变构是调节蛋白质功能的重要机制,对许多生物过程至关重要。变构调节剂比正构剂具有更高的特异性和更低的毒副作用,这使得变构药物设计比正构药物设计有更多的优势。变构位点的发现是变构药物设计的前提,目前实验上获得的变构位点多是...变构是调节蛋白质功能的重要机制,对许多生物过程至关重要。变构调节剂比正构剂具有更高的特异性和更低的毒副作用,这使得变构药物设计比正构药物设计有更多的优势。变构位点的发现是变构药物设计的前提,目前实验上获得的变构位点多是偶然所得,因此亟待发展有效的理论方法来预测蛋白质变构位点。本工作提出了一种集成的机器学习方法AllosEC用于预测蛋白质变构口袋,该方法除了考虑口袋的理化性质外,还加入了口袋的二级结构信息、深度指数(DPX)和突出指数(CX)特征。另外,为了克服正负样本极度不平衡的问题,本工作使用欠采样方法来平衡训练数据集。在独立测试集上,AllosEC在多个评价指标上优于现有的其他方法,SEN、SPE、PRE和MCC分别为0.708、0.915、0.405和0.486。这样,本工作提供了性能良好的蛋白质变构位点预测方法AllosEC。Allostery is an important mechanism for regulating protein functions, which is essential for many biological processes. Compared with orthosteric regulators, allosteric regulators have higher specificity and lower toxicities, which makes allosteric drug design have more advantages than orthosteric drug design. The discovery of allosteric sites is a prerequisite for allosteric drug design. Currently, experimentally obtained allosteric sites are mostly obtained by chance, and therefore there is an urgent need to develop effective theoretical methods to predict protein allosteric sites. Here, we present an ensemble machine learning method AllosEC for protein allosteric pocket prediction, where besides the pockets’ physicochemical properties, their secondary structure information, depth indexes (DPXes) and protrusion indexes (CXes) are considered. In order to overcome the problem of extreme imbalance between positive and negative samples, this work uses an under sampling method to balance the training dataset. AllosEC outperforms other existing methods in multiple evaluation metrics on the independent test set, with SEN, SPE, PRE and MCC of 0.708, 0.915, 0.405 and 0.486, respectively. Thus, this work provides a good method AllosEC for protein allosteric site prediction.展开更多
由于乙型肝炎病毒(hepatitis B virus,HBV)的共价闭合环状DNA的持续存在和病毒介导的宿主免疫反应钝化,导致慢性乙型肝炎病毒感染很难治愈。现有的核苷(酸)类似物或聚乙二醇干扰素疗法难以实现高比率的HBV表面抗原清除。目前正在研发的...由于乙型肝炎病毒(hepatitis B virus,HBV)的共价闭合环状DNA的持续存在和病毒介导的宿主免疫反应钝化,导致慢性乙型肝炎病毒感染很难治愈。现有的核苷(酸)类似物或聚乙二醇干扰素疗法难以实现高比率的HBV表面抗原清除。目前正在研发的核心蛋白变构调节剂有望大幅度降低血清表面抗原。本文就HBV核心蛋白的结构、功能以及核心蛋白变构调节剂的分类、应用前景等方面进行综述。展开更多
[Objective]The aim was to study the effect of herbicide on the growth of early seedlings of rye（Secale cereale）.[Method]Effect of two kinds of herbicide（Atrazine and APM）on seedling growth of rye was investigated ...[Objective]The aim was to study the effect of herbicide on the growth of early seedlings of rye（Secale cereale）.[Method]Effect of two kinds of herbicide（Atrazine and APM）on seedling growth of rye was investigated at the physiological,biochemical and cellular level.[Result]The Atrazin significantly decreased the contents of chlorophyll a,b and soluble proteins.Rye seeds were treated with 0.01-1 mg/L Atrazine for 16 h,the contents of chlorophyll a and b decreased from 1.26（a）,0.49（b）mg/g FW（control）to 1.15（a）,0.46（b）mg/g FW（0.1 mg/L）and 0.81（a）,0.33（b）mg/g FW（1.0 mg/L）.The content of soluble protein decreased with the increasing concentration of Atrazin.Atrazin had no significant influence on the cell division and chromosome structure variation.The contents of chlorophyll a,b and soluble proteins had no significantly change under the treatment of APM,but the number of chromosome structure variation such as chromosome bridge,multipolar division cells,lagging chromosome and unequal division cells increased significantly.[Conclusion]The critical concentration of Atrazine was 0.1-1.0 mg/L and 4 mg/L of APM in rye.展开更多
Oytoskeletal changes in transformed cells (LM-51) exhibiting obviously metastatie eapabilities were investigated by utilization of double-fluorescent labelling through combinations of:(1) tubulin indirect immunofluore...Oytoskeletal changes in transformed cells (LM-51) exhibiting obviously metastatie eapabilities were investigated by utilization of double-fluorescent labelling through combinations of:(1) tubulin indirect immunofluoresoenoe plus Khodamine-phalloidin staining of F-artins;(2) indirect immunofluorescent staining with α-aotinin polyolonal- and vinoulin monoclonal antibodies. The LM-51 cells which showed metastatic index of >50% were derived from lung metastasis in nude mice after subcutaneous inoculation of human highly metastatic tumor DNA transfected NIH3T3 cell transformants. The parent NIH3T3 cells exhibited well-organized miorotubu-les, prominent stress fibers and adhesion plaques while their transformants showed remarkable oytoskeletal alterations: (1) reduced microtubules but increased MTOC fluorescence; (2) disrupted stress fibers and fewer adhesion plaques with their protein components redistributed in the cytoplasm; (3) F-aotin-and α-actinin/vinculin aggregates appeared in the cytoplasm. These aggregates were dot-like, varied in size (0.1-0.4u,m) and number, located near the ventral surface of the cells. TPA-induced aotin/vinoulin bodies were studied too. Indications that aotin and α-actinin/vinoulin redistribution might be important alterations involved in the expression of metastatio capabilities of LM-51 transformed cells were discussed.展开更多
Dengue virus(DENV) nonstructural protein 1(NS1) is a highly conserved 46-kDa protein that contains 2 glycosylation sites(Asn-130 and Asn-207) and 12 conserved cysteine(Cys) residues. Here, we performed site-directed m...Dengue virus(DENV) nonstructural protein 1(NS1) is a highly conserved 46-kDa protein that contains 2 glycosylation sites(Asn-130 and Asn-207) and 12 conserved cysteine(Cys) residues. Here, we performed site-directed mutagenesis to generate systematic mutants of viral strain TSV01. The results of the subsequent analysis showed that an alanine substitution at the second N-linked glycan Asn-207 in NS1 delayed viral RNA synthesis, reduced virus plaque size, and weakened the cytopathic effect. Three mutants at Cys sites(Cys-4, Cys-55, Cys-291) and a C-terminal deletion(ΔC) mutant significantly impaired RNA synthesis, and consequently abolished viral growth, whereas alanine mutations at Asn-130 and Glu-173 resulted in phenotypes that were similar to the wild-type(WT) virus. Further analysis showed that the Asn-207 mutation slightly delayed viral replication. These results suggest that the three conserved disulfide bonds and the second N-linked glycan in NS1 are required for DENV-2 replication.展开更多
ATP-binding cassette exporters transport many substrates out of cellular membranes via alternating between inward-facing and outward-facing conformations. Despite extensive research efforts over the past decades, unde...ATP-binding cassette exporters transport many substrates out of cellular membranes via alternating between inward-facing and outward-facing conformations. Despite extensive research efforts over the past decades, understanding of the molecular mechanism remains elusive. As these large-scale conformational movements are global and collective, we have previously performed extensive coarse-grained molecular dynamics simulations of the potential of mean force along the conformational transition pathway [J. Phys. Chem. B 119, 1295(2015)]. However, the occluded conformational state, in which both the internal and external gate are closed, was not determined in the calculated free energy profile. In this work, we extend the above methods to the calculation of the free energy profile along the reaction coordinate, d1-d2, which are the COM distances between the two sides of the internal(d1)and the external gate(d2). The potential of mean force is thus obtained to identify the transition pathway, along which several outward-facing, inward-facing, and occluded state structures are predicted in good agreement with structural experiments. Our coarse-grained molecular dynamics free-energy simulations demonstrate that the internal gate is closed before the external gate is open during the inward-facing to outward-facing transition and vice versa during the inward-facing to outward-facing transition. Our results capture the unidirectional feature of substrate translocation via the exporter, which is functionally important in biology. This finding is different from the previous result, in which both the internal and external gates are open reported in an X-ray experiment [Proc. Natl. Acad. Sci. USA 104,19005(2007)]. Our study sheds light on the molecular mechanism of the state transitions in an ATP-binding cassette exporter.展开更多
文摘变构是调节蛋白质功能的重要机制,对许多生物过程至关重要。变构调节剂比正构剂具有更高的特异性和更低的毒副作用,这使得变构药物设计比正构药物设计有更多的优势。变构位点的发现是变构药物设计的前提,目前实验上获得的变构位点多是偶然所得,因此亟待发展有效的理论方法来预测蛋白质变构位点。本工作提出了一种集成的机器学习方法AllosEC用于预测蛋白质变构口袋,该方法除了考虑口袋的理化性质外,还加入了口袋的二级结构信息、深度指数(DPX)和突出指数(CX)特征。另外,为了克服正负样本极度不平衡的问题,本工作使用欠采样方法来平衡训练数据集。在独立测试集上,AllosEC在多个评价指标上优于现有的其他方法,SEN、SPE、PRE和MCC分别为0.708、0.915、0.405和0.486。这样,本工作提供了性能良好的蛋白质变构位点预测方法AllosEC。Allostery is an important mechanism for regulating protein functions, which is essential for many biological processes. Compared with orthosteric regulators, allosteric regulators have higher specificity and lower toxicities, which makes allosteric drug design have more advantages than orthosteric drug design. The discovery of allosteric sites is a prerequisite for allosteric drug design. Currently, experimentally obtained allosteric sites are mostly obtained by chance, and therefore there is an urgent need to develop effective theoretical methods to predict protein allosteric sites. Here, we present an ensemble machine learning method AllosEC for protein allosteric pocket prediction, where besides the pockets’ physicochemical properties, their secondary structure information, depth indexes (DPXes) and protrusion indexes (CXes) are considered. In order to overcome the problem of extreme imbalance between positive and negative samples, this work uses an under sampling method to balance the training dataset. AllosEC outperforms other existing methods in multiple evaluation metrics on the independent test set, with SEN, SPE, PRE and MCC of 0.708, 0.915, 0.405 and 0.486, respectively. Thus, this work provides a good method AllosEC for protein allosteric site prediction.
文摘由于乙型肝炎病毒(hepatitis B virus,HBV)的共价闭合环状DNA的持续存在和病毒介导的宿主免疫反应钝化,导致慢性乙型肝炎病毒感染很难治愈。现有的核苷(酸)类似物或聚乙二醇干扰素疗法难以实现高比率的HBV表面抗原清除。目前正在研发的核心蛋白变构调节剂有望大幅度降低血清表面抗原。本文就HBV核心蛋白的结构、功能以及核心蛋白变构调节剂的分类、应用前景等方面进行综述。
基金Supported by Key Project for Science Researches of Ministry of Education(02010)~~
文摘[Objective]The aim was to study the effect of herbicide on the growth of early seedlings of rye（Secale cereale）.[Method]Effect of two kinds of herbicide（Atrazine and APM）on seedling growth of rye was investigated at the physiological,biochemical and cellular level.[Result]The Atrazin significantly decreased the contents of chlorophyll a,b and soluble proteins.Rye seeds were treated with 0.01-1 mg/L Atrazine for 16 h,the contents of chlorophyll a and b decreased from 1.26（a）,0.49（b）mg/g FW（control）to 1.15（a）,0.46（b）mg/g FW（0.1 mg/L）and 0.81（a）,0.33（b）mg/g FW（1.0 mg/L）.The content of soluble protein decreased with the increasing concentration of Atrazin.Atrazin had no significant influence on the cell division and chromosome structure variation.The contents of chlorophyll a,b and soluble proteins had no significantly change under the treatment of APM,but the number of chromosome structure variation such as chromosome bridge,multipolar division cells,lagging chromosome and unequal division cells increased significantly.[Conclusion]The critical concentration of Atrazine was 0.1-1.0 mg/L and 4 mg/L of APM in rye.
文摘Oytoskeletal changes in transformed cells (LM-51) exhibiting obviously metastatie eapabilities were investigated by utilization of double-fluorescent labelling through combinations of:(1) tubulin indirect immunofluoresoenoe plus Khodamine-phalloidin staining of F-artins;(2) indirect immunofluorescent staining with α-aotinin polyolonal- and vinoulin monoclonal antibodies. The LM-51 cells which showed metastatic index of >50% were derived from lung metastasis in nude mice after subcutaneous inoculation of human highly metastatic tumor DNA transfected NIH3T3 cell transformants. The parent NIH3T3 cells exhibited well-organized miorotubu-les, prominent stress fibers and adhesion plaques while their transformants showed remarkable oytoskeletal alterations: (1) reduced microtubules but increased MTOC fluorescence; (2) disrupted stress fibers and fewer adhesion plaques with their protein components redistributed in the cytoplasm; (3) F-aotin-and α-actinin/vinculin aggregates appeared in the cytoplasm. These aggregates were dot-like, varied in size (0.1-0.4u,m) and number, located near the ventral surface of the cells. TPA-induced aotin/vinoulin bodies were studied too. Indications that aotin and α-actinin/vinoulin redistribution might be important alterations involved in the expression of metastatio capabilities of LM-51 transformed cells were discussed.
基金supported by Important National Science & Technology Specific Projects (2012ZX10004219, 2012ZX10004403)the National Natural Scientific Fund of China (81072675)the Wuhan Key Laboratory on Emerging Infectious Diseases and Biosafety
文摘Dengue virus(DENV) nonstructural protein 1(NS1) is a highly conserved 46-kDa protein that contains 2 glycosylation sites(Asn-130 and Asn-207) and 12 conserved cysteine(Cys) residues. Here, we performed site-directed mutagenesis to generate systematic mutants of viral strain TSV01. The results of the subsequent analysis showed that an alanine substitution at the second N-linked glycan Asn-207 in NS1 delayed viral RNA synthesis, reduced virus plaque size, and weakened the cytopathic effect. Three mutants at Cys sites(Cys-4, Cys-55, Cys-291) and a C-terminal deletion(ΔC) mutant significantly impaired RNA synthesis, and consequently abolished viral growth, whereas alanine mutations at Asn-130 and Glu-173 resulted in phenotypes that were similar to the wild-type(WT) virus. Further analysis showed that the Asn-207 mutation slightly delayed viral replication. These results suggest that the three conserved disulfide bonds and the second N-linked glycan in NS1 are required for DENV-2 replication.
基金supported by the National Natu-ral Science Foundation of China(No.21073170 and No.21273209).
文摘ATP-binding cassette exporters transport many substrates out of cellular membranes via alternating between inward-facing and outward-facing conformations. Despite extensive research efforts over the past decades, understanding of the molecular mechanism remains elusive. As these large-scale conformational movements are global and collective, we have previously performed extensive coarse-grained molecular dynamics simulations of the potential of mean force along the conformational transition pathway [J. Phys. Chem. B 119, 1295(2015)]. However, the occluded conformational state, in which both the internal and external gate are closed, was not determined in the calculated free energy profile. In this work, we extend the above methods to the calculation of the free energy profile along the reaction coordinate, d1-d2, which are the COM distances between the two sides of the internal(d1)and the external gate(d2). The potential of mean force is thus obtained to identify the transition pathway, along which several outward-facing, inward-facing, and occluded state structures are predicted in good agreement with structural experiments. Our coarse-grained molecular dynamics free-energy simulations demonstrate that the internal gate is closed before the external gate is open during the inward-facing to outward-facing transition and vice versa during the inward-facing to outward-facing transition. Our results capture the unidirectional feature of substrate translocation via the exporter, which is functionally important in biology. This finding is different from the previous result, in which both the internal and external gates are open reported in an X-ray experiment [Proc. Natl. Acad. Sci. USA 104,19005(2007)]. Our study sheds light on the molecular mechanism of the state transitions in an ATP-binding cassette exporter.
