The in vitro cytotoxicities and apoptosis-inducing properties of these complexes are extensively studied. Complexes 1 and 2 exhibit potent antiproliferative activities against a panel of human cancer cell lines. The cell cycle analysis shows that complexes 1 and 2 exhibit effective cell growth inhibition by triggering G0/G1 phase arrest and inducing apoptosis by mitochondrial dysfunction. The in vitro DNA binding properties of the two complexes are investigated by different spectrophotometric methods and viscosity measurements.Two new Ru(II) complexes, [Ru(bpy) Since cisplatin and its platinum complexes were first used as anticancer agents successfully, more active transition metal complexes with better anticancer activity have been studied. Transition metal complexes have potential advantages, such as variable coordination numbers and geometries, accessible redox states, rich physicochemical properties, and extensive structural diversity, over common organic-based drugs. Therefore, the metal-based cancer drug is a focus of research in bioinorganic chemistry H imidazol-2-yl)hydrazone]dihydrochloride, which has been evaluated in clinical phase I studies.15 However, the research of metal complexes based on analogs of anthracene is lacking. The development of metal complexes with bioactive molecules as ligands enables the synthesis of novel drugs that are more active and desirable than the ligands themselves 2(FAMP)](ClO4)21 and [Ru(phen)2(FAMP)](ClO4)22, which were obtained by the combination of Ru(II) polypyridyl moieties with anthracene derivative. The antitumor activity, cell cycle arrest and apoptosis, DNA binding, and photocleavage properties of the two complexes were studied.A large number of anthracene analogs have been reported as important DNA-intercalating agents with antitumor activity 3·xH2O was purchased from the Kunming Institution of Precious Metals. 1, 10-phenanthroline was obtained from the Guangzhou Chemical Reagent Factory. Cell lines of BEL-7402 (human hepatocellular carcinoma cell line), A549 , MG-63 (human osteosarcoma cell line), and SKBR-3 (human breast cancer cell line) were purchased from the American Type Culture Collection. Agarose and ethidium bromide were obtained from Aldrich. Doubly distilled water was used to prepare buffers (5 mM Tris(hydroxymethylaminomethane)-HCl, 50 mM NaCl, pH = 7.2). A solution of calf thymus DNA in the buffer gave a ratio of UV absorbance at 260 and 280 nm of ca. 1.8∼1.9∶1, indicating that the DNA was sufficiently free of protein –1 cm–1) at 260 nm All reagents and solvents were purchased commercially and used without further purification unless specially noted. Ultrapure MilliQ water was used in all experiments. Calf thymus DNA (CT DNA) was obtained from the Sino-American Biotechnology Company. pBR 322 DNA was obtained from Shanghai Sangon Biological Engineering&Services Co., Ltd. Dimethyl sulfoxide (DMSO), RPMI 1640 , 9, l0-bis(chloromethyl)anthracene and 2-nitro-propane were purchased from Sigma. RuClm/z values are for the major peaks in the isotope distribution. 1H NMR spectra were recorded on a Varian-500 spectrometer with DMSO [D6] as solvent and tetramethylsilane (TMS) as an internal standard at 500 MHz at room temperature. UV/Vis spectra were recorded on a Perkin- Elmer Lambda 850 spectrophotometer and emission spectra were recorded on a Perkin-Elmer LS 55 spectrofluorophotometer at room temperature.Microanalyses  were carried out with a Perkin-Elmer 240Q elemental analyzer. Fast atom bombardment (FAB) mass spectra were recorded on a VG ZAB-HS spectrometer in a 3-nitrobenzyl alcohol matrix. Electrospray ionization mass spectra (ES-MS) were recorded on a LCQ system  using methanol as mobile phase. The spray voltage, tube lens offset, capillary voltage and capillary temperature were set at 4.50 KV, 30.00 V, 23.00 V and 200°C, respectively, and the quoted 3) was refluxed with stirring for 2 h. The cooled solution was then diluted with water and neutralized with concentrated aqueous ammonia. The precipitate was collected and purified by column chromatography on silica gel (60–100 mesh) with ethanol as eluent to give the compound as a yellow powder. Yield: 0.51 g, 80%. Anal. Calcd for C28H16N4O: C, 79.23; H, 3.80; N, 13.20. Found: C, 79.14; H, 3.91; N, 13.27%. FAB-MS: m/z = 425 (M+1).A mixture of 9, l0-Anthracenedicarboxaldehyde  cis-[Ru(bpy)2Cl2]·2H2O  3) was refluxed under argon for 8 h to give a clear red solution. Upon cooling, a red precipitate was obtained by dropwise addition of saturated aqueous NaClO4 solution. The crude product was purified by column chromatography on a neutral alumina with a mixture of MeCN-toluene  as eluent. The red band was collected. The solvent was removed under reduced pressure and a red powder was obtained. Yield: 0.36 g, 70%. C48H32N8Cl2O9Ru: C, 55.61; H, 3.11; N, 10.81%; Found: C, 54.78; H, 3.21; N, 10.68%. ES-MS : 837.2 ([M–2ClO4–H]+), 419.1 ([M–2ClO4]2+). 1H NMR : δ 14.16 , 13.08 , 9.08 , 8.94 , 8.87 , 8.83 , 8.76 , 8.51 , 8.21 , 8.13 , 8.08 , 7.97 , 7.84 , 7.57–7.61 , 7.35 .A mixture of 1, with cis-[Ru(phen)2Cl2]·2H2O cis-[Ru(bpy)2Cl2]·2H2O Yield: 0.35 g, 65%. C52H32N8Cl2O9Ru: C, 57.57; H, 2.97; N, 10.33%; Found: C, 57.65; H, 3.04; N, 10.28%. ES-MS : 885.2 ([M–2ClO4–H]+), 443.1 ([M–2ClO4]2+). 1H NMR : δ 14.38 , 13.52 , 8.97 , 8.87 , 8.76 , 8.67 , 8.62 , 8.54 , 8.31 , 8.21 , 8.15 , 8.06 , 7.94 , 7.84 , 7.75 .This complex was synthesized by an identical method as described for complex Perchlorate salts of metal compounds with organic ligands are potentially explosive, and only small amounts of the material should be prepared and handled with great care.3 cells per well) and grown overnight at 37°C in a 5% CO2 incubator. Complexes were tested then added to the wells to achieve final concentrations ranging from 10–6 to 10–4 M. Control wells were prepared by addition of culture medium (100 µL). The plates were incubated at 37°C in a 5% CO2 incubator for 48 h. Upon completion of the incubation, stock MTT dye solution  was added to each well. After 4 h, buffer (100 µL) containing N, N-dimethylformamide (50%) and sodium dodecyl sulfate (20%) was added to solubilize the MTT formazan. The optical density of each well was then measured with a microplate spectrophotometer at a wavelength of 490 nm. The IC50 values were determined by plotting the percentage viability vs. concentration on a logarithmic graph and reading off the concentration at which 50% of cells remain viable relative to the control. Each experiment was repeated at least three times to obtain the mean values. Four different tumor cell lines were the subjects of this study: BEL-7402, A549, MG-63 and SKBR-3.MTT -2,5-diphenyltetraazolium bromide) assay procedures were used 5 cells per well and incubated for 24 h. The cells were cultured in RPMI 1640 supplemented with 10% of fetal bovine serum (FBS) and incubated at 37°C and 5% CO2. The medium was removed and replaced with medium  containing the complex (12.5 µM) for 24 h. The medium was removed and the cells were washed with ice-cold PBS, and fixed with formalin . Cell nuclei were counterstained with Hoechst 33258 (10 µg·mL–1 in PBS) for 10 min. Then the cells were observed and imaged by a fluorescence microscope  with excitation at 350 nm and emission at 460 nm.BEL-7402 cells were seeded onto chamber slides in six-well plates at a density of 2×105 cells per well and incubated for 24 h. The cells were cultured in RPMI 1640 supplemented with fetal bovine serum  and incubated at 37°C and 5% CO2. The medium was removed and replaced with medium  containing complexes 1 and 2 (6.25 µM). After incubation for 24 h, the cell layer was trypsinized and washed with cold phosphate buffered saline (PBS) and fixed with 70% ethanol. The fixed cells are rinsed with PBS and then stained with the DNA fluorochrome PI in a solution containing Triton X-100 as well as RNase, keep 15 min at 37°C. Then the samples were analyzed with a FACSCalibur flow cytometer . The number of cells analyzed for each sample was 10000 BEL-7402 cells were seeded into six-well plates at a density of 2×10–1 of JC-1  in culture medium at 37°C in the dark. Cells were immediately centrifuged to remove the supernatant. Cell pellets were suspended in PBS and then photographed under a microscope.Cells were treated for 24 h with complexes in 12-well plates and were then washed three times with cold PBS. The cells were then detached with trypsin-EDTA solution. Collected cells were incubated for 20 min with 1 µg·mLThe DNA-binding and photoactivated cleavage experiments were performed at room temperature. Buffer A  was used for absorption titration, luminescence titration and viscosity measurements. Buffer B  was used for DNA photocleavage experiments.Kb, based on the absorption titration, were measured by monitoring the changes of absorption in the MLCT band with increasing concentration of DNA using the equation The absorption titrations of the complex in buffer were performed using a fixed concentration (10 µM) for complex to which increments of the DNA stock solution were added. The intrinsic binding constants Tm) was taken as themid-point of the hyperchromic transition. The melting curves were obtained by measuring the absorbance at 260 nm for solns of CT-DNA (100 µM ) in the absence and presence of the RuII complex (10 µM) as a function of the temp. The temp was scanned from 30 to 95°C at a speed of 1°C min–1. The data were presented as (A – 0A)/(fA – 0A) vs. temp., where A, 0A, and fA are the observed, the initial, and the final absorbance at 260 nm, resp.Thermal denaturation studies were carried out with a Perkin-Elmer Lambda 35 spectrophotometer equipped with a Peltier temperature-controlling programmer (±0.1°C). The melting temp (η = (t–0t)/0tη/0η)1/3 versus binding ratio Viscosity measurements were carried out using an Ubbelodhe viscometer maintained at a constant temperature at 25.0 (±0.1) °C in a thermostatic bath. DNA samples approximately 200 base pairs in average length were prepared by sonicating in order to minimize complexities arising from DNA flexibility For the gel electrophoresis experiment, supercoiled pBR322 DNA (0.1 µg) was treated with the Ru(II) complexes in buffer B, and the solution was then irradiated at room temperature with a UV lamp . The samples were analyzed by electrophoresis for 1.5 h at 80 V on a 0.8% agarose gel in TBE . The gel was stained with 1 µg/ml ethidium bromide and photographed on an Alpha Innotech IS–5500 fluorescence chemiluminescence and visible imaging system.1 and 2 is presented in 1 and 2 were synthesized and high yield was obtained by the direct reaction of FAMP with appropriate precursor complexes in ethanol. The desired Ru(II) complexes were isolated as perchlorates and purified by column chromatography. In the ES–MS spectra of the Ru(II) complexes, the signals of [M-2ClO4-H]+ and [M-2ClO4]2+ were observed. The ES–MS results are provided in the experimental section. The measured molecular weights were consistent with expected values.An outline of the synthesis of complexes 1 and 2 on BEL-7402, A549, MG-63, and SKBR-3 cell lines to determine their potential as anticancer agents. Cisplatin, the ligand FAMP and similar compound [Ru(phen)2(Hecip)](ClO4)2 were included for comparison, and were tested under identical conditions. The above cell lines were treated with different concentrations of complexes 1 and 2 for 48 h. The IC50 values for complexes 1 and 2 against these cell lines are summarized in 50 values, the newly synthesized complexes 1 and 2 were more active against SKBR-3 cells than against other cancer cells. The ligand FAMP showed less toxicity than complexes 1 and 2. The IC50 values of complexes 1 and 2 toward BEL-7402 cells were lower than that of cisplatin, which indicates that complexes 1 and 2 show higher cytotoxic effect on BEL-7402 cells than cisplatin under identical conditions. Comparing the IC50 values, we also found that complex 1 was effective against BEL-7402 cells. Additionally, complex 2 shows higher cytotoxic activity than complex 1 against A549, MG-63, and SKBR-3 cell lines. The two complexes displayed different cytotoxic effect against different tumor cell lines. At the same time, the compound [Ru(phen)2(Hecip)](ClO4)2 shows lower IC50 values than complex 2 against BEL-7402, A549 and MG-63 cell lines. Based on the results, we speculate that the antitumor activity of complex may be related to the specific molecular shape of the complex and the chemical structure and nature of the inserted ligand.The MTT assay procedures were used to evaluate the in vitro cytotoxicity of complexes 1 and 2 induced chromatin condensation and fragmentation, which are recognized morphological features of apoptosis, BEL-7402 cell line was treated with complexes 1 and 2 at 12.5 µM for 24 h. As shown in 1 and 2 can induce apoptosis in BEL-7402 cells.To determine whether or not complexes 1 for 24 h. After treatment, cell apoptosis analyses were performed using flow cytometry. As shown in 1 follows a pathway from the viable cells to the early apoptotic cells and then to the necrotic or late apoptotic cells. This pathway suggests that induced cell death occurs mainly through apoptosis. Furthermore, the effect increases significantly in a dose-dependent manner.To determine the percentage of the necrotic or late apoptotic cells and early apoptotic BEL-7402 cells, the cells were treated with 6.25, 12.5 and 25 µM of complex 1 and 2 on the cell cycle progression of BEL-7402 cells. The cell cycle distribution after treating BEL-7402 cells with complexes 1 or 2 at 6.25 and 12.5 µM for 24 h is shown in 1 and 11.24% and 12.90% for complex 2 at 6.25 and 12.5 µM, respectively, which was accompanied by a corresponding reduction in the percentage of cells in the S and G2/M phases. Obviously, complex 1 caused more pronounced changes in the cell cycle than complex 2 in the G0/G1 phase under identical conditions. The antiproliferative mechanism induced by complexes 1 or 2 on BEL-7402 cells was G0/G1 phase arrest, and the changes in percentages at G0/G1 and S phases were concentration dependent.Flow cytometry was used to determine the effects of complexes 1 and 2. At high mitochondrial membrane potential, JC-1 forms aggregates, which have a red fluorescence emission peak. At low mitochondrial membrane potential, JC-1 forms monomers, which emit a green fluorescence peak. As shown in 1 and 2 for 24 h, JC-1 showed green fluorescence (JC-1 monomers) corresponding to low mitochondrial membrane potential. The change from red to green fluorescence suggests the decrease of mitochondrial membrane potential in the presence of complexes 1 and 2, which indicates that complexes 1 and 2 induced apoptosis in BEL-7402 cells through the mitochondrial signal transduction pathway.JC-1 was used as a fluorescence probe in detecting the change of mitochondrial membrane potential induced by complexes 1 and 2 in the presence of increasing amounts of calf thymus DNA (CT DNA) are shown in 1 and 447 nm for complex 2 were assigned to the metal-to-ligand charge transfer transition. The bands in the range of 300 nm to 400 nm were attributed to π–π* transitions. The bands below 300 nm were attributed to intraligand π–π* transitions. As the concentration of CT DNA increased, the MLCT transition band of complexes 1 and 2 exhibited hypochromism of 14.68% and 17.20%, respectively. The values of K for complexes 1 and 2 were 0.80±0.24×105 M-1 (s = 1.88) and 1.70±0.25×105 M-1 (s = 2.59), respectively. These values were smaller than those of [Ru(bpy)2(dppz)]2+ (4.9×106 M-1) 2(dppz)]2+ (5.1×106 M-1) 2(dppz)]2+ (3.2×105 M-1) and Λ-[Ru(phen)2(dppz)]2+ (1.7×105 M-1) 4 M-1 to 4.8×104 M-1). The complexes intercalatively bind to DNA, which involves a strong stacking interaction between the aromatic chromophore and the base pairs of the DNA. Additionally, the larger DNA binding constant of complex 2 reflects its higher binding affinity and deeper intercalation than complex 1.The absorption spectra of complexes 1 and 2. In the absence of DNA, complexes 1 and 2 can emit luminescence in Tris buffer at ambient temperature, with maximum luminescence appearing at 598 and 605 nm, respectively. With CT DNA addition, the emission intensities of complexes 1 and 2 increased to approximately 1.3 and 1.5 times the original intensities, respectively . To identify this transition process, the melting temperature Tm, defined as the temperature when half of the total base pairs is unbonded, is usually introduced. A previous report Tm. The melting curves of CT DNA in the absence and presence of the complex are presented in Tm of 60.5±0.2°C under our experimental conditions. The observed melting temperature in the presence of complexes 1 and 2 was 69.0±0.1°C and 74.2±0.2°C, respectively, at a concentration ratio of r = 0.10 (r = [Ru]/[DNA]). The ΔTm values (8.5°C and 13.7°C) of complex 1/2–DNA adducts are comparable to that observed for classical intercalators ethidium bromide , Δ-[Ru(phen)2(dppz)]2+ , and Δ-[Ru(phen)2(dppz)]2+  1 and 2 to DNA.DNA melting experiments can establish the extent of intercalation because the intercalation of the complex into DNA base pairs causes stabilization of base stacking, and therefore, raises the melting temperature of double-stranded DNA 1 and 2 on the viscosity of CT-DNA are shown in 2> complex 1. The results are consistent with our conclusions.Relative viscosity measurements have been effective as a method for the assignment of the mode of binding compounds to DNA. In classical intercalation, the DNA helix lengthened as base pairs are separated to accommodate the binding ligand that increases DNA viscosity, whereas a partial, non-classical ligand intercalation causes a bend in the DNA helix reducing its effective length, and consequently, its viscosity. The viscosity studies provide a stronger argument for intercalation 1 and 2 was monitored by agarose gel electrophoresis. The DNA cleaving efficiencies of these complexes are shown in II complex in the dark. In the presence of different concentrations of the complexes, the Form I amount of pBR322 DNA decreased, whereas that of Form II increased. These results indicate that these complexes can effectively cleave pBR322 DNA upon irradiation.When circular plasmid DNA is subject to electrophoresis, relatively fast migration is observed for the intact supercoiled form (Form I). If scission occurs on one strand (nicked), the supercoiled form will relax to generate a slower-moving, open circular form (Form II) 1 and 2 can suppress tumor cells proliferation. Notably, the antiproliferative activities of complexes 1 and 2 are much higher than that of the ligand FAMP, suggesting that the significantly higher anticancer activity is due to the complexation with Ru(II) polypyridyl moieties. The apoptotic study indicates that complexes 1 and 2 can effectively induce apoptosis of BEL-7402 cells and inhibit the proliferation in the G0/G1 phase on BEL-7402 cells. Additionally, complexes 1 and 2 induce a decrease of the mitochondrial membrane potential. The complexes induce apoptosis of BEL-7402 cells through the mitochondrial signal transduction pathway. The DNA-binding behavior show that the two complexes interact with CT DNA through intercalative mode. Upon irradiation, these complexes can effectively cleave pBR322 DNA. These results will help in designing highly cytotoxic Ru(II) complexes and DNA-binding reagents.Two new Ru(II) complexes were synthesized and characterized. The cytotoxicity assay shows that complexes Figure S11 and 2.Synthetic route of complexes (TIF)Click here for additional data file.


We evaluated the short-term spontaneous fluctuations of HBV DNA and HBsAg levels in Senegalese patients with chronic infection with hepatitis B virus and normal ALT and determined factors related to these fluctuations.A total of 87 patients with persistent normal ALT values were enrolled in the study. Serum samples were obtained at three different visits, with an interval of 2 months , and without initiating anti HBV treatment. Levels of HBV DNA, quantitative HBsAg, ALT and AST, genotyping and viral DNA mutations were analyzed.10 IU/mL [2.2-3.4], 2.7 log10 IU/mL [2.1-3.6] and 2.7 log10 IU/mL [2.1-3.4] at M0, M2 and M4, respectively. The values ranged from <1.1 to 7 log10 IU/mL and 55 (63%) had HBV DNA fluctuations ≥ 0.5 log10 IU/mL between two visits. Patients in whom HBV DNA fluctuated ≥0.5 log10 IU/mL between M0 and M2 also had significant fluctuations between M2 and M4, while patients with stable HBV DNA between M0 and M2 showed a stable viral load between M2 and M4. The only factor found to be associated with HBV DNA fluctuations ≥ 0.5 log10 IU/mL was a low BMI (<21 kg/ m2). HBsAg levels were not correlated with HBV DNA levels.Among the 87 patients, genotype E was predominant (75%). The median HBV DNA level was 2.9 logSixty-three percent of the enrolled Senegalese population showed a large, short-term fluctuation of HBV DNA levels. Such fluctuations may have an impact on therapeutic management, requiring closer monitoring. Hepatitis B virus (HBV) infection is a global epidemic, with more than 350 million of chronically infected carriers of the virus surface antigen (HBsAg) . Without10 IU/mL), or serum alanine aminotransferase (ALT) levels above the upper limit of normal (ULN) and liver biopsy suggestive of significant fibrosis (≥F2) 0.5-1]1-2] and >2 log10 IU/mL. Friedman’s test was used to compare HBsAg fluctuations between the three values. Correlation between viral load fluctuations was based on the Spearman coefficient.Continuous variables were expressed as median and interquartile ranges (IQR), and categorical variables were expressed as percentages. Univariate analyses were based on Fisher’s exact test for categorical variables and Mann–Whitney test for continuous variables. The HBsAg  were included in a backward stepwise logistic regression model. A P value of ≤0.05 was considered to denote statistical significance. Statistical analyses were performed using STATA software version 12.0 .All variables associated with HBV DNA fluctuations > 0.5 log2. All patients maintained normal ALT, with a median of 0.5xULN  [0.4-0.6] over the three visits. Genotype E was predominant (75%), the remaining belonging to genotype A. The median HBV DNA level was 2.9, 2.7 and 2.7 log10 IU/mL for the first, second and third visits, respectively (Table 10 IU/mL. Thirty-six subjects (41%) had at least one visit with a viral load ≥3.2 log10 IU/mL. Only 15 patients (17%) were above ≥3.2 log10IU/mL and five patients (6%) had an undetectable HBV DNA (<1.1 log10 IU/mL) throughout the three visits.The characteristics of the studied population are summarized in Table Ninety percent of patients had a detectable viral load at baseline (M0) and among them, two patients (5%) had a positive HBeAg. Ninety four percent of patients had at least one visit with detectable viral load, while 6% were always undetectable (<1.1 log) over all the visits.10 IU/mL between two visits. In 8 patients (9%) the difference was >2 log10 IU/mL (max 2.8 log10 of difference) (Table Sixty-three percent of subjects (n = 55) experienced a change in viral load of at least 0.5 log2   , mainly due to the difference observed between the second and third samples, with respective medians of 4546 and 3473 IU/mL Table .10 IU/mL  to HBV DNA (log10 IU/mL) ratio was significantly higher in samples with HBV DNA values ≤3 log10 IU/mL (1.48 [1.24-1.73]), compared to high HBV DNA values (1.04 [0.91-1.15] for]3-4] log10 IU/mL (p < 0.001), 0.94 [0.67-0.95] for]4-5] log10 IU/mL (p = 0.05) and 0.63 [0.58-0.67] for more than 5 log10 IU/mL, (p < 0.001))  among Senegalese patients with normal transaminases, as well as their possible impact on clinical characterization of the disease and its therapeutic management. The lack of association between these fluctuations and demographic parameters  or biochemical (transaminases) or virological data  could be explained by a lack of power because of the small number of subjects included in the study. Low BMI < 21 kg/mividuals .The stability of the biochemical markers contrasts with the volatility of the HBV DNA.The lower replicativity among HBeAg negative patients does not explain the HBV DNA variability observed in our study.The frequency and magnitude of DNA fluctuations and their possible impact on clinical characterization of the disease and its therapeutic management must be emphasized. Guidelines recommend that to be eligible for treatment, patients must have a viral load greater than or equal to HBV DNA 3.2 log IU/mL. Only 17% of our patients constantly exceeded this threshold in the three samples tested . Forty-two percent of patients would have been eligible or ineligible for treatment according to the viral load value at a given time. It therefore seems necessary to measure viral load for multiple occasions before deciding to treat.More recently, studies have reported that the management of chronic hepatitis B can be optimized with HBsAg quantification, used as a biomarker for stratifying the risk of disease progression  and for In clinical practice, HBsAg quantification cannot replace viral load measurement. Combining both measures has been shown to be important for monitoring the natural history of the disease and treatment outcome . We obseSince HBeAg-negative patients with higher HBsAg levels were more susceptible to developing active disease, quantification of HBsAg in such patients merits further investigation.


Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRβ bias. Using a retrogenic model of TB10.44-11-specific CD8+ T cells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including  Mycobacterium tuberculosis infection, attempts to prevent tuberculosis by vaccines designed to elicit memory T cells have only been partially successful. Several vaccine candidates are in clinical trials, but progress has been slow because their ability to prevent disease must be empirically tested. There is little understanding of why certain antigens are targets of protective immunity. We have characterized an immunodominant CD8+ T cell response to the M. tuberculosis antigen TB10.4 (EsxH). CD8+ T cells specific for the TB10.44–11 epitope are primed early during infection and account for 30–50% of lung CD8+ T cells during chronic infection. Now we have used deep sequencing to characterize the TCR repertoire of TB10.44-11-specific CD8+ T cells in the lungs of infected mice. Interestingly, TB10.44-11-specific CD8+ T cells exhibit extreme clonal expansion of certain TCRβ with common structural features, most likely because of affinity selection. Affinity selection of T cells is more important when antigen presentation is limiting. Although the lung contains numerous bacteria during infection, antigen-presentation by infected APC may be limiting, mimicking a “low antigen” state. Thus, even T cells that have the potential to mediate protection may function inefficiently because of suboptimal T cell activation.While T cells are required for protection against  Here entropy, a measure of diversity within a complex data set, is also known as the Shannon-Wiener index, Shannon’s diversity index or Shannon’s entropy [For TCRβ high-throughput sequencing, genomic DNA was purified from sorted cell populations and sequenced by Adaptive Biotechnologies Corp.  using the ImmunoSEQ assay . Positive and negative fractions were then surface stained with anti-MHC II, CD11b, CD19, and CD4 as a “dump” channel, and anti-CD8α, CD3 and CD44. Flow cytometry counting beads were added immediately before samples were collected by the cytometer to determine the fraction of tetramer+ events collected and used to determine the total number and frequency of tetramer+ cells in each animal. For analysis purposes, naïve cells were cells that were present in the bound fraction, costained with PE- and APC-conjugated tetramers, and did not express CD44.Analysis of the precursor frequency of naïve T cells was performed as previously described . Briefly+CD8+Tet+CD19-CD4-CD11b-CD11c- as individual cells into wells of 96-well PCR plates containing 10 μl of reverse transcriptase buffer , 2% Triton X-100, 500 μM dNTP with 1 μg BSA, 50 ng of oligo(dT) (12–18), 500 μM dTT, 50 μM of TCRα-specific primer, 50 μM of TCRβ-specific primer, 8 U of RNaseOUT, and 30 U of Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies). The plates were incubated for 90 min at 37°C, then heat inactivated for 20 min at 80°C. 2 μl of the cDNA were used for each of the nested PCRs for TCRα or TCRβ , and 2.5 mM MgCl2), 500 μM dNTP, 0.3 U of Taq polymerase, 50 μM of TRACext- or TRBCext-specific primer for the constant region and an oligonucleotide mixture of 23 TRAVext or 19 TRBVext primers . For the second round of the nested reaction, 2 μL of the first reaction were combined with 18 μL of Taq buffer , and 2.5 mM MgCl2), 500 μM dNTP, 0.6 U of Taq polymerase, 50 μM of TRACint- or TRBCint-specific primer for the constant region and an oligonucleotide mixture of 23 TRAVint or 19 TRBVint primers . The PCR conditions for the first round of PCR were 94°C for 3 min followed by 35 cycles of 94°C for 20 s, 52°C for 45 s, and 72°C for 60 s, with a final extension at 72°C for 7 min. For the second round of PCR conditions were the same, but only for 26 PCR cycles. Contamination was monitored for all steps , by leaving 16 control wells empty per 96-well PCR plate sorted.Live lymphocytes were sorted as TCRβFor TCR product sequencing, a total of 12 μl of the products from the second round of the nested PCR amplification was combined with 1.5 μl of 10X shrimp alkaline phosphatase reaction buffer (200 mM Tris-HCl (pH 8.0) and 100 mM MgCl2), 1 U of shrimp alkaline phosphatase (Amersham Biosciences), and 1 U of exonuclease I (New England Biolabs), and water to total 15 μl. The reaction was then heated to 37°C 30 min, 80°C 10 min, and cooled to 4°C, and the product was subjected to automated sequencing (Dana-Farber/Harvard Cancer Center High-Throughput Sequencing Core). The sequences of the four TCRs cloned are shown in samuel.behar@umassmed.edu). Retroviral-mediated stem cell gene transfer was performed as previously described [TCR retroviral constructs were generated as 2A-linked single open reading frames using PCR and cloned into a murine stem cell virus-based retroviral vector with a GFP marker as previously described ,56. Detaescribed ,56.5 cells were plated in each well of a round bottom 96-well plate and incubated in the presence of TB10.44–11 peptide . Incubation in the presence of αCD3/αCD28  or in the absence of stimuli were used as positive and negative controls, respectively. Cells were incubated for 1 h at 37° C, at which point Golgi Stop solution  was added to each well for the remaining 4 h. Cells were collected after the 5 h stimulation and then surface stained with the antibodies described above, followed by intracellular staining for IFNγ (clone) using BD Permwash Kit  as per manufacturer’s instructions.5×10In vitro cytotoxicity was determined using peptide- coated EL4 target cells differentially labeled with the cell proliferation dye efluor 450 (eBiosciences) as previously described . Briefly+ T cells were purified from each suspension using the CD8+ T cell isolation kit and magnetic separation . After purification, cells were counted and transferred via the tail vein into congenically marked recipients (CD90.1), which had been infected 6–7 d earlier with virulent M. tuberculosis (Erdman) via the aerosol route. For priming experiments, 104 to 105 cells were transferred into each recipient. For competition experiments, cells were mixed at a 1:1 ratio  and then transferred into each recipient.Single cell suspensions of pools of spleens and LNs from naive retrogenic mice (6 to 10 wks post reconstitution) were prepared. CD8For analysis of cell proliferation of retrogenic cells after adoptive transfer, bead-purified naïve Rg cells (see above) were labeled with 10 μM of the cell proliferation dye efluor 450 (eBiosciences) in PBS for 20 min at room temperature, followed by extensive washing.+ T cells were purified from each suspension using the CD8+ T cell isolation kit and magnetic separation . After purification, cells were counted and mixed at a 1:1 ratio with peptide coated APCs in media containing 100U/mL of IL-2 and 10U/mL of IL-12. APCs used were red blood cell lysed splenocytes from naïve C57Bl6/j mice, pulsed with 10 μM of TB10.4 or SIINFEKL peptide at 37°C for 1 h in complete medium, followed by irradiation with 3200 Rads from a cesium-137 source and extensive washing. 2x106 cells were plated, 1 mL into each well of a 24 well plate. After 24 h, cells were fed with 1 mL of fresh media, containing 100U/mL of IL-2 and 10U/mL of IL-12. 48 h after the initial stimulation, cells were fed by removing 1 mL of culture media and addition of 1 mL of fresh media, containing 100U/mL of IL-2. 60–72 h after the initial stimulation, cells were extensively washed with complete media, and used for adoptive transfer experiments.Single cell suspensions of pools of spleens and LNs from naive retrogenic or OT-I mice were prepared. CD8M. tuberculosis infection as previously described [4 to 106 bead-purified activated Rg T cells  were transferred via the tail vein. Mice were infected with virulent M. tuberculosis (Erdman) via the aerosol route within 24 h of the adoptive T cell transfer. Three weeks after infection, the bacterial burden in the lung and spleen was determined. For survival experiments, naïve T cells (105 per mouse) were transferred into TCRα KO mice via the tail vein; following transfer, mice were infected with virulent M. tuberculosis (Erdman) via the aerosol route.An adoptive transfer model was used to analyze the ability of T cells to mediate protection against pulmonary escribed . BrieflyU test was used. To compare more than 2 groups, one-way ANOVA, followed by Bonferroni post-hoc test was performed. Differences with a p<0.05 were considered significant and represented by *.Population medians were used to compare TCR frequencies. Other data are represented as mean + SEM. For data with a verified for Gaussian distribution, a t-test was performed to compare two groups; otherwise, a Mann-Whitney S1 DataSummary of deep sequencing data of human TCRβs from lung granulomas and lymph node resected from subjects with tuberculosis.(PDF)Click here for additional data file.S2 DataA. The Vβ gene distribution is shown for two samples: (top) CD8+ T cells from a human lung granulomas; and (B) PBMC from a normal donor. One can see how differences in the Vβ gene use affects the calculation of clonality can be visualized. Numbers do not add to 100% because only productive recombination events are plotted. Width axis: V gene; Depth Axis: CDR3 length; Height axis: frequency. B. The Vβ gene distribution is shown for four samples from a single subject (#23): lung sample A, B, and C; and LN. Although there are considerable differences between the samples, particularly with respect to TCR diversity Click here for additional data file.S3 DataThe TCRβ frequencies, derived for the frequency of unique DNA sequences, in each sample analyzed by deep sequencing. The bar is the median and the error bars denote the interquartile range. For comparison, the TCRβ frequencies in PBMC from three normal donors are shown. Only productive recombination events are plotted.(PDF)Click here for additional data file.S4 Data10 transformation showed that these differences were significant (P<0.001) for all pairwise combinations by one-way ANOVA with Tukey’s multiple comparison test. These data show that TCRβ chains with the “DREN” CDR3 motif are increased in frequency among CD8+ T cells in tuberculous lung granulomas compared to peripheral blood T cells from healthy controls.The frequency of the “DREN” motif in the CDR3 region of TCRβ chains sequenced from human granulomas or PBMC samples from normal individuals. Multiple unique sequences were detected in these samples. The frequency of “DREN” motifs was elevated in lung granuloma compared to peripheral blood. Statistical testing after log(PDF)Click here for additional data file.S5 DataA. Schematic of the Vα2var locus of Vα2var mice. B. The TB10.44-11-specific CD8+ T cell response following aerosol infection of WT vs. Vα2var mice. C. Survival of C57BL/6, Vα2var, or TCRα knockout mice after low dose aerosolized Mtb.(PDF)Click here for additional data file.S6 Data4-11-specific CD8+ T cells. Testing using the Chi-square with Yates' correction shows that the frequency of R, D, or RD in the clonally expanded sequences is significantly increased in the four cloned sequences compared to the normal splenic repertoire of B6 mice (P<0.0001).To determine whether the enrichment of “R” or “D” was significant, we compared their occurrence in the normal TCRβ repertoire. We queried the splenic TCRβ repertoire from three C57BL/6 mice representing over 1.1 million reads and ~53,000 unique sequences each. The average frequency of “R”, “D”, or “RD” at CDR3β position 6, 7, or 6–7, was 10.1%, 7.5%, and 2.5% respectively, indicating “R,” “D,” and “RD,” were significantly enriched in the clonally expanded TB10.4(PDF)Click here for additional data file.S7 DataRg3 CD8+ T cells were transferred to Mtb infected mice as described in the methods. Three to six weeks after aerosolized Mtb infection, lung cells were stimulated with the TB10.4 epitope. Intracellular staining for IFNγ, IL-2, TNF, and granzyme were performed. The results shown are gate on Rg3 CD8+ T cells identified by their co-expression of GFP and Vα2(PDF)Click here for additional data file.S8 DataA. Protection mediated by 106, 105 or 104 in vitro activated Rg4  CD8+ T cells. T cells were transferred by the IV route into sublethally irradiated mice and challenged with Mtb. CFU were determined ~ 4 wks after infection. Protection (Δlog10) is the lung CFU in mice that did not receive Rg T cells minus the lung CFU in mice that did receive Rg T cells. Two independent experiments are shown each with 5 mice. One way ANOVA with Tukey’s post test to compare differences in CFU. *, p<0.05; ***, p<0.001. B. The level of TCRα staining on Rg3 vs. Rg4 CD8+ T cells from the lungs of infected mice. T test showed the difference was not significant. C. Priming of naïve Rg4 and Rg3 CD8+ T cells in the pulmonary LN of Mtb infected mice occurs with similar kinetics. Rg3 and Rg4 CD8+ T cells were co-transferred or Rg3 CD8+ T cells were transferred alone. Priming is indicated by acquisition of an activated (CD44+CD62Llo phenotype.(PDF)Click here for additional data file.S9 DataList of the TCRα and TCRβ primers used for nested PCR for the single cell analysis of TCRs used by TB10-specific CD8+ T cells from the lungs of Mtb-infected mice.(PDF)Click here for additional data file.S10 DataTCR sequences and 2A peptide cloned into retroviral vectors. Sequences of four TCR sequences as assembled and cloned into the retroviral vectors. The first sequence is color-coded. VαJα (blue); Cα (purple); P2A (grey); VβDJβ (yellow); Cβ (green).(PDF)Click here for additional data file.


Benzoquinazoline compounds have been reported to exhibit an interesting antiviral activity. This work aims to study and evaluate the antiviral activity of a newly prepared 2-thioxo-benzoquinazolin-4(3H)-one agents.The obtained results gave us some useful insights about the characteristic requirements for future trials to build up and design more active and selective antiviral 2-thioxo-benzo[ In contrast to HSV-1 and 2 which classified as enveloped double-stranded DNA viruses, CVB-4 is non-enveloped RNA viruses. They are common human pathogens and considered a significant worldwide health concern -isoquinoline-1,3-diones have been reported as antiherpetic agents quinazolin-4(3H)-one derivatives against HSV-1, HSV-2 and CVB4 viruses.In view of these evidences and an extension of our ongoing research on benzoquinazolines chemistry, we herein report the antiviral evaluation of a new series of 2g]quinazolines 1–28  with regard to ribavirin (16.38). However, 6, 7, 8, 11, 16, 17, 18, 22, 25, 27 and 28 exhibited moderate activity against CVB4, with SI values in the range of 2.05‒3.31. Moreover, compound 5 demonstrated good activity against HSV-1 and HSV-2  and 24 was active against HSV-1 (SI = 2.61) in relation to ribavirin (41.93 and 24.69).We previously reported our findings regarding the antiviral activity of isoquinazoline and triazoloquinazoline derivatives. The results suggested that quinazolines can be good platform for designing a new antiviral agent –13. Here1–3 afforded new structural features (5–28) with a wide range of effects against the HSV and CVB4 viruses. For instance, S-alkylated products 7–28 exhibited significant activity against Coxsackie B4. In particular, compounds 7–9, 11, 15–18, 21, 22, 24, 25, 27 and 28 were more active than their parents 1–3. Moreover, variations in the type of the N-alkyl and S-alkyl  groups resulted in variations of the activity, in which compound 9 represented against CVB4 as the most active among the S-alkylated compounds (SI = 5.57). Compounds 15, 21, 24 and 28 showed a pronounced activity against CVB4 . In regard to anti-herpes activity, compound 1 was inactive, but its S-alkylated products 7–15 exhibited slight activity. Similarly, the parents 2 and 3 appeared less active than their chemically transformed products 16–21 and 22–28, respectively. However, hydrazino products 5 and 6 offered more advantages in terms of activity against HSV and CVB4 viruses. Depending on the values of the SI-parameter, 5 gave rise to the greatest activity against HSV-1 (4.28), followed by HSV-2 (5.18) and CVB4 (6.27). Moreover, the presence of the butyl group at the “R” position provided a significant effect against CVB4 and HSV viruses. This effect can be seen in both S-alkylated and hydrazino derivatives. However, the “R1” position requires a hydrophobic moiety to provide a selective antiviral activity against CVB4, as in compound 9. On the other hand, compound 5 exhibited a non-specific antiviral activity against CVB4 and HSV viruses. This effect also can be seen with compound 24 that has a 3-cyanobenzyl moiety at “R1” position but with a phenyl group instead of butyl at “R” position.In outlining the results in Table 1–28 against the X-ray crystal structure of Coxsackievirus B4 2A proteinases (Protein Data Bank (PDB): 1Z8R)  to each well, including the untreated controls. The 96-well plates were then incubated at 37 °C and 5 % COle cells , 22, 26.50 was estimated with respect to the virus control from the graphic plots, using STATA modelling software and (SI) calculated from the ratio of CC50 to EC50 [The viral inhibition rate was calculated as follows: to EC50 , 26.2 for 4 h. An 85 μL aliquot of the medium was removed from the wells, and 50 μL of DMSO were added to each well, mixed thoroughly with the pipette, and incubated at 37 °C for 10 min. Then, the optical density was measured at 590 nm with the microplate reader  to determine the number of viable cells. Without added stain, all obtained findings were corrected for background absorbance detected in wells. In the absence of the tested compounds, treated samples were compared with the cell controls. All experiments were carried out in triplicate. The cytotoxicity of each tested compound was calculated [The procedure for seeding and incubation of Vero cells was explained in details in previous research , 23, 27.lculated , 27, 28.®, is as follows: 50.The percentage cell viability, calculated using Microsoft ExcelStatistical analysis was done using a one-way ANOVA test . All exphttp://www.chemaxon.com, 2013) with MM force field, and saved in Tripos mol2 file format. MolDock score functions were used with a 0.3 A° grid resolution. Prior to the calculation of the MolDock scores of the tested compounds, the MVD software was benchmarked docking ribavirin [The modelling studies were done by a PC with Intel© Core™ i7-3630 QM CPU  operating under the Windows 7 Professional Operating System . The modibavirin .


Background: HSF1 is the major eukaryotic transcription factor that regulates expression of HSP genes.Results: We identify two novel HSF1 isoforms and show that HSF1 isoforms differentially regulate chaperone gene transcription.Conclusion: HSF1 isoforms work synergistically, and the ratio of HSF1 isoforms determines chaperone gene transcription levels.Significance: Our findings unravel an additional layer of chaperone gene regulation through modulation of HSP expression by HSF1 isoform ratios. Hsf1γ isoforms are expressed in various mouse tissues and are translated into protein. Furthermore, after heat shock, HSF1γ isoforms are exported from the nucleus more rapidly or degraded more quickly than HSF1α or HSF1β. We also show that each individual HSF1 isoform is sufficient to induce the heat shock response and that expression of combinations of HSF1 isoforms, in particular HSF1α and HSF1β, results in a synergistic enhancement of the transcriptional response. In addition, HSF1γ isoforms potentially suppress the synergistic effect of HSF1α and HSF1β co-expression. Collectively, our observations suggest that the expression of HSF1 isoforms in a specific ratio provides an additional layer in the regulation of heat shock protein gene transcription.The heat shock response, resulting in the production of heat shock proteins or molecular chaperones, is triggered by elevated temperature and a variety of other stressors. Its master regulator is heat shock transcription factor 1 (HSF1). Heat shock factors generally exist in multiple isoforms. The two known isoforms of HSF1 differ in the inclusion (HSF1α) or exclusion (HSF1β) of exon 11. Although there are some data concerning the differential expression patterns and transcriptional activities of HSF2 isoforms during development, little is known about the distinct properties of the HSF1 isoforms. Here we present evidence for two novel HSF1 isoforms termed HSF1γα and HSF1γβ, and we show that the HSF1 isoform ratio differentially regulates heat shock protein gene transcription.  Organisms often face temperature changes in environmental conditions. Molecular chaperones, also termed heat shock proteins, are induced as a cellular response to this proteotoxic stress. Eukaryotes possess a family of transcription factors that regulate this stress response program: the heat shock transcription factors. To date seven members of this family have been identified: HSF1,5–11–HSF1, HSF2, and HSF4 are all known to be expressed as two isoforms with tissue specific patterns of expression , 18. UnsHSF1 mRNA does seem to be constitutively expressed and transcription is not induced by heat stress  (m1Ijb/J)  were purm1Ijb/J) . GenomicR)-2-amino-7-((R)-4-fluoro-2-(6-methoxypyridin-2-yl)phenyl)-4-methyl-7,8-dihydropyridopyrimidin-5(6H)-one) (NVP-HSP990 ((6H)-one)  was obtaHsf1 transgenic animals were interbred to obtain homozygous Hsf1 knock-out and wild type littermates. Tissue from two ear punches from the same animal for each cell line was sterilized with 70% ethanol (v/v), chopped into small pieces, and put in a 12.5-ml flask with fibroblast medium . The tissue pieces were incubated at 37 °C until they attached and cells started to migrate. This was defined as passage 0. Cells were subsequently incubated with trypsin solution and transferred into fresh medium. All experiments were performed between passages 3 and 6.Heterozygous Flasks or multiwell plates were sealed with Parafilm and submerged in a water bath. Heat shock treatment was 42 °C for 45 min unless otherwise stated. Flasks or plates were put back at 37 °C to let the cells recover from heat shock. Non-induced cells were maintained at 37 °C.18) oligonucleotide. 5% of the cDNA reaction was used as template for the isoform RT-PCR assay with primers HSF1iso-t2_f (5′-TCAGCGTAGCCTGCCTAGACAA), HSF1iso-t2_r (5′-GCTCTTGTGGAGACAGAAGCTCC), GAPDH_new_r (5′-GTGGGTGCAGCGAACTTTATT), and GAPDH_111_f (5′-CACTGAGCATCTCCCTCACAATT). The PCR conditions were: 95 °C for 2 min, 30 cycles of 95 °C 20 s, 60 °C 20 s, 72 °C 45 s, and a final 6 min at 72 °C. PCR products were separated on a 3% MetaPhor agarose gel (Lonza). Bands were digitally visualized on a Benchtop UV transilluminator (UVP) and quantified using the Image Studio Lite version 3.1 software (LI-COR).RNA from tissue or cells was extracted using QIAZOL together with RNeasy mini kits (Qiagen) according to the manufacturer's instructions. 1 μg of RNA was reversed-transcribed using an oligo(dT) (THsf1 isoforms were amplified from oligo(dT) reverse-transcribed wild type RNA. Primers for untagged isoforms were mHSF1_kozak_f (5′-GCCGCCATGGATCTGGCCGTGGGCC), and primers for FLAG-tagged isoforms were mHSF1_FLAG_kozak_f (5′-GCCGCCATGGATTACAAGGATGACGATGACAAGGGTCTTTTAATGGATCTGGCCGTGGGCC), both together with mHSF1_rev (5′-CTAGGAGACAGTGGGGTCCTTGG). PCR products were cloned into pCR8/GW-TOPO vector (Life Technologies). Both untagged and FLAG-tagged versions of Hsf1α and Hsf1γα were obtained. To generate the other isoforms, the α/β-cassette (exon 10 to exon 13) was exchanged by PstI/SacI digest. All plasmids were verified by sequencing, with National Center for Biotechnology Information (NCBI) accession number NC_000081.6 from base pairs 76477389 to 76500978 being used as a reference. Hsf1α isoforms contain a silent mutation in exon 9 (A1068G from the ATG start codon), Hsf1γα isoforms contain a silent mutation in exon 11 (G1323A from the ATG start codon), and Hsf1β and Hsf1γβ have no mutations. The gateway entry vectors were subsequently inserted into pT-RExT-DEST30 to give the final expression plasmids. All plasmids are driven by the CMV promoter. The firefly luciferase gene was cloned with primers FFLuc_for (5′-GCGCTCGAGAAGCTTGGATCCGCGGTACCATGGAAGACGCCAAAAACATAAAG) and FFLuc_rev (5′-CGCGAGCTCTTACAATTTGGACTTTCCGCC) into pcDNA3 to give plasmid pcFFLuc. The Hspa1a/b promoter was cloned into pcFFLuc with primers hsp70_for (5′-GCGCTCGAGCCCCAGAAACCTCTGGAGAGT) and hsp70_rev (5′-CGCGGTACCGCGCTCTGCTTCTG). All plasmids were transfected with jetPRIME (Polyplus-transfection SA) according to the manufacturer's instructions. The amount of DNA for each transfection, including the isoform combinations, was kept constant . Until otherwise noted, all experiments were performed 48 h after transfection.m Tris-Cl, pH 7.4, 150 mm NaCl, 0.1% (w/v) Tween 20) for 5 h at room temperature. HSF1γ preimmune serum was used at 1:5000 in TBS-T for 5 h at room temperature. Other antibodies and dilutions were GAPDH  1:1000, HSF1  1:500, HSF2  1:75, HSF3  1:100, HSF4  1:200, β-actin (ACTB)  1:10000, HSP70  1:1000, HSP40  1:1000, and HSP25  1:1000. Blocking buffer was 5% (w/v) skimmed milk powder in TBS-T. Secondary antibodies were purchased from LI-COR, and Western blots were visualized on an Odyssey Sa (LI-COR) and analyzed with the Image Studio Lite version 3.1 software (LI-COR). Membranes were stripped from bound antibody by incubation at 55 °C for 30 min with three changes of buffer during this time . After stripping, membranes were extensively washed in TBS-T and reblocked.Anti-HSF1γ antibody was raised against peptide LARAPQMSGVARLFPCPSS in rabbit (Davids Biotechnologie GmbH) and used 1:150 in TBS-T ), used immediately, and never frozen. Sample concentration was determined by measuring absorbance at 280 nm or using a BCA assay (Thermo Scientific). About 5 mg of total protein was incubated with either 3 μg of anti-HSF1 (Abcam) antibody or 3 μg of rabbit IgG . Immunoprecipitation (IP) was carried out overnight at 4 °C. On the next day, 10 μl of preblocked  magnetic Dynabeads protein G beads (Life Technologies) was added, and the IPs were incubated for another 1.5 h at 4 °C. IPs were washed four times with 0.5 ml of cold lysis buffer.Mouse tissue was homogenized in ice-cold lysis buffer (PBS (Life Technologies), 1% (w/v) Triton X-100, 1 ml-lysine-coated coverslips in 12-well plates. Cells were washed with PBS and fixed for 16 min at room temperature with 4% formaldehyde (Sigma) in PBS. Cells were washed twice with PBS followed by permeabilization with 0.1% Triton X-100 (w/v) in PBS for 5 min. Cells were washed with PBS and blocked for 20 min at room temperature with 3% skimmed milk powder (w/v) in PBS. Coverslips were incubated overnight at 4 °C with anti-FLAG antibody  1:220 in block buffer with 0.5% Tween 20. Cells were washed three times with TBS-T and incubated for 1 h at room temperature with A488-coupled goat-anti-rabbit secondary antibody (1:1000 in PBS with 1 μg/ml DAPI). Cells were washed three times with TBS-T and mounted in VECTASHIELD mounting medium (Vector Laboratories). Images were taken on an Eclipse Ti-E inverted CSU-X1 spinning disk confocal with the NIS-Elements C software (Nikon).Cells were grown directly on poly-Hspa1a/b, Dnajb1, and Hspb1 were purchased from Eurofins MWG. Sequences are as follows: Hspa1a/b-F, 5′-GGTGGTGCAGTCCGACATG; Hspa1a/b-R, 5′-TTGGGCTTGTCGCCGT; Hspa1a/b-P, 5′-CACTGGCCCTTCCAGGTGGTGAA; Dnajb1-F, 5′-CCCCATGCCATGTTTGCT; Dnajb1-R, 5′-GCGCTGCCCAAAAAAGG; Dnajb1-P, 5′-TCTTCGGTGGCAGAAACCCCTTTGA; Hspb1-F, 5′-CACTGGCAAGCACGAAGAAAG; Hspb1-R, 5′-GCGTGTATTTCCGGGTGAAG; and Hspb1-P, 5′-ACCGAGAGATGTAGCCATGTTCGTCCTG.TaqMan RT quantitative PCR, and the evaluation of the data was performed as described previously . The quaHsf1 expression plasmid, HSP promoter-driven firefly luciferase , and thymidine kinase promoter-driven Renilla (Renilla reniformis) luciferase. The amount of DNA for each transfection, including the isoform combinations, was kept constant. The Dual-Luciferase reporter assay system (Promega) was used according to the manufacturer's instructions. Luminescence signals were read on an Orion II microplate luminometer (Berthold Detection Systems).Cells were seeded in white 96-well plates (Thermo Scientific) and transfected with 100 ng of each p values less than 0.05 were considered as statistically significant.Data were screened for outliers using Grubb's test (GraphPad). Statistical significance was calculated using SPSS (IBM). Analysis of variance with Tukey's post hoc test was used to calculate significance. Hsf1α transcript. This novel exon was present in ∼10% of the clones that were analyzed. Sequencing allowed us to map this exon to a genomic sequence of Hsf1 at base pairs 21688 to 21771 (NCBI accession 15499) from the transcription start site (A). We termed the two novel transcripts Hsf1γα and Hsf1γβ, with Hsf1γα containing exon 9a and exon 11 and Hsf1γβ containing only exon 9a. The genetic structure of the four Hsf1 isoforms is shown in Fig. 1B. Exon 9a contains 84 bp and consequently does not cause a frameshift when included. The amino acid sequence encoded by exon 9a is shown in Fig. 1C. In Fig. 1C, the asterisks mark hydrophobic amino acids in a potential heptad repeat spacing. Although cDNA clones containing exon 9a sequences can be found in the NCBI database, this sequence was never assigned an official HSF1 exon status. A BLAST analysis of the exon 9a amino acid sequence against the NCBI database for mammals found that the murine exon 9a is highly similar to other mammalian homologues .We first discovered the additional exon 9a, which gives rise to the two novel HSF1 isoforms, during the cloning of a full-length art site A. We terHsf1 isoforms in different tissues, we used an RT-PCR assay, which can distinguish between all four isoforms (A). This was run on cDNA from six peripheral tissues and eight brain regions from 8-week-old wild type mice (B). The identity of the isoform bands was confirmed by sequencing. In addition to the four major Hsf1 isoforms , we could detect multiple additional bands, all specific for Hsf1 sequences as confirmed by sequencing. The three asterisks in Fig. 2B mark isoforms, with retained partial intronic sequences that were not differentially expressed across tissues. In contrast, the two immature isoforms labeled as unspliced 1 (Hsf1α with retained intron 11) and unspliced 2 (Hsf1α with retained introns 10 and 11) did show differences in expression levels between tissues . The expression levels of the four mature Hsf1 isoforms and the two immature isoforms were quantified in Fig. 2C. The intensities of all bands in each tissue were normalized to the GAPDH band intensity, and the mean expression of the normalized intensities between experiments was calculated. Either the sum of all Hsf1 isoform band intensities for each tissue was set to 100% , or only the sum for the four major Hsf1 isoform was set to 100% (D and E). Some of the peripheral tissues showed a very high level of the immature Hsf1 isoforms . Hsf1 isoform ratios in brain regions were much more homogenous. Overall, the γ isoforms constituted about 10% of all Hsf1 isoforms in the peripheral tissues and about 15% in the brain regions. The proportion of the Hsf1γα isoform was quite variable in peripheral tissues, with testes showing exceptionally high levels of about 22% . The Hsf1γβ isoform was expressed at almost 3-fold higher levels in brain regions than in peripheral tissues . The periphery also showed a slightly lower percentage of α isoforms and a slightly higher level of β isoforms. These Hsf1 isoform ratios remained highly comparable in tissues from mice that had been treated with HSP990 to induce the heat shock response (compare D and E). The only significant difference  was a lower percentage (about 2-fold) of the Hsf1γβ isoform in the hippocampus, striatum, and brain stem after HSP990 dosing (compare D and E).To further analyze the expression of isoforms A. This wype mice B. The idHsf1 isoforms were translated into protein in vivo, we generated an antibody against the exon 9a peptide. Validation of the anti-HSF1γ antibody in an Hsf1 knock-out cell line showed that it indeed recognized the two HSF1γ isoforms, but not HSF1α nor HSF1β . A commercially available antibody against HSF1 , which was raised against full-length HSF1 (epitope: amino acids 288–439), recognized all four isoforms . We detected the highest level of total Hsf1γ expression with our RT-PCR assay in testis. Therefore, we immunoprecipitated HSF1 from the testes of 8-week-old wild type and Hsf1 knock-out mice and probed with the anti-HSF1γ antibody . We could detect a signal in the IP of HSF1 in wild type mice, but not in the knock-out mice. The very small difference in size of only 2.3 kDa between HSF1γα and HSF1γβ made it impossible to separate the two isoforms on a gel. This paradigmatic result demonstrates that HSF1γ isoforms are translated into protein in vivo.To determine whether the two novel Hsf1 knock-out mice. Using the same RT-PCR assay as before, we analyzed the isoform ratios in the wild type fibroblast lines (A). Only miniscule amounts of the Hsf1γ isoforms could be detected. The ratio of Hsf1α to Hsf1β was about 2 to 1 and did not significantly change after heat shock (A). Furthermore, we analyzed the expression of HSF2, HSF3, and HSF4 in the fibroblast lines and found that there was no apparent change in the basal expression of any HSFs between the wild type and Hsf1 knock-out lines, nor after heat shock (B). On the other hand, the induction of heat shock proteins, as reported previously (B). Interestingly, HSP70 (Hspa1a/b) and HSP40 (Dnajb1) levels under basal conditions were not affected by the Hsf1 knock-out . Because the duplicate cell lines for both the wild type and the Hsf1 knock-out cell lines were identical regarding isoform ratio and induction of heat shock proteins, we only focused on one line for each genotype in the following experiments.We next developed a cell model system to characterize the individual HSF1 isoforms in more detail. We established two fibroblast lines each from wild type and from st lines A. Only mat shock A. Furtheat shock B. On theeviously , dependseviously B. IntereHsf1 knock-out cell line resulted in readily detectable signals at the same apparent molecular weight as in the wild type cell line . This implied a much higher expression level of the individual HSF1 isoforms when compared with their endogenous counterparts, most likely because their expression was under the control of the very strong CMV promoter. However, all isoforms were expressed to a similar level when compared with each other . HSF1 activation by heat stress induces a shift of the HSF1 bands toward higher molecular weight (hypershift) due to extensive post-translational modifications . This hypershift was still visible even 4 h after heat shock, although at this time point, the very high molecular weight signals had diminished, indicating a deactivation of HSF1 . Taken together, all HSF1 isoforms were extensively post-translationally modified after heat shock and were activated with similar kinetics when compared with each other and with the wild type situation.Individual isoforms expressed in the ell line C, basal.ications . A time Hsf1 knock-out fibroblasts (data not shown). Hence, we used the N-terminal FLAG epitope-tagged HSF1 isoforms to analyze their intracellular distribution. When we analyzed the transfected fibroblast lines, we observed small DAPI-positive foci appearing . However, this did not occur when DNA was omitted, i.e. when only the components of the transfection system were used . We concluded that the polyethylenimine-DNA complexes were very stable and could be stained with DAPI. In the non-induced situation, the HSF1α isoform showed a relatively high level of signal in the nucleus, whereas the other isoforms showed almost exclusive cytoplasmic staining . Consistent with the observed hypershift due to extensive post-translational modifications (C), the HSF1 isoforms could only be detected in the nucleus immediately after heat shock , and a shorter heat shock of only 15 min at 42 °C was also sufficient to induce nuclear translocation of the HSF1 isoforms . At 2 h after heat shock, we started to detect a cytoplasmic signal for the HSF1γ isoforms, which became more pronounced at 3 h after heat shock . At both time points, HSF1α and HSF1β were still predominantly localized to the nucleus . The HSF1γβ isoforms could not be detected in the nucleus from 3 h after heat shock onwards . At 4 h after heat shock, a cytoplasmic signal was also detectable for HSF1α and HSF1β . However, HSFα, HSF1β, and HSF1γα still had a rather large fraction of signal visible in the nucleus . At 6 h after heat shock, the intracellular distribution of the isoforms was comparable with the non-induced conditions .We used immunofluorescence to study the intracellular localization of HSF1 isoforms in non-induced conditions or after heat shock. Our attempt to detect endogenous HSF1 with different antibodies ; Santa Cruz Biotechnology ; StressGen (ADI-SPA-901); NeoMarkers (Ab-3 10H8)), in a variety of conditions, was unsuccessful as in each case, we observed a positive signal in the ications C, the HSHspa1a/b (Hsp70) and Dnajb1 (Hsp40) was not significantly changed between the Hsf1 knock out line and wild type cells, and the comparative expression levels did not change on expression of the individual isoforms (A and C). Only Hspb1 (HSP25) showed a significantly lower amount of transcripts in the knock-out line when compared with wild type . This reduced level could not be rescued by the expression of individual isoforms . However, expression of combinations of Hsf1 isoforms under non-induced conditions resulted in higher levels of transcripts for all three HSP genes, when compared with the expression of individual isoforms . The amount of DNA used for transfections was kept constant for all conditions, and the relative isoform proportions were (given in %): α and β = 68.7 and 31.3; α, β and γα = 64.2, 29.2, and 6.6; α, β, and γβ = 67.3, 30.6, and 2.1; α, β, γα, and γβ = 62.7, 28.6, 6.6, and 2.1. These proportions were derived from the average levels in mouse peripheral tissues (with testis excluded) as determined in Hsf1γα isoform and 2.1% for the Hsf1γβ isoform. Similar to the induction of heat shock proteins in the wild type fibroblast line , HSP genes were highly induced 2 h after heat shock (Hsf1+/+). Consistent with previous publications . However, each individual isoform was sufficient to significantly induce transcription at the Hspa1a/b locus after heat shock . Hsf1α, Hsf1β, and Hsf1γα were sufficient to significantly induce Dnajb1, and for the Hspb1 gene, Hsf1α and Hsf1γα were sufficient to significantly induce transcription . As for non-induced conditions, expression of Hsf1 isoform combinations resulted in higher levels of transcripts for the respective HSP gene after heat shock when compared with expression of the individual isoforms . Although this was not statistically significant, we observed a clear trend for this synergistic enhancement of transcription induction for all three HSP genes .Under basal conditions, expression of isoforms , A and CHsf1 combinations were used, which was more pronounced when the cells were heat-shocked . Furthermore, as for the endogenous Hspa1a/b gene, expression of the individual Hsf1 isoforms was sufficient to significantly induce transcription after heat shock (H). In this assay, the individual isoforms induced transcription of the luciferase reporter after heat shock to a similar extent as the wild type cell line . There was no significant difference in luciferase signal between the individual isoforms, although the α isoform showed a trend toward higher activity . Expression of a combination of the Hsf1α and Hsf1β isoform in a ratio of about 2:1 (see above) resulted in a highly significant synergistic induction after heat shock when compared with the signal for the individual isoforms . Interestingly, the addition of either of the Hsf1γ isoforms to the Hsf1α and Hsf1β mixture significantly reduced the signal after heat shock . The signal for the combination of all four HSF1 isoforms was reduced when compared with the Hsf1α and Hsf1β combination, although not significantly . In summary, our data indicate that the ratio of HSF1 isoforms mediates the level of HSP expression in non-induced and heat shock conditions and that HSF1 isoforms can synergistically work to induce transcription of HSP genes.Generally, we noticed a higher variability in transcript levels when -shocked . This coons used G. Furtheat shock H. In thiThe mammalian heat shock factor 1 was first cloned 23 years ago . It soonHsf1 isoforms in different tissues were not fully comparable on an absolute level because the GAPDH signals might differ between tissues, However, Fig. 2C clearly shows considerable differences in the ratios of the four major isoform transcripts as well as some immature isoforms between tissues. Interestingly, tissues that induce the heat shock response to a much lower extent, such as e.g. the brain regions when compared with skeletal muscle . The high levels of immature Hsf1 transcripts in some tissues (e.g. spleen) open the possibility that HSF1 protein levels are not regulated by changes in transcription rates, but rather through modulation of splicing. This observation is consistent with previous reports that HSF1 expression is stable and not influenced by stress  F1. In this study, we analyzed the Hsf1 isoform ratios in 8-week-old mice. It would be very interesting to determine whether these ratios change during aging, which could explain some of the changes in the proteostasis capacity of aged tissue . This additional sequence could therefore easily influence the regulation of HSF1 activity, as well as its DNA binding status or protein stability. The activation of HSF1 isoforms after heat shock, which is composed of nuclear import concomitant with post-translational modifications (C and 5B), seems to be similar between the isoforms. We could detect extensive and comparable post-translational modifications for each isoform as indicated by a hypershift on Western blot (C). However, we cannot exclude the possibility that individual isoforms are modified with distinct patterns of post-translational modifications. A decrease in the hypershift begins to occur between 2 and 4 h after heat shock, indicating an attenuation of HSF1 activation . At the same time points, the HSF1 isoforms start to appear in the cytoplasm (B). Interestingly, for the two HSF1γ isoforms, a cytoplasmic signal is evident by 2 h after heat shock when compared with 4 h for the α and β isoforms (B). At 4 h after heat shock, all HSF1 isoforms exhibit a strong cytoplasmic signal, yet they are still to some extent post-translationally modified . These modifications likely represent inhibitory signals for the transcriptionally active HSF1, e.g. phosphorylation on Ser-303 followed by sumoylation on Lys-298 . The steady state localization of HSF1 is however somewhat controversial. Very often, predominant nuclear localization of HSF1 has been reported . Many studies also rely on GFP-fused constructs to detect intracellular localization, with the problem of GFP having an intrinsic propensity to localize to the nucleus . Taken together, these data are in good agreement with the proposed constitutive nuclear import of HSF1 isoforms . The activation temperature of HSF1 has been shown to be dependent on the average environmental temperature in which the organism lives (–B) might contribute to an adaption mechanism to fast changing environmental temperatures.Intriguingly, mouse testes showed a very high level of the 1 levels D. The acsm lives –49, but m lives –, 50, 51.m lives –, 50. Thum lives –B might cHsf1 does not lead to significantly changed levels of Hspa1a/b (HSP70) or Dnajb1 (HSP40). Hspb1 (HSP25), however, is down-regulated at both transcript and protein levels (see Ref. B and 6E). The expression of many HSP genes depends on the concerted action of multiple transcription factors, e.g. members of the V-maf musculoaponeurotic fibrosarcoma oncogene homolog (MAF) family, PAX6, NF-Y, NF-κB, or cAMP-response element-binding protein (CREB) (C) does not result in elevated levels of HSP transcripts under basal conditions (Knock-out of see Ref. B and 6E.n (CREB) , 53. Thin (CREB) C does nonditions , basal.Hspa1a/b in non-induced cells, we observed a statistically significant higher expression when combinations of isoforms, in particular Hsf1α and Hsf1β, were expressed (A). We also observed a similar pattern of Dnajb1 expression, although this did not reach statistical significance (C). Furthermore, expression of the individual isoforms was not sufficient to rescue the reduced level of Hspb1 in non-induced conditions (E). However, expression of HSF1 isoform combinations restored the basal expression levels of Hspb1 to be similar to wild type levels (E). In general, the HSF1α isoform seems to have the highest transcription activation potential of the four isoforms, although we did not observe statistically significant differences between the individual isoforms , a synergistic effect for HSF1 isoform combinations under basal conditions (G). However, 2 h after heat shock, we saw a dramatic, highly statistically significant synergistic enhancement when expressing HSF1α and HSF1β together when compared with the expression of only the individual isoforms (H). It is intriguing that the average ratio of Hsf1α to Hsf1β is ∼2 to 1 (4A). Therefore, stochastically each HSF1β isoform could heterotrimerize with two HSF1α isoforms. This 2 to 1 ratio could potentially create a different structure in the trimeric transcription activation domain and thereby influence HSF activation, for example by inhibiting binding of HSP70 and HSP40  by almost completely abolishing the synergistic effects of Hsf1α and Hsf1β co-expression. Such antagonistic functions of isoforms generated from the same transcript have been previously described for other transcription factors, e.g. acute myeloid leukemia 1 (AML1)   and cAMP1 (AML1) . Hence, 1 (AML1)  could beTaken together, our data provide proof for the existence of two novel HSF1 isoforms, which are expressed across a wide range of mouse tissues. We have also shown for the first time that the ratio of HSF1 isoforms determines the rate of heat shock protein gene transcription, under both non-stressed and heat shock conditions. Furthermore, expression of isoforms in a distinct ratio leads to a synergistic enhancement of transcription of HSP genes. In contrast, the physiological function of the HSF1γ isoforms might be to attenuate the level of transcriptional activation by the two major isoforms, HSF1α and HSF1β. We propose that HSF1 isoform ratios are a major factor in the regulation of the expression of heat shock protein genes and that modulation of this ratio could influence the stress response capacity of various tissues.


OUT) is compatible with scanning mRNA for the AUG start codon. Base pairing with AUG is thought to promote isomerization to a more stable conformation (PIN) that arrests scanning and promotes dissociation of eIF1 from the 40S subunit. Here, we present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 Å resolution with initiator tRNA in the PIN state, prior to eIF1 release. The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2. The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2α, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon.During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (P  •Structure of a partial yeast 48S translation initiation complex at 4.0 Å resolution•A distorted initiator tRNA is trapped in the act of recognizing the start codon•The codon-anticodon duplex is stabilized by the N-terminal tail of eIF1A•eIF2α makes contacts with key nucleotides at the −2 and −3 positions of the mRNA i in the act of recognizing the start codon prior to eIF1 release. This snapshot of the transition from scanning to codon recognition provides a framework for numerous genetic and biochemical findings underlying the current model for eukaryotic translation initiation.The cryoEM structure of a partial yeast 48S preinitiation complex at 4.0 Å resolution shows initiator tRNA The 43S PIC subsequently binds the 5′ end of the mRNA, a step promoted by eIF4F, eIF4B, and eIF3, and scans the 5′ untranslated region (UTR) of the mRNA for an AUG codon in suitable sequence context. Recognition of the AUG codon by the anticodon of tRNAi leads to conversion of eIF2 to its GDP-bound form, release of eIFs, and joining of the 60S subunit to form the elongation-competent 80S ribosome with tRNAi and the start codon base paired in the 40S P site.Eukaryotic translation initiation involves at least 12 initiation factors (eIFs) . The facOUT) that allows tRNAi to sample successive triplets entering the P site for complementarity to the anticodon. The unstructured C-terminal tail (CTT) of eIF1A plays a key role in stabilizing this open/POUT PIC conformation (i) is blocked by the presence of eIF1 in the complex. Base pairing of tRNAi with an AUG triplet evokes a rearrangement of factors in the PIC—including displacement of eIF1 and possibly the eIF1A CTT from their locations near the P site—and movement of the eIF1A CTT toward the GAP domain of eIF5  included eIF3, TC, and RNA helicase Dhx29 but lacked eIF1, eIF1A, and eIF5  containing eIF1, eIF1A, mRNA, and TC at an overall resolution of 4.0 Å. The structure shows tRNAKluyveromyces lactis, initiation factors eIF1, eIF1A, eIF3, eIF5, and TC  from Saccharomyces cerevisiae, and an unstructured 25 nucleotide mRNA containing an AUG codon but lacking a 5′ cap. Although eIF4F, eIF4B, and eIF3 are not required for PIC assembly with this model mRNA (K. lactis and S. cerevisiae are closely related species (S. cerevisiae initiation factors form a well-defined PIC with K. lactis 40S subunits in vitro (K. lactis because they better tolerate the slightly acidic pH (6.5) chosen to minimize deacylation of Met-tRNAi  from the canonical P/P state, the P/I state observed in the pm48S (i (eP/I state) observed in the pm43S   B. Most shenotype , indicathenotype . MoreoveIN state .IN state and blocks initiation at non-AUG codons, whereas changing its identity to U31:A39 , in a loop of eIF2α-D1 close to mRNA  was commercially synthesized by Integrated DNA Technologies. The py48S was reconstituted by incubating 120 nM 40S with eIF1, eIF1A, TC, eIF3, eIF5, and mRNA in the ratio of 40S:eIF1:eIF1A:TC:eIF3:eIF5:mRNA::1:2.5:2.5:1.5:1.2:2.5:2. The sample was directly used to make cryo-EM grids without further purification.rlier in . eIF3 anescribed . Mutant escribed . An unst−/Å2) using a defocus range of 1.6–4.0 μm. Images were recorded manually on a back-thinned FEI Falcon II detector at calibrated magnification of 104,478 (yielding a pixel size of 1.34 Å). An in-house system was used to intercept the videos from the detector at a speed of 16 frames/s exposures, as described earlier  estimated as in (Statistical movie processing was also done as described previously  in ordered as in .S. cerevisiae 40S ribosome (PDBID 3U5B and 3U5C; The atomic model of i was derived from tRNAs from the archaeal TC (PDB 3V11; 4KZZ; 3D models of eIFs were generated with I-TASSER . An initT.H., J.L.L., I.S.F., A.M., and P.M.-M. made the samples. T.H., J.L.L., and C.G.S. collected the data. T.H. and J.L.L. performed the structure determination and analyzed the data. J.R.L., A.G.H., and V.R. supervised the work and helped to write the manuscript.


We reveal the identification of a polybasic motif necessary for polyphosphoinositide interaction and nucleolar targeting of ErbB3 binding protein 1 (EBP1). EBP1 interacts directly with phosphatidylinositol-triphosphate and their association is detected in the nucleolus, implying regulatory roles of nucleolar processes. P3 together with EBP1 in the nucleolus. Using NMR, we further demonstrate an association between EBP1 and PtdInsP3 via both electrostatic and hydrophobic interactions. Taken together, these results show that EBP1 interacts directly with PPIns and associate with PtdInsP3 in the nucleolus. The presence of p110β and PtdInsP3 in the nucleolus indicates their potential role in regulating nucleolar processes, at least via EBP1.Polyphosphoinositides (PPIns) are present in the nucleus where they participate in crucial nuclear processes, such as chromatin remodelling, transcription and mRNA processing. In a previous interactomics study, aimed to gain further insight into nuclear PPIns functions, we identified ErbB3 binding protein 1 (EBP1) as a potential nuclear PPIn-binding protein in a lipid pull-down screen. EBP1 is a ubiquitous and conserved protein, located in both the cytoplasm and nucleolus, and associated with cell proliferation and survival. In the present study, we show that EBP1 binds directly to several PPIns via two distinct PPIn-binding sites consisting of clusters of lysine residues and positioned at the N- and C-termini of the protein. Using interaction mutants, we show that the C-terminal PPIn-binding motif contributes the most to the localization of EBP1 in the nucleolus. Importantly, a K372N point mutation, located within the C-terminal motif and found in endometrial tumours, is sufficient to alter the nucleolar targeting of EBP1. Our study reveals also the presence of the class I phosphoinositide 3-kinase (PI3K) catalytic subunit p110β and its product PtdIns P2 and PtdInsP2, the other five PPIns have been detected within the nucleus deprived of their nuclear envelope, by radiolabelling and mass assays [P2 and this interaction regulates E-actin association to the complex [P2 present in nuclear speckles binds to and regulates the activity of the poly(A) polymerase Star-PAP [nuclear speckle targeted PIPKIα regulated-poly(A) polymerase]  . FurtherA export . Regardig pocket –41 and tg pocket . PtdIns, alias proliferation-associated protein 2G4 (PA2G4). EBP1 is conserved through evolution and ubiquitously expressed [PA2G4 encodes two splice variants, a long and predominant form p48, and a shorter and minor form p42 (homologue to p38-2G4) which differs in its N-terminus by the lack of the first 54 amino acids [These studies clearly show the importance of PPIns in the nucleus but they represent only a few examples of nuclear PPIn-binding proteins. To further define the global significance of nuclear PPIns, we have previously established a quantitative proteomic method to identify nuclear PPIn-interacting proteins . Using Sxpressed –49. Humaxpressed  and as txpressed . PA2G4 eno acids . The twono acids ,53, wherno acids ,54. p48-no acids  or upon no acids .P3 via its C-terminal PBR and this association is localized in the nucleolus. The C-terminal PBR is mutated in endometrial cancer and we showed that this partially prevents PtdInsP3-interaction as well as its nucleolar localization. These data demonstrate that the EBP1 PBRs have a dual function as a PPIn interaction motif and nucleolar localization signal, and imply that the regulation of EBP1-mediated nucleolar processes is potentially regulated by PtdInsP3.In the present study, we show that EBP1 binds directly to several PPIn species via two different PPIn interaction sites consisting of lysine-rich PBRs located in the two termini of the protein. The two PBRs have a different PPIn-interaction profile and contribute to EBP1 nucleolar localization, albeit differently. EBP1 interacts particularly with PtdInsThe phospholipase Cδ1 pleckstrin homology domain (PLCδ1-PH) cloned in pGEX-4T was obtained from Dr A.Z. Gray  and the general receptor for phosphoinositides-1 PH domain (GST-GRP1-PH) cloned in pGEX-4T3 was from Dr J. Hastie . pGEX-4T2-hEBP1 and pEGFP-C2-hEBP1 were from M. Squatrito . The N- Escherichia coli BL21-RIL DE3 and bacterial cultures were grown at 37°C and further induced with 0.5 mM isopropyl-E-D-thiogalactopyranoside for 3 h at 37°C. Bacterial pellets were resuspended in 50 mM Tris pH 7.5, 2 mM EDTA, 1 mM DTT and 1x Sigma protease inhibitor cocktail, sonicated three times for 30 s at 4°C and centrifuged at 4400 g for 10 min at 4°C. GST-tagged proteins were purified with glutathione-agarose 4B beads from an overnight pull down, eluted with 50 mM Tris pH 8.0, 100 mM NaCl, 0.5 mM DTT and 10 mM reduced glutathione, and analysed by SDS/PAGE and Coomassie staining for purity. For NMR studies, E.coli were grown in M9 minimal medium, supplemented with 6 g/l Na2HPO4, 3 g/l KH2PO4, 0.5 g/l NaCl, 0.25 g/l MgSO4, 1 g/l 98%-enriched (15NH4)2SO4 and/or 4 g/l 13C6-glucose to produce 15N- and/or 13C-uniformly labelled GST-C-terminal domain of EBP1. Further protein expression and purification was performed using the same protocol as described above.GST-PLCδ1-PH and GST-GRP1-PH were expressed and purified as described previously . GST-EBPP3  followed by anti-mouse IgG-HRP (1:10000), both diluted in blocking buffer made in PBS-T . Six washes of 5 min each with TBS-T or PBS-T were performed after incubations with protein and antibody. The protein–lipid interactions were visualized using a west pico or femto chemiluminescent substrate and a Bio-Rad ChemiDoc™ XRS+Imaging System from Bio-Rad and the ImageLab™ Software Version 3.0.Lipid overlay assay was carried out using PIP Strips™ (Echelon Biosciences) spotted with 100 pmol of each of the 7 PPIns in addition to other phospholipids, and PIP Arrays™ spotted with 1.56–100 pmol of PtdIns or each of the 7 PPIns. PIP strips™ and arrays were incubated with blocking buffer (3% fatty acid-free BSA (Sigma A6003) in TBS-T  for 1 h at room temperature. PIP Strips™ were incubated with 1.5 μg/ml GST-tagged protein or dialysed neomycin extracts in the same buffer overnight at 4°C. Detection of GST-tagged proteins and EBP1 (from neomycin extracts) was performed with an anti-GST-HRP conjugated antibody  and an anti-EBP1 antibody  respectively, both diluted in blocking buffer. PIP arrays™ were incubated with anti-PtdInshttp://rsb.info.nih.gov/ij). The data were normalized to background signals for each blot.Relative binding of EBP1 to the phospholipids was quantified by densitometry using ImageJ software . 1H, 13C and 15N backbone resonance assignments for the protein were determined using CARA (Computer Aided Resonance Assignment) version 1.8.4.2 [1H–15N heteronuclear single quantum coherence (HSQC) spectra acquired in the absence and presence of 0.08 mM diC16-PtdInsP3 or 0.33 mM diC8-PtdInsP3 (Echelon Biosciences) were subsequently compared and analysed. The peak intensities were measured in CARA using peak-fitting algorithm [The NMR sample contained 0.14 mM uniformly  1.8.4.2 . Seconda 1.8.4.2 . To monilgorithm . The rob2. AU565 cells were cultured under the same conditions but in RPMI-1640 medium. For transfections, cells were plated in six-well plates and transfected with 1–2 μg DNA and XtremeGene 9 (Roche) at 3:1 ratio for 24 h. Treatment with the pan-PI3K inhibitor LY294002 (10 μM) or DMSO (0.2% (v/v)) was performed 4 h post-transfection.MEL cells were cultured with DMEM supplemented with 10% foetal bovine serum (FBS) and antibiotics (50000 units of both penicillin and streptomycin) at 37°C with 5% CO2, 340 mM sucrose, 10% glycerol). Cells were resuspended in buffer A containing 0.1% Triton X-100, 1 mM DTT, 5 μg/ml leupeptin and 5 μg/ml aprotinin, left to swell for 5 min on ice and centrifuged at 1300 g for 5 min at 4°C. Nuclei were washed quickly with retention buffer . NucleiProteins were resolved by SDS/PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk, incubated with primary antibodies overnight at 4°C and with secondary antibodies conjugated to HRP for 1 h at room temperature. Protein detection was performed by chemiluminescence using the SuperSignal West Pico Chemiluminescent Substrate (Pierce) and imaged using the Molecular Imager® ChemiDoc™ XR+Imaging System and the ImageLab™ Software Version 3.0 (Bio-Rad).P3  diluted in blocking buffer, followed by incubation with anti-mouse IgG antibody conjugated to Alexa-594 (1:200) or Alexa-488 (1:400) (Life Technologies) diluted in blocking buffer for 1 h at room temperature. Washes were performed with 0.05% PBS-T after incubation with each antibody. The cover slips were mounted in ProLong® Gold Antifade Reagent containing 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies). Control staining with secondary antibody alone under the same staining and exposure conditions showed no unspecific staining. Images were acquired with a Leica DMI6000B fluorescence microscope using ×40 or ×100 objectives or with a Leica TCS SP5 confocal laser scanning microscope using a 63×/1.4, oil immersion lens. Images were processed with the Leica application suite v4.0 and Adobe Photoshop CS5.AU565 cells were seeded on 12 mm glass coverslips placed in 12-well plates and cultivated for 24 h before fixation. The cells were washed two times in PBS, fixed in 3.7% paraformaldehyde for 10 min at room temperature. Following fixation, cells were washed three times with PBS, permeabilized with 0.25% Triton X-100 in PBS pH 7.2 for 10 min at room temperature and blocked with 5% goat serum in PBS containing 0.05% Triton X-100 for 1 h at room temperature. Cells were incubated overnight at 4°C with anti-EBP1 , anti-NPM , anti-nucleolin , anti-p110β , anti-PtdInsP2-binding protein by combining PtdInsP2 pull down from neomycin-displaced nuclear proteins and quantitative MS [P2. Binding to PtdInsP2, PtdInsP2, phosphatidic acid (PA) and phosphatidylserine (PS) is weaker and not always detected P3 were also performed using specific probes, i.e. GST-fused PH domains of PLC-δ1 and GRP1 respectively. As shown in P2, whereas very weak binding was observed for PtdIns3P, Ptdins5P and PtdInsP2. The PH domain of GRP1 interacted strongly with PtdInsP3 and very weakly with both PtdInsP2 and PtdInsP2. Considering that EBP1 interacts with most anionic phospholipids, we tested the effect of increasing NaCl concentration on these interactions P2. These results suggest that the interactions with PtdIns3P, PtdIns5P and particularly with PtdIns4P and PtdInsP2 have the highest affinity for the FL EBP1 protein.We have previously identified EBP1 as a potential PtdInsative MS . The pepdetected C and 1D.ractions E and 1F.n=3–7)-KXKK, have also been implicated in PPIn-binding via electrostatic interactions [364RKTQKKKKKK373) [65KKEKEMKK72), named N-term PBR (87KRKRK91 → 87KAAAK91) led to a significant decrease in binding to monophosphorylated PPIns [368KAAAAK373 (FL-C-K4A). FL-C- K4A was tested for its PPIn-binding properties by lipid overlay assay in parallel with the wild-type (WT) FL GST-EBP1. This mutant did not show significant change in PPIn-binding compared with WT except for a variable decrease in binding to PtdInsP2 (results not shown). A mutant was also produced in the N-terminal PBR motif residing on the protruding loop . These results suggested that both motifs may act independently in binding to PPIns. An N-terminal fragment (amino acids 1–351), harbouring the N-term PBR motif and a C-terminal fragment (amino acids 352–394) containing the C-term PBR, were therefore created  , named Cterm PBR A and 1B.ed PPIns . In ordeing loop B, result created D. As shoonstruct D. The N-onstruct D. FinallEBP1 has previously been shown to localize in the nucleolus of HeLa and NIH-3T3 cells  and we cP3 from isolated nuclei [P3, at least among other PPIns P3-specific probe GRP1-PH, fused it with NLS-EGFP and examined its localization by immunofluorescence. In contrast with NLS-EGFP, which was predominantly diffuse in the nucleus, the NLS-EGFP-GRP1-PH was found in 81% of cells in the nucleus and strongly in the nucleolus together with nucleolin P2 . Furthermore, the localization of p110β, one of the class IA phosphoinositide 3-kinase (PI3K) catalytic subunits producing PtdInsP3, has previously been reported to be present in the nucleus [P3 antibody utilized, and that the nucleolar sites detected by the antibody are PtdInsP3. The presence of p110β in nuclei further substantiates the existence of nucleolar PtdInsP3.EBP1-p48 has previously been shown to bind to NPM . Consided nuclei , and thaer PPIns D, we argucleolin A and 4B. nucleus . PtdInsP3 within nucleoli, suggesting therefore a potential association ociation A. Bindiner PPIns D and 2D er PPIns B. The paantibody C. Taken P3 at the molecular level, high resolution NMR was used. The C-term PBR was examined since we have shown that it binds better to PtdInsP3 in lipid overlay assays and it strongly affects the nucleolar localization of EBP1, where EBP1 and PtdInsP3 co-localize. 1H-15N HSQC fingerprint spectrum of the 15N isotopically labelled GST-C-terminal fragment of EBP1 . In order to first assign each amide signal to a specific residue in the protein, standard NMR experiments were performed to detect 13C chemical shifts and sequential connectivities. Heteronuclear 3D NMR experiments led to the chemical shift assignment of 1H, 15N and 13C nuclei for 37 out of 43 (86%) residues in the C-terminal fragment of EBP1. Ala352, Glu353, Leu354, as well as the following residues from the PBR, Lys369, Lys370, Lys371, were not assigned due to signal broadening beyond detection, which could be attributed to the intermediate solvent or conformational exchange rates of the protein. Altogether, the number of amide proton cross-peaks, as well as nearly complete assignment of the protein, demonstrated that the observed signals originated almost exclusively from the target protein and not from GST . The narrow signal dispersion of the spectrum, observed for the backbone amide protons (cross-peaks positioned within 8.7–7.7 ppm range in the 1H dimension), indicates an overall disordered state of the protein. For proteins without a stable, folded structure, chemical shifts are essential parameters encoding local conformational propensities of the protein in solution [P3 . Similar effects were observed for the PBR residues when diC8-PtdInsP3 was added, but less intensity changes were overall detected in residues in the N-terminal part. These results validate that protein–lipid contacts involve most importantly electrostatic forces between lysines and phosphates. Hydrophobic interactions between nonpolar N-terminal residues and with long acyl chains, notably in the region covered by 356ALL358, may also be involved, particularly with long hydrocarbon chains.To establish further the interaction of EBP1 with PtdInssolution ,68. To psolution . Figure tion [P3 E. Additiss-peaks D and 6F,PA2G4, the gene encoding EBP1, is mutated at a very low frequency . A mutation was however identified in the C-term PBR, i.e. K372N P2 and PA , as well as prostate and breast cancer –73. Conse. K372N A, in an e. K372N B and PPI2 and PA C and 7D.nX=3-7)-K-X-K/R-K/R were implicated in PPIn-binding of nuclear proteins, suggesting a mode of interaction for PPIn-interacting proteins localized in the nucleus [P2. In addition the C-terminal PBR was more promiscuous and interacted with all the other PPIns albeit more weakly. This pattern of interaction is consistent with other studies showing the importance of PBRs for PPIn binding in other nuclear proteins such as ING2 [P2 interacts with Pf1 and TAF3. The consistency of the pattern of interaction of these PBRs with certain PPIns is rather interesting and may hence point to specificity due to the position of the phosphate on the inositol ring for short basic motifs. Clusters of basic residues in PBRs may hence preferentially accommodate interaction with either one phosphate or PtdInsP2 with sufficiently spaced phosphates. Interestingly, SAP30L binds to mono-phosphorylated PPIns via a stretch of three basic residues within its PBR which also functions as an NLS adjacent to its DNA binding domain. The C-terminal PBR of EBP1, which contains six adjacent lysines, binds more extensively to PPIns and includes in addition the other bis-phosphorylated as well as PtdInsP3. The higher number of lysines in this motif may hence offer more binding probabilities and alternatives to several PPIns with differently spaced phosphates on the inositol ring, at least in vitro.We have previously reported that short motifs consisting of basic residues or PBRs following the sequence motif K/R-P3 was demonstrated in several ways: (1) the C-term PBR binds to this PPIn species in vitro, as shown by lipid overlay assays and NMR analyses, (2) endogenous nuclear EBP1 has binding capacity as shown by lipid overlay assays. Secondly, partial co-localization of endogenous EBP1 with PtdInsP3 within the nucleolus was observed, and this could be consistent with a functional association. In addition, NMR studies revealed that other residues located N-terminal of the PBR, including the nonpolar 356ALL358 motif, made contact with PtdInsP3 containing 16-carbon but not 8-carbon acyl chains. This would suggest that the acyl chains contribute to the interaction together with the head group. The participation of acyl chains in protein–PPIn interaction is a mechanism that has previously been suggested in several nuclear proteins identified to bind diC16-PtdInsP3 without the involvement of structured PI-binding domain [The molecular mode of retention of EBP1 in the nucleolus is not clear but it is tempting to suggest that PtdInsg domain . Thus, tP3 in the nucleus has been reported previously [P2 has previously been detected in the nucleolus of asynchronously growing cells [P3 production in this nuclear site. We have initially identified EBP1 as a potential PtdInsP2-binding protein by pull down assay and mass spectrometry [P2 and PtdInsP3, the two PPIns found to be present in the nucleolus. EBP1 may therefore associate with PtdInsP2 as well as PtdInsP3 in this nuclear site. We observed that the pattern of detection of nucleolar PtdInsP3 varies in asynchronous AU565 P2 under certain conditions of the cell cycle. Consistently p110β has been previously reported to be active during G1 to S phase transition of the cell cycle [P3 in the nucleolus and nucleoplasm and regulate the function of EBP1 and we are currently pursuing this line of study.The presence of PtdInseviously  and in peviously  but the ng cells ,82–84, wtrometry  and we hus AU565 C and HeLll cycle . We suggP3, another mode of retention of EBP1 is possible via rRNA-interaction. The C-terminal region of EBP1 spanning amino acids 361–394 was shown to be necessary for RNA interaction [P3 and rRNA compete for binding to the C-terminal PBR. This scenario has indeed been reported for the HIV-1 viral protein Gag where binding of RNA to a highly basic region was demonstrated as a mechanism to prevent PtdInsP2-mediated binding to the plasma membrane [P3 interacts with NPM via lysine residues [P3 binds ALY, a protein regulating mRNA export via basic residues [P3 may regulate protein–RNA interaction utilizing basic motifs in disordered regions which have dual functions in PPIn and RNA interaction.Although the localization of EBP1 in the nucleolus can be due to the interaction of its C-terminal PBR with PtdInseraction , includieraction . This remembrane . In addiresidues , which aresidues . Finallyresidues , and thiresidues . In lighP3 as well as the class I PI3K catalytic subunit p110β in the nucleolus. EBP1 binds PtdInsP3 and this association is detected in the nucleolus. Our data imply novel regulation of nucleolar functions by PtdInsP3, which are lines of research that we are currently pursuing.In conclusion, we have shown that the PBRs identified in EBP1 have a dual function as they contribute to PPIn interaction, potentially via electrostatic interactions, as well as nucleolar localization. Considering that sub-cellular localization often correlates with function, PBRs may provide a molecular mechanism allowing EBP1 to switch between different sub-cellular compartments and functions due to their interaction with PPIns. In addition, this is the first report providing evidence of the presence of PtdIns