Pancreatic ductal adenocarcinoma (PDA) has a poor prognosis, in part, due to the therapy-recalcitrant nature of the disease. Loss of the CDK4/6 inhibitor CDKN2A is a signature genetic event in PDA. Therefore, PDA may be amenable to treatment with pharmaceutical CDK4/6 inhibitors. Surprisingly, response to CDK4/6 inhibition was highly variable in PDA models, and associated with differential suppression of gene expression. Mitotic genes were repressed and FOXM1 was uniformly attenuated; however, genes involved in DNA replication were uniquely suppressed in sensitive models. Aberrant induction of Cyclin E1 was associated with resistance, and knockdown demonstrated synergistic suppression of the cell cycle with CDK4/6 inhibition. Combination therapies are likely required for the effective treatment of disease, and drug screening demonstrated additive/antagonistic interactions with CDK4/6 inhibitors. Agents dependent on mitotic progression (taxanes/PLK1 inhibitors) were antagonized by CDK4/6 inhibition, while the response to 5-FU and gemcitabine exhibited drug specific interactions. PI3K/MTOR and MEK inhibitors potently cooperated with CDK4/6 inhibition. These agents were synergistic with CDK4/6 inhibition, blocked the aberrant upregulation of Cyclin E1, and yielded potent inhibition of tumor cell growth. Together, these data identify novel mechanisms of resistance to CDK4/6 inhibitions and provide a roadmap for combination therapies in the treatment of PDA. Pancreatic ductal adenocarcinoma (PDA) has a terrible prognosis with a 5-year survival of approximately 6% -3. The aThe CDKN2A gene encodes the p16ink4a protein that is a potent inhibitor of Cyclin Dependent Kinases 4 and 6 (CDK4/6). Physiologically, p16ink4a represents a key barrier to oncogenic transformation, as it is induced by oncogenic stress and leads to senescence in multiple disease relevant settings . In the While multiple CDK-inhibitory agents have been evaluated in clinical trials, only recently have highly specific CDK4/6 inhibitory drugs been developed , 30. ConHere we find that CDK4/6 inhibition can have a potent impact on PDA models. While some models exhibit a durable response, acquired/intrinsic resistance can bypass the action of CDK4/6 inhibition in the majority of models analyzed via a novel mechanism involving induction of Cyclin E1. Drug screening reveals a complex and mechanism specific impact of CDK4/6 inhibitors on drug-sensitivity. However, specific combination therapies clearly expand the therapeutic potency of CDK4/6 inhibition for the treatment of PDA.Eight pancreatic cancer cell lines were screened for their proliferative response to CDK4/6 inhibition. In addition to multiple PDA signature mutations  these cell lines carried a non-functional CDKN2A gene Fig. . In ordeTo interrogate the downstream signaling from CDK4/6 inhibition, we analyzed the expression of RB/E2F target genes using a high-density RBTARGET Affymetrix array . Using a 2-fold cutoff and p<0.05 we found that there were many RB/E2F targets genes that were repressed potently with CDK4/6 inhibition Fig. . SpecifiIn an attempt to decipher the mechanism underlying the specific resistance to CDK4/6 inhibition, we interrogated the expression of critical CDK and Cyclins that are important for progression through G1/S. These data demonstrated that certain genes are repressed irrespectively (e.g. Cyclin A2) Fig. . HoweverIt is widely recognized that combination approaches to therapy will be required for the success of targeted agents in the treatment of pancreatic cancer. Given the modest response to CDK4/6 inhibitors we interrogated the impact of PD-0332991 on 304 anti-cancer compounds . Drug screening was performed in PL45, PL5 and MIAPACA2 PDA models, which are relatively resistant to PD-0332991. Cells were treated singly with the drug library or with concurrent PD-0332991. Cell survival was determined by CTG analysis and representative data is depicted in the scatter plots Fig. . To globThe drug screening indicated that CDK4/6 inhibition has the potential to antagonize the effect of various types of chemotherapies. We speculated that the cytostatic effect of CDK4/6 inhibition may represent the principle mechanism leading to a decreased efficacy of drugs targeting the mitotic machinery. As shown, PLK1 inhibitors were antagonized by co-treatment with PD-0332991 Fig. . Since sIn addition to anti-mitotic drugs, PD-0332991 treatment significantly antagonized Gemcitabine activity in drug screening. Since the anti-metabolites Gemcitabine and 5-FU are both used in the treatment of PDA, we specifically evaluated response to these drugs. Initially, we simply recapitulated the antagonism of Gemcitabine-mediated toxicity Fig. . These sAs a whole, PI3K/MTOR inhibitors showed cooperative effects in concert with CDK4/6 inhibitor treatment assessed by viability assay in drug screening. In order to examine the mechanisms, BrdU incorporation was assessed after treatment with BEZ235, GDC0980 and AZD0855 in combination with CDK4/6 inhibitor Fig . ResultsIn addition to PI3K/MTOR, MEK inhibitors also had additive effects in concert with CDK4/6 suppression. We demonstrated the cooperation at the level of cell cycle progression by BrdU incorporation and found that similar suppression of proliferation with the combination treatment Fig. . The bioThe treatment of pancreatic cancer is a particular challenge and new therapeutic approaches are urgently needed. One appealing means of intervention is to target specific genetic features of disease, and CDKN2A loss is one of the most common genetic alterations in PDA. Ostensibly, the loss of the kinase inhibitor results in aberrant CDK4/6 activation that could be targeted through the use of CDK4/6 inhibitors. Here we challenged this simple precept in preclinical models.Interestingly, and contrary to multiple other tumor models, PDA models exhibited disparate responses to CDK4/6 inhibition that were not dependent on the canonical RB-pathways. In breast cancer, HCC, glioma, melanoma and other models, the response to CDK4/6 inhibition is simply dependent on the presence of RB , 33, 51.The realization that such uncoupling can occur necessitated a highly focused analysis of CDK genes/proteins that drive the G1/S transition. Our group and others have shown that CDK4/6 inhibitors can lead to the accumulation of Cyclin D1 levels , 33. TheIn recognition that many PDA models did not harbor a particularly durable response to CDK4/6 inhibition, combination treatments were interrogated. This work revealed that CDK4/6 inhibition has complex interactions on the response to multiple agents. In particular, and consistent with others , CDK4/6 In contrast with chemotherapy, we observed highly reproducible additive effects across a relatively large panel of MEK and PI3K/MTOR inhibitors with CDK4/6 inhibition. These findings are particularly important since MEK and PI3K/MTOR inhibitors have been considered for the treatment of pancreatic cancer . As showHuman pancreatic cancer cell lines PL45, MIAPACA-2, PANC1, CAPAN2, BXPC3, HS 766T, ASPC1, PL5 were purchased from the American Type Culture Collection (ATCC). All cells except for MIAPACA2 were cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics. MIAPACA2 cells were grown in DMEM with fetal bovine serum and horse serum to a final concentration of 10% and 2.5% respectively plus antibiotics. Drugs used in this study were purchased from Selleck Chemicals. Compounds were diluted in DMSO to 5 mM stock concentrations. PD-0332991 was employed at a concentration range of 0.5-2 μM. Drugs screen was performed at 2.5 μM. Antibodies against CDK2, CCNE1, CCNA2, GAPDH, total ERK, p-ERK, lamin B, DCK, TS and p27 were purchased from Santa Cruz biotechnology. While, antibodies against total RB, p-RB(780), total Akt, p-Akt (ser473) were purchased from Cell signaling. Cyclin D1 (AB3) was from NeoMarkers.2. The next day, cells were treated with the drug library to final concentration of 2.5 uM, and incubated at 37° and 5.0 CO2 for 72 hours. Following treatment, viability was assessed using Celltiter Glo(Promega). The relative fluorescence units were detected and analyzed by a BIOTEK plate reader and Gen5 software. Data analysis was performed using Drug-decode macro .A library of 304 drugs from Selleck Chemicals was used for drug screening. Cells were grown in 96-well plates at 2500 cell per well. Subsequent to plating, cell were treated with 1μM PD-0332991 or DMSO for 24 hours at 37° and 5.0 COCell were trypsinized and fixed in 70% Ethanol post a 2 hour incubation with Brdu labeling solution(Invitrogen). Fixed cells were incubated in 2M HCl with 0.3mg/ml pepsin solution for 30 minutes followed by addition of 0.1 M sodium tetraborate solution(pH 8.5). After acid neutralization, cells were washed twice with IFA(10mM HEPES(pH 7.4), 25 mM NACI, 4% FBS) buffer. Cells were then incubated with FITC labeled anti-Brdu(BD Pharmingen) at 1:10 dilution for 30 min at room temperature. After Brdu labeling, Propidium iodine(20ug/ml) and RNAse(4ug/ml) was added and incubated for 15 minutes in Dark. BrdU incorporation was assessed using a Facscalibur Instrument and data was analyzed using FlowJo.tm Protein Assay(BIO-RAD) using manufacture's protocol. These samples were separated on SDS polyacrylamide gels and electroblotted on to Immobilon-P Transfer Membrane. Membranes were washed with wash buffer . Followed by incubation with blocking solution , followed by incubation with rabbit primary antibodies (1/500 dilution) at 4°C overnight. The membranes were then washed in Wash buffer and incubated with horseradish peroxidase conjugated secondary antibodies (Jackson ImmunoResearch) for one hour at room temperature. Antibody detection was performed with an enhanced chemiluminescence reaction  as directed by manufacture.Pancreatic cell lines were lysed in RIPA buffer, 1.5M NaCI, 5% sodium doxycholate, 10mM EDTA) containing both complete protease inhibitor cocktail tablets (Roche) and phosphatases inhibitors (Roche). Protein concentrations were subsequently measured with the BCA protein assay DCProtein lysates were prepared from cell treated with vehicle or 1 μM PD-0332991 for 48 hours. Preparation of the lysates were as directed by the MD Anderson RPPA shared service, and the analysis of the signals were performed using their highly standardized procedures. The difference in normalized log protein values are presented in the analysis.4 cell/well in 6 well plate and left to adhere for 3 hours at 37 °C. Cells were then treated with individual drug or drug combinations at 1uM concentrations and incubated for 24-72 hours. For long-term experiment, cell were seeded at 5×103 cell/ well and drugs were replenished every 3 days for 10 days. Following long term studies, they were assessed by cystal violet.For short term treatments, pancreatic cell lines were seeded at 5×105 cells per well and transfected with siRNA against either CCND1 (Santa Cruz biotechnology), CCNE1  and PLK1 at a final concentration 50nmol/L with Lipofectamine RNAiMAX(Invitrogen) according to manufacture's instructions. Transfections with a control sRNA(TMEM) served as a negative control.Pancreatic cell lines were seeded in a 6-well plate at a desity of 5×10Cells were treated with vehicle or 1μM PD-0332991 for 48 hours (RBTARGET) or 120 hours (Agilent Array). The RBTARGET array is a custom Affymetrix Gene Chip that encompasses genes regulated by RB and highly correlated genes together with additional gene for normalization, full list of the probes on the RBTARGET platform are provided in the supplemental data. Total RNA was recovered and hybridized using standard procedures. Data analysis was performed on normalized data. To define differentially expressed genes, a cutoff of 2-fold and p<0.05 was utilized.


We then carried out combination studies with four everolimus resistant triple-negative breast cancer cell lines, and found an unexpectedly high degree of synergy between everolimus and the other inhibitors tested. The level of potentiation of everolimus inhibitory activity  was found to be cell line-specific for all the kinase inhibitors tested. The results suggest that judicious combination of mTOR inhibitors with different modes of action could have beneficial effects in the treatment of breast cancer.The mammalian target of rapamycin (mTOR), a vital component of signaling pathways involving PI3K/AKT, is an attractive therapeutic target in breast cancer. Everolimus, an allosteric mTOR inhibitor that inhibits the mTOR functional complex mTORC1, is approved for treatment of estrogen receptor positive (ER+) breast cancer. Other mTOR inhibitors show interesting differences in target specificities: BEZ235 and GSK2126458 are ATP competitive mTOR inhibitors targeting both PI3K and mTORC1/2; AZD8055, AZD2014 and KU-0063794 are ATP competitive mTOR inhibitors targeting both mTORC1 and mTORC2; and GDC-0941 is a pan-PI3K inhibitor. We have addressed the question of whether mTOR inhibitors may be more effective in combination than singly in inhibiting the proliferation of breast cancer cells. We selected a panel of 30 human breast cancer cell lines that included ER and PR positive, HER2 over-expressing, and “triple negative” variants, and determined whether signaling pathway utilization was related to drug-induced inhibition of proliferation. A significant correlation ( PIK3CA deregulate the PI3K/AKT/mTOR pathway and are frequent in breast cancer [PIK3CA mutations show higher response rates to PI3K/AKT/mTOR inhibitors than do patients whose tumours lack PIK3CA mutations [mTOR  is a component of two distinct cell signaling complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), each of which plays an essential role in the control of cell proliferation. Activating mutations in A number of inhibitors of the PI3K/AKT/mTOR pathway have now been identified. Everolimus, an allosteric mTORC1-specific inhibitor, has been used clinically to treat ER+ breast cancer ,5. KU-00We have chosen everolimus to address the question of whether anti-proliferative activity is related to inhibition of the mTOR signaling pathway, as measured by p70S6K phosphorylation. We have utilized 30 human breast cancer cell lines, including those that could be classified as ER and PR positive, HER2 over-expressing, or “triple negative”. We have also investigated whether the dual PI3K/mTOR inhibitors BEZ235 and GSK2126458, or the dual mTORC1/mTORC2 inhibitor (AZD8055), can sensitize everolimus resistant breast cancer cell lines to everolimus by reducing AKT phosphorylation. We selected four everolimus resistant triple-negative breast cancer cell lines  to answer the question of whether ATP competitive mTORC1/2 inhibitors synergize with everolimus in their effects on cell proliferation. We have tested for possible interactions between everolimus and both dual PI3K/mTOR inhibitors and the pan-PI3K inhibitor GDC-0941 . These eCulture conditions have been described in detail previously ,20; MCF-Tamoxifen was purchased from Sigma . Everolimus, AZD8055 and AZD2014, KU-0063794 and GDC-0941 were purchased from Selleck Chemicals . BEZ235 ,25 and G3H-thymidine was added to each well and incubated for 6 h; the cells were harvested on glass fiber filters using an automated TomTec harvester. Filters were incubated with Betaplate Scint and thymidine incorporation measured in a Trilux/Betaplate counter. Effects of inhibitors were determined relative to the incorporation of 3H-thymidine into DNA of control (non-drug-treated) cells.As described in detail previously , prolifeCells were seeded as described in cell proliferation assay in the presence of varying concentration of inhibitors for 3 days. AlamarBlue (Life Technologies) cell viability reagent was used to assess cell viability by adding 10% of the sample volume into breast cancer cells in culture media, followed by a 3 hours incubation at 37°C. The resulting fluorescence was assessed on a fluorescence plate reader with excitation at 570 nm and fluorescence emission was read at 585 nm.As described in detail previously , breast 6 cells) were grown in 3.5 cm Petri dishes and incubated with inhibitors for 24 h. They were harvested, washed with 1% FCS/PBS, resuspended in 200 μl of PBS, fixed in 2 ml of ice-cold 100% ethanol and stored overnight at −20°C. The cells were washed and resuspended in 1 ml of 3% FCS/PBS containing RNase (1 μg/ml) and propidium iodide (PI) (10 μg/ml) for 30 min at room temperature. DNA content was determined using forward scatter (FSC) intensity by PI staining based on a total 30,000 acquired events by FACScan cytometry.As described in detail previously , cells  in the experimentally measured fractional inhibition (Expt) and Ebliss are defined as synergism (Expt < Ebliss), additivity (Expt = Ebliss), and antagonism (Expt > Ebliss). Bliss = 0 would indicate the combination is additive; Bliss > 0 would indicate the percentage increase in maximal inhibition above additivity (synergy); and Bliss < 0 would indicate the percentage decrease in maximal inhibition below additivity (antagonism).The Bliss additivism model  was usedR) and statistical significance (P) using SigmaPlot. Values of P<0.05 were considered to be statistically significant.As described in detail previously , data we50 values) using a 3-day 3H-thymidine incorporation assay (p = 0.005) was found between everolimus IC50 values and p70S6K phosphorylation, but not between everolimus IC50 values and AKT or ERK phosphorylation. Estrogen receptor positive breast cancer cell lines showed significantly higher sensitivity to everolimus than did receptor negative lines (p = 0.034). However, no significant correlation was observed between PIK3CA mutation status and either sensitivity to everolimus (p > 0.05) or degree of p70S6K phosphorylation (p > 0.05).A series of breast cancer cell lines was assayed for sensitivity to everolimus  showed relative resistance. However, no significant correlation was observed between the degree of p70S6K phosphorylation, AKT phosphorylation, ERK phosphorylation and everolimus sensitivity  MCF-7 tamoxifen-resistant sub-lines ,20,23,27sitivity .3H-thymidine incorporation assay  . The Blis tested .3-thymidine incorporation  (We next assessed drug effects using Alamar Blue (to assess cell viability) and compared them with the results of the thymidine incorporation assay (which measures DNA synthesis) . Viabilix 10−18) .In a third method to investigate the effects of the inhibitors, MDA-MB-231 cells were treated with everolimus, BEZ235, or the combination and analyzed by flow cytometry. A time dependent decrease in the proportion of S-phase cells was observed in the treatment groups as compared to the control using , consistThe synergism in growth inhibition observed with the above drug combinations indicated that targeting mTOR alone is sufficient for sensitizing the cell lines to everolimus. We therefore applied the same strategy to test the effects of two other mTOR specific inhibitors (KU-0063794 ) and onep < 0.01), HCC1143 (p = 0.01) and MDA-MB-231 (p = 0.023) using multiple comparisons and MDA-MB-436 as the control group.Although synergy was observed in all cell lines tested, the level of synergy of drug combination  ; Fig 9 w50 values, varied over a range of more than 570-fold. Sensitivity did not seem to be related to PIK3CA mutational status or ER status  (p = 0.005), but not with the phosphorylation of AKT or ERK, consistent with the mTOR pathway being a principal target of action of this drug.In a broad series of breast cancer cell lines, sensitivity to the mTOR allosteric inhibitor everolimus, as measured by ICR status , in agret cancer . Triple-50 values 2.2, 2.8, and 3.1 nM, respectively, suggesting high ER expression is not required for everolimus response. Other mechanisms may explain the clinical observation that inhibition of ER can sensitize breast cancer cells to everolimus [The ER+ MCF-7 line showed high sensitivity to the mTOR inhibitor everolimus , possiblerolimus .The main aim of our study was to examine growth inhibitory effects of combinations of inhibitors. We found that inhibitors targeting the same PI3K/mTOR pathway sensitize everolimus resistant breast cancer cell lines to growth inhibition , regardlin vitro and in vivo despite these important signaling differences [The signaling response of concurrent targeting of both PI3K and mTOR pathways, as measured by phosphorylation of AKT and p70S6K respectively, did not predict growth inhibitory effects in MDA-MB-231, MDA-MB-436, BT20 and HCC1143 breast cancer cell lines. Everolimus alone efficiently inhibited the p70S6K pathway downstream of mTORC1, and activated AKT phosphorylation only in the HCC1143 cell line. Due to a negative feedback loop , inhibitferences . Here, aferences .Since the mTOR pathway regulates protein translation  and mTORIn this study, MDA-MB-436 showed the least utilization of the mTOR signaling pathway , and also showed least synergism (in terms of growth inhibition) when treated with drug combinations Figs  and 9. CWe also examined the growth inhibitory effect of a pan-PI3K inhibitor GDC-0941), in comparison with inhibitors of mTOR specific kinase (AZD2014 and KU-0063794) to determine whether the observed synergistic effects are PI3K independent. Synergy was previously observed with the mTOR selective inhibitor KU-006394 , in comp. They cain vitro. Our results provide the rationale to design in vivo studies in the future, which may represent a novel strategy to enhance the efficacy of mTOR-targeted breast cancer therapy. Measurement of p70S6K phosphorylation can potentially identify human breast cancers that would benefit from therapy with the mTOR allosteric inhibitor everolimus. The suitability of such candidate biomarkers will need to be confirmed in future clinical trials.It was suggested that reducing the degree of mTORC1 signaling (as assessed by the phosphorylation of substrates) would decrease the drug concentration required to fully inhibit mTORC1 activity . While iS1 FigImmunoblots with antibodies specific for phosphorylated ERK proteins and total ERK protein are indicated. Actin is the loading control.(EPS)Click here for additional data file.S2 FigCells were treated with drugs for 24 h and signaling pathway usage was measured by protein phosphorylation of AKT, p70S6K, rpS6, 4E-BP1 and ERK in the four cell lines. Immunoblots with antibodies specific for phosphorylated proteins and total proteins are indicated. Actin is the loading control.(EPS)Click here for additional data file.S3 FigCells were treated with drugs for 24 h and signaling pathway usage were measured by protein phosphorylation of AKT, p70S6K, rpS6, 4E-BP1 and ERK in the four cell lines. Immunoblots with antibodies specific for phosphorylated proteins and total proteins are indicated. Actin is the loading control.(EPS)Click here for additional data file.


Background: AMP-activated protein kinase (AMPK) is a current drug target. AMPK can attach to glycogen granules.Results: Autophosphorylation of AMPK prevents its association with glycogen.Conclusion: Subcellular localization of AMPK is affected by the kinase autophosphorylation status.Significance: Understanding the regulation of AMPK at subcellular level is crucial for the currently pursued drug targeting approach. The mammalian AMP-activated protein kinase (AMPK) is an obligatory αβγ heterotrimeric complex carrying a carbohydrate-binding module (CBM) in the β-subunit (AMPKβ) capable of attaching AMPK to glycogen. Nonetheless, AMPK localizes at many different cellular compartments, implying the existence of mechanisms that prevent AMPK from glycogen binding. Cell-free carbohydrate binding assays revealed that AMPK autophosphorylation abolished its carbohydrate-binding capacity. X-ray structural data of the CBM displays the central positioning of threonine 148 within the binding pocket. Substitution of Thr-148 for a phospho-mimicking aspartate (T148D) prevents AMPK from binding to carbohydrate. Overexpression of isolated CBM or β1-containing AMPK in cellular models revealed that wild type (WT) localizes to glycogen particles, whereas T148D shows a diffuse pattern. Pharmacological AMPK activation and glycogen degradation by glucose deprivation but not forskolin enhanced cellular Thr-148 phosphorylation. Cellular glycogen content was higher if pharmacological AMPK activation was combined with overexpression of T148D mutant relative to WT AMPK. In summary, these data show that glycogen-binding capacity of AMPKβ is regulated by Thr-148 autophosphorylation with likely implications in the regulation of glycogen turnover. The findings further raise the possibility of regulated carbohydrate-binding function in a wider variety of CBM-containing proteins. Many enzymes involved in the synthesis and degradation of complex carbohydrate molecules, such as cellulose or glycogen, contain a domain called the carbohydrate-binding module (CBM)AMPK is a heterotrimer consisting of (i) a catalytic subunit α (α1 or α2) carrying the kinase domain, (ii) a regulatory subunit β (β1 or β2) with the CBM and the C-terminal region tethering α and γ together, and (iii) a regulatory subunit γ  responsible for the adenine nucleotide sensing , 10. AMP11–Considering the micromolar affinity of recombinant AMPK complexes and CBM for small cyclic and linear oligosaccharides , 16, theBacterial expression plasmids encoding for hexahistidine-tagged wild type (WT) AMPK  , 25, nonTo obtain activated AMPK phosphorylated at Thr-172, each of the tricistronic WT and kinase-deficient coding sequences were combined with the LKB1-MO25-STRADα coding sequence in a single hexacistronic plasmid, allowing for co-expression of both protein complexes and the subsequent purification of the hexahistidine-tagged AMPK alone  as also described recently .For expression in mammalian cells, the cDNA of WT β1-CBM (amino acids 68–163) or β2-CBM (amino acids 67–163) was amplified by PCR and ligated in frame into the pAcGFP expression vector (Clontech) via the EcoRI and SalI restriction sites, resulting in expression constructs for β1-CBM-WT-GFP or β2-CBM-WT-GFP, respectively. The cDNA of full-length β1 was amplified by PCR and subcloned either into the pmCherry expression vector (Clontech) via EcoRI and SalI restriction sites or into the pCMV5–3xHA expression vector  via HindFor retroviral overexpression, GFP-tagged β1-CBM or β2-CBM (either WT or T148D) was subcloned, using an oligonucleotide linker, into the EcoRI and SalI restriction sites of the pBMZ-ires-neo retroviral backbone . Primer sequences are available upon request. All of the constructs were verified by sequencing.Escherichia coli cells (Novagen). Bacteria were grown in autoinduction medium, and proteins were purified as described with few modifications  and lysed using a high pressure homogenizer. After centrifugation, the supernatant was incubated for 1 h at 4 °C on a roller bank in the presence of either 1 ml of nickel-Sepharose HP  for the hexahistidine-tagged proteins or 1 ml of glutathione-Sepharose 4B  for the GST-tagged proteins. After centrifugation, the resin was washed three times with wash buffer , and then the protein was eluted in elution buffer , frozen in liquid nitrogen, and kept at −80 °C until use.Proteins were expressed in Rosetta 2 (DE3) ications . Briefly2+-affinity-purified AMPK was rebuffered into assay buffer  using PD-10 columns , and 500 μl (25 μg/ml) of this AMPK solution (I fraction) was incubated with 50 μl of pre-equilibrated β-cyclodextrin resin in a 0.7-ml reaction tube  with gentle agitation for 15–20 min. The supernatant containing the non-bound material (S fraction) was removed after centrifugation  and the resin was washed twice in assay buffer. The wash fractions were discarded. Bound AMPK was eluted by incubation in 500 μl of elution buffer (5 mm β-cyclodextrin in assay buffer) for 5 min with gentle agitation. After centrifugation the supernatant (P fraction) was collected, the resin was washed twice with elution buffer, and supernatants were discarded. Finally, the resin was incubated with SDS-sample buffer at 95 °C to obtain the precipitated fraction (L fraction). For analyses, 5 μl of each fraction were subjected to SDS-PAGE and Western blotting and further processed with AMPKα and phospho-AMPK (Thr(P)-172) antibodies .β-Cyclodextrin was immobilized on epoxy-activated Sepharose 6B  according to the manufacturer's protocol. Briefly, Niin vitro by upstream kinases as described previously (AMPK was activated eviously . Brieflym) (Gibco), supplemented with 10% (v/v) heat-inactivated fetal calf serum  and penicillin/streptomycin (Invitrogen), unless otherwise stated. HL-1 cardiomyocyte cell line was kindly provided by Dr. W. Claycomb , cultured on fibronectin /gelatin  coating in Claycomb medium , 0.1 mmol/liter noradrenaline, 2 mmol/liter l-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin) at 37 °C and 5% CO2.The human hepatocyte HepG2 and human embryonic kidney 293T cell line (HEK293T) were cultured in DMEM with high glucose  24 h before transfection. Cells were co-transfected with plasmid DNA  using Lipofectamine 2000 (Invitrogen) in antibiotic-free culture medium. Six to eight hours after transfection, transfection medium was replaced by normal growth medium. At 24–48 h after transfection, cells were either harvested or fixed.m glucose and 10% iFCS) or treated with forskolin  in the same medium or glucose-deprived  for 16 h.For the glycogen depletion experiments, cells were either maintained in high glucose medium (DMEM with 25 mm glucose) for 16 h and subsequently treated with 5-aminoimidazole-4-carboxamide riboside , oligomycin , A769662 (100 μm), phenformin (1.5 mm), vehicle (DMSO), or high glucose medium for 45–60 min. Insulin  treatment for 15 min was done in order to stimulate glycogen synthesis.In order to activate cellular AMPK, cells were serum-starved , supplemented with protease and phosphatase inhibitor mixtures (Roche Applied Science). Endogenous AMPK was immunoprecipitated using a combination of AMPKα1 and AMPKα2 antibodies raised in sheep (kindly provided by G. Hardie). Myc-tagged AMPK was immunoprecipitated using a Myc tag antibody . The primary antibody was incubated top-over-top with 350 μg of protein lysate at 4 °C for 16 h, followed by incubation with protein G-Sepharose beads for 3–4 h at 4 °C. The immune complexes were then collected by centrifugation. The immunoprecipitated proteins were electrophoresed by SDS-PAGE and analyzed by Western blot analysis. Immunoblot analysis was carried out with the following primary antibodies: Myc tag, AMPKα, phospho-AMPK-Thr-172, Akt, phospho-Akt-Ser-473, and GS . GS-Ser(P)-7 antibodies were a kind gift from G. Hardie. In order to detect changes in Thr-148 phosphorylation, a phospho-specific AMPKβ-Thr-148 antibody was produced (peptide sequence AMPKβ2(142–154) Thr(P)-148, VTSQLGINNLI)  assay kit (Sigma), according to the manufacturer's instructions.Extraction of glycogen from 293T cells was adapted from a method described by McMahon and Frost . BrieflyRetroviral systems and Phoenix helper-free retrovirus producer cell lines were used as described –34. Amph–3 (0.03%) or washed three times with PBS+/+ (Gibco) and directly used for immunocytochemistry. Subsequently, cells were permeabilized (0.1% Triton X-100 and 0.2% BSA in PBS) for 15 min and blocked (2% BSA-PBS) for 30 min at room temperature. Primary and secondary antibodies (2% BSA-PBS) were incubated for 1 h at room temperature. Coverslips were washed and mounted onto glass slides using DABCO-glycerol medium (Sigma-Aldrich) containing DAPI  in order to counterstain nuclei. The anti-glycogen antibody (1:500) was courtesy of Dr. O. Baba , and the secondary antibody was goat anti-mouse IgM Alexa647  or goat anti-mouse IgM Alexa488 .Cells were grown in 12-well plates (Greiner Bio-One) on coverslips  to 60–80% confluence. Cells were washed twice with PBS and fixed with 4% formaldehyde in PBS for 10 min at room temperature. Fixed cells were stored at 4 °C in PBS-NaNFixed cells were imaged using a Leica TCS SPE confocal laser scanning microscope (Leica Microsystems GmbH) equipped with an air-cooled argon-krypton mixed gas laser, using oil immersion objectives . Optical sections were recorded with three scans for each image. ImageJ software was used to process and analyze the images. Image brightness and contrast was adjusted to the same settings where needed.t test and statistical analysis software Prism version 4 . A p value of <0.05 was considered statistically significant.All bar graph data are presented as means ± S.E. Statistical analysis was performed by using Student's A, immobilized β-cyclodextrin retains the AMPK complex in the column and could therefore be used to affinity-purify recombinant AMPK. On the contrary, when no β-cyclodextrin was immobilized to the Sepharose, the overexpressed AMPK was not retained, confirming that AMPK retention is dependent on β-cyclodextrin. In further experiments, this procedure was simplified by allowing the recombinant AMPK to bind to the β-cyclodextrin resin in a batch set-up  instead of using a column. Fractions of the protein that could not bind to the resin stayed in the supernatant (S), whereas fractions that bound to the resin were eluted from this initial pellet fraction (P) by inclusion of soluble β-cyclodextrin in the buffer. The remaining resin-bound proteins were solubilized by extraction with Laemmli buffer (L) and considered as nonspecific precipitates. All fractions were collected and analyzed by SDS-PAGE followed by Coomassie Brilliant Blue stain or Western blotting. This assay was performed both with the isolated CBM  and a kinase-dead (KD) mutant of AMPK  . As expected, both CBM isoforms as well as both catalytically inactive AMPK complexes were able to bind to the resin and were consequently found in the pellet fraction.To verify the carbohydrate-binding ability of AMPK, β-cyclodextrin was coupled to Sepharose beads, and recombinant AMPK (α1β1γ1) was expressed in bacteria, purified, and loaded onto the packed column. As shown in Fig. 1D). AMPKα is the only protein of the plasmid constructs carrying a His tag that resulted in purified AMPK complexes when using the His tag purification protocol. Heterotrimeric α1β1γ1 and α1β2γ1 AMPK complexes, as well as the AMPK α1β1γ1 complex lacking the N-terminal CBM (α1β1-ΔCBMγ1), were expressed and purified in their inactive and active state . As shown in Fig. 1F, the α1β1-ΔCBMγ1 complex was expectedly found in the supernatant fraction and therefore did not retain the ability to bind to carbohydrates . As seen before with the KD mutants, the inactive forms of the AMPK complexes containing full-length β1 or β2 normally bound to β-cyclodextrin because these were found in the pellet fraction. In contrast, the active AMPK isoforms showed a loss of binding affinity to the β-cyclodextrin resin similar to the truncated AMPK complex . These findings suggest that activation of AMPK prevents its later binding to carbohydrates, such as β-cyclodextrin.To obtain Thr-172-phosphorylated active AMPK, the tricistronic plasmid for AMPK expression , 25 was Fig. 1A). Thus, phosphorylation of AMPK at Thr-172 by an upstream kinase is insufficient to trigger the loss of carbohydrate binding, suggesting that AMPK enzyme activity is required.To test whether phosphorylation of Thr-172 in AMPKα was sufficient to cause the loss of carbohydrate-binding, a KD mutant of AMPK α1β1γ1 was bacterially expressed in the presence or absence of the LKB1-Mo25α-Stradα complex and subsequently purified. In contrast to WT AMPK, both Thr-172-phosphorylated and non-phosphorylated KD complexes were found in the pellet fraction, indicating that catalytically inactive AMPK did not lose its ability to bind to the β-cyclodextrin when co-expressed with the LKB1-Mo25α-Stradα complex A. Thus, i.e. without co-expression of the LKB1-Mo25-Stradα complex) for subsequent activation in vitro before or during the β-cyclodextrin binding assay. Bacterial expression of recombinant CaMKK2 yields higher purity than LKB1 complex and was therefore used as an alternative upstream kinase of AMPK for these assays. As shown by the phosphorylation of Thr-172, all AMPK isoforms were activated and, as expected, mostly lost their binding ability to the resin material (B), indicating that activation of AMPK leads to loss of its carbohydrate-binding ability irrespective of the identity of the upstream kinase used, CaMKK2 or LKB1. However, when inactive AMPK was initially allowed to bind to the β-cyclodextrin resin and was subsequently incubated with CaMKK2, both AMPK isoforms retained their ability to bind to the resin because the proteins were predominantly found in the pellet fraction, despite phosphorylation at Thr-172 (C). The β2-isoform showed stronger retention upon activation compared with the β1 complex, which corresponds to a higher binding affinity of the β2-CBM for β-cyclodextrin (D–F). The data, together with the retention of carbohydrate binding for the phosphorylated KD mutant, underline that phosphorylation of AMPK at Thr-172 is compatible with its binding capacity to carbohydrates. Further, the binding of AMPK to β-cyclodextrin prior to activation is protective against the loss of binding ability.To confirm this, AMPK α1β1γ1, α1β2γ1, and α1β1-ΔCBMγ1 were expressed as inactive kinases . However, in the presence of ATP, both AMPK isoforms were found in the supernatant fraction, indicating their loss of binding ability to the β-cyclodextrin resin despite the lack of Thr-172 phosphorylation by upstream kinases. The presence of the small fraction of active AMPK was confirmed by prolonged exposure . These results show that a catalytic amount of enzymatically active AMPK is sufficient to prevent inactive AMPK from binding to β-cyclodextrin in the presence of ATP, suggesting that a bimolecular autophosphorylation event precludes AMPK from β-cyclodextrin binding.We speculated that autophosphorylation could be responsible for the loss of binding to carbohydrates, because autophosphorylation is a consequence of the initial activation of kinases. Moreover, autophosphorylation at multiple sites has been demonstrated for AMPK upon activation , 36. ExpFig. 2A). Using point mutagenesis, this site was mutated to an alanine (phosphorylation-resistant mutant T148A) in both inactive and active α1β1γ1 complexes. In the β-cyclodextrin binding assay, the inactive mutant conserved its binding capacity (B). However, in contrast to the active WT (F), the active T148A mutant partially conserved its carbohydrate-binding capacity upon co-expression of AMPK with the LKB1-Mo25-Stradα complex (B), implying a protective role of the T148A mutation.Considering that the binding of β-cyclodextrin prior to AMPK activation is protective from loss of binding, we speculated that the autophosphorylation event takes place directly in the carbohydrate-binding pocket of the CBM. Inspection of the x-ray structure of the β1-CBM identified Thr-148 as centrally located in the carbohydrate binding pocket A. Using capacity B. Howevective WT F, the ac complex B, implyiC). In addition, a phosphorylation site-specific Thr-148 (Thr(P)-148) antibody was developed, allowing for detection of the modification in AMPK by Western blotting. The recombinant activated WT AMPK α1β1γ1 and α1β2γ1 complexes showed a Thr-148 phospho-specific signal, whereas neither the inactive counterparts nor inactive/active α1β1ΔCBMγ1 showed detectable signals (D). Moreover, the Thr-172-phosphorylated KD and T148A mutants of AMPK α1β1γ1 did not show a Thr(P)-148 signal either (E), thus indicating the specificity of the Thr(P)-148 antibody. Therefore, the Thr-148 site is indeed autophosphorylated, and this modification directly correlates with the inability of AMPK to bind to the β-cyclodextrin resin. Taken together, these results indicate that autophosphorylation of AMPK at Thr-148 mediates its loss of carbohydrate-binding capacity.To further establish the involvement of Thr-148 phosphorylation in the carbohydrate-binding ability of AMPK α1β1γ1, a phosphorylation-mimicking T148D mutant was incubated with the β-cyclodextrin resin. Although not being activated, the T148D mutant was unable to bind to the β-cyclodextrin resin C. In add signals D. Moreovl either E, thus iA) and β2-CBM in HL-1 (B), both co-localizing with glycogen. In contrast, the β1- and β2-CBM T148D presented a diffuse pattern despite the presence of glycogen, as shown by a glycogen-specific antibody. Similar results were obtained using the HEK293T cell line stably expressing the β1-CBM (C); the speckled pattern of the WT CBM co-localized with glycogen, whereas the diffuse pattern of the mutant was incongruent with the observed glycogen staining. We further assessed the glycogen localization of mCherry-tagged full-length AMPKβ1-WT and T148D mutant after transient transfection of AMPK subunits in HEK293T cells. Results shown in Fig. 4D again revealed co-localization with glycogen for the WT, whereas the T148D mutant was diffusely located in the cytosol. Altogether, these findings indicate that the CBM of AMPK, expressed as an isolated domain or as a full-length protein forming part of the AMPK heterotrimer, naturally binds to glycogen, whereas the phospho-mimicking T148D mutation prevents this binding from occurring.Next, we investigated whether the Thr-148 phospho-mimicking mutation (T148D) triggers a loss of binding of AMPK to glycogen, its natural ligand, both in the β1- and β2-isoforms. Our initial analysis concentrated on the isolated WT and T148D β-CBMs. Stable cell lines were established that expressed GFP-tagged CBM variants in the human hepatic cell line HepG2 and cultured mouse HL-1 cardiomyocytes. Immunofluorescence showed a marked speckled pattern of the WT GFP-tagged constructs, β1-CBM in HepG2 A and β2- in HL-1 B, both ce β1-CBM C; the spA and B). Importantly, phosphorylation of Thr-148 could be detected in both cell lines, although under different conditions. In HEK293T, only the treatment with AICAR plus insulin led to the detection of the Thr-148 phosphorylation (A), and this treatment also showed the highest level of Thr-172 phosphorylation. In HepG2, however, Thr-148 phosphorylation occurred independently of AMPK activation changes (B). Hence, the data verify Thr-148 phosphorylation as an endogenous posttranslational modification. In addition, the differences of Thr-148 phosphorylation between cell types and conditions underline the control and dynamics of this post-translational modification.In order to investigate the occurrence of Thr-148 phosphorylation in cells and its detection with the Thr(P)-148 antibody, we immunoprecipitated endogenous AMPK from treated and untreated HEK293T or HepG2 cells , A and Brylation A, and th changes B. Hence,A) and compared β1-WT- with β1-T148D mutant AMPK-expressing cells (B). HEK293T cells were either triple-transfected (as before) with AMPK γ1, Myc-tagged AMPKα1, and mCherry-tagged AMPKβ1 (WT or T148D) or left untransfected and then treated for 16 h with a high glucose medium with or without forskolin, a drug that promotes glycogen degradation (B). In all of the three culturing conditions, the glycogen content was similar in untransfected, β1-WT, and β1-T148D mutant-expressing cells. Notably, Thr-148 phosphorylation was specifically induced by glucose deprivation but not forskolin, although both treatments depleted from glycogen (A). The Thr(P)-148 signal was absent in immunoprecipitates of T148D cells, reassuring that the antibody specifically recognizes the phosphorylation modification on the Thr-148 residue. Given the fact that increased AMPK activation by glucose deprivation is correlated with increased phosphorylation of Thr-148, these data support the cellular AMPK-mediated Thr-148 autophosphorylation mechanism.To investigate the signals leading to AMPK Thr-148 autophosphorylation, we tested whether glycogen depletion induced AMPKβ-Thr-148 phosphorylation A and comng cells B. HEK293radation , or withradation B. In allglycogen A. The ThA). Further, the Thr(P)172 levels were paralleled by similar increases in Thr-148 phosphorylation with the Thr(P)-148 signal virtually absent in high glucose-treated cells and highest upon phenformin treatment. Cellular glycogen content generally decreased upon AMPK activation in accordance with the expected AMPK-induced shift toward catabolism (C). Statistically significant reductions were observed between vehicle and AICAR or oligomycin-treated WT-transfected cells. Interestingly, in A769662 or AICAR-treated cells, glycogen content was significantly higher in T148D versus WT-transfected cells, which may relate to AMPKβ1-T148D being absent from glycogen and thus unable to inhibit glycogen synthesis. Indeed, the AMPK-dependent inhibition of glycogen synthase (GS) by phosphorylation at serine 7 (pS7) was strongly increased in WT-transfected cells upon oligomycin and A769662, whereas the respective Ser(P)-7 signals were fainter in the T148D-transfected cells (B). Moreover, the glycogen content of WT-transfected cells treated with phenformin showed a difference neither with the vehicle-treated WT cells nor with the phenformin-treated T148D cells (C). Interestingly, this absence of difference correlated with the highest Thr-148 phosphorylation (A), suggesting that the autophosphorylation was sufficient to mimic the effect of the mutation. Taken together, Thr-148 is phosphorylated upon AMPK activation and thus prevents AMPK from binding to glycogen, which affects glycogen metabolism.To further explore the physiological significance of Thr-148 phosphorylation, we treated cells with different AMPK activators and measured the resulting glycogen content. To allow for comparison of β1-WT with β1-T148D mutant AMPK, we again employed the HEK293T triple transfection model. As expected, in serum-starved cells, the treatment with oligomycin, A769662, and phenformin resulted in activation of AMPK in β1-WT as well as in β1-T148D cells, as observed by an increase in phosphorylation of Thr-172 A. Furthetabolism C. Statised cells B. Moreov8D cells C. Intererylation A, suggesIn this study, we investigated the molecular mechanism causing AMPK to refrain from binding to glycogen, thereby affecting glycogen turnover. Our results demonstrate that bimolecular AMPK autophosphorylation at β-subunit Thr-148 interferes with its carbohydrate binding capacity. The Thr-148 site is located within the center of the carbohydrate-binding pocket, thereby controlling the attachment to glycogen. Accordingly, autophosphorylation at Thr-148 is prevented by carbohydrate occupancy. We also provide evidence that WT isolated CBMs and full-length β1-subunit as part of the AMPK heterotrimer localize to glycogen particles, whereas the respective T148D mutants do not bind cellular glycogen. In addition, we show that activation of AMPK enhances cellular Thr-148 phosphorylation and provide first evidence for involvement of β1Thr-148 phosphorylation in preventing AMPK from glycogen metabolism regulation.It is established that many protein kinases catalyze their own activation by autophosphorylation . AutophoOur investigation revealed that little amounts of active AMPK were sufficient to prevent catalytically inactive AMPK from binding to glycogen, suggesting that a bimolecular autophosphorylation event is involved in this process. Therefore, it seems possible that few activated AMPK molecules are able to prevent glycogen binding of the entire pool of AMPK molecules, which would explain why the correlation of Thr-172 and Thr-148 phosphorylation was not always seen in cells. However, we can expect that the cell will have preventive measures, such as dephosphorylation of Thr-148, in order to restore glycogen binding. The phosphatase responsible for dephosphorylation therefore awaits identification.E). Therefore, based on our data, we cannot draw conclusions on isoform-specific differences of cellular Thr-148 phosphorylation.Although some proteins can indirectly associate with glycogen via interacting with other glycogen-binding proteins , most prFig. 3B). This observation could be related to the fact that insulin promotes glycogen synthesis  could be interpreted as an augmented rate of glycogenesis due to loss of AMPK glycogen localization and thus exclusion from glycogen synthesis inhibition. In any case, the unraveling of a new mechanism allowing for AMPK to stay unbound in the cytosol has consequences for our understanding of the kinase function, especially in the myocellular cell types, where glycogen is a major fuel resource (The precise roles of AMPK associated with glycogen are still subject to debate. On the one hand, AMPK stimulates GLUT4-mediated myocellular glucose uptake , 50 and et al.  delimite (et al.  showed a (et al.  showed t (et al. . In conttivation C could bresource . Hence, In summary, using cell-free systems and cellular models, we shed light on a novel molecular mechanism that is responsible for the loss of AMPK-glycogen interaction. Thr-148 autophosphorylation blocks the carbohydrate-binding pocket of the β-CBM, thus adding a new layer of complexity and identifying an unexpected switch regulating AMPK subcellular localization with relevance for glycogen metabolism.


Schistosoma mansoni. Recombinant antigens were produced in Escherichia coli and IgG1, IgG4, and IgE responses against them measured in a cohort of people (n = 222) infected with S. mansoni. All allergen-like antigens were targeted by IgE responses in infected subjects, whilst IgE responses to the non-allergen-like antigens, SmAK, SmUbiquitin, and Sm14-3-3 were essentially absent being of both low prevalence and magnitude. Two new IgE-binding Pfam domain families, not previously described in allergen family databases, were also found, with prevalent IgE responses against SmDLC (PF01221) and SmAldolase (PF00274). Finally, it was demonstrated that immunoregulatory serological processes typically associated with allergens also occurred in responses to allergen-like proteins in S. mansoni infections, including the production of IgG4 in people responding with IgE and the down-regulation of IgE in response to increased antigen exposure from S. mansoni eggs. This study establishes that structures of known allergens can be used to predict IgE responses against homologous parasite allergen-like molecules  and that serological responses with IgE/IgG4 to parallergens mirror those seen against allergens, supporting our hypothesis that allergenicity is rooted in expression of certain protein domain families in metazoan parasites.The IgE response has been associated with both allergic reactions and immunity to metazoan parasites. Recently, we hypothesized that all environmental allergens bear structural homology to IgE-binding antigens from metazoan parasites and that this homology defines the relatively small number of protein families containing allergenic targets. In this study, known allergen structures (Pfam domains) from major environmental allergen families were used to predict allergen-like  and non-allergen-like  proteins in  The role of IgE in allergic immune responses is well established and extensive research has characterized ≈3000 known allergens , 2. FurtWe have therefore suggested a new paradigm, which defines a common characteristic of potentially allergenic structural protein families, and explains why they are restricted in number . Potent Examination of the “top 10” allergen families in the context of the human antibody responses against infecting metazoan parasites revealed examples of parasite proteins with known IgE-binding domains or “parallergens” in three allergen families; EF-hand domains in Tegumental-Allergen-Like (TAL) proteins from schistosomes , TropomyS. mansoni life cycle and their localization in specific tissues may be important in determining the levels of exposure of a protein to the immune system and the corresponding antibody responses. The human life cycle stages of S. mansoni include infective skin-penetrating cercaria, host tissue-migrating schistosomule, blood-dwelling adult worms, and eggs  of Pfam domains associated with the “top 10” allergen families listed in Allfam domains , 31. Simoteomics ] were usS. mansoni proteins with known 3D structure were used to search AllergomeE-value cut-off of <1 × 10−4 were searched for corresponding known 3D structures in Protein Data BankTo compare known parasite and allergen structures, protein sequences of E. coli cells by heat shock. Sanger sequencing was used to characterize cloned sequences. The sequences of SmLipocalin, Sm phosphoglycerate kinase (SmPGK), SmCyclophilin, and SmAldolase were found to be identical to previously cloned and sequenced cDNAs; GenBank Accession numbers M60895.1, L36833.1, L46884.1, and L38658.1. For molecules that had not been previously cloned and sequenced or differed from previous submissions, sequences were uploaded to GenBank with the following accession numbers; SmProfilin, KJ545485; Sm14-3-3, KM281669; SmUbiquitin, KM281670; SmAK, KM281671; SmThioredoxin, KM281672; Sm superoxide dismutase (SmSOD), KM281673; and SmHsp20, KM281674.SmTAL1 (GenBank M37003.1), SmTAL2 (GenBank M67506.1), Sm triosephosphate isomerase (SmTPI) (GenBank M83294.1), and SmTropomyosin II (GenBank KC904504.1) were produced using the pGEX family of plasmids during other studies , 24, 33.E. coli containing the relevant plasmid were diluted 1:10, expanded to OD 0.4–0.8 and induced for 3–5 h with 1 mM IPTG. For SmSOD, TG2 E. coli cultures were also supplemented with 0.02 mM CuCl2 and 0.02 mM ZnCl2 and induced with 0.1 M IPTG as defined by Cardoso et al.  as per the manufacturer’s instructions. Bacterial protein contamination was removed by Q-Sepharose ion exchange using a HiTrap Q (FF) column. Proteins were separated by elution on a 0–1 M NaCl gradient, and fractions containing only SmVAL-6 (120–350 mM NaCl) reserved. Proteins were tested for GST and bacterial contamination by in-house enzyme-linked immunosorbent assay (ELISA) assay using rabbit anti-GST antisera (Sigma) and rabbit anti-E. coli lysate antisera as previously described . For this study, we focused on 222 individuals  for whom full serological and parasitological data were available .Venous blood samples were collected from volunteers living in the fishing village of Namoni, on the shores of Lake Victoria in the Mayuge district of South East Uganda. Transmission of schistosomiasis was consistently high throughout the year and the community received annual treatment as part of the Ugandan national control program. Three hundred seventy-two individuals  with detectable infection  were randomly selected from the village. Blood samples were collected, immediately prior to chemotherapy with PZQ (40 mg/kg body mass) and 5 weeks post-treatment, into heparinized tubes. Plasma was isolated by centrifugation at 2000 × The Ugandan National Council of Science and Technology provided ethical clearance. Forms were translated in the local language and informed written assent was obtained from all adults and written consent from the parents/legal guardians of all children under 15.Antigen-specific IgG1, IgG4, and IgE levels were measured in the plasma of infected individuals in duplicate by ELISA as described previously . The folData management and statistical analysis was performed using STATA for Mac version 12.1. Change in the magnitude of antibody levels following treatment with PZQ were calculated as follows:Significance in the changes of magnitudes were calculated by Wilcoxon matched-pairs signed-ranks test. Prevalences/proportions of responders were calculated following the application of assay sensitivity cut-offs (mean + 3σ) determined using sera from 26 normal European controls. Final cut-off values were determined following correction for outliers (NES responses greater than mean + 3σ) using in iterative process in which outliers were removed and the threshold recalculated until no outliers were found . Comparison of proportions of individuals responding with IgE alone vs. those responding with IgE and IgG4 together was performed by two-sample test of proportions. Significance in the difference in of antigen expression levels by microarray was calculated by one way ANOVA with Bonferroni post-tests for antigen-to-antigen differences. Correlations between IgE and egg expression levels were calculated using linear regression followed by Cook’s distance and leverage-vs.-squared-residual plot regression diagnostics to assess the impact of influential outliers. Regression diagnostics were performed to determine that assumptions were valid including, linearity, normal distribution of errors, and homoscedasticity of variance.S. mansoni proteins with functional domain sequence similarity (similar Pfam domains) to known allergens. Firstly, we searched for examples of S. mansoni proteins with Pfam domain similarity to allergens from the “top 10” Allfam allergen families. Proteins with similarity to the EF-Hand, Profilin, CAP, Tropomyosin, Lipocalin, and Subtilisin-like Serine Protease families listed in Allfam were found in S. mansoni  containing the CAP domain was expressed. For each protein, coding sequences were cloned from parasite cDNA by PCR. These were inserted into either the pGEX-KG or pET-30a vector and sequenced. Following expression and extensive purification molecular weights of the recombinant proteins were estimated by SDS-PAGE  with PZQ within varying portions of the population Figure . IgE resOf the five antigens that did not contain known allergenic domains, three of the antigens, SmAK, SmUbiquitin, and Sm14-3-3 were found to have low prevalences of IgE responders; 1.3/3.1%, 2.7/0.9%, and 0.9/0.45% pre-/post-RX, respectively Figure , 24.S. mansoni allergen-like proteins can be induced as part of the regulatory response to IgE-mediated inflammation and should therefore be considered as part of an allergy-like response , SmSOD (1TO4), and SmThioredoxin (2XBI), were compared for gross structural similarity to allergens with known 3D structure including Dermatophagoides farinae Der f 13 (2A0A), Solanum lycopersicum Superoxide dismutase (3HOG), and Litopenaeus vannamei Thioredoxin (3ZZX), respectively. All three proteins were found to have overall structural similarity with their counterpart allergens with RMSD scores of 0.9 Å, 0.6 Å, and 1.5 Å for SmThioredoxin/LvThioredoxin, SmSOD/SlSOD, and SmLipocalin/Der f 13, respectively  and some significantly higher than others . SmVAL-6 and SmDLC, both transcribed at low-levels in eggs  showed marked boosts in antibody levels following treatment, whilst those more strongly expressed in egg, such as SmSOD, SmUbiquitin, and Sm14-3-3 (between 47.5 and 82.5% of adult transcription levels) all showed marked decreases in antibody levels post-treatment  an association that strengthened post-treatment with PZQ  as shown in Figure In order to determine if constant exposure of antigens from the death of short-lived eggs in host tissues influenced levels of IgE, we compared the level of antigen expression in eggs with antigen-specific IgE levels in infected individuals both pre- and post-treatment with PZQ. As previous studies have demonstrated that the age of infected individuals, their level of IgG4 antibodies and intensity of infection (as measured by number of eggs in feces) may confound the levels of IgE antibodies, these variables were included in univariate regression models explaining variation in IgE magnitude Figure  17, 29,–38. RegrS. mansoni.Allergy and atopic spectrum diseases have been extensively studied over a number of decades providing a detailed description of many of the protein allergens that are associated with IgE-mediated allergic immune reactions , 3. We hS. mansoni parallergen examples of abundant common allergens and to predict parallergens in proteins abundant in S. mansoni. Nine potential IgE-binding antigens were cloned in E. coli alongside, three known IgE targeted S. mansoni antigens, and five other abundant S. mansoni antigens that were predicted not to bind IgE due to the lack of known similar proteins in allergy. We then tested if these antigens were targets for IgE, IgG4, and IgG1 in individuals infected with the parasite.We used a bioinformatic approach to search for S. mansoni and along with previously identified hookworm Na-ASP-2 and SmVAL-4 parallergens is a further example of a parallergen with structural similarity to allergens from the CAP family  as allergens, whilst SmAldolase is a class-I aldolase (Pfam Acc. PF00274) with distinctive structure associated with a separate Pfam domain (Zea mays) is targeted by IgE in maize allergic individuals, confirming SmAldolase as an allergen homolog  with IgE responses down regulated and replaced with IgG4 antibody responses in older individuals 23), respividuals , 29. It ividuals , 47, in Importantly, all of antigens tested were immunogenic, with differing proportions of infected individuals within the population producing an IgG1 response against each of the antigens. For several of these , responsiveness has been shown previously in infected humans and mice , 48–52. Alignments of known parallergen and allergen structures including examples of Superoxide dismutase, Thioredoxin, and Lipocalin allergen families demonstrated that, despite relatively low sequence identity between matched parallergens and allergens, there was high gross structural similarity. This observation lends further weight to our hypothesis, which suggests that the structural similarity between allergens and metazoan parasite IgE-binding molecules defines the restriction of allergen families to a small proportion of structural domains. We predict that more extensive bioinformatic, crystallographic, and serological studies of whole parasite proteomes and the five remaining allergen super families  may reveal further parallergens in support of this hypothesis.S. mansoni infection described here and elsewhere highlight the similarities between the immune responses to metazoan parasites and allergens and provide further evidence for the evolutionary origins of allergy-like responses in the immune reactions against metazoan parasites.Studies on the SmTAL family of antigens suggest that sequestration of antigens may influence their overall levels of exposure to the immune system and consequently antibody levels . In schiS. mansoni antigens, guided by a structural bioinformatics pipeline based on known allergen structures, may provide a clearer picture as to the complex relationship between IgE production, regulatory IgG4 responses, and protective responses to the parasite and help elucidate the mechanisms of IgE targeting in both parasitic disease and allergy.Our data, supporting the hypothesis that molecular structures associated with metazoan parasites determine the allergen landscape, have broad implications for the evolution and function of the IgE response consistent with its role of protection against these organisms. Endo- and ecto-macroparasites would have placed strong evolutionary pressure on the immune system, and specialized molecular mechanisms are likely to have developed to rapidly expand IgE responsiveness during parasite challenge and regulate harmful IgE-mediated immune responses. Whether operating through an antigen-specific mechanism, or invoking a new set of pathogen receptors, this hypothesis provides a novel, testable, explanation of allergy and allergens . In thisThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The Review Editor Adam Cunningham declares that, despite having collaborated on a publication in the last 2 years with author Rick M. Maizels, the review process was handled objectively.http://www.frontiersin.org/Journal/10.3389/fimmu.2015.00026/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.Click here for additional data file.Click here for additional data file.


The blood-brain barrier (BBB) is known to protect healthy brain cells from potentially dangerous chemical agents, but there are many evidences supporting the idea that this protective action is extended to tumor cells. Since the process of angiogenesis in brain tumors leads to BBB breakdown, biochemical characteristics of the BBB seem to be more relevant than physical barriers to protect tumor cells from chemotherapy. In fact, a number of resistance related factors were already demonstrated to be component of both BBB and tumor cells. The enzyme glutathione S-transferases (GST) detoxify electrophilic xenobiotics and endogenous secondary metabolites formed during oxidative stress. A role has been attributed to GST in the resistance of cancer cells to chemotherapeutic agents. This study characterized 8-methoxypsoralen (8-MOP) as a human GST P1-1 (hGST P1-1) inhibitor. To identify and characterize the potential inhibitory activity of 8-MOP, we studied the enzyme kinetics of the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with GSH catalyzed by hGST P1-1. We report here that 8-MOP competitively inhibited hGST P1-1 relative to CDNB, but there was an uncompetitive inhibition relative to GSH. Chromatographic analyses suggest that 8-MOP is not a substrate. Molecular docking simulations suggest that 8-MOP binds to the active site, but its position prevents the GSH conjugation. Thus, we conclude that 8-MOP is a promising prototype for new GST inhibitors pharmacologically useful in the treatment of neurodegenerative disorders and the resistance of cancer to chemotherapy. The Brain Blood Barrier (BBB) protects central nervous system (CNS) against chemical and biological insults. It was described as composed mainly of highly specialized endothelial cells with tight junctions, and astrocyte endfeets with anchoring transmembrane proteins , multifunctional enzymes which are mainly involved in phase II metabolism and antioxidant cell systems   were obtained from the average potential energies of the structures resulting from the MD through the equation Ebind = Ecomplex − (EGST + Eligand), in which Ecomplex, EGST and Eligand correspond to the average potential energies of the complex ligand-GST, the enzyme alone and the ligand alone respectively. PyMOL  was used for visual interpretation of the results.A docking study of the 8-MOP in the GST structure (Protein Data Bank code 3IE3) was conducted. In this structure the enzyme is complexed with 6-hexanol (NBDHEX)  for 1 h. Subsequently, cells were rinsed three times with PBS, incubated with the secondary antibody  and finally observed by fluorescence microscopy (Olympus AX70 microscope—green filter). Nuclei were stained by the dye Hoechst 33258 (ex/em 340/510 nm)  assay . Cell viability was measured by the 3--2,5-diphenyltetrazolium bromide (MTT) method , and expressed as percentage of cells in the first day. Changes in cell morphology were observed by contrast phase microscopy and nuclear morphology was assessed by Hoechst 33258 staining.In order to evaluate the chemosensitizer potential of 8-MOP, cells were seeded in 96-well plates at a density of 3.1 × 10Mosmann, . In shorMosmann,  in cultuData were showed as mean with SEM or median with range according to their distribution, analyzed by Shapiro-Wilk normality test and Skewness  and Kurtosis  calculation. Parametric or non-parametric statistic tests were also chosen according to the distribution. The most appropriate test for each experiment was used and this information is in the respective figure legends. At least three independent experiments were done for each assay.50value of 0.092 mM  bind to the same region of the active site. However, when 0.1 mM 8-MOP was tested varying GSH concentrations both Km and Vmax values decreased, which can be interpreted as an uncompetitive inhibition . Therefore, these data support the idea that 8-MOP-SG is not formed or it is formed in a very low extent.Since 8-MOP inhibits competitively GST-π activity, we hypothesized that this drug could be a substrate, just as CDNB. If it was true, a new compound “8-MOP-SG”   Figure . Substra) Figure . Additio) Figure , even atin vitro antitumor effect. It promoted significant reduction of cell viability in tumor cells, but rat astrocytes used as normal cells control were not affected to the same extent  pre-treated with a sub-toxic concentration of 8-MOP or vehicle Figure  were expt Figure . Microsct Figure . A low ct Figure . No apopThe overexpression of GSTs in many kinds of tumors encourages studies with agents able to promote down regulation of these enzimes  of the enzyme, keeping the glutathione binding site (G-site) free. Since inhibition is competitive, 8-MOP could simply act as a substrate. However, spectrophotometric and chromatographic analyses showed no formation of the 8-MOP/GSH conjugate. Additionally, in silico tests showed that 8-MOP alone interacts with the active site more favorably than the inhibitor NBDHEX.8-MOP concentration-dependently reduced human GST-π activity Finally, the results from experiment with monochlorobimane (MCB) suggest that 8-MOP does not act as substrate of GST from any class, since no GSH depletion occurred at all. Whereas conjugation of MCB with GSH is catalyzed by many cytosolic GSTs  were described. The hGSTP1*C variant being expressed at a higher frequency in gliomas than in normal cells  was associated with resistance to etoposide (Depeille et al., In conclusion, we have shown new application for a well-established drug. 8-MOP can represent a molecule of a novel class of GST inhibitors. The therapeutic potential of these inhibitors is not restricted to cancer treatment, they could also have application in treatment of infectious diseases since GST activity has been reported in many pathogenic parasites (Mahajan and Atkins, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It is generally accepted that ATP regulates mitochondrial function through the AMPK signaling pathway. However, the AMPK-independent pathway remains largely unknown. In this study, we investigated ATP surplus in the negative regulation of mitochondrial function with a focus on pyruvate dehydrogenase (PDH) phosphorylation and protein acetylation. PDH phosphorylation was induced by a high fat diet in the liver of obese mice, which was associated with ATP elevation. In 1c1c7 hepatoma cells, the phosphorylation was induced by palmitate treatment through induction of ATP production. The phosphorylation was associated with a reduction in mitochondria oxygen consumption after 4 h treatment. The palmitate effect was blocked by etomoxir, which inhibited ATP production through suppression of fatty acid β-oxidation. The PDH phosphorylation was induced by incubation of mitochondrial lysate with ATP in vitro without altering the expression of PDH kinase 2 (PDK2) and 4 (PDK4). In addition, acetylation of multiple mitochondrial proteins was induced by ATP in the same conditions. Acetyl-CoA exhibited a similar activity to ATP in induction of the phosphorylation and acetylation. These data suggest that ATP elevation may inhibit mitochondrial function through induction of the phosphorylation and acetylation of mitochondrial proteins. The results suggest an AMPK-independent mechanism for ATP regulation of mitochondrial function. Mitochondria utilize glucose and free fatty acid (FFA) in the production of ATP. As a product of mitochondria, ATP may inhibit mitochondrial function in the feedback regulation. This ATP activity may play a role in the mechanism of insulin resistance under over-supply of free fatty acids (FFAs). Specifically, ATP may mediate FFA signal to inhibit glucose utilization in mitochondria to cut down glucose demand in cells in the mechanism of insulin resistance as suggested by Randle, et al. years ago . In the The PDK-PDH pathway represents an AMPK-independent pathway in the regulation of mitochondrial function . PDH is Lysine acetylation is an important mechanism in the regulation of enzyme activities in variety of metabolic pathways –14. The We hypothesize that ATP may inhibit mitochondria function through direct induction of phosphorylation as well as acetylation of mitochondrial proteins. This possibility was investigated in the liver of diet-induced obese (DIO) mice and 1c1c7 hepatoma cells in this study.Male C57BL/6 mice were purchased from the Jackson Laboratory  at 6 wks of age. The mice were maintained in the animal facility of the Pennington Biomedical Research Center with a 12:12-h light-dark cycle, constant room temperature 22–24°C), free access to water and diet. Mice were fed a high fat diet  at 8 wks (C57BL/6J) to generate a diet-induced obese (DIO) model as described elsewhere [–24°C, frHepatic steatosis was determined by liver size, lipid content of hepatocytes in histology and triglyceride content per gram liver tissue. Hematoxylin and eosin (H&E) staining of liver tissue and triglyceride assay was performed using protocols as described elsewhere .™) was purchased from the American Type Culture Collection . The cells were maintained in DMEM supplemented with 10% fetal calf serum. Sodium palmitate (P9767), etomoxir (E1095), calyculin (C5552), acetyl-CoA (A2056) and ATP (A6559) were purchased from Sigma . Antibodies to phosphorylated PDH e1 alpha subunit (S293) (ab177461), PDHe1 alpha subunit (ab110330), PDK2 (ab68164), PDK4 (ab89295), and tubulin (ab7291) and anti-voltage dependent anion channel  were obtained from Abcam . Antibody to acetylated lysine (#9814) was from Cell Signaling .Cell line Hepa-1c1c7  was used to inhibit β-oxidation with 4 h treatment. Calyculin (20 nM) was used to induce phosphorylation in cells with 1 h treatment. Mitochondrial lysate was made from freshly-isolated mitochondria and kept in -70°C until experiment. The lysate was incubated with ATP (1–10 μM/g) or acetyl-CoA (1–500 μM) for 30 min at 37°C to induce protein phosphorylation and acetylation.2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EGTA and 0.2% fatty acid-free BSA, pH 7.2 at 37°C. A stock solution (2X) of MAS was prepared for dilution of substrates, ADP and respiration reagents. Stocks of succinate and ADP were made in H2O and adjusted to pH 7.2 with potassium hydroxide. Stocks of 10 mM FCCP (carbonyl cyanide 4-trifluoromethoxy phenylhydrazone), 2 mM rotenone, 5 mg/ml oligomycin and 40 mM antimycin A were made in DMSO.Mitochondria were isolated from live cells or fresh liver tissue using the standard protocol . Mitochog for 10 minutes at 4°C. The supernatant was then centrifuged for 10 minutes at 7,000 g at 4°C, and the pellet was collected as mitochondria after washing with ice-cold buffer. Mitochondria were suspended and then used in functional analysis. Mitochondria were sonicated in lysis buffer in the preparation of mitochondrial lysate. Protein concentration was determined by the BCA (bicinchoninic acid) method using BSA as a standard.Cells were homogenized using a glass-Teflon potter, and centrifuged at 600 Liver tissues were collected from DIO mice at 12 wks on HFD. Mitochondrial lysate was used at 50 μg protein/sample and resolved on 8% SDS-PAGE gel by electrophoresis. The protein was blotted onto nitrocellulose membrane. Blots were then blocked in 5% nonfat milk buffer containing 0.05% Tween-20, rinsed in PBS (pH 7.4) as described elsewhere , The mem2 and H+ levels via a sensor cartridge that is equipped with four reagent injectors. Mitochondrial substrates and compounds were prepared at 10× final concentrations in 1× MAS buffer. The reagents were loaded into the injection ports before mitochondria loading to the XF plate. The loaded cartridge was calibrated before the assay. To minimize variability among the wells, mitochondria were first diluted by 10 times in cold MAS buffer with substrate (succinate), and then diluted again to the final concentration. The mitochondrial suspension (50 μl) was delivered into each well of XF plate (except for background correction wells) on ice. The plate was centrifuged at 2000 g for 20 minutes at 4°C, and then loaded with 450 μl/well of substrate in 1× MAS. The mitochondria were observed briefly under a microscope to ensure consistent adherence to the well bottom, then placed at 37°C for 6–8 minutes to allow the plate to warm up. The plate was used in the OCR measurement according to the protocol.All OCR assays were performed using XF24 Extracellular Flux Analyzer . The assay is based on fluorimetric detection of OATP was measured using the ATP Determination Kit  immediately after mitochondria isolation. For all experiments, ATP standard curves were run in the range of 1 μM to 1mM and the correlation coefficient was 0.990 or higher. The samples were added to the reaction mixture and the luminescent signal was measured by the luminometer . The ATP concentration was normalized with the mitochondrial protein.3. The CoASH Quencher and Quencher remover were added into the sample to correct the background generated by free CoASH and succ-CoA. The sample was then diluted with the reaction mix, and the fluorescence signal was measured at Ex/Em = 535/589 nm with Spectra max Gemini XPS . The relative acetyl-CoA concentration was normalized with the mitochondrial protein.The acetyl-CoA content was determined immediately after isolation of the mitochondria using the PicoProbe acetyl-CoA assay kit . Briefly, acetyl-CoA standard curve was made in the range of 0–100 pM and the correlation coefficient was 0.990 or higher. Protein was removed in the sample using the perchloric acid protocol and the supernatant was neutralized with 3 M KHCOPDH activity was measured according to the protocol by PDH activity assay kit . Mitochondria were diluted and added into the microplate. After incubation in the plate for 3 hours at room temperature, the samples were stabilized and incubated with assay buffer. The fluorescence was measured at 450 nm for 30 minutes with 30 seconds interval among each measurement, and the slope of the line indicated the PDH activity.In this study, the data were presented as the mean ± SEM from multiple samples. In cell culture study and immunoblotting, the experiments were repeated at least three times. The representative immunoblot blot is presented. In the statistical analysis, two-tailed, unpaired Student’s t test was used in analysis of in vitro data, and one-way ANOVA was used in analysis of in vivo data with significance P <0.05.Diet-induced obese mouse is a well-established model in the study of insulin resistance. We use the model routinely in our laboratory in the study of type 2 diabetes. ATP was tested in the liver of DIO mice at 12 weeks on HFD. In the fresh liver tissue, ATP was increased by 2 folds . The incTo test the possible role of ATP in the induction of PDH phosphorylation, ATP was tested in cells, in which 1c1c7 hepatoma cells were treated with palmitate to induce intracellular ATP. Palmitate was conjugated with BSA and used as a free fatty acid in the study. An increase in ATP was observed in the cells at 1 h of palmitate treatment and the peak was observed at 16 h . An induAbove data suggest that ATP may contribute to PDH hyper-phosphorylation in cells. To test the possibility, the loss-of-function strategy was employed by pre-treatment of cells with the β-oxidation inhibitor, etomoxir (ET), which blocks acetyl-CoA production from palmitate. β-oxidation is required for ATP production from FFA. ATP was reduced by the ET pre-treatment . The treTo test ATP in the induction of PDH phosphorylation, we treated mitochondrial lysate with ATP in vitro. Fresh mitochondria were isolated from cells and used in the preparation of mitochondria lysate. The internal ATP was depleted in the lysate by keeping the lysate at -70°C overnight. The lysate was then incubated with ATP at multiple concentrations at 37°C for 30 minutes to induce the phosphorylation. ATP was used at physiology relevant concentrations between 2–10 μM/g. PDH phosphorylation was induced by ATP at 2 μM/g and the ATP effect was reduced at the higher concentration . The mecAcetylation of mitochondrial proteins was examined in the mitochondrial lysate using the anti-acetyl-lysine antibody in Western blot. The acetylation was induced in 1c1c7 cells by palmitate treatment between 1–24 h. The induction was observed at multiple proteins as indicated by the band patterns . The chaAbove data suggest that the FFA effects are dependent on β-oxidation which breaks FFA down to generate acetyl-CoA. If this is true, acetyl-CoA should exhibit a similar activity to ATP in the induction of phosphorylation and acetylation. To test the possibility, acetyl-CoA was determined in the whole cell lysate after cell treatment by palmitate. An increase was observed at 1 h and the strong increase was observed at 16 h with the palmitate treatment . Acetyl-The phosphorylation and acetylation may contribute to mitochondrial inhibition by palmitate in our system. To test this possibility, oxygen consumption rate (OCR) was examined using the Seahorse system to determine the mitochondrial function. Mitochondria were isolated from cells immediately after palmitate treatment and tested for OCR in the presence of substrate of succinate. OCR was induced at 1 h, but suppressed at 4 h and 16 h by palmitate . The palOur data suggest that ATP induces PDH phosphorylation independently of PDK expression. This mechanism may be used by FFA to inhibit glucose utilization in mitochondria given that FFA has a high density in energy. The inhibition is mediated by PDH hyper-phosphorylation as suggested by our data. PDH is subject to reversible phosphorylation in physiological conditions upon changes in fuel supply. The phosphorylation is catalyzed by PDK. Hyper-phosphorylation of PDH is a mechanism of enzyme inhibition in the liver and muscle in starvation and diabetes –8, whichIn addition to phosphorylation, ATP promoted protein acetylation in mitochondria in our system. Hyperacetylation represents a mechanism of inhibition of glucose utilization by mitochondria in SIRT3 knockout mice –17. In cIn summary, we investigated molecular mechanism by which ATP inhibits mitochondrial activity with a focus on posttranslational modification of proteins by phosphorylation and acetylation. The role of ATP is supported by several lines of evidence including those from DIO mice, palmitate-treated cells, β-oxidation inhibition by etomoxir, and ATP treatment of mitochondrial lysate. The data consistently suggest that ATP is able to induce the phosphorylation and acetylation. Although the nature of acetylated proteins and acetyltransferase remains unknown in this study, this does not influence our conclusion about ATP in the inhibition of mitochondrial function. It is well known that AMPK is down-regulated by ATP surplus in the feedback regulation of mitochondria function. Current study does not exclude the role of the AMPK pathway. However, we propose that as an AMPK-independent pathway, the PDH response may contribute to the feedback regulation as well. In addition, the acetylation of mitochondrial proteins may represent another AMPK-independent feedback mechanism. The study provides a mechanism for insulin resistance in the condition of ATP surplus in human and mouse , 11. Our