Role of importin-beta in coupling Ran to downstream targets in microtubule assembly

The guanosine triphosphatase Ran stimulates assembly of microtubule asters and spindles in mitotic Xenopus egg extracts. A carboxyl-terminal region of the nuclear-mitotic apparatus protein (NuMA), a nuclear protein required for organizing mitotic spindle poles, mimics Ran's ability to induce asters. This NuMA fragment also specifically interacted with the nuclear transport factor, importin-beta. We show that importin-beta is an inhibitor of microtubule aster assembly in Xenopus egg extracts and that Ran regulates the interaction between importin-beta and NuMA. Importin-beta therefore links NuMA to regulation by Ran. This suggests that similar mechanisms regulate nuclear import during interphase and spindle assembly during mitosis.     

固醇调节元件结合蛋白-1c在肝脂肪合成中转录调节

固醇调节元件结合蛋白 -1c(SREBP -1c)是脂肪合成基因的重要转录调节因子 ,又称脂肪细胞决定和分化因子 1(AdipocyteDetermi nationandDifferentiationfactor -1,ADD1) ,它是动物肝脏中主要的转录物 ,主要调节动物脂肪合成和葡萄糖代谢相关基因的表达。研究发现 ,SREBP -1c的过度表达与肝脏等非脂肪组织的脂质积聚相关。SREBP -1c调节体内的脂肪合成主要是通过改变自身的mRNA水平来实现的 ,即生脂酶的转录调节是由SREBP- 1cmRNA的数量控制的。另一方面 ,LXRs、胰岛素、胰高血糖素等因素对SREBP- 1c的转录有调节作用。     

外源反-10，顺-12共轭亚油酸对体外培养牛乳腺上皮细胞SREBP-1基因表达和蛋白质合成的影响

 【目的】旨在观察反-10,顺-12,共轭亚油酸（t10c12 CLA）对牛乳腺上皮细胞中甾醇调控元件结合蛋白（SREBP-1）基因的表达和蛋白质加工的影响。【方法】本试验使用组织块培养法获得奶牛乳腺上皮细胞,使用不同浓度t10c12 CLA处理乳腺上皮细胞后,用油红染色法检测胞质内的脂滴分布,Trizol法提取细胞总RNA,荧光定量PCR法测定相关基因的表达量,试剂盒法提取细胞总蛋白,进行Western blotting。【结果】随t10c12 CLA添加量的增加,细胞质内甘油三酯的含量逐渐降低。与对照组相比,75、112.5和150 μmol•L-1 t10c12 CLA均对胞质内甘油三酯的积累有显著抑制作用（P＜0.05）。t10c12 CLA 的添加对SREBP-1基因和参与SREBP-1蛋白加工调节基因的表达均无显著性影响（P＞0.05）。根据蛋白质电泳结果推测,t10c12 CLA对SREBP-1前体蛋白的合成并无影响,可能对SREBP-1的加工活化产生抑制作用或对其分解产生促进作用。【结论】t10c12 CLA能抑制胞质内甘油三酯的积累,对SREBP-1基因的表达、蛋白质翻译均无影响,可能影响其加工活化过程和活性蛋白的降解过程。     

Spatial coordination of spindle assembly by chromosome-mediated signaling gradients

During cell division, chromosomes are distributed to daughter cells by the mitotic spindle. This system requires spatial cues to reproducibly self-organize. We report that such cues are provided by chromosome-mediated interaction gradients between the small guanosine triphosphatase (GTPase) Ran and importin-beta. This produces activity gradients that determine the spatial distribution of microtubule nucleation and stabilization around chromosomes and that are essential for the self-organization of microtubules into a bipolar spindle.     

Structure and allostery of the PKA RIIβ tetrameric holoenzyme

In its physiological state, cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) is a tetramer that contains a regulatory (R) subunit dimer and two catalytic (C) subunits. We describe here the 2.3 angstrom structure of full-length tetrameric RIIβ(2):C(2) holoenzyme. This structure showing a dimer of dimers provides a mechanistic understanding of allosteric activation by cAMP. The heterodimers are anchored together by an interface created by the β4-β5 loop in the RIIβ subunit, which docks onto the carboxyl-terminal tail of the adjacent C subunit, thereby forcing the C subunit into a fully closed conformation in the absence of nucleotide. Diffusion of magnesium adenosine triphosphate (ATP) into these crystals trapped not ATP, but the reaction products, adenosine diphosphate and the phosphorylated RIIβ subunit. This complex has implications for the dissociation-reassociation cycling of PKA. The quaternary structure of the RIIβ tetramer differs appreciably from our model of the RIα tetramer, confirming the small-angle x-ray scattering prediction that the structures of each PKA tetramer are different.     

BAR domains as sensors of membrane curvature: the amphiphysin BAR structure

The BAR (Bin/amphiphysin/Rvs) domain is the most conserved feature in amphiphysins from yeast to human and is also found in endophilins and nadrins. We solved the structure of the Drosophila amphiphysin BAR domain. It is a crescent-shaped dimer that binds preferentially to highly curved negatively charged membranes. With its N-terminal amphipathic helix and BAR domain (N-BAR), amphiphysin can drive membrane curvature in vitro and in vivo. The structure is similar to that of arfaptin2, which we find also binds and tubulates membranes. From this, we predict that BAR domains are in many protein families, including sorting nexins, centaurins, and oligophrenins. The universal and minimal BAR domain is a dimerization, membrane-binding, and curvature-sensing module.     

Atomic structure of thymidylate synthase: target for rational drug design

The atomic structure of thymidylate synthase from Lactobacillus casei was determined at 3 angstrom resolution. The native enzyme is a dimer of identical subunits. The dimer interface is formed by an unusual association between five-stranded beta sheets present in each monomer. Comparison of known sequences with the Lactobacillus casei structure suggests that they all have a common core structure around which loops are inserted or deleted in different sequences. Residues from both subunits contribute to each active site. Two arginine side chains can contribute to binding phosphate on the substrate. The side chains of several conserved amino acids can account for other determinants of substrate binding.     

脂肪酸对小鼠前体脂肪细胞分化中脂代谢基因mRNA表达的影响

采用RT-PCR技术,分别研究短链和长链脂肪酸及P38 MAPK通路抑制剂SB20358对小鼠前体脂肪细胞分化过程中脂代谢相关基因转录表达的影响及其调控机制.结果表明:短链脂肪酸可提高PPARγ2和C/EBPα mRNA表达量(P<0.05),而长链脂肪酸上调PPARγ2、C/EBPα、FAS、SREBP-1c mRNA表达量(P<0.05),但脂肪酸对TGH表达量影响不显著(P>0.05);当P38MAPK抑制剂存在时,PPARγ2、FAS和SREBP-1c mRNA表达量显著下降(P<0.05),而C/EBPα和TGH表达量变化不显著(P>0.05).表明脂肪酸可通过P38MAPK通路促进PPARγ2增量表达从而促进小鼠前体脂肪细胞分化.     

Crystal structure of the dynein motor domain

Dyneins are microtubule-based motor proteins that power ciliary beating, transport intracellular cargos, and help to construct the mitotic spindle. Evolved from ring-shaped hexameric AAA-family adenosine triphosphatases (ATPases), dynein's large size and complexity have posed challenges for understanding its structure and mechanism. Here, we present a 6 angstrom crystal structure of a functional dimer of two ~300-kilodalton motor domains of yeast cytoplasmic dynein. The structure reveals an unusual asymmetric arrangement of ATPase domains in the ring-shaped motor domain, the manner in which the mechanical element interacts with the ATPase ring, and an unexpected interaction between two coiled coils that create a base for the microtubule binding domain. The arrangement of these elements provides clues as to how adenosine triphosphate-driven conformational changes might be transmitted across the motor domain.     

Dimerization of human growth hormone by zinc

Size-exclusion chromatography and sedimentation equilbrium studies demonstrated that zinc ion (Zn2+) induced the dimerization of human growth hormone (hGH). Scatchard analysis of 65Zn2+ binding to hGH showed that two Zn2+ ions associate per dimer of hGH in a cooperative fashion. Cobalt (II) can substitute for Zn2+ in the hormone dimer and gives a visible spectrum characteristic of cobalt coordinated in a tetrahedral fashion by oxygen- and nitrogen-containing ligands. Replacement of potential Zn2+ ligands (His18, His21, and Glu174) in hGH with alanine weakened both Zn2+ binding and hGH dimer formation. The Zn(2+)-hGH dimer was more stable than monomeric hGH to denaturation in guanidine-HCl. Formation of a Zn(2+)-hGH dimeric complex may be important for storage of hGH in secretory granules.     

The structure of the kinesin-1 motor-tail complex reveals the mechanism of autoinhibition

When not transporting cargo, kinesin-1 is autoinhibited by binding of a tail region to the motor domains, but the mechanism of inhibition is unclear. We report the crystal structure of a motor domain dimer in complex with its tail domain at 2.2 angstroms and compare it with a structure of the motor domain alone at 2.7 angstroms. These structures indicate that neither an induced conformational change nor steric blocking is the cause of inhibition. Instead, the tail cross-links the motor domains at a second position, in addition to the coiled coil. This "double lockdown," by cross-linking at two positions, prevents the movement of the motor domains that is needed to undock the neck linker and release adenosine diphosphate. This autoinhibition mechanism could extend to some other kinesins.     

The role of individual cysteine residues in the structure and function of the v-sis gene product

The v-sis oncogene encodes a platelet-derived growth factor (PDGF)-related product whose transforming activity is mediated by its functional interaction with the PDGF receptor. PDGF, as well as processed forms of the v-sis gene product, is a disulfide-linked dimer with eight conserved cysteine residues in the minimum region necessary for biologic activity. Site-directed mutagenesis of the v-sis gene revealed that each conserved cysteine residue was required directly or indirectly for disulfide-linked dimer formation. However, substitution of serine for cysteine codons at any of four positions had no detrimental effect on transforming activity of the encoded v-sis protein. These results establish that interchain disulfide bonds are not essential in order for this protein to act as a functional ligand for the PDGF receptor. The remaining four substitutions of serine for cysteine each inactivated transforming function of the molecule. In each case this was associated with loss of a conformation shown to involve intramolecular disulfide bonds. These studies provide insight into the role of individual cysteine residues in determining the structure of the sis/PDGF molecule critical for biological activity.     

Quantum reflection of He2 several nanometers above a grating surface

Quantum reflection allows an atom or molecule to be reflected from a solid before it reaches the region where it would encounter the repulsive potential of the surface. We observed nondestructive scattering of the helium dimer (He(2)), which has a binding energy of 10(-7) electron volt, from a solid reflection grating. We scattered a beam containing the dimer as well as atomic helium and larger clusters, but could differentiate the dimer by its diffraction angle. Helium dimers are quantum reflected tens of nanometers above the surface, where the surface-induced forces are too weak to dissociate the fragile bond.     

Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease

The rational design of drugs that can inhibit the action of viral proteases depends on obtaining accurate structures of these enzymes. The crystal structure of chemically synthesized HIV-1 protease has been determined at 2.8 angstrom resolution (R factor of 0.184) with the use of a model based on the Rous sarcoma virus protease structure. In this enzymatically active protein, the cysteines were replaced by alpha-amino-n-butyric acid, a nongenetically coded amino acid. This structure, in which all 99 amino acids were located, differs in several important details from that reported previously by others. The interface between the identical subunits forming the active protease dimer is composed of four well-ordered beta strands from both the amino and carboxyl termini and residues 86 to 94 have a helical conformation. The observed arrangement of the dimer interface suggests possible designs for dimerization inhibitors.     

Structure of a tyrosine phosphatase adhesive interaction reveals a spacer-clamp mechanism

Cell-cell contacts are fundamental to multicellular organisms and are subject to exquisite levels of control. Human RPTPmu is a type IIB receptor protein tyrosine phosphatase that both forms an adhesive contact itself and is involved in regulating adhesion by dephosphorylating components of cadherin-catenin complexes. Here we describe a 3.1 angstrom crystal structure of the RPTPmu ectodomain that forms a homophilic trans (antiparallel) dimer with an extended and rigid architecture, matching the dimensions of adherens junctions. Cell surface expression of deletion constructs induces intercellular spacings that correlate with the ectodomain length. These data suggest that the RPTPmu ectodomain acts as a distance gauge and plays a key regulatory function, locking the phosphatase to its appropriate functional location.     

葡萄血红蛋白的序列特征、结构建模及分子进化

对葡萄血红蛋白基因的结构、进化关系、序列特征及蛋白结构进行剖析。基因结构和蛋白序列特征分析表明:尽管葡萄3个血红蛋白基因外显子个数和保守基序组织结构变异较大,但功能结构域具有高度保守性。结构模拟显示,葡萄VvHb1以单体形式存在,而VvHb2和VvHb3则以二聚体形式存在,尽管它们三维构象以螺旋结构为主,但结合血红素分子活性不同。进化分析揭示,植物血红蛋白具有4个类群,其中葡萄的血红蛋白落入主要类群外,并且单独形成了一个进化分支,说明葡萄血红蛋白基因在物种形成后发生了快速进化和基因扩增现象。     

A photoinduced persistent structural transformation of the special pair of a bacterial reaction center

Structural modification of photosynthetic reaction centers is an important approach for understanding their charge-separation processes. An unprecedented persistent structural transformation of the special pair (dimer) of bacteriochlorophyll molecules can be produced by light absorption alone. The nonphotochemical hole-burned spectra for the reaction center of Rhodopseudomonas viridis show that the phototransformation leads to a red shift of 150 wave numbers for the special pair's lowest energy absorption band, P960, and a comparable blue shift for a state at 850 nanometers, which can now be definitively assigned as being most closely associated with the upper dimer component. Additional insights on excited-state electronic structure include the identification of a new state.     

Structure of a legume lectin with an ordered N-linked carbohydrate in complex with lactose

The three-dimensional structure of the lactose complex of the Erythrina corallodendron lectin (EcorL), a dimer of N-glycosylated subunits, was determined crystallographically and refined at 2.0 angstrom resolution to an R value of 0.19. The tertiary structure of the subunit is similar to that of other legume lectins, but interference by the bulky N-linked heptasaccharide, which is exceptionally well ordered in the crystal, forces the EcorL dimer into a drastically different quaternary structure. Only the galactose moiety of the lactose ligand resides within the combining site. The galactose moiety is oriented differently from ligands in the mannose-glucose specific legume lectins and is held by hydrophobic interactions with Ala88, Tyr106, Phe131, and Ala218 and by seven hydrogen bonds, four of which are to the conserved Asp89, Asn133, and NH of Gly107. The specificity of legume lectins toward the different C-4 epimers appears to be associated with extensive variations in the outline of the variable parts of the binding sites.