Mapping the locations of B1 substitutions on a capsid protein structure shows that VP1/290S, VP1/308N, and VP2/222T are located on the surface of the virion A and couEnterovirus genomes encode a single open reading frame that is translated into one long polyprotein, which is subsequently cleaved by viral proteases (3C and 2A) into functional mature peptides. Substitution 2C/1G is distinct in subclade B1 and also observed in poliovirus Fig. 2. TPicornaviruses have a conserved and highly structured 5′UTR region, indicating the importance of this region in the virus life cycle . An inteIn some cases, not all AFM/AFP isolates carry the same substitutions (e.g. 5′UTR/669T in the CA/AFP/v12T00346 isolate). The analysis performed using BaTS allows us to reject the null hypothesis that there is no association between the neurovirulence trait and the phylogenetic topology, so we based the polymorphism analysis on subclade B1, which includes all AFM/AFP isolates. However, there are exceptions to a strict residue association. For example, the CA/AFP/v12T00346 isolate differs from the other AFM/AFP isolates at four positions in the 5′UTR region . This stIn summary, our results suggest that three EV-D68 clades were co-circulating in 2013–2014; isolates associated with AFM/AFP belong to a distinct phylogenetic cluster designated as B1; the majority of the B1 isolates have 21 unique nucleotide and amino acid substitutions that distinguish them from other EV-D68 isolates, among which 12 are observed at equivalent positions in other enteroviruses associated with neurovirulent phenotypes. Based on these results, we hypothesis that unique B1 substitutions might contribute to the increased incidence of neuropathology associated with the 2014 outbreak.Picornaviridae family (http://www.viprbrc.org/brc/home.spg?decorator=picorna).All sequences are available in the Virus Pathogen Resource (ViPR) – Supplementary DataClick here for additional data file.


Recombinant immunotoxins (RITs) are genetically engineered proteins being developed to treat cancer. They are composed of an Fv that targets a cancer antigen and a portion of a protein toxin. Their clinical success is limited by their immunogenicity. Our goal is to produce a new RIT that targets mesothelin and is non-immunogenic by combining mutations that decrease B- and T-cell epitopes. Starting with an immunotoxin that has B-cell epitopes suppressed, we added mutations step-wise that suppress T-cell epitopes. The final protein (LMB-T14) has greatly reduced antigenicity as assessed by binding to human anti-sera and a greatly decreased ability to activate helper T-cells evaluated in a T-cell activation assay. It is very cytotoxic to mesothelioma cells from patients, and to cancer cell lines. LMB-T14 produces complete remissions of a mesothelin expressing cancer (A431/H9) xenograft. The approach used here can be used to de-immunize other therapeutic foreign proteins. Pseudomonas exotoxin A (PE38).SS1P was developed to treat a variety of mesothelin expressing tumors; these include mesothelioma, ovarian, pancreatic, lung, stomach and cervical cancer [Recombinant immunotoxins (RITs) are antibody-toxin fusion proteins developed for cancer therapy. SS1P is a RIT composed of a Fv that targets mesothelin and a 38-kDa fragment of l cancer . In a phl cancer . Howeverl cancer . This inFormation of anti-drug antibodies is a major problem in the development of protein therapeutics  and specMouse models have shown that elimination of murine B-cell epitopes can significantly reduce the formation of anti-drug antibodies (ADA) against therapeutic foreign proteins  and specElimination of T-cell epitopes is also a well-accepted strategy to de-immunize protein therapeutics. Yeung et al. showed that elimination of a T-cell epitopes in the protein IFNβ resulted in elimination of ADA response in BALB/c mice . SimilarWe previously reported the location of the eight human T-cell epitopes in the PE38 portion of immunotoxins  and usedPseudomonas exotoxin A (PE38). PE38 is made up of two domains; domain II (amino acids 253-364) contains a furin cleavage site necessary for toxin processing, and domain III (amino acids 395-613) contains the ADP ribosylation activity. Previous work showed that modifying SS1P by deletion of the majority of domain II and retaining the 11 amino acid furin cleavage site followed by a GGS spacer results in a RIT (SS1-LR-GGS) with high cytotoxic activity on many cell lines and decreased nonspecific toxicity in mice [To construct the new de-immunized RITLMB-T14) we used the dsFv and toxin present in SS1P, deleted most of domain II and made mutations in domain III as shown Figure 4 we used in mice .50 of 1.7 pM, which is similar to that of the parent RIT that has no point mutations (SS1-LR-GGS) when evaluated on A431/H9 cells. LMB-T20 , and when combined, it moderated the decrease in activity to 2.5-fold.LMB-T14 Figure  is the m50s that were less than 100 pM on NCI-Meso16, NCI-Meso19, NCI-Meso21, and NCI-Meso29, .Because SS1P has shown anti-tumor activity in patients with mesothelioma , 17, we , Figure . Figure , Figure  (p < 0.0p < 0.01 in one way ANOVA with Dunn's multiple comparison). A431/H9 cells were the only cells that displayed a different pattern in which SS1P had a significantly better or similar activity to all of the variants. This is a transfected cell line that does not normally express mesothelin.We also compared the activity of LMB-T14 with the three other RITs on several mesothelin expressing cancer cell lines Figure . We foun50was calculated for each time point. We found that LMB-T14 was very stable with no loss in activity after 24 hr .LO10R also had significantly reduced binding compared to SS1P (p < 0.05 in Friedman's test and Dunn's multiple comparisons)  Figure . These fin vitro expansion, the cells that were stimulated with SS1P were re-stimulated with 111 peptides spanning the sequence of PE38 and the cells that were stimulated with LMB-T14 or LMB-T20 were re-stimulated with 76 peptides spanning the sequence of LMB-T14 or LMB-T20, respectively. T-cell activation was detected using IL-2 ELISpot. As expected, both de-immunized RITs (LMB-T20 and LMB-T14) had a significant reduction in the number of IL-2 specific spots. The decrease was 61% with LMB-T14 and 81% with LMB-T20 (p < 0.01 in Wilcoxon matched-pairs signed rank test)  induced formation of new T-cell epitopes, we stimulated peripheral blood mononuclear cells (PBMC) from 10 normal donors with SS1P or the variants LMB-T20 and LMB-T14. After 14 days of ) Figure .Unexpectedly, 4/10 donors had a new and significant response to peptides 63-65 in LMB-T14 Figure  and searWe describe here the properties of a new RIT, LMB-T14, that has greatly reduced immunogenicity because it contains mutations that suppress both T and B-cell epitopes. The new protein, which contains a large deletion of domain II and 10 amino acid mutations in domain III, is very cytotoxic to mesothelioma cells as well as other cancer cell lines, is well tolerated by mice and produces complete remissions of mesothelin expressing cancers in mice.We have previously described immunotoxins with mutations in either B- or T-cell epitopes designed to decrease immunogenicity , 14. In Clinical trials with immune suppressive regimens also indicate a need to address both the B- and T-cells arms of the adoptive immune system. Hassan et al. treated patients with Rituximab to induce depletion of all circulating B-cells and followed with RIT treatment . They foIn the process of combining six mutations designed to eliminate B-cell epitopes and the six mutations designed to diminish T-cell epitopes, we found that two point mutations (R427A and R505A) diminished both B- and T-cell mediated immunity. R505A and R427A have very large ASAs (150Å and 142Å) indicating that the arginines are located on the surface of the protein. Since B-cell epitopes are known to contain bulky hydrophilic amino acid like arginine , 20, 21,Our finding that the mutations (D463A and R467A) created a new T-cell epitope was unexpected, because alanine substitutions are frequently found to reduce the binding of a peptide to an HLA molecule due to loss of non-polar side chains , 26 and When incorporating multiple point mutations into a molecule, there is the risk in decreasing its activity. There is a delicate balance between the decrease in activity the molecule will endure and the benefit of the de-immunization. This tradeoff is demonstrated in Table PBMC were isolated from apheresis samples from patients who were previously treated with a PE38-containing RIT and from naïve donors were collected under research protocols approved by the NIH Review Board (08-C-0026) and (99-CC-0168), respectively with informed consent. PBMC were isolated using gradient density separation by Ficoll-Hypaque  according to manufacturer's instructions. PBMC were frozen in 10% human AB serum  RPMI media  containing 7.5% DMSO  for 12 months in liquid nitrogen. Human sera were obtained under protocols 01-C-0011, 03-C-0243, and 08-C-0026.Peptides for T-cell assays were synthesized by American Peptides . All peptides were purified to 95% homogeneity by HPLC and confirmed by mass spectrometry.All RIT described in this work are composed of a heavy-chain Fv fused to LR-PE24 (VH-PE24) disulfide-linked to the light-chain Fv (VL) of SS1 antibody . The dif50 values were calculated from the binding curves. The IC50 values indicate the concentration of RIT that inhibits 50% of the antibody reactivity with SS1P. The binding ratio was calculated from each IC50 value.Binding of RITs to antibodies present in patients sera was assayed as previously described . BrieflyIn vitro expansion using whole RIT and T-cell activation detection using IL-2 ELISpot were performed as previously described [6cells/ml and 50 μl were plated in pre-coated ELISpot plates (Mabtech). The enriched cells were then restimulated with peptide pools; cells that were expanded with SS1P were restimulated with 22 peptide pools spanning the sequence of WT PE38. Cells that were expanded using LMB-T14 or T20 were restimulated with 15 peptide pools spanning the sequence of the deimmunized RIT. Peptide pools that had a positive responses as defined [escribed . Briefly defined  were fin3cells/well) cells were seeded in a 96 well plate and 24 hr later were treated with various concentrations of the RITs.Early passage mesothelioma cells from the ascites or pleural fluid of four patients with mesothelioma seen at the National Cancer Institute on Institutional Review Board-approved protocols (08-C-0026) . Frozen 3/well, KLM1, L55, MKN74, MKN45, and HAY cells at 5 ×103/well) and 24hr later were treated with various concentrations of the RITs. The A431/H9 cell line was transfected in our laboratory and previously described [Cells were seeded in a 96 well plate at optimal cell concentrations  according to manufacturer's instructions. Color change was evaluated at O.D. 450nm.Cell viability was normalized between 0-100 percent. Complete cell death (0%) was obtained by treating the cells with Cyclohexamideor Staurosporine and 100% by no treatment.3 cells/well and 24 hr later were treated using serial dilutions of the treated RITs in six replicas. Cell viability was detected 72 hr later as described above.LMB-T20, LO10R and LMB-T14 were diluted in D-PBS to concentrations of 0.5 mg/ml. RIT variants were distributed in five aliquots and placed in an incubator at 37°C. Vials were taken out of the incubator and placed on dry ice for 15 min and transferred to −80°C at the following time points: 0, 1, 2, 6 and 24 hr. A431/H9 cells were plated in a concentration of 2.5 ×10RIT variants were diluted to a concentration of 15 μg/ml in 100% human AB Serum (Gemini Bio-products). Five aliquots of 60 μl each were made for each protein and placed in 37°C.Vials were taken out of the incubator and placed in −20°C in the following time points: 0, 1, 6, 24 and 72 hr. Functional activity was evaluated as described above.6 A431/H9 cells in 0.2 mL RPMI with 4 mg/mL Matrigel (BD Biosciences) on day 0. After 7 days, when the tumors reached 100 mm3, mice were injected IV with RITs in the indicated concentrations and the indicated schedules. Body weight and tumor size were observed for 30 days. Mice were euthanized if they experienced rapid weight loss or tumor burden greater than 10% body weight. No animals were excluded from statistical analysis. Tumor-size evaluation was evaluated blindly using a caliper.All animal experiments were performed in accordance with NIH guidelines and approved by the NCI Animal Care and Use Committee. Female athymic nude mice were injected subcutaneously in the flank with 1.0 × 10Nonspecific toxicity was evaluated by IV injections of indicated doses to Swiss mice.Statistical analysis and plots were done using Graph Pad Prism software. For comparisons between two parametric variables we used Student T test. For comparisons between two non- parametric variables we used Wilcoxon matched rank test. For comparisons of multiple parametric variables we used One way ANOVA followed by Holm-Sidak's multiple comparisons test. For comparisons of multiple non-parametric variables we used Friedman test followed by Dunn's multiple comparison test. For comparisons between different cell lines and different RIT we used two way ANOVA with multiple comparisons with Dunnet test.


Interestingly, GAPDH aggregates induced necrotic cell death via a permeability transition pore (PTP) opening. The expression of either WT- or C152A-GAPDH did not affect other cell death pathways associated with protein aggregation, such as proteasome inhibition, gene expression induced by endoplasmic reticulum stress, or autophagy. Collectively, these results suggest that NO-induced GAPDH aggregation specifically induces mitochondrial dysfunction via PTP opening, leading to cell death.Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein that also mediates cell death under oxidative stress. We reported previously that the active-site cysteine (Cys-152) of GAPDH plays an essential role in oxidative stress-induced aggregation of GAPDH associated with cell death, and a C152A-GAPDH mutant rescues nitric oxide (NO)-induced cell death by interfering with the aggregation of wild type (WT)-GAPDH. However, the detailed mechanism underlying GAPDH aggregate-induced cell death remains elusive. Here we report that NO-induced GAPDH aggregation specifically causes mitochondrial dysfunction. First, we observed a correlation between NO-induced GAPDH aggregation and mitochondrial dysfunction, when GAPDH aggregation occurred at mitochondria in SH-SY5Y cells. In isolated mitochondria, aggregates of WT-GAPDH directly induced mitochondrial swelling and depolarization, whereas mixtures containing aggregates of C152A-GAPDH reduced mitochondrial dysfunction. Additionally, treatment with cyclosporin A improved WT-GAPDH aggregate-induced swelling and depolarization. In doxycycline-inducible SH-SY5Y cells, overexpression of WT-GAPDH augmented NO-induced mitochondrial dysfunction and increased mitochondrial GAPDH aggregation, whereas induced overexpression of C152A-GAPDH significantly suppressed mitochondrial impairment. Further, NO-induced cytochrome  In addi7–ide (NO) . Nuclearide (NO)  and polyide (NO) . Additioide (NO) –15.in vitro and in vivo  and apoptosis-inducing factor (AIF) 28–or (AIF) . From thc and nuclear translocation of AIF via PTP opening, in NO-induced necrotic cell death mediated by GAPDH aggregation.We demonstrated previously that NO-induced cell death is attenuated by a GAPDH mutant with a substitution of Cys-152 to alanine (C152A-GAPDH) in a dominant-negative manner . Therefo50 for NOC18-induced decrease of cell viability in SH-SY5Y cells was ∼200 μm (A). Treatment with NOC18 caused GAPDH oligomer formation in cells and in insoluble fractions in a concentration-dependent manner (B). To validate the effect of GAPDH aggregation on mitochondria, we assessed mitochondrial membrane potential in cells using the anion dye rhodamine 123 (Rho 123). Compared with vehicle treatment, 200 μm NOC18 significantly increased Rho 123 fluorescence, indicating a disruption of the mitochondrial membrane, which was enhanced in a concentration-dependent manner with NOC18 (C). We next examined the correlation between the formation of GAPDH oligomers and the disruption of mitochondrial membrane potential; the result clearly indicated a strong correlation . These results indicate a relation between NO-induced GAPDH aggregation and mitochondrial dysfunction. Thus, in the following experiments, sufficient aggregates of GAPDH and mitochondrial dysfunction were observed after 48 h of treatment with 200 μm NOC18.As an oxidant, we selected NOC18, an NO generator . The IC5 ∼200 μm A. Treatmt manner B. To valth NOC18 C. We nexA). Successful mitochondrial fractionation was confirmed by the presence of cytochrome c oxidase (complex IV (CIV)) and the absence of histone H2B (a marker for nuclear fraction) and triosephosphate isomerase (a marker for cytosolic fraction). A large amount of GAPDH was present in the mitochondrial fraction, as reported previously  . Similarly, we used immunofluorescence to assess whether GAPDH was localized near the mitochondria using the cell-permeable mitochondrial-selective dye MitoTracker Red . Whereas the juxtaposition of the GAPDH signal was around the mitochondria in vehicle-treated cells, NOC18-treated cells showed punctate GAPDH-positive signals that were consistent with aggregates of GAPDH, which partially merged with MitoTracker Red, indicating mitochondrial localization of GAPDH aggregates . To identify where the aggregates of GAPDH occurred in cells, we examined the amounts of oligomeric GAPDH in mitochondrial fractions with or without DTT, which reduces GAPDH oligomers and causes dissociation to monomers . These results suggest that NO-induced aggregates are derived from mitochondrial GAPDH.To investigate the origin of the aggregates of GAPDH that induce mitochondrial dysfunction, we used Western blotting to study whether these aggregates exist within mitochondrial fractions in NOC18-treated SH-SY5Y cells A. Succest panel) . Furthermonomers . In the A, left panel). The absence of GAPDH in the isolated mitochondria was ascertained by Western blotting  and by measuring GAPDH enzyme activity . We showed recently that the addition of the GAPDH mutant C152A-GAPDH inhibits NO-induced amyloidogenic aggregation of wild type GAPDH (WT-GAPDH) in vitro  relative to vehicle treatment . The mixture containing aggregates of WT- and C152A-GAPDH significantly reduced the degree of swelling compared with that of WT-GAPDH alone . We also found that treatment with aggregates of WT-GAPDH caused robust mitochondrial membrane depolarization, indicated by an increase in Rho 123 fluorescence , and the mixture containing aggregates of WT- and C152A-GAPDH significantly attenuated this mitochondrial membrane depolarization . Further, we morphologically examined the effects of aggregates of GAPDH on isolated mitochondria using transmission electron microscopy , and mitochondrial swelling was assessed quantitatively . Compared with vehicle treatment, marked mitochondrial swelling was observed after treatment with aggregates of WT-GAPDH, whereas treatment with a mixture containing aggregates of WT- and C152A-GAPDH attenuated but did not abolish the formation of this abnormal morphology . Similar to treatment with aggregates of WT-GAPDH, the swelling of isolated mitochondria was observed after treatment with 1 mm Ca2+, which was used as a positive control. These results indicate that NO-induced GAPDH aggregation directly participates in mitochondrial dysfunction.We next evaluated whether GAPDH aggregation leads directly to mitochondrial dysfunction. It has been reported that the detectable amount of GAPDH bound to mitochondria differs depending on the method of isolation . TherefoFig. 3in vitro . TherefoF and G). Together, these results suggest that aggregates of GAPDH directly evoke mitochondrial dysfunction, resulting in the swelling and depolarization of mitochondria via the opening of PTP.One of the most convincing proposed mechanisms underlying mitochondrial swelling and depolarization is the PTP-induced mitochondrial swelling model . Based oA). In addition, NOC18-induced mitochondrial membrane depolarization was measured in these cells by Rho 123 fluorescence (B). Similar to the results shown in Fig. 3D, NOC18-induced mitochondrial membrane depolarization was significantly increased (by 2-fold) in cells overexpressing WT-GAPDH but inhibited by C152A-GAPDH overexpression (by 0.6-fold) (B). These results indicate that NO-induced aggregation of overexpressed GAPDH induces mitochondrial dysfunction in cells.To investigate the involvement of aggregates of GAPDH in mitochondrial dysfunction at the cellular level, we used doxycycline (DOX)-inducible WT- or C152A-GAPDH-overexpressing SH-SY5Y cells. Western blotting performed with an anti-GAPDH antibody showed that NOC18-induced GAPDH aggregation in mitochondrial fractions was augmented by overexpression of WT-GAPDH (by 2-fold) but was significantly attenuated by C152A-GAPDH overexpression (by 0.8-fold) A. In addrescence B. Simila.6-fold) B. These c, which goes into the cytosol, and AIF, which translocates into the nucleus . Further, NOC18-induced release of cyt c was significantly augmented by the overexpression of WT-GAPDH, whereas the release was decreased by overexpression of C152A-GAPDH (B). This result was also observed by immunofluorescence. In the absence of DOX, cyt c partially merged with MitoTracker Red. This mitochondrial cyt c decreased, likely by diffusion into the cytosol, by overexpression of WT-GAPDH in DOX-treated cells. However, the overlap of MitoTracker Red and cyt c was retained by overexpression of C152A-GAPDH (C). Similarly, we confirmed that NOC18 induced nuclear translocation of AIF in SH-SY5Y cells (D). The levels of AIF in the nuclear fraction from cells treated with NOC18 were significantly increased by overexpression of WT-GAPDH and decreased by overexpression of C152A-GAPDH (E). Immunofluorescence showed that overexpression of WT-GAPDH reduced the merging of AIF signals with those of MitoTracker Red and increased nuclear localization of AIF following NOC18 treatment (F). Moreover, the merging of AIF and MitoTracker Red signals was retained by overexpression of C152A-GAPDH, which did not induce nuclear translocation of AIF. Additionally, CsA treatment significantly inhibited NOC18-induced cytosolic cyt c release (G) and AIF nuclear translocation (H). These results imply that GAPDH aggregation participates in the cytosolic release of cyt c and the nuclear translocation of AIF through PTP-dependent mechanisms under oxidative stress.Initiation of cell death induced by oxidative stress from NO involves mitochondrial membrane depolarization and causes the subsequent release of two cell death mediators from the mitochondria: cyt  nucleus . Therefoncreased A. Furthe2A-GAPDH B. This r2A-GAPDH C. Simila5Y cells D. The le2A-GAPDH E. Immunoreatment F. Moreov release G and AIFlocation H. These A). To determine the type of cell death induced by GAPDH aggregates, we employed either WT- or C152A-GAPDH-overexpressing cells. Necrosis was significantly increased in cells overexpressing WT-GAPDH but was decreased by C152A-GAPDH overexpression (B and C). Notably, there were no significant differences in other types of cell death (B and C). Greater necrosis following WT-GAPDH overexpression was completely abolished by CsA treatment (D), indicating that NOC18-induced GAPDH aggregates cause necrotic cell death via PTP opening.We next evaluated the type of cell death induced by GAPDH aggregates using propidium iodide (PI)/annexin V staining. A large percentage of cell death induced by NOC18 treatment was necrotic , whereas early apoptosis (PI−/annexin V+ cells) and late apoptosis/necrosis (PI+/annexin V+ cells) were hardly seen A. To detpression , B and Cll death , B and Creatment D, indicaA). However, the decline of these proteasome activities induced by NOC18 treatment was not altered by overexpression of either WT- or C152A-GAPDH .In addition to mitochondrial dysfunction, several lines of evidence suggest that the proteasome is also involved in the toxicity of amyloidogenically aggregated proteins, such as amyloid-β and α-synuclein, and these aggregated proteins attenuate the activity of proteasomes in neurons and contribute to cell death , 40. TheFig. 7A), which was not altered in cells overexpressing WT- or C152A-GAPDH .We also evaluated the influence of GAPDH aggregation on ER stress, which is considered critical for aggregated protein-induced cell death, by assessing the expressions of the ER chaperone glucose-regulated protein 78  and the ER-related proapoptotic protein C/EBP homologous protein (CHOP) , 42. An Fig. 8A). The conversion from LCS-I to LCS-II is considered a marker of autophagy (B), which accumulates in autophagic vacuoles (C and D). Additionally, the viability of WT- and C152A-GAPDH-overexpressing cells was tested in the presence or absence of 3-methyladfenine (3-MA), an autophagy inhibitor. Similar to results from our previous report (E). Together, these observations indicate that proteasome activity, ER stress-related protein expression, and autophagy do not contribute to mechanisms that mediate GAPDH aggregate-induced cell death with NOC18 treatment.Lastly, we examined the effects of GAPDH aggregation on autophagy. In SH-SY5Y cells, the induction of autophagy by treatment with NOC18 was confirmed by modification of microtubule-associated protein 1 light chain 3B (LC3), observed as an increase in the amount of the faster migrating form, LC3-II A. The coutophagy . Concomiutophagy B, which vacuoles . By contvacuoles , C and Ds report , we obses report E. Togethc and nuclear translocation of AIF . Several studies have reported on the mechanisms of mitochondrial dysfunction induced by other aggregated proteins such as amyloid-β, α-synuclein, and mutant huntingtin via PTP  of GAPDH aggregates using Saville's method described in Ref. S-nitrosothiols (0.10 ± 0.02 SNOs/tetramer), similar to soluble GAPDH ). To further investigate the ability of GAPDH aggregate to act as a mitochondrial trans-S-nitrosylase, SNO levels of mitochondrial proteins were examined using isolated mitochondria and the biotin switch method. Compared with mitochondria incubated with vehicle, those treated with GAPDH aggregates did not show different SNO-protein band patterns . Although it is possible that the trans-S-nitrosylase activity of GAPDH may influence intracellular mitochondrial function, taking into consideration that GAPDH aggregates directly cause dysfunction in isolated mitochondria . Interestingly, a large population of cell death induced by GAPDH aggregates was necrotic . However, our results indicate that neither NO-induced proteasome inhibition nor ER stress is affected by altering the amount of GAPDH aggregation . 2A-GAPDH  and is r2A-GAPDH . Nonethe2A-GAPDH .supplemental Fig. S2). Treatment of naive SH-SY5Y with NOC18 induced distinct nuclear translocation of GAPDH . The amount of nuclear GAPDH was increased by overexpressing WT-GAPDH but not by overexpressing C152A-GAPDH . Further, we assessed the viability of WT- and C152A-GAPDH-overexpressing cells in the presence or absence of deprenyl, an inhibitor of GAPDH nuclear translocation, and showed that nuclear translocation of GAPDH does not regulate cell viability . Consequently, these results support those from our previous study showing that high levels of oxidative stress are unrelated to the nuclear translocation of GAPDH  from BD Biosciences; rabbit polyclonal anti-Myc , rabbit polyclonal anti-CHOP , and rabbit polyclonal anti-His  from Santa Cruz Biotechnology; mouse monoclonal anti-CIV (1D6E1A8) from Invitrogen; monoclonal anti-KDEL (GRP78) (10C3) from Stressgen; monoclonal anti-β-actin (AC15) from Sigma; mouse monoclonal anti-LC3 (2G6) from NanoTools; and polyclonal anti-triosephosphate isomerase (TPI) prepared in house  and rabbit polyclonal anti-histone H2B  from Millipore; rabbit polyclonal anti-AIF (AF1475) from R&D Systems; mouse monoclonal anti-cyt in house .The cloning of human WT GAPDH cDNA was performed as reported previously . For bacm glutamine, and antibiotics-antimycotics (Invitrogen) at 37 °C in a 5% CO2 humidified incubator. Generation of stable cell lines for inducible expression of GAPDH was established as reported previously (Human neuroblastoma SH-SY5Y cells (ATCC) were grown in DMEM/F12 supplemented with 10% FBS, 2 meviously . SH-SY5Yeviously .n NaOH) or the NO generator NOC18 (Dojindo), cells were washed twice with PBS and then incubated for 5 min in ice-cold PBS containing 40 mm iodoacetamide to protect unmodified thiols from oxidation during fractionation. All subsequent steps were performed at 4 °C. For isolation of the insoluble fraction, cells were scraped in 500 μl of buffer A ). After 30 min, the suspensions of cells were homogenized vigorously for 15 s with rocking. The total lysates were centrifuged at 20,400 × g for 10 min, and both the total cell lysates and pellets were collected to prepare insoluble fractions. To detect insoluble GAPDH oligomers in cells, the pellets obtained from total cell lysates were resuspended in 200 μl of buffer B ), washed three times by centrifugation (3000 × g for 10 min), and again resuspended in the buffer. After the addition of 100 μl of buffer B, the pellets were sonicated for 30 s and finally obtained as insoluble fractions. These fractions were stored at −80 °C for further use. Protein concentrations of the samples were determined using the Bradford assay (Bio-Rad).Subcellular fractionations were carried out according to the following procedures. After a 48-h treatment with either control vehicle . Homogenates were centrifuged at 5000 × g for 20 min at 4 °C, and then the cell pellet was resuspended in isolation buffer. The suspensions were homogenized and centrifuged (2000 × g for 5 min at 4 °C). Supernatants were further centrifuged at 11,000 × g for 10 min at 4 °C, and the resultant pellets were used as mitochondrial fractions.Mitochondria were isolated from cells as reported previously  with somm Rho 123 for 30 min at 37 °C with 5% CO2. Cells were washed with PBS and stained with Hoechst 33342. The fluorescence of Rho 123 was detected by a confocal scanning microscope . For semiquantification of fluorescence of Rho 123, four to seven microscopic fields were selected randomly, and the fluorescence values  were measured using an Ez-C1 free viewer (Nikon). Concomitantly, the total number of cells stained with Hoechst 33342 (2 μg/ml) in the same field was counted, and substantial values of fluorescence of Rho 123 were normalized.This procedure was performed as described previously  with minm Tris-HCl, pH 6.8, 0.5% SDS, 10% glycerol, and 0.002% bromphenol blue) and then heated at 100 °C for 5 min. These samples were separated by 5–20% non-reducing SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad). The membranes were incubated for 1 h with Blocking One  to block nonspecific binding. The membrane was then incubated for 2 h at room temperature with an anti-GAPDH monoclonal antibody (1:300) in 10% Blocking One-PBST (0.05% Tween 20 and 0.02% NaN3 in PBS) followed by incubation for 1 h at room temperature with a peroxidase-conjugated affinity-purified secondary antibody (Invitrogen). The signals were detected using both SuperSignal West Pico chemiluminescent substrate  and LAS3000 . To assess the purity of mitochondria isolated from cells, Western blotting was performed using an anti-CIV monoclonal antibody , an anti-histone H2B polyclonal antibody , or an anti-TPI polyclonal antibody ; membranes were also blotted with an anti-GAPDH antibody.To detect aggregates of GAPDH in insoluble and mitochondrial fractions, each fraction was mixed with low SDS sample buffer  for 30 min at 37 °C with 5% CO2. Cells were then washed with PBS and fixed with 4% paraformaldehyde in PBS, pH 7.4, for 10 min at room temperature. The cells were permeabilized for 5 min with PBS containing 0.1% Triton X-100 and incubated with 10% BSA in PBS for 1 h at room temperature. The cells were incubated overnight at 4 °C with an anti-GAPDH (1:1000), anti-AIF , or anti-cyt c (1:200) antibodies in 10% Blocking One-PBST. After three washes with PBST, the specific signal was visualized by staining the cells with an Alexa Fluor 568-conjugated secondary antibody  using a confocal scanning microscope .Immunofluorescence we performed as described previously  with minEscherichia coli strain W3CG (The pBAD-HisA vector carrying WT- or C152A-GAPDH cDNA was transformed into GAP(−) ain W3CG . Expressain W3CG , 15. Prom NOR3 ((±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide) in G2′ buffer . The prepared aggregates were applied immediately to various experiments in the present study.The aggregates of GAPDH were prepared as described in the published protocols . Brieflym Tris-HCl, 0.25 m sucrose, and 0.1 mm EDTA). After centrifugation at 80 × g for 7 min, the supernatant was layered on the same amount of solution B, pH 7.4, containing 10 mm Tris-HCl, 0.35 m sucrose, and 0.1 mm EDTA and centrifuged at 700 × g for 10 min. The supernatant was centrifuged at 7000 × g for 10 min. The pellet was resuspended in solution C, pH 7.4 (10 mm Tris-HCl and 0.25 m sucrose), and centrifuged again at 7000 × g for 10 min. The final mitochondrial pellet was resuspended in solution C. The solution containing the purified mitochondria was used immediately.Liver mitochondria from mice  were isolated as reported previously  with somg for 10 min. The pellet containing the mitochondria was fixed in 2.5% buffered glutaraldehyde and postfixed in 1% buffered osmium tetroxide. The samples were gradually dehydrated and then embedded in epoxy resin for 48 h at 60 °C. Thin sections (80 nm) stained with uranyl acetate and lead citrate were examined with an electron microscope (Hitachi H-7500) at 75 kV. A mitochondrial cross-sectional area was quantified by using ImageJ software .Isolated mitochondria were treated with aggregates of GAPDH for 30 min at room temperature and then centrifuged at 7000 × m triethanolamine, pH 8.9, 0.2 mm EDTA, and 50 mm K2PO4) was mixed with 1 mm NAD+ and 10 μg of sample. The enzyme activity was initiated by the addition of 2 mm glyceraldehyde-3-phosphate. GAPDH activity was measured for 1 min at 25 °C via the change in absorbance at 340 nm using a VERSAmax microplate reader .An assay solution . After adding aggregates of GAPDH, mitochondrial swelling was measured by the decrease in absorbance at 540 nm for 150 min. The mitochondrial depolarization assay was performed as described previously (m). After a 5-min incubation, aggregates of GAPDH were added, and the fluorescence  was measured using an ARBO multilabel plate reader (PerkinElmer Life Sciences). CsA (1 μm) was added 2 min before treatment with GAPDH aggregates for PTP inhibition.The mitochondrial swelling assay was carried out as described previously . The isoeviously . The isom NOC18 for 48 h, washed twice with PBS, and then incubated for 10 min in ice-cold hypotonic buffer . CsA (1 μm) was added 1 h before treatment with NOC18 for PTP inhibition. The lysates were centrifuged at 3200 × g for 5 min, and the supernatants and pellets were collected. The supernatants were further centrifuged at 100,000 × g for 60 min, and these supernatants were obtained as the cytosolic fraction. The pellets were suspended in isotonic buffer , washed twice followed by centrifugation at 3200 × g for 1 min, and resuspended in the buffer. After the addition of hypertonic buffer , the pellets were sonicated for 30 s and obtained as the nuclear fraction. Western blotting was performed as described above using polyclonal anti-AIF  and monoclonal anti-cyt c (1:500) antibodies. The membranes were also reprobed with an anti-TPI antibody (1:4000) as a cytosolic marker or an anti-histone H2B polyclonal antibody (1:5000) as a nuclear marker.Cells were treated with or without 200 μThe apoptosis assay was conducted using the MEBCYTO apoptosis kit  according to the manufacturer's instructions with minor modifications. In brief, cells were washed and resuspended in binding buffer. Hoechst 33342 (2 μg/ml), annexin V-FITC, and PI were added to the cell suspension, and then the mixture was incubated for 15 min in the dark at room temperature. Thereafter, the suspension was analyzed using a confocal scanning microscope . Quantitative analysis was performed by counting more than 700 cells in each examination.m Tris-HCl, pH 7.4, 0.1 mm EDTA, 1 mm 2-mercaptoethanol, 5 mm ATP, 20% glycerol, and 0.04% Nonidet P-40). Freeze-thaw cycles were repeated four times, and lysates were centrifuged at 13,000 × g for 15 min at 4 °C. Protein concentrations of the samples were determined by the Bradford assay. The reaction mixture contained 50 mm HEPES-KOH, pH 8.0, 5 mm EGTA, and 100 μm Boc-LRR-MCA or Suc-LLVY-MCA. The reaction was performed at 37 °C for 60 min and stopped by the addition of 1% SDS. The fluorescence was detected by an ARBO multilabel plate reader (PerkinElmer Life Sciences) at an excitation wavelength of 380 nm and emission wavelength of 440 nm.This assay was performed as described previously  with minm Tris-HCl, pH 7.5, 0.5% Nonidet P-40, 150 mm NaCl, 1 mm EDTA, 1 mm PMSF, 100 μm sodium vanadate, 1 mm sodium fluoride, and protease inhibitor mixture). After rotating for 30 min at 4 °C, the lysates were centrifuged at 20,400 × g for 15 min at 4 °C, and the supernatants were subjected to Western blotting as described above using monoclonal anti-KDEL (GRP78), polyclonal anti-CHOP, and monoclonal anti-β actin  antibodies.Cells were washed twice with PBS and lysed in buffer  and centrifuged at 16,400 × g for 20 min. The supernatants were subjected to SDS-PAGE and transferred to PVDF membranes. Western blotting was carried out with anti-LC3 (1:1000)  and anti-GAPDH antibodies. For MDC staining, the cells were treated with either vehicle or NOC18 and then stained with 200 μm MDC for 45 min. The cells were washed with PBS, and fluorescence was detected as described for cell immunofluorescence.For the detection of LC3, cells were lysed in lysis buffer (50 mn) in each figure legend (t tests or Dunnett's multiple tests after one-way analysis of variance, respectively.All data are presented as the mean ± S.D. of independent experiments as indicated (e legend –9. For sH. N. designed the study. M. I., T. K., A. K., N. H., R. Y., and H. N. performed the biochemical and cell-based studies. M. I., T. I., and M. K. performed the transmission electron microscopy. M. I., T. K., H. N., A. K., Y. A., and R. Y. analyzed the data. H. N. and M. I. wrote the paper, and H. N. and T. T. supervised the study.


PA-N155 and PA-N182 proteins were translated from the 11th and 13th start codon AUG of the RNA polymerase acidic protein (PA) mRNA of H5N1 influenza A virus (IAV), which plays an important role in viral replication. Little is known about the interactions between PA-N155 and PA-N182 and the host proteins. This study investigated the interaction landscape of PA-N155 and PA-N182 of H5N1 IAV in chicken cells while their interacting complexes were captured by immunoprecipitation and analyzed by mass spectrometry. A total of 491 (PA-N155) and 302 (PA-N182) interacting proteins were identified. Gene ontology and pathway enrichment analyses showed that proteins of the two interactomes were enriched in RNA processing, viral processing and protein transport, and proteins related to signaling pathways of proteasome, ribosome, and aminoacy1-tRNA biosynthesis were significantly enriched, suggesting their potential roles in H5N1 IAV infection. Comparative analysis of the interactome of PA, PA-N155, and PA-N182 identified UBA52 as a conserved host factor that interacted with all three viral proteins. UBA52 is a fusion protein consisting of ubiquitin at the N terminus and ribosomal protein L40 at the C terminus. Knockdown of UBA52 significantly decreased the titer of H5N1 IAV in chicken cells and was accompanied with attenuated production of proinflammatory cytokines. Our analyses of the influenza–host protein interactomes identified UBA52 as a PA interaction protein for virus replication. Influenza A virus (IAV) is responsible for seasonal epidemics and represents a serious threat to public health and the poultry industry . Avian iThe RNA polymerase system of IAV consists of PA, PB1, and PB2 . Many stin vivo to impede nuclear transport of PA and hence halt viral replication. IAV–host interactions in chicken cells, however, remain incompletely explored.Influenza A virus relies on host factors to support its life cycle, including transcription, viral RNA transportation, and packaging , so the In the present study, we investigated the PA-N155 and PA-N182 interactome of H5N1 IAV in chicken cells by affinity purification-MS (AP-MS); 491 (PA-N155) and 302 (PA-N182) interacting proteins were identified in chicken DF1 cells. Of these, 222 were common host proteins shared by PA-N155 and PA-N182, including many crucial genes involved in important biological processes. UBA52 was identified as a conserved host factor that interacted with PA, PA-N155 and PA-N182, and was a promotive factor for virus replication. Our study describes the fundamental landscape of PA-N155 and PA-N182 interacting proteins in host cells and promotes a better understanding of the mechanisms of IAV infection.2. Highly pathogenic H5N1 strain A/mallard/Huadong/S/2005 (SY) (Chicken embryonic fibroblast (DF1) cells and Madin–Darby canine kidney (MDCK) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco), 100 μg/ml streptomycin and 100 U/ml penicillin at 37°C, under a humidified atmosphere of 5% CO005 (SY)  was propPA-N155 and PA-N182 genes were amplified by high-fidelity DNA polymerase (Transgen) using cDNA derived from H5N1 virus (A/Chicken/ShanXi/2/2006) as a template. A 3× FLAG tag was inserted into the C terminus of the pcDNA 3.1 vector and the PA-N155 and PA-N182 genes were cloned upstream of the FLAG tag using the Seamless Assembly Cloning Kit (CloneSmarter) to make FLAG-tagged C-terminal fusion proteins. All expression vectors were validated by sequencing.Transfection with plasmids was performed with Lipofectamine 3000 (Life Technologies). Cells were harvested for protein extraction 48 h after transfection. The experiments were repeated three times independently, and cells grown in 3 cm × 10 cm dishes were used for transfection.The anti-FLAG, anti-MYC, and anti-β-Actin mouse monoclonal antibodies were obtained from Abmart.Transiently transfected cells were washed twice with PBS and lysed in RIPA buffer . Whole cell lysate was cleared with protein A/G slurry (Millipore) and then incubated with 40 μl anti-FLAG affinity gel (Sigma-Aldrich) for 2 h at 4°C. Immunoprecipitated samples were washed with RIPA buffer four times and twice with 54K buffer . FLAG tag associated proteins were eluted with 250 ng/μl FLAG peptide (Sigma) by rocking using a tilted rotator for 2 h at 4°C.For western blotting, after SDS-PAGE, proteins were transferred to polyvinylidene fluoride membranes. Membranes were blocked and incubated with corresponding antibodies. Proteins were visualized using an Immobilon Western Chemiluminescent HRP Substrate (Millipore).Strict experimental controls were used for the MS analysis. Immunoprecipitated samples from empty FLAG-transfected cells (empty FLAG control) and protein complexes that were pulled down by normal IgG (IgG control) were also subjected to MS for identification. All proteins identified in these two sets of controls were excluded as interacting proteins of PA-N155 and PA-N182. Authentic FLAG-precipitated proteins for PA-N155 and PA-N182 were examined in triplicate. Proteins that were enriched by co-IP were separated by SDS-PAGE and the entire lane was cut and sent for tryptic digestion.4OH) and B  were used: 5–8% B, over 5 min; 8–18% B, to 25 min; 18–32% B, to 32 min; 32–95% B, over 2 min; 95% B for 6 min; and 95–5% B over 5 min. The peptides were monitored at 214 nm and 1-min fractions were collected, dried and reconstituted in 20 μl of 0.1% (v/v) formic acid in water for nano-LC-MS/MS analyses. The fractions were further separated on a C18 column  with a flow rate of 250 nl/min. Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 0.1% formic acid and 100% acetonitrile. The Orbitrap Q-Exactive mass spectrometer was operated in the data-dependent acquisition mode using Xcalibur 3.0 software. The scan range was from m/z 300 to 1800 with resolution 70,000 at m/z 400. A full-scan followed by 20 data-dependent MS/MS scans were acquired with collision-induced dissociation with normalized collision energy of 35%. The MS/MS spectra from each LC–MS/MS run were searched against the protein database using the Proteome Discovery searching algorithm. Precursor ion mass tolerance was set to be 20 ppm and the fragment ion mass tolerance was 20 mmu. One missed trypsin cleavage event was allowed. The FDR rate of the LC–MS/MS is 1%. Oxidation (Met) was chosen as variable modifications. Carbamidomethyl (Cys) and TMT6plex were chosen as the fixed modifications.Proteins pulled down by anti-FLAG beads were digested with trypsin for 20 h at room temperature. Peptides were extracted twice with 50% aqueous acetonitrile containing 0.1% formic acid, dried in a Speed Vac and then desalted using Sep-Pak C18 cartridges. TMT reagents (Thermo Fisher) were used to label the purified peptides. The TMT labeling reagents in anhydrous acetonitrile were carefully added to the desalted peptides, incubated for 1 h at room temperature, and the reactions were stopped by hydroxylamine. The TMT-labeled peptides were separated by reverse phase (RP) chromatography. The first dimension RP separation by microLC was performed by an Ultimate 3000 System (Thermo Fisher) using a XbridgeC18 RP column . Mobile phases A .All proteins that interacted with PA were entered into The Search Tool for the Retrieval of Interacting Genes/Proteins STRING;  and thesAll the siRNAs used in this study were designed and synthesized by Guangzhou Ruibo . DF1 cells, at a confluence of 90% in 6-well plates, were transfected with 100 nM effective siRNA specific for chicken UBA52 gene (Gene ID: 395958); siUBA52-1, sense 5′-CCAAGAAGAAGGTCAAATA-3′. siUBA52-2, sense 5′-GGAGCCCAATGACACCATC-3′. The negative control siRNA was a scrambled siRNA for UBA52 . All siRNAs were transfected into DF1 cells using Lipofectamine 3000 (Life Technologies).The TCID50 assay was used to evaluate progeny virus production. After transfection with siRNAs, DF1 cells were infected with the SY virus at a multiplicity of infection of 0.1 PFU. At 1 h post infection (1 hpi), the medium was replaced with DMEM without fetal bovine serum. Conditioned media were collected at 12 and 24 hpi for measuring viral titers. Viral titers were determined by agglutination assay after growth in MDCK cells. The MDCK cells were seeded in 96-well plates and infected after reaching 85% confluence. Cells were washed with phosphate-buffered saline (PBS) twice and infected with a series of dilutions of viruses, and incubated for 72 h, as described above. Agglutination assays were performed in a round-bottomed 96-well plate using 1% chicken red blood cells in PBS.-ΔΔct method.The efficiency of UBA52 expression knockdown was confirmed by quantitative real-time PCR. Total RNA was isolated from DF1 cells using TRIzol reagent (Tiangen). One microgram of total RNA per sample was reverse transcribed into cDNA using FastQuant RT Kit (Tiangen). We next used the ABI Prism 7500 system (Applied Biosystems) in conjunction with One Step SYBR PrimeScript RT-PCR Kit II (TaKaRa) to analyze the expression of UBA52. The expression level of each gene, relative to that for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was calculated using the 2Levels of the selected cytokines and chemokines  in the supernatant of infected DF1 cells were determined using ELISA kits (Bio-Swamp). All samples were measured in triplicate, and the experiments were repeated three times independently.t-test was used to analyze the data for TCID50. For all the tests, p ≤ 0.05 was considered as being significant.The viral titers are shown as means ± standard deviations (SD) from three independent experiments. Independent-samples Figure 1A). AP was carried out against the FLAG tag to immunoprecipitate PA-N155 and PA-N182 associated host proteins. Protein complexes pulled down by immunoprecipitation were denatured and separated by SDS-PAGE and the gels were visualized using silver staining . Clear PA-N155 and PA-N182 bands were observed at the expected molecular weight, along with the arrays of immunoprecipitated host proteins. PA-N155, PA-N182 and their interaction partners were not observed in the empty FLAG control, indicating the specific enrichment of PA-N155- and PA-N182-associated proteins.The exogenous FLAG-tagged PA-N155 and PA-N182 were highly expressed in chicken DF1 cells, as revealed by western blotting (Table 1.The interacting proteins that remained after excluding those in the controls and then having a SAINT score > 0.89 were considered to be bona fide PA-N155 or PA-N182 interactors. This resulted in 491 (PA-N155) and 302 (PA-N182) interacting proteins being identified in chicken DF1 cells. Of these, 222 were common host proteins shared by PA-N155 and PA-N182 .To identify the specific protein families that associate with PA-N155 and PA-N182, we analyzed the protein–protein interactions among the PA-N155 and PA-N182 host-interacting factors using the STRING database . We thenFigure 2). Most sub-networks were found in both the PA-N155– and PA-N182–host networks, such as Coatomer subunits, and MCM components. MCM3, MCM4, MCM5, and MCM6 were found to interact with PA-N155 and PA-N182 . Eukaryotic translation initiation factor (eIF) subunits including eIF2S1, eIF2S3, eIF3B, eIF4A3, eIF2AK2, and eIF3M formed a network interacting with PA-N155 . Similarly, eIF2S1, eIF2S3, eIF3B, eIF4A3, eIF2S2, eIF3A, and eIF3D formed a network interacting with PA-N182 . The ribosomal proteins including RPS2, RPS3, RPS3A, RPS4X, RPS24, RPS15A, RPL18A, RPL26L1, RPL27, and RPL21 also interacted with PA-N155 as a protein complex.Many proteins associated with fundamental biological process were identified as PA-N155- and PA-N182-interacting proteins in DF1 chicken cells. Many proteins gathered forming several sub-networks in the PA-N155–host and PA-N182–host interactomes, implying a close relationship among them to form functional protein complexes (Table 2).Gene ontology (GO) analysis of the 491 PA-N155 and 302 PA-N182 host-interacting proteins was done with DAVID. Gene ontology enrichment of PA-N155 and PA-N182 host-interacting proteins included viral process, RNA processing, protein transport and cell cycle. Many other different terms, such as NIK/NF-κB signaling and Ribosome were significantly enriched only in the host proteins interacting with PA-N155 . Ribosome, aminoacyl-tRNA biosynthesis and proteasome were enriched in the interacting chicken proteins of both viral proteins . These results indicated that proteins involved in DNA replication, ribosomes, RNA transport, and proteasomes might be related to IAV infection and participate in IAV replication.All host factors interacting with PA-N155 and PA-N182 were analyzed for pathway enrichment using KOBAS . PathwayFigure 4). A total of 26 protein domains including HELICc, AAA, Cation_ATPase_N, DEXDc, MCM, RAP, and CDC48_2 were over-represented in both sets of host proteins associated with PA-N155 and PA-N182 . To determine the effect of UBA52 on the replication of IAV, we knocked down the expression by siRNA . The mixture of the two pairs of siRNA was used in the following experiment. The titers of progeny viruses were quantified after the knock down of UBA52. There was a significant reduction in the progeny viral titer in the supernatant of the UBA52-knockdown cells at 12 and 24 hpi , indicating a promotive role of UBA52 in IAV infection. To further investigate the effect of UBA52-knockdown on immune response upon virus infection, we tested the production of some cytokines and chemokines in the supernatant of infected DF1 cells. The infected UBA52-knockdown cells showed significantly reduced expression of IFN-β, IL-6, TNF-α, and CCL-4 at 12 hpi compared to the control group .UBA52  was shared by PA, PA-N155, and PA-N182. UBA52 occupied an important position in the interaction networks of RNA processing and viral process . The active interaction sources analyzed by STRING included text mining, experiments, databases, co-expression, neighborhood, gene fusion and co-occurrence, with minimum interaction scores exceeding 0.7. On the other hand, most cellular proteins perform biological functions in the form of functional protein complexes, so whether the interaction between viral proteins and host proteins mentioned above is direct or not remains to be further verified.Many studies have characterized the biological functions of the PA proteins, but fewer on the two truncated PA proteins and notably, the virus–host factors remain unknown. PA-N155 and PA-N182 are important for efficient viral replication . The intWD repeats (MDRs) are minimally conserved regions of ∼40 amino acids typically bracketed by Gly-His and Trp-Asp (GH-WD) that may facilitate the formation of heterotrimeric or multiprotein complexes. Members of this family are involved in a variety of cellular processes, including cell cycle progression, signal transduction, apoptosis, and gene regulation . WDR82 nRNF185 showed interaction with PA-N155. Much is known of RNF185, especially about its role associated with viral infections. As a mitochondrial outer membrane ubiquitin E3 ligase, RNF185 is involved in the regulation of selective autophagy . RNF185 Many host factors form a sub-network to interact with viral proteins. MCM3, MCM4, MCM5, and MCM6 were identified to have interactions with both PA-N155 and PA-N182. MCM functions as a scaffold between the nascent RNA chains and viral RNA polymerase . MCM2 toOne of the most important finding of our study is that UBA52 interacts with PA, PA-N155, and PA-N182 and promotes the replication of highly pathogenic H5N1 avian IAV. PA contains 716 amino acids, under the action of trypsin, which can be hydrolyzed into two parts: the N-terminal domain of PA  and the C-terminal domain of PA  . PA-N plQW, QL, TL, JW, DP, and GZ: conceived and designed the experiments. QW, QL, and ZS: performed the experiments. QW and QL: analyzed the data. ZG, FW, XY, RL, MZ, HC, GbC, GhC, HZ, and HL: contributed reagents/materials/analysis tools. QW, QL, and GZ: wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Binding ability of ligand-L to DNA/Cu(II) was evaluated using a plethora of biophysical techniques which revealed ligand-L-DNA and ligand-L-Cu(II) interaction. Competitive displacement assay and docking confirmed non-intercalative binding mode of ligand-L with ctDNA. Cyclic voltammetry confirmed ligand-L causes quasi reversible Cu(II)/Cu(I) conversion. Further, acute toxicity studies revealed no toxic effects of ligand-L on mice. To evaluate the chemotherapeutic potential and anticancer mechanism of ligand-L, DNA damage via pBR322 cleavage assay and reactive oxygen species (ROS) generation were studied. Results demonstrate that ligand-L causes DNA cleavage involving ROS generation in the presence of Cu(II). In conclusion, ligand-L causes redox cycling of Cu(II) to generate ROS which leads to oxidative DNA damage and pro-oxidant cancer cell death. These findings will establish ligand-L as a lead molecule to synthesize new molecules with better copper chelating and pro-oxidant properties against different malignancies.Despite substantial research on cancer therapeutics, systemic toxicity and drug-resistance limits the clinical application of many drugs like cisplatin. Therefore, new chemotherapeutic strategies against different malignancies are needed. Targeted cancer therapy is a new paradigm for cancer therapeutics which targets pathways or chemical entities specific to cancer cells than normal ones. Unlike normal cells, cancer cells contain elevated copper which plays an integral role in angiogenesis. Copper is an important metal ion associated with chromatin DNA, particularly with guanine. Thus, targeting copper via copper-specific chelators in cancer cells can serve as an effective anticancer strategy. New pharmacophore di(2-picolyl)amine (DPA)-3(bromoacetyl) coumarin (ligand-L) was synthesized and characterized by IR, ESI-MS,  DNA is the genetic material which regulates vital processes such as transcription, recombination, proliferation and cell survival. Targeting DNA to modify or inhibit its activity is a subject of extensive research for designing new anticancer agents against different malignancies . Among vTo minimize the toxicity and side effects of metal-containing drugs, new chemotherapeutic agents and therapies against different malignancies need to be developed. Targeted cancer therapy is a relatively new form of therapy which involves the use of drugs that block the growth of cancerous tissue by interfering with specific molecules/pathways and spare the normal cells . The basOne major difference between normal and cancer cells is the levels of copper which are elevated in malignant cells –11. CoppH-1-benzopyran-2-one; 1,2-benzopyrone; cis-o-coumarinic acid lactone; coumarinic anhydride) are a large group of naturally occurring organic compounds synthesized by plants, bacteria and fungi . Anal. Calcd. for C23H19N3O3: C, 71.67; H, 4.97; N, 10.90. Found: C, 71.64; H, 4.98; N, 10.91.Ligand-L crystallized from CHCl260nm / A280nm. Absorbance ratio was between 1.8 and 1.9, and therefore no further purification was required. DNA concentrations used in different experiments were determined using average molar extinction coefficient value of 6600 M-1 cm-1 of a single nucleotide at 260 nm  and Δ 4.15 (-CO-CH2-N-) correspond to four and two methylene protons (-CH2), respectively. A doublet at Δ 7.81 ppm for two protons has been assigned to aromatic H-5 and H-8 protons of coumarin nucleus. A pair of multiplet resonating at Δ 7.64–7.68 and Δ 7.20–7.53 have been allocated to H-6, H-7 (coumarin nucleus) (2 protons) and pyridine nucleus (8 protons), respectively. In 13C NMR spectral study, the absorption bands resonating at Δ 181.79 and Δ 166.81 have been assigned to α, β-unsaturated carbonyl and lactone carbonyl groups, respectively (2) resonating at Δ 64.20, 61.35 and 61.34, confirming the condensation of two substrate moieties to form ligand-L. The absorption bands Δ 116.53–157.85 have been assigned to other aromatic ring carbons of coumarin and pyridine nucleus. The mass spectral analysis was found to be in good conformity with the proposed structure.Ligand-L was typically synthesized perature . The com28 cm-1) . In 1H N (= C-H) . This apectively . The speTo understand the interaction of ligand-L with ctDNA and Cu(II) ions, UV-Vis spectroscopy was performed. Results showed that ligand-L exhibits maximum absorbance at ~292 nm . Additiomax emission of ligand-L (Fluorescence spectroscopy has been widely used to determine the interaction and binding mode of drugs/molecules to DNA due to high sensitivity and accuracy . In the ligand-L . This qusv) was obtained from Stern-Volmer equation  is the concentration of ctDNA or Cu(II) ions in the solution. Ksv was determined by plotting the ratio of fluorescence intensity (F0/F) in the absence and presence of ctDNA or Cu(II) ions as a function of increasing concentrations of DNA or Cu(II) ions. Ksv values were calculated from the slopes of 3 M-1 and (8.53 ± 0.01) × 103 M-1 for ctDNA and Cu(II) ions, respectively. Ligand-L-DNA complex exhibits a quenching constant value lower than the classical intercalators  is the concentration of DNA or Cu(II) used in the experiments. The values of binding stoichiometry and binding constant were determined by the slope and intercept of the plot log [(F0 –F)/F] vs log [Q], respectively. The results so obtained are summarized in Later, steady state fluorescence data was also used to calculate binding stoichiometry (n) and binding constant (K) of ligand-L-DNA and ligand-L-Cu(II) complexes using the equation:0) and entropy change (ΔS0) are the main quantities that determine the interaction forces: (1) ΔH0 > 0 and ΔS0 > 0, hydrophobic forces; (2) ΔH0 < 0 and ΔS0 < 0, van der Waals force’s and hydrogen bonds; (3) ΔH0 << 0 and ΔS0 > 0, electrostatic forces. The values of entropy change and enthalpy change were calculated at different temperatures using the van’t Hoff equation:−3 kcal mol-1 K-1). The value of ΔH0 and ΔS0 were obtained from the slope and intercept of the plot ln k vs 1/T, respectively  are given in 0 and ΔS0 values indicate that the major interaction forces in ligand-L-DNA complexation are hydrogen bonding and van der Waals forces. Negative ΔH0 and positive ΔS0 confirm the presence of electrostatic interactions between ligand-L and Cu(II). The negative values of ΔG0 confirmed that the interaction of ligand-L to DNA/Cu(II) is spontaneous in nature.Thermodynamic parameters were also calculated to determine the nature of binding forces between ligand-L and DNA/Cu(II). There are several kinds of binding forces that exist in interactions between drug and biomolecules such as hydrophobic forces, van der Waals forces, electrostatic interactions and hydrogen bonds , 63. Entectively  Figs. ΔGCompetitive binding experiments are extensively used to reveal the binding mode of small molecules to DNA. In order to decipher the binding mode, various DNA binding dyes such as EtBr  or Hoechst 33258 (groove binder) are used. This assay is based on the principle that any small molecule/drug that displaces the bound dye from DNA is expected to bind in a similar fashion as bound dye –67. TherTo further confirm the non-intercalative mode of binding, Hoechst dye based displacement assay was performed. Hoechst dye holds an ability to bind to the minor groove region of DNA and increases fluorescence intensity on binding to DNA , 67. Res0 = -5.03 × 102 kcal/mol), which suggests that the binding of ligand-L with ctDNA is enthalpy driven. It can also be seen from results in 0) for ligand-L-DNA complex which indicates that the interaction process proceeds spontaneously. It is also important to note that intercalative binding interactions are entropy driven whereas groove binding mode is enthalpy driven  is the concentration of KI in the solution. Ksv is the quenching constant obtained from the slope of F0/F vs [Q] plot. Ksv values determine the type of binding of molecules with DNA. Decrease in Ksv occurs in intercalation and it remains unchanged when interaction is electrostatic or when molecules bind to the groove. As evident from results, no significant difference in Ksv values is observed in the absence and presence of DNA for ligand-L  and lower inhibition constant (21.69 μM) confirm strong binding of ligand-L with B-DNA. Further, higher hydrogen bonding interaction number (5 H-bonds) confirms the greater stability of ligand-L with B-DNA. Finally, it is interesting to note that both the experimental binding free energy -ligand-L solution are shown in 0½ = 0.48 V . The separation between the cathodic and anodic peak current (Ipa/Ipc = 0.84) clearly confirms the formation of redox couple Cu(II)/Cu(I) via one electron transfer redox process.Cyclic voltammetry is an important technique for characterizing the redox properties of metal complexes . The cycx couple , 84 with2; (5) Hydrogen bond acceptors ≤ 10; (6) Hydrogen bond donors ≤ 5. Ligand-L under investigation follows all the parameters under ‘Rule of five’ (no violations) calculated via molinspiration and chemicalize.org servers, and revealed higher tendency of ligand-L towards drug-likeness , it should follow Lipinski ‘Rule of five’ which includes the following parameters: (1) Mass < 500; (2) Octanol-water partition coefficient (logP) ≤ 5; (3) Rotatable bonds ≤ 10; (4) Polar surface area ≤ 150 Ålikeness .in vitro RBC lysis testing  and hydroxyl radical (OH.) which are harmful to bio-macromolecules of cells. Superoxide anion and hydroxyl radicals were estimated using NBT assay and ctDNA substrate assay -ligand-L system was assessed on supercoiled pBR322 plasmid DNA. As seen from the agarose gel pattern, increasing concentrations of ligand-L alone (25–75 μM) and Cu(II) alone (25–75 μM) did not cause any DNA cleavage . On the in vitro and in vivo amino}acetyl)-stant, K  is high.ft model –94.ROS generation and DNA cleavage assays are suggestive of a role of Cu(II)-ligand-L interaction in ROS generation that causes DNA breakage. Further, experiments using neocuproine (copper chelator) and ROS scavengers have shown inhibition of DNA cleavage and ROS generation by Cu(II)-ligand-L complex, suggesting that ligand-L toxicity requires interaction with copper. Thus, copper ions act as a molecular target of ligand-L in mediating redox cycling that leads to ROS generation  and selective cytotoxicity against cancer cells.Ligand-L in the presence of transition metals like copper generates ROS via Fenton-like reaction. Such a quasi reversible Cu(II)/Cu(I) redox couple decomposes hydrogen peroxide to generate hydroxyl radicals, which act as proximal DNA cleaving agent .We believe that copper-dependent ROS generation by ligand-L is the primary step in inducing oxidative stress and DNA damage. Further, up-regulation of p53 mitochondrial gene, ATM/ATR pathway, down-regulation of anti-apoptotic protein (Bcl-2) and cell cycle related proteins  might be the secondary downstream effects of ROS generation induced by Cu(II)-ligand-L interaction . These eAmong oxygen radicals generated by Cu(II)-ligand-L system, hydroxyl radicals are the most electrophilic with high reactivity and also possess small diffusion radius. For hydroxyl radicals to cause strand scission, ROS should be generated in close proximity of cellular DNA –99. For To establish the role of ROS in cancer treatment, it is important to note that tumor cells have higher basal levels of intra-cellular ROS as compared to normal cells . StudiesIn summary, we synthesized, characterized and examined the biological relevance of coumarin nucleus-based DPA derivative (ligand-L). A series of biophysical and docking studies were implemented to predict the binding characteristics and interaction of ligand-L with ctDNA and Cu(II). Results clearly confirmed the formation of ligand-L-DNA and ligand-L-Cu(II) complex. Ligand-L engages in Cu(II) redox cycling to generate ROS that leads to DNA damage (nicking). Thus, ligand-L in all probabilities acts as pro-oxidant in the presence of Cu(II), leading to its cytotoxic action. Since tumor cells have elevated copper than normal cells, this provides a window of opportunity for ligand-L to be used as robust therapeutic agent for selective cytotoxic action against different malignancies with copper as the molecular target. Further studies are in progress in our laboratory to provide a deep insight into the molecular mechanism of anticancer activity of ligand-L in malignant cells. We expect this study to establish ligand-L as a lead molecule to identify or synthesize new anti-cancer drugs with better copper chelating and pro-oxidant properties.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 FigTreatment of plasmid pBR322 DNA with increasing concentrations of ligand-L alone (25–100 μM) (Lanes 1–3) and Cu(II) ions alone (25–100 μM) (Lanes 4–6). Lane C represents untreated (control) plasmid. Ligand-L and Cu(II) treatment alone were ineffective in plasmid DNA cleavage.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableVirtual screening of ligand-L showing drug-likeliness by (A) Molinspiration (B) chemicalize.org servers.(PDF)Click here for additional data file.