Functional Analysis of Homodimer Formation between Movement Proteins in Barley Yellow Dwarf Virus

[Objective] The aim was to investigate the dimer formation between the movement proteins（MP）in barely yellow dwarf virus by using the technology of bimolecular fluorescence complementation technology and to further study the relationship between MP homodimerization and viral movement.[Method] The DNA sequence of bimolecular fluorescent complementary vector containing cloning multiple cloning sites,35S promoter and terminator was cloned into the expression vector pCAMBIA1300,which replicates at a higher copy number in E.coli.Then,the BYDV-MP gene fragment was amplified in the presence of the whole BYDV-PAV cDNA sequence as template and the primers designed according to the BYDV-MP gene sequence from GenBank,cloned into the modified bimolecular fluorescent complementary vectors pCAMBIA1300-NE and pCAMBIA1300-CE.The resulting vectors were transformed into Agrobacterium by electroporation method and infiltrated into the tobacco leaf.Protein interactions were observed under fluorescence microscope.[Result] Yellow fluorescence could be viewed in the leaves co-infiltrated with Agrobacterium carrying pCAMBIA1300NE-MP and pCAMBIA1300CE-MP at 2-5 d post-infiltration,while yellow fluorescence could not be observed in negative control groups.[Conclusion] BYDV-MP formed homodimers in plant cells.The results can provide theoretical basis for further in-depth research about the movement process and mechanism of BYDV.

Small changes in the length of diselenide bond-containing linkages exert great influences on the antitumor activity of docetaxel homodimeric prodrug nanoassemblies

Homodimeric prodrug-based self-assembled nanoparticles,with carrier-free structure and ultrahigh drug loading,is drawing more and more attentions.Homodimeric prodrugs are composed of two drug molecules and a pivotal linkage.The influence of the linkages on the self-assembly,in vivo fate and antitumor activity of homodimeric prodrugs is the focus of research.Herein,three docetaxel(DTX)homodimeric prodrugs are developed using different lengths of diselenide bond-containing linkages.Interestingly,compared with the other two linkages,the longest diselenide bond-containing linkage could facilitate the self-delivery of DTX prodrugs,thus improving the stability,circulation time and tumor targeting of prodrug nanoassemblies.Besides,the extension of linkages reduces the redox-triggered drug release and cytotoxicity of prodrug nanoassemblies in tumor cells.Although the longest diselenide bond-containing prodrug nanoassemblies possessed the lowest cytotoxicity to 4T1 cells,their stable nanostructure maintained intact during circulation and achieve the maximum accumulation of DTX in tumor cells,which finally“turned the table”.Our study illustrates the crucial role of linkages in homodimeric prodrugs,and gives valuable proposal for the development of advanced nano-DDS for cancer treatment.

Phylogeny derived from homodimeric endonuclease correlates with its pre-RNA substrates

Amongst endonuclease, the homodimeric variety is found in many prokaryotes for processing of the introns out from pre-RNAs. But as the variety and the complexity of introns rise with evolution, do the homodimeric endonuclease adapt to the changes? The correlations between evolving pre-RNAs and adapting homodimeric endonuclease in lower prokaryotes is investigated in this paper. First, we construct and observe the appearance of a long branch in the phylogeny based on homodimeric endonuclease. To appreciate the finer aspects of accelerating evolution near this long branch, we delve deeper into the pre-RNA substrates of the endonuclease. Computational evidence of an as-yet-unreported noncoding RNA gene then emerges from this study. The capabilities of homodimeric endonuclease and the complexities of its pre-RNA substrates appear to evolve in steps together.

Arabidopsis ABCG14 forms a homodimeric transporter for multiple cytokinins and mediates long-distance transport of isopentenyladeninetype cytokinins

Cytokinins(CKs),primarily trans-zeatin(tZ)and isopentenyladenine(iP)types,play critical roles in plant growth,development,and various stress responses.Long-distance transport of tZ-type CKs meidated by Arabidopsis ATP-binding cassette transporter subfamily G14(AtABCG14)has been well studied;however,less is known about the biochemical properties of AtABCG14 and its transporter activity toward iP-type CKs.Here we reveal the biochemical properties of AtABCG14 and provide evidence that it is also required for long-distance transport of iP-type CKs.AtABCG14 formed homodimers in human(Homo sapiens)HEK293T,tobacco(Nicotiana tabacum),and Arabidopsis cells.Transporter activity assays of AtABCG14 in Arabidopsis,tobacco,and yeast(Saccharomyces cerevisiae)showed that AtABCG14 may directly transport multiple CKs,including iP-and tZ-type species.AtABCG14 expression was induced by iP in a tZ-type CKdeficient double mutant(cypDM)of CYP735A1 and CYP735A2.The atabcg14 cypDM triple mutant exhibited stronger CK-deficiency phenotypes than cypDM.Hormone profiling,reciprocal grafting,and 2H6-iP isotope tracer experiments showed that root-to-shoot and shoot-to-root long-distance transport of iP-type CKs were suppressed in atabcg14 cypDM and atabcg14.These results suggest that AtABCG14 participates in three steps of the circular long-distance transport of iP-type CKs:xylem loading in the root for shootward transport,phloem unloading in the shoot for shoot distribution,and phloemunloading in the root for root distribution.We found that AtABCG14 displays transporter activity toward multiple CK species and revealed its versatile roles in circular long-distance transport of iP-type CKs.These findings provide newinsights into the transport mechanisms of CKs and other plant hormones.

Structural characterizations of protonated homodimers of amino acids:Revealed by infrared multiple photon dissociation(IRMPD) spectroscopy and theoretical calculations

Infrared multiple photon dissociation （IRMPD） spectroscopic and theoretical studies of protonated homodimers of amino acids generated by electrospray ionization in the gas phase have been reviewed. Results show that proton affinity （PA） may be applied as a probe to predict their structural type： salt- bridged or charge-solvated. Proline can be viewed as a reference. For an amino acid with a PA value higher than that of proline, the most stable conformation of its protonated homodimer tends to prefer salt- bridged conformation; otherwise, charge-solvated conformation is expected to be the most stable. However, side chain effects may cause the inaccuracy in structural determination due to the strong interactions with the charge, which makes the charge-solvated structure more stable even for species with high PA values. Temperature effect on distribution of different isomers is also very important. In lots of cases, the coexistence of multiple isomers is general, which makes the explanation of an overall IRMPD spectrum difficult. So a statistical view on the distribution of optimized isomers is very helpful.

Cysteine residues 87 and 320 in the amino terminal domain of NMDA receptor GluN2A govern its homodimerization but do not influence GluN2A/GluN1 heteromeric assembly

N-Methyl-D-aspartate receptors（NMDARs） play a central role in various physiological and pathological processes in the central nervous system.And they are commonly composed of four subunits,two GluN1 subunits and two GluN2 or GluN3 subunits.The different subunit compositions make NMDARs a heterogeneous population with distinct electrophysiological and pharmacological properties and thus with different abilities to conduct neuronal activities.The subunit composition,assembly process,and final structure of assembled NMDARs have been studied for years but no consensus has been achieved.In this study,we investigated the role of the amino terminal domain（ATD） of GluN2A in regulating NMDAR assembly.The ATD of GluN2A was first expressed in heterogeneous cells and the homodimer formation was investigated by fluorescent resonance energy transfer and non-reducing SDS- PAGE electrophoresis.Each of the three cysteine residues located in the ATD was mutated into alanine,and the homodimerization of the ATD or GluN2A,as well as the heteromeric assembly of NMDARs was assessed by non-reducing SDS- PAGE electrophoresis,co-immunoprecipitation and immunocytochemistry.We found that two cysteine residues,C87 and C320,in the ATD of the GluN2A subunit were required for the formation of disulfide bonds and GluN2A ATD homodimers.Furthermore,the disruption of GluN2A ATD domain dimerization had no influence on the assembly and surface expression of NMDARs.These results suggest that the two ATD domains of GluN2A are structurally adjacent in fully-assembled NMDARs.However,unlike GluN1,the homomerization of the ATD domain of GluN2A is not required for the assembly of NMDARs,implying that GluN2A and GluN1 play unequal roles in NMDAR assembly.

鼠表皮生长因子受体胞外段原核表达、纯化与复性

用多聚酶链反应 (PCR)方法从既往构建质粒扩增鼠表皮生长因子受体 (EGFR)胞外段基因及适宜酶切位点 ,进而构建原核表达载体。克服质粒丢失的不足 ,在大肠杆菌中表达目的蛋白 ,并在变性条件下通过 Ni2 +柱亲和层析纯化表达蛋白 ,聚丙烯酰胺凝胶电泳 (SDS- PAGE)示纯度在 95 %以上。去除组氨酸标记的纯化蛋白在梯度尿素中透析复性。复性蛋白在表皮生长因子的作用下可以同源二聚化而在非还原 SDS- PAGE及蛋白印迹分析中呈二聚体状态 ,其所产生抗体可以识别复性蛋白及表达于肺癌细胞上的 EGFR,提示复性后大肠杆菌表达鼠EGFR胞外段重组蛋白已获得一定空间结构 ,并具有免疫原性 ,可以进一步用于

吲哚基二聚体菁类探针同步荧光测定蛋白质的研究

基于蛋白质对双嵌吲哚染料具有良好的荧光增强作用,以新型水溶性吲哚基同型二聚体探针I,建立了一种灵敏的蛋白质同步荧光分析体系。实验考察了吲哚探针的荧光特征、吲哚探针浓度、缓冲体系pH、盐浓度等参数对体系荧光的影响。在酸性条件下,蛋白质分子与探针I发生结合作用,同步荧光明显增强并向长波方向发生红移,且同步荧光强度与蛋白质浓度成良好的线性关系。在最优条件下,牛血清白蛋白BSA的线性响应范围5.00×10-7～2.50×10-5g.mL-1,检测限(3σ/K)为3×10-8g.mL-1;测定了血清蛋白BSA的合成样品,不同浓度BSA样品回收率为98.6%～103.0%,相对标准偏差1.1%～1.9%;与蛋白质紫外标准测定法比较,测定偏差为0.4%～3.9%。

在同源二聚体转录因子识别螺旋相对运动中发现平面摆动现象

目的:考察同源二聚体转录因子的2个识别螺旋在自由状态下和结合状态下相对运动的特征。方法:从蛋白质结构数据库(PDB)得到1R4I、3JXB、1KB2、1LE8等4个复合物结构文件,分别在自由状态和结合状态下用NAMD进行16 ns分子动力学模拟,将2个识别螺旋所成角分解为垂直偏移角和水平偏移角,并做散点图。结果:2个中心对称结合的同源二聚体转录因子在2种状态下,2个偏移角都相等;串联结合的同源二聚体在结合状态下2个偏移角相等。结论:同源二聚体转录因子识别螺旋相对运动存在平面摆动现象。

吲哚菁类探针与血清蛋白的相互作用研究

目的应用荧光光谱法,研究了新型水溶性吲哚基同型聚合体菁类探针I与牛血清蛋白(BSA)的相互作用。方法探索吲哚探针I-BSA的静态荧光猝灭机制和静电相互作用,系统考察了吲哚探针I与BSA的结合常数、结合位点数、热力学函数。结果确定了两者在不同温度下的结合常数分别为3.72×105(293K)、3.59×105(298K)、3.40×105(303K);结合位点数分别为1.02、0.99、0.98,吲哚探针I和BSA结合作用的ΔH,ΔS和ΔG分别为-2.242kJ.mol-1,98.80kJ.mol-1和-31.69kJ.mol-1(298K)。结论吲哚探针I与牛血清蛋白之间的结合作用力主要为静电作用。

CD4同源二聚化或寡聚化的间接检测及其介导的生物学功能的初步分析

近年来的研究表明 ,CD4分子于细胞膜表面不仅以单体形式存在还可以通过其D4和D1形成同源二聚化及寡聚化 ,并且二聚化及寡聚化的CD4分子才能稳定地与MHC Ⅱ类分子结合。通过分析CD4分子以及CD4缺失突变体分子融合Fas基因片段所诱导的转染靶细胞的凋亡 ,以及携带绿色荧光蛋白的CD4分子转染HEK2 93细胞所筛选出的稳定克隆的不同荧光强度和与MHC Ⅱ类分子阳性细胞Raji之间的不同黏附效应间接鉴定CD4同源二聚体或寡聚体的存在 ,并对二聚化或寡聚化CD4分子所介导的生物学功能进行初步分析。

新的吲哚类同型二聚体荧光探针测定蛋白质的分析研究

采用新的水溶性吲哚类同型二聚体探针I,建立了一种新的蛋白质荧光分析体系。在酸性条件下,蛋白质分子能与吲哚探针发生结合作用,体系荧光明显增强并向长波方向发生红移,荧光强度与蛋白质质量浓度呈良好的线性关系,在最优条件下,牛血清白蛋白BSA的线性响应范围0.80-25.00μg/mL,检出限（3σ/K）为0.05μg/mL;用于模拟样品的测定,1.00,2.00和5.00μg/mL的样品测定回收率为99.0%-102.0%,相对标准偏差1.8%-2.8%;与国家标准方法比较,结果测定偏差为1.0%-4.1%。

单体异柠檬酸脱氢酶的研究进展

异柠檬酸脱氢酶是三羧酸循环的关键酶之一,依赖金属离子Mg2+(或Mn2+)和辅酶NAD(P),催化异柠檬酸氧化脱羧生成α-酮戊二酸及CO2,为细胞新陈代谢提供能量和生物合成的前体物质。根据亚基的组成,原核生物IDH可分为单体IDH和同源二聚体IDH。本文主要对单体IDH的空间结构、催化机制及分子进化机制等研究进展进行了综述。

大麦黄矮病毒运动蛋白形成同型二聚体的功能研究

[目的]利用荧光双分子互补技术研究大麦黄矮病毒运动蛋白(BYDV-PAV,MP)形成二聚体的可能性,并研究MP同型二聚体与病毒运动之间的关系。[方法]将双分子荧光互补载体中包含多克隆位点、35S启动子及终止子的DNA片段构建到拷贝数较高的植物表达载体pCAMBIA1300上,然后以BYDV-PAV的全长cDNA为模板,根据GenBank中登记的BYDV-MP的基因序列设计引物,通过PCR扩增得到目的基因片段BYDV-MP,克隆到经过改造的双分子荧光互补载体pCAMBIA1300-NE、pCAMBIA1300-CE上,得到了重组的双分子荧光互补载体。电击转化农杆菌,利用农杆菌渗透注射技术注射到烟草叶片,荧光显微镜下观察植物体内的蛋白互作现象。[结果]农杆菌渗透注射后,2～5d观察双分子荧光互作现象,MP蛋白互作组及正对照组叶片产生黄色荧光,负对照组未有荧光现象。[结论]BYDV-MP在植物体内形成同源二聚体,该研究结果为深入开展BYDV运动过程和机理等研究提供了依据。

(+)/(-)-Yanhusuosines A and B, two dimeric benzylisoquinoline-protoberberine alkaloid atropo-enantiomers featuring polycyclic skeletons from Corydalis yanhusuo

(+)/(-)-Yanhusuosines A(1) and B(2), two pairs of trace benzylisoquinoline-protoberberine atropoenantiomeric homodimers featuring an unprecedented 6/7/6/6/6/6 hexacyclic skeleton, were isolated from the tubers of Corydalis yanhusuo. The structures of(+)/(-)-1 and(+)/(-)-2 were elucidated using spectroscopic and quantum-chemical calculation approaches.(+)/(-)-Yanhusuosines A(1) and B(2)represent a new class of alkaloid dimers biogenetically constructed by a molecule of benzylisoquinoline with a unit of protoberberine via an intermolecular [4 + 3] cycloaddition. Their plausible biosynthetic pathways are discussed, and compound 2 exerted moderate inhibitory activity of NO formation in LPS induced RAW264.7 macrophages.

Self-assembled nanoformulations of paclitaxel for enhanced cancer theranostics

Chemotherapy has occupied the critical position in cancer therapy,especially towards the post-operative,advanced,recurrent,and metastatic tumors.Paclitaxel(PTX)-based formulations have been widely used in clinical practice,while the therapeutic effect is far from satisfied due to off-target toxicity and drug resistance.The caseless multi-components make the preparation technology complicated and aggravate the concerns with the excipients-associated toxicity.The self-assembled PTX nanoparticles possess a high drug content and could incorporate various functional molecules for enhancing the therapeutic index.In this work,we summarize the self-assembly strategy for diverse nanodrugs of PTX.Then,the advancement of nanodrugs for tumor therapy,especially emphasis on monochemotherapy,combinational therapy,and theranostics,have been outlined.Finally,the challenges and potential improvements have been briefly spotlighted.

雷帕霉素介导的caspase 9同源二聚化调控人类多能干细胞自杀

人诱导多能干细胞(human induced pluripotent stem cells,hiPSCs)在再生医学领域具有广阔的应用前景。然而,多能干细胞(pluripotent stem cells,PSCs)具有肿瘤化风险,成为其临床应用最主要的安全性问题。雷帕霉素是一种安全和广泛使用的免疫抑制药物,通过诱导FKBP12与FRB片段的异源二聚起作用。为了保障hiPSCs治疗的安全性,本研究将雷帕霉素诱导的caspase 9(riC9)基因插入到AAVS1安全位点,构建了含有EF1α启动子、FRB-FKBP-Caspase9(CARD结构域)融合蛋白和嘌呤霉素抗性基因的供体,并与sgRNA/Cas9载体共转染hiPSCs。用嘌呤霉素筛选2周后,收集单个克隆进行基因和表型分析。最后,用雷帕霉素诱导caspase9同源二聚化,激活工程细胞的凋亡。通过对筛选获得的5个hiPSCs克隆鉴定,表明供体DNA准确敲入内源性AAVS1位点。hiPSCs保持正常的多潜能状态和增殖能力。雷帕霉素可诱导caspase9同源二聚化,并激活细胞凋亡程序。本研究通过药物精确调控caspase9的同源二聚化来启动细胞自杀,实现了可控的hiPSCs存活,这为保证hiPSCs治疗的安全性提供了新的策略。

来自普哥滨珊瑚的一株Talaromyces sp.真菌的活性次生代谢产物

利用硅胶柱层析、制备型HPLC和重结晶等手段从普哥滨珊瑚分离的一株Talaromycessp.真菌C21-1中筛选得到2个活性化合物,运用核磁共振、质谱和圆二色谱等技术鉴定这两个化合物分别为(R)-(-)-hydroxysydonic acid(1)和homodimeric WIN 64821(2),补充完善了化合物2的核磁共振信号归属,并对化合物进行抗菌活性和乙酰胆碱酯酶抑制活性的测定,发现化合物1对白色假丝酵母Canidia albicans和耐甲氧基青霉素的金黄色葡萄球菌Methicillin-resistantStaphylococcusaureus(MRSA)具有一定的抑制作用,其最低抑菌浓度(MIC)分别为0.075mmol/L和0.2mmol/L,对副溶血弧菌Vibrio parahemolyticus的抑制活性较弱,在0.2mmol/L浓度下的抑制率为17%;化合物2最大浓度0.2mmol/L条件下对这3种菌均没有明显的抑制效果。化合物2表现出剂量依赖的乙酰胆碱酯酶抑制活性,0.5mmol/L时抑制率达到35.1%。

Crystal structures of NAC domains of human nascent polypeptide-associated complex (NAC) and its αNAC subunit

Nascent polypeptide associated complex(NAC)and its two isolated subunits,αNAC and βNAC,play important roles in nascent peptide targeting.We determined a 1.9Åresolution crystal structure of the interaction core of NAC heterodimer and a 2.4Åresolution crystal structure ofαNAC NAC domain homodimer.These structures provide detailed information of NAC heterodimerization and αNAC homodimerization.We found that the NAC domains of αNAC and βNAC share very similar folding despite of their relative low identity of amino acid sequences.Furthermore,different electric charge distributions of the two subunits at the NAC interface provide an explanation to the observation that the heterodimer of NAC complex is more stable than the single subunit homodimer.In addition,we successfully built a βNAC NAC domain homodimer model based on homologous modeling,suggesting that NAC domain dimerization is a general property of the NAC family.These 3D structures allow further studies on structurefunction relationship of NAC.

Structural studies on MRG701 chromodomain reveal a novel dimerization interface of MRG proteins in green plants

MRG proteins are conserved during evolution in fungi, flies, mammals and plants, and they can exhibit diversified functions. The animal MRGs were found to form various complexes to activate gene expression. Plant MRGI/2 and MRG702 were reported to be involved in the regulation of flowering time via binding to H3K36me3- marked flowering genes. Herein, we determined the crystal structure of MRG701 chromodomain （MRG701CD）. MRG701co forms a novel dimerization fold both in crystal and in solution. Moreover, we found that the dimerization of MRG chromodomains is conserved in green plants. Our findings may provide new insights into the mechanism of MRGs in regulation of gene expression in green plants.