The prevalence of allergic diseases are increasing worldwide, emphasizing the need to elucidate their pathogeneses. The aims of this study were to use a two-stage design to identify DNA methylation levels at cytosine–phosphate–guanine (CpG) sites across the genome associated with atopy and high serum immunoglobulin E (IgE), then to replicate our findings in an independent cohort.Atopy was assessed via skin prick tests and high serum IgE. Methylation levels were measured from whole blood using the Illumina Infinium HumanMethylation450 BeadChip from 18-year-old women (n = 245) and men (n = 122) in the Isle of Wight birth cohort. After data cleaning and processing, and removing probes with possible single nucleotide polymorphisms, DNA methylation levels from 254,460 CpG sites from the 245 women were subjected to recursive Random Forest feature selection for stage 1. The sites selected from stage 1 were tested in stage 2 for associations with atopy and high IgE levels (>200 kU/L) via logistic regression adjusted for predicted cell-type proportions and sex. Sites significantly associated with atopy in stage 2 underwent replication tests in the independent Swedish birth cohort BAMSE (n = 464).P-value range 6.5E−9 to 1.4E−5) and 12 associated with high IgE levels  at the Bonferroni adjusted alpha (0.05/62 = 0.0008). Of the 19 available sites, 13 were replicated.In stage 1, 62 sites were selected, of which 22 were associated with atopy in stage 2 .We identified 13 novel epigenetic loci associated with atopy and high IgE that could serve as candidate loci for future studies; four were within genes with known roles in the immune response (cg04983687 in the body of The online version of this article (doi:10.1186/s13073-015-0213-8) contains supplementary material, which is available to authorized users. The prevalence of allergic disease is increasing worldwide; approximately 40 % of the population of industrially developed countries are considered to be affected . Many ofEpigenetics, changes in gene activity not caused by alterations to the sequence of DNA, may clarify some of these mechanisms because much of cell lineage and tissue-specific gene expression is tightly regulated by epigenetic programming . One of Atopy is defined as a positive reaction to a skin prick test (SPT) or immunoglobulin E (IgE) production in response to allergens . IgE plan-large-p (few observations relative to the number of predictors) and because traditional methods are not optimized for identifying complex biological processes. Because of the large-scale data generated for each completed methylation assay, techniques to select a subset of informative variables are needed   . Prior tP-values were compared against an α-level of 0.05. We implemented a two-stage genome-wide approach  and fx5  . Atopy was defined as a positive Phadiatop or a positive fx5 test with specific IgE antibody levels ≥0.35 kUA/L. Furthermore, epigenome-wide DNA methylation was measured in 472 children using DNA extracted from blood samples collected at the 8 year follow-up [Sites that were significantly associated with atopy in stage 2 analyses were selected for replication in the Children, Allergy, Milieu, Stockholm, Epidemiology (BAMSE), a prospective population-based cohort study of children recruited at birth and followed during childhood. Details of the study design, inclusion criteria, enrolment, and data collection are described elsewhere . In brieollow-up . For thiollow-up . This stThe randomForest package in R was used to implement the RF algorithm . The outsampsize, mtry, and ntree, so these could be optimally set for the recursive RF implementation described below. The sampsize parameter controls whether to use balanced or imbalanced sampling to generate the training datasets; mtry specifies the number of variables to be randomly selected and tested at each node of each tree; and ntree determines the number of trees to be grown in a forest. Using the default values for mtry  and ntree (500), we compared the OOB-ER and class-specific misclassification rates for an imbalanced RF grown without sampsize and a balanced RF grown with sampsize = . Specifying sampsize =  meant that 50 observations from those with atopy and 50 observations from those without atopy were randomly selected when creating the training set for each tree. Once we determined whether or not to utilize the sampsize parameter, we tracked the prediction accuracy of the RF at different combinations of mtry  and ntree . Once the optimal parameter values were selected, the recursive RF was implemented.Prior to implementing the recursive RF  describeThe general methodology of the recursive RF for feature selection has been proposed and utilized elsewhere , 35, 37,The sites selected from the recursive RF were then tested for univariate associations with atopy via logistic regression. Given that methylation levels vary significantly by cell type, peripheral blood samples are composed of multiple different cell types, and allergic diseases often influence the proportions of circulating immune cell types, we considered the potential confounding effect of cell-type differential for each participant. However, logistical limitations prevented the acquisition of cell differential at the original time of blood sample collection. Thus, we utilized the methylation data to predict cell differential , 39, theBoxplots of β values stratified by atopy status were used to ensure that the distributions of methylations levels in the female and male samples were similar and could be combined. Two observations (in cg12819873 and in cg13168187) were identified as strong outliers, and re-coded as missing. The joint sample was then used for all stage 2 analyses in which each CpG was tested for its individual association with atopy and high IgE, adjusted for important covariates. These tests were conducted with logistic regression in which high IgE and atopy were the dependent variables and M-values for CpGs were the primary independent variables, while cell type proportions and sex were included as covariates. CpG sites that were significantly associated with both atopy and high IgE at the Bonferroni corrected α were subjected to set analyses, used to show the combined effect of DNA-M on atopy .P-value < 0.05. These sites were submitted for functional annotation analyses in DAVID [For each site that was significantly associated with atopy in stage 2, we conducted multiple logistic regression models in the BAMSE cohort. Atopy status was the dependent variable and M-values for CpGs were the independent variables, while cell type proportions, sex, asthma treatment within the last 12 months, and batch (bisulfite treatment date) were included as covariates (identified as confounders in the regression model). Successful replication was defined as having the same direction of association and a in DAVID , 42.P-value = 0.0972) between the female sample, used in stage 1, and the male sample which was pooled with the female sample in stage 2. However, prevalence of high serum IgE (≥200 kU/L) was significantly  lower in the female sample (23.8 %) than in the male sample (41.9 %). These differences in high IgE did not affect the analytic methods because serum IgE was only used as a secondary and alternate biomarker of atopy to provide further confidence in our top results  that survived data cleaning, pre-processing, and removal of probes potentially containing SNPs. At each step in the reduction, the dataset was reduced by half; by the 15th iteration the data was reduced to a total of 15 CpG sites. The OOB-ER achieved its lowest point  at the 11th iteration, which included 248 CpGs . Prior to implementing stage 2 analyses, we tested whether any of the selected sites may have been selected due to confounding by cell type. Many of the associations were altered by adjusting for proportions of CD8+ T cells, CD4+ T cells, natural killer cells, B cells, monocytes, and granulocytes   in the IOW. Set analyses [Of the 62 sites, 22 had statistically significant associations with atopy  with atopy in BAMSE and had comparable ORs , cg18219873 in proteoglycan 2 (PRG2), cg07908654 (intergenic), cg06824199 in EF-hand calcium binding domain 14 , cg27469152 in eosinophil peroxidase (EPX), cg27468224 (intergenic), cg13233042 in atlastin GTPase 3 (ATL3), cg13197551 in SCD6 homolog B , cg09332506 in coatomer protein complex, subunit alpha (COPA), g07970948 in zinc finger protein 862 (ZNF862), cg25854298 in activating signal cointegrator 1 complex subunit 1 (ASCC1), and cg17971837 in parvalbumin (PVALB). For all 13 sites, persons with atopy or high IgE had lower methylation levels compared to those without atopy or with lower IgE. All nine sites that were associated with both atopy status and high IgE in IOW after adjusting for multiple tests  were successfully replicated in BAMSE, whereas the remaining four replicated sites , cg25854298 in ASCC1, and cg17971837 in PVALB) had nominal  associations with high IgE in IOW. Interestingly, almost all sites (five out of six) that were not replicated in BAMSE did not have even nominal associations with high IgE in IOW.Finally, 19 of the 22 sites (data on three sites were not available in BAMSE) associated with atopy in IOW were studied in an independent cohort. Of the 19 sites tested, 13 were significantly associated  associated with the 13 replicated CpG sites (Table EPX and PRG2) in the KEGG pathway for asthma  and associated with the eosinophils annotation .The 13 replicated sites were investigated for functional annotation in DAVID and for individual biological relevance via literature review. Functional annotation of the 10 genes  and non-atopics (from 19.87 % to 7.22 % error) when compared to the RF on the full dataset. Of particular note was the large proportion of CpG loci that were statistically significant at a Bonferroni-adjusted α for atopy (35 %) and high IgE (19 %) within the IOW sample and the large proportion (13 of 19) of sites that were successfully replicated in the BAMSE cohort.Our findings are the latest in a series of recent work that supports the application of RF for genome-wide association studies (GWAS) and in allergic diseases. The recursive RF process we utilized was similar to methods proposed elsewhere , 17, 35.ZFPM1, PRG2, KIAA0494, EPX, ATL3, LSM14B, COPA, ZNF862, ASCC1, and PVALB. For each of these genes, we performed a search of the literature for possible roles in atopy and conducted functional annotation in DAVID.The true challenge with high-throughput techniques is in connecting the results to biological processes, which are complex and can involve combinations of many genes working together. We investigated the biological roles of the ten genes associated with the 13 replicated CpGs sites: ZFPM1  is a binding factor for the transcription factor GATA-1 and has been primarily studied for its role in the differentiation of erythroid, megakaryocyte, and mast cells [FOG-1 expression appear to be dependent on its cellular origin and the biochemical surroundings, which can determine whether FOG-1 acts as a repressor or co-activator of GATA-1 [. Recently, ZFPM1 was shown in an in vitro study to down-regulate IL-4 and therefore facilitate TH1 differentiation [ZFPM1 were recently identified in association with asthma [ZFPM1 in immune-cell activity and allergic disease suggest that this is an interesting yet possibly overlooked gene in atopy and atopic diseases.Among the replicated loci, a number of their associated genes were involved in intriguing processes that may have a role in atopy. st cells . Howeverf GATA-1 , 44. Recntiation . Also, th asthma . The mulEPX encodes eosinophil peroxidase, a protein expressed by eosinophils. Previous investigations found that both serum and urine levels of EPX were elevated in children who had positive SPTs, as well as those with allergic diseases such as asthma, allergic rhinoconjunctivitis and atopic dermatitis [PRG2, which were associated with high versus low total IgE, primarily driven by eosinophils. Interestingly, this study also found that the methylation levels in isolated eosinophils differed among asthmatics with high total IgE, asthmatics with low total IgE, and controls, suggesting that eosinophils from persons with allergic hypersensitivity or asthma may have different epigenetic profiles compared to eosinophils from non-allergic individuals [PRG2 expression was up-regulated in response to dust-mite exposure, suggesting a possible role in the adaptive immune response [Eosinophils are subtypes of granulocytes that are heavily involved in inflammatory responses and atopic asthma through the mechanism of airway inflammation . EPX encrmatitis –51. A reividuals . Also, aresponse .COPA, though these did not achieve genome-wide significance [COPA have been linked to an autoimmune disease characterized by high-titer autoantibodies, interstitial lung disease, and inflammatory arthritis [COPA also appears to drive CD4+ T-cells toward TH17 phenotype via increased expression of IL-1β, IL-6 and IL-23 [COPA may play a role in hypersensitivity, though further research is necessary to elucidate this role.A GWAS of atopic asthma implicated SNPs that were in linkage disequilibrium with SNPs in ificance . More rerthritis . These mnd IL-23 . Thus, mLSM14B may be involved in mRNA translation [EPX and PRG2 in eosinophil activity and in the KEGG pathway for late hypersensitive responses in asthma. Some of the genes  did not have any apparent role in immune response.nslation , 57. SomThese findings should be interpreted within the limitations of the study. Although we provide evidence in support of associations between 13 CpG sites and atopy, variations in methylation at these sites may not cause allergic sensitization. The cross-sectional nature of this sample prohibited us from distinguishing between which DNA-M variations at CpG sites may have caused, been caused by, or just been markers of sensitization. However, associations in any of these directions may yield important insights into the development, persistence, and consequences of allergic sensitization. Some of the CpG sites that were selected could not be replicated and some that were replicated were not involved in any known biological processes related to atopy or allergy. The unsuccessful replication could be due to false-positive findings from the discovery analyses, or differences in how atopy was assessed between the discovery and replication cohorts. The lack of biological roles for these CpG sites could be explained by selected CpG sites possibly being highly correlated with other CpG sites that truly influence atopy status, or by CpG sites having roles in unknown, but still important, biological pathways involved in atopy.n and very small p, statistical interactions, or correlations between predictors. Despite the issues of correlated predictors, such variables can still provide useful information. DNA-M loci that are merely surrogates of actual CpGs associated with atopy can still serve as biomarkers of disease, but do not serve to improve our understanding of the etiology of atopy.Correlated predictors may present an issue that we were unable to address , 17. If Some of the CpGs that we identified with the recursive RF but that did not meet our replication criteria may in fact be biologically relevant in atopy. We would not expect all biologically relevant findings to be included in the functional annotation results of our gene list for two reasons: first, our gene list of 10 genes is quite small because DAVID is optimized for lists between 100 and 2,000 genes in length ; and sec+ T cells, CD4+ T cells, natural killer cells, B cells, monocytes, and granulocytes) in our regression analyses, the predicted cell proportions for the low-frequency cell types, such as T-cell subtypes, may be less accurate than those of the higher frequency cell types and these predictions did not distinguish eosinophils from other granulocytes. Given the importance of T-cell subtypes  and eosinophils (a subset of granulocytes) in atopic responses, this may have resulted in some residual confounding. However, given the inability to collect actual cell differentials in this study, the predictions we used likely accounted for the majority of cellular heterogeneity in our blood samples.Despite correcting for cell proportions (CD8The lack of independence between the samples used for RF feature selection (stage 1) and the samples used for determinations of statistical significance (stage 2) was another limitation, and may have led to some over-fitting during stage 2 analyses. Also, 12 CpG sites that were selected in stage 1 were not present in the male sample. Although these were still evaluated in the stage 2 analyses, the lack of full methylation data reduced the power to identify significant findings at these 12 sites. However, the strong replication results in the BAMSE cohort would suggest that the majority of our findings were not due to random chance or over-fit to the IOW sample.A/L to any allergen in BAMSE. The associations with high serum IgE in the IOW support that at least some of the unsuccessful replications may have been due to these differences in measurement. All 13 sites that replicated in BAMSE had at least nominal associations with high serum IgE in IOW , whereas only one of the six sites that did not replicate in BAMSE had an association with high serum IgE in IOW . These findings suggest that the only sites that could be replicated in this study may be involved in IgE-mediated allergic sensitization. Also, some atopy-associated CpG sites in IOW, which were measured at 18 years old, may not have been able to replicate in BAMSE, measured at 8 years of age, because methylation levels can be age dependent [ACOT7, which was strongly associated with both atopy and high IgE in the IOW cohort, is particularly interesting because others have identified differentially methylated regions within this gene associated with asthma [Not all of our findings were replicated; six sites that were tested did not successfully replicate and three sites could not be tested because the data were unavailable. The six non-replicated sites may represent false-positives from our stage 2 analyses or could be due to differences in the measurement of atopy status between the two cohorts. One limitation of the replication study was that atopy was defined as at least one positive SPT to any allergen in the IOW; whereas atopy was defined as specific IgE antibody ≥ 0.35 kUependent . It is ph asthma ; thus, iZFPM1, cg12819873 in the 5′UTR of PRG2, cg07908654 (intergenic), cg06824199 in the body of KIAA0494, cg27469152 in the 3′UTR of EPX, cg27468224 (intergenic), cg13233042 in the body of ATL3, cg13197551 in the 3′UTR of LSM14B, cg09332506 in the body of COPA, cg17041511 (intergenic), cg07970948 in the body of ZNF862, cg25854298 in the body of ASCC1, and cg17971837 in the TSS1500 of PVALB. Three of the 22 sites associated with atopy in IOW were not available for testing in the BAMSE cohort, so may be of interest for follow-up in future studies of DNA-M and atopy: cg09249800 in the body of ACOT7, cg07765167 in the TSS1500 of MRPL45, and cg24836822 in the body of KCNH2. These CpG sites and their associated genes could be treated as under-studied candidates for future studies of atopy; particularly cg04983687 in ZFPM1, cg12819873 in PRG2, cg27469152 in EPX, and cg09332506 in COPA. Furthermore, we showed that recursive RF data reduction can be an effective approach for epigenome-wide DNA-M studies, and may be considered by other investigators as it has now been successful in multiple studies with large-scale data.Utilizing a two-stage design with a well-characterized but sparsely implemented RF feature selection method followed by logistic regression for both atopy and an alternate marker of atopy (high IgE), we identified a number of CpG sites associated with atopy. Most importantly, 13 sites were replicated in an independent cohort for atopy status: cg04983687 in the body of


E. coli DNA gyrase B subunit reveal two discrete monovalent cation-binding sites that could have functional roles.New structures of the N-terminal 43 kDa fragment of the  Escherichia coli DNA gyrase have been determined. One of these, solved in the presence of K+, is the highest resolution structure reported so far for this domain and, in conjunction with the three other structures, reveals new insights into the function of this domain. Evidence is provided for the existence of two monovalent cation-binding sites: site 1, which preferentially binds a K+ ion that interacts directly with the α-phosphate of ATP, and site 2, which preferentially binds an Na+ ion and the functional significance of which is not clear. The crystallographic data are corroborated by ATPase data, and the structures are compared with those of homologues to investigate the broader conservation of these sites.Four new crystal structures of the ATPase domain of the GyrB subunit of  The focus of this study is the ATPase domain of Escherichia coli DNA gyrase, located in the B subunit, which functions as an ATP-operated clamp that captures the T segment prior to its passage through the G segment.DNA topoisomerases are essential enzymes that are found across all kingdoms of life and, as a consequence, they represent valuable drug targets for both antimicrobial and anticancer therapies , comprising residues 2–393 of the 804-amino-acid wild-type sequence (UniProtKB/Swiss-Prot entry P0AES6), was produced using a modification of a previously published protocol  pLysS and a 5 ml overnight culture was used to inoculate 1 l Luria–Bertani medium containing 100 mg ampicillin and 30 mg chloramphenicol. The cells were grown at 310 K to an OD600 of 0.4–0.6. Protein expression was induced by the addition of isopropyl β-d-1-thiogalactopyranoside to a final concentration of 0.5 mM and the culture was left shaking overnight at 298 K. The harvested cells were resuspended in TGED  and then lysed by freeze–thawing with liquid nitrogen. The cell debris was removed by centrifugation at 84 000g for 60 min at 277 K. The sample was subsequently purified using an ÄKTA FPLC system  using a two-column procedure. The sample was maintained at 277 K throughout and fractions that contained GyrB43 were identified using SDS–PAGE. The cell lysate was passed through a Q Sepharose column pre-equilibrated with TGED, which was then washed with excess TGED. The remaining bound protein was eluted with a 0.0–1.0 M NaCl gradient in TGED. Solid ammonium sulfate was added to the protein fraction containing the GyrB43 to a final concentration of 1.5 M. This was then loaded onto a 1 ml Phenyl Sepharose column pre-equilibrated with three column volumes of 1.0 M ammonium sulfate in TGED. The column was then washed with one column volume of 1.5 M ammonium sulfate in TGED. Any remaining bound protein was subsequently eluted by applying a 1.0–0.0 M ammonium sulfate gradient in TGED buffer. Eluted fractions containing GyrB43 were dialysed overnight against excess TGED containing 30%(v/v) glycerol. The dialysed protein was then aliquoted, flash-cooled in liquid nitrogen and stored at 193 K.The N-terminal 43 kDa fragment (exact molecular weight 43 254.8 Da) of M and then concentrated to ∼10 mg ml−1 using an Amicon Ultra centrifugal filter unit with a 10 kDa molecular-weight cutoff.Prior to crystallization, the protein was buffer-exchanged into TGED containing the nonhydrolysable ATP analogue 5′-adenylyl-β,γ-imidodiphosphate (ADPNP) at a concentration of 40 µ2.2.et al., 1991v/v) PEG 3350, 2 mM MgCl2, 100 mM Tris–HCl pH 8.0. In subsequent experiments, crystals were grown from the same conditions with the addition of either 100 mM KCl or 100 mM NaCl or both 100 mM KCl and 100 mM NaCl. Prior to mounting, the crystals were cryoprotected in the corresponding precipitant solution supplemented with 10%(v/v) glycerol and then flash-cooled in liquid nitrogen using LithoLoops (Molecular Dimensions). The mounted crystals were stored in Unipuck cassettes (MiTeGen) prior to transport to the synchrotron. Crystals were subsequently transferred robotically to the goniostat on one of three beamlines at the Diamond Light Source  with a reservoir volume of 1 ml and drops consisting of 0.6 µl protein solution and 0.6 µl precipitant. Crystallization conditions were optimized from those reported previously , was used to solve and refine the first structure. One monomer (together with its associated ADPNP and magnesium ion) was taken from the deposited structure of E. coli GyrB43 . Initially, solvent molecules were introduced automatically using ARP/wARP  server  to further probe the identities of metal ions through the use of anomalous difference Fourier maps. These were produced using phases calculated from the K-only model after the removal of all metal ions, simulated annealing as described above and re-refinement against the K-only data set. The resultant maps were inspected for significant positive peaks of electron density associated with atomic positions, which could then be assigned as anomalously scattering atoms. At a wavelength of 1.91 Å, the theoretical anomalous signals associated with the non-H atoms that could be present would be ranked as follows: K > Cl > S > P > Mg > Na >> O, N, C, where the theoretical Na signal would be roughly one eighth of the K signal (Supplementary Table S1).CCP4mg  glycerol to remove the KCl present in the storage buffer. Briefly, assays were performed in microtitre plates with a reaction volume of 100 µl and a constant GyrB43 concentration of 20 µM. The absorbance at 340 nm was measured continuously in a CLARIOstar plate reader (BMG LABTECH) and used to evaluate the oxidation of NADH (using an extinction coefficient of 6.22 mM−1 cm−1), which relates stoichiometrically to the production of ADP.ATPase assays were carried out using a linked assay as described previously . As such, this offers the best prospect so far for the unambiguous identification of any metal ions bound to this protein fragment. Indeed, during refinement it became apparent that two sites occupied by water molecules could be convincingly remodelled as metal ions. In the first site, lying adjacent to the nucleotide-binding site at the base of a surface loop termed the ‘ATP-lid’ . Whilst the average interaction length of 2.8 Å was comparable to that expected for a hydrogen bond to a water molecule, the refined temperature factor was significantly lower than the average value for the interacting atoms, suggestive of a species that is significantly more electron-dense than a water molecule. Taken together, the evidence indicated that site 1 was occupied by a K+ ion. In support of this conclusion, subsequent refinement with a K+ ion in this position gave a temperature factor comparable to the average value of the interacting atoms (Supplementary Table S3). In the second site, located towards the tip of the ATP-lid and hereafter referred to as ‘site 2’, the ‘water’ molecule was also octahedrally coordinated by six O atoms, but in this case only two of these were provided by backbone carbonyls and the remaining four were provided by other water molecules .The K-only model was determined to 1.75 Å resolution, representing the highest resolution structure reported so far for GyrB43, or indeed for any equivalent topoisomerase fragment . Overall the map was quite noisy, but of the top 11 peaks two were associated with ADPNP phosphates and four were associated with Met or Cys S atoms; peak 10 corresponded to a ‘water’ molecule and peak 11 corresponded to the species present in site 2 . The height of the latter was ∼35% that of the site 1 peak and therefore was too significant to result from an Na+ ion. We concluded that this site was most likely partially occupied by a K+ ion. However, given that the refined geometrical parameters of the modelled ion were entirely consistent with Na+, a mixed-occupancy site was not modelled. The ‘water’ molecule corresponding to peak 10 had three amine groups as ligands at distances of 3.2–3.5 Å, strongly indicative of it most likely representing a chloride ion, which was subsequently modelled satisfactorily in this position. Indeed, a chloride ion was inserted here in the ensuing structures. As expected, there were no peaks associated with the Mg2+ ions, which would have a relatively low anomalous signal at this wavelength (Supplementary Table S1).When the K-only model was superimposed on an anomalous difference Fourier map calculated using data collected from a similarly prepared crystal but at a longer wavelength of 1.91 Å (K-anom data), the highest peak in the map corresponded to the species present in site 1, consistent with it being a KRwork and Rfree values of 0.179 and 0.204, respectively, and satisfactory geometrical parameters, with MolProbity . The resultant model was essentially identical to the K-only model at the nucleotide-binding site, site 2 and the chloride site. However, the electron density at site 1 was elongated and weaker than expected for a K+ ion. The latter could however be modelled satisfactorily as a disordered Na+ ion occupying two discrete locations, each with 50% occupancy, within the binding site . Overall the structure was closely similar to the K-only structure, giving an r.m.s. deviation of only 0.16 Å after superposition (Supplementary Table S2).The second structure was determined using a data set collected at 1.90 Å resolution from a crystal grown in 100 mM KCl and 100 mM NaCl (‘K+Na’ data set) gave a model that was essentially equivalent to the original K-only structure, i.e. containing a K+ ion in site 1 and an Na+ ion in site 2. After superposition, the two structures gave an r.m.s. deviation of only 0.13 Å (Supplementary Table S2). Moreover, the geometric parameters of the monovalent cation-binding sites were closely similar to those of the K-only model (Supplementary Tables S3 and S5). An anomalous difference Fourier map calculated using data collected from a similarly prepared crystal at a wavelength of 1.91 Å (K+Na-anom data) gave peaks corresponding to the site 1 species, the ADPNP phosphates , several Met/Cys S atoms and the previously mentioned chloride ion. However, this time there was no peak associated with site 2 (not shown), which was indicative of this site being fully occupied with an Na+ ion.Refinement against a data set collected at 1.92 Å resolution from a crystal grown in the presence of both 100 m+ ion, and indicated an ordered species, in contrast to the elongated density that was attributed to a discretely disordered Na+ ion in the Na-only model. The distances to five of the six O atoms that form the surrounding octahedral coordination sphere lay in the range 2.75–3.22 Å, being entirely consistent with hydrogen bonds to a water molecule. The distance of 3.38 Å to the sixth potential ligand  was somewhat outside the normal range expected for hydrogen bonds and is therefore unlikely to contribute to binding the water molecule . Refining the bound species as a water molecule gave a temperature factor comparable to the average for the surrounding liganding atoms. Despite the exclusion of NaCl from the crystallization and cryoprotection solutions, site 2 was again modelled satisfactorily with a fully occupied Na+ ion (Supplementary Table S6). Overall the structure was closely similar to the three structures described above, giving r.m.s. deviations of only 0.242, 0.225 and 0.188 Å after superposition for the K-only, Na-only and K+Na structures, respectively (Supplementary Table S2).The last structure was determined using a data set collected at 1.95 Å resolution from a crystal grown using a precipitant solution containing neither KCl nor NaCl . This time the density at site 1 was relatively weak, less than would be expected for a Kle Fig. 2c. Refin3.2.DALI server , whilst at the same time screening for the presence of cations in locations corresponding to sites 1 and 2 of GyrB43. Within the remaining structures, the architecture of site 1 was generally well conserved and frequently contained a bound water molecule, although only in the structure of E. coli MutL , yeast DNA topoisomerase II (UniProtKB/Swiss-Prot entry P06786), E. coli MutL (UniProtKB/Swiss-Prot entry Q3YUH3), Caenorhabditis elegans heat-shock protein 90 , Thermotoga maritima chemotaxis protein CheA (UniProtKB/Swiss-Prot entry Q56310), human mismatch-repair endonuclease PMS2 (UniProtKB/Swiss-Prot entry P54278) and rat branched-chain α-ketoacid dehydrogenase kinase . Only the BCK search yielded new hits: a total of 12 structures with K+ in the equivalent of site 1, all of which were structures of rat BCK  structures from yeast and human , the GHKL domains superpose well and reveal that, like topo II, topoisomerase VIB also has a Lys side chain occupying the equivalent of the site 1 cavity adjacent to the bound nucleotide  it is occupied by a water molecule. Analysis of B factors and interatomic distances suggests that this could also be modelled satisfactorily as a chloride ion.In the previously deposited e Table 3. In the 3.3.M) to the assay mixture. We then tested the metal-ion preference with a competition ATPase assay, keeping a constant concentration of 500 mM NaCl in each reaction mixture while increasing the KCl concentration.We evaluated the effect of different metal ions on the ATPase activity of GyrB43 by the addition of increasing concentrations of either KCl or NaCl . However, when only NaCl is present Na+ is bound, giving rise to elongated electron density that can be modelled as two discrete half-occupancy Na+ sites . In the absence of either KCl or NaCl, the site can be occupied by a water molecule . The respective binding of K+, Na+ and water in this site in the K-only (and the closely similar K+Na model), Na-only and No-salt models is consistent with the evidence from the atomic B factors in the refined models. In all cases, the final B factor of the bound species is within 15% of the average value for the six potential liganding atoms (Supplementary Tables S3–S6).Here, we have shown that there are two monovalent metal ion-binding sites in the ATPase domain of the DNA gyrase B protein. Site 1 corresponds to the monovalent metal-ion site reported previously  proposed for K+—O distances . As result, there is no drawing in of the liganding O atoms and the cavity volume is similar to that seen when water is bound. If, as an approximation, we assume that the binding pocket is spherical, with a radius derived from the average interatomic distances of diametrically opposed O atoms , this value is 1.38 Å in the K-only structure . Nevertheless, the appearance of a minor peak in an anomalous difference Fourier map calculated using the K-anom data set suggested a minor contamination with K+ in site 2 when this is the only monovalent cation present in the crystallization and cryoprotection solutions . Initially, we were at a loss to explain why site 2 contained an Na+ ion in the K-only and No-salt structures, as this site would only be formed after the addition of ADPNP owing to the ATP-lid adopting a different conformation (or being disordered) in the absence of nucleotide. However, having looked back through all the reagents used, we realised that the EDTA component of the TGED buffer was in fact the disodium salt. Therefore, given that EDTA is at 1 mM in TGED, the protein would have been exposed to 2 mM Na+ ion when the ADPNP was added prior to the crystallization stage. Nevertheless, no Na+ ions were present in the cryoprotectant used for the K-only and No-salt data sets. We therefore propose that in these structures the species present in site 2 was a tightly bound Na+ ion that was not removed by the cryoprotection process.Since the occupant of site 2 makes direct contact with only two protein ligands, the remaining four interactions being with water molecules Fig. 3, one wouα positions and values not exceeding 0.18 Å in pairwise comparisons of the respective ATP-lids . In the crystal, the ATP-lid is not constrained by crystal contacts, therefore if the species occupying site 1 has a direct influence on the stability of the domain, one might expect this to be reflected in the B factors of the ATP-lid. However, plots of averaged residue main-chain B factors normalized to the overall main-chain average were somewhat similar for the four structures  and that GyrB43 will preferentially utilize K+ ions over Na+ ions. Since our structural data reveal only a single K+-specific site in GyrB43, i.e. site 1, we propose that K+ ions stimulate ATPase activity through binding to this site. Given the proximity of the site to the nucleotide, it seems probable that K+ binding has a direct effect on catalysis rather than simply having a structural role. The triphosphate moiety of the nucleotide is bound almost exclusively by amide groups provided by both the protein backbone and the side chains of Asn46, Lys103, Gln335 and Lys337 in GyrB. Together with the Mg2+ ion, these protein ligands will enhance ATP hydrolysis through electrostatic stabilization of the transition state of the reaction. It seems likely that K+ also contributes to this effect. However, given its juxtaposition to the α-phosphate, rather than the γ-phosphate, its principal role may be to withdraw negative charge from the latter, thereby deshielding the P atom and increasing its susceptibility to the water-mediated nucleophilic attack that initiates hydrolysis . Similar substitutions are observed elsewhere in the wider phosphotransferase family. For example, whilst bovine Hsc70 binds K+ adjacent to the nucleotide (PDB entry 1hpm; Wilbanks & McKay, 19951atn; Kabsch et al., 1990et al., 2003The observation that eukaryotic topo II enzymes employ the positively charged primary amine group of a Lys side chain in place of a KE. coli GyrB. One site lies at the dimer interface and in our hands preferentially binds a sodium ion; the functional significance of this site is unclear. The other site shows a preference for binding a potassium ion, which interacts directly with the α-phosphate of the nucleotide, and we postulate that it is responsible for the potassium-stimulated increase in the ATPase activity of DNA gyrase that we observe biochemically.In summary, we present evidence for two monovalent cation-binding sites in the vicinity of the Mg-ATP site in the ATPase domain of 5.et al. (1990et al. (2007The following references are cited in the Supporting Information for this article: Kabsch 1976; Altschu al. 1990 and Suze al. 2007.E. coli DNA gyrase B, 4wubN-terminal 43 kDa fragment of PDB reference: 4wucPDB reference: 4wudPDB reference: 4xtjPDB reference: 10.1107/S1399004715002916/dw5123sup1.pdfSupporting Information.. DOI:


The MN Genus-MTBC FISH assay uses an orange fluorescent probe specific for the Mycobacterium tuberculosis complex (MTBC) and a green fluorescent probe specific for the Mycobacterium and Nocardia genera (MN Genus) to detect and distinguish MTBC from other Mycobacteria and Nocardia. A complementary MTBC-MAC FISH assay uses green and orange fluorescent probes specific for the MTBC and M. avium complex (MAC) respectively to identify and differentiate the two species complexes. The assays are performed on acid-fast staining bacteria from liquid or solid cultures in less than two hours. Forty-three of 44 reference mycobacterial isolates were correctly identified by the MN Genus-specific probe as Mycobacterium species, with six of these correctly identified as MTBC with the MTBC-specific probe and 14 correctly as MAC by the MAC-specific probe. Of the 25 reference isolates of clinically relevant pathogens of other genera tested, only four isolates representing two species of Corynebacterium gave a positive signal with the MN Genus probe. None of these 25 isolates were detected by the MTBC and MAC specific probes. A total of 248 cultures of clinical mycobacterial isolates originating in India, Peru and the USA were also tested by FISH assays. DNA sequence of a part of the 23S ribosomal RNA gene amplified by PCR was obtained from 243 of the 248 clinical isolates. All 243 were confirmed by DNA sequencing as Mycobacterium species, with 157 and 50 of these identified as belonging to the MTBC and the MAC, respectively. The accuracy of the MN Genus-, MTBC-and MAC -specific probes in identifying these 243 cultures in relation to their DNA sequence-based identification was 100%. All ten isolates of Nocardia,  tested were detected by the MN Genus-specific probe but not the MTBC- or MAC-specific probes. The limit of detection for M. tuberculosis was determined to be 5.1x104 cfu per ml and for M. avium 1.5x104 cfu per ml in liquid cultures with the respective MTBC- and MAC-specific probes in both the MN Genus-MTBC and MTBC-MAC FISH assays. The only specialized equipment needed for the FISH assays is a standard light microscope fitted with a LED light source and appropriate filters. The two FISH assays meet an important diagnostic need in peripheral laboratories of resource-limited tuberculosis-endemic countries.Two rapid dual color fluorescence  Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis [Mycobacterium bovis that together comprise the Mycobacterium tuberculosis complex or MTBC, clinically relevant infections with non-tuberculous mycobacteria (NTM) also occur in the USA and elsewhere [Mycobacterium avium (the M. avium complex or MAC), but other Mycobacteria such as M. kansasii, M. fortuitum, M. xenopi, M. abscessus, and M. simiae can be responsible in some instances [Norcadia species also needs to be differentiated from MTB in human lung disease [Norcadia are Gram positive, partially acid fast-staining bacteria closely related to the genus Mycobacterium. They are found in soil and water and norcardiosis can manifest in pulmonary, cutaneous or disseminated forms [e.g. due to HIV infection, cancer and diabetes [The World Health Organization (WHO) estimates that one third of the human population is infected with rculosis . A smallrculosis . An addirculosis . While mlsewhere , 3. The nstances –8. Althonstances , 3, it instances –8. Norca disease . Norcadied forms . An estidiabetes .e.g. with the Ziehl-Neelsen stain, in sputum or tissue [® MTB/RIF system , a test based on PCR amplification and detection of the relevant DNA sequences in MTB, is recommended by the WHO as an initial diagnostic test for MTB and rifampicin resistance in the sputum of adults and children presumed to have tuberculosis [Norcardia species in clinical samples and cultures derived from them in resource-limited countries can therefore enhance the worldwide effort to control tuberculosis [The identification of MTB, following a presumption of tuberculosis on clinical criteria, conventionally relies on microscopic examination for acid-fast staining bacilli, r tissue . Howeverrculosis –13. Althrculosis . The corrculosis , and addrculosis –14. The rculosis –14 S1 Tarculosis –14 S1 Tarculosis –14.in situ hybridization (FISH) is a cytogenetic technique that utilizes short complementary probes labeled with fluorescent molecules to localize and detect specific target DNA or RNA sequences. Delong et al. demonstrated in 1989 that FISH can be used to detect a single microbial cell using fluorescently labeled oligodeoxynucleotides complementary to 16S ribosomal RNA (rRNA), and that with appropriately-targeted probes labeled with different fluorescent dyes, FISH can distinguish closely related organisms [M. avium-specific PNA FISH probe has also been applied to detect M. avium in water samples and biofilms [Plasmodium falciparum and Plasmodium vivax [Nocardia from liquid and solid cultures, and which also meet the criteria for application in resource-limited, tuberculosis-endemic countries.Fluorescence rganisms . The prerganisms  or peptirganisms –20 direcrganisms . The MTBrganisms  had a seum vivax . Diagnosum vivax –14. We rMycobacterium species and three of Nocardia species  solid media slants  and as described in the ATCC product inserts, except that Bacillus subtilis and all Corynebacterium species were grown on blood agar plates . The reference MTBC, two Bacillus subtilis (ATCC 23059 and 6633) and the five Corynebacterium clinical isolate cultures were grown in the Microbiology Laboratory, University of California San Francisco, San Francisco, CA. Other reference cultures were grown on LJ solid media slants in the ID-FISH laboratory.Forty-four clinically relevant reference cultures of Nocardia identified as N. brasiliensis, N. farcinica, N. wallacei, N. cyriacigeorgica, N. pseudobrasiliensis, N. nova and N. otitidiscaviarum were obtained from Banner Health, Phoenix, AR.A total of 248 cultures derived from clinical mycobacterial samples were tested. These comprised 174 samples cultured on LJ solid media slants obtained from the Kasturba Medical College, Mangalore, India, the Laboratorio de Investigación en Enfermedades Infecciosas of the Universidad Peruana Cayetano Heredia, Iquitos, Peru and the Microbiology Laboratory at the University of California San Francisco, CA, as well as 74 liquid cultures in Mycobacteria Growth Indicator Tubes  from the Kasturba Medical College, Mangalore, India. All the samples originated from digested, decontaminated sputum from patients suspected to have tuberculosis. Additionally, phenol-ethanol fixed smears prepared from solid cultures of seven clinical isolates of A loopful of colony was first transferred from the solid medium into distilled water to yield a McFarland standard of 0.5 to 1.0. Several sterile 3 mm glass beads were added to the mixture which was then vortexed for 30 s to facilitate homogenization of the cell suspension. Ten μl of the cell suspension was pipetted onto a pre-cleaned glass microscope slide to make a 15 mm-diameter smear. Smears were then air dried and chemically fixed with either 100% methanol for all samples from Peru and the USA, or a solution of 5% v/v phenol in 70% v/v ethanol in the case of samples from India, for 5 min according to the established procedures in the respective laboratories. This was followed by a rinse with water and air-drying. Fixation in 5% v/v phenol in 70% v/v ethanol renders the fixed smears non-infectious and safe for handling outside a biosafety facility .TM MGIT TM 320 detection system . Smears for microscopy were prepared usually within a day of a positive signal being detected in the BACTEC TM MGIT TM 320 machine. The presence of acid-fast staining bacteria was confirmed prior to preparing smears. The Mycobacterium Processing Kit  was used to process the liquid cultures according to the manufacturer’s instructions provided in the kit. The kit contained two reagents, a sample processing solution containing a strong reducing agent and MycoSPR containing a chaotropic agent, that help retain optimal specimen morphology in the FISH assay. In the first step, 40 μl of the undiluted culture was added to 40 μl sample processing solution provided in the kit, mixed and incubated for 5–10 min at room temperature, followed by the addition of 10 μl of MycoSPR. Ten μl of the resulting mixture was placed on a pre-cleaned glass microscope slide to generate a 15mm-diameter circular smear. The smears were dried and then fixed with a solution of 5% v/v phenol in 70% v/v ethanol for 5 min at room temperature.Liquid cultures were grown at the Kasturba Medical College, Mangalore, India by inoculating either processed sputum samples or stock cultures into MGIT tubes and placed in the BACTEC Mycobacterium/Nocardia Genus (MN Genus) and MTBC combination FISH test kit  and the MTBC and MAC combination FISH test kit  were obtained from ID-FISH Technology Inc., Palo Alto, CA. The two test kits contained the relevant rRNA-specific DNA probes, pretreatment buffer, pretreatment rinse buffer, hybridization solution, wash buffer and mounting medium. The MN Genus-specific probe is complementary to a 16S rRNA sequence that is common to the Mycobacterium and Nocardia genera. The MTBC and MAC-specific probes hybridize to 23S rRNA sequences that are unique to each of the two species complexes. The tests were performed according to the manufacturer’s instructions provided with the kits.The In the MN Genus-MTBC FISH assay, the MN Genus-specific DNA probe is conjugated with a green fluorescent dye Atto 480 and the MTBC-specific DNA probe is conjugated with an orange fluorescent dye Atto 550. In the MTBC-MAC FISH test, the MTBC-specific DNA probe is conjugated with Atto 480 and the MAC-specific DNA probe is conjugated with Atto 550.Norcardia are summarized in All FISH assays were performed in the ID-FISH laboratory in Palo Alto, CA, USA. Two slides from each sample were processed with one slide used for the MN Genus-MTBC FISH assay and the other for the MTBC-MAC FISH assay. After the addition of 1 ml of pre-treatment buffer to each fixed smear, the slides were placed in a humid chamber at 37°C for 15 min. The slides were then removed from the incubator, rinsed with pre-treatment rinse buffer and air-dried. After the subsequent addition of 10 μl of appropriate hybridization solution containing the DNA probe mix, the smear was covered with a plastic cover-slip and placed in a 37°C humid chamber for 15 min for hybridization. After hybridization, each smear was washed three times for 2 min each with wash buffer at room temperature and then dried in complete darkness. A drop of mounting medium was then added and the smear was covered with a glass cover-slip. Smears were read at 1000x magnification using a LED light source with custom filter sets for viewing green  and orange fluorescence  obtained from Fraen Corp, Cusago, Italy, fitted to an Olympus BX light microscope. A workflow diagram for performing the FISH assay is provided in 5’-AGAATGAGCCTGCGAGTCAG-3’ and Myco-R, 5’-ACARCTCATCCCCTCAGTCT-3’. The approximately 200bp PCR products were subsequently sequenced using the same Myco-F and Myco-R primers. The resulting sequences were compared by BLAST with published Mycobacterium 23S rDNA sequences to identify the mycobacterial species.A sample was taken from a smear made from each of the 248 mycobacterial cultures and processed to obtain purified DNA using the Generation Capture Column kit . The relevant DNA region was then amplified by PCR using the following forward and reverse primers: Myco-F, 6 to 107 cells per ml from MGIT cultures, serial ten-fold dilutions in MGIT broth up to a dilution of 10−7 were prepared from three different replicate M. tuberculosis (ATCC 25177) and three different replicate M. avium (ATCC 25291) cultures. At each dilution, 10 μl samples were plated on Middlebrook 7H10 agar plates and incubated at 37°C, 5% v/v CO2 for up to four weeks. Colonies were counted after sufficient growth was obtained to determine the number of colony forming units (cfu) at each dilution. Three replicate smears prepared in parallel from each of the same dilutions were tested in both MN Genus-MTBC FISH and MTBC-MAC FISH assays. Three hundred fields were examined at 1000x magnification before recording a negative reaction for a smear in the limit of detection (LOD) determination. The LOD was defined as the lowest concentration of bacteria at which the three replicate smears for each original culture tested gave a positive result with relevant probes in the two FISH assays. The final LOD was calculated as the mean of the three LODs derived for the three replicate starting cultures for each species.With an approximate starting concentration of 10The diagnostic sensitivity, specificity, positive predictive value and negative predictive value and their 95% confidence intervals were calculated using an online statistical calculator  for eachM. tuberculosis, M. avium and M. kansasii, and with the MTBC-MAC FISH test on a smear from an artificially mixed culture of reference strains of M. tuberculosis and M. avium are shown in The dual color fluorescence observed in the two FISH assays with individual culture smears of reference strains of Mycobacterium species cultures, except for M. wolinskyi, tested positively with the MN Genus-specific probe and only the six M. tuberculosis complex cultures reacted positively with the MTBC- specific probe in the MN Genus—MTBC FISH assay. Of the same 44 Mycobacterium species cultures tested in the MTBC-MAC FISH assay, only the six M. tuberculosis complex cultures reacted positively with the MTBC-specific probe and only the 14 M. avium complex cultures reacted positively with the MAC–specific probe  and MGITs (74 cultures) were tested in both the MN Genus-MTBC FISH and MTBC-MAC FISH assays. In addition, all cultures were subjected to PCR amplification and sequencing of 23S rDNA. Of the 248 cultures tested in the two FISH assays, 247, 158 and 50 were detected by the MN Genus-, MTBC- and MAC-specific probes respectively. PCR amplification was successful with 243 of the 248 cultures tested . In the M. immunogenum by DNA sequencing . However, only 47 of the 50 were confirmed as belonging to MAC by 23S rDNA sequencing in the present study. All these 47 specimens tested positive for MAC in the MTBC-MAC FISH assay. The remaining 3 were negative in the MTBC-MAC FISH assay and were confirmed to be quencing . In totamegmatis . The proNocardia species  were positive with the MN Genus-specific probe and negative with the MTBC- and MAC-specific probes in the FISH assays, consistent with the expectation summarised in All seven clinical specimens of Mycobacterium clinical isolate cultures that could be identified by DNA sequencing  and intermediate (district and sub-district) level laboratories will help reduce delays in diagnosing and initiating treatment of patients in many tuberculosis-endemic countries. We suggest in conclusion that the FISH assays described here meet this important diagnostic need.The FISH assays reported here therefore offer many advantages over other common assays used for characterizing clinical mycobacterial cultures Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Altered mitochondrial metabolism acts as an initial cause for cardiovascular diseases and metabolic intermediate succinate emerges as a mediator of mitochondrial dysfunction. This work aims to investigate whether or not extracellular succinate accumulation and its targeted G protein-coupled receptor-91 (GPR91) activation induce cardiac injury through mitochondrial impairment. The results showed that extracellular succinate promoted the translocation of dynamin-related protein 1 (Drp1) to mitochondria via protein kinase Cδ (PKCδ) activation, and induced mitochondrial fission factor (MFF) phosphorylation via extracellular signal-regulated kinases-1/2 (ERK1/2) activation in a GPR91-dependent manner. As a result, enhanced localization of MFF and Drp1 in mitochondria promoted mitochondrial fission, leading to mitochondrial dysfunction and cardiomyocyte apoptosis. We further showed that inhibition of succinate release and GPR91 signaling ameliorated oxygen–glucose deprivation-induced injury in cardiomyocytes and isoproterenol-induced myocardial ischemia injury in mice. Taken together, these results showed that in response to cardiac ischemia, succinate release activated GPR91 and induced mitochondrial fission via regulation of PKCδ and ERK1/2 signaling branches. These findings suggest that inhibition of extracellular succinate-mediated GPR91 activation might be a potential therapeutic strategy for protecting cardiomyocytes from ischemic injury. Although inflammation, oxidative stress and endoplasmic reticulum stress are involved in cardiac injury, accumulating evidence demonstrates that mitochondrial dysfunction is an initial cause for these events3. Alterations in mitochondrial morphology increases the susceptibility of the heart to ischemia/reperfusion injury5, indicating the important role of mitochondrial integrity in the protection of cardiac function.In cardiomyocytes, mitochondrial homeostasis plays a key role in maintaining heart function in response to metabolic stress6. Drp1 activation is regulated by phosphorylation modification. Protein kinase Cδ (PKCδ) and extracellular signal-regulated kinase-2 (ERK2) are shown to increase Drp1 translocation to mitochondria and promote mitochondrial fission by phosphorylation of Drp1 at Serine 6168. Moreover, phosphorylation of MFF may regulate the association of Drp1 with mitochondria. This concept is supported by the enhanced binding of Drp1 to MFF when MFF is phosphorylated by AMP-activated protein kinase (AMPK)9.Mitochondrial morphology is dynamically controlled by continuous fission and fusion. Dynamin-related protein 1 (Drp1) is a central regulator in mitochondrial fission. Drp1 is primarily located in the cytosol. Upon activation, Drp1 is recruited from the cytoplasm to the mitochondrial outer membrane, where it binds to mitochondrial fission factor (MFF) to trigger mitochondrial fission10. Although succinate is normally produced in mitochondria, the accumulated succinate can be released to extracellular space in response to pathological status, such as ischemia, diabetes and hypertension12. It has been demonstrated that extracellular succinate exerts a paracrine and endocrine effector through activation of its specific G protein-coupled receptor-91 (GPR91)10. Signaling pathways triggered by GPR91 include activation of PKC and ERK1/214. GPR91 is highly expressed in the heart15, mediating succinate-induced cardiomyocyte death16. Moreover, upholding levels of serum succinate can cause cardiac hypertrophy through activation of GPR9117. Given the important role of mitochondrial function in cardioprotection3, this work aims to investigate whether or not extracellular succinate accumulation and its targeted GPR91 receptor activation induce cardiac injury through mitochondrial impairment.Succinate is an important metabolic intermediate in the citric acid cycle and emerging evidence demonstrates that dysregulation of succinate generation is involved in cardiovascular diseases and metabolic disorders+/K+-ATPase 1) (14418-1-AP) and anti-ATP5A1 (66037-1-lg) from Proteintech Group ; anti-ERK1/2 (CY5487) and anti-phospho-ERK1 (T202/Y204)+ERK2 (T185/Y187) (CY5277) from Abways Technology .Sodium succinate dibasic hexahydrate, dimethyl malonate, rottlerin and tetramethylrhodamine ethyl ester perchlorate (TMRE) were purchased from Sigma-Aldrich . Mito Tracker Red CMXRos (M7512) was obtained from Molecular Probes . Cell Tracker CM-Dil and dihydroethidium (DHE) were purchased from Beyotime Institute of Biotechnology . U0126-EtOH was purchased from Apex Bio . These agents were dissolved in dimethyl sulfoxide (DMSO) to obtain stock solutions and the final working concentration of DMSO was <0.1% (v/v). Antibodies were purchased from the following companies: anti-phospho-Drp1 (#4494), anti-phospho-MAPK Substrates Motif [PXpTP] (#14378), anti-Bax (#2772) and anti-HK-II (#2867) from Cell Signaling Technology ; anti-Drp1 (ab184247), anti-prohibitin (ab75771), anti-PKC-delta (ab182126) and anti-β-Actin (ab8226) and anti-PKC-epsilon from Abcam ; anti-MFF (sc-32577) from Santa Cruz Biotechnology, Inc. ; anti-GPR91 (BS2961), Goat Anti-Rabbit IgG (H+L), HRP (BS13278), anti-GAPDH (AP0063) and Goat Anti-Mouse IgG (H+L) (Alexa Fluor 488) (BS12478) from Bioworld Technology ; Alexa Fluor 647 AffiniPure Donkey Anti-Mouse IgG (H+L) antibody from Yeasen ; anti-ATP1A1  and ICR male mice (18–22 g) were purchased from the Laboratory Animal Center of Nanjing Qinglongshan. The care and treatment of animals were consistent with the Animal Ethics Committee of China Pharmaceutical University.18. Briefly, NRVMs were isolated from 1–2-day-old Sprague-Dawley rat, digested with 0.08% collagenase and purified by differential adhesion method. Myocytes were incubated overnight in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) and 0.1 mmol/L 5-bromo-2-deoxyuridine at 37 °C in a humidified incubator of 5% CO2 atmosphere. For oxygen–glucose deprivation (OGD) insult, the NRVMs were exposed to 1% O2 with glucose deprivation for 4 h. For succinate treatment, NRVMs were incubated with sodium succinate (200 μmol/L) for 2 h.Neonatal rat ventricular myocytes (NRVMs) were prepared as previously described2.H9c2 cells  were maintained in DMEM containing 10% FBS, supplemented with antibiotics (100 U/mL penicillin G and 100 μg/mL streptomycin sulfate), at 37 °C in a humidified atmosphere of 5% COMice were treated with intraperitoneal injection of dimethyl malonate (160 mg/kg) or saline 1 h before isoproterenol injection  for 2 consecutive days. At 24 h after the last injection, mice were killed for collection of hearts. For western blot analysis, left ventricle regions were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer to extract protein. For immuofluorescent assay and apoptosis assay, the whole heart was directly fixed in 4% paraformaldehyde or 15% sucrose. The infarct size of the hearts was viewed by 2,3,5-triphenyltetrazolium chloride (TTC) staining.2+ was monitored with a Ca2+ assay kit. NRVMs were lysed with deionized water followed by 3 times of freezing and thawing cycle. After centrifuging at 10,000 rpm for 10 min at 4 °C, the supernatant was collected for the determination of intracellular Ca2+ contents according to the manufacturer’s instructions (Jiancheng Bioengineering Institute).Intracellular CaAfter stimulation with indicated agents, NRVMs were loaded with 5 μM DHE working solution (Beyotime Institute of Biotechnology) at 37 °C for 30 min. After washing, cells were fixed in 4% paraformaldehyde for 5 min at 4 °C, and ROS production was measured by a microplate reader.6) or cardiac tissue (10 mg) were rapidly homogenized on ice in 100 μL of succinate assay buffer. Then, the homogenate was centrifuged at 10,000 × g for 5 min to collect the supernatant. Intracellular succinate was detected using a Succinate Colorimetric Assay Kit (Sigma-Aldrich).After stimulation, NRVMs  referring to the instructions. The samples were moderately homogenized and then centrifuged at low speed to abandon nucleus and intact cells. The supernatants were centrifuged at 14,000 × g. The supernatants were centrifuged at 11,000 × g for 10 min at 4 °C to obtain mitochondrial pellets. The remaining supernatant was taken as the cytosol fraction. Then, the mitochondrial pellets were lysed to extract mitochondrial protein using lysis buffer.Cytosolic and mitochondrial fractions were prepared from NRVMs using Cell Mitochondria Isolation Kit (Beyotime Institute of Biotechnology) according to the manufacturer’s protocol. Briefly, cells were collected and homogenized with a glass homogenizer in mitochondrial isolation buffer on ice. Unlysed cells and nuclei were pelleted by spinning for 10 min at 600 × g for 20 min at 4 °C. Protein concentration was quantified using Enhanced BCA Protein Assay Kit . Equal amount of protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride membranes and then blocked at room temperature for 2 h. The membranes were immunoblotted with primary antibodies at 4 °C overnight. After washing, blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 2 h. Protein bands were visualized with chemiluminescence reagent and quantified by Image-ProPlus 6.0 software.Cells were lysed in ice-cold RIPA buffer to obtain protein by centrifugation at 12,000 × g for 20 min and the soluble fraction was collected. Next, anti-Drp1 antibody or anti-MFF antibody was immunoprecipitated overnight at 4 °C and then with protein A+G agarose beads (Beyotime Institute of Biotechnology) for another 2 h. After rinsing with the lysis buffer or phosphate-buffered saline, the beads complexes were then boiled in 1% SDS loading buffer for western blot analysis with the indicated antibodies.For immunoprecipitation, collected cells or cardiac tissues were incubated in the ice-cold cell lysis buffer for western and IP (Beyotime Institute of Biotechnology) for 30 min. Cell lysates were then centrifuged at 12,000 × To specifically suppress GPR91 expression, H9c2 cells of 60-80% confluence were transfected with small interfering RNA (siRNA) duplex specific for rat GPR91  or control siRNA (sc-37007) by siRNA transfection reagent (sc-29528), according to the manufacturer’s instructions. For silencing of PKCδ or MFF expression, H9c2 cells were transfected with rat PKCδ siRNA or rat MFF siRNA (Genomeditech) by Lipofectamine 2000 Reagent , using control siRNA (Genomeditech) as the control. After 48 h post transfection, the cells were treated with sodium succinate for 2 h and then collected and processed for immunofluorescence microscopy and western blot.After treatment, NRVMs were stained with 200 nmol/L Mito Tracker Red CMXRos  to label mitochondria. NRVMs were then fixed in 4% paraformaldehyde for 20 min, and permeabilized with 0.2% Triton X-100 for 10 min at room temperature. After blocking with 3% bovine serum albumin, specimens were then labeled with specific primary antibodies overnight at 4 °C in a humidified chamber, followed by incubation with fluorescent secondary antibodies for 1 h at 37 °C. After washing, specimens were examined with a confocal scanning microscope (Zeiss LSM 700).NRVMs were stained with 200 nmol/L Mito Tracker Red CMXRos for 30 min at 37 °C. The structure of mitochondria was viewed using confocal microscopy (Zeiss LSM 700). Mitochondria with a predominantly intact network of tubular were identified as normal. Cells with disrupted and predominantly spherical mitochondria were identified as having mitochondrial fission.To detect mitochondrial morphology in heart, cardiac tissues were fixed with 4% paraformaldehyde, dehydrated and embedded in Tissue-Tek O.C.T. Compound  and then cut into slices. The slices were stained with ATP5A1 antibody followed by secondary antibody. Mitochondrial morphology was visualized by confocal microscopy (Zeiss LSM 700).Treated NRVMs were loaded with the potentiometric dye TMRE (500 nmol/L) for 30 min at 37 °C. Fluorescence intensities were determined at the single-cell level using confocal microscopy (Zeiss LSM 700).2) in the presence or absence of dimethyl malonate (5 mmol/L). Cell cytosolic and mitochondrial fractions were collected and the concentration of cytochrome C was assayed with commercial enzyme-linked immunosorbent assay (ELISA) kits .NRVMs were stimulated with sodium succinate (200 μmol/L) with or without mdivi-1 (10 μmol/L), or treated with hypoxia  system . The staining was detected by NanoZoomer 2.0-RS . In addition, cell apoptosis in NRVMs was detected with TUNEL assays kit  by a confocal scanning microscope (Zeiss LSM 700).p < 0.05 is considered statistically significant.Data are presented as mean values ± SD. The significance of differences was analyzed by one-way analysis of variance followed by the Tukey's test. A value of 2+ concentration is the feature of GPR91 activation19. We observed that sodium succinate effectively increased intracellular Ca2+ contents at concentrations ranging from 100 μM to 1 mM with half-maximal effective concentration (EC50) of 153.9 μM   Fig. . Thus, sion Fig. , furtherion Fig. . PKC actria Fig. . Correspria Fig. . PKCδ in22. Sodium succinate increased Drp1 phosphorylation (Ser 585)  promotes Drp1 translocation to mitochondria and induces mitochondrial fission85) Fig.  and prom85) Fig. , whereas85) Fig. . To dete85) Fig. . These r85) Fig. . To prov85) Fig. .Fig. 2Su14. In cardiomyocytes, sodium succinate insult increased ERK1/2 phosphorylation and promoted its translocation to mitochondria  for ERK1/2 consensus phosphorylation motifs. Search result revealed that rat MFF contains ERK1/2 consensus phosphorylation sites at T112  activationOGD Fig. . As expeOGD Fig.  and therOGD Fig. . MeanwhiOGD Fig. . Malonatsis Fig. . Althoug10, these results suggested that accumulated succinate in mitochondria was released to activate membrane GPR91. Subsequently, malonate treatment attenuated colocalization of MFF and Drp1 in mitochondria and then prevented mitochondrial fission  signaling cascades31. Mitochondrial morphology is dynamically controlled by Drp1 and the sub-mitochondrial localization of Drp1 is essential for Drp1 to mediate mitochondrial fission. Two sites are phosphorylated on Drp11. Phosphorylation of Drp1 at Ser 616 residue in humans, corresponding to Ser 585 in rats, is able to activate Drp122, while Ser 637 phosphorylation can inhibit Drp1 activation32. In the present study, upon succinate stimulation, PKCδ combined with Drp1 in the cytoplasm, leading to Drp1 phosphorylation and then translocation of PKCδ with phosphorylated Drp1 to mitochondria to induce mitochondrial fission. Knockdown of GRP91 reduced PKCδ translocation to mitochondria and prevented mitochondrial fission, providing evidence that succinate-mediated GRP91 activation induced mitochondrial fission through regulation of PKCδ. This regulation is consistent with the published study which shows that PKCδ induced aberrant mitochondrial fragmentation in neuronal cells7.GPR91 is a plasma membrane receptor presenting in many tissues, including the heart33. Our previous study also showed that in response to succinate-mediated GPR91 activation, PKCδ was activated and translocated to mitochondria, impairing pyruvate dehydrogenase activity in cardiomyocytes20. Herein, we identified another pathological mechanism by which PKCδ activation impairs mitochondrial integrity in cardiomyocytes. The defects in mitochondrial dynamics can impair energy metabolism, leading to mitochondrial dysfunction34. Therefore, we reasoned that PKCδ is involved in both mitochondrial morphological and functional regulation in cardiomyocytes. In fact, PKCδ has multiple protein substrates. By phosphorylating such diverse substrates at different subcellular locations, PKCδ mediates a variety of cellular responses35.Intriguingly, we found that PKCδ but not PKCε is activated in response to GPR91 stimulation. PKCδ and PKCε, the principal members of the novel group of PKCs, are opposed by their principal effects. In the heart, PKCε is favorably involved in the preconditioning protection against ischemic insult, while PKCδ serves as a mediator for the ischemic damage9. This evidence indicates that MFF phosphorylation facilitates Drp1 binding to mitochondria to influence the dynamic change of mitochondrial morphology. In cardiomyocytes, we showed that MFF contains ERK1/2 consensus phosphorylation sites, indicative of the potency to be regulated by ERK1/2. Importantly, MAPK/ERK signaling is considered to be a downstream regulation by GRP91 activation14. Succinate-mediated GPR91 activation increased ERK1/2 phosphorylation and promoted ERK1/2 translocation to mitochondria, where it phosphorylated MFF to facilitate Drp1 localization on mitochondria, contributing to mitochondrial fission. Even though it is documented that ERK can directly phosphorylate Drp1 to promote mitochondrial fission37, our work demonstrated another pathway to regulate Drp1 activation by phosphorylation of MFF. Together with the regulation of PKCδ, our study elucidated that PKC/ERK signaling cascades linking GPR91 activation mediated mitochondrial fission upon extracellular succinate stimulation.Upon activation, Drp1 recruits to mitochondria, where it binds to MFF to assemble fission sites. Evidence shows that phosphorylation of MFF by AMPK may increase the association of Drp1 with mitochondria to induce mitochondrial fission38. The structural and functional integrity of mitochondria is essential for mitochondrial HK-II binding which is associated with permeability barrier of inner mitochondrial membrane39. Succinate-induced mitochondrial fission resulted in the HK-II dissociation from mitochondria, leading to the collapse of mitochondrial membrane potential. Bax and Drp1 colocalize together at scission sites on mitochondria, enabling cytochrome C release from mitochondria41. We observed that succinate insult increased Bax expression in mitochondrial fraction and promoted cytochrome C release from mitochondria, and subsequently induced cell apoptosis in cardiomyocytes. These alterations were reversed by mitochondrial fission inhibitor mdivi-1, demonstrating that mitochondrial fission contributed to succinate-mediated mitochondrial dysfunction and cell death.Aberrant mitochondrial fragmentation is the early stages of apoptosis42, mediating oxidative stress43. A recent study shows that accumulated intracellular succinate acts as a potential electron store that fuels to drive ROS formation from mitochondrial complex I by reverse electron transport during reperfusion24. Differently, the present work suggested that under hypoxic conditions, excessive extracellular succinate could induce ROS production in response to GPR91-induced mitochondrial fission, which may contribute to exaggerating ROS production to induce oxidative damage during subsequent reperfusion.The hypoxic succinate accumulation within cells is a universal metabolic signal in heartTo translate our findings in vivo, we observed the protective effects of SDH inhibitor malonate in isoproterenol-induced cardiac ischemia model. We observed that succinate substantially accumulated in the heart and induced GPR91 activation. As expected, SDH inhibitor malonate reduced succinate accumulation in the heart and decreased GPR91 activation against ischemic insult, a regulation likely due to the limited succinate release. Consequently, malonate blocked MFF and Drp1 colocalization in mitochondria and prevented mitochondrial fission. The attenuated heart injury by malonate treatment provided in vivo evidence to support the conclusion that inhibiting succinate-mediated GPR91 signaling may protect the heart from ischemic injury, at least in part.Taken together, we showed that hypoxic succinate accumulation and release could activate membrane GPR91 and induce mitochondrial fission through PKCδ/ERK1/2 signaling pathways Fig. . These r


Living systems use fuel-driven supramolecular polymers such as actin to control important cell functions. Fuel molecules like ATP are used to control when and where such polymers should assemble and disassemble. The cell supplies fresh ATP to the cytosol and removes waste products to sustain steady states. Artificial fuel-driven polymers have been developed recently, but keeping them in sustained non-equilibrium steady states (NESS) has proven challenging. Here we show a supramolecular polymer that can be kept in NESS, inside a membrane reactor where ATP is added and waste removed continuously. Assembly and disassembly of our polymer is regulated by phosphorylation and dephosphorylation, respectively. Waste products lead to inhibition, causing the reaction cycle to stop. Inside the membrane reactor, however, waste can be removed leading to long-lived NESS conditions. We anticipate that our approach to obtain NESS can be applied to other stimuli-responsive materials to achieve more life-like behaviour. Several cell functions are based on the fuel-driven assembly and disassembly of supramolecular polymers under non-equilibrium conditions. Here, the authors show controlled formation and breaking of a supramolecular polymer by enzymatic phosphorylation and dephosphorylation of a building block by continuously adding ATP fuel and removing waste products. Dissipative supramolecular polymerization is commonplace in living cells such as in the actin and microtubule networks, where chemical fuels control spatiotemporal behaviour, endowing the cell with exquisite responsivity, motility, adaptability and replicationArtificial supramolecular assemblies have been developed in the recent years where a monomer can be switched from assembling to nonassembling or vice versa by direct chemical modification23567910112361112Here we demonstrate that it is possible to keep supramolecular polymers in various sustained non-equilibrium steady states (NESS) by a continuous influx of chemical fuel and outflux of waste products. We control our supramolecular polymerization by phosphorylation/dephosphorylation mediated by kinase and phosphatase enzymes, respectively. The phosphorylation is driven by the fuel ATP that adds a charged phosphate group to our self-assembling molecule resulting in polymer growth and switching of supramolecular chirality. We show that in batch conditions transient self-assembly occurs upon addition of ATP, but poisoning due to phosphatase inhibition limits the number of assembly/disassembly cycles. The poisoning can be overcome by using a home-built membrane reactor, where different NESS can be maintained depending on the level of fuel supplied and waste removed under continuous flow conditions. The result is that the assembly/disassembly process can be kept continuously working for days as long as the fuel is supplied. Maintaining NESS conditions has been heralded as one of the requirements to obtain life-like systems and materialsPDI in PDI can be phosphorylated at both serine residues by PKA consuming ATP, resulting first in p-PDI and finally in p2-PDI  are produced as waste products in a complete phosphorylation/dephosphorylation cycle , in turn affecting the structure and stability of their supramolecular polymers.Perylenediimide derivatives are prone to self-assemble in water due to π−π stacking, the hydrophobic effect and electrostatic interactions18PDI, p-PDI and p2-PDI) occurs through irreversible enzyme-catalysed reactions. Thus, the solution composition is only dictated by the relative rates of competing phosphorylation and dephosphorylation, but not by their thermodynamic stability  and 31P-nuclear magnetic resonance to verify that PKA can indeed quantitatively phosphorylate PDI to p2-PDI, using two equivalents of ATP  at which both the enzymes can work, and at commensurate speeds. The optimized buffer is a compromise between the optimal ones for each of the two enzymes and is referred to as the reaction buffer (see Methods).Beyond verifying the ability of PKA and λPP to catalyse the (de)phosphorylation of PDI and p2-PDI in the reaction buffer to determine the changes induced by phosphorylation on their supramolecular polymers. To this end, we performed phosphorylation and dephosphorylation in two separate steps  due to the S0 → S1 transition of the perylenediimide chromophore, with two maxima at 504 and 540 nm (A504 nm and A540 nm) (A504 nm/A540 nm of 1.46 strongly indicates PDI aggregation through π–π stacking (H-aggregation) into helical stacks of molecules with rotational offset between them (see below)1819PDI has a A504 nm/A540 nm ratio of 0.65 (c=0.13 μM) and ATP ) to the PDI solution (200 μM), a continuous increase of A504 nm/A540 nm over time was observed, up to a plateau at 1.65 after ∼100 min  . The rat of 0.65 . Upon ad∼100 min . LC–MS antensity  indicatePDI and p2-PDI was studied by temperature-dependent ultraviolet–visible experiments , suggesting that the polymers are equilibrium structures with exchange dynamics on the second timescale. The A504 nm/A540 nm versus temperature curves could be described with an isodesmic  polymerization model25PDI and p2-PDI. This analysis (p2-PDI polymers (Keq (298 K)=3.8 × 104 M−1, Tm (230 μM)=368.9±0.1 K) are more stable as compared with PDI polymers (Keq (298 K)=2.1 × 104 M−1, Tm (230 μM)=358.5±0.1 K). Perylenediimide derivatives are well known for their propensity to assemble into one-dimensional columnar aggregates1819p2-PDI we could observe bundles of long fibrous aggregates by atomic force microscopy  in samples drop cast on mica (g(1)(τ) of p2-PDI (red squares) is clearly shifted to higher lag time τ when compared with that of PDI (black circles), corresponding to hydrodynamic radii RH of 690±40 and 440±10 nm for p2-PDI and PDI, respectively (at 1 mM). In addition, the corresponding relaxation Γ showed a slope of 2 versus scattering vector squared q2  that is known to lower solubility and diminish electrostatic repulsion, leading to increased supramolecular polymerization.We explain the observed increase in size and stability of the supramolecular assemblies upon phosphorylation by considering the overall charge of the monomer. Specifically, PDI solutions feature an asymmetric positive CD couplet at the main absorption of the perylenediimide chromophore (black line in 282930p2-PDI (red line in 282930PDI and p2-PDI polymers in solution (important later on) was verified by self-sorting experiments32PDI and p2-PDI spectra corresponding to their molar fraction, suggesting that they do not form mixed assemblies32p2-PDI polymers that not only have a higher degree of polymerization, but also opposite helicity  spectroscopy, we found that PDI and p2-PDI we could use time-dependent CD measurements to follow the phosphorylation and dephosphorylation in a stepwise manner (c=0.13 μM) to a solution of PDI (c=200 μM) and ATP  resulted in a progressive change (inversion) of the CD spectra over time  caused a fast decrease of the ellipticity at 479 nm to its initial value, with the CD spectrum reverting to the shape characteristic for PDI (again LC–MS confirmed complete dephosphorylation). Note that the latter occurred in the absence of ATP, since it was completely consumed in the prior phosphorylation. So far, we have shown that it is possible to reversibly phosphorylate and dephosphorylate our self-assembling peptide-perylenediimide derivatives, and that phosphorylation leads to longer polymers with opposite chirality that coexist in solution with nonphosphorylated polymers.Due to the unique CD spectra of e manner . For insver time . The latver time . After cPDI→p-PDI→p2-PDI→p-PDI→PDI) in a nonstirred batch reactor  to a PDI solution (c=100 μM) containing PKA (c=0.13 μM) and λPP (c=0.3 μM). Interestingly, a rather complex behaviour was observed for monophosphorylated p-PDI, showing two maxima during a single transient experiment  is due to the competition between the two phosphorylation reactions PDI→p-PDI and p-PDI →p2-PDI, while the second maximum (t≈750 min) results from the competition between the phosphorylation and dephosphorylation of p2-PDI, that is, p-PDI→p2-PDI and p2-PDI→p-PDI  consumes two equivalents of ATP, and produces the same molar amount of ADP and phosphate Pi. The latter is a potent λPP inhibitorp2-PDI. The poisoning due to accumulation of waste is a ubiquitous problem for chemically fuelled supramolecular systems in (semi)batch conditions2611t=2,160 min) against fresh buffer, choosing a membrane with a suitable molecular weight cutoff (MWCO=2 kD). Encouragingly, it was possible to get rid of the waste (ADP+Pi), and as a result the system returned back completely to the starting nonphosphorylate state PDI as confirmed by LC–MS and CD , as well as the two enzymes PKA and λPP, are compartmentalized inside the cassette, whereas ATP and the waste  can easily pass through the membrane  by constantly flowing 1 mM ATP  PDI solutions used in the experiments. The stock solutions were freshly prepared the same day of the experiment.The enzymatic reactions were performed, when not differently specified, in 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM MgClPDI solution , followed by the addition of PKA (1–3 μl of the supplied 13 μM solution) and gentle shaking. The equivalents of ATP reported in the paper are with respect to PDI molecules . The dephosphorylation was triggered by the addition of an aliquot of λPP (1–3 μl of the supplied 20 μM solution) to a p2-PDI solution, prepared as described above, in the absence of unreacted ATP. The enzymatic reactions were performed in screw cap sample vials equipped with a glass insert (when followed by LC–MS), or in a 1 mm quartz cuvette .In a typical phosphorylation experiment, an aliquot of freshly prepared ATP  was added to the PDI (or p2-PDI) solutions versus time (or temperature), from which we calculated the ratio of the two maxima at 504 and 540 nm (A504 nm and A540 nm). Note that the absorbances of the real maxima were considered to calculate A504 nm/A540 nm .Ultraviolet–visible spectroscopy was used to follow (in time) the changes in aggregation upon phosphorylation , as wellPDI and p2-PDI assemblies phosphorylation in both the stepwise  and batcsemblies . For thesemblies , the phoPDI, p-PDI and p2-PDI in the batch and steady state experiments reported in m/z ±2), from which their relative mole fraction were calculated.Liquid chromatography–mass spectrometry (electrospray ionization–ion trap) was used to both check qualitatively the (de)phosphorylation and to calculate the fraction of the three species PDI solution in the reaction buffer was split in two parts, and ATP  was added to both the resulting solutions. After that the p2-PDI sample was prepared by adding PKA to one of the two solutions. After complete phosphorylation (run overnight and checked by LC–MS) both samples were measured by DLS. The intensity cross-correlation functions n is the refractive index of the solution. The relaxation dynamics were analysed by the inverse Laplace transformation D was calculated with the slope of Γ/q2 obtained from experimental g(1). Hydrodynamic radii RH were then calculated using the Stokes–Einstein relation A 1 mM PDI solution containing PKA (0.085 μM) and λPP (0.10 μM) were injected in the dialysis cassette, and remaining air was carefully removed. After that, the 1 mM ATP solution was flowed through the chambers by using a pressure-driven pump (Elveflow). We started flowing ∼ 20 ml at a flow rate of ∼4 ml min−1, after that we set the flow rate at ∼ 150 μl min−1. At defined times, small samples (∼40 μl) of the reaction solution were taken from the cassette, analysed by LC–MS (5 μl injection) and the remaining part put back in the cassette. At each change of the ATP concentration, the flow chambers were washed with the new solution by flowing at least ∼25 ml at a flow rate of ∼5 ml min−1, before setting the flow at ∼150 μl min−1.The cassette holder in the continuous flow device .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a key enzyme for the metabolism of many toxic aldehydes such as acetaldehyde, derived from alcohol drinking, and 4HNE, an oxidative stress-derived lipid peroxidation aldehyde. Post-translational enhancement of ALDH2 activity can be achieved by serine/threonine phosphorylation by epsilon protein kinase C (εPKC). Elevated ALDH2 is beneficial in reducing injury following myocardial infarction, stroke and other oxidative stress and aldehyde toxicity-related diseases. We have previously identified three εPKC phosphorylation sites, threonine 185 (T185), serine 279 (S279) and threonine 412 (T412), on ALDH2. Here we further characterized the role and contribution of each phosphorylation site to the enhancement of enzymatic activity by εPKC.Each individual phosphorylation site was mutated to a negatively charged amino acid, glutamate, to mimic a phosphorylation, or to a non-phosphorylatable amino acid, alanine. ALDH2 enzyme activities and protection against 4HNE inactivation were measured in the presence or absence of εPKC phosphorylation in vitro. Coevolution of ALDH2 and its εPKC phosphorylation sites was delineated by multiple sequence alignments among a diverse range of species and within the ALDH multigene family.We identified S279 as a critical εPKC phosphorylation site in the activation of ALDH2. The critical catalytic site, cysteine 302 (C302) of ALDH2 is susceptible to adduct formation by reactive aldehyde, 4HNE, which readily renders the enzyme inactive. We show that phosphomimetic mutations of T185E, S279E and T412E confer protection of ALDH2 against 4HNE-induced inactivation, indicating that phosphorylation on these three sites by εPKC likely also protects the enzyme against reactive aldehydes. Finally, we demonstrate that the three ALDH2 phosphorylation sites co-evolved with εPKC over a wide range of species. Alignment of 18 human ALDH isozymes, indicates that T185 and S279 are unique ALDH2, εPKC specific phosphorylation sites, while T412 is found in other ALDH isozymes. We further identified three highly conserved serine/threonine residues  in all 18 ALDH isozymes that may play an important phosphorylation-mediated regulatory role in this important family of detoxifying enzymes.εPKC phosphorylation and its coevolution with ALDH2 play an important role in the regulation and protection of ALDH2 enzyme activity.The online version of this article (doi:10.1186/s12929-016-0312-x) contains supplementary material, which is available to authorized users. The mitochondrial aldehyde dehydrogenase 2, ALDH2, is known for its role in ethanol metabolism, mediating the rate-limiting step of metabolizing acetaldehyde to acetic acid . HoweverThe functional ALDH2 is a homotetramer . In humaThe α,β-unsaturated reactive 4HNE is well-known for its genotoxicity and cytotoxicity, causing DNA damage and proteins inactivation –18. 4HNEEnhancing the catalytic activity of ALDH2 and/or protecting ALDH2 enzyme activity from 4HNE-induced inactivation has recently emerged as new strategy for the development of therapeutics , 27. OurAnother way to enhance ALDH2 activity is by post-translational phosphorylation of the enzyme. We previously found that activation of epsilon protein kinase C (εPKC) at the mitochondria increases ALDH2 activity in the heart by ~40%, thus protecting the heart from ischemic injury , 32; phoUsing site-directed mutagenesis of the three possible εPKC phosphorylation sites, we set out to determine their role in enzyme activity, phosphorylation, folding, and resistance to 4HNE inactivation. We also explored the conservation of these sites with εPKC in evolution, as a means to demonstrate their importance in regulating ALDH2.+ to NADH at λ340 nm. All the assays were carried out in a 96-well plate in triplicates at 30 °C in 50 mM sodium pyrophosphate buffer, pH = 8.8, 2.5 mM NAD+ and 10 mM acetaldehyde as the substrate, as described [Enzymatic activity of ALDH2 was determined spectrophotometrically, using purified recombinant protein to measure the reductive reaction of NADescribed . ALDH2 aHuman recombinant ALDH2 wild type and ALDH2*2 mutant enzymes were expressed in bacteria as previously described . The PKC2 with 0.24 mg/ml phosphatidylserine , 0.04 mg/ml 1,3-s-n-dioleylglycerol  at 37 °C for 30 min as described in Chen et al. [For in vitro kinase reaction, recombinant εPKC  and each ALDH2 protein (8 μg) were incubated in the presence of 20 mM Tris–HCl pH 7.5, 200 μM ATP, 20 mM MgCln et al. .Sequences for members of the ALDH family and the ALDH2 protein from multiple species were found through the NCBI protein database  and ALDH sequences with the following GenBank Accession numbers: ALDH2 (GI: 48146099), ALDH1A1 (GI: 16306661), ALDH1A2 (GI: 119597936), ALDH1A3 (GI: 153266822), ALDH1B1 (GI: 119578656), ALDH1L1 (GI: 393195306), ALDH1L2 (GI:166198355), ALDH3A1 (GI: 206597441), ALDH3A2 (GI:73466520), ALDH3B1 (GI:125950429), ALDH3B2 (GI: 73695881), ALDH4A1 (GI: 23271000), ALDH5A1 (GI: 21708023), ALDH6A1 (GI: 119601566), ALDH7A1 (GI: 49117277), ALDH8A1 (GI: 88683005), ALDH9A1 (GI: 119611164), ALDH16A1 (GI: 223972651). For longer sequences of ALDH isozymes, both N- and C-terminal sequences were truncated and small sequence gaps were introduced to obtain the best fitted alignment against the published ALDH2 protein sequence.19 different, functional ALDH genes are known in the human genome . Since A+ [The common East Asian ALDH2*2 single point mutation (E487K), which is away from the catalytic site, causes a >95% loss of activity in ALDH2 due to structural changes that affect both the dimerization of the enzyme and binding of the cofactor, NAD+ . To dete+ , 35. ThrWe have reported previously that in vitro phosphorylation of wild type ALDH2 recombinant protein increases its enzymatic activity . We obseWe set out to determine which of the phosphorylation sites contributes to εPKC-mediated activation of ALDH2 enzymatic activity. Site-directed mutagenesis was carried out for each of the putative εPKC phosphorylation sites, Thr185, Ser279 and Thr412 on ALDH2. Since phosphomimetic of an amino acid is a good estimation for the function of phosphorylation, we first mutated the three phosphorylation sites individually to a charged amino acid residue, glutamate, to mimic the function of the negatively charged phosphate group . We founNext, we determined whether further activation of the enzymatic activity could be achieved by εPKC phosphorylation of each of the single phosphomimetic or non-phosphorylatable alanine substitution mutants. We reasoned that since further activation by εPKC phosphorylation on the specific phosphomimetic or non-phosphorylatable alanine substitution was no longer possible, such experiments will help to identify the true phosphorylation site(s) contributing the enhanced ALDH2 enzyme activity by εPKC. We found that five out of six possible amino acid substitutions, T185A/E and T412A/E and S279A mutants, were significantly activated by εPKC-mediated phosphorylation, resulting in an increase of 50–150% above their basal activity Fig. . The phoBecause 4HNE causes a fast inactivation of ALDH2 by adduct formation with the critical catalytic Cys302 , 36, we We also used multiple sequence alignments to determine whether the three ALDH2 phosphorylation sites were conserved among species and co-evolved with εPKC. We reasoned that if εPKC-mediated phosphorylation of ALDH2 is critical for the regulation of ALDH2 activity, the critical phosphorylation sites should co-evolve with εPKC. We aligned multiple sequences of ALDH2 from a wide range of eukaryotic species that express εPKC, and compared ALDH2 sequence conservation with species that do not express this protein kinase  to mimic phosphorylation . We founWe also aligned and compared amino acid sequences of all 19 identified and functional ALDH isozymes within the human genome and determined how the positions equivalent to T185, S279 and T412 are conserved among the human ALDH supergene family . This implies that the co-evolution relationship was uniquely maintained between ALDH2 and εPKC and these three phosphorylation sites may be preferentially regulated by εPKC. We also identified three other serine/threonine residues, T384, T433 and S471 that were extremely well conserved across all the ALDH gene family members. Based on the alignment of 16 known ALDH sequences, Sheikh et al., also identified T384 and S471 as critical conversed amino acids . T384 isMitochondrial ALDH2 is a key detoxifying enzyme guarding the integrity and health of this important organelle . As mostThe role of three serine/threonine phosphorylation sites by εPKC on ALDH2 were characterized. Site-directed mutagenesis and in vitro phosphorylation revealed that S279 was a critical εPKC phosphorylation site for the activation of ALDH2. Whereas, phosphorylation of T185, S279 and T412 conferred protection against reactive aldehyde, 4HNE, inactivation of ALDH2. Alignment across a wide range of diverse biological species and of 18 known human ALDH multigene family members showed that the three phosphorylation sites co-evolved tightly with species that expressed εPKC. Such alignment also identified both unique and conserved serine/threonine on ALDH2 and its isozymes. Our findings indicated that εPKC phosphorylation and its coevolution with ALDH2 played an important role in the regulation and protection of ALDH2 enzyme activity.