Identification of the cellular receptor for anthrax toxin.

The tripartite toxin secreted by Bacillus anthracis, the causative agent of anthrax, helps the bacterium evade the immune system and can kill the host during a systemic infection. Two components of the toxin enzymatically modify substrates within the cytosol of mammalian cells: oedema factor (OF) is an adenylate cyclase that impairs host defences through a variety of mechanisms including inhibiting phagocytosis; lethal factor (LF) is a zinc-dependent protease that cleaves mitogen-activated protein kinase kinase and causes lysis of macrophages. Protective antigen (PA), the third component, binds to a cellular receptor and mediates delivery of the enzymatic components to the cytosol. Here we describe the cloning of the human PA receptor using a genetic complementation approach. The receptor, termed ATR (anthrax toxin receptor), is a type I membrane protein with an extracellular von Willebrand factor A domain that binds directly to PA. In addition, a soluble version of this domain can protect cells from the action of the toxin.     

炭疽毒素及其克隆受体的研究进展

综述炭疽毒素研究的最新进展，炭疽毒素由3种蛋白组成：致死因子（L ethal factor,LF)，水肿因子(edema factor,EF)和保护性抗原（protective antigen,PA)，本文主要讲述了炭疽毒素的关键致病因子－致死因子（LF）的结构和功能，炭疽素受体（anthrax toxin receptor,ATR）的结构及其特征性功能基团，介绍了通过基因互补对ATR进行克隆的方法，并讨论了ATR和ATR的cDNA克隆在炭疽治疗的可能应用。     

Anthrax toxins cooperatively inhibit endocytic recycling by the Rab11/Sec15 exocyst

Bacillus anthracis is the causative agent of anthrax in humans and other mammals. In lethal systemic anthrax, proliferating bacilli secrete large quantities of the toxins lethal factor (LF) and oedema factor (EF), leading to widespread vascular leakage and shock. Whereas host targets of LF (mitogen-activated protein-kinase kinases) and EF (cAMP-dependent processes) have been implicated in the initial phase of anthrax, less is understood about toxin action during the final stage of infection. Here we use Drosophila melanogaster to identify the Rab11/Sec15 exocyst, which acts at the last step of endocytic recycling, as a novel target of both EF and LF. EF reduces levels of apically localized Rab11 and indirectly blocks vesicle formation by its binding partner and effector Sec15 (Sec15-GFP), whereas LF acts more directly to reduce Sec15-GFP vesicles. Convergent effects of EF and LF on Rab11/Sec15 inhibit expression of and signalling by the Notch ligand Delta and reduce DE-cadherin levels at adherens junctions. In human endothelial cells, the two toxins act in a conserved fashion to block formation of Sec15 vesicles, inhibit Notch signalling, and reduce cadherin expression at adherens junctions. This coordinated disruption of the Rab11/Sec15 exocyst by anthrax toxins may contribute to toxin-dependent barrier disruption and vascular dysfunction during B. anthracis infection.     

Crystal structure of a complex between anthrax toxin and its host cell receptor

Anthrax toxin consists of the proteins protective antigen (PA), lethal factor (LF) and oedema factor (EF). The first step of toxin entry into host cells is the recognition by PA of a receptor on the surface of the target cell. Subsequent cleavage of receptor-bound PA enables EF and LF to bind and form a heptameric PA63 pre-pore, which triggers endocytosis. Upon acidification of the endosome, PA63 forms a pore that inserts into the membrane and translocates EF and LF into the cytosol. Two closely related host cell receptors, TEM8 and CMG2, have been identified. Both bind to PA with high affinity and are capable of mediating toxicity. Here, we report the crystal structure of the PA-CMG2 complex at 2.5 A resolution. The structure reveals an extensive receptor-pathogen interaction surface mimicking the non-pathogenic recognition of the extracellular matrix by integrins. The binding surface is closely conserved in the two receptors and across species, but is quite different in the integrin domains, explaining the specificity of the interaction. CMG2 engages two domains of PA, and modelling of the receptor-bound PA63 heptamer suggests that the receptor acts as a pH-sensitive brace to ensure accurate and timely membrane insertion. The structure provides new leads for the discovery of anthrax anti-toxins, and should aid the design of cancer therapeutics.     

Coiled-coil fibrous domains mediate ligand binding by macrophage scavenger receptor type II

The macrophage scavenger receptor, which has been implicated in the pathogenesis of atherosclerosis, has an unusually broad binding specificity. Ligands include modified low-density lipoprotein and some polyanions (for example, poly(I) but not poly(C]. The scavenger receptor type I (ref. 3) has three principal extracellular domains that could participate in ligand binding: two fibrous coiled-coil domains (alpha-helical coiled-coil domain IV and collagen-like domain V), and the 110-amino-acid cysteine-rich C-terminal domain VI. We have cloned complementary DNAs encoding a second scavenger receptor which we have termed type II. This receptor is identical to the type I receptor, except that the cysteine-rich domain is replaced by a six-residue C terminus. Despite this truncation, the type II receptor mediates endocytosis of chemically modified low-density lipoprotein with high affinity and specificity, similar to that of the type I receptor. Therefore one or both of the extracellular fibrous domains are responsible for the unusual ligand-binding specificity of the receptor.     

磷脂酰肌醇转移蛋白质家族的研究进展

脂类的单体转移是由一娄蛋白质来执行的，这组蛋白质把脂类结合到疏水腔，从而使脂娄避开了含水环境、其中的这样一组蛋白质是磷脂酰肌醇转移蛋白质家族(PITPs)，能结合磷脂酰肌醇和磷脂酰胆碱，把它们从一个膜区转移到另一膜区．PITPs是在单细胞和多细胞组织中发现的，但在细菌中没有发现．在鼠和人类中，人们发现负责脂类转移的PITP结构域有五个蛋白，按照序列分成两类：类型I PITPs由两个家族成员α，β构成，它们是小蛋白35kDa，有一个PITP结构域，可以普遍表达；类型Ⅱ A PITPs(RdgBαI和Ⅱ)是很大的蛋白质，有另外的结构域，把蛋白质靶向膜，仅能结合脂类，但不能介导转移．类型Ⅱ B PITP(RdgBβ)与类型I在大小(38kDA)上相似，也是普遍表达的．类型Ⅲ PITPs，以secl4P家族为代表，是在酵母和植物中发现的，但是在序列和结构上与类型I和类型Ⅱ PITPs相似．讨论了PITP蛋白是被动转运蛋白辽是调节蛋白，在行使肌醇酯类和膜转换的专门的生物功能时，能否把转运和结合性质偶连起来．     

Synaptic vesicle-associated Ca2+/calmodulin-dependent protein kinase II is a binding protein for synapsin I.

Synapsin I is a synaptic vesicle-associated phosphoprotein that is involved in the modulation of neurotransmitter release. Ca2+/calmodulin-dependent protein kinase II, which phosphorylates two sites in the carboxy-terminal region of synapsin I, causes synapsin I to dissociate from synaptic vesicles and increases neurotransmitter release. Conversely, the dephosphorylated form of synapsin I, but not the form phosphorylated by Ca2+/calmodulin-dependent protein kinase II, inhibits neurotransmitter release. The amino-terminal region of synapsin I interacts with membrane phospholipids, whereas the C-terminal region binds to a protein component of synaptic vesicles. Here we demonstrate that the binding of the C-terminal region of synapsin I involves the regulatory domain of a synaptic vesicle-associated form of Ca2+/calmodulin-dependent protein kinase II. Our results indicate that this form of the kinase functions both as a binding protein for synapsin I, and as an enzyme that phosphorylates synapsin I and promotes its dissociation from the vesicles.     

Silencing of TGF-beta signalling by the pseudoreceptor BAMBI.

Members of the transforming growth factor-beta (TGF-beta) superfamily, including TGF-beta, bone morphogenetic proteins (BMPs), activins and nodals, are vital for regulating growth and differentiation. These growth factors transduce their signals through pairs of transmembrane type I and type II receptor kinases. Here, we have cloned a transmembrane protein, BAMBI, which is related to TGF-beta-family type I receptors but lacks an intracellular kinase domain. We show that BAMBI is co-expressed with the ventralizing morphogen BMP4 (refs 5, 6) during Xenopus embryogenesis and that it requires BMP signalling for its expression. The protein stably associates with TGF-beta-family receptors and inhibits BMP and activin as well as TGF-beta signalling. Finally, we provide evidence that BAMBI's inhibitory effects are mediated by its intracellular domain, which resembles the homodimerization interface of a type I receptor and prevents the formation of receptor complexes. The results indicate that BAMBI negatively regulates TGF-beta-family signalling by a regulatory mechanism involving the interaction of signalling receptors with a pseudoreceptor.     

参附注射液对大鼠心肌缺血再灌注细胞凋亡的影响

通过观察参附注射液对缺血再灌注心肌超微结构和细胞凋亡参数的影响 ,探讨参附注射液拮抗心肌缺血再灌注损伤的作用机制 .使用结扎 /松解左冠状动脉前降支缺血 60min,再灌注 2 4 0min复制缺血再灌注动物模型 .Sprague_Dawley(SD)大鼠 40只随机分为 5组 :对照组(Ⅰ组 ,n =8) ,缺血再灌注组(Ⅱ组 ,1 /R组 ,n =8) ,参附组 (Ⅲ组 ,n=8) ,红参组 (Ⅳ组 ,n =8) ,附子组 (Ⅴ组 ,n =8) .Ⅲ、Ⅳ、Ⅴ各治疗组在缺血前 1 0min经静脉分别注射参附注射液 (1 0mg/kg)、红参注射液 (9mg/kg)、附子注射液 (1mg/kg) ,Ⅰ组、Ⅱ组在缺血前 1 0min经静脉注射同等容积生理盐水 .测定心肌组织丙二醛 (MDA)和超氧化物歧化酶 (SOD)值 ;电镜观察心肌细胞超微结构变化 ;TUNEL法原位标记凋亡心肌细胞 ,免疫组化和图像分析技术检测心肌细胞内Bcl_2、Bax蛋白表达 .与Ⅱ组比较 ,Ⅲ、Ⅳ、Ⅴ各治疗组 ,心肌组织MDA明显降低 ,有极显著差异(P <0 .0 1 ) .III组与IV组、V组之间比较 ,MDA降低 ,有显著性差异 (P <0 .0 5) .SOD活性明显升高 ,有显著差异 (P <0 .0 5) .心肌超微结构病变明显改善、减轻 .心肌细胞凋亡指数和Bax蛋白含量显著减少(P <0 .0 1 ) ,Bcl_2蛋白含量显著增多 (P <0 .0 1 ) .参附注射液具有抗大鼠心肌缺血再灌注损伤作用 ,     

Inhibition of the EGF receptor by binding of MIG6 to an activating kinase domain interface

Members of the epidermal growth factor receptor family (EGFR/ERBB1, ERBB2/HER2, ERBB3/HER3 and ERBB4/HER4) are key targets for inhibition in cancer therapy. Critical for activation is the formation of an asymmetric dimer by the intracellular kinase domains, in which the carboxy-terminal lobe (C lobe) of one kinase domain induces an active conformation in the other. The cytoplasmic protein MIG6 (mitogen-induced gene 6; also known as ERRFI1) interacts with and inhibits the kinase domains of EGFR and ERBB2 (refs 3-5). Crystal structures of complexes between the EGFR kinase domain and a fragment of MIG6 show that a approximately 25-residue epitope (segment 1) from MIG6 binds to the distal surface of the C lobe of the kinase domain. Biochemical and cell-based analyses confirm that this interaction contributes to EGFR inhibition by blocking the formation of the activating dimer interface. A longer MIG6 peptide that is extended C terminal to segment 1 has increased potency as an inhibitor of the activated EGFR kinase domain, while retaining a critical dependence on segment 1. We show that signalling by EGFR molecules that contain constitutively active kinase domains still requires formation of the asymmetric dimer, underscoring the importance of dimer interface blockage in MIG6-mediated inhibition.     

Unfolding of C2A Domain of Synaptotagmin I in the Presence of Guanidine Hydrochloride

The C2 domain originally referred to the second of four constant structural motifs in protein kinase C (PKC). Now this domain represents a large structural family sharing a homologous dimensional structure in many proteins that play important roles in many organisms. The C2A domain is one of the two C2 domains of synaptotagmin I involved in the Ca^2 regulation of exocytosis. This domain is mostly composed of β-sheet except for a small fraction of α-helix, and therefore provides an ideal model for a protein folding study. In this report, the unfolding equilibrium of the C2A domain in guanidine hydrochloride (GdnHCI) containing solutions has been studied using ultraviolet (UV) difference spectrum, fluorescence spectrum, size exclusion chromatography (SEC), and circular dichroism (CD) spectrum. The results suggest that unfolding of the C2A domain occurs as a two-state process during GdnHCI titration. By examining the changes of both tertiary structure and secondary structure, no intermediates could be detected during this unfolding study. However, it has been found that the native state of the C2A domain has a large hydrophobic surface. This result suggests that as a fragment of a protein, the C2A domain itself may exist in a state with large hydrophobic surface. This hydrophobic surface may be the molecular basis for interaction between domains in the whole protein.Furthermore, the hydrophobic behavior may play a role during the oligomerization of svnaptotagmin.     

Characterization of 64-, 123- and 182-base-pair exons in the mouse alpha 2(IV) collagen gene

Genes encoding types I, II and III collagens (fibrillar collagens) contain many discrete-size exons, most of them 54 base pairs (bp) long, in addition to the 45-, 99-, 108- and 162-bp exons. It has been suggested that these collagen genes evolved from an ancestral coding unit of 54 bp. Type IV collagen is a specific component of basement membranes and contains two genetically distinct polypeptides, the alpha 1(IV) and alpha 2(IV) chains. It differs from the types I-III collagens in that it contains interruptions in the Gly-X-Y repeat sequence and does not form ordered fibrillar structures. We have isolated complementary DNA and genomic clones for the mouse alpha 2(IV) collagen chain and here characterize 64-, 123- and 182-bp exons in the Gly-X-Y coding domain of the gene. The data suggest that the alpha 2(IV) collagen gene may have evolved differently from those encoding the fibrillar collagens.     

Stokes方程的边界元区域分裂算法及其应用

用边界元求解不规则凹凸区域时,积分误差很大,区域分裂法是将不规则凹凸区域上的求解问题化的多个不重叠凸区域上的求解问题,在公共的边界上用Dirichlet条件,Neumann条件交替迭代得到全区域上的解。该方法计算精度高、适用于并行计算。作者给出了Stokes方程边界元求解不规则凹凸区域的区域分裂算法,并给出了将该算法用在贵阳市阿哈水库的流场计算的算例。     

A CHASE domain containing protein kinase OsCRL4, represents a new AtCRE1-like gene family in rice

AtCRE1 is known to be a cytokinin receptor inArabidopsis. The AtCRE1 protein contains CHASE domain at the N-terminal part, followed by a transmitter (histidine kinase) domain and two receiver domains. The N-terminal CHASE domain of AtCRE1 contains putative recognition sites for cytokinin. Five CHASE domains containing proteins were found in rice, OsCRLla, OsCRLlb, OsCRL2, OsCRL3, and OsCRL4. OsCRL1a, OsCRL1b, OsCRL2 and OsCRL3 contain the four domains existing in CRE1, whereas OsCRL4 only contains the CHASE domain and a putative Ser/Thr protein kinase domain The authors cloned the encoding gene OsCRL4 and found that it represents a new member of the cytokinin receptor protein in rice.     

Structural basis for EGFR ligand sequestration by Argos

Members of the epidermal growth factor receptor (EGFR) or ErbB/HER family and their activating ligands are essential regulators of diverse developmental processes. Inappropriate activation of these receptors is a key feature of many human cancers, and its reversal is an important clinical goal. A natural secreted antagonist of EGFR signalling, called Argos, was identified in Drosophila. We showed previously that Argos functions by directly binding (and sequestering) growth factor ligands that activate EGFR. Here we describe the 1.6-A resolution crystal structure of Argos bound to an EGFR ligand. Contrary to expectations, Argos contains no EGF-like domain. Instead, a trio of closely related domains (resembling a three-finger toxin fold) form a clamp-like structure around the bound EGF ligand. Although structurally unrelated to the receptor, Argos mimics EGFR by using a bipartite binding surface to entrap EGF. The individual Argos domains share unexpected structural similarities with the extracellular ligand-binding regions of transforming growth factor-beta family receptors. The three-domain clamp of Argos also resembles the urokinase-type plasminogen activator (uPA) receptor, which uses a similar mechanism to engulf the EGF-like module of uPA. Our results indicate that undiscovered mammalian counterparts of Argos may exist among other poorly characterized structural homologues. In addition, the structures presented here define requirements for the design of artificial EGF-sequestering proteins that would be valuable anti-cancer therapeutics.     

领域分析方法研究及应用

通过对传统的OOA技术和面向特征的思想在领域分析中优缺点的比较分析,介绍了一种将两者结合的领域分析方法,即基于特征的面向对象领域分析方法。描述了此方法的分析和建模过程,以及所产生的领域模型。采用该方法对集团化企业供应物流领域进行分析与建模,界定了该领域的范围、子领域,建立了采购计划子领域的领域模型。此方法有利于获取领域业务构件,提高软件复用度。     

酵母培养物对土杂鸡生产性能及营养物质代谢率的影响

本试验旨在研究酵母培养物对土杂鸡生产性能及营养物质代谢率的影响,试验选择28日龄健康、体重相近的土杂鸡200只,随机分为5组,设1个对照组,饲喂基础日粮,其余4个为试验组(Ⅰ、Ⅱ、Ⅲ、Ⅳ组),在基础日粮中分别添加0.15%、0.20%、0.25%、0.30%的酵母培养物。结果表明:与对照组相比,各酵母培养物组肉鸡的平均日增体质量和平均日采食量均显著高于对照组(P0.05);试验Ⅱ、Ⅲ、Ⅳ组的料重比均显著低于对照组(P0.05),其中试验Ⅱ组的最低;与对照组相比,所有试验组粗蛋白质(CP)代谢率均明显提高(P﹤0.01);粗脂肪(EE)代谢率有提高趋势,但差异不显著(P0.05);试验I、Ⅱ组的钙、磷代谢率明显提高(P0.05);试验Ⅱ、Ⅲ、Ⅳ组的表观代谢率(ME)显著提高(P0.05),且试验Ⅱ的最高。结论:综合分析,本试验在土杂鸡日粮中酵母培养物的适宜添加量为0.2%。     

Dynamic measurement of myosin light-chain-domain tilt and twist in muscle contraction.

A new method is described for measuring motions of protein domains in their native environment on the physiological timescale. Pairs of cysteines are introduced into the domain at sites chosen from its static structure and are crosslinked by a bifunctional rhodamine. Domain orientation in a reconstituted macromolecular complex is determined by combining fluorescence polarization data from a small number of such labelled cysteine pairs. This approach bridges the gap between in vitro studies of protein structure and cellular studies of protein function and is used here to measure the tilt and twist of the myosin light-chain domain with respect to actin filaments in single muscle cells. The results reveal the structural basis for the lever-arm action of the light-chain domain of the myosin motor during force generation in muscle.     

Novel role of PKR in inflammasome activation and HMGB1 release

The inflammasome regulates the release of caspase activation-dependent cytokines, including interleukin (IL)-1β, IL-18 and high-mobility group box 1 (HMGB1). By studying HMGB1 release mechanisms, here we identify a role for double-stranded RNA-dependent protein kinase (PKR, also known as EIF2AK2) in inflammasome activation. Exposure of macrophages to inflammasome agonists induced PKR autophosphorylation. PKR inactivation by genetic deletion or pharmacological inhibition severely impaired inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminium, rotenone, live Escherichia coli, anthrax lethal toxin, DNA transfection and Salmonella typhimurium infection. PKR deficiency significantly inhibited the secretion of IL-1β, IL-18 and HMGB1 in E. coli-induced peritonitis. PKR physically interacts with several inflammasome components, including NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), NLRP1, NLR family CARD domain-containing protein 4 (NLRC4), absent in melanoma 2 (AIM2), and broadly regulates inflammasome activation. PKR autophosphorylation in a cell-free system with recombinant NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC, also known as PYCARD) and pro-caspase-1 reconstitutes inflammasome activity. These results show a crucial role for PKR in inflammasome activation, and indicate that it should be possible to pharmacologically target this molecule to treat inflammation.     

编码ATR的胞外区基因cDNA的克隆、测序及表达

目的克隆并在大肠杆菌中表达编码炭疽毒素受体(anthrax toxin receptor,简称ATR)的胞外区基因。方法收集CHO-K1细胞,提取其总RNA,经反转录成单链cDNA,以此为模板PCR扩增出编码ATR胞外区基因,将该基因克隆入载体pUC19中,测序正确后,亚克隆入表达载体pMal-c2x中进行表达。结果利用所设计的引物扩增出完整的编码ATR胞外区基因。以大肠杆菌为宿主在IPTG的诱导下获得表达,激光薄层扫描显示表达蛋白占总蛋白的39%。结论获得了编码ATR胞外区基因cDNA及其原核表达产物,为进一步研究炭疽毒素致病机理和炭疽病的防治奠定了基础。