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991.
Yu Liu Feiya Du Wei Chen Minya Yao Kezhen Lv Peifen Fu 《Experimental cell research》2013,319(20):3140-3149
BackgroundBreast cancer is the major cause of cancer-related deaths in females world-wide. Doxorubicin-based therapy has limited efficacy in breast cancer due to drug resistance, which has been shown to be associated with the epithelial-to-mesenchymal transition (EMT). However, the molecular mechanisms linking the EMT and drug resistance in breast cancer cells remain unclear. Dual specificity phosphatase 4 (DUSP4), a member of the dual specificity phosphatase family, is associated with cellular proliferation and differentiation; however, its role in breast cancer progression is controversial.MethodsWe used cell viability assays, Western blotting and immunofluorescent staining, combined with siRNA interference, to evaluate chemoresistance and the EMT in MCF-7 and adriamycin-resistant MCF-7/ADR breast cancer cells, and investigate the underlying mechanisms.ResultsKnockdown of DUSP4 significantly increased the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin, and MCF-7/ADR cells which expressed high levels of DUSP4 had a mesenchymal phenotype. Furthermore, knockdown of DUSP4 reversed the EMT in MCF-7/ADR cells, as demonstrated by upregulation of epithelial biomarkers and downregulation of mesenchymal biomarkers, and also increased the chemosensitivity of MCF-7/ADR cells to doxorubicin.ConclusionsDUSP4 might represent a potential drug target for inhibiting drug resistance and regulating the process of the EMT during the treatment of breast cancer. 相似文献
992.
Recent development of titratable coions has paved the way for realizing all-atom molecular dynamics at constant pH. To further improve physical realism, here we describe a technique in which proton titration of the solute is directly coupled to the interconversion between water and hydroxide or hydronium. We test the new method in replica-exchange continuous constant pH molecular dynamics simulations of three proteins, HP36, BBL, and HEWL. The calculated pKa values based on 10-ns sampling per replica have the average absolute and root-mean-square errors of 0.7 and 0.9 pH units, respectively. Introducing titratable water in molecular dynamics offers a means to model proton exchange between solute and solvent, thus opening a door to gaining new insights into the intricate details of biological phenomena involving proton translocation.Solution pH is an important factor in biology. Although neutral pH in extracellular medium accounts for balanced electrostatics and proper folding of protein structures, pH gradients across cell membranes induce large conformational changes that are necessary for biological functions, such as ATP synthesis and efflux of small molecules out of the cell. To gain detailed insights into pH-dependent conformational phenomena, several constant pH molecular dynamics (pHMD) methods, based on either discrete or continuous titration coordinates, have been developed in the last decade (1–4). In the continuous pHMD (CpHMD) framework (2,4), a set of titration coordinates {λi} are simultaneously propagated along with the conformational degrees of freedom. Although the original CpHMD method based on the generalized Born (GB) implicit-solvent models (2,4) offers quantitative prediction of pKa values and pH dependence of folding and conformational dynamics of proteins (5), its accuracy and applicability to highly charged systems and those with dominantly hydrophobic regions are limited due to the approximate nature of the underlying implicit-solvent models.Motivated by the above-mentioned need, three groups have made efforts to develop a CpHMD method using exclusively the explicit-solvent models (6–8). In our development, the titration of acidic and basic sites is coupled with that of coions to level the total charge of the system (8). To further improve physical realism, here we replace the coions by titratable water molecules, which not only absorb the excess charge but also enable direct modeling of solute-solvent proton exchange in classical molecular dynamics simulations.To illustrate the utility of the new methodology, we applied it to the titration simulations of three proteins that were previously used to benchmark the GB-based CpHMD. Although this work does not explore specific interactions between titratable waters and proteins, the methodology can be further tested or improved to provide a rigorous way for modeling proton transfer in molecular dynamics, which is a computationally efficient alternative to the empirical valence-bond theory-based methodologies (9,10).We define titration of water as:
Open in a separate windowaTaken from Wallace and Shen (12). The pKa''s of BBL were recalculated.bSampling time per pH replica.Breaking the simulations in two halves, we noticed that the second 5-ns sampling gave better agreement with experiment. The RMS deviation is reduced from 1.2 to 0.9 pH units, while the average absolute deviation is reduced from 1.0 to 0.6 pH units. The linear regression against experimental data is also improved, with the slope decreasing from 1.4 to 1.1 although R2 remains the same. Comparing these second-half results with the GB-based simulations, we find that the RMS and average absolute deviations are about the same as the GB-CpHMD results; however, the all-atom simulations show a small systematic overestimation (regression slope >1), whereas GB simulations show a systematic underestimation (regression slope <1).The improvement in the second halves of the simulations are seen mainly for residues involved in attractive electrostatic interactions, including Asp44 and Asp46 of HP36, Asp129 of BBL, and Asp48, Asp66, and Asp87 of HEWL. These residues are initially locked in salt-bridges or hydrogen bonds. However, in the second 5 ns, the attractive interactions weakened, leading to a decrease in the calculated pKa shifts relative to the model values and better agreement with experiment. For instance, Asp44 was initially in a salt-bridge distance from Arg55. However, the salt-bridge positions were sampled less often in the second 5 ns (see Fig. S5), which explains the 1-unit reduction in the calculated pKa shift. Significant fluctuation in ion-pair interactions was also observed in the work by Alexov (11). The carboxyl oxygen of Asp46 was a hydrogen-bond acceptor with both the backbone amide and hydroxyl of Ser43. These hydrogen bonds were less frequently sampled in the second 5 ns (see Fig. S6), leading to a decrease of the pKa shift for Asp46 by 1.3 units. These results indicate that extensive conformational sampling is necessary to give an accurate estimate of the ratio between the charged and neutral populations.Limited conformational sampling is also a contributing factor to the overestimation of the pKa shifts for buried residues (Fig. S7 and Fig. S8). The increase in SASA is correlated with the more frequent sampling of the states with λ close to 1, i.e., the deprotonated form (see Fig. S9). However, because Glu35 was buried in the starting conformation and the transition between buried and exposed states is slow compared to the simulation length, the exposed state may not be sufficiently sampled, leading to overestimation of the pKa shift.In contrast to Glu35, the SASA of Asp52 in HEWL is almost identical for both protonation states. The lack of conformational fluctuation is due to the strong hydrogen bonding with the side-chain amino group of Asn46 and Asn59 (data not shown). Overestimation of the pKa shifts for buried residues can also be attributed to the limitation of the additive force field which underestimates dielectric response in protein environment (more discussion see Supporting Material) of the pKa shifts for buried residues.Finally, to ascertain if the presence of hydroxide/hydronium introduces artifacts, we studied the interaction between hydroxide/hydronium and the titratable sites/ions. Comparing the hydroxide/hydronium with respective chloride/sodium ions, we find that the spatial distributions are nearly identical (see plots of distance distributions and radial distribution functions in Figs. S10–S13). However, the relative occupancy of the hydroxide around the neutral Asp/Glu, positive histidine, or sodium ion is 2–3 times as that of a chloride. The water-bridged interaction between sodium and chloride ions becomes much weaker when chloride is replaced by hydroxide or sodium is replaced by hydronium. By contrast, the occupancy of the hydronium around the solute is similar to that of the sodium. Furthermore, similar pKa results for these proteins were obtained when coions were used instead of titratable waters (data not shown). Thus, we believe that potential artifacts related to the ionized forms of water are negligible. Work is underway to further understand the limitations of the methodology and to explore applications to protein dynamics coupled to proton transfer.In summary, we have developed and tested titratable water models for use in all-atom CpHMD simulations. Although the benchmark pKa calculations indicate a comparable accuracy as the GB-CpHMD method, the all-atom method offers physical rigor and most importantly, it is applicable to systems that cannot be studied with GB-based simulations such as lipids and nucleic acids. We anticipate that the accuracy of this methodology can be further improved by incorporating the new-generation force fields that account for polarization. The coupling between proton titration of water and solute offers a computationally efficient way to model proton transfer in molecular mechanics simulations. 相似文献
- 1.Loss of a proton to give a negatively charged hydroxide,
- 2.Gain of a proton to give a positively charged hydronium,
Table 1
Calculated and experimental pKa values of three proteinsResidue | Experimenta | GBa | All-atom CpHMD | ||
---|---|---|---|---|---|
Time (ns)b | 0–1 | 0–5 | 5–10 | 0–10 | |
HP36 | |||||
Asp44 | 3.10 (0.01) | 3.2 (0.1) | 2.0 | 3.0 | 2.6 (0.5) |
Glu45 | 3.95 (0.01) | 3.5 (0.1) | 4.3 | 4.5 | 4.4 (0.1) |
Asp46 | 3.45 (0.12) | 3.5 (0.1) | 2.4 | 3.7 | 3.1 (0.6) |
Glu72 | 4.37 (0.03) | 3.5 (0.1) | 4.4 | 4.4 | 4.4 (0.0) |
BBL | |||||
Asp129 | 3.88 (0.02) | 3.2 (0.0) | 2.2 | 3.2 | 2.7 (0.5) |
Glu141 | 4.46 (0.04) | 4.3 (0.0) | 4.0 | 4.4 | 4.2 (0.2) |
His142 | 6.47 (0.04) | 7.1 (0.0) | 5.9 | 5.8 | 5.8 (0.0) |
Asp145 | 3.65 (0.04) | 2.8 (0.2) | 3.0 | 3.1 | 3.1 (0.0) |
Glu161 | 3.72 (0.05) | 3.6 (0.3) | 4.2 | 3.9 | 4.0 (0.2) |
Asp162 | 3.18 (0.04) | 3.4 (0.3) | 2.9 | 3.5 | 3.2 (0.3) |
Glu164 | 4.50 (0.03) | 4.5 (0.1) | 5.7 | 4.6 | 5.2 (0.6) |
His166 | 5.39 (0.02) | 5.4 (0.1) | 4.4 | 4.4 | 4.4 (0.0) |
HEWL | |||||
Glu7 | 2.6 (0.2) | 2.6 (0.1) | 3.6 | 3.4 | 3.5 (0.1) |
His15 | 5.5 (0.2) | 5.3 (0.5) | 5.1 | 5.1 | 5.1 (0.0) |
Asp18 | 2.8 (0.3) | 2.9 (0.0) | 2.5 | 3.3 | 2.9 (0.4) |
Glu35 | 6.1 (0.4) | 4.4 (0.2) | 8.5 | 8.7 | 8.6 (0.1) |
Asp48 | 1.4 (0.2) | 2.8 (0.2) | −0.1 | 1.1 | 0.6 (0.6) |
Asp52 | 3.6 (0.3) | 4.6 (0.0) | 5.4 | 5.6 | 5.5 (0.1) |
Asp66 | 1.2 (0.2) | 1.2 (0.4) | −0.6 | 0.8 | 0.3 (0.7) |
Asp87 | 2.2 (0.1) | 2.0 (0.1) | 0.8 | 2.1 | 1.5 (0.7) |
Asp101 | 4.5 (0.1) | 3.3 (0.3) | 6.1 | 5.7 | 5.9 (0.2) |
Asp119 | 3.5 (0.3) | 2.5 (0.1) | 3.0 | 3.3 | 3.2 (0.1) |
Maximum absolute deviation | 1.8 | 2.4 | 2.6 | 2.5 | |
Average absolute deviation (RMS deviation) | 0.5 (0.7) | 1.0 (1.2) | 0.6 (0.9) | 0.7 (0.9) | |
Linear fit R2 (slope) | 0.7 (0.8) | 0.8 (1.4) | 0.7 (1.1) | 0.8 (1.2) |
993.
Molecular dynamics trajectories 2 μs in length have been generated for the pH-activated, tetrameric M2 proton channel of the influenza A virus in all protonation states of the pH sensor located at the His37 tetrad. All simulated structures are in very good agreement with high-resolution structures. Changes in the channel caused by progressive protonation of His37 provide insight into the mechanism of proton transport. The channel is closed at both His37 and Trp41 sites in the singly and doubly protonated states, but it opens at Trp41 upon further protonation. Anions access the charged His37 and by doing so stabilize the protonated states of the channel. The narrow opening at the His37 site, further blocked by anions, is inconsistent with the water-wire mechanism of proton transport. Instead, conformational interconversions of His37 correlated with hydrogen bonding to water molecules indicate that these residues shuttle protons in high-protonation states. Hydrogen bonds between charged and uncharged histidines are rare. The valve at Val27 remains on average quite narrow in all protonation states but fluctuates sufficiently to support water and proton transport. A proton transport mechanism in which the channel, depending on pH, opens at either the histidine or valine gate is only partially supported by the simulations. 相似文献
994.
995.
Wei‐Li Quan Xia‐Lin Zheng Xin‐Xin Li Xing‐Miao Zhou Wei‐Hua Ma Xiao‐Ping Wang 《Entomologia Experimentalis et Applicata》2013,146(3):398-403
Although parasitoids ultimately kill their host, koinobiont parasitoids must protect not only themselves but also their hosts against extreme environments. In this study, the parasitism rate of Chilo suppressalis Walker (Lepidoptera: Pyralidae) was investigated, and the average body weights, supercooling points, and concentrations of glycerol (acting as a cryoprotectant) in the hemolymph were compared between parasitized and non‐parasitized larvae. Five species of koinobiont endoparasitoids parasitized the overwintering C. suppressalis larvae and the total parasitism rate was 47.6% (n = 1 537). Average body weight of parasitized larvae was significantly lower than that of non‐parasitized larvae, and the parasitism rate of the lighter group (20–30 mg) was highest. The supercooling point of parasitized C. suppressalis larvae (?15.7 ± 0.3 °C) was significantly lower than that of the non‐parasitized larvae (?14.3 ± 0.2 °C). In addition, supercooling points were not correlated with body weights between parasitized and non‐parasitized larvae, indicating that cold hardiness of parasitized larvae was enhanced by endoparasitoids. Furthermore, the concentration of glycerol in the hemolymph was significantly higher in parasitized larvae (205.0 ± 7.1 μmol ml?1) than in non‐parasitized larvae (169.8 ± 14.4 μmol ml?1), which suggests that the mechanism that decreases the supercooling point of parasitized larvae was associated with glycerol. All these results indicated that the cold hardiness of parasitized C. suppressalis larvae was enhanced by their endoparasitoids, which benefitted overwintering endoparasitoids. 相似文献
996.
Effects of Commercial Harvesting on Population Characteristics and Rhizome Yield of Anemone altaica. Commercial harvesting constitutes a direct threat to numerous non–timber forest products (NTFPs), but its ecological effects have not been well documented. Anemone altaica Fisch. ex C. A. Mey, a spring ephemeral plant found in temperate forests of Eurasia, is a traditional Chinese herb. Owing to medicinal value, its rhizomes have been harvested for commercial purposes in northwestern China for many years. This paper addresses the ecological effects of commercial harvesting on A. altaica populations under different harvest intensities. The results show that size–selective harvesting of rhizomes can increase population densities by asexual propagation. Currently, two– to three–year–old individuals derived from asexual propagation are the main targets of commercial harvesting. The increased demand in recent years has resulted in earlier and more intensive harvesting activities largely impacting the natural recovery of the harvested populations. For sustainable use of this traditional medicinal species, we recommend that a periodic harvest strategy of three to four years be adopted. 相似文献
997.
Shi‐Ze Zhang Hong Huang Hong‐Wei Shan Fan Zhang Fang‐Hao Wan Tong‐Xian Liu 《Entomologia Experimentalis et Applicata》2013,147(3):293-300
Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) and Pieris rapae L. (Lepidoptera: Pieridae) are serious pests of vegetables, that can occur simultaneously on a single cabbage plant [Brassica oleracea var. capitata L. (Brassicaceae)]. We determined whether pre‐feeding or infestation by B. tabaci on cabbage could induce physiological and biochemical responses of the plant against P. rapae. Developmental time, length, and weight of immature P. rapae, and defense‐related plant compounds (SOD, superoxide dismutase; POD, peroxidase; CAT, catalase; APX, ascorbate peroxidase) were measured. Development of P. rapae larvae was 21% slower on B. tabaci‐pre‐infested plants than on plants without B. tabaci infestation. When feeding on B. tabaci‐pre‐infested plants, 22% of P. rapae larvae pupated as compared with 83% on B. tabaci‐free plants. Weights of P. rapae from first to fourth instars that fed on B. tabaci‐pre‐infested plants were also reduced, whereas those of fifth instars and pupae were not. Similarly, body length of P. rapae from first to fourth instars was affected by B. tabaci pre‐infestation, whereas that of the fifth instars was not. Peroxidase and APX activities of the B. tabaci‐pre‐infested plants increased more than SOD and CAT. Peroxidase and SOD activities of B. tabaci and P. rapae co‐infested plants increased as compared with those of P. rapae‐infested plants; however, CAT and APX activities were not different between B. tabaci‐ and P. rapae‐infested plants. These results showed that B. tabaci infestation had a negative effect on P. rapae when they occurred simultaneously on the same host plant. The implications of the induced plant changes on the herbivore are discussed. 相似文献
998.
Liping Zhang Hong Zhang Yining Zhao Zhe Li Shangke Chen Jing Zhai Yunyun Chen Wei Xie Zhong Wang Qing Li Xuehua Zheng Xiaopeng Hu 《FEBS letters》2013
The antineoplastic target aldo–keto reductase family member 1B10 (AKR1B10) and the critical polyol pathway enzyme aldose reductase (AKR1B1) share high structural similarity. Crystal structures reported here reveal a surprising Trp112 native conformation stabilized by a specific Gln114-centered hydrogen bond network in the AKR1B10 holoenzyme, and suggest that AKR1B1 inhibitors could retain their binding affinities toward AKR1B10 by inducing Trp112 flip to result in an “AKR1B1-like” active site in AKR1B10, while selective AKR1B10 inhibitors can take advantage of the broader active site of AKR1B10 provided by the native Trp112 side-chain orientation. 相似文献
999.
Can-Jie Guo Qin Pan Hua Xiong Yu-Qi Qiao Zhao-Lian Bian Wei Zhong Li Sheng Hai Li Lei Shen Jing Hua Xiong Ma Jing-Yuan Fang 《FEBS letters》2013
In our previous study, miR-126 was identified as one of the leading miRNAs that is downregulated during activation of hepatic stellate cells (HSCs). However, the roles and related mechanisms of miR-126 in HSCs are not understood. In this study, we compared expression of miR-126 during HSC activation both in vitro and in vivo. We also applied RNA interference to analyze the role and mechanism of miR-126∗ in the activation of HSCs. Restoring HSCs with Lv-miR-126∗ resulted in decreased proliferation, accumulation of extracellular matrix components, and cell contraction, while also negatively regulating the vascular endothelial growth factor (VEGF) signal transduction pathways by partially targeted VEGF-A. Thus, we postulate that miR-126 may be a biological marker for the activation of HSCs, and useful for reducing intrahepatic vascular resistance and improving the sinusoidal microcirculation in chronic liver diseases. 相似文献
1000.
Y. Ding W. Wu W. Wei R.E. Davis I.‐M. Lee R.W. Hammond J.P. Sheng L. Shen Y. Jiang Y. Zhao 《The Annals of applied biology》2013,162(1):131-139
Phytoplasmas are phloem‐inhabiting, cell wall‐less bacteria that cause numerous plant diseases worldwide. Plants infected by phytoplasmas often exhibit various symptoms indicative of hormonal imbalance. In this study, we investigated the effects of potato purple top (PPT) phytoplasma infection on gibberellin homeostasis in tomato plants. We found that PPT phytoplasma infection caused a significant reduction in endogenous levels of gibberellic acid (GA3). The decrease in GA3 content in diseased plants was correlated with down regulation of genes responsible for biosynthesis of bioactive GAs ( GA20ox1 and GA3ox1) and genes involved in formation of GA precursors [geranyl diphosphate synthase (GPS) and copalyldiphosphate synthase (CPS)]. Exogenous application of GA3 at 200 µmol L?1 was able to restore the GA content in infected plants to levels comparable to those in healthy controls, and to attenuate the characteristic ‘big bud’ symptoms induced by the phytoplasma. The interesting observation that PPT phytoplasma‐infected plants had prolonged low expression of key GA biosynthesis genes GA20ox1 and GA3ox1 under GA deficiency conditions led us to hypothesise that there was a diminished sensitivity of the GA metabolism feedback regulation, especially GA biosynthesis negative feedback regulation, in those affected plants, and such diminished sensitization in early stages of infection may represent a central element of the phytoplasma‐induced disruption of GA homeostasis and pathogenesis. 相似文献