Phanerochaete chrysosporium, which is known for its versatile pollutant-biodegradation ability, possesses an extraordinarily large repertoire of P450 monooxygenases in its genome. However, the majority of these P450s have hitherto unknown function. Our initial studies using a genome-wide gene induction strategy revealed multiple P450s responsive to individual classes of xenobiotics. Here we report functional characterization of a cytochrome P450 monooxygenase, CYP5136A3 that showed common responsiveness and catalytic versatility towards endocrine-disrupting alkylphenols (APs) and mutagenic/carcinogenic polycyclic aromatic hydrocarbons (PAHs). Using recombinant CYP5136A3, we demonstrated its oxidation activity towards APs with varying alkyl side-chain length (C3-C9), in addition to PAHs (3–4 ring size). AP oxidation involves hydroxylation at the terminal carbon of the alkyl side-chain (ω-oxidation). Structure-activity analysis based on a 3D model indicated a potential role of Trp129 and Leu324 in the oxidation mechanism of CYP5136A3. Replacing Trp129 with Leu (W129L) and Phe (W129F) significantly diminished oxidation of both PAHs and APs. The W129L mutation caused greater reduction in phenanthrene oxidation (80%) as compared to W129F which caused greater reduction in pyrene oxidation (88%). Almost complete loss of oxidation of C3-C8 APs (83–90%) was observed for the W129L mutation as compared to W129F (28–41%). However, the two mutations showed a comparable loss (60–67%) in C9-AP oxidation. Replacement of Leu324 with Gly (L324G) caused 42% and 54% decrease in oxidation activity towards phenanthrene and pyrene, respectively. This mutation also caused loss of activity towards C3-C8 APs (20–58%), and complete loss of activity toward nonylphenol (C9-AP). Collectively, the results suggest that Trp129 and Leu324 are critical in substrate recognition and/or regio-selective oxidation of PAHs and APs. To our knowledge, this is the first report on an AP-oxidizing P450 from fungi and on structure-activity relationship of a eukaryotic P450 for fused-ring PAHs (phenanthrene and pyrene) and AP substrates.The model white rot fungus  However, little is known about the specific enzymes capable of oxidizing these chemicals as part of the biodegradation pathways in these microorganisms. Fungi have been shown to possess the capability to oxidize PAHs via P450-mediated mechanisms Polycyclic aromatic hydrocarbons (PAHs) and alkylphenols (APs) are two important groups of environmentally persistent and highly toxic chemicals that have received much attention for their slow biodegradation in the environment. Because of their persistence, these chemicals have the potential to bioaccumulate in the food-chain, causing adverse health effects in humans. PAHs are mutagenic and/or carcinogenic, and APs are endocrine-disrupting chemicals (EDCs) which can cause reproductive and other adverse health effects. Among the few microorganisms capable of oxidizing these persistent chemicals, fungi possess an extraordinary ability to completely degrade and/or mineralize (to COPhanerochaete chrysosporium (henceforth abbreviated as Pc), which belongs to the white rot group of the wood decaying fungi, is the most intensively studied white-rot fungus for its extraordinary ability to oxidize and/or mineralize a broad range of environmental toxicants, including PAHs and alkyl-substituted aromatics P. chrysosporiumIn light of these findings, the current study focuses on CYP5136A3 to assess its catalytic versatility and structure-activity relationship in terms of critical catalytic amino acid residues for biodegradation of PAHs and APs. The resulting information could improve strategies using P450-based biocatalysts for biotechnological and bioremediation applications. Additionally, considering the overall low sequence homology (12–23%) of this fungal P450 to its orthologs in humans and animals P. chrysosporium P450s (Pc-P450s) are heterologously expressed in the active form along with the homologous cytochrome P450 reductase (Pc-CPR) in Pichia pastorisin vitro enzyme reconstitution assays where uncoupling is the major factor that interferes in estimation of the turnover of the substrates. Hence, in this study, the whole cell biocatalyst strategy was employed using the recombinant yeast to characterize the catalytic properties of CYP5136A3 and its mutants  towards APs and PAHs. Phenanthrene and pyrene were used as the model compounds to understand the PAH catalysis, whereas APs with varying alkyl-chain length (C3-C9) were tested individually as model alkylphenols, in addition to the technical grade nonylphenol (tNP) that is comprised of more than 100 NP isomers containing a quaternary α-carbon Given the difficulties in heterologous expression of P450 genes of this fungus tNP mixture. The results provide experimental evidence on the role of Trp129 and Leu324 in governing the oxidation capacity and/or regio-selectivity of CYP5136A3 for oxidation of PAHs and APs.We observed broad substrate specificity of CYP5136A3 towards linear APs (C3-C9) and the E. coli TOP10 and Pichia pastoris KM71H (henceforth abbreviated as PP) were as described in the Pichia expression manual . PAH compounds (phenanthrene and pyrene), technical grade nonylphenol (tNP), 4-n-nonylphenol (4-n-NP) and 4-n-octylphenol (4-n-OP), 9-phenanthrol, and 1-hydroxypyrene were obtained from Sigma-Aldrich . 4-n-Heptylphenol (4-n-HTP), 4-n-pentylphenol (4-n-PTP), 4-n-butylphenol (4-n-BP), and 4-n-propylphenol (4-n-PP) were purchased from Fisher Scientific . 3-Phenanthrol was purchased from Toronto Research Chemicals, Canada and 4-phenanthrol from Chiron AS, Norway.The microbial strains, plasmids and primers used in this study are listed in P. pastoris.A binary vector containing CYP5136A3 and homologous reductase partner (Pc-CPR) cDNAs, generated in our recent study The binary vector containing the original coding sequence was used as a template for creating the W129A, W129L, W129F, and L324G mutants of CYP5136A3. The corresponding primers shown in PmeI-linearized binary vector constructs (PP using the Pichia Transformation Kit (Invitrogen). Co-presence of CYP5136A3 (mutated) and Pc-CPR in zeocin-resistant colonies resulting from the integrative transformation was confirmed by genomic DNA PCR. Site-specific genomic integration of the mutated CYP5136A3 and Pc-CPR was analyzed using three sets of primers  form of CYP5136A3 protein along with the homologous reductase Pc-CPR (named as PP WT) and a PP clone containing the empty vector (named as PP C), both generated in our recent study nstructs  containi primers . The binPP clones generated above was carried out per manufacturer's instructions  using slight modifications PP clone was inoculated into buffered minimal glycerol (BMG) medium (100 ml) and the cultures incubated at 30°C using 250 rpm speed until the optical density (OD) at 600 nm reached 2.0. Cells were pelleted by centrifugation (5000 g for 5 min), washed once with buffered minimal solution and resuspended in buffered minimal methanol (BMY) medium (21 ml) containing 0.5% methanol and aminolevulenic acid (2 mM) followed by incubation at 30°C and 250 rpm for 48 h. The cells were again pelleted and resuspended in 5 ml of lysis buffer . The cells were disrupted by vortexing  using an equal volume of acid-washed glass beads. The total volume was made up to 30 ml with lysis buffer and centrifuged at 5,000 g for 10 min at 4°C to remove cell debris. Supernatant was collected and centrifuged at 20,000 g for 30 min at 4°C to remove mitochondria. The resulting supernatant was again centrifuged at 100,000 g for 3 h to pellet. The pellet was resuspended in 4 ml of lysis buffer and the resulting microsomal preparation was used for confirming the expression of CPR and CYP5136A3 (mutant and wild type) proteins. P450 and CPR expression analysis in microsomes was done based on reduced CO-difference spectrum and NADPH-dependent cytochrome c reducing activity, respectively, as described elsewhere Recombinant protein expression in the PP C, PP WT, PP W129A, PP W129L, PP W129F, and PP L324G) was cultured as described above until the optical density (OD) at 600 nm reached 2.0. Cells were pelleted and resuspended in buffered minimal (BM) medium (21 ml) containing aminolevulenic acid (2 mM). The cell suspension was subdivided equally (7 ml each) into three conical flasks. Each flask was spiked with the test xenobiotic compound dissolved in methanol. All compounds were tested at 20 ppm final concentration and the concentration of the vehicle (methanol), which was also the carbon source in the medium, did not exceed 0.5% (v/v). An uninoculated control for estimation of the initial level of the compound and for assessing the degree of any abiotic degradation was prepared using the same medium and run along side the test flasks. All treatments were performed in triplicate. The cultures were incubated  for 48 h. At 24 h time-point, the cultures were spiked with methanol at 0.5% final concentration. At 48 h, 5 ml culture aliquots were removed aseptically and extracted (3×) with an equal volume of methylene chloride. The solvent extracts were dried on anhydrous sodium sulfate and resuspended in acetonitrile. Simultaneously, 1 ml of the final culture was centrifuged and the cell pellet dried to estimate the dry biomass weight, as described elsewhere To assess the capability of wild type and mutant forms of the CYP5136A3 enzyme, we performed whole cell-based P450 oxidation activity towards phenanthrene, pyrene and APs. The method employed for analysis of the oxidation activity was the same as described previously for PAHs PP clones incubated with different xenobiotic substrate compounds as described above, were filtered through 0.45-µm glass fiber filters and analyzed using Prostar 210/215 HPLC system  equipped with a C18 reverse-phase column (4.6 mm×250 mm) and a UV detector. Separation was achieved using a 20-min linear gradient of acetonitrile (ACN) in water  or 25-min linear gradient of ACN in water  at a flow rate of 2 ml/min. HPLC gradient for phenanthrene and pyrene metabolites was as follows: 50% methanol for 5 min, 50–75% methanol for 50 min, followed by a hold at 100% methanol for 2 min and 50% methanol for 3 min. PAHs and their metabolites were detected at 254 nm whereas APs and their metabolites were detected at 277 nm, and both groups of compounds were quantified based on standard curves generated using their known concentrations of parent compounds.Organic extracts, prepared for individual -n-NP and 4-n-OP; 50% to 90% for 4-n-HTP; 50% to 80% for 4-n-PTP; 50% to 60% for 4-n-BP and 4-n-PP), followed by a hold at 100% methanol for 6 min and 50% methanol for 4 min. A survey MS scan from m/z (100–500) at 30,000 resolution in the Orbitrap was paralleled by data dependent MS2, MS3, and MS4 scans at normalized collision energies of 25, 35 and 45, respectively. The results were analyzed using XCalibur (Thermo) and the experimental masses of metabolites were calculated by averaging 25 scans. The fragmentation patterns obtained from the MSn scans were compared with those reported in the literature APs and their P450-hydroxylated metabolites were analyzed by liquid chromatography-electrospray mass spectrometry (LC-ESI/MS) using the LTQ-Orbitrap coupled with a surveyor MS Pump Plus (Thermo Finnigan) as described previously Two 3D homology-based models of CYP5136A3 were generated using the Phyre server t test using GraphPad QuickCalcs software package .The substrate oxidation activities and the ratios of metabolites for different clones were analyzed for means and standard deviations and compared for statistical differences by Student tNP showed almost complete (98%) reduction of the parent compound (tNP) peak and appearance of a metabolite peak in PP WT  compared to PP C (P. pastoris clone containing empty PPICZB vector)  . There wtNP was difficult as it contains a mixture of over 100 p-isomers PP WT cells oxidized a number of APs with linear alkyl side-chain length ranging from C3 to C9, albeit to a variable extent. The C3-C5 chain-length APs including 4-n-propylphenol (PP), 4-n-butylphenol (BP), and 4-n-pentylphenol (PTP) were oxidized to a greater extent (93–95%) as compared to the C9 congener 4-n-nonylphenol . APs with C8  and C7  alkyl-chain lengths showed 78% and 89% oxidation, respectively. The PP C and PP WT cultures grown in the presence of different APs yielded the same biomass, indicating that there is no stimulatory or inhibitory effect of heterologous P450 expression on growth rate, and that the observed oxidation activity differences are due to the expressed CYP5136A3.Analysis of the catalytic specificity of CYP5136A3 towards tNP  , C2-C3 (120), C3-C4 (134/135), C4-C5 (147/148), C5-C6 (161), C6-C7 (173/175), C7-C8 (189/190) and C8-C9 (203)  from the parent ion of all the AP metabolites suggested that hydroxylation occurred at the terminal carbon atom of the alkyl-chain in different AP metabolites (ω-) hydroxylations in yeasts yield aldehyde metabolites due to the activity of the resident alcohol dehydrogenases on the P450-hydroxylated intermediate As observed in the HPLC chromatograms for tNP , the LC extracts . The ideextracts . The expmpounds) . In ordeC9 (203) . In addiabolites . The for3. This volume includes the putative substrate access channel. The volume of the cavity in the second model (based on 1A2) was estimated to be 2428.2 Å3, as this model did not indicate presence of a substrate access channel. In this study, we have chosen the 3A4-based model, as this template had a relatively higher overall sequence identity with CYP5136A3 as compared to other templates. In addition, sequence homology within the SRS regions between CYP5136A3 and the mammalian CYP3A4 template constitutes 34% sequence identity (“identity”) and 58% conservative identity (“similarity”). Major elements of the CYP5136A3 topology are indicated in Three-dimensional homology-based models of CYP5136A3 were built using the Phyre web-server -n-nonylphenol, were docked into the putative active site cavity. Contacting residues were identified from the top-scoring poses of the substrates (The CYP5136A3 3D model was subsequently used for ligand docking simulations by AutoDock 4 bstrates  using thbstrates . Out of PP clones with the control-positive (PP WT) and control-negative (PP C) clones.The effects of the mutations in terms of the expression levels of the P450 (CYP5136A3) and its oxidation capacity towards xenobiotic compounds were investigated by comparing the mutant PP clones expressing WT, W129A, W129L, W129F, and L324G forms of CYP5136A3 showed characteristic reduced CO-difference P450 spectrum as opposed to no such spectrum observed in the microsomes prepared from PP C  showed high CPR activity at comparable levels (PP C microsomes showed negligible native yeast CPR activity (50 nmol/min/mg protein). In order to further verify that the higher levels of CPR activity in the recombinant clones are due to the expression of cloned Pc-CPR, SDS-PAGE profiles of the microsomal proteins were compared. An intense band corresponding to Pc-CPR  is due to actual expression of the cloned Pc-CPR.Microsomes from all five e levels , in the o Pc-CPR  in the mPP WT and mutant PP clones allowed assessment of the catalytic role of Trp129 and Leu324 residues of CYP5136A3 in the oxidation of different APs and PAHs (phenanthrene and pyrene).Whole cell catalysis assays using the PP W129L showed higher reduction (81%) in phenanthrene oxidation activity as compared to PP W129F (56%), and both clones showed a significant decrease (76% by PP W129L and 88% by PP W129F) in pyrene oxidation (p<0.05). For APs, PP W129F showed a substantial loss in APs oxidation (60% for C9 AP and 28–41% for C3-C8 APs) whereas PP W129L showed an almost complete loss of oxidation activity for C3-C8 APs (83–90%) and a comparable loss in oxidation activity for C9 AP.HPLC profiles of the reaction extracts showed no considerable effect (p>0.05) of the W129A mutation on the extent of oxidation of either PAHs or APs . HoweverPP W129A, PP W129F, PP W129L, and PP L324G) yielded the same biomass as the wild type (PP WT) and the control (PP C) yeast clones, indicating (i) no adverse effect of the P450 mutations on the rates of growth, and (ii) that the oxidation activity differences among the clones are due to the P450 mutations.The L324G mutant caused a nearly complete impairment of NP oxidation (95%) when compared to the WT form. Furthermore, it showed almost 50% lower level of oxidation activity in case of OP, HTP and PP as compared to WT . OxidatiPP W129A, PP W129L, PP W129F, and PP L324G showed differences in metabolite profiles for phenanthrene and pyrene as compared to PP WT (The metabolite profiles for PAHs (phenanthrene and pyrene) and APs  were compared between WT and the mutants. The HPLC and LC-ESI/MS analysis of the xenobiotic reaction extracts prepared using to PP WT .viz. 3-phenanthrol, 4-phenanthrol and 9-phenanthrol in a ratio of 5.8∶40.7∶53.5. A different ratio (4.4∶32.0∶63.6) was observed in case of W129A mutant indicating that this mutation caused a decrease in the formation of 3-phenanthrol (−1.4%) and 4-phenanthrol (−8.7%), and an increase in the formation of 9-phenanthrol (+10.1%). Interestingly, in contrast with W129A, the W129F mutation  caused a decrease in 9-phenanthrol (−5.5%) level, and an increase in 3-phenanthrol (+0.8%) and 4-phenanthrol (+5.5%) levels compared to the WT form. W129L also followed the same trends as W129F and caused a higher level of 4-phenanthrol formation (+6.3%) and a lower level of 9-phenathrol (+4.1%) formation as compared to WT. On the other hand, the L324G mutant showed a large difference in the ratio of phenanthrene metabolites as compared to W129A and WT; the observed 0.1∶0.9∶99.0 ratio indicated an increased formation of 9-phenanthrol (+45.5%) and a decreased formation of 3- and 4-phenanthrols  when compared to WT. Interestingly, when calculated separately, just for the 3-phenanthrol∶4-phenanthrol ratio, it was comparable among the WT, W129A, and W129F, but appeared shifted for L324G towards a higher proportion of 4-phenanthrol  pointed to the promising potential of the CYP5136A3 enzyme as a catalyst for use in bioremediation of tNP. Our results showed that CYP5136A3 possesses a broad substrate-specificity towards linear APs with alkyl-chain length ranging form C3 to C9. Based on the results of this work, CYP5136A3 appears to preferentially target primarily short alkyl-chain APs, such as PTP, BP, and PP, over the longer alkyl-chain APs, such as NP and OP. Activity towards HTP was moderate .Our recent report on inducibility of CYP5136A3 by P. pastoris) dehydrogenase activity is involved in further oxidation of the hydroxylated AP metabolite generated by the recombinant fungal P450  in AP-metabolite is critical. Mass fragmentation of the metabolite showed that the aldehyde group is located at the terminal carbon of the alkyl-chain of each tested AP metabolite, confirming that CYP5136A3 is an alkyl side-chain ω-hydroxylase (ω-hydroxylation of varying chain-length (C3-C9) APs implied that this P450 plays a role in fungal biodegradation of APs via sequential shortening of the side-chain. This is consistent with our whole fungus-based studies on another structurally-related compound, the linear alkylbenzene sulfonate by P. chrysosporium, wherein sequential shortening of the 12-carbon linear alkyl chain was demonstrated In the whole cell P450 biocatalysis assays for APs, an aldehyde metabolite accumulated, rather than the expected hydroxylated AP, suggesting that the resident host yeast  and hydrophobic Leu324 with polar Gly . Collectively, these mutations were intended to confirm our homology model-based predictions on the catalytic role of these amino acids. This also allowed us to understand the catalytic relevance (specificity and/or regio-selectivity) of these residues in the fungus compared to their mammalian counterparts in this catalytic group of P450s  and Leu324 (L324G) residues in CYP5136A3 caused no adverse effect on the active expression of the mutant forms of CYP5136A3 as revealed by the unaltered typical CO-difference spectrum  versus the control clone (PP WT) enabled uniform supply of electrons for the wild type and mutant forms  of CYP5136A3. This directly supported the interpretation that the observed differences in the oxidation profile of PAHs and APs are due to the effect of mutations and not because of the differences in supply of electrons for the oxidation reaction, nor of the unfolding of the enzyme.Mutation of Trpspectrum . ComparaP. chrysosporium. Substitution of Trp129 with Leu (W129L) or Phe (W129F) caused loss of oxidation of APs. W129L was deleterious, as it resulted in complete loss of AP oxidation. This suggested that W129 of CYP5136A3 plays a key role in the oxidation of APs. Like W129L and W129F, the Leu324 mutation (L324G) was also found to be critical for the AP oxidation activity. Notably, L324G mutation caused a complete loss of oxidation activity towards 4-n-nonylphenol, the most environmentally recalcitrant congener among the APs, indicating a key role of L324 residue in alkylphenol oxidation activity. All these mutations had no effect on regio-selectivity of hydroxylation of APs as the metabolite profile was the same for the mutant forms and the WT form. This is the first report across the phylogenetic ranks on identification of critical residues in a P450 enzyme involved in AP oxidation.Although information on P450 oxidation of alkylphenols is available for certain mammalian P450s, studies on the P450 structure-activity relationship with respect to this group of compounds have not been reported. Here we identified two amino acid residues that play a key role in the oxidation of alkylphenols by the P450 enzyme CYP5136A3 from the model white-rot basidiomycetous fungus trans-dihydrodiols, . The three phenanthrols detected in the study were likely formed either by dehydration of the trans-dihydrodiols Cunninghamella eleganstrans-9,10-dihydrodiol is the predominant metabolite, CYP5136A3 showed almost comparable proportions of trans-9,10-dihydrodiol and trans-3,4-dihydrodiol (54∶46), as indicated by the relative amounts of 9-phenanthrol versus 3- and 4-phenanthrols  had no effect on the oxidation activity of CYP5136A3 towards phenanthrene, substitution with Leu (W129L) and Phe (W129F) decreased the oxidation of phenanthrene, with nearly a complete loss of oxidation for W129L. Although W129A had no effect on the overall oxidation level of phenanthrene, it altered the regio-selectivity of CYP5136A3 towards 9-phenanthrol formation. Interestingly, in contrast with the W129A mutation, W129F and W129L mutations led to high levels of 4-phenanthrol and low levels of 9-phenanthrol formation. This suggests that Trp129 of CYP5136A3 plays a key role in regio-selective oxidation of phenanthrene at both K-region  and non-K region .While substitution of Trp129 and Leu324 play a key role in the positioning of the phenanthrene towards the CYP5136A3 heme oxidation center. L324G converts the fungal P450 regio-selectivity into the mammalian P450-like regio-selectivity for phenanthrene . Taken together, our observations suggest that the two residues Trp129 and Leu324 are critical in modulating the regio-selectivity of PAH oxidation.L324G reduced the phenanthrene oxidation activity by half and favored the formation of 9-phenanthrol compared to the WT enzyme. While formation of 3- and 4-phenanthrols was slightly lowered in W129A, L324G mutation led to negligible levels of these phenanthrols. However, the ratio between 3- and 4-phenanthrols in the W129A, W129F, and L324G mutants still followed the NIH shift mechanism as in case of the WT form of the enzyme . This suK region of pyrene. Interestingly, to our knowledge, no other P450 across the prokaryotes and eukaryotes has been reported to yield any additional pyrenol (other than 1-pyrenol) from pyrene Pseudomonas putida129 (W129A and W129L) and Leu324 (L324G) favored the formation of 2-pyrenol, its amount was predominant in L324G as compared to W129A and W129L, suggesting the role of these amino acid residues in the regio-selectively of pyrene (1- C position) oxidation. Because of the low level of pyrene oxidation by W129F mutation and difficulty in quantification of its pyrene metabolites, the role of this substitution in regio-selective oxidation of pyrene is not clear.CYP5136A3 oxidized pyrene into two monohydroxylated metabolites, 1-pyrenol and 2-pyrenol in a ratio of 64∶36  suggesti129 and Leu324) involved in substrate specificity and/or regio-selectivity for the oxidation activity. This marks the beginning of our understanding of the catalytically significant amino acid residues governing the oxidation of alkylphenols and the tested fused-ring PAHs phenanthrene and pyrene in this class of eukaryotic P450s In conclusion, this study led to functional characterization of the first fungal P450 (CYP5136A3) with a broad catalytic ability to oxidize both alkylphenols  and PAHs, the two highly toxic environmental contaminants. The information generated is significant considering that the identified P450 is part of the overall enzymatic apparatus in this versatile fungal organism, and thus could be targeted for improved bioremediation applications including those involving mixture of these chemicals. Structure-activity analysis led to the identification of two key catalytic amino acid residues . The primer sequences used are listed in Schematic representation of the strategy employed for screening of (TIF)Click here for additional data file.Figure S2P. pastoris PP WT whole cell assays. Extracted ion chromatograms for APs constructed with cut-off molecular mass of 233.0–233.5 (NP metabolite), 219.0–219.2 (OP metabolite), 205.00–205.13 (HTP metabolite), 177.05–177.10 (PTP metabolite), 163.05–163.10 (BP metabolite), and 149.05–149.10 (PP metabolite). Inset: Extracted ion chromatograms-mass spectrum for the respective AP metabolites. Abbreviations: NP, 4-n-nonylphenol; OP, 4-n-octylphenol; HTP, 4-n-heptylphenol; PTP, 4-n-pentylphenol; BP, 4-n-butylphenol; PP, 4-n-propylphenol.LC-ESI/MS analysis profile of the alkylphenols (APs) oxidation products from (TIF)Click here for additional data file.Figure S3Structure alignment  of the two 3D models of CYP5136A3 built based on human P450s CYP3A4 (PDB ID 1tqn) and CYP1A2 (PDB ID 2hi4) using the Phyre server. Aligned tertiary structures of the models differ by 3.2 Å root mean square deviation (RMSD). Rendered in blue is the model based on the 3A4 template (t3A4); the yellow is based on 1A2 (t1A2).(TIF)Click here for additional data file.Figure S4Structure-based alignment of amino acid sequences and corresponding secondary structure (SS) states of the CYP5136A3 models. SS were computed by the DSSP program and reduced to 3 states: H – α-helices, E – β-sheets, and L – loops. Structurally non-equivalent residues are in lowercase. P450 topology (based on t3A4) is labeled alphabetically for helices and numerically for sheets. Residues labeled with asterisk were mutated in this study (W129A and L324G).(TIF)Click here for additional data file.Figure S5PP WT) and mutant form of CYP5136A3 (PP W129A). Oxidation of PAHs and APs by PP W129A clone was comparable with PP WT. The values represent means ± standard deviations for three biological replicates. Abbreviations: Phe, phenanthrene; Pyr, pyrene; NP, 4-n-nonylphenol; OP, 4-n-Octylphenol; HTP, 4-n-heptylphenol; PTP, 4-n-pentylphenol; BP, 4-n-butylphenol; PP, 4-n-propylphenol.PAH- and APs-oxidizing activities of the wild-type CYP5136A3 ((TIF)Click here for additional data file.


It is this form that is competent for specific encapsidation of the viral genome. Second, the P protein binds to oligomeric N in the nucleoprotein complex (N-RNA-P complex), and thereby facilitates the recruitment of the viral polymerase (L) onto its template. All previous attempts to study these complexes relied on co-expression of the two proteins in diverse systems. In this study, we have characterised these different modes of N-P interaction in detail and for the first time have been able to reconstitute these complexes individually in vitro in the chandipura virus (CHPV), a human pathogenic NNS RNA virus. Using a battery of truncated mutants of the N protein, we have been able to identify two mutually exclusive domains of N involved in differential interaction with the P protein. An unique N-terminal binding site, comprising of amino acids (aa) 1–180 form the N0-P interacting region, whereas, C-terminal residues spanning aa 320–390 is instrumental in N-RNA-P interactions. Significantly, the ex-vivo data also supports these observations. Based on these results, we suggest that the P protein acts as N-specific chaperone and thereby partially masking the N-N self-association region, which leads to the specific recognition of viral genome RNA by N0.The nucleocapsid protein (N) and the phosphoprotein (P) of nonsegmented negative-strand (NNS) RNA viruses interact with each other to accomplish two crucial events necessary for the viral replication cycle. First, the P protein binds to the aggregation prone nascent N molecules maintaining them in a soluble monomeric (N Rhabdoviridae in the order Mononegavirales, which also includes vesicular stomatitis virus (VSV) and rabies virus (RAV). This virus belongs to the broader group of negative-strand RNA viruses (NSRVs), which includes many pathogenically significant viruses, like avian influenza, measles, and Ebola. CHPV has repetitively caused severe outbreaks of encephalitis in parts of India Chandipura virus (CHPV) is a prototype member of the family 0-P) CTD). It showed that decameric structures of N enwrapping a 90 nt RNA which remains associated with 5 molecules of P protein (N∶P molar ratio of 2∶1) Encapsidation of the genome RNA by N protein is not only essential for protecting the viral genome from RNase action, but, is also believed to play a major role in the switching of the viral transcription to replication mode 0-P), the interacting domains of N, and the nature of the interaction at large, remains uncharacterised. One of the major obstacles for such biochemical and structural studies has been the unavailability of soluble N0 protein. However, recently Leyrat et al. has reported the structure of a N-terminal 21 aa deleted version of the VSV N protein with the first 60 aa of VSV P (NΔ210-P60) 0 interacting domains of the P protein. Chen et. al. has shown that for VSV, N-terminal 11–30 amino acids of P protein are essential in keeping N in soluble form (N0-P complex formation) 0-binding region of VSV P was shown to be globally disordered and encompasses the transient α-helices 0-P interactions 0 and N-RNA.However, for the monomer N-P complex . This helps sheds light upon the intricate molecular machinery that controls encapsidation in CHPV and other rhabdoviruses at large. We have also suggested a model that helps to explain the molecular basis of the N specific chaperone like activity of P protein, and subsequent specific encapsidation of the viral RNA by N.In this study, we chose to characterise the P binding region(s) of CHPV N. Recently, we have delineated the self-association and RNA binding domains of CHPV N To monitor the interaction of CHPV N and P proteins in living cells, in isolation of other viral proteins, the two proteins were either expressed individually or co-expressed in different molar ratios in Vero-76 cell line. Immunofluorescence against untagged N (pCDNA3.1(+) N) and P protein (pCDNA3.1(+) P) reveals that, when expressed alone, N protein exhibits a punctate distribution, while P protein demonstrated a homogeneous distribution, throughout the cytoplasm . TransfeCo-transfection of the plasmids encoding EGFP-N and untagged P proteins in a 1∶1 ratio resulted in redistribution of the punctate structures of N into a complete homogenous distribution . Among cth fraction; showing considerably higher sedimentation value than decameric N . It seems that this population represents a low molecular weight complex, consisting of single subunits of N0-P proteins, as depicted by sedimentation values. Although, it is difficult to determine the exact stoichiometry of N to P, this experiment clearly shows the formation of two distinct complexes among N and P when co-expressed. In addition, presence of RNA in the two complexes was tested by measuring their A280 nm/A260 nm ratio. Higher RNA content should be reflected by a lower A280 nm/A260 nm ratio compared to fractions with lesser or no RNA content. The larger complex (7th fraction) corresponding to decameric N-P populations, exhibited a much lower A280 nm/A260 nm ratio compared to the 17th fraction, which corresponds to N0-P complexes (data not shown). This confirms that the larger complex is an N-RNA-P complex, where decameric N have encapsidated cellular RNAs non-specifically. The smaller N0-P complex seems to be devoid of any cellular RNA.To further validate this data, cells expressing either EGFP-N alone or EGFP-N and P together in different ratios were lysed and centrifuged at 13,000 rpm. Supernatant and pellet fractions were analyzed for N and P using specific antibodies . As expeNext, a cell free assay system was employed to further characterize the different N-P interactions and the detail stoichiometry involved in it. To this end, we have performed size-exclusion chromatography through Superdex-200 column using bacterially expressed, purified N and P proteins th ml fraction  results in the dissociation of the N oligomers into monomers fraction , the estin vitro. The purified truncated proteins were subjected to size-exclusion chromatography through S-200 column to ensure their oligomerization status as was reported previously Next, to establish the domain(s) of the N protein involved in its interaction with P, we have employed a set of deletion mutants of N previously described in reference CTD complex which demonstrated the presence of such C-terminal P binding domain active only in the ring shaped decameric structures of the protein Next, His-tag co-elution assay was employed to determine the interacting ability of these truncated N proteins with the full length His-P. To this end, His-P was incubated either with wild-type or different truncated N proteins and the resulting mixture was then subjected to binding with Ni-NTA resin and subsequently eluted as described in material methods . Co-elut0-P complex after removal of DOC. This phenomenon was used to verify the ability of the C-terminal deletion mutants N(1–320), N(1–220) and the middle deletant N(1/180–265/422) to interact with His-P protein in their monomeric form. It is noteworthy that, although these mutants retain the proposed N-terminal P binding domain, they failed to interact with P protein in their native self-associated form  forms trimers along with higher oligomers .From the data presented so far, it is evident that in addition to the C-terminal P binding domain, the CHPV N possesses another unique N-terminal P interacting region which is functional only in the monomeric form of the protein. Now, the self-association domain of N has been reported to reside in the chymotrypsin resistant N-terminal 320 amino acids of the protein 0 and one P. However, complexes of other stoichiometry are also observed. This certainly suggests that a major fraction of N(1–320) is retained in its monomer form by forming a 1∶1 complex with P. DOC treatment does not have any permanent effect upon the oligomerization ability of the protein as was evident from the identical gel-filtration profile of the DOC removed and untreated sample (not shown). In agreement with the His-tag co-elution data, P fails to interact with oligomeric N(1–320) (DOC untreated) and therefore, does not have any effect upon its gel-filtration profile . Thus, the N-terminal 320 amino acids of CHPV N have all the necessary contacts to interact with P under monomeric conditions.To validate our hypothesis, we have chosen the mutant N(1–320) which lacks the C-terminal P interacting region but possesses the N-terminal one. Also, this is the largest possible C-terminal deletion of the protein retaining the ability to form characteristic oligomeric structures like the wild-type N ex-vivo, we monitored the effect of co-expression of P upon the cellular distribution of GFP-N(1–320). As evident from in vitro gel-filtration analysis.Next, to substantiate this observation 0) in specific encapsidation of the viral genome in presence of large excess of cellular RNAs, during viral replication 0-P complex CTD) 0 binding domain at the N-terminal region of P 0 that participate in this interaction. This is primarily due to the lack of soluble preparations of monomeric N (N0), which till date has been a major hurdle to structural and biochemical study of the N0-P complex.Recent work with the CHPV N protein has put an emphasis on the obligatory role of monomer N (N0-P). It appears that, within the system, these two complexes remain in equilibrium, and probably for this reason, it is difficult to modulate the abundance of one of these complexes at the expense of the other. Here we have been able to reconstitute the formation of both oligomer N-P and monomer N-P complexes in vitro, independent of one another. Incubation of individually expressed  soluble N and P proteins together, resulted in a high molecular weight complex, composed of octameric/decameric nucleocapsid like structures of N associated with P in 2∶1 ratio. This complex vastly resembles the VSV N-P complex encountered by Green et al. while co-expressing N-P in E. coli00-P complex. Moreover, formation of this complex resulted in inhibition of N-N self-association, re-establishing the role of P in retaining N in its monomeric form. Interestingly, these in vitro results are in accordance with our ex-vivo data, where dual mode of N-P interaction is reflected when co-expressed in live cells. According to our observations, co-transfection of N and P in a 1∶1 ratio resulted in solubilisation of aggregated N, and co-localization of the two proteins together, indicating N0-P complex formation. However, co-transfection in a 1∶0.5 ratio resulted in co-localization of the otherwise homogenous P, with punctate N, which we deem to be representative of oligomer N-P complexes. A similar observation has been described by Omi-Furutani et al. for the Nipah virus Co-expression of VSV N and P proteins in different expression systems results in formation of multiple N-P complexes of various stoichiometries 0-P). Primary indications for the possibility of these two independent domains came from His-tag co-elution assays with different truncated versions of the N protein, either untreated or pre-treated with 1% DOC. It has been found that DOC untreated oligomer N possesses a C-terminal P binding domain residing between residues 320–390. However, this P binding domain is only active in the proper nucleocapsid like decameric structures of N, as evident from the incapability of mutants N(180–422) and N(1/180–265/422), to interact with P under similar conditions. These mutants although retain their C-terminal domain intact, lacks the ability to form proper nucleocapsid like structures like the wild-type protein. Presence of such P binding site in the nucleocapsid has also been observed previously by the crystal structure analysis of VSV nucleocapsid-RNA-P complex In the present work, we have successfully identified two unique P interacting regions in the CHPV N protein. While a C-terminal region has been found to bind P only under oligomeric conditions, another N-terminal region has been identified, which mediates interaction of monomeric N with P , N(1–220), N(1/180–265/422) in their monomer forms generated by prior treatment with DOC. N(1/180–265/422) is interesting in this respect because, though it does not form decameric structures, it is capable of forming dimers  in the His-tag co-elution assay, opens up the possibility of a new P interacting region within N. This is because, N(48–422) is incapable of oligomerization and exists solely as a monomer, and thus, does not have a functional Pg dimers , which, 0). Support in favour of this mechanism came when DOC treated N(1–320) was allowed to form oligomers in presence of the P protein. It must be remembered that N(1–320) is the smallest possible C-terminal truncation of the N protein retaining its ability to form characteristic oligomeric structures like the wild-type protein 0-P complex. Furthermore, co-expression of N(1–320) with P in live cells in 1∶1 ratio resulted in redistribution of the punctate structures of this protein into a homogeneous form, confirming the chaperone like activity of P upon N(1–320). Together, these data confirms that interaction of P with the N-terminal portion of N is sufficient to block the N-N self-association and subsequently inhibit the oligomerization process. Our data validates the prediction made by Curran et al. 0-P from aggregation. However, in a pre-formed nucleocapsid this domain is involved in interaction with neighbouring N subunits and hence unavailable for interaction with P. In such form, the C-terminal domain represents the only binding site for P. A similar observation has been reported recently by Leyrat et al., where they have shown that the N0-binding region of P competes with the N terminal arm of a neighbouring N molecule, thus preventing N-N self assembly A challenging question that remains is how interaction with P inhibits the self-association of N and therefore, maintains it in its soluble form. We have previously shown that formation of helical nucleocapsid like structure of CHPV N is dependent upon the interaction of the N-terminal arm (residues 1–47) of one N monomer with the central region (residues 180–265) of another adjacent N moiety 0) by its interaction with the P protein (by N0-P complex formation). In this context, P masks the self-association domain of N and thus maintains it in a monomer form. Previously, we have shown that CHPV N0 is capable of specifically recognizing the viral leader sequence and the C-terminal 102 amino acids are essential for this recognition. On the other hand, upon oligomerization, a new RNA binding cavity is formed utilizing the N-terminal arm (1–47 aa) and the central region of N 00 binding site could be a thrust to replace the N from P. Release of P from N0 unmask its self-association domain, hence can trigger the N-N self-association to begin, leading to helical nucleocapsids. During this self-association, a new RNA binding surface is generated using the N-terminal two-third of N, which is capable of accommodating diverse RNA sequences in the elongation phase of encapsidation Finally, we present a model  that expRhabdoviridae family Mononegalovirales as well. This newly elucidated P binding region may serve as a potential target for designing novel therapeutics interventions.It is interesting to note that CHPV nucleocapsid not only shares common architecture with other viruses in the Oligonucleotides used in this study were purchased from Integrated DNA Technologies (IDT), USA. All column chromatography materials were from GE Healthcare. Ni-NTA resin was from Qiagen. Foetal Bovine Serum, antibiotics and Trypsin-EDTA needed for cell culture purpose were supplied by Invitrogen™ (GIBCO). Radioactive biomolecules were from BRIT, India. All other chemicals and biochemicals were of analytical grade.2 incubator at 5% CO2, 80% humidity and 37°C temperature.Vero-76 cell lines supplied by NCCS, Pune were grown as monolayers in DMEM enriched with 10% FBS, 100 U/ml Penicillin-Streptomycin, 2 mM Glutamine in tissue culture treated flask (BD Biosciences) in CODifferent GFP tagged, truncated versions of CHPV N were created by PCR amplification from pET3a-NC E. coli, BL21(DE3) or in BL21(DE3) pLysS. The expression and purification of full-length untagged protein was carried out as described earlier All full-length and deletion mutants of N were expressed in 5 Vero-76 cells per well. Cells were transiently transfected with 2 µg of either pEGFP-C1 containing cDNA of wild-type or mutant forms of CHPV N alone, or co-transfected with 2 µg (or 1 µg) pCDNA 3.1(+) containing cDNA of CHPV P. Transfection were performed with 6 µl of TurboFect™ in vitro Transfection Reagent (Fermentas) according to manufacturer's protocol.One day prior to performing transient transfection, 35 mm tissue culture plates (containing coverslips for immunofluorescence) were seeded with 4×10Polyclonal anti-CHPV N and anti-CHPV P has been described previously At 24 hrs of post-transfection, cells were washed with phosphate buffer saline (PBS) and lysed in lysis buffer (20 mM Tris-HCl (pH 8), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% TritonX-100, 5 mM DTT) and sonicated on ice with a 5 second pulse. After centrifugation at 13000 rpm at 4°C for 30 minutes, the supernatant containing the soluble protein pool were layered over a 10–60% sucrose step gradient and were centrifuged for 16 hours at 32000 rpm in a SW44 Beckman rotor at 4°C. 0.5 ml fractions were collected from the bottom of the gradients, precipitated with Trichloroacetic acid (TCA) and resolved in a 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were Western blotted (WB) with N and P antibodies to check their distribution pattern, according to standard protocols. Densitometric analysis of WB were performed with ImageQuant™ TL software  within the linear dynamic range of detection.Equimolar amounts of bacterially expressed, purified N and P proteins were incubated together in 100 mM NaCl containing TET buffer , at 4°C for 30 minutes. The resulting complex was then subjected to gel-filtration chromatography through S-200 (10/300) column , pre-equilibrated in the same buffer. For dissociation of the N oligomers, N was pre-treated with 1% Sodium Deoxycholate (DOC) at room temperature for 30 minutes followed by overnight dialysis against 100 mM NaCl containing TET buffer in presence of equimolar amounts of P protein. The resulting adduct were then subjected to gel-filtration chromatography as mentioned above. 0.5 ml fractions were collected, and resolved on 12% SDS-PAGE. Bands were visualised by silver staining and densitometric analysis of bands were performed with ImageQuant™ TL software  within the linear dynamic range of detection.Wild-type or truncated N proteins were incubated with 6×His-P protein in binding buffer , at 4°C for 30 minutes and the resulting complexes were allowed to bind to Ni–NTA agarose pre-equilibrated in the same buffer. A 10 µl aliquot of the complex was kept aside before addition of Ni-NTA, as loading sample (L). Binding was allowed for 1 hour, and the flow-though (FT) was collected. After consecutive washings with 10 mM (W), 20 mM and 50 mM Imidazole, the proteins were finally eluted with 250 mM Imidazole (E). L, FT, W and E were resolved in 12% SDS-PAGE and visualised by Coomassie Blue Staining. Alternatively, N variants were treated with 1% DOC and dialysed against the binding buffer in presence of P protein. The complexes formed were subjected to the co-elution assay mentioned above.35S labelled methionine and cysteine  in the same medium, for 2 hrs. Cells were then washed with PBS and lysed as mentioned previously. Lysates were incubated overnight with polyclonal anti-P antibody (1∶400 dilution) and the protein complexes were immunoprecipitated using Protein-A Sepharose™ CL-4B  for one hour, according to manufacturer's instructions. After subsequent washing with lysis buffer, the sepharose beads were boiled with 1× protein loading dye for 5 minutes and samples were resolved in a 10% SDS-PAGE and subjected to autoradiography.pCDNA 3.1-N and pCDNA 3.1-P were co-transfected into Vero-76 cells, grown in 35 mm culture plates. At 24 hours of post-transfection, cells were starved in DMEM lacking methionine and cysteine (PAN Biotech) for another 30 min and then exposed to 50 µCi/ml of Figure S1CHPV P proteins acts as an N-specific chaperone when transfected at a 1∶1 ratio in Vero-76 cell lines. Upon transfection of Vero-76 cells with GFP-N and P encoding constructs in a 1∶1 ratio, about 90% cells exhibited homogenization of the otherwise punctated distribution of GFP-N. The remaining 10% cells, were found to be lacking in the expression of P, and therefore showed characteristic punctated structures of N. This image shows the three possible types of cells in one field. Cells co-transfected with both GFP-N and P plasmids resulted in homogenization of N aggregates (NGFP+P). Cells that were transfected with GFP-N alone continued to exhibit punctated pattern of GFP-N distribution (NGFP), while cells that received P alone, showed typical homogenous distribution of P throughout the cytoplasm (P-IF). Immunofluorescence against P was performed with P Ab and anti-rabbit TRITC conjugated secondary antibody. Images were captured on a laser scanning confocal microscope (Carl Zeiss). The bar represents 5 µm.(DOC)Click here for additional data file.


Pseudomonas syringae strains possess an uncharacterized cluster of genes encoding putative components of a second T3SS (T3SS-2) in addition to the well characterized Hrc1 T3SS which is associated with disease lesions in host plants and with the triggering of hypersensitive response in non-host plants. The aim of this study is to perform an in silico analysis of T3SS-2, and to compare it with other known T3SSs.The central role of Type III secretion systems (T3SS) in bacteria-plant interactions is well established, yet unexpected findings are being uncovered through bacterial genome sequencing. Some P. syringae pv. phaseolicola strain is grouped with a second T3SS found in the pNGR234b plasmid of Rhizobium sp. These additional T3SS gene clusters define a subgroup within the Rhizobium T3SS family. Although, T3SS-2 is not distributed as widely as the Hrc1 T3SS in P. syringae strains, it was found to be constitutively expressed in P. syringae pv phaseolicola through RT-PCR experiments.Based on phylogenetic analysis and gene organization comparisons, the T3SS-2 cluster of the P. syringae T3SS-2 to a second T3SS from the pNGR234b plasmid of Rhizobium sp., member of subgroup II of the rhizobial T3SS family, indicates common ancestry and/or possible horizontal transfer events between these species. Functional analysis and genome sequencing of more rhizobia and P. syringae pathovars may shed light into why these bacteria maintain a second T3SS gene cluster in their genome.The relatedness of the  Gram-negative proteobacteria deploy various types of protein secretion systems for exporting selected sets of proteins to the cell surface, the extracellular space or into host cells ,2. Type The T3SS apparatus comprises three distinct parts: a) the basal body, which forms a cylindrical base that penetrates the two bacterial membranes and the periplasmic space; b) the extracellular part with the needle or the pilus as its main feature which is formed through the polymerization of specialized protein subunits that are T3SS substrates themselves; and c) the cytoplasmic part, which forms the export gate for secretion control. This apparatus is built by specific core proteins encoded by a conserved subset of genes tightly organized in gene clusters with counterparts in the bacterial flagellum ,7.Ysc-T3SS family, named after the archetypal Yersinia system, is present in α-, β-, γ-, and δ- proteobacteria. At least in α-proteobacteria the system confers resistance to phagocytosis and triggers macrophage apoptosis. (2) The Ssa-Esc-T3SS family is named after the archetypal T3SS of enteropathogenic and enterohemorrhagic E.coli. (3) The Inv-Mxi-Spa-T3SS family named after the Inv-Spa system of Salmonella enterica and the Inv-Mxi T3S system of Shigella spp. The family members trigger bacterial uptake by nonphagocytic cells.(4) The Hrc-Hrp1- and (5) the Hrc-Hrp2-T3SS families are present in plant pathogenic bacteria of the genus Pseudomonas, Erwinia, Ralstonia and Xanthomonas. The two families are differentiated on the basis of their genetic loci organization and regulatory systems. (6) The Rhizobiales-T3SS family (hereafter referred to as Rhc-T3SS) is dedicated to the intimate endosymbiosis serving nitrogen fixation in the roots of leguminous plants. (7) Finally the Chlamydiales-T3SS is present only in these strictly intracellular nonproteobacteria pathogens . The other is the region from hrpIIQ to PSPPH_2522 which, however, is inverted in the Rhizobium sp. NGR234 pNGR234b T3SS cluster relative to those in the pseudomonads. The coding frame for the RhcU/HrcU/YscU/FhlB homolog in the NGR234 cluster is transposed in relation to the Pseudomonas cluster (position which is maintained in the R.etli and B. japonicum clusters). In subgroup II of Rhc-T3SS gene clusters an hrcIIC2 gene can be identified in synteny to the subgroup I cluster. A common property of subgroups II and III of Rhc-T3SS gene clusters is the presence of hrpK-like genes.The T3SS-2 clusters of the Rhc-T3SS subgroups is the absence of a hrpP/yscP –like gene which usually resides between the hrpO/yscO-like gene and the hrcQ/yscQ homolog gene. A hrpO/yscO-like gene is absent from the subgroup III cluster. Subgroup I and III clusters maintain synteny with the P. syringae T3SS-2 clusters for most of the core T3SS ORFs. Finally, a gene coding for a HrpW homolog is found only in the R. etli clusters.Common to all R. etli Rhc T3SS (subgroup III) and the T3SS-2 of P. syringae pathovars (but not of the Rhizobium sp. NGR234 T3SS-2) is the presence of an ORF coding for a hypothetical translocator protein: The PSPPH_2540 locus of the P. syringae pv phaseolicola 1448a T3SS-2 codes for a large protein of 1106 residues. The C-terminal part of this protein (residues 421 – 1106) is homologous to the HrpK proteins of the Hrc-Hrp1 T3SS family based on Psi-BLAST searches (25% identity with HrpK of Erwinia amylovora). HrpK shares low similarity with the putative translocator, HrpF, from Xantomonas campestris pv vesicatoria. Furthermore, the C-terminal part of the protein coded by PSPPH_2540 also possesses two predicted transmembrane α-helices comprising residues 879–898 and 1029–1047 . The subgroup I Rhc T3SS lacks a hrpK ortholog. The HrpK protein was initially identified as a component of the Hrc-Hrp1 family of T3S systems  revealed the total absence of a T3SS-2 like cluster.In our phylogenetic analysis proteins encoded in the T3SS-2 cluster of P. syringae pv phaseolicola 1448a, P. syringae pv oryzae str.1_6, P. syringae pv tabaci and of Rhizobium sp. NGR234, is also present in P. syringae pv aesculi (strains NCPPB 3681 and 2250), ref|ZP 04589253.1| HrcIIC1 Pseudomonas syringae pv. oryzae str. 1_6], ref|YP 002824487.1| RhcIIC Rhizobium sp. NGR234], ref|NP 444156.1| NolW Rhizobium sp. NGR234], ref|NP 106861.1| NOLW Mesorhizobium loti MAFF303099], ref|NP 768451.1| RhcC1 Bradyrhizobium japonicum USDA 110] and B) RhcC2 proteins  RClick here for fileIIO) indicates that this hypothetical protein belongs to the HrpO/YscO/FliJ family of T3SS proteins [Rhizobium sp. NGR342. Residues predicted in α-helical conformation are indicated in yellow and unfolded regions in red. Green areas indicate ordered regions. Residues for which a high propensity for coiled-coil formation is predicted are indicated in blue rectangular. Here α-helix prediction was performed with PsiPRED, disordered prediction with FOLDINDEX and coiled coils prediction with COILS. Accession numbers or loci numbers are: AAC25065 (HrpO), P25613 (FliJ), AAB72198 (YscO), PSPPH_2532 (HrpIIO), NGR_b22960 (RhcZ), NGR234_462 (Y4yJ).Sequence analysis for HrpO-like proteins. The analysis of PSPPH_2532 (Hrpproteins . The samClick here for fileCodon Usage Bias Table. Click here for file


Antrodia cinnamomea is a traditional healthy food that has been demonstrated to possess anti-inflammatory, antioxidative, and anticacer effects. The purpose of this study was to evaluate whether the ethanolic extract of A. cinnamomea (EEAC) can affect the efflux function of P-glycoprotein (P-gp) and the effect of ABCB1 genetic variants on the interaction between EEAC and P-gp. To investigate the mechanism of this interaction, Flp-In™-293 cells stably transfected with various genotypes of human P-gp were established and the expression of P-gp was confirmed by Western blot. The results of the rhodamine 123 efflux assay demonstrated that EEAC efficiently inhibited wild-type P-gp function at an IC50 concentration of 1.51±0.08 µg/mL through non-competitive inhibition. The IC50 concentrations for variant-type 1236T-2677T-3435T P-gp and variant-type 1236T-2677A-3435T P-gp were 5.56±0.49 µg/mL and 3.33±0.67 µg/mL, respectively. In addition, the inhibition kinetics of EEAC also changed to uncompetitive inhibition in variant-type 1236T-2677A-3435T P-gp. The ATPase assay revealed that EEAC was an ATPase stimulator and was capable of reducing verapamil-induced ATPase levels. These results indicate that EEAC may be a potent P-gp inhibitor and higher dosages may be required in subjects carrying variant-types P-gp. Further studies are required to translate this basic knowledge into clinical applications. Antrodia cinnamomea, a well-known medicinal mushroom, has attracted lots of attentions to public due to the potential therapeutic efficacy of liver function protection, antihypertension and anti-inflammation A. cinnamomea has been demonstrated to exhibit anticancer and chemopreventive effects ABCB1 gene, belongs to the ATP-binding cassette superfamily and is constitutively expressed in the apical membranes of the liver, kidney, intestines, lung, and adrenal glands, as well as in the luminal surface of the blood-brain, blood-testis, and blood-ovarian barriers P-glycoprotein (P-gp), encoded by the ABCB1 gene ABCB1 gene is a highly polymorphic gene and several genetic polymorphisms have been demonstrated to affect P-gp expression, protein conformation, and efflux function ABCB1 1236C>T, 3435C>T, and 1199G>A with irinotecan concentrations in patients To overcome MDR to chemotherapeutic agents, several P-gp inhibitors have been generated and evaluated both in vitro and clinically A. cinnamomea induces a wide range of biological activities, including antiinflammation, antioxidation, liver function protection, and antitumor effects A. cinnamomea exhibited cytotoxic effects on prostate, breast, bladder, and lung cancers A. cinnamomea bodies on P-gp efflux function have not been investigated.The systemic toxicities of synthetic P-gp inhibitors have elicited an increasing amount of attention to the potential use of natural products. Among traditional healthy natural products, increasing evidence supports the hypothesis that ABCB1 gene on the potency of P-gp inhibition. Furthermore, we explored the underlying molecular mechanisms of the P-gp inhibition effects.In the present study, we evaluated whether EEAC inhibits P-gp and investigated the influences of genetic polymorphisms in the A.cinnamomea was a kindly gift from Cosmox Biomedical Co., Ltd. . The preparation of the ethanolic extracts of fruiting bodies of A.cinnamomea (EEAC), methods of HPLC and MS analysis were the same as our previous studies ABCB1cDNA in pMDRA1 was provided by the Riken BRC DNA bank (RDB No. 1372) .The 2. The pcDNA5/ABCB1 containing different genotypes of ABCB1cDNA were constructed in our previous studies ABCB1 plasmid and pOG44 (the Flp recombinase expression plasmid) were co-transfected into the Flp-In™-293 cells. Stable transfected with wild-type P-gp (1236C-2677G-3435C) and variant types P-gp (1236T-2677A-3435T and 1236T-2677T-3435T) cell lines were selected on the basis of hygromycin B resistance. The protein and mRNA expressions of P-gp were confirmed by Western blot analysis and real-time quantitative RT-PCR, respectively, in our previous studies Flp-In™-293 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and selected with 100 µg/mL zeocin at 37°C, 95% humidity and 5% COCytotoxicities of EEAC at concentrations used in the present study were determined by MTT assay. Briefly, the cells were seeded into 24-well plates and cultured overnight. Various concentrations of EEAC (from 1 µg/mL to 5 µg/mL) were then added and incubated for 72 hours. After incubation, the medium was removed and added fresh culture medium 200 µl containing 0.5 mg/ml MTT and then incubated at 37°C for 4 hours. The medium was removed and 200 µl DMSO was added. After incubation for 30 minutes at room temperature, 100 µl of solution was transferred to a 96-well plate and absorbance was measured using an ELISA plate reader  at 570 nm with a reference wavelength of 650 nm.The effect of EEAC on P-gp ATPase activity was examined by Pgp-GIO assay system  according to manufacturer’s protocol. To be brief, in a 96-well untreated white plate, 25 µg recombinant human Pgp was incubated with P-gp-GIO assay buffer (untreated control) or 200 µM verapamil (positive control of drug induced P-gp ATPase activity) or 100 µM sodium orthovanadate (selective inhibitor of P-gp ATPase activity) or EEAC (from 0.01 µg/mL to 200 µg/mL). The reaction was initiated by adding 5 mM MgATP and incubated for 40 min at 37°C. The luminescence was initiated by adding 50 µl of ATP detection reagent after 20 min of incubation at room temperature and read on the SpectraMax Gemini XS microplate spectrofluorometer . Each experiment was performed at least three times, each in triplicate on different days.5 cells were harvested after trypsinization by centrifugation and resuspended in fresh culture medium. Rhodamine123  was added and the cells were incubated in a water bath at 37°C in the dark. Cells were pelleted at 1, 5, 10, 15, 30, 45, 60, and 90 min. After washed with ice-cold PBS, cells were resuspended in 1-ml, ice-cold PBS prior to FACS analysis using CellQuest software . Each cell type was performed at least three experiments, each in triplicate on different days.For FACS analysis, 5×105 cells were seeded on 96-well plates and cultured for 24 hours. After incubation, cells were washed with warm Hanks’ balanced salt solution (HBSS) and pre-incubated with HBSS for 30 min, and subsequently incubated in 1 µM rhodamine123 for 30 min with or without EEAC in triplicate. After washed with warm PBS, cells were allowed to efflux rhodamine123 for 10 min at 37°C in the incubator. Samples (150 µl) were collected and transferred to 96-well black plates. Rhodamine123 concentrations were measured using a set of standards (from 62.5 nM to 8 µM) in HBSS analyzed by the SpectraMax Gemini XS microplate spectrofluorometer  with the excitation set at 485 nm and the emission set at 535 nm. The measured rhodamine123 concentrations were normalized to the total protein content in the cells. Total protein concentrations were measured by a microplate assay protocol , and bovine serum albumin was used as the standard. Each experiment was repeated at least three times across cell passages to ensure stability of transfection and consistency and reproducibility of the experiments.For efflux assays, 1×105 cells were placed on 96-well black plates and cultured for 24 hours. Before the uptake assay, cells were washed with warm Hanks’ balanced salt solution (HBSS) and pre-incubated with HBSS for 30 min, and subsequently with EEAC for 30 min in triplicate. After pre-incubation, calcein AM  was added and incubated at 37°C in the incubator for 30 min. After washed by ice-cold HBSS, cells were lysed with 1% Triton X-100 and calcein fluorescence generated within the cells was analyzed by the SpectraMax Gemini XS microplate spectrofluorometer  with the excitation set at 485 nm and the emission set at 535 nm. Each experiment was performed at least three times, each in triplicate on different days.For calcein-AM uptake study, 1×105 cells were harvested after trypsinization by centrifugation and resuspended in fresh culture medium. Fluorescent doxorubicin  was added and the cells were incubated in a water bath at 37°C in the dark. Cells were pelleted at 1, 5, 10, 15, 30, 45, 60, 90, 120, 150 and 180 min. After washed with ice-cold PBS, cells were resuspended in 1 ml ice-cold PBS prior to FACS analysis using CellQuest software . Each cell type would be used to perform at least three experiments, each in triplicate on different days.For FACS analysis, 5×105 cells were seeded on 96-well plates and cultured for 24 hours. After incubation, cells were washed with warm Hanks’ balanced salt solution (HBSS) and preincubated with HBSS for 30 min, and subsequently incubated in 10, 20 or 30 µM fluorescent doxorubicin for 3 hours with or without EEAC in triplicate. After being washed with warm PBS, cells were allowed to efflux fluorescent doxorubicin for 2 hours at 37°C in the incubator. Samples (100 µl) were collected and transferred to 96-well black plates. Fluorescent doxorubicin concentrations were measured using a set of standards (from 0.5 µM to 10 µM) in HBSS analyzed by the SpectraMax Gemini XS microplate spectrofluorometer  with the excitation set at 485 nm and the emission set at 590 nm. The measured fluorescent doxorubicin concentrations were normalized to the total protein content in the cells. Total protein concentrations were measured by a microplate assay protocol , and bovine serum albumin was used as the standard. Each experiment was repeated at least three times across cell passaging to ensure stability of transfection and consistency and reproducibility of the experiments.For efflux assays, 1×10For ATPase Assay data analysis, basal P-gp activity, verapamil-stimulated P-gp ATPase activity, EEAC effect on P-gp ATPase activity and fold stimulation by EEAC were calculated as following equations:Basal P-gp activity:Verapamil-stimulated P-gp ATPase activity:EEAC effect on P-gp ATPase activity:Fold stimulation by EEAC:50 values were calculated by the following equation:For evaluation of inhibitor potency, IC0, the efflux in the absence of inhibitor; I, the inhibitor concentration; IC50, the concentration that caused 50% inhibition of the maximal drug effect and s is the slope factor.Where E is the observed efflux in the presence of inhibitor; EFor kinetic studies, kinetic parameters were estimated by nonlinear regression using Scientist v2.01  according to the following equation:max, the maximal efflux rate; Km, the Michaelis-Menten constant and C is the substrate concentration. Statistical comparisons among different cells were performed by using SYSTAT v10 . Statistical differences were evaluated by ANOVA and post hoc analysis (Tukey’s test) and the statistical significance was set at p value <0.05.Where V denoted the efflux rate; VABCB1 gene stable transfected cells expressed large quantities of human P-gp, whereas little expression of ABCB1 mRNA and protein was found in the Flp-In™-293 cells (host cells). There was no significant difference among the mRNA and protein expression levels of wild type ABCB1 and the two variant types as showed in our previous studies ABCB1 mRNA and protein expression remained unchanged in wild type ABCB1 and the two variant types , ABCB1 1236T-2677T-3435T and ABCB1 1236T-2677A-3435T were 21.27±1.26, 29.31±1.72, 20.60±0.94 and 22.74±1.04 µg/ml, respectively (The nt types . The cytectively f).M and Vmax values for rhodamine 123 efflux in wild-type human P-gp were estimated to be 20.17±4.45 µM and 11.22±0.73 nmole/mg protein/10 min, respectively, whereas the non-specific first-order rate constant was estimated to be 10.08±1.12 µL/mg protein/10 min. Since the intrinsic clearance, Vmax/KM, of rhodamine 123 was 556.3 µL/mg protein/10 mins, non-specific transport accounted for only 1.8% of the total efflux. The KM values for rhodamine 123 efflux in wild-type and variant-types P-gp were similar, while the Vmax value for rhodamine 123 efflux was significantly lower in variant type 1236T-2677A-3435T P-gp than wild-type 1236C-2677G-3435C P-gp and variant type 1236T-2677T-3435T P-gp  values for EEAC in wild-type P-gp, variant type 1236T-2677T-3435T P-gp, and variant type 1236T-2677A-3435T P-gp were 1.51±0.08 µg/mL, 5.56±0.49 µg/mL and 3.33±0.67 µg/mL, respectively  were incubated with recombinant human P-gp. EEAC was demonstrated to stimulate P-gp ATPase activity by two fold higher than basal ATPase activity and inhibited 200 µM verapamil-stimulated P-gp ATPase activity at 5 µg/mL .The inhibition kinetics of EEAC was further investigated in the wild type and the variant types of P-gp by Lineweaver-Burk plot . In the As for the kinetics of P-gp mediated doxorubicin efflux, the EEAC was demonstrated to inhibit doxorubicin transport by distinctive kinetics among different types P-gp. In the wild-type P-gp, when the concentration of EEAC increased, the maximum rate of doxorubicin efflux (Vmax) remaining unchanged with affinity (Km) increased . These r50, whereas the variant-type 1236T-2677T-3435T P-gp was the least sensitive to EEAC at the highest concentration of IC50. Regarding the inhibition kinetics, EEAC inhibited wild-type P-gp and variant-type 1236T-2677T-3435T P-gp through noncompetitive inhibition, whereas it inhibited variant-type 1236T-2677A-3435T P-gp through uncompetitive inhibition. In our previous study, we have reported the P-gp inhibition kinetics of methadone The present study demonstrated that EEAC may inhibit human P-gp efflux function through ATPase stimulation. The common genetic polymorphisms of human P-gp influence the inhibition kinetics and inhibition potency of EEAC. The wild-type human P-gp was the most sensitive to EEAC at the lowest concentration of ICAntrodia camphorate (AC-SS) exhibited adjuvant antiproliferative effects on hepatoma cells and on xenografted cells in tumor-implanted nude mice ABCB1 gene was demonstrated to be reduced by AC-SS treatment in two hepatoma cell lines (C3A and PLC/PRF/5) ABCB1 gene expression. The inhibition of human P-gp efflux function by EEAC occurred rapidly, within 30 min, through non-competitive inhibition and lasted for more than 2 h. These results provide explanations for the mechanism of the significant inhibiting effect of EEAC on P-gp function. Similar to verapamil, a well-known P-gp inhibitor, EEAC acted as an ATPase stimulator and reduced the ATPase stimulating effect of verapamil. These results indicate that EEAC interacted with verapamil on the same binding site of ATPase and exerted a significant P-gp-inhibiting effect at low concentrations (1–5 µg/mL). Potential drug-drug interactions may occur when a P-gp inhibitor, such as EEAC, co-administered with P-gp substrates, such as digoxin and fexofenadine A previous study reported on how P-gp expression was influenced by this traditional natural product ABCB1 genetic polymorphisms on the P-gp-inhibition potency of EEAC, the results of this study indicate that EEAC exhibited 2-fold lower and 4-fold lower inhibition potency toward variant-type 1236T-2677A-3435T and 1236T-2677T-3435T P-gp than toward wild-type P-gp, respectively. Moreover, the inhibition kinetics also changed between the wild-type and variant-type P-gps. These results correspond with those of a previous study that identified the functional effect of ABCB1 1236C>T, 2677G>T/A, and 3435C>T Regarding the influences of ABCB1 genotypes and chemotherapy responses have been reported previously. Patients with the 1236C-2677G-3435C haplotype were associated with poor responses to imatinib treatment for chronic myeloid leukemia The associations between A. cinnamomea has been demonstrated to be a potential anticancer, antiinflammatory, and antioxidative agent. In this study, we demonstrated that A. cinnamomea may possess the ability to overcome MDR.In our previous study, the index compounds within EEAC were measured and quantified using HPLC, LC/MS, and LC/MS/MS and used for controlling the quality of the extraction procedure for each batch ABCB1 gene influence the binding kinetics of EEAC to human P-gp. Additional preclinical and clinical studies are required to verify whether EEAC can be developed as an MDR-reversing agent.In conclusion, we demonstrated that EEAC inhibited the efflux function of human P-gp through an ATPase stimulation mechanism. The common SNPs on the Figure S1Representative HPLC and mass spectrometry of the index compounds in EEAC. The relative content of adenosine, cordycepin, and zhankuic acid A in EEAC were calculated according to the concentration of standard compounds. The index compounds  were confirmed by ESI-MS and ESI-MS/MS within EEAC. (A) HPLC data for adenosine and cordycepin. (B) HPLC data for zhankuic acid A. (C) LC/MS spectra of cordycepin. (D) LC/MS spectra of zhankuic acid A. (E) LC/MS/MS spectra of cordycepin. (F) LC/MS/MS spectra of zhankuic acid A.(PDF)Click here for additional data file.


Streptococcus pneumoniae is a major cause of pneumonia and meningitis. Several genetic polymorphisms have been described to explain differences in susceptibility and severity of encapsulated pathogen-related diseases. Among them, a functional FCGR2A polymorphism leading to amino acid change of histidine (H) to arginine (R) at position 131 appears to be a major candidate in adult invasive pneumococcal diseases (IPD). However, previous reports needed confirmation in a large well-defined population.S. pneumoniae from a normally sterile site ). DNA from all Caucasian patients with IPD (pneumonia and/or meningitis) was genotyped for the FcγRIIa-R/H131polymorphism.A prospective genetic association study in a 24-bed medical ICU of a tertiary teaching hospital over 7 years. Patients were retrospectively selected from two prospective cohorts generated between January 2001 and December 2008. All Caucasian subjects with proven IPD and hospitalized in the ICU were included. Cases of IPD were defined by the isolation of P = 0.004). See Figure Two hundred and forty-three patients with proven IPD were enrolled, 202 (82%) with pneumonia and 55 (22%) with meningitis. Mean age was 61 years, mean SAPS2 was 50.4, one-half of the patients had bacteremia, 84% of the cohort was mechanically ventilated and the hospital mortality rate was 31%. In the IPD group, distribution of the FcγRIIa-R/H131 genotypes  was comparable with the distribution in the Caucasian control group. Comparison of the FcγRIIa-R/R131 and the (FcγRIIa-R/H131 + FcγRIIa-H/H131) groups did not demonstrate any difference for age, SAPS2, origin of sepsis and other co-morbid conditions. However, the variant FcγRIIa-R/R131 genotype was independently associated with decreased hospital mortality (OR = 0.251, CI 0.098 to 0.645; In a well-defined population of IPD patients, the frequency of the variant FcγRIIa-R131 does not differ from other critically ill patients. However, the FcγRIIa-R/R131 genotype was independently associated with increased survival regardless of the site of infection.


According to the prevailing view, soluble oligomers or small fibrillar fragments are considered to be the most toxic species in prion diseases. To test this hypothesis, two conformationally different amyloid states were produced from the same highly pure recombinant full-length prion protein (rPrP). The cytotoxic potential of intact fibrils and fibrillar fragments generated by sonication from these two states was tested using cultured cells.C) at high levels confirming that cytotoxicity was in part PrPC-dependent. Silencing of PrPC expression by small hairpin RNAs designed to silence expression of human PrPC (shRNA-PrPC) deminished the deleterious effects of the two amyloid states to a different extent, suggesting that the role of PrPC-mediated and PrPC-independent mechanisms depends on the structure of the aggregates.For one amyloid state, fibril fragmentation was found to enhance its cytotoxic potential, whereas for another amyloid state formed within the same amino acid sequence, the fragmented fibrils were found to be substantially less toxic than the intact fibrils. Consistent with the previous studies, the toxic effects were more pronounced for cell cultures expressing normal isoform of the prion protein  molecules within aggregated states. Depending on the structure, a decrease in size of amyloid fibrils can either enhance or abolish their cytotoxic effect. Regardless of the molecular structure or size of PrP aggregates, silencing of PrP Sc) were found to exhibit the highest specific prion infectivity Etiology and progression of several neurodegenerative diseases including Alzheimer's, Parkinson's, Huntington's and prion diseases are linked to the accumulation of protein aggregates in the form of large amyloid fibrils/plaques, or small oligomers or fibrillar fragments The current studies were designed to test the hypothesis about the relationship between prion protein fibril dimension and their cytotoxic potential and specifically, to address the question of whether fragmentation of fibrils into smaller fragments or oligomers always enhances toxic potential. To address this question, two conformationally different fibrillar amyloid states referred to as R- and S-fibrils were produced from highly-pure, full-length Syrian hamster rPrP. The cytotoxic potential of intact fibrils and small fibrillar fragments generated by sonication was tested using cultured cells. For one amyloid state, fibril fragmentation was found to enhance its cytotoxic potential, whereas for another amyloid state formed within the same amino acid sequence, the fragmented fibrils were found to be less toxic than the intact fibrils. These studies show that molecular structure of the amyloid state controls the relationship between fibrillar size and toxicity.The R- and S-fibrils were formed from full-length rPrP encompassing residues 23–231 under identical solvent conditions but different agitation modes as previously described CC expression but not by the cell type. We chose two SKMEL cell lines, SKMEL-2 and SKMEL-28 that express PrPC at very low or high levels, respectively  showed very modest cytotoxic responses. The toxic effects tend to be higher in the lines with high levels of PrPC expression (SKMEL-28 and transfected CHO cells)  . Second,O cells) . In factte trend : the toxte trend , the forte trend . This obC expression, all cell lines showed similar rank orders with respect to their susceptibilities to intact or sonicated R- or S-fibrils within each individual cell line . As judged from Western blotting, expression of PrPC was silenced in cells treated with shRNA-PrPC#1 lentivirus, but not in cells transduced with shRNA-PrPC#2 or with scrambled shRNA lentiviruses  was expressed and purified as previously described C. pcDNA5/FRT/PrP plasmid was constructed and used for transfection as previously described C in non-transfected CHO cells was found to be below detectible levels, whereas the CHO cells transfected with pcDNA5/FRT/PrP plasmid expressed PrPC in amounts comparable to those found in normal hamster brains (7 cells for each cell line) were lysed in ProteoJET Mammalian Cell Lysis Reagent  with a protease inhibitor cocktail . After centrifugation at 16,000×g for 15 min at 4°C, the supernatant was transferred to a new tube and protein concentrations were determined by absorbance spectroscopy. Twelve micrograms of total protein were loaded to 12% SDS-PAGE, and then analyzed by Western blotting using mouse anti-PrP 3F4 or mouse anti-β-actin antibodies .FLP-In™ CHO cells  cells were transfected with pcDNA5/FRT/PrP plasmid  containing the gene for expression of full-length wild type Syrian hamster PrPr brains . FLP-In™2, 95% air at 37°C. Intact or fragmented fibrils were added to the cultured cells at concentrations equivalent to 1 µM  and incubated for 24 h. The cytotoxic effect was analyzed using Trypan Blue  or XTT  assays according to the manufacturer's procedure. Briefly, for the Tryptan Blue assay, after treatment with fibrils, cells were trypsinized, incubated with 0.2% trypan blue solution  for 2 minutes, and counted using light microscopy. For the XTT assay, XTT -2H-tetrazolium-5-carboxanilide) was added to the amount equal to 20% of the culture medium volume, the cells were then incubated for four hours in 5% CO2 at 37°C, and the dehydrogenase activity was measured by monitoring absorbance at 450 and 690 nm according to the manufacturer's procedure.The cytotoxic potential of rPrP fibrils was assessed using Chinese hamster ovary cell (CHO) or human melanoma SKMEL cell lines. CHO or SKMEL lines were cultured in F-12 medium  or RPMI 1640 (Invitrogen), respectively, both supplemented with 10% fetal bovine serum (Invitrogen) in a humidified atmosphere of 5% COPrnp gene (GenBank BC022532). CAATAGGGAGACAATCTAA  and GCAATGTTATTATTGGCTT  were selected as target sequences for silencing the expression of PrPC, the scrambled sequence GAATGCAATAACGAGAGTA was used as a negative control for testing the effects of non-specific shRNA. To avoid off-target effects, a homology search was performed using BLAST (http://blast.ncbi.nlm.nih.gov) to ensure that only the PrPC mRNA sequence was targeted. Two complementary oligonucleotides necessary to create the hairpin insert for pENTR-H1/TO vectors were designed using SiRNA Scales software Target sequences were derived from the 3′ UTR region of human C#1-Top, 5′-CACCGAATAGGGAGACAATCTAACGAATTAGATTGTCTCCCTATTC-3′shRNA-PrPC#1-Bot., 5′-AAAAGAATAGGGAGACAATCTAATTCGTTAGATTGTCTCCCTATTC-3′shRNA-PrPC#2-Top, 5′-CACCGCAATGTTATTATTGGCTTCGAAAAGCCAATAATAACATTGC-3′ shRNA-PrPC#2-Bot., 5′-AAAAGCAATGTTATTATTGGCTTTTCGAAGCCAATAATAACATTGC-3′ shRNA-Scrmbl-Top, 5′-CACCGAATGCAATAACGAGAGTACGAATACTCTCGTTATTGCATTC-3′ shRNA-Scrmbl-Bot., 5′-AAAAGAATGCAATAACGAGAGTATTCGTACTCTCGTTATTGCATTC-3′shRNA-PrP6), and virus-containing supernatants were harvested 72 hours post-transfection. The lentiviruses were added to the SKMEL-28 cell line at a multiplicity of infection (MOI) of 2–5, and cells were cultured using a complete medium containing 50 µg/ml Zeocin  for 14 days to establish stable cell lines.After annealing, each double-stranded oligonucleotide (5 nM) was cloned into pENTR/H1/TO vector . To construct shRNA-expression vectors, recombination reactions of pENTR/H1/TO vectors that included specific target sequences with pLenti4/BLOCK-iT-DEST vector  were performed. For producing lentivirus, the resulting pLenti4/BLOCK-iT-DEST vectors were mixed with ViraPower Packaging Mix , transfected into 293FT cells (6×10


Klotho was first identified in 1997 and has been considered as an anti-aging gene. Emerging evidence demonstrates that klotho has a close relationship with cancers, including lung cancer, breast cancer, etc, by inhibiting the proliferation and promoting apoptosis of cancer cells. Cisplatin has been the most widely used drug in the first-line chemotherapy. However, the increase in cisplatin-resistant cancer cells has become a major obstacle in clinical management of cancers. In our study, we for the first time demonstrated that klotho could attenuate the resistance of lung cancer to cisplatin based chemotherapy and the apoptosis of the resistant cells with klotho overexpression was markedly increased. However, klotho knockdown cells showed enhanced resistance to chemotherapy. Further analysis showed that inhibition of PI3K/Akt pathway with specific inhibitor (LY294002) attenuated the promotive effects on cancer growth following interfering with klotho shRNA. Moreover, we demonstrated that klotho modulated the resistance to cisplatin in a xenograft nude mice model. These observations suggested that klotho could improve the resistance of lung cancer cells to chemotherapy and may serve as a potential target for the gene therapy of lung cancers resistant to cisplatin based chemotherapy. Non-small cell lung cancer (NSCLC) accounts for 75–85% of lung cancer cases and chemotherapy plays an important role in the treatment of lung cancer Klotho is a newly found anti-aging gene and was originally identified in klotho homozygous mutant mice (kl-/-) which showed a human-like aging-related syndrome and develop multiple disorders such as hypogonadism, ectopic calcification, osteoporosis, skin atrophy, and pulmonary emphysema In this study, we hypothesized klotho could inhibit the PI3K/Akt pathway and further to alleviate the resistance of lung cancer cells to cisplatin and may serve a potent candidate for the gene therapy of lung cancer.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Nanjing medical university (Permit Number: 2120474). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.2 at 37 °C. To maintain drug resistance, A549/DDP and H460/DDP cells were grown in RPMI 1640 containing 2 µg/ml DDP and then in DDP free RPMI 1640 two days before experiments. A549DDP cells reaching 80% confluence were transfected with pCMV6-MYC-KL and its specific shRNAs in the presence of lipofectamine 2000 (Invitrogen). The shRNAs were as follows:Human lung cancer cell line (A549 cells) was obtained from the American Type Culture Collection (ATCC). The H460 cells and cisplatin-resistant A549 and H460 (A549DDP and H460DDP) cells were kindly provided by the Prof. Zhou in Shanghai Pulmonary Hospital. All cell lines were maintained in RPMI 1640  containing 10% fetal bovine serum (FBS) , 100 U/mL penicillin, 100 U/mL streptomycin, 2 mM glutamine in a humidified atmosphere with 5% COCCTAAGCTCTCACTGGATCAATCCTCGAA;sh-1: CTGAGGCAACTGCTTTCCTGGATTGACCT;sh-2: GGTCACTCACTACCGCTTCTCCATCTCGT;sh-3: GTTACAGCATCAGGCGTGGACTCTTCTAT;sh-4: All plasmids were purchased from the OriGene .4 cells per well), and treated with cisplatin at different concentrations for 48 h. After incubation, the media was replaced with 50 µL of MTT reagent (2 mg/mL) followed by further incubation in an atmosphere with 5% CO2 at 37 °C for 2 h. Then, the media were removed and dimethylsulfoxide (DMSO) (150 µL) was added to each well. The optical density (OD) of each well was measured using a microplate reader at 560 nm.The half maximal inhibitory concentration (IC50) of A549DDP and A549 cells were determined by the MTT  assay (Sigma). Cancer cells were seeded into 96-well plates  cisplatin for 8 h, and 60 µM for H460DDP, and then washed once in phosphate buffer saline (PBS) followed by fixation in cold methonal: acetone (1:1) for 5 min. After washing thrice in PBS for 5 min, these cells were treated with 4 µg/ml 4′, 6-diamidine-2′-phenylindole dihydrochloride (DAPI) (Sigma) for 10 min at room temperature. Cells were then examined by fluorescence microscopy. Cells were randomly selected for examination at a high magnification (×400) and photographed. Both normal cells  and the apoptotic cells (nuclear shrinkage and hyperchromatic nuclei) were counted under each field.Cells were harvested and gently disaggregated to a single cell suspension. Staining was made according to the manufacturer's protocol. The rate of apoptosis induced by anticancer regimens was analyzed by flow cytometry using an annexin V-FITC/PI kit  following the manufacturer's instructions.Total RNA was extracted from cells with TRIzol reagent  according to the manufacturer's instructions, and quantified by measuring the absorbance at 260 nm. Gene expressions were detected by quantitative real-time RT-PCR (qRT-PCR) using the standard SYBR Green RT-PCR kit  according to the manufacture's instructions. The cDNA was synthesized using the RevertAid First-Strand cDNA Synthesis kit  according to the manufacturer's instructions. Primer sequences were designed with Origene Technologies, Inc. The primers used were as follows:klotho sense: 5′-GCTCTCAAAGCCCACATACTG -3′;5′-GCAGCATAACGATAGAGGCC -3′;antisense: β-actin sense: 5′-TGACGTGGACATCCGCAAAG-3′;5′-CTGGAAGGTGGACAGCGAGG-3′;antisense: PCR conditions consisted of pre-denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at at 94 °C for 30 sec, annealing at 60 °C for 30 sec and extension at 72 °C for 30 sec and a final extension at 72 °C for 10 min.24 h and 48 h after transfection, cells were washed twice in ice-cold PBS and proteins were extracted from cells by lysis in RIPA buffer , 50 mM Tris HCl [pH 8], 10 mM EDTA, and 1 mM PMSF [Sigma]) for 30 min at 4 °C and subsequent centrifugation for 15 min at 12,000 g. Protein concentration was determined with a BCA kit (Pierce) according to the manufacturer's instructions. Then, 60 µg of protein was loaded onto a 10% (w/v) SDS-polyacrylamide gel, electrophoresed, and transferred onto a PVDF membrane  which was then blocked for 2 h at room temperature with blocking buffer (TBS containing 0.1% [v/v] Tween 20 [Sigma] and 5% [w/v] milk powder). Primary antibodies  were: anti-phospho-akt,anti-akt, , anti-Bax,anti-klotho, and anti-GAPDH (Santa Cruz). Antibodies were diluted to 1:1,000, except for the anti-GAPDH antibody (1:500). Thereafter, membranes were incubated for 2 h with HRP-conjugated secondary antibodies  . Visualization was done by using an ECL kit, and the signals were quantified by scanning densitometry.CGCGTCCCCCTGAGGCAACTGCTTTCCTGGATTGACCTTCAAGAGAGGTCAATCCAGGAAAGCAGTTGCCTCAGTTTTTGGAAAT-3′5′-.The lentiviral vectors expressing short hairpin RNAs (shRNAs) were successfully constructed by OriGene  as previously described. The effectively constructed klotho-shRNA oligonucleotide sequences were as follows, sense: mulI and claI enzyme sites of pLVTHM vector, respectively. For recombination reaction using pCMV-dR8.74 and pCMV-VSV-G vectors , lentiviral vector DNAs and packaging vectors were then transfected into 293T cells. After 48 hr transfection, supernatants containing lentiviruses were harvested, centrifuged at 1800 g for 10 min, and filtered through a 0.45 µm poly (vinylidene difluoride) Durapore membrane  to remove any nonadherent packaging cells. Resistant lung cancer cell line (A549DDP) was infected with the lentivirus. Then, the infected cells were selected by puromycin (0.4 µg/ml) for 14 days. A549DDP cells infected with lentivirus-mediated shRNA targeting klotho (sh-klotho) or lentivirus-mediated shRNA (scramble) were named A549DDP-lenti-sh-2 or A549DDP-lenti-scramble, respectively.The sequences were inserted into the For in vivo experiments, Male athymic nude mice  were used. They were injected subcutaneously with A549DDP-lenti-sh-2 or A549DDP-sh-scramble cells. When tumors measured an average volume of 100 mm3, the mice (6 per group) were treated with cisplatin  or PBS for 21 days. Tumors were measured with calipers. Tumor volumes were calculated by the following formula: tumor volume (mm3)  =  0.5*length (mm)*width (mm)*width(mm).P<0.05 was considered statistically significant.Data were expressed as mean ± standard deviation (SD). Experiments were performed at least in triplicate. Comparisons were done with two-tailed Student's t test or ANOVA. A value of To confirm the resistance of A549DDP and H460DDP cells, MTT assay was done to measure the IC50 of A549 cells and A549DDP cells. Results showed the IC50 was 17.7±1.93 µM in A549 cells and 86.6±13.2 µM in A549DDP cells,while H460DDP and its parent cell line were 63.5±3.8 and 27.7±2.0 respectively . The expTo determine the role of klotho in the cisplatin-resistance of cisplatin resistant cells, we first detected the p-AKT expression in A549, H460 and their cisplatin resistant cells. Results demonstrated that the expression of phosphorylated Akt was significantly lowered in the A549 and H460 cells . Then, cFurther, we investigated the phenotype of the drug-resistant cells and the parent cells, and we found that ERCC1 was down-regulated in the klotho transfected cells, while the p-gp, a gene related to the drug efflux, and contributed to the most cisplatin resistance, was not affected by klotho . These rTo further investigate the role of klotho in the resistance of the cells to chemotherapy, klotho knockdown was performed in A549DDP and H460DDP cells with specific shRNAs. Four shRNAs were designed and transfected into A549DDP or H460DDP cells as described in our previous study in which results demonstrated that the sh-2 showed the best interference efficiency Furthermore, the expression of total-Akt, phospho-Akt (p-Akt), Bax and bcl-2 were examined in klotho shRNA transfected cells. Western blot assay showed that the klotho knockdown led to the significant increase in the expression of phospho-Akt  and bcl-To further explore whether klotho could improve the resistance of the cisplatin resistant cells to cispaltin through regulating the p-Akt expression, cells transfected with klotho specific shRNA were treated with 10 µM and 40 µM LY294002, a phosphoinositide-3 kinase inhibitor, to elucidate the role of Akt in the increased resistance to chemotherapy following klotho knockdown. LY294002 significantly inhibited the expression of p-Akt in the shRNA transfected cells, especially after treatment with 40 µM LY294002 , as compin vitro experiment of klotho in the cisplatin resistance of lung cancers, we further examined if klotho expression affects the cispaltin sensitivity in vivo, A549DDP-lenti-sh-2 and A549DDP-lenti-scramble cells were injected subcutaneously into the right flank of nude mice. Cisplatin could significantly inhibit the tumor growth in mice injected with A549DDP-lenti-scramble cells compared to the phosphate buffer control, however, mice injected with A549DDP-lenti-sh-2 cells showed no significantly inhibition compared to the control group , mainly through inhibiting the IGF-1/R pathway in vitro, we performed an in vivo experiment in nude mice. We found that the decreased antitumor efficacy in vivo was associated with the inhibition of klotho expression and the activated PI3K/Akt pathway. These results confirmed that klotho, an anti-aging gene, could alleviate resistance of A549DDP and H460DDP to cisplatin by inhibiting Akt signal pathway.In the present study, cisplatin resistant NSCLC cells (A549DDP and H460DDP cells) and its parent cells (A549 and H460 cells) were employed for experiments. First, the klotho expression was measured in both cell lines, and results showed the klotho expression at both mRNA and protein levels in the resistant cells was significantly lowered as compared to the parent cells (P<0.05). However, the expression of p-Akt in the cisplatin resistant cells was significantly higher than that in the parent cells. In addition, the high IC50s were significantly reduced in A549DDP and H460DDP cells after klotho up-regulation. However, klotho down-regulation by transfection with specific shRNA increased the resistance to cisplatin, accompanied by an increase in p-Akt expression. Previous studies have identified Akt as an important protein in the IGF-1 mediated cell survival The regulation of apoptosis is complicated and a number of genes are involved in this process, including Bax/bcl-2, cell-regulatory proteins, etc In summary, our results demonstrate that the klotho, an anti-aging gene, is associated with the resistance of lung cancer cells to cisplatin and may alleviate the resistance mainly through modulating the PI3K/Akt signal pathway and the expression of bax/bcl-2. Moreover, our results may have important clinical implications in the chemotherapy of lung cancer because the therapeutic efficacy is often impeded by the presence of cisplatin resistance. However, our results are derived from in vitro experiment, and the specific mechanism in vivo is required to be further investigated.