A kinase-regulated PDZ-domain interaction controls endocytic sorting of the beta2-adrenergic receptor.

A fundamental question in cell biology is how membrane proteins are sorted in the endocytic pathway. The sorting of internalized beta2-adrenergic receptors between recycling endosomes and lysosomes is responsible for opposite effects on signal transduction and is regulated by physiological stimuli. Here we describe a mechanism that controls this sorting operation, which is mediated by a family of conserved protein-interaction modules called PDZ domains. The phosphoprotein EBP50 (for ezrinradixin-moesin(ERM)-binding phosphoprotein-50) binds to the cytoplasmic tail of the beta2-adrenergic receptor through a PDZ domain and to the cortical actin cytoskeleton through an ERM-binding domain. Disrupting the interaction of EBP50 with either domain or depolymerization of the actin cytoskeleton itself causes missorting of endocytosed beta2-adrenergic receptors but does not affect the recycling of transferrin receptors. A serine residue at position 411 in the tail of the beta2-adrenergic receptor is a substrate for phosphorylation by GRK-5 (for G-protein-coupled-receptor kinase-5) and is required for interaction with EBP50 and for proper recycling of the receptor. Our results identify a new role for PDZ-domain-mediated protein interactions and for the actin cytoskeleton in endocytic sorting, and suggest a mechanism by which GRK-mediated phosphorylation could regulate membrane trafficking of G-protein-coupled receptors after endocytosis.     

Dynamin is a GTPase stimulated to high levels of activity by microtubules.

Dynamin was initially identified in calf brain tissue as a protein of relative molecular mass 100,000 which induced nucleotide-sensitive bundling of microtubules. Purified dynamin showed only trace ATPase activity. But in combination with an activating factor removed during the purification, it exhibited microtubule-activated ATPase activity and dynamin-induced bundles showed evidence of ATP-dependent force production. Dynamin is the product of the Drosophila gene shibire, which has been implicated in synaptic vesicle recycling and, more generally, in the budding of endocytic vesicles from the plasma membrane. Dynamin also shows extensive homology with proteins that participate in vacuolar protein sorting and spindle pole-body separation in yeast, and in interferon-induced viral resistance in mammals. All members of this family contain consensus sequence elements consistent with GTP binding near their amino termini, although none has been shown to have GTPase activity. We report here that dynamin is a specific GTPase which can be stimulated to very high levels of activity by microtubules.     

Re-routing of a secretory protein by fusion with human growth hormone sequences

Cells with electron-dense secretory vesicles use them to store only specialized secretory products such as peptide hormones; other types of secreted proteins are externalized by an alternative, constitutive route. One possible mechanism for such segregation is that proteins destined for dense secretory vesicles contain unique 'sorting domains' that allow for selective targeting. Here, we set out to determine whether a constitutively secreted protein could be diverted to the dense secretory vesicles by attachment to a peptide hormone sequence. We made use of the ability of the mouse pituitary tumour cell, AtT-20, to correctly sort exogenous secretory proteins introduced into them by DNA transfection. We constructed a plasmid encoding a hybrid protein in which a constitutively secreted viral protein was fused to the carboxy terminus of human growth hormone (hGH). Cells expressing the hybrid protein were found to target it to dense secretory vesicles with an efficiency close to that observed for the parental hGH. These results support the hypothesis that sorting domains on peptide hormones direct their packaging into dense secretory vesicles. The results also suggest that proteins secreted by the constitutive pathway either do not contain any sorting domain, or their sorting signals can be overridden by those which direct peptide hormones.     

Dependence on pH of polarized sorting of secreted proteins

The plasma membranes of epithelial cells are divided into apical and basolateral domains. These two surfaces are characterized by markedly different protein compositions, reflecting the ability of the cell to target newly synthesized membrane proteins to specific regions of the cell surface. This targeting capability is also apparent in the polarized release of secretory products. Recent studies using canine renal tubule (MDCK) cells have suggested that distinct sets of secretory proteins are released from their apical and basolateral poles. We report experiments designed to examine secretory protein sorting by MDCK cells. We have shown that secretion of basement membrane components (laminin and heparan sulphate proteoglycan (HSPG] takes place from the basolateral cell surface and that this polarized release results from active sorting. The sorting process which mediates this polarized secretion requires an acidic intracellular compartment. MDCK cells treated with NH4Cl to raise the pH of their intracellular compartments, secrete laminin and HSPG by a default pathway which leads to their release in roughly equal quantities into the medium of both the apical and basolateral compartments.     

Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis.

Dynamin was discovered in bovine brain tissue as a nucleotide-sensitive microtubule-binding protein of relative molecular mass 100,000. It was found to cross-link microtubules into highly ordered bundles, and appeared to have a role in intermicrotubule sliding in vitro. Cloning and sequencing of rat brain dynamin complementary DNA identified an N-terminal region of about 300 amino acids which contained the three consensus elements characteristic of GTP-binding proteins. Extensive homology was found between this domain and the mammalian Mx proteins which are involved in interferon-induced viral resistance, and with the product of the VPS1 locus in Saccharomyces cerevisiae, which has been implicated both in membrane protein sorting, and in meiotic spindle pole separation. Dynamin-containing microtubule bundles were not observed in an immunofluorescence study of cultured mammalian cells, but a role for a GTP-requiring protein in intermicrotubule sliding during mitosis in plants has been reported. We report here that Drosophila melanogaster contains multiple tissue-specific and developmentally-regulated forms of dynamin, which are products of the shibire locus previously implicated in endocytic protein sorting.     

Microfluidic sorting in an optical lattice

The response of a microscopic dielectric object to an applied light field can profoundly affect its kinetic motion. A classic example of this is an optical trap, which can hold a particle in a tightly focused light beam. Optical fields can also be used to arrange, guide or deflect particles in appropriate light-field geometries. Here we demonstrate an optical sorter for microscopic particles that exploits the interaction of particles-biological or otherwise-with an extended, interlinked, dynamically reconfigurable, three-dimensional optical lattice. The strength of this interaction with the lattice sites depends on the optical polarizability of the particles, giving tunable selection criteria. We demonstrate both sorting by size (of protein microcapsule drug delivery agents) and sorting by refractive index (of other colloidal particle streams). The sorting efficiency of this method approaches 100%, with values of 96% or more observed even for concentrated solutions with throughputs exceeding those reported for fluorescence-activated cell sorting. This powerful, non-invasive technique is suited to sorting and fractionation within integrated ('lab-on-a-chip') microfluidic systems, and can be applied in colloidal, molecular and biological research.     

Polarized sorting of glypiated proteins in hippocampal neurons.

Our recent studies suggested that neurons and epithelial cells sort viral glycoproteins in a similar manner. The apical influenza virus haemagglutinin was preferentially delivered to the axon of hippocampal neurons in culture, whereas the basolateral vesicular stomatitis virus glycoprotein was sorted to the dendrites. To investigate whether other membrane proteins showed similar sorting in neurons and epithelial cells, we have analysed the localization of a glypiated (glycosylphosphatidylinositol anchored) protein, Thy-1, in hippocampal neurons in culture. In MDCK and other epithelial cells, endogenous glycosylphosphatidylinositol (GPI)-anchored proteins, as well as mutated exogenous proteins containing the GPI-attachment signal, undergo preferential delivery to the apical surface. This polarized sorting of GPI-anchored proteins has been proposed to occur by the same mechanisms as the sorting of glycolipids to the apical surface. We report here that the neuronal GPI-protein Thy-1 is present in hippocampal neurons in culture and is exclusively located on the axonal surface. This finding further strengthens our hypothesis that the mechanisms of sorting of surface components may be similar in neurons and epithelial cells.     

Clathrin self-assembly is mediated by a tandemly repeated superhelix.

Clathrin is a triskelion-shaped cytoplasmic protein that polymerizes into a polyhedral lattice on intracellular membranes to form protein-coated membrane vesicles. Lattice formation induces the sorting of membrane proteins during endocytosis and organelle biogenesis by interacting with membrane-associated adaptor molecules. The clathrin triskelion is a trimer of heavy-chain subunits (1,675 residues), each binding a single light-chain subunit, in the hub domain (residues 1,074-1,675). Light chains negatively modulate polymerization so that intracellular clathrin assembly is adaptor-dependent. Here we report the atomic structure, to 2.6 A resolution, of hub residues 1,210-1,516 involved in mediating spontaneous clathrin heavy-chain polymerization and light-chain association. The hub fragment folds into an elongated coil of alpha-helices, and alignment analyses reveal a 145-residue motif that is repeated seven times along the filamentous leg and appears in other proteins involved in vacuolar protein sorting. The resulting model provides a three-dimensional framework for understanding clathrin heavy-chain self-assembly, light-chain binding and trimerization.     

一种基于最优局部信息融合的蛋白质-亚细胞定位预测方法

 基于蛋白质的合成及分选机制,提出了一种新的蛋白质亚细胞定位预测方法。先采用遍历搜索技术,找出各种亚细胞蛋白质序列分选信号和成熟蛋白质之间的最佳分割位点,把蛋白质序列分为两条子序列,计算这两条子序列中的氨基酸组份并将它们融合起来作为整条蛋白质序列的特征,然后构造用于识别每类蛋白质的最佳子分类器,再根据最大化原则组建集成分类器。在NNPSL数据集上,采用5重交叉验证方法对本文方法进行测试,原核和真核两个蛋白质序列子集分别取得94.1%和87.5%的总体预测精度。同时,此方法在一些蛋白质序列中找到的分割位点与真实生物现象相吻合,能为预测蛋白质序列的剪切位点提供参考信息。     

蛋白质肽链的翻译后加工

综述了蛋白质翻译和翻译后所发生的诸多生化事件 :肽链的卷曲、肽链穿越 ER膜、肽链定位分拣、投送、肽链中残基的修饰及在蛋白质成熟过程中的作用和影响     

基于微粒群优化的快速K-近邻分类算法

随着全球信息化的出现,手工分类索引已经不适用于大规模信息的处理,自动分类的研究得到迅速发展。K-近邻法是具有一定效率的自动分类算法。本文将其与智能优化技术结合,用于基于机器学习的文本分类过程中。实验结果表明,对于庞大的文档集合分类,该算法提高了分类的速度和精度。     

电动汽车锂离子动力电池分选方法研究

为了改善电动汽车电池组的不一致性,提高电池组的可用功率和容量利用率,以电池的不一致性机理分析为基础,分别对电池进行了传统分选、主因子分选和总因子分选.单体试验计算结果表明,总因子分选方法是最优的.接着在总因子分选结果的基础上运用模糊C均值聚类算法对电池进行了动态特性分选,试验结果表明:此分选方法能有效地改善电池组的不一致性.     

Structure of the ESCRT-II endosomal trafficking complex

The multivesicular-body (MVB) pathway delivers transmembrane proteins and lipids to the lumen of the endosome. The multivesicular-body sorting pathway has crucial roles in growth-factor-receptor downregulation, developmental signalling, regulation of the immune response and the budding of certain enveloped viruses such as human immunodeficiency virus. Ubiquitination is a signal for sorting into the MVB pathway, which also requires the functions of three protein complexes, termed ESCRT-I, -II and -III (endosomal sorting complex required for transport). Here we report the crystal structure of the core of the yeast ESCRT-II complex, which contains one molecule of the Vps protein Vps22, the carboxy-terminal domain of Vps36 and two molecules of Vps25, and has the shape of a capital letter 'Y'. The amino-terminal coiled coil of Vps22 and the flexible linker leading to the ubiquitin-binding NZF domain of Vps36 both protrude from the tip of one branch of the 'Y'. Vps22 and Vps36 form nearly equivalent interactions with the two Vps25 molecules at the centre of the 'Y'. The structure suggests how ubiquitinated cargo could be passed between ESCRT components of the MVB pathway through the sequential transfer of ubiquitinated cargo from one complex to the next.     

Structural basis for selective recognition of ESCRT-III by the AAA ATPase Vps4

The AAA+ ATPases are essential for various activities such as membrane trafficking, organelle biogenesis, DNA replication, intracellular locomotion, cytoskeletal remodelling, protein folding and proteolysis. The AAA ATPase Vps4, which is central to endosomal traffic to lysosomes, retroviral budding and cytokinesis, dissociates ESCRT complexes (the endosomal sorting complexes required for transport) from membranes. Here we show that, of the six ESCRT--related subunits in yeast, only Vps2 and Did2 bind the MIT (microtubule interacting and transport) domain of Vps4, and that the carboxy-terminal 30 residues of the subunits are both necessary and sufficient for interaction. We determined the crystal structure of the Vps2 C terminus in a complex with the Vps4 MIT domain, explaining the basis for selective ESCRT-III recognition. MIT helices alpha2 and alpha3 recognize a (D/E)xxLxxRLxxL(K/R) motif, and mutations within this motif cause sorting defects in yeast. Our crystal structure of the amino-terminal domain of an archaeal AAA ATPase of unknown function shows that it is closely related to the MIT domain of Vps4. The archaeal ATPase interacts with an archaeal ESCRT-III-like protein even though these organisms have no endomembrane system, suggesting that the Vps4/ESCRT-III partnership is a relic of a function that pre-dates the divergence of eukaryotes and Archaea.     

一种分类机械手的研制与开发

提出了一种用在晶体角分类机上替代工人来完成卸料及分类工作的机械手.该分类机械手是机械、电气传动、气动控制、嵌入式微控制器和微电子器件相结合的产物.机械部分构成了分类机械手的骨架;步进电机等使分类机械手实现了准确运动;气动元件使分类机械手变得灵巧、简捷.借助于系统状态变换等方法,以微控制器C8051F206为核心的分类机械手控制器的软件给予了分类机械手某些简单的人工智能,使其能准确无误地重复完成卸料及分类任务.分类机械手的基准定位与步进电机步进计数控制的结合可使其长期运行无积累误差.经在工业现场的长期使用证明,该分类机械手设计合理、工作可靠.     

O（N）时空复杂度排序器的设计

用特别设计的自动检索电路与一般存储器一起构成一个独立的硬件排序单元．其时间、空间复杂度均是Ｏ（Ｎ）．它既可以作为主ＣＰＵ的协处理器，也可以作为高性能计算系统（如并行处理系统）或高速数据库查询系统（如数据库机）的一个专用功能单元     

bicoid RNA localization requires specific binding of an endosomal sorting complex

bicoid messenger RNA localizes to the anterior of the Drosophila egg, where it is translated to form a morphogen gradient of Bicoid protein that patterns the head and thorax of the embryo. Although bicoid was the first localized cytoplasmic determinant to be identified, little is known about how the mRNA is coupled to the microtubule-dependent transport pathway that targets it to the anterior, and it has been proposed that the mRNA is recognized by a complex of many redundant proteins, each of which binds to the localization element in the 3' untranslated region (UTR) with little or no specificity. Indeed, the only known RNA-binding protein that co-localizes with bicoid mRNA is Staufen, which binds non-specifically to double-stranded RNA in vitro. Here we show that mutants in all subunits of the ESCRT-II complex (VPS22, VPS25 and VPS36) abolish the final Staufen-dependent step in bicoid mRNA localization. ESCRT-II is a highly conserved component of the pathway that sorts ubiquitinated endosomal proteins into internal vesicles, and functions as a tumour-suppressor by removing activated receptors from the cytoplasm. However, the role of ESCRT-II in bicoid localization seems to be independent of endosomal sorting, because mutations in ESCRT-I and III components do not affect the targeting of bicoid mRNA. Instead, VPS36 functions by binding directly and specifically to stem-loop V of the bicoid 3' UTR through its amino-terminal GLUE domain, making it the first example of a sequence-specific RNA-binding protein that recognizes the bicoid localization signal. Furthermore, VPS36 localizes to the anterior of the oocyte in a bicoid-mRNA-dependent manner, and is required for the subsequent recruitment of Staufen to the bicoid complex. This function of ESCRT-II as an RNA-binding complex is conserved in vertebrates and may clarify some of its roles that are independent of endosomal sorting.     

选择排序算法的一个改进及分析

针对少量记录排序的应用，对直接选择排序算法进行了挖掘，通过增加记忆功能，使算法性能得到明显提高。改进后的算法在大量记录排序时，较原算法的速度提高1倍以上；在少量记录排序时，是基于比较和移位的排序算法中总体表现最佳的；并且对原序列的有序程度很敏感，原序列相对有序时，速度能大幅度提高。结果表明：该算法很适合少量记录排序、部分排序、较有序记录的排序，以及与快速排序算法的混合使用。     

基于群体智慧的排序任务多聚合方法比较研究

为解决排序任务聚合方法效果不明确的问题, 在群体智慧概念下, 对排序任务问题群体智慧效应发生的条件即集中化机制进行聚合方法的比较研究, 试图找到已知基本事实排序任务问题中使群体智慧效应最显著的排序聚合方法。 创新性提出评估群体智慧的概念即群体智慧效度。 运用实证研究方法对群体进行排序任务重建实验, 以实验测试的排序数据为基础, 使用 7 种不同的排序聚合方法对排序任务问题的所有个体排序进行聚合, 得到相应的群体排序。 测量7 种群体排序和真实排序之间的偏差, 并根据群体智慧效度大小判断不同聚合方法的表现。 实验结果显示, 在基于群体智慧效度的重建排序任务聚合方法中, 众数法的表现最好, 中位数法和 K-Y 方法表现次之。