A range of novel carboxamide fungicides, inhibitors of the succinate dehydrogenase enzyme  is currently being introduced to the crop protection market. The aim of this study was to explore the impact of structurally distinct carboxamides on target site resistance development and to assess possible impact on fitness.Mycosphaerella graminicola, a key pathogen of wheat to compare the nature, frequencies and impact of target mutations towards five subclasses of carboxamides. From this screen we identified 27 amino acid substitutions occurring at 18 different positions on the 3 subunits constituting the ubiquinone binding (Qp) site of the enzyme. The nature of substitutions and cross resistance profiles indicated significant differences in the binding interaction to the enzyme across the different inhibitors. Pharmacophore elucidation followed by docking studies in a tridimensional SDH model allowed us to propose rational hypotheses explaining some of the differential behaviors for the first time. Interestingly all the characterized substitutions had a negative impact on enzyme efficiency, however very low levels of enzyme activity appeared to be sufficient for cell survival. In order to explore the impact of mutations on pathogen fitness in vivo and in planta, homologous recombinants were generated for a selection of mutation types. In vivo, in contrast to previous studies performed in yeast and other organisms, SDH mutations did not result in a major increase of reactive oxygen species levels and did not display any significant fitness penalty. However, a number of Qp site mutations affecting enzyme efficiency were shown to have a biological impact in planta.We used a UV mutagenesis approach in Using the combined approaches described here, we have significantly improved our understanding of possible resistance mechanisms to carboxamides and performed preliminary fitness penalty assessment in an economically important plant pathogen years ahead of possible resistance development in the field. Mycosphaerella graminicola (Fückel) J. Schröt. in Cohn (anamorph Septoria tritici) is the causal agent of Septoria leaf blotch (STB), a major threat to bread and durum wheat worldwide Cytb gene of the target enzyme Cytochrome C reductase also known as the respiratory channel complex III M. graminicola population carries the G143A mutation, making its control highly reliant on C14-demethylase inhibitors (DMI) usage targeting the ergosterol biosynthesis pathway and on the multisite fungicide chlorothalonil (CTN). Gradual shifts in DMI sensitivity observed as an incremental reduction in sensitivity of pathogen population towards DMIs over time www.FRAC.info) for this fungicidal class is “SDHIs” for succinate dehydrogenase inhibitors. At the molecular level, carboxamides inhibit ubiquinone reduction by binding to the ubiquinone binding site (Qp site) of the SDH enzyme Escherichia coliGallus gallus (chicken) Sus scrofa (pig) The introduction of novel carboxamide fungicides has delivered a major mode of action to almost all fungicide market segments including fruits, vegetables and cereals. These molecules display fungicidal activity by disrupting the mitochondrial tricarboxylic acid cycle (TCA) through inhibition of the succinate dehydrogenase (SDH) enzyme , EC 1.3.5.1). The official term, as stated by the Fungicide Resistance Action Committee  M. graminicola (SDHB_H267L/Y) Aspergillus oryzae  Coprinus cinereus (SDHC_N80K) Ustilago nuda in France, Canada and Italy and the resistance mechanism although not elucidated was reported as monogenic Botrytis cinereaAlternaria alternataCorynespora cassiicola and Podosphaera xanthii show that striking lack of cross resistance can be found across novel carboxamides M. graminicola mutants which were selected on Carboxin only confirmed this is also true in M. graminicolaCarboxin, was the first carboxamide to be developed for crop protection and was used as seed treatment displaying mainly a basidiomycete spectrum of control M. graminicola by exploring the impact of target mutations on compound binding, enzyme efficiency and pathogen fitness. To this end M. graminicola was subjected to random UV mutagenesis and 5 structurally distinct carboxamides were used for selection. The characterization of more than 480 mutants enabled the identification of as many as 27 substitution types affecting in total 18 positions in the SDHB, SDHC and SDHD-Qp site forming proteins. Characterization of the mutants enabled the identification of substitution types that display selectivity to structurally distinct carboxamides. Docking studies, using a homology model of M. graminicola SDH, offer valuable insight to some of the experimental findings. Characterization of enzyme efficiencies showed that the ubiquinone reduction step was impaired in all mutants. Using transgenic strains expressing mutated copies of the enzyme SDH subunits we showed that very low levels of SDH activity is required for the establishment of resistance in vivo. Finally, homologous recombinant gene replacements for the most relevant substitutions types enabled preliminary fitness studies in vitro and in planta to be performed. Homologous recombinant strains developed in this haploid pathogen, correspond to the introduction of a single mutation in the whole genome enabling us to perform a very clean comparison of likely biochemical factors affecting fitness. Using these homologous recombinant strains we unexpectedly found that M. graminicola Qp site mutations did not significantly impact reactive oxygen species (ROS) production in vivo. However, in planta virulence was affected suggesting that these carboxamide selected Qp site mutations have an impact on the biology of this pathogen. Our study nicely complements recent results reported with M. graminicola Carboxin-selected mutants The primary aim of this study was to understand possible target site resistance mechanisms to a range of newly introduced subclasses of carboxamides in Compounds used for selection following UV mutagenesis were chosen both for structural diversity and relevance to the crop protection market .−2 or 50 mJ cm−2 leading to 80% and 50% lethality after UV treatment respectively. Frequency of resistance development ranged between 7.2 E−7 for Carboxin at the minimal selective concentration . Target mutations were identified in all three Qp site encoding subunits and as many as 27 different substitution types were identified. Rare cases of double substitutions were observed in SDHB , SDHC (Isopyrazam selection) and SDHD (Carboxin selection). However, no mutants carrying substitutions in more than one subunit simultaneously were obtained. The absence of target mutation was only observed for 8 strains (1.8%). Additional controls performed on these strains including repeated isolation under selective conditions and re-sequencing of the four SDH encoding genes confirmed non target site resistance mechanisms can be selected under low compound selection pressure.Following primary selection, 482 colonies were picked from the primary selection plates  and re-iSelected substitution types appeared to be both compound and concentration dependent. Interestingly, clear decreases in resistance frequencies were observed for Fluopyram and Carboxin upon shifting selection from MSC to 5× MSC . These wAmong the 3 subunits composing the Qp site, SDHB is by far the most conserved, displaying 57% identity across ascomycetes sequences used in the alignment . By contOur mutagenesis screen for carboxamide resistance led to the identification of 18 substituted amino acids, among which 13 were affecting highly conserved residues of the Qp site. A great proportion of these residues has been shown to be functionally involved in ubiquinone binding and reduction catalysis in other species Two regions prone to substitution could be identified on SDHB, one between amino acids S218 and P225 and the other between amino acids R265 and N271. All these SDHB amino acid positions are strictly conserved across species. On SDHC, one major region prone to amino acid substitution was identified between amino acid T79 and V88, with a continuous stretch of substituted positions between S83 and V88. This later contains the S83 and R87 highly conserved Qp site residues and four neighbouring positions which are not conserved across species . The nature of the residues present at these positions may have an influence on Qp site structure and properties and thus, on the biological profile of carboxamides. Finally, the H145 and H152 histidines, which are also strictly conserved across species, were both found substituted with arginines. On SDHD, D129 was the only substituted position; together with its neighbouring Y130 these 2 positions represent the only conserved residues across the whole protein in the SDHD alignment.in vivo using liquid culture growth (AE media) and in vitro using purified mitochondria and the succinate: Q0/DCPIP activity test  to 39.7 (SDHD_D129S) for Carboxin selection, 22.9 (SDHD_D129G) to 464.4 (SDHC_N86K) for Isopyrazam selection, 5 (SDHB_P220L) to 126.8 (SDHC_S83G) for Fluopyram and 21.7 (SDHB_H267Q) to 564.3 (SDHC_S83G) for Boscalid. Because of the very poor succinate: Q0/DCPIP activity displayed by some mutants in vitro, we calibrated our test by adjusting enzyme concentrations at a similar initial velocity . Similar discrepancies between the in vivo and in vitro rankings can be observed for all of the carboxamides tested. However, these data enable a comparison of the differential behaviours displayed towards the 4 carboxamides tested by a given mutated enzyme. Qp site mutations are well known to influence SDH enzyme activity and stability upon catalysis, in vivo and in vitro sensitivity data suggested that the impact of substitutions on enzymatic activity needed to be addressed to have a fuller understanding of the factors influencing the resistance displayed in vivo.To explore the sensitivity profiles conferred by amino acid substitutions at the cellular and mitochondrial level, we determined sensitivity towards 4 different compounds for 26 of the identified substitution types both ity test . To ensucity see . As IC50E. coli SQR). In the crystal structure 1NEN the quinone ring is sandwiched by B_Pro160 and C_Ile28 et al. who divided the binding pocket into a Q1 and Q2 sites E. coli SQR (2WDQ) enzymes demonstrated a 180° flipped oxathiin ring M.graminicola, C_Ser27 in E.coli) in ubiquinone binding and catalysis In order to obtain a clearer understanding of the structural basis for the differential resistance profiles observed across carboxamides in the SDH mutants, a prediction of the binding modes for the carboxamides used in this study was required. In order to generate putative binding modes, all publicly available crystal structures were analysed in a first step to identify key pharmacophoric features see . All cryin vivo) observed across all ligands with the SDHC_S83G mutant. The acid part of all carboxamides  is sandwiched between C_Arg87 and B_Ile269 and form Van-der-Waals interactions to SDHB_I269, and cation-π interaction to SDHC_R87. The SDHC_R87C mutation abolishes the cation-π interaction; in vivo, this mutation conferred only limited carboxamide sensitivity decrease (RFs between 3.2 and 18.8). In vitro, the effect of the SDHC_R87C substitution on enzyme inhibition looked more complex as only partial inhibition could be reached at maximal concentration of the inhibitor despite of an otherwise WT like response. The SDHB_I269V mutation also conferred limited resistance factors (ranging between 4 to 20 fold in vitro). A valine can still compensate for some of the lipophilic interactions carried out by SDHB_I269. The ortho substituents of the acid parts  are pointing towards SDHB_S221 which corresponds to the orientation suggested by Ruprecht et alUtilizing observed protein ligand interactions, putative binding modes were generated for Boscalid, Isopyrazam, and Fluopyram . All thrG. gallus and S. scrofa SQR crystal structures) or to one heme propionate . By analyzing the electron density for the histidine sidechain it was suggested that this amino acid can adopt multiple conformations M. graminicola SDH, SDHB_H267 makes a direct hydrogen bond to SDHD_D129 and edge to face stacking to the four different inhibitors. However, an alternative binding mode can be hypothesized, where a direct hydrogen bond of the histidine to the pyridine of Boscalid, to the pyrazole of Isopyrazam, and to the oxathiin of Carboxin occurs. In this case SDHB_H267 cannot make direct hydrogen bonds to SDHD_D129 or one heme propionate anymore. This alternative is supported by the recently solved crystal structure of the E. coli SQR enzyme containing the histidine to threonine substitution (PDB code 2WP9). In this study, the complex II was shown to remain stable and active in a case where neither a hydrogen bond to the heme propionate nor to SDHD_D129 are formed in vitro resistance factors for Boscalid (RF = 473.8), Isopyrazam (RF = 142.6), and Carboxin (RF = 65.9). In order to understand this in more detail a model for M. graminicola SQR with the SDHB_H267Y substitution was generated (in vivo RF = 14.1) is equipped with the weakest accepting group followed by Isopyrazam and Boscalid.SDHB_R265 and SDHD_D129 are not involved in direct interactions to the ligands according to the binding models but form a hydrogen bond network with the heme and the imidazole ring of SDHB_H267. SDHB_H267 is positioned at the bottom of the Qp site. Depending on the organism this residue either makes a direct hydrogen bond to SDHD_D129  and Boscalid (RF = 28) with the SDHB_P220T mutant. SDHC_A84 is predicted to interact via Van-der-Waals forces with the aliphatic linker of Fluopyram and the pyridine ring. This aliphatic linker is a specific feature of Fluopyram compared to the other carboxamides analysed in this study. The linker is in the z-dimension sterically more demanding and is getting closer to SDHC_A84 compared to phenyl ring systems of Carboxin, Boscalid, and Isopyrazam. In agreement with these predictions, Fluopyram binding is found to be specifically affected by the SDHC_A84V/I substitutions as shown by over 30 fold higher RFs in vitro compared to the phenyl ring carboxamides. Furthermore, higher resistance factors are observed with the bulkier amino acids substitution, SDHC_A84I which is in line with the proposed interaction. Interestingly, the amino acid at SDHC_A84 position is not conserved across fungal species and may contribute to some of the biological profile observed for these carboxamide subclasses.SDHB_P220 is a fully conserved Qp site residue sandwiching the quinone ring together with a residue corresponding to SDHC_A84 in all ubiquinone-SDH co-crystal structures E. coli, modification of the coordinated histidine impairs ubiquinone binding and catalysis efficiency of the SDH enzyme M. graminicola may influence heme properties and/or positioning which in turn might have a negative impact on inhibitor binding.The SDHB_W223, SDHC_L71, and SDHC_W80 residues, are all in Van-der-Waals distance to the amine part of all ligands. However, no substitutions at these positions were observed in this study. The substituted residues, SDHB_S218, SDHB_N225, SDHB_N271; SDHC_T79, SDHC_L85, SDHC_N86, SDHC_V88, SDHC_H145, and SDHC_H152 are not predicted to directly interact with carboxamides but amino acid substitutions more distant from the binding pocket may influence the shape and electronics of the Qp site and therefore have a negative impact on ligand binding. A putative long-range effect and/or a rearrangement of a large zone affecting part of the Qp site is particularly likely for the SDHC_H145 position which was substituted by an arginine upon Carboxin selection as this is the furthest substituted residue from the Qp site. Despite a distance of ∼10 Å to the closest atom of docked Carboxin, the SDHC_H145 is a ligand of the heme and therefore may influence the Qp site structure and/or properties. According to available SDH crystal structures, the propionate side chains of the heme can be considered part of the Qp site. Furthermore, it was shown that the heme itself directly participates in electron transfer during catalysis M. graminicola tridimensional SDH model, these residues are not predicted to directly interact with the inhibitor yet are in the close vicinity to the heme, again making a direct influence on heme positioning possible.SDHC_N86K and SDHC_H152R were the most frequent substitutions upon Isopyrazam and compound A selection. Both were also selected by Boscalid but less frequently and the SDHC_N86K substitution was also found upon Fluopyram selection. In the M. graminicola SDHB antibodies (data not shown).To compare specific activities conferred by the different amino acid substitutions we first determined the level of SDH enzyme in mitochondrial preparations for each of the resistant strains . SDH enzM. graminicolaThe malonate sensitive succinate: PMS/MTT activity test is classically considered as a measurement of the SDHA-B dimer In vivo, the electrons derived from succinate have to be transferred to its acceptor (ubiquinone) to enable the enzymatic oxidizing of novel molecules of succinate. The succinate: Qo/DCPIP activity is a measure of the succinate: ubiquinone reductase activity, which is the most relevant one biologically. In vitro, full inhibition of the WT enzyme can be reached using all four carboxamides compared in this test.All mutants displayed weaker ubiquinone reductase activity compared to the wild type. The weakest effect was detected for the SDHC_A84V mutant which was 87% as active as the WT. The strongest impairment was displayed by the SDHB_N271K mutant with only 5% residual activity. As might be expected, different substitutions at the same residue can result in differential impact on enzyme efficiency. This effect seems to be linked to the degree of steric or physico chemical conservation displayed by the substitutive amino acid. For example, the SDHD_D129E conservative substitution maintains 42% of WT activity whiles the non conservative SDHD_D129G/S/T substitutions impact enzyme activity much more strongly (from 6 to 23% remaining activity). The same observation can be made for the SDHC_A84V variant which is more active (87%) than the SDHC_A84I (51%) counterpart which carries a larger substituent.in vivo log IC50 estimates and in vitro log IC50 estimates across the different strains for any given compound displayed reasonable correlation for each of the four compounds considered here  compared to the one reached by the corresponding homologous recombinant HR_H267L strains. This effect was not restricted to SDHB, as similar results were obtained with the SDHC_A84V mutation towards Fluopyram , a herbicide reported to induce oxidative stress through the generation of superoxide anions and a continuous 100% oxygen atmosphere simulating hyperoxia. Paraquat supplemented media had a similar impact on growth of both the homologous recombinant strains and the WT  . As could be expected from the lack of hypersensitivity to oxidative stresses in previous in vivo tests, comparison of our subset of homologous recombinant strains showed no clear evidence for a difference across the WT and the target mutants (p>0.1)  . This re (p>0.1) . Howeverin planta growth, a smaller subset of the recombinant strains was compared for necrosis symptoms displayed in planta. For comparing the diverse SDH mutants we used various homologous recombinant strains carrying SDHB_H267L, SDHC_H152R, SDHC_N86K and SDHC_A84V mutations. Homologous recombinant strains were similarly inoculated on the sensitive WT plant Riband and in planta symptoms assessed visually 14 days after inoculation , B. cinerea (SDHB_H272Y) and A. alternata (SDHB_H277Y) (C. cassiicola and P. xanthiiM. graminicola SDHB_H267Y mutant compared to the WT (data not shown). Given the degree of conservation for cross resistance profiles seen with this particular mutant it seems that the depicted interaction is consistently conserved across species.Our mutagenesis study led us to identify 27 different substitution types affecting 18 positions in 3 of the 4 subunits encoding the Qp site of the target SDH enzyme. The pattern and frequency of mutations selected was found to be highly dependent on the compound used for selection. Accordingly, sensitivity profiles are substitution dependent, as a result of specific interaction of different classes of inhibitors to specific structural features of the enzyme. The large majority of the mutations lead to a sensitivity decrease across all carboxamides sistance . Therefoin vivo survival upon carboxamide treatment is a balance between a negative impact brought by decreased enzyme activity/stability caused by substitutions affecting the Qp site and a positive one brought by poorer binding of carboxamide inhibitors resulting in weaker inhibition of the enzyme. From a cellular perspective and considering the central role of SDH for energy production, it seems logical that the remaining SDH activity, which is necessary to maintain an active TCA cycle, is the driver for survival.Using transformation we evidenced that the remaining SDH activity present in the cells at a given inhibitor concentration is responsible for survival. Interestingly, very low levels of SDH activity were sufficient for the establishment of resistance, as confirmed by the selection of substitutions leading to over 90% loss in activity. This suggests that for each mutant, E. coli, SDHD_Y83F and S. cerevisae SDHD_Y89F substitutions impair 85% and 95% of the ubiquinone reductase activity respectively M. graminicola MgSDHD gene using site directed mutagenesis and found that ectopic transformants expressing SDHD_Y130F are more sensitive to carboxamides compared to the WT (data not shown). The absence of any mutation at this residue for all carboxamides tested might indicate that substitutions at this position could not confer selective advantage in the balance between catalysis and inhibition.A balance between substrate and inhibitor binding would explain why some highly conserved residues of the Qp site which are predicted to be essential for carboxamide inhibitor binding in the tridimensional model were neither found substituted in our screen nor reported yet in field populations. Notably the fully conserved Qp site residues SDHB_W224 and SDHD_Y130 which are predicted to hydrogen-bond to the amide oxygen of carboxamides in vivo. Furthermore, similar Qp site substitutions have been shown to have biological influence on the lifespan of organisms through the increased production of ROS by the mutated SDH enzyme M. graminicola. One explanation for this result may be that our initial selection for carboxamide resistance is strongly biased against the selection of mutants which display high level of oxidative stress. This could occur if the presence of ROS producing mutations impacted growth as shown with many yeast Qp site mutants on non-fermentable media M. graminicola SDH is 10 times lower compared to that of S. cerevisiae SDH M. graminicola is a slow growing pathogen and might have a much lower mitochondrial ROS production rate compared to fast growing fungi like S. cerevisiae. Secondly, M. graminicola is a hemibiotrophic pathogen and therefore has to survive plant induced oxidative burst in colonized plant tissue. The pathogen requires and induces an exceptionally efficient ROS detoxification enzyme toolset in order to survive the necrotrophic stage of its lifecycle M. graminicola itself and ROS signalling may also contribute to pathogenicity In vitro tests with a purified mitochondrial fraction will be required to measure the potentially increased production of ROS by the mutated M. graminicola SDH enzyme in more detail M. graminicola and other species.Because SDH enzyme activity was impaired in all mutants we expected to find some degree of fitness penalty in planta virulence tests performed with some of the HR strains showed that some of the SDH mutations can lead to increased symptoms in the absence of carboxamide treatment. The reason for this remains unclear, but the increased symptomology observed in the climatic room is unlikely to result in increased fitness in the field as nature would have selected such SDH variants during the course of evolution if it was the case. One explanation for the observed phenotype is that the presence of less efficient SDH enzyme might lead to increased intracellular levels of succinate as observed in yeast mutants M. graminicola WT upon treatment with sub-lethal doses of carboxamides (data not shown). In yeast, perturbations of the succinate dehydrogenase function have a large impact on metabolism. Moreover, it was shown that succinate dehydrogenase mutations displaying similar impairment in quinone reductase activity can lead to very distinct metabotypes M. graminicola SDH enzyme may lead to developmental effects on in planta growth and symptoms development as complex regulation of metabolic fluxes have been shown to play a major role in the infection process in planta remain elusive, our data indicate that SDH mutations can have an influence on the biology of the pathogen. Aside additional fitness penalty and resistance studies to be performed in planta, the question as to know whether this phenomenon is influenced by ROS or TCA metabolites or both will require further evaluations now rendered possible by the homologous recombinants generated during the course of this study.In our test conditions, in planta growth point towards some mutations for which sensitive molecular tests will be designed and applied in the monitoring of field populations. These results combined with the resistance situation in other pathogens further stress the importance of a proper anti-resistance strategy for the SDHIs fungicides. At this point in time and in order to prolong the efficacy of this class of fungicide in wheat, recommendations include restrictions in the number and timing of applications as well as the mandatory usage of mixtures (see www.FRAC.info).Finally, our data clearly shows that a combination of factors might have to be considered for a diagnosis of mutations likely to occur in the field. In our mutagenesis screen, high resistance factors, frequency of occurrence and maintained M. graminicola isolate IPO323 was used for all experiments E. coli. Agrobacterium. tumefaciens strain EHA105 A. tumefaciens mediated transformation.M. graminicola strain after inoculation of AE agar plates with 107 cells followed by incubation for 2 weeks at 20°C in the dark. Selection plates were produced after adding 1% of a 100× DMSO solution of the active ingredients to pre-melted AE-agar. A higher concentration corresponding to 5× MSC was applied with Carboxin, Isopyrazam, Fluopyram and Boscalid and 2.5× MSC with the Isopyrazam pyrrole analogue compound A.Minimal selective concentration (MSC) is the minimal concentration of carboxamide necessary to fully inhibit growth of the wild type IPO323 7 cells were spread onto carboxamide amended AE agar plates and mutagenized using UV Stratalinker 2400 (Stratagene) with 75 mJ cm−2 or 50 mJ cm−2. These UV treatments produce lethality of 80% and 50% respectively. Selection plates were incubated in the dark at 20°C for 14 days. Visible colonies were picked from the primary selection plate and re-grown on fresh selection media for 2 additional weeks under the same conditions.Cells were harvested from 4 days old V8 agar plates and conidial cells were suspended in liquid AE. 10M. graminicola IPO323 genome sequence Strains were grown in 5 ml liquid TSM40 on a reciprocal shaker  for 5 days prior to genomic DNA extraction using a Qiagen mini plant kit (Qiagen). PCR primer design was based on the A. nidulans gpda  promoter to drive the expression MgSDH gene were amplified from genomic DNA using PfuTurbo-DNA polymerase (Stratagene) and two rounds of amplification with primers listed in MgSDHB_TrpCter, pNOV_gpda_MgSDHB-H267L_TrpCter, pNOV_gpda_MgSDHC_TrpCter, and pNOV_gpda_MgSDHC-A84V_TrpCter plasmids.For ectopic integration of SDH subunit expression cassettes, the multisite binary pNOV2114_gateway vector was used with the SDH gene without the start codon but containing the mutation(s) leading to the specified amino acid substitution plus ∼800 bp down-stream of the gene were PCR amplified using primers described in For homologous recombination, genomic fragments containing part of the M. graminicola transformation procedure has been described previously MgSDH gene and hygromycin resistance gene. For homologous recombinants, primer pairs were generated to verify the unique presence of the mutated copy using differential restriction profiles displayed by the mutated allele versus the non mutated allele. In addition primer pairs were designed to screen for the absence of T-DNA downstream of the integrated fragment. All strains which passed PCR verification were sequence verified. All PCR primers and details mentioned above are listed in For ectopic transformants, primer pairs were designed to screen for the presence and completeness of the gpda promoter, SDHB and SDHD transformants; BamH1/EcoR1 for SDHC transformants. Digested DNA was electrophoresed and subsequently blotted and UV cross-linked to Hibond™ Nylon membranes  SDH genes, followed by washes at 55°C (SDHC and SDHD) or 60°C (SDHB). Luminescence signal was generated with CDP-Star  and visualized on Kodak Biomax light films (Sigma).The copy number of the SDH gene in the transformed strains was assessed by Southern blot. Hybridization probes were produced by PCR amplification of the SDH gene of interest and labeled using the AlkPhos Direct™ system . Genomic DNA (∼10 µg) were digested using BamH1 for 600 nm and grown on a reciprocal shaker  for 5 days. Cells were harvested by vacuum filtration and disrupted in liquid nitrogen using a mortar and pestle. Resultant powder was re-suspended to 10% w/v in mitochondrial extraction buffer . Extract was clarified by centrifugation , intact mitochondria were then pelleted at 10,000 g for 20 minutes at 4°C. The pink-reddish pellet was re-suspended in a small volume  of extraction buffer. This volume was transferred to conical 1.5 ml tubes and re-centrifuged, 10,000 g for 10 minutes at 4°C. The pellet separates into two layers; the upper reddish colored being easily re-suspended by pipetting is composed of nearly pure mitochondria as assessed by microscopy. Subsequent sucrose gradient centrifugation did not improve specific activity of the mitochondrial fractions; therefore the purification procedure was stopped at this stage. Mitochondrial suspensions were brought to a final concentration of 10 mg ml−1 in extraction buffer and stored at −80°C until used. SDH activity was found to remain stable for months.Strains were grown in 1 liter TSM40 units. Cultures were inoculated at 0.05 ODMitochondrial suspensions were incubated at 30°C for 30 minutes in extraction buffer supplemented with 10 mM succinate prior to enzymatic measurements.3, 10 mM succinate) supplemented with 140 µM Dichlorophenolindophenol (DCIP) and 1 mM of 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q0), from a 0.1 M DMSO stock solution. A reaction plate was equilibrated for 10 minutes at 30°C before starting the reaction. DCPIP reduction was conducted at 30°C and monitored at 595 nm using a HTS7000 microtiterplate reader (Perkin Elmer). For calculations a molecular extinction coefficient of ε = 17 mM−1 cm−1 was used Succinate: ubiquinone/DCPIP measurements were performed by adding 5 µl of pre-activated mitochondria to 100 µl assay buffer . Reactions were started with the addition of 5 µl of preactivated mitochondrial suspension to 100 µl of the reaction mixture at 30°C using an experimental setup similar to DCPIP measurements. The MTT reduction was monitored by reading the absorbance increase at 570 nm using a HTS7000 microtiterplate reader (Perkin Elmer). For calculations a molecular extinction coefficient of ε = 21 mM−1.cm−1 was used The succinate: PMS (Phenazine methosulfate) coupled reduction of MTT -2,5-diphenyltetrazolium bromide) measurements were performed using final concentrations of 400 µM PMS and 150 µM MTT freshly diluted in assay buffer  following conditions described above. Inhibitor concentrations ranged between 1 mM and 0.95 nM with uniform 4× dilution factor steps (11 inhibitor concentrations+DMSO control). 96 wells plates were pre equilibrated at reaction temperature (30°C) for 10 minutes before the reactions were started by the addition of 5 µl preactivated M. graminicola mitochondrial suspension. Calculated absorbance slopes (OD/hour) were used for IC50 calculations using Graphpad Prism 5.0 software non linear curve fitting against log inhibitor concentrations.In the succinate: ubiquinone/DCPIP test, activity displayed by some mutants was so low that an adjustment for enzyme activity was necessary for accurate IC50 determination. For this reason, all mitochondrial suspensions were adjusted to a similar initial velocity  G. gallus (chicken) SQR in complex with the inhibitor carboxin resolved at 2.10 Å (PDB code 2FBW) S. scrofa (pig) SQR cocrystallized with ubiquinone-2 at 2.40 Å (PDB code 1ZOY) The four M. graminicola SDH protein sequences with the bacterial (1NEK), the avian (2FBW) and the porcine (1ZOY) protein subunits. The alignments were analyzed and edited in the Prime 1.5 workflow within the Maestro environment  M. graminicola displayed limited identity with the sequences of the three crystallographic templates. Therefore, to further improve the alignments, the multiple template strategy together with a secondary structure prediction  and superimposed onto each other. The Qp binding sites of the superimposed structures with a resolution higher than 3 Å were visually analyzed in more detail for common pharmacophoric features .MgSDH with Moloc G.gallus SQR complex crystal structure 2WQY. In a second step the protein ligand complex was minimized using Moloc MAB force field M. graminicola SQR protein rigid.Carboxin was manually placed in the Qp binding site of M. graminicola SQR Qp binding site. Interactions to key residues which were determined through the pharmacophore elucidation (see results) were formed. In a second step the protein ligand complexes were minimized using Moloc MAB force field M. graminicola SQR protein rigid.Boscalid, Fluopyram, and Isopyrazam were manually docked into the M. graminicola SQR homology model with the “mutate residue” procedure included in Maestro , followed by a contact analysis involving multiple rotamers and a geometry sanitation within the OPLS2005 force field.Mutations were generated within the 5 cells ml−1 in AE media. Sterile 96 wells microtiter plates with lids (Costar) were inoculated with 198 µl of this cell suspension (105 cells per well) and 2 µl of inhibitor (100× in DMSO). Final inhibitor concentrations were between 0.95 nM and 1 mM with uniform 4× dilution factor steps (11 inhibitor concentrations+1 DMSO control). Plates were incubated for 6 days at 19°C in the dark before cells were re-suspended and measured for growth (OD600 nm). IC50 values were determined by non linear curve fitting on the basis of measured OD values versus log inhibitor concentrations (GraphPad Prism 5.0 software).Conidial cells were collected from 4 day old V8 agar plates (cultured at 18°C in the dark) using liquid AE and a cell spreader. Cell density was determined and cells re-suspended to a concentration of 5×107 cells ml−1 in assay prior to their incubation for 2.5 hours in the dark in the presence of 5 µM MitoSOX red (Invitrogen). After the incubation period, 200 µl aliquots of each cell suspension were distributed (3 replicates per strain) into 96well plates (AcroWell™ 96 filter plates) equipped with 0.45 µm filters . First step of centrifugation 500 g for 3 minutes eliminated non incorporated fluorescent dye. Cells were resuspended in 200 µl assay buffer and same centrifugation procedure was applied. Finally, cells were resuspended in 110 µl assay buffer and 100 µl were transferred to clear bottom/black framed 96 wells Optilux™ plates (Becton Dickinson) for measurement. Fluorescence was measured with an Enspire 2300 multimode reader (Perkin Elmer) using λexc of 510 nm and λem of 580 nm .Conidial cells were collected from 4 day old V8 agar plates (cultured at 18°C in the dark) using 2 ml of assay buffer  and a cell spreader. Cell density was determined and cells re-suspended to a concentration of 3×106 cells ml−1 in de-ionized water supplemented with 0.05% Tween 20. Each spore suspension was used for the inoculation of three pots of two weeks old wheat (var. Riband) plantlets (3 to 4 plants/pot). The plants were homogenously sprayed and covered with a Plexiglas chamber for incubation at 100% humidity for 48 hours at 24°C in the dark. After this incubation period, pots were placed in a climatic room with alternation periods of 8 hours darkness (18°C), and 16 hours light (24°C), with 60% constant humidity. Evaluation of symptoms was performed 16 days after inoculation by visually estimating the percentage of 2nd leaf area covered with pathogen–related necrosis symptoms. Negative controls (uninfected plants) confirmed the absence of necrosis at the evaluation time.Conidial cells were collected from 4 day old V8 agar plates (cultured at 18°C in the dark) using 2 ml of de-ionized water. Cell density was determined and cells re-suspended to a concentration of 10Table S1Oligonucleotides list. Oligonucleotides that were used for cloning, sequencing and primary screening of successful homologous recombination events.(XLSX)Click here for additional data file.Table S2Protein identifiers and source databases used in  sequences alignments. Protein identifiers are hyperlinked to corresponding feature web page in the right column.(XLSX)Click here for additional data file.


PKC plays a pivotal role in mediating monocyte adhesion; however, the underlying mechanisms of PKC-mediated cell adhesion are still unclear. In this study, we elucidated the signaling network of phorbol ester PMA-stimulated human monocyte adhesion. Our results with pharmacological inhibitors suggested the involvement of AMPK, Syk, Src and ERK in PKC-dependent adhesion of THP-1 monocytes to culture plates. Biochemical analysis further confirmed the ability of PMA to activate these kinases, as well as the involvement of AMPK-Syk-Src signaling in this event. Direct protein interaction between AMPK and Syk, which requires the kinase domain of AMPK and linker region of Syk, was observed following PMA stimulation. Notably, we identified Syk as a novel downstream target of AMPK; AICAR can induce Syk phosphorylation at Ser178 and activation of this kinase. However, activation of AMPK alone, either by stimulation with AICAR or by overexpression, is not sufficient to induce monocyte adhesion. Studies further demonstrated that PKC-mediated ERK signaling independent of AMPK activation is also involved in cell adhesion. Moreover, AMPK, Syk, Src and ERK signaling were also required for PMA to induce THP-1 cell adhesion to endothelial cells as well as to induce adhesion response of human primary monocytes. Taken together, we propose a bifurcated kinase signaling pathway involved in PMA-mediated adhesion of monocytes. PKC can activate LKB1/AMPK, leading to phosphorylation and activation of Syk, and subsequent activation of Src and FAK. In addition, PKC-dependent ERK activation induces a coordinated signal for cytoskeleton rearrangement and cell adhesion. For the first time we demonstrate Syk as a novel substrate target of AMPK, and shed new light on the role of AMPK in monocyte adhesion, in addition to its well identified functions in energy homeostasis. Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase, comprising two N-terminal Src homology 2 (SH2) domains, a linker region, and one kinase domain in its C-terminal region Besides the well identified signaling pathway that links Syk indirectly to PKC via PLCgamma, which induces phosphoinositide turnover to generate diacylglycerol for PKC activation, direct activation of PKC by Syk was demonstrated. In FcRI-stimulated mast cells, PKCbetaI and PKCalpha are activated by Syk-mediated tyrosine phosphorylation at Tyr662 and Tyr658, respectively AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase composed of a catalytic alpha subunit and regulatory beta and gamma subunits PKC plays a pivotal role in mediating monocyte adhesion; however, the downstream mechanisms mediating its function are not fully elucidated. Thus, in this study, using phorbol 12-myristate 13-acetate (PMA)-stimulated human monocytic leukemia cell line THP-1 as a model system in most experiments, we investigated the signaling network among PKC, Syk and AMPK, and explored their functional relevance in monocyte adhesion.Previous reports have demonstrated that human monocytic THP-1 leukemia cells can be induced to differentiate along the monocytic lineage following exposure to PMA, a potent tumor promoter capable of activating conventional and novel PKC isoforms. PMA treatment resulted in adherence, loss of proliferation, phagocytosis of latex beads, and expression of CD11b and CD14 To further identify the downstream signaling elements, we analyzed the phosphorylation status of Syk, Src and AMPK. Phosphorylation of human Syk at Y525/526 , Src at Y419  and AMPKalpha at T172 are activation indexes of the respective kinases. Next we examined whether inhibition of PKC-dependent adhesion by pharmacological inhibitors is due to off-target effects. Since targeting PKC to the plasma membrane is a prerequisite for activation, we examined the membrane translocation of PKCalpha and PKCbeta after PMA stimulation. As shown in After observing Syk, Src and AMPK activation are downstream events of PKC, we investigated the order of the signaling cascade. We found that PMA-induced phosphorylation of Src at Tyr419 was dramatically inhibited by PP2, Syk inhibitor I and compound C. Consistent with this finding, results using antibody against Tyr216 phosphorylation on c-Src, which is another index of c-Src activation, also exhibited the same phenomena . These dContinuing to use pharmacological tools to dissect the relationship between AMPK, Syk and Src, we found that PMA-induced AMPK activation was inhibited by compound C, but not by PP2 and Syk inhibitor I. In contrast, PMA-induced Syk activation was inhibited by Syk inhibitor I and compound C, but not by PP2 . In vitrTo verify the finding that AMPK is responsible for Syk activation, we examined the protein interaction between both molecules. Given that PKC mediates AMPK activation, we next explored the molecular mechanism underlying this event. To understand whether AMPK is a direct substrate of PKC, AMPK was immunoprecipitated, then probed with anti-phosphoserine PKC substrate Ab. After observing the association of Syk and AMPK under conditions of PKC activation, we would like to map their respective interaction domains. To this end, we made various Flag-tagged DNA constructs of Syk  . Conversin vitro autophosphorylation assay. As shown in SEQIV) containing the amino acid sequences of Syk at 167–182 was synthesized as substrate in an in vitro kinase assay. We found that AICAR- and PMA-activated AMPK indeed caused Syk peptide phosphorylation , which interfere with the interaction through fibronectin and vitronectin respectively, on PMA-stimulated monocyte adhesion to culture dishes and HUVEC In this study, we demonstrate that PKC, Syk, AMPK, Src and ERK kinases play critical roles in monocyte adhesion, and propose a novel signaling mechanism of this event. Previous studies have implicated PKC and Syk in the adhesive functions of integrins Besides the relatively well identified role of AMPK in metabolic processes, this energy sensor kinase is also implicated as an anti-inflammatory target. AICAR, the pharmacological activator of AMPK, has been reported to exert anti-inflammatory and immunomodulatory effects in various models of inflammation Despite the above reports implicating AMPK as an anti-inflammatory molecule, contrary findings of AMPK-dependent inflammatory responses are also documented. In this respect, AICAR and adiponectin can stimulate IL-6 production in fibroblasts via an AMPK-dependent pathway Leukocyte adhesion is the essential step required for circulating monocytes to traverse multiple tissue barriers, and to perform their functions in host defense. Both lower expression of proinflammatory cytokines, chemokines, and adhesion molecules, and upregulation of eNOS activity in endothelial cells might contribute to the AMPK-mediated inhibition of leukocyte-endothelial cell interaction SEQIV), we further confirmed the phosphorylation site by AMPK at the Ser178 residue of Syk. Fourth, phosphorylated Syk in turn exerts its activation and propagates downstream signals to Src and FAK for cell adhesion. The cross-activation of Syk and Src has been demonstrated to exert the cellular functions of immunoreceptors and integrins In this study, we proved that the upstream kinase of AMPK, LKB1, is a substrate of PKC in THP-1 monocytes stimulated by PMA. We ruled out that AMPK has a direct interaction with PKC or involvement of CaMKK in this event. Moreover, we also provide evidence to support that Syk is a novel biological substrate of AMPK, thus extending the functional impact of AMPK in regulation of leukocyte adhesion. First, Syk can directly interact with AMPKalpha2 via the linker region of Syk and the kinase domain of AMPK. Second, the direct interaction between Syk and AMPKalpha2 is induced upon AMPK activation. Third, Syk-AMPKalpha2 interaction leads to AMPK-mediated phosphorylation of Syk at Ser178, which is located at the C terminal region of SH2 domain. Using Syk synthetic peptides as a substrate  were purchased from ScienCell . Primary monocytes were prepared from the blood of healthy human donors and cultured in DMEM as described previously DMEM, RPMI 1640, FBS, penicillin, and streptomycin were obtained from Gibco BRL. The enhanced chemiluminescence detection agents were purchased from Amersham Biosciences. Lipofectamine™ LTX (Cat No. 15338-100) and Plus™ Reagent (Cat. No. 11514-015) were obtained from Invitrogen. Inhibitor of alpha5beta1 and anti-CD11b-FITC were purchased from Santa Cruz Biotechnology. Antibodies specific for AMPKalpha, Syk, ERK and phosphorylated AMPKalpha (Thr172), Syk (Tyr525/526), ERK1/2 (Thr202/Tyr204), GFP, tubulin and Na/K-ATPase were purchased from Cell Signaling. Antibody against human Src  was purchased from Invitrogen. Anti-Flag bead antibody (M2-beads), anti-Flag antibody, STO-609 and other chemicals were obtained from Sigma Aldrich. Integrin–alphaVbeta3 blocking antibody (LM609) was obtained from Chemicon . Anti-CD14-PE was purchased from Bioscience. All materials for SDS-PAGE were obtained from Bio-Rad. Ro318220, GF109203X, PMA, Compound C, Syk inhibitors , PP2, Go6983, Ro320432, A769662 and U0126 were purchased from Calbiochem. LY333531 was purchased from Alexis Biochemicals. Human fibronectin and vitronectin were purchased from R&D systems . PKC activity kit was purchased from ENZO Life Sciences .4 cells) and human primary monocytes (2×104 cells) in complete culture medium containing 10% FBS were plated to tissue culture 96-well plate , and pretreated with the indicated kinase inhibitors for 30 min, followed by stimulation with PMA (1–100 nM) for different periods. Nonadherent cells were removed by gentle washing with PBS before fixation with methanol for 10 min, followed by addition of 0.1% (v/v) crystal violet in PBS for 15 min. After washing with distilled water three times, acetic acid was added to a final concentration of 33% (v/v) to solubilize cell lysates, followed by absorbance measurement at 550 nm. In some experiments to determine the adhesive response to HUVEC, THP-1 cells were pre-labeled with 0.1 µg/ml BCECF-AM for 1 h at 37°C. Then cells were washed and treated with indicated agents including kinase inhibitors and/or PMA for 1 h. Cells were washed twice with growth medium, and equal amount of THP-1 cells were added to EC monolayer cultured in 96-wells for 1 h. After removal of culture medium and non-adherent THP-1 cells, the number of adherent cells was determined by measuring the fluorescence intensity.THP-1 cells (5×105′-CTC TCG GGA AGA AGC TGA GCA AAT TGT-3′) and primer 2 (5′-ACA ATT TGC TCA GCT TCT TCC CGA GAG) for hSyk (S178A) mutation.Myc-AMPKalpha2, Myc-AMPKalpha2 (1–312), Myc-AMPKalpha2 (1–398), and Myc-AMPKalpha2 (303–552) were provided from Dr. Kelly A. Wong . Flag-tagged wild type human Syk (1–635) was constructed by ligating the PCR-derived DNA fragment spanning the open reading frame region of Syk with the EcoRI/SalI digested pCMV2-FLAG vector. The regions covering 107–635, 260–635 or 1–369 amino acids of hSyk cDNA were amplified by PCR and inserted to EcoRI/SalI digested pCMV2 FLAG to generate the N-SH2-del (dNSH2), SH2-del (dSH2) or kinase domain-del (dKD) of hSyk. The C-SH2-del hSyk (dCSH2) construct was amplified by PCR covering 1–106 and 260–635 amino acids of hSyk. To generate S178A Syk, we used the Quickchange site-directed mutagenesis kit (Stratagene), and serine 178 in the human Syk cloned in the pBlue-script vector was substituted with alanine by changing the codon from ATC to AGC. The mutated primers used were primer 1 (6 cells/well) were transfected using lipofectamine™ and Plus™ Reagent with 3 µg plasmid for 4 h, and then changed to complete medium. After 24 h transfection, cells were harvested, followed by collection of cell lysates for immunoprecipitation or western blot. GFP-tagged constitutively active adenovirus-AMPKalpha2-CA and adenovirus-DN kinase-dead (K45R) variant of AMPKalpha2 provided by Dr. Benoit Viollet  and Dr. Morris Birnbaum  respectively were prepared in 293F cells and purified by BD-Adeno-X™ Virus Purification kits. THP-1 cells were infected with virus at M.O.I. of a 20∶1 ratio for 48 h.HEK293T cells (1×106 cells/well) were transfected using lipofectamine™ LTX and Plus™ Reagent with 3 µg plasmid for 4 h, and then changed to complete medium for 48 h. The combination of both reagents can enhance transfection efficiency in THP-1 cells as mentioned by manufacturer. Using pEGFP-C2 expression and fluorescent microscopy as index, we found that the transfection rate in THP-1 cells was 60%.THP-1 cells (1×106 cells/well) were harvested, fixed with 2% paraformaladehyde, stained extracellularly in 1% FBS/PBS with equal concentrations of control IgG conjugated with fluorochrome (FITC or PE), FITC-conjugated CD11b, or PE-conjugated CD14 antibody for 1 h and then analyzed by flow cytometry.THP-1 monocytes  and once with the kinase reaction buffer . The beads were then incubated for 30 min at 30°C in 20 µl kinase reaction buffer supplemented with 10 µCi of γ-32P-ATP. The precipitated complexes were added with 5 µl 5× Laemmli sample buffer and heated at 95°C for 5 min. After centrifuging the samples, the supernatants were run on 10% SDS-PAGE, followed by autoradiography.To determine in vitro kinase activities of Syk, 500 µg of total protein extracts from stimulated cell lysates were pre-cleared for 30 min and then immunoprecipitated with 1 µg anti-Syk antibody overnight, followed by addion of 10 µl protein A/G-agarose beads and rotation at 4°C or another 1 h. The immunocomplexes were washed 3 times with cold lysis buffer  for 1 h. PKC kinase activity was measured using the PKC kinase activity kit according to the manufacturer's instructions.Values are expressed as the mean ± SEM of the least three experiments. ANOVA and Dunnett's tests were used to assess the statistical significance of the differences, with P values of less than 0.05 being considered statistically significant.


In vascular tissues, phosphorylation of the PKC hydrophobic site, but not turn motif site, as well as phosphorylation of PDK at S241 increased in response to phenylephrine. Calponin knockdown inhibits autophosphorylation of cellular PKC in response to phenylephrine, confirming results with recombinant PKC. Thus these results show that autophosphorylation of PKC is regulated in dVSM and calponin is necessary for autophosphorylation of PKC in VSM.Protein kinase C (PKC) is a key enzyme involved in agonist-induced smooth muscle contraction. In some cases, regulatory phosphorylation of PKC is required for full activation of the enzyme. However, this issue has largely been ignored with respect to PKC-dependent regulation of contractile vascular smooth muscle (VSM) contractility. The first event in PKC regulation is a transphosphorylation by PDK at a conserved threonine in the activation loop of PKC, followed by the subsequent autophosphorylation at the turn motif and hydrophobic motif sites. In the present study, we determined whether phosphorylation of PKC is a regulated process in VSM and also investigated a potential role of calponin in the regulation of PKC. We found that calponin increases the level of in vitro PKC In recent years, it has become clear that multiple, redundant signaling pathways are responsible for the fine-tuning of smooth muscle contractility . This co2+, DAG, and PS. Novel PKCs (nPKC) have a nonligand binding C2 domain and are activated by DAG and PS but not Ca2+. Atypical PKCs contain a nonligand binding C1 domain and are activated by PS but by neither Ca2+ nor DAG. The conventional PKCα and the novel PKCε are the best studied isoforms in the contractile, fully differentiated vascular smooth muscle cell (dVSMC) [PKC isoforms are grouped into 3 classes based on the domain composition of the N-terminal half of the molecule –6. The C (dVSMC) –10. In t (dVSMC) , but, th In general, all members of the AGC kinase family are now thought to require a series of ordered phosphorylations that are thought to “prime” the kinase and convert it into a mature form capable of being activated by the allosteric activators. The first event is a phosphorylation by PDK at a conserved threonine in the activation loop , 12. Thiα in a variety of cell types in culture have been consistent with the completion of such phosphorylation events only during enzyme maturation; however, for novel PKCs at least 2 groups have reported that the phosphorylations are dynamically regulated [ε and PKCδ in cardiomyocytes in primary culture appear to undergo regulated and regulatory phosphorylation of the activation loop and the hydrophobic motif even in the absence of allosteric regulators [The phosphorylation of PKC is thought by some to occur during maturation of the newly synthesized enzyme –18. Howeegulated , 16, 19.gulators . Thus thα-agonist induced activation vascular tissue [Protein kinase C is a key enzyme involved in signaling during smooth muscle contraction . Previour tissue . This ra2, 0.01% NaN3, 1 mM ATP, and 15 mM β-mercaptoethanol. Recombinant PKCα or recombinant PKCε (Panvera) was incubated in solution with either 25 μM BSA, 25 μM protamine, or 25 μM calponin for 15 seconds at 37°C. Calponin was prepared from turkey gizzard. The reaction was stopped by the addition of sample buffer followed by heating. The phosphorylation of PKC was detected by western blot using specific site phosphorylation antibodies.PKC phosphorylation was carried out in the following solution: A: 150 mM NaCl, 10 mM imidazole, 2 mM MgCl2-5% CO2 physiological salt solution (PSS) composed of (in mM) 120 NaCl, 5.9 KCl, 25 NaHCO3, 11.5 dextrose, 1 CaCl2, 1.4 MgCl2, and 1.2 NaH2PO4, pH 7.4. The aorta was cut into circular strips, attached to a force transducer, and allowed to equilibrate for at least 1 hour before being challenged with a depolarizing saline solution replacing 51 mM NaCl with 51 mM KCl, to confirm viability and for normalization. The tissues were washed and allowed to relax for 1 hour before beginning the experiment. The muscle strips were frozen at the desired time points following agonist stimulation by using a dry ice acetone, TCA slurry, and stored at −80°C for further study. Calponin knockdown was performed by 4 days of exposure of vascular strips to phosphorothioate antisense or a scrambled version of the ferret CaP sequence (5′-AT GTT TTC CAG CTG GTG CCA A-3′), or a scrambled version of that sequence (5′-CG TGG TAT AAA ACC GAT CAC G-3′) in serum free organ culture, as previously describe [All procedures in this study were performed according to protocols approved by the Institutional Care and Use Committee. Ferrets  were euthanized with either chloroform or isoflurane in a ventilation hood. The aorta was quickly removed and immersed in oxygenated 95% Odescribe .The previously frozen samples were homogenized at 4°C in a buffer containing 50 mM Tris-HCl, 10% glycerol, 140 mM NaCl, and 1% Triton X-100. Sample homogenates were subjected to immunostaining. Blots were visualized with a supersignal west peroxide solution (Pierce). The images were detected with a chemiluminescence imaging screen with a Bio-Rad molecular Imager phosphor imager and quantified with Multi-Analyst software or with an Odyssey infrared imaging system (LI-COR Biosciences).410) and which cross-reacts with the activation-loop Thr497 of all PKCs (Upstate Biotechnology) detected phosphorylation of PKC at the PDK site (1 : 500). A phosphospecific antibody against PKC Thr638 of PKC  detected the phosphorylation of PKC at turn motif (1 : 2000). A phosphospecific antibody against Ser719 of PKC (Upstate biotechnology) detected the phosphorylation of PKCε at hydrophobic site (1 : 1000). A phosphospecific antibody against Ser657 of PKC (Upstate biotechnology) detected the phosphorylation of PKC at hydrophobic site (1 : 1000). Alexa Fluor 680 goat anti-mouse IgG and Alexa Flour 680 goat anti-rabbit IgG were all from Molecular Probes. IR-Dye 800 goat anti-rabbit IgG and IR-Dye 800 goat anti-mouse IgG were both from Rockland.A phosphospecific antibody against the activation loop of PKCzeta . Interestingly, protamine, a known direct activator of PKC [We have previously reported  and confbic site . We now bic site . In contr of PKC , 25, als−5 M), and the level of PKC phosphorylation at the hydrophobic and turn motif sites was determined by immunoblot .Aortic tissue strips were quick-frozen at different time points after the addition of the vasoconstrictor phenylephrine  to ferret aorta. Secondly, ERK 1/2 is known to be downstream of PKC in a pathway activated by phenylephrine in this tissue [α and PKCε, at the hydrophobic site, peaking at 2 minutes after PE addition has an appropriate timecourse to cause the activation of PKC sufficiently early to lead to the subsequent direct phosphorylation of α-adducin as well as the downstream signaling to ERK and CaD   . In paran strips . Thus, t2+ levels and subsequent myosin light chain phosphorylation levels peaking at 30 sec ~ 1 min after the addition of PE in this tissue [α-adducin (Ser662) which has been monitored previously as a surrogate reporter of cellular PKC kinase activity [α and PKCε at the hydrophobic site occurs, within the resolution of our methods, at essentially the same time point, consistent with PKC autophosphorylation causing full activation of the kinase. It is of interest to compare the relative temporal profile of signaling events reported here and in past work on this same tissue, aorta of the ferret. We have previously reported that there is a largely transient increase in intracellular ionized Cas tissue , 33 whics tissue , 35. Theactivity , 30, 36.α-adducin does not show this delay, presumably because it is associated with the membrane cytoskeleton [Of interest also is the fact that subsequent events previously shown to be downstream of PKC activation in this tissue, ERK1/2 phosphorylation and CaD phosphorylation (Ser789) reach the maximum much later, with CaD phosphorylation being the slowest event. Thus, additional factors are clearly involved, perhaps including the fact that PKC is activated at the cell membrane whereas smooth muscle CaD is spatially removed, residing on the contractile filaments in the core of the cell. The phosphorylation of skeleton .α and PKCε is regulated by an α-agonist in dVSM and that the smooth muscle differentiation marker, CaP, is necessary for this autophosphorylation. We have previously shown that CaP is necessary for maximal α-agonist-induced contraction of aorta [α-agonists trigger a translocation of CaP from the contractile filaments to the cortex of the dVSMC [α-agonist and that CaP is necessary for this process.The main findings of the present study are that autophosphorylation of PKCof aorta  and thathe dVSMC . CaP coihe dVSMC , 23, 31.2+/CaM/myosin light chain kinase (MLCK) mediated phosphorylation of the 20 kDa myosin light chains (LC20) is an important mechanism of regulation of smooth muscle contraction and is known to be tightly controlled by regulation of both MLCK and myosin phosphatase by multiple mechanisms [Cachanisms . It has chanisms , 40. Phochanisms . ApparenThere are three key phosphorylation sites for PKC functions, known as the activation loop, hydrophobic-motif, and turn-motif. Interestingly, PDK was phosphorylated and PKC hydrophobic motif phosphorylation was increased by phenylephrine stimulation in dVSM, but there were no detectable changes in the level of phosphorylation of the turn motif site in this study, neither invitro nor in vivo. Many studies reported that phosphorylation of the activation loop, catalyzed by the PDK, is critical for activation of PKC. Mutation of phosphorylatable residues in the activation loop abolished PKC activity , 43. Howα-adducin, reported to be a PKC-specific substrate that can be used as an in-cell monitor of PKC kinase activity [α-adducin occurs quite early, but the phosphorylation of CaD is delayed by several minutes and presumably is due to time delays involved in the yet to be determined process by which ERK moves from the cell cortex, where it is activated, to the location of the ERK substrate, CaD, on the contractile filaments in the core of the cell. The delay of several minutes is surprising from a kinetic point of view but may point to diffusion slowed by the viscosity of the cytosol in contractile smooth muscle, multiple ERK binding sites in the cell and other ERK sequestering mechanisms yet to be determined.To further determine the time course of the onset of actual kinase activity of PKC in vivo, we monitored the phosphorylation level of activity , 30, 46.2+ levels and subsequent myosin light chain phosphorylation in this tissue [Thus, in summary, the present study demonstrates that the vasoconstrictor, phenylephrine activates, in aortic dVSM, a pathway involving CaP-dependent PKC autophosphorylation and activation followed by a much-delayed ERK activation, CaD phosphorylation and contraction. This PKC-dependent pathway occurs in parallel with the previously described transient spike in intracellular Cas tissue , 47, 48,


These features make them attractive for genome engineering and synthetic genetics, although the basis for DNA site selection is poorly understood. Here we show that attP selection is determined through multiple proofreading steps that reject non-attP substrates, and that discrimination of attP and attB involves two critical site features: the outermost 5–6 base pairs of attP that are required for Int binding and recombination but antagonize attB function, and the “discriminators” at positions −15/+15 that determine attB identity but also antagonize attP function. Thus, although the attachment sites differ in length and sequence, only two base changes are needed to convert attP to attL, and just two more from attL to attB. The opposing effect of site identifiers ensures that site schizophrenia with dual identities does not occur.Phage-encoded serine integrases mediate directionally regulated site-specific recombination between short  attP and attB, the attachment sites for the phage and bacterial host, respectively. Although the DNA sites are relatively small (<50 bp), the reaction is highly selective for these sites and is also strongly directional. Here, we address the question of what sequences within attP are required for it to act as an attP site and identify the key sequence features that are required not just for Integrase binding but also for synapsis and post-synapsis events. We also have identified the key determinants of attP and attB identity, and although the sites are different in sequence and length, they can be interconverted with just two base changes in each of the half sites.Site-specific recombinases catalyze recombination between two specific DNA sites to generate the products of recombination. The Integrase encoded by mycobacteriophage Bxb1 is a member of the serine-recombinase family and catalyzes strand exchange between  Integration is catalyzed by a phage-encoded Integrase protein (Int) mediating site-specific recombination between phage and bacterial attachment sites , Bxb1 gp47 , but somewhat tighter to attL and attR (Kd 15 nM); CTD binds as a monomer to each half site with similar affinities for the B, B′ and P half sites (∼120 nM), and a somewhat lower affinity for the P′ half site The serine-integrase (Int) coded by mycobacteriophage Bxb1 is relatively large (500 aa) and contains an N-terminal catalytic domain (∼150 aa) common to all serine-recombinases, and a C-terminal domain  that binds DNA xb1 gp47  [7]. attattP and attBatt sites with synapsis requiring compatible configurations attL and attR in the presence of the RDF is orientation dependent, suggesting that an Int protomer bound at a B-type half site (either B or B′) can only productively interact with a P-type half site (P or P′) attB site of φC31 show that specific DNA sequences are also important for post-synaptic events Selection of cognate sites that support recombination involves multiple steps in the reaction attP site and to functionally distinguish attP and attB. We show that there are two critical site components. One is the outermost flanks of attP that are required for Int binding and for recombination, but which also prevent usage as attB. The second is a key discriminator position at positions −15 and +15 where a T∶A/A∶T base pair  is required for both Int binding and recombination as an attB site, but which interferes with attP functionality. The identities of attP and attB are mutually exclusive, but they can be interconverted with mutations in the critical discriminator and flanking motifs.Here, we investigate what specific sequences are required for Bxb1 Int to recognize its attP, we initially constructed a series of altered half-site substrates with transition mutations at each of 23 positions within the P half site, and examined the binding of the Bxb1 Integrase CTD  for their ability to support integrative recombination  are deleterious for recombination, even though Int binding to most of these substrates is only mildly affected , and the −4/+4 positions that have a modest contribution to CTD binding, but are critical for recombination. A summary of mutant site activities is shown in We surveyed all of the P-mutants  and only cleavage of the bottom strand is required to generate a dsDNA cleaved product  base pair at both −15 and +15  behaves similarly , then CTD binds well with efficient formation of complex 2 (attL-like substrate. We observe that both CTD and Int (Int Kd = 10 nM) bind to Hybrid-6 with similar patterns to attL (attL for recombination (attP or attB. The full Int protein binds slightly less well to Hybrid-5 (Kd = 120 nM) but Hybrid-5 has acquired the ability to function as an attB site, albeit inefficiently, and lost the ability to function as attP  to a half-site imilarly . Howeveromplex 2 . If thes to attL , and Hybbination ; it does as attP . These eattP with G-15A/G+15A and G-15T/G+15A mutations, but with wild-type attP flanking sequences (Hybrid-7 and Hybrid-8 respectively). Int binds well to both substrates (Kd = ∼10 nM for both), but neither function as attB, and both work only poorly as attP, with Hybrid-8 working substantially worse than Hybrid-7  albeit with reduced activity, and eliminates attP identity. We note that although inclusion of the T∶A/A∶T base pair  at both −15 and +15 in attP site with proper flanks (Hybrid-8) is not sufficient to switch from attP to attB identity, it severely impairs attP function, and thus antagonizes attP identity. Most other substitutions at the −15/+15 positions in attP that we tested have little impact on binding or recombination.The second architecture feature is the key discriminator positions at −15 and +15 (which we refer to as Discriminator-L and Discriminator-R). The T∶A/A∶T  base pair is critical for Int binding to entities . FurtherattP that have no counterpart in attB. Flank-L and Flank-R  does not prevent the site from acting as attB, but considerably impairs it, showing that these not only are required for attP function, but are also somewhat anti-attB. We note that the flanking sequences of φC31 attB are also important for efficient recombination by φC31 Int, although these are all encompassed within the site length requirements for attPThe third architectural feature is the two flanking sequences of attP is longer than attBattP flanking sequences to confer attP identity is a common feature. The use of the −15/+15 discriminator position in other systems is unclear, although we predict that it may be a common site feature, with different systems using different positions for this function. The way in which Int recognizes these features are unclear and no structural information is available. However, we propose that a common DNA binding feature within CTD recognizes the inner parts of both attP and attB, and we predict that this lies within the N-terminal part [CTDa; attP and attB similarly. A zinc-finger motif common to serine integrases – and proposed to be involved in DNA recognition attP flanking sequences.In all large serine-recombinase systems in which the site requirements have been examined, rt CTDa;  of CTD (attL site (Hybrid-6), and introducing the same mutations to convert the P′ site into a B′-like site generates attB identity. A second pathway involves addition of the short attP flanking sequences to attB (Hybrid-2) which then retains attB identity but functions poorly. Adding GC base pairs at the −15 and +15 positions then results in a switch to attP function. It is noteworthy that none of the inter-conversion pathways we have described generate substrates that can act as both attP and attB, although this is perhaps not unexpected considering that the key identifiers (attP flanks and the discriminators) antagonize one identity while promoting the other. We also recognize that there are clearly additional contributions to site identity and function, as substrates such as Hybrid-7 and Hybrid-8 function as attP, but relatively inefficiently. It seems likely that a combination of activities and integration of several site components will be common to other serine-integrase systems, although because there is so much sequence diversity among the sites, often without substantial symmetry and with few positions shared between attP and attB, understanding site selection and identity in other serine integrase systems will likely require empirical determination.A striking conclusion from these studies is the simplicity with which site identities can be changed with only a few mutations . There aDrosophila, and Plasmodium genomes Serine-integrases are attractive systems for genome manipulation in heterologous systems as well as for construction of synthetic genetic circuits attB, 200 bp of attP and 376 bp of attR, respectively, have been described previously attP sites were prepared by annealing complementary oligonucleotides. Mutant attP DNAs containing a single gpInt binding site were prepared by either mutating a half-site  or by eliminating a half-site . These sites are obtained by annealing the necessary pairs of oligonucleotides (attB (50 bp) was prepared as described earlier Plasmids pMY1, pMOS-attB, pMOS-attP and pMOS-attR containing 343 bp and 50 bp of leotides . MutatioBxb1 integrase, CTD and gp47 were purified as described earlier DNA substrates were prepared by 5′ end labeling of one oligonucleotide of each pair and annealing. Approximately 0.1 pmol of labeled DNA was incubated with either gpInt and CTD in a buffer containing 20 mM Tris (pH-7.5), 25 mM NaCl, 10 mM EDTA, 10 mM Spermidine, 1 mM DTT, and 1 µg Calf Thymus DNA, in a total volume of 10 µl. Reactions were incubated at 37°C for one hour and the protein-DNA complexes separated on a native 5% (unless otherwise stated) polyacrylamide gel at 4°C. Gels were dried, exposed to a phosphorimager screen overnight and scanned (Fuji Phosphoimager). Kd was determined as the Int or CTD concentration in which one half of maximal binding was observed. If multiple complexes were observed the apparent Kd was deduced from the protein concentration at which half of the DNA remained unbound.In vitro integrative recombination assays were performed as described previously attP DNA. The integration reactions were incubated at 37°C for up to 1 h and heat inactivated at 75°C for 15 min. The products were separated by electrophoresis in 0.8% agarose in 1× TBE running buffer and visualized by ethidium bromide staining.attR in pMOS-attR and linear attL (50 bp) in the above recombination buffer, gpInt and gp47 were added as indicated. The reaction were carried out at 25°C for 2 hours and separated on a 0.8% agarose gel.In vitro excision were carried out between 376 bp of attB (50 bp) DNA was incubated with Int and attP DNA under the same conditions as for DNA-binding. After 1 hour incubation at 37°C reactions were heat inactivated at 75°C for min 15 min. For cleavage assays reactions were treated with 1 mg/ml Proteinase K and 0.2% SDS at 55°C for 15 min.For synaptic complex formation and cleavage assays, 5′-end labeled suicide Figure S1attP mutant substrates. (A) CTD binding to half site substrates containing mutations in the P site as indicated. The concentrations of CTD used are 0, 5, 15, 45, 135, 405 and 1215 nM. (B) Complexes formed between CTD and full length attP substrates containing mutations in the P site as indicated are shown. The concentrations of CTD are 0, 15, 45, 135, 405 and 1215 nM. (C) Separation of complexes formed with Int and attP DNA containing mutations in P site are shown. The concentrations of Int used are 0, 15, 45, 135, 405 and 1215 nM.Binding titrations of Bxb1 CTD and Int to P half site and (PDF)Click here for additional data file.Figure S2attP substrates with P′ site mutations. (A) Separation of complexes formed by CTD with attP substrates containing mutations in P′ site. (B) Separation of complexes formed by Int with attP substrates containing mutations in P′ site are shown. The concentrations of Int and CTD used are 0, 15, 45, 135, 405 and 1215 nM.Binding titration of Int and CTD to (PDF)Click here for additional data file.Figure S3attP substrates. (A–C) Recombination activities of attP substrates having mutations either in P (A), P′ (B), or in both half sites (C) (double mutants). The Int concentrations in panel (A) are 0, 18.75, 37.5, 75, 150 nM and in panel (B–C) are 0, 37.5, 75, 150, 300 nM.Recombination of mutant (PDF)Click here for additional data file.Figure S4attP substrates. attP mutant substrates are incubated with radiolabeled suicide attB substrate and varying concentration of gpInt to form synaptic complexes. The concentrations of gpInt are 0, 45, 135, 405, 1215 nM.Synaptic complex formation by mutant (PDF)Click here for additional data file.Table S1attP sites.Binding affinities by Bxb1 Int for P′ mutant (PDF)Click here for additional data file.Table S2Oligonucleotides used in this study.(PDF)Click here for additional data file.


Mycobacterium tuberculosis contains eleven Ser/Thr kinases, two of which, PknA and PknB, are key signaling molecules that regulate cell division/morphology. One substrate of these kinases is Wag31, and we previously showed that partial depletion of Wag31 caused morphological changes indicative of cell wall defects, and that the phosphorylation state of Wag31 affected cell growth in mycobacteria. In the present study, we further characterized the role of the Wag31 phosphorylation in polar peptidoglycan biosynthesis.Sensing and responding to environmental changes is a central aspect of cell division regulation. wag31 alleles  is caused by, at least in part, dissimilar nascent peptidoglycan biosynthesis. The phosphorylation state of Wag31 is found to be important for protein-protein interactions between the Wag31 molecules, and thus, for its polar localization. Consistent with these results, cells expressing a phosphomimetic wag31 allele have a higher enzymatic activity in the peptidoglycan biosynthetic pathway.We demonstrate that the differential growth among cells expressing different Mtb phosphorylation is a novel molecular mechanism by which Wag31Mtb regulates peptidoglycan synthesis and thus, optimal growth in mycobacteria.The Wag31 Mycobacterium tuberculosis is able to remain dormant in the human host without causing active disease for prolonged periods. Despite the importance of latency in the epidemiology and pathology of TB, it is not clear how M. tuberculosis controls the latent state in human host. However, to achieve, maintain, or escape from the latent state, M. tuberculosis must carefully regulate cell division by sensing and responding to specific signals in the host environment. To successfully complete this essential process, the M. tuberculosis genome contains a wide variety of transcription regulators, surface receptors, and signaling molecules including eleven "eukaryotic-type" Ser/Thr protein kinases (STPKs) GlcNAc, and then the reaction product of MurG ([14C]GlcNAc-MurNAc-(pentapeptide)-diphosphoryl-undecaprenol, lipid II) was analyzed by TLC GlcNAc (Perkin Elmer Life Sciences) and incubated at 28°C. After 1 hr, reactions were terminated by addition of 20 volumes of CHCl3/CH3OH (2:1), centrifuged at 3,000 × g for 10 min at room temperature, and the supernatant was mixed with 0.6 ml of dH2O in a new tube. The resulting biphasic solution was centrifuged again and the upper, aqueous phase was discarded. The bottom, organic phase was washed with 1.5 ml of CHCl3/CH3OH/H2O (3:47:48), dried under a stream of N2 and re-dissolved in CHCl3/CH3OH/H2O/NH4OH (65:25:3.6:0.5). The recovered radioactive materials were applied to a silica gel TLC plate, which was developed with CHCl3/CH3OH/H2O/NH4OH (5.6:4.2:0.68:0.27). The location and quantity of radiolabeled lipid II ([14C]GlcNAc-MurNAc-(pentapeptide)-diphosphoryl-undecaprenol) on the TLC plate was determined by using a Molecular Dynamics Typhoon 8600 Phosphoimager (Molecular Dynamics).For enzymatic assay, reaction mixtures containing 2 mg of P60 protein from each strain, 50 μM UDP-MurNAc-pentapeptide and 100 μM ATP in a reaction volume of 300 μl with buffer A were incubated for 5 min at 28°C. Reactions were initiated by adding 1 μCi of UDP-[Mycobacterium tuberculosis; Msm: Mycobacterium smegmatis; MurNAc-(pentapeptide): N-acetylmuramyl-(pentapeptide); GlcNAc: N-acetylglucosamine; DIC: Differential interference contrastMtb: CJ, HE, and JJL participated in the design and conduct of the study, data analysis, and drafting of manuscript. In particular, CJ conducted Van-Alexa568 staining and localization of Wag31 in cells expressing different mutant form of Wag31. HE conducted the enzymatic assay of Mur proteins. JJL performed the yeast two-hybrid experiments and Van-Alexa568 and localization of wild-type Wag31 in the presence of kinase overexpression. KH and MBS participated in culture and isolation of P60 samples for Mur enzyme assays and Raman spectrometry. JSH, SN and JYL constructed plasmids for localization and yeast two-hybrid assay. JWS, SHL, and SJR participated in the data analysis, and drafting and revision of the manuscript. DCC participated in the conception and design of the study, general supervision of the Mur enzyme assays. CMK participated in the design of the study, general supervision of the research, and critical revision of the manuscript. All authors read and approved the final version of the manuscript.Table A1: List of strains and plasmids used in this study. List of plasmid constructs and strains made for this study.Click here for fileM. smegmatis expressing gfp aloneFig. A1: Control . A control experiment in M. smegmatis to show that GFP-Wag31 localization is not due to the non-specific localization of GFP itself.Click here for fileMtbpknA-overexpressionFig. A2: Localization of Wag31 and nascent peptidoglycan biosynthesis in the presence or absence of . Examination of wild-type Wag31 localization and polar peptidoglycan biosynthesis when pknA is overexpressed in M. smegmatis.Click here for fileTable A2: Primers used in this study. List of primers used to make plasmid constructs for this study.Click here for file


To elucidate the anti-obesity mechanism of grape skin, we investigated the effects of grape skin extract (GSE) and resveratrol, one of the phytochemicals in GSE, on FAS and FAS over-expressed 3 T3-L1 preadipocyte.Grape skin, a rich source of phytochemicals, has been reported to possess remarkable anti-obesity activity. Fatty acid synthase (FAS) is a key enzyme catalyzing the synthesis of fatty acid Purified FAS was obtained from chicken liver. Dried grape skin was extracted by 50% ethanol and partitioned by ethyl acetate. Inhibitory effects of GSE and resveratrol on FAS including fast-binding inhibition, time-dependent inhibition, and enzyme kinetics were determined. Inhibitory effects of GSE and resveratrol on 3T3-L1 preadipocyte were also measured.50 values of 4.61 μg/ml and 20.3 μg/ml. For inhibition by resveratrol, the relevant IC50 values were 11.1 μg/ml and 21.9 μg/ml, respectively. And both GSE and resveratrol showed time-dependent inhibition for FAS, with the kobs values of 0.028 min-1, and 0.040 min-1 respectively. They inhibited the overall reaction of FAS competitively with acetyl-CoA, noncompetitively with malonyl-CoA and in a mixed manner with NADPH. Moreover, the inhibition on KR domain by resveratrol was time-dependent with kobs value of 0.106 min-1. In 3 T3-L1 preadipocytes, resveratrol reduced lipid accumulation remarkably.GSE inhibited the overall reaction and β-ketoacyl reductase (KR) reaction of FAS with ICGSE and resveratrol are potent FAS inhibitors and they bound reversibly to the KR domain of FAS to inhibit the reduction of the saturated acyl groups in fatty acid synthesis. Based on the valid data and deliberate analysis, we proposed that GSE and resveratrol have great medical potential and officinal value in treating obesity and related diseases. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL . All other reagents were local products with purity of analytical grade. Grape Air-dried grape skin (100 g) was added to 2000 ml of 50% ethanol and extracted for 4 h at room temperature. Grape skin was then removed from the ethanol extract by centrifugation and filtration. The recovered ethanol extracts were evaporated under reduced pressure to yield 25.3 g. A portion (1 g) of the ethanol extracts were suspended in water and partitioned with petroleum ether, ethyl acetate (EtOAc), and n-butanol sequentially to yield four fractions. Among them, EtOAc-soluble fraction (GSE) was chosen and dissolved in DMSO for this study.The FAS used was obtained from chicken liver , since the amino acid sequence of chicken FAS has 63% identity with that of humans . The FASThe overall reaction of FAS and β-ketoacyl reduction catalyzed by KR were determined with an Amersham Pharmacia Ultrospec 4300 pro UV–vis spectrophotometer at 37°C by following the decrease of NADPH at 340 nm. The overall reaction mixture contained potassium phosphate buffer, 100 mM, pH 7.0; EDTA, 1 mM; DTT, 1 mM; Ac-CoA, 6 μM; Mal-CoA, 12 μM; NADPH, 37.5 μM and chicken liver FAS 10 μg in a total volume of 2.0 ml .50 value, which was obtained from a plot of residual activity versus inhibitor concentration.Fast-binding inhibition was determined by adding the inhibitor into the reaction system before FAS initiated the reaction. This inhibition is generally caused by the non-covalent loading on the enzyme, and is fast and reversible. The final concentration of ethanol did not exceed 0.2% (v/v) in the reaction mixture, so the ethanol did not affect the FAS activity. The extent of inhibition by the addition of inhibitor was measured by reference to the ICt/A0 = - kobs t. The At/A0 expresses the remaining activity at t time, and kobs is the observed first-order rate constant, which is equal to k2 [I]. The k2 is the second-order rate constant, which is equal to kobs/[I] and shows the inhibitory capability. Based on earlier studies  values, namely the second-order rate constant k2, were 0.009 (min · mg/ml)-1, 0.013 (min · mg/ml)-1, and 0.035 (min · mg/ml)-1 for these three inhibitions. These results illustrated that resveratrol was one of the effectives in GSE when inhibiting FAS, and it took effect via reacting irreversibly with KR domain. In a word, the time-dependent inhibition of GSE on FAS was mainly due to the reaction between resveratrol and the KR domain.Figure The possible interference manner by resveratrol at each substrate-binding site on FAS was kinetically determined. The results of double-reciprocal plots showed that resveratrol inhibited FAS overall activity competitively with respect to Ac-CoA Figure A and nonTo ensure that the doses of the inhibitors were not generally cytotoxic, 3 T3-L1 preadipocytes were incubated with resveratrol for 48 h, after which a MTT cytotoxicity assay was performed. Resveratrol showed nearly no cytotoxicity to the preadipocytes at doses up to 50 μM, whereas little influence at 75 μM or 100 μM  [GSE and resveratrol are very potent inhibitors of the overall reaction of FAS. Compared with the first reported FAS inhibitor, cerulenin, the IC0 μg/ml) . The str50 = 12.2 μg/ml) [50 = 20 μg/ml) [50 = 18 μg/ml) [We noticed that the inhibitory activity of GSE on FAS was a little stronger than that of resveratrol, thus there must be some other active components in GSE or had combined effect with resveratrol on FAS. So we proposed that, besides resveratrol, GSE as a whole had also a great prospective to be investigated as FAS inhibitors. It was reported in our previous study that teas and tea-polyphenols, which have been considered as functional foods for treating obesity, inhibited FAS activity ,34,36. B0 μg/ml)  and ECG in vivo application.Figure Unlike C75 but allied with EGCG, the KR domain is a major reaction site on FAS by the irreversible time-dependent inhibition of resveratrol. However, the resveratrol molecule does not contain a carboxyl group of gallated ester, which has been identified as an active group of EGCG in irreversible time-dependent inhibition . TherefoAs the major FAS inhibitor in grape skin, resveratrol mainly reacts with the KR domain on FAS. The kinetic study of resveratrol suggested that it exerted its inhibition on the site where NADPH binds to the KR domain. This is similar to EGCG , which sIt is worth noting that extracts of grape flesh expressed no inhibition to FAS (data not shown). This result is consistent with the finding that resveratrol is distributed mainly in grape skin but is scarce in grape flesh. It is also further demonstrated that grape skin plays an important role in the curative effect of grapes, which should not be neglected in nutritional planning and in the processing of grape products. Taking flavor and customs into account, it will be quite acceptable for grape skins to be processed into more food forms without losing its beneficial effects, as exemplified by red wine.Previous studies on cell lines, animal models, and human epidemiological trials have certified the potential of dietary polyphenols as anti-proliferation agents ,39. Polyin vivo synthesis. High fat in the diet depresses the expression of FAS, and decreases fat synthesis, while excessive absorption of sugar accelerates the expression of FAS [+ ratio could help organisms obtain more energy. Therefore, inhibition of FAS leads to the control of the ingestion of energy, the reduction of endogenetic fat, and the promotion of in vivo energy production. Consequently, as effective FAS inhibitors, GSE and resveratrol have great potential for clinical treatment of obesity.Fat in the animal bodies comes from two main sources: absorption from food and n of FAS . In contn of FAS . Furthern of FAS ,45,46. Tn of FAS ,47. Ac-CIn conclusion, GSE and resveratrol could inhibit FAS activity in both reversible and irreversible manner. Kinetic results confirmed that the main active domain that GSE and resveratrol acted was KR. Since grape and resveratrol are reported to have the ability of treating obesity, we now speculate that they possibly perform, at least in part, by affecting FAS activity.Ac-CoA: Acetyl-CoA; EtOAc: Ethyl acetate; FAS: Fatty acid synthase; GSE: Ethyl acetate extract of grape skin; IC50: The half inhibition concentration; KR: β-ketoacyl reductase; Mal-CoA: malonyl-CoA.The authors declare that they have no competing interest.WT, as the principal investigator, was responsible for the concept and design of the study. XM conducted the research and wrote the manuscript. YL did the whole experiments of the study. All authors participated in the preparation of, and have approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/13/361/prepub