拟南芥AHA2基因的生物信息学分析

利用生物信息学方法对拟南芥AHA2(H+-ATPase 2)基因编码蛋白的理化性质、保守结构域、疏水性/亲水性、信号肽、跨膜结构域、磷酸化修饰、二级结构和系统进化等进行了预测和分析。结果表明:AHA2属于拟南芥H+-ATPase家族成员;拟南芥AHA2基因编码的蛋白与荠菜的亲缘关系最近,具有较高的相似性。本研究的结果将为进一步研究其生物学功能提供一定的参考依据。     

水稻OsRacD与OsRhoGDI2基因共表达载体的构建

水稻OsRacD是从水稻幼穗中分离的Rho基因,OsRhoGDI2是通过酵母双杂交筛选到的与OsRacD相互作用蛋白的编码基因,已有的研究表明OsRacD与OsRhoGDI2之间可能存在相互作用,并参与水稻育性调控过程.为了鉴定二者的相互作用,本研究构建了OsRacD与OsRhoGDI2基因共表达载体,为采用免疫共沉淀验证二者在体内的相互作用,解析OsRacD与OsRhoGDI2基因参与水稻育性控制的分子机制奠定了基础.     

Rewiring cellular morphology pathways with synthetic guanine nucleotide exchange factors

Eukaryotic cells mobilize the actin cytoskeleton to generate a remarkable diversity of morphological behaviours, including motility, phagocytosis and cytokinesis. Much of this diversity is mediated by guanine nucleotide exchange factors (GEFs) that activate Rho family GTPases-the master regulators of the actin cytoskeleton. There are over 80 Rho GEFs in the human genome (compared to only 22 genes for the Rho GTPases themselves), and the evolution of new and diverse GEFs is thought to provide a mechanism for linking the core cytoskeletal machinery to a wide range of new control inputs. Here we test this hypothesis and ask if we can systematically reprogramme cellular morphology by engineering synthetic GEF proteins. We focused on Dbl family Rho GEFs, which have a highly modular structure common to many signalling proteins: they contain a catalytic Dbl homology (DH) domain linked to diverse regulatory domains, many of which autoinhibit GEF activity. Here we show that by recombining catalytic GEF domains with new regulatory modules, we can generate synthetic GEFs that are activated by non-native inputs. We have used these synthetic GEFs to reprogramme cellular behaviour in diverse ways. The GEFs can be used to link specific cytoskeletal responses to normally unrelated upstream signalling pathways. In addition, multiple synthetic GEFs can be linked as components in series to form an artificial cascade with improved signal processing behaviour. These results show the high degree of evolutionary plasticity of this important family of modular signalling proteins, and indicate that it may be possible to use synthetic biology approaches to manipulate the complex spatio-temporal control of cell morphology.     

The structural basis of Arfaptin-mediated cross-talk between Rac and Arf signalling pathways

Small G proteins are GTP-dependent molecular switches that regulate numerous cellular functions. They can be classified into homologous subfamilies that are broadly associated with specific biological processes. Cross-talk between small G-protein families has an important role in signalling, but the mechanism by which it occurs is poorly understood. The coordinated action of Arf and Rho family GTPases is required to regulate many cellular processes including lipid signalling, cell motility and Golgi function. Arfaptin is a ubiquitously expressed protein implicated in mediating cross-talk between Rac (a member of the Rho family) and Arf small GTPases. Here we show that Arfaptin binds specifically to GTP-bound Arf1 and Arf6, but binds to Rac.GTP and Rac.GDP with similar affinities. The X-ray structure of Arfaptin reveals an elongated, crescent-shaped dimer of three-helix coiled-coils. Structures of Arfaptin with Rac bound to either GDP or the slowly hydrolysable analogue GMPPNP show that the switch regions adopt similar conformations in both complexes. Our data highlight fundamental differences between the molecular mechanisms of Rho and Ras family signalling, and suggest a model of Arfaptin-mediated synergy between the Arf and Rho family signalling pathways.     

Cdc42的生物活性

Cdc42是Rho家族蛋白(Rho GTPase)中的一种,是Rho家族中研究得较多的一个,具有GTP酶活性。Cdc42参与细胞周期调控和基因转录的调节,在调节细胞骨架、肿瘤表达、细胞极性等方面发挥重要作用。     

Nucleotide sequence of the rat skeletal muscle actin gene

The actins constitute a family of highly conserved proteins found in all eukaryotic cells. Their conservation through a very wide range of taxonomic groups and the existence of tissue-specific isoforms make the actin genes very interesting for the study of the evolution of genes and their controlling elements. On the basis of amino acid sequence data, at least six different mammalian actins have been identified (skeletal muscle, cardiac muscle, two smooth muscle actins and the cytoplasmic beta- and gamma-actins). Rat spleen DNA digested by the EcoRI restriction enzyme contains at least 12 different fragments with actin-like sequences but only one which hybridized, in very stringent conditions, with the skeletal muscle cloned cDNA probe. Here we describe the sequence of the actin gene in that fragment. The nucleotide sequence codes for two amino acids, Met-Cys, preceding the known N-terminal Asp of the mature protein. There are five small introns in the coding region and a large intron in the 5'-untranslated region. Comparison of the structure of the rat skeletal muscle actin gene with available data on actin genes from other organisms shows that while the sequenced actin genes from Drosophila and yeast have introns at different locations, introns located at codons specifying amino acids 41, 121, 204 and 267 have been preserved at least from the echinoderm to the vertebrates. A similar analysis has been done by Davidson. An intron at codon 150 is common to a plant actin gene and the skeletal muscle acting gene.     

玫瑰微球菌海藻糖合成相关酶基因的克隆和序列分析

从玫瑰微球菌QS412中克隆出海藻糖生成相关酶――麦芽寡糖基海藻糖基水解酶的基因treZ ,测定了其核苷酸序列并进行了表达。treZ 编码的蛋白质有624个氨基酸、分子质量为68 kD. 它们与已报道的其他微生物的海藻糖生成相关酶的基因进行同源性比较，treZ 的同源性分别为33.0%(耐放射异常球菌)；10.1%(硫矿硫化叶菌KM1)；51.9%(节杆菌Q36)；52.8%(根瘤菌M11)；48.5% (短杆菌)。经过氨基酸序列比较分析还发现，所有的海藻糖生成相关酶都含有糖苷酶家族13个中几个高度保守的α-淀粉酶催化活性区，推测这些海藻糖生成相关酶都可能有着共同的进化来源。     

甲胎蛋白分子演化的生物信息学分析

甲胎蛋白（AFP）是类白蛋白家族成员蛋白之一，在生物个体发育以及肝癌发生发展中起重要作用. 为了研究AFP的系统发生，并以此探讨系统发育与个体发育的关系，运用生物信息学手段，分析了不同物种中类白蛋白家族成员的进化关系，并对各分子的白蛋白结构域进行了进化归类.同时，对不同物种的白蛋白（ALB）和AFP之间包含启动子、抑制子和增强子在内的约14?kb的核苷酸序列进行了比较分析. 研究从系统发育的角度阐释了AFP分子及其基因调控机制的演化方式，为更好的认识AFP在机体生理和病理状态的作用建立了理论分析的基础.     

Moesin functions antagonistically to the Rho pathway to maintain epithelial integrity

Two prominent characteristics of epithelial cells, apical-basal polarity and a highly ordered cytoskeleton, depend on the existence of precisely localized protein complexes associated with the apical plasma membrane, and on a separate machinery that regulates the spatial order of actin assembly. ERM (ezrin, radixin, moesin) proteins have been proposed to link transmembrane proteins to the actin cytoskeleton in the apical domain, suggesting a structural role in epithelial cells, and they have been implicated in signalling pathways. Here, we show that the sole Drosophila ERM protein Moesin functions to promote cortical actin assembly and apical-basal polarity. As a result, cells lacking Moesin lose epithelial characteristics and adopt invasive migratory behaviour. Our data demonstrate that Moesin facilitates epithelial morphology not by providing an essential structural function, but rather by antagonizing activity of the small GTPase Rho. Thus, Moesin functions in maintaining epithelial integrity by regulating cell-signalling events that affect actin organization and polarity. Furthermore, our results show that there is negative feedback between ERM activation and activity of the Rho pathway.     

Structural and mechanistic insights into the interaction between Rho and mammalian Dia

Formins are involved in a variety of cellular processes that require the remodelling of the cytoskeleton. They contain formin homology domains FH1 and FH2, which initiate actin assembly. The Diaphanous-related formins form a subgroup that is characterized by an amino-terminal Rho GTPase-binding domain (GBD) and an FH3 domain, which bind somehow to the carboxy-terminal Diaphanous autoregulatory domain (DAD) to keep the protein in an inactive conformation. Upon binding of activated Rho proteins, the DAD is released and the ability of the formin to nucleate and elongate unbranched actin filaments is induced. Here we present the crystal structure of RhoC in complex with the regulatory N terminus of mammalian Diaphanous 1 (mDia1) containing the GBD/FH3 region, an all-helical structure with armadillo repeats. Rho uses its 'switch' regions for interacting with two subdomains of GBD/FH3. We show that the FH3 domain of mDia1 forms a stable dimer and we also identify the DAD-binding site. Although binding of Rho and DAD on the N-terminal fragment of mDia1 are mutually exclusive, their binding sites are only partially overlapping. On the basis of our results, we propose a structural model for the regulation of mDia1 by Rho and DAD.     

Cdc42 and mDia3 regulate microtubule attachment to kinetochores

During mitosis, the mitotic spindle, a bipolar structure composed of microtubules (MTs) and associated motor proteins, segregates sister chromatids to daughter cells. Initially some MTs emanating from one centrosome attach to the kinetochore at the centromere of one of the duplicated chromosomes. This attachment allows rapid poleward movement of the bound chromosome. Subsequent attachment of the sister kinetochore to MTs growing from the other centrosome results in the bi-orientation of the chromosome, in which interactions between kinetochores and the plus ends of MTs are formed and stabilized. These processes ensure alignment of chromosomes during metaphase and their correct segregation during anaphase. Although many proteins constituting the kinetochore have been identified and extensively studied, the signalling responsible for MT capture and stabilization is unclear. Small GTPases of the Rho family regulate cell morphogenesis by organizing the actin cytoskeleton and regulating MT alignment and stabilization. We now show that one member of this family, Cdc42, and its effector, mDia3, regulate MT attachment to kinetochores.     

拟南芥、水稻和杨树ACTIN家族全基因组分析

鉴定了覆盖拟南芥、水稻和杨树3种模式植物全基因组的20个拟南芥、18个水稻、22个杨树ACTIN蛋白基因,对其染色体定位、基因结构、基因复制等进行了综合分析.并在系统进化分析基础上,将ACTIN基因家族分为12个亚家族,有助于揭示植物ACTIN基因家族的进化历史,为后续ACTIN基因家族的功能提供线索,对研究植物ACTIN基因家族功能和进化上的多样性提供理论基础.     

白介素24融合蛋白的表达、纯化和促伤口愈合功能的鉴定

本研究通过优化小鼠白介素24(mIL-24)基因序列,构建以Fc-tag为标签的mIL-24-Fc融合蛋白.对CHO进行MTX分级加压筛选,获得了稳定、高表达mIL-24-Fc蛋白的阳性克隆,同时优化了CHO细胞无血清驯化与稳定表达蛋白体系.经protein A亲和层析纯化mIL-24-Fc蛋白并利用AP binding实验检测生物学活性后,小鼠皮内注射mIL-24-Fc蛋白和伤口愈合实验证明:注射mIL-24-Fc蛋白后,小鼠表皮产生2~3层细胞增生,伤口愈合速度加快.     

Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1

The principal guanine nucleotide exchange factors for Rho family G proteins contain tandem Dbl-homology (DH) and pleckstrin-homology (PH) domains that catalyse nucleotide exchange and the activation of G proteins. Here we have determined the crystal structure of the DH and PH domains of the T-lymphoma invasion and metastasis factor 1 (Tiam1) protein in complex with its cognate Rho family G protein, Rac1. The two switch regions of Rac1 are stabilized in conformations that disrupt both magnesium binding and guanine nucleotide interaction. The resulting cleft in Rac1 is devoid of nucleotide and highly exposed to solvent. The PH domain of Tiam1 does not contact Rac1, and the position and orientation of the PH domain is markedly altered relative to the structure of the uncomplexed, GTPase-free DH/PH element from Sos1. The Tiam1/Rac1 structure highlights the interactions that catalyse nucleotide exchange on Rho family G proteins, and illustrates structural determinants dictating specificity between individual Rho family members and their associated Dbl-related guanine nucleotide exchange factors.     

人CREB4转核机制的初步研究

cAMP 反应元件结合蛋白（cAMP response element-binding proteins，CREB）是一个哺乳动物转录因子家族，通过cAMP 反应元件（cAMP response element，CRE）调节cAMP和钙离子依赖性基因的表达.CREB4是CREB转录因子家族的一员.经酵母双杂交筛选人胎脑文库发现CREB4^215-279aa可能与核转运因子kayopherinα2相互作用，提示karyopherinα2可能参与CREB4的跨膜转运过程.亚细胞定位结果显示，CREB4全长定位于细胞质，而缺失C端假定转膜结构域的CREB4^1-279aa蛋白则转移至细胞核内.荧光共定位进一步显示，CREB4和karyopherinα2共定位于细胞质中，CREB4^1-279aa和karyopherinα2共定位于细胞核中.结果提示C端被切除之后，CREB4被karyopherinα2转运到核内发挥转录作用.     

Disruption of the actin cytoskeleton in yeast capping protein mutants

Capping protein controls the addition of actin subunits to the barbed end of actin filaments and nucleates actin polymerization in vitro. Capping protein has been identified in all eukaryotic cells examined so far; it is a heterodimer with subunits of relative molecular masses 32,000-36,000 (alpha-subunit) and 28,000-32,000 (beta-subunit). In skeletal muscle, capping protein (CapZ) probably binds the barbed ends of actin filaments at the Z line. The in vivo role of this protein in non-muscle cells is not known. We report here the characterization of CAP2, the single gene encoding the beta-subunit of capping protein in Saccharomyces cerevisiae. Yeast cells in which the CAP2 gene was disrupted by an insertion or a deletion had an abnormal actin distribution, including the loss of actin cables. The mutant cells were round and large, with a heterogeneous size distribution, and, although viable, grew more slowly than congenic wild-type cells. Chitin, a cell wall component restricted to the mother-bud junction in wild-type budding yeast, was found on the entire mother cell surface in the mutants. The phenotype of CAP2 disruption resembled that of temperature-sensitive mutations in the yeast actin gene ACT1, indicating that capping protein regulates actin-filament distribution in vivo.     

Requirement of yeast fimbrin for actin organization and morphogenesis in vivo

The SAC6 gene was found by suppression of a yeast actin mutation. Its protein product, Sac6p (previously referred to as ABP67), was independently isolated by actin-filament affinity chromatography and colocalizes with actin in vivo. Thus Sac6p binds to actin in vitro, and functionally associates with actin structures involved in the development and maintenance of cell polarity in vivo. We report here that Sac6p is an actin-filament bundling protein 43% identical in amino-acid sequence to the vertebrate bundling protein fimbrin. This yeast fimbrin homologue contains two putative actin-binding regions homologous to domains of dystrophin, beta-spectrin, filamin, actin-gelation protein and alpha-actinin. Mutants lacking Sac6p do not form normal actin structures and are defective in morphogenesis. These findings demonstrate an in vivo role for the well-documented biochemical interaction between fimbrin and actin.     

hASB-8相互作用蛋白的初步研究

hASB-8基因是对肿瘤细胞生长具有明显抑制作用的人类新基因．其编码蛋白属于人ASB蛋白家族中的一个成员,与小鼠中的ASB-8蛋白同源性达96％．保守结构域分析显示hASB-8在N端包含4个Ankyrin repeats,在C端包含了一个SOCS box．利用酵母双杂交技术,筛选了人的胎盘(Placenta)cDNA文库,获得了与KASB-8相互作用的2个蛋白,Elongin C和CDK4 binding protein;并在二倍体酵母体内进行了验证．这些试验提示hASB-8蛋白可能介导肿瘤细胞中靶蛋白和泛素复合体之间的相互作用,并与肿瘤细胞靶蛋白转录调节有关．     

富含半胱氨酸分泌蛋白的结构及其离子通道方面作用的研究进展

富含半胱氨酸分泌蛋白（Cystein—rich secretory protein，简称CRISP），在进化上非常保守，大部分成员功能尚不明了．研究发现，CRISP除广泛存在于哺乳动物、一些爬行动物和软体动物体内，近来我们还在日本七鳃鳗口腔腺中发现新的CRISP家族成员．对这些CRISP家族成员做简要综述．