Dishevelled 2 recruits beta-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4

Wnt proteins, regulators of development in many organisms, bind to seven transmembrane-spanning (7TMS) receptors called frizzleds, thereby recruiting the cytoplasmic molecule dishevelled (Dvl) to the plasma membrane.Frizzled-mediated endocytosis of Wg (a Drosophila Wnt protein) and lysosomal degradation may regulate the formation of morphogen gradients. Endocytosis of Frizzled 4 (Fz4) in human embryonic kidney 293 cells was dependent on added Wnt5A protein and was accomplished by the multifunctional adaptor protein beta-arrestin 2 (betaarr2), which was recruited to Fz4 by binding to phosphorylated Dvl2. These findings provide a previously unrecognized mechanism for receptor recruitment of beta-arrestin and demonstrate that Dvl plays an important role in the endocytosis of frizzled, as well as in promoting signaling.     

The amyloid precursor protein has a flexible transmembrane domain and binds cholesterol

C99 is the transmembrane carboxyl-terminal domain of the amyloid precursor protein that is cleaved by γ-secretase to release the amyloid-β polypeptides, which are associated with Alzheimer's disease. Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy show that the extracellular amino terminus of C99 includes a surface-embedded "N-helix" followed by a short "N-loop" connecting to the transmembrane domain (TMD). The TMD is a flexibly curved α helix, making it well suited for processive cleavage by γ-secretase. Titration of C99 reveals a binding site for cholesterol, providing mechanistic insight into how cholesterol promotes amyloidogenesis. Membrane-buried GXXXG motifs (G, Gly; X, any amino acid), which have an established role in oligomerization, were also shown to play a key role in cholesterol binding. The structure and cholesterol binding properties of C99 may aid in the design of Alzheimer's therapeutics.     

Bright Yellow 2烤烟热激蛋白90生物信息学分析

[目的]分析Bright Yellow 2烤烟热激蛋白90(HSP90)的生物学信息,为揭示烘烤过程中烟叶的烘烤特性提供理论依据.[方法]从NCBI中获取Bright Yellow 2烤烟HSP90蛋白序列NtHSP-1、NtHSP-2和NtHSP-3(GenBank登录号分别为BAL42333、BAL42332和BAM24708),利用生物信息学软件对各序列进行理化特性、跨膜区、信号肽、亚细胞定位、二级结构和三级结构预测分析.[结果]Bright Yellow 2烤烟NtHSP-1、NtHSP-2和NtHSP-3的氨基酸数量分别为812、811和699个,均以酸性氨基酸含量较多,其中谷氨酸(Glu)含量最高,理论等电点均小于5.00,二级结构均以α-螺旋(H)和无规则卷曲(C)为主.NtHSP-1和NtHSP-2的亲水性较高,具有较高的稳定性,属于分泌蛋白,具有锚定结构,定位于内质网,其三级结构呈现"r"形,N端内部具有Mg2+结合位点、HATPase_c(ATP酶)和top6b(DNA拓扑异构酶VI)功能域;NtHSP-3为非分泌型蛋白,定位于叶绿体,三级结构呈现"i"形,N端含有Mg2+结合位点和HATPase_c功能域.[结论]NtHSP-1和NtHSP-2可能与烟叶耐烤性有关,而NtHSP-3可能与烟叶易烤性有关.     

A model for the tertiary structure of p21, the product of the ras oncogene

A model was developed for the structure of p21, the protein with a molecular weight of 21,000 that is produced by the ras genes. This model predicts that p21 consists of a central core of beta-sheet structure, connected by loops and alpha helices. Four of these loops comprise the guanine nucleotide binding site. The phosphoryl binding region is made up of amino acid sequences from 10 to 16 and from 57 to 63 of p21. The latter sequence may contain a site for magnesium binding. Amino acids defining guanine specificity are Asn-116 and Asp-119, and sequences around amino acid 145 may contribute to guanine binding. The model makes it possible to visualize how oncogenic mutations of p21 affect interaction with guanine nucleotides.     

A specific amino acid binding site composed of RNA

A specific, reversible binding site for a free amino acid is detectable on the intron of the Tetrahymena self-splicing ribosomal precursor RNA. The site selects arginine among the natural amino acids, and prefers the L- to the D-amino acid. The dissociation constant is in the millimolar range, and amino acid binding is at or in the catalytic rG splicing substrate site. Occupation of the G site by L-arginine therefore inhibits splicing by inhibiting the binding of rG, without inhibition of later reactions in the splicing reaction sequence. Arginine binding specificity seems to be directed at the side chain and the guanidino radical, and the alpha-amino and carboxyl groups are dispensable for binding. The arginine site can be placed within the G site by structural homology, with consequent implications for RNA-amino acid interaction, for the origin of the genetic code, for control of RNA activities, and for further catalytic capabilities for RNA.     

Three-dimensional structure of favin: saccharide binding-cyclic permutation in leguminous lectins

The three-dimensional structure of favin, the glucose- and mannose-binding lectin of Vicia faba (vetch, broad bean), has been determined at a resolution of 2.8 angstroms by molecular replacement. The crystals contain specifically bound glucose and provide the first high-resolution view of specific saccharide binding in a leguminous lectin. The structure is similar to those of concanavalin A (Con A) and green pea lectin; differences from Con A show that minimal changes are needed to accommodate the cyclic permutation in amino acid sequence between the two molecules. The molecule is an ellipsoidal dimer dominated by extensive beta structures. Each protomer contains binding sites for two divalent metal ions (Mn2+ and Ca2+) and a specific saccharide. Glucose is bound by favin in a cleft in the molecular surface and has noncovalent contacts primarily with two peptide loops, one of which contains several metal ion ligands. The specific carbohydrate-binding site is similar to that of Con A in location and general peptide folding, despite several differences in specific amino acid residues.     

Convergent solutions to binding at a protein-protein interface

The hinge region on the Fc fragment of human immunoglobulin G interacts with at least four different natural protein scaffolds that bind at a common site between the C(H2) and C(H3) domains. This "consensus" site was also dominant for binding of random peptides selected in vitro for high affinity (dissociation constant, about 25 nanomolar) by bacteriophage display. Thus, this site appears to be preferred owing to its intrinsic physiochemical properties, and not for biological function alone. A 2.7 angstrom crystal structure of a selected 13-amino acid peptide in complex with Fc demonstrated that the peptide adopts a compact structure radically different from that of the other Fc binding proteins. Nevertheless, the specific Fc binding interactions of the peptide strongly mimic those of the other proteins. Juxtaposition of the available Fc-complex crystal structures showed that the convergent binding surface is highly accessible, adaptive, and hydrophobic and contains relatively few sites for polar interactions. These are all properties that may promote cross-reactive binding, which is common to protein-protein interactions and especially hormone-receptor complexes.     

甘蔗ATP结合蛋白基因克隆及其干旱胁迫下的表达特性分析

[目的]克隆甘蔗ATP结合蛋白基因,分析其基本生物学信息及在干旱胁迫下的表达特性,为ATP结合蛋白的功能研究提供理论依据.[方法]从乙烯诱导甘蔗差异表达转录组和水分胁迫下甘蔗cDNA文库中获取ATP结合蛋白基因序列,设计其引物进行RT-PCR扩增,利用生物信息学软件进行序列分析及其蛋白结构和功能预测,并采用实时荧光定量PCR(qPCR)检测干旱胁迫处理下的甘蔗ATP结合蛋白基因的表达情况.[结果]克隆获得的甘蔗ATP结合蛋白基因全长2145bp,编码714个氨基酸,其蛋白分子质量为79.430kD,理论等电点(pI)为9.33,不稳定系数为44.38;甘蔗ATP结合蛋白与玉米ATP结合蛋白的核苷酸序列及其推导氨基酸系列同源性最高,均达93%.甘蔗ATP结合蛋白主要由无规则卷曲(42.02%)、α螺旋(25.63%)和延伸链(22.27%)结构组成,具有蛋白激酶催化结构域、ATP结合位点、核苷酸结合位点(NBS)、糖基化位点、激酶磷酸化结合位点及核定位信号等;甘蔗ATP结合蛋白可能主要定位在细胞核、叶绿体、线粒体和过氧化物酶体中,起中间代谢调控作用,或参与转录调控、免疫应答、胁迫应答及信号转导等生物反应.qPCR检测结果显示,在干旱胁迫7d后,甘蔗ATP结合蛋白基因的表达量明显下调,约为对照的30%,恢复正常供水(复水)后,表达量回升.[结论]甘蔗ATP结合蛋白基因表达受干旱胁迫诱导,可能参与甘蔗对干旱逆境胁迫响应的代谢调控.     

瓦氏黄颡鱼肝脂酶基因cDNA序列的克隆与序列分析

利用RT-PCR方法克隆瓦氏黄颡鱼(Pelteobagrus vachelli)肝脂酶cDNA序列片段,并对其基因结构和系统进化关系进行分析.瓦氏黄颡鱼肝脂酶cDNA片段长1065 bp,编码353个氨基酸.肝脂酶氨基酸序列同源性分析结果表明,瓦氏黄颡鱼与其他鱼类的同源性为62%-100%.瓦氏黄颡鱼肝脂酶氨基酸残基包含糖基化位点、催化位点、催化中心三联体位点、脂质结合位点和多肽"盖"等主要结构区域.系统进化树分析表明,瓦氏黄颡鱼肝脂酶与草鱼、鳙、斑马鱼肝脂酶聚为一支.因此,瓦氏黄颡鱼的肝脂酶在鱼类进化中较为保守.     

An insertion in the human thyrotropin receptor critical for high affinity hormone binding

Thyrotropin (TSH), luteinizing hormone (LH), and chorionic gonadotropin (CG) are structurally related glycoprotein hormones, which bind to receptors that share a high degree of sequence similarity. However, comparison of the primary amino acid sequences of the TSH and LH-CG receptors reveals two unique insertions of 8 and 50 amino acids in the extracellular domain of the TSH receptor. The functional significance of these insertions were determined by site-directed mutagenesis. Deletion of the 50-amino acid tract (residues 317 to 366) had no effect on TSH binding or on TSH and thyroid-stimulating immunoglobulin (TSI) biological activities. In contrast, either deletion or substitution of the eight-amino acid region (residues 38 to 45) abolished these activities. This eight-amino acid tract near the amino terminus of the TSH receptor appears to be an important site of interaction for both TSH and TSI.     

新疆南疆驴源H3N8亚型EIVHA基因序列分析

为明确新疆南疆驴是否感染马流感病毒(Equine influenza virus,EIV)及驴源EIV HA基因特征,根据GenBank登录的EIV基因序列,设计合成1对引物,采集新疆南疆地区2个驴屠宰点33份驴肺组织样品,采用RT-PCR扩增驴源EIV HA基因片段,并对扩增产物进行测序与序列分析。结果表明,中国新疆南疆驴源EIV A/donkey/Xinjiang/1/2015(H3N8)株HA基因长1 704bp,编码567个氨基酸。同源性分析结果显示,与国内外H3N8亚型EIV参考毒株核苷酸和氨基酸同源性分别为88.9%~99.9%和88.3%~99.8%,与国内毒株和哈萨克斯坦毒株在一个进化分支内。与GenBank数据库中唯一1个驴源毒株A/donkey/Xinjiang/5/2007(H3N8)的比对结果显示,2个毒株裂解位点、糖基化位点、抗原位点和受体结合位点氨基酸完全一致,只是错位2个氨基酸。确定新疆南疆存在驴感染EIV,可能为国内疫情的延续或周边国家传入。     

Cloning of Thymidine Kinase Gene of Duck Plague Virus Using Degenerate PCR

The DNA of duck plague virus （DPV） thymidine kinase （TK） gene was cloned and sequenced from a vaccine virus in the study. Degenerate oligonucleotide primers for the consensus site of herpesvirus UL24, TK, and glycoprotein H（gH） gene were used in the polymerase chain reaction （PCR） to amplify DNA product with 3 741-base-pairs （bp） in size. DNA sequence analysis revealed a 1 077-base-pairs （bp） open reading frame （ORF） encoding a 358 amino acid polypeptide homologous to herpesvirus TK proteins. The predicted TK protein shared 31.2, 41.3, 35.7, 37.4, and 28.4% identity with herpes simplex virus typel, equine herpesvirus type 4, Marek＇s disease virus 2, herpesvirus turkey, and infectious laryngotracheitis virus, respectively. Comparison of the amino acid sequences of other herpesvirus TK proteins showed that these proteins were not conserved on the whole, otherwise the portion of the TK proteins corresponding to the nucleotide binding domain and the nucleoside binding site were highly conserved among herpesvirus. Comparison with the amino acid sequences of the conserved nucleotide and nucleoside binding domains of other eleven herpesvirus TK proteins to the predicted DPV peptide confirmed its identity as the DPV TK protein.     

A synaptic vesicle protein with a novel cytoplasmic domain and four transmembrane regions

Complementary DNA and genomic clones were isolated and sequenced corresponding to rat and human synaptophysin (p38), a major integral membrane protein of synaptic vesicles. The deduced amino acid sequences indicate an evolutionarily highly conserved protein that spans the membrane four times. Both amino and carboxyl termini face the cytoplasm, with the latter containing ten copies of a tyrosine-rich pentapeptide repeat. The structure of synaptophysin suggests that the protein may function as a channel in the synaptic vesicle membrane, with the carboxyl terminus serving as a binding site for cellular factors.     

α?芋螺毒素LvIA第11位氨基酸的新突变体合成及其靶点活性

为了进一步探究第11位氨基酸的性质对LvIA靶点结合活性的影响，设计了LvIA的2个新型突变体[D11R]LvIA和[D11H]LvIA，即用2个碱性氨基酸?精氨酸（R）和组氨酸（H）分别替换原来的酸性氨基酸D。先人工合成了这2个新突变体的线性肽，然后采用2步氧化法进行折叠，以获得在第1位和第3位半胱氨酸（Cys 1～3）、第2位和第4位半胱氨酸（Cys 2～4）之间定点连接形成二硫键。经高效液相色谱分离纯化和质谱鉴定，合成了含有Cys(1～3, 2～4)二硫键连接方式的多肽，其分子质量正确，纯度在95%以上。利用双电极电压钳电生理学技术对这2种突变体与α3β2 nAChR的结合活性进行了检测。结果发现，当该位点的氨基酸性质由酸性转换为碱性后，对LvIA的活性影响巨大，直接导致对α3β2 nAChR的阻断活性丧失。[D11R]LvIA和[D11H]LvIA的活性与野生型LvIA相比分别降低了574.38%和408.62%。由此表明，第11位氨基酸的酸碱性对LvIA的活性至关重要。     

Mechanisms of antibody binding to a protein

The mechanisms of antibody binding to a protein were studied by an analysis of specific amino acid residues critical to nine antigenic sites on myohemerythrin. Rabbit antisera to the whole protein were assayed for binding to more than 1500 distinct peptide analogs differing from the protein sequence by single amino acid replacements. The results, combined with information from the three-dimensional crystallographic structure, were used to evaluate probable mechanisms of antibody binding at individual sites. The data from all sites examined indicate that initial binding to solvent-exposed amino acid residues may promote local side-chain displacements and thereby allow the participation of other, previously buried, residues.     

西花蓟马气味结合蛋白的cDNA克隆、序列分析及时空表达

【目的】鉴定西花蓟马(Frankliniella occidentalis)气味结合蛋白（odorant binding protein,OBP）并对其序列特征及分布情况进行研究,为阐明该虫嗅觉识别的机制、利用干扰昆虫嗅觉识别来进行害虫防治提供依据。【方法】利用RT-PCR和RACE技术克隆西花蓟马气味结合蛋白基因,用DNAMAN软件进行序列分析,用BLAST进行同源性比较,并用MEGA6.0的邻接法（neighbor-joining）构建系统进化树,应用服务器Chou & Fasman预测蛋白的二级结构,通过实时荧光定量PCR检测该基因在西花蓟马不同发育期以及成虫不同组织中的分布情况。【结果】克隆了一个西花蓟马气味结合蛋白基因,命名为FoccOBP1（GenBank登录号：KM527948）;该基因cDNA序列全长660 bp,其中开放阅读框450 bp,编码149个氨基酸,3′端非编码区长172 bp,具有真核生物polyA加尾结构,5′末端非编码区长38 bp,预测成熟蛋白分子量为16.39 kD,等电点为7.46,N端有22个氨基酸组成的信号肽序列,具有气味结合蛋白典型的6个保守半胱氨酸位点特征,其排列方式为C1-X₂₆-C2-X₃-C3-X₄₀-C4-X₉-C5-X₈-C6,符合典型OBP 6个保守的半胱氨酸位点结构模型。氨基酸序列中有3个亲脂性区域,第74-83位的氨基酸残基形成的亲脂性口袋尤为明显,可能是脂溶性气味分子的结合位点;FoccOBP1氨基酸序列与3个半翅目和10个同翅目昆虫OBP聚在同一分支,其中与绿盲蝽（Apolygus lucorum）的AlucOBP8（GenBank登录号为AFJ54049.1）、苜蓿盲蝽（Adelphocoris lineolatus）的AlinOBP5（GenBank登录号为ACZ58031.1）、牧草盲蝽（Lygus lineolaris）的LlinOBP2（GenBank登录号为AHF71029.1）同源相似性最高,其次是棉蚜（Aphis gossypii）的AgosOBP7（GenBank登录号为AGE97637.1）、大豆蚜（Aphis glycines）的AglyOBP7（GenBank登录号为AHJ80893.1）、禾谷缢管蚜（Rhopalosiphum padi）的RpadOBP7（GenBank登录号为AHL30243.1）等昆虫的OBP,表明FoccOBP1与这些基因的亲缘关系也较近;经FoccOBP1蛋白的二级结构预测,FoccOBP1以α-螺旋为主,其次是β-折叠,转角含量较少;经不同发育阶段检测发现,FoccOBP1在西花蓟马若虫期和初羽化成虫期的表达量较高,且随成虫羽化后天数的延长表达量逐渐降低,在雌雄成虫间的表达量也有差异;在初羽化成虫的不同组织检测发现,FoccOBP1几乎在初羽化成虫触角中特异性表达;构建了重组表达质粒pET-30a/FoccOBP1。【结论】明确了FoccOBP1的核苷酸、氨基酸序列特征,分析了该蛋白的二级结构特征,根据FoccOBP1在西花蓟马中的时空表达情况,推测该基因可能在西花蓟马嗅觉识别、定位寄主植物、信息素合成或性行为方面扮演重要角色;成功构建了重组表达质粒pET-30a/FoccOBP1,为下一步该蛋白表达纯化及其功能的深入研究打下基础。     

Three-dimensional structure of an oncogene protein: catalytic domain of human c-H-ras p21

The crystal structure at 2.7 A resolution of the normal human c-H-ras oncogene protein lacking a flexible carboxyl-terminal 18 residue reveals that the protein consists of a six-stranded beta sheet, four alpha helices, and nine connecting loops. Four loops are involved in interactions with bound guanosine diphosphate: one with the phosphates, another with the ribose, and two with the guanine base. Most of the transforming proteins (in vivo and in vitro) have single amino acid substitutions at one of a few key positions in three of these four loops plus one additional loop. The biological functions of the remaining five loops and other exposed regions are at present unknown. However, one loop corresponds to the binding site for a neutralizing monoclonal antibody and another to a putative "effector region"; mutations in the latter region do not alter guanine nucleotide binding or guanosine triphosphatase activity but they do reduce the transforming activity of activated proteins. The data provide a structural basis for understanding the known biochemical properties of normal as well as activated ras oncogene proteins and indicate additional regions in the molecule that may possibly participate in other cellular functions.     

Human apolipoprotein B: structure of carboxyl-terminal domains, sites of gene expression, and chromosomal localization

Apolipoprotein (apo-) B is the ligand responsible for the receptor-mediated catabolism of low density lipoproteins, the principal cholesterol-transporting lipoproteins in plasma. The primary structure of the carboxyl-terminal 30 percent (1455 amino acids) of human apo-B (apo-B100) has been deduced from the nucleotide sequence of complementary DNA. Portions of the protein structure that may relate to its receptor binding function and lipid binding properties have been identified. The apo-B100 messenger RNA is about 19 kilobases in length. The apo-B100 gene is expressed primarily in liver and, to a lesser extent, in small intestine, but in no other tissues. The gene for apo-B100 is located in the p24 region (near the tip of the short arm) of chromosome 2.     

Immunoglobulin structure: amino terminal sequences of mouse myeloma proteins that bind phosphorylcholine

The amino terminal sequences of five light and heavy immunoglobulin chains from myeloma proteins of the BALB/c mouse with binding activity to phosphorylcholine are presented. Except for a single substitution in position 4, all five heavy chains have identical amino terminal sequences through the first hypervariable region. Proteins which share unique (idiotypic) antigenic determinants are identical through the first hypervariable region of their light and heavy chains. Proteins with differing idiotypic determinants have light chains of differing amino acid sequence. These observations suggest that the heavy chain plays a more important role than the light chain in determining the phosphorylcholine binding site.     

Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase

The crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase complexed with a 20-amino acid substrate analog inhibitor has been solved and partially refined at 2.7 A resolution to an R factor of 0.212. The magnesium adenosine triphosphate (MgATP) binding site was located by difference Fourier synthesis. The enzyme structure is bilobal with a deep cleft between the lobes. The cleft is filled by MgATP and a portion of the inhibitor peptide. The smaller lobe, consisting mostly of amino-terminal sequence, is associated with nucleotide binding, and its largely antiparallel beta sheet architecture constitutes an unusual nucleotide binding motif. The larger lobe is dominated by helical structure with a single beta sheet at the domain interface. This lobe is primarily involved in peptide binding and catalysis. Residues 40 through 280 constitute a conserved catalytic core that is shared by more than 100 protein kinases. Most of the invariant amino acids in this conserved catalytic core are clustered at the sites of nucleotide binding and catalysis.