The assembly of a GTPase-kinase signalling complex by a bacterial catalytic scaffold

The fidelity and specificity of information flow within a cell is controlled by scaffolding proteins that assemble and link enzymes into signalling circuits. These circuits can be inhibited by bacterial effector proteins that post-translationally modify individual pathway components. However, there is emerging evidence that pathogens directly organize higher-order signalling networks through enzyme scaffolding, and the identity of the effectors and their mechanisms of action are poorly understood. Here we identify the enterohaemorrhagic Escherichia coli O157:H7 type III effector EspG as a regulator of endomembrane trafficking using a functional screen, and report ADP-ribosylation factor (ARF) GTPases and p21-activated kinases (PAKs) as its relevant host substrates. The 2.5?? crystal structure of EspG in complex with ARF6 shows how EspG blocks GTPase-activating-protein-assisted GTP hydrolysis, revealing a potent mechanism of GTPase signalling inhibition at organelle membranes. In addition, the 2.8?? crystal structure of EspG in complex with the autoinhibitory Iα3-helix of PAK2 defines a previously unknown catalytic site in EspG and provides an allosteric mechanism of kinase activation by a bacterial effector. Unexpectedly, ARF and PAKs are organized on adjacent surfaces of EspG, indicating its role as a 'catalytic scaffold' that effectively reprograms cellular events through the functional assembly of GTPase-kinase signalling complex.     

Structural basis for the assembly of a nuclear export complex

The nuclear import and export of macromolecular cargoes through nuclear pore complexes is mediated primarily by carriers such as importin-beta. Importins carry cargoes into the nucleus, whereas exportins carry cargoes to the cytoplasm. Transport is orchestrated by nuclear RanGTP, which dissociates cargoes from importins, but conversely is required for cargo binding to exportins. Here we present the 2.0 A crystal structure of the nuclear export complex formed by exportin Cse1p complexed with its cargo (Kap60p) and RanGTP, thereby providing a structural framework for understanding nuclear protein export and the different functions of RanGTP in export and import. In the complex, Cse1p coils around both RanGTP and Kap60p, stabilizing the RanGTP-state and clamping the Kap60p importin-beta-binding domain, ensuring that only cargo-free Kap60p is exported. Mutagenesis indicated that conformational changes in exportins couple cargo binding to high affinity for RanGTP, generating a spring-loaded molecule to facilitate disassembly of the export complex following GTP hydrolysis in the cytoplasm.     

Structure of the nociceptin/orphanin FQ receptor in complex with a peptide mimetic

Members of the opioid receptor family of G-protein-coupled receptors (GPCRs) are found throughout the peripheral and central nervous system, where they have key roles in nociception and analgesia. Unlike the 'classical' opioid receptors, δ, κ and μ (δ-OR, κ-OR and μ-OR), which were delineated by pharmacological criteria in the 1970s and 1980s, the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP, also known as ORL-1) was discovered relatively recently by molecular cloning and characterization of an orphan GPCR. Although it shares high sequence similarity with classical opioid GPCR subtypes (～60%), NOP has a markedly distinct pharmacology, featuring activation by the endogenous peptide N/OFQ, and unique selectivity for exogenous ligands. Here we report the crystal structure of human NOP, solved in complex with the peptide mimetic antagonist compound-24 (C-24) (ref. 4), revealing atomic details of ligand-receptor recognition and selectivity. Compound-24 mimics the first four amino-terminal residues of the NOP-selective peptide antagonist UFP-101, a close derivative of N/OFQ, and provides important clues to the binding of these peptides. The X-ray structure also shows substantial conformational differences in the pocket regions between NOP and the classical opioid receptors κ (ref. 5) and μ (ref. 6), and these are probably due to a small number of residues that vary between these receptors. The NOP-compound-24 structure explains the divergent selectivity profile of NOP and provides a new structural template for the design of NOP ligands.     

Structural basis for the subunit assembly of the anaphase-promoting complex

The anaphase-promoting complex or cyclosome (APC/C) is an unusually large E3 ubiquitin ligase responsible for regulating defined cell cycle transitions. Information on how its 13 constituent proteins are assembled, and how they interact with co-activators, substrates and regulatory proteins is limited. Here, we describe a recombinant expression system that allows the reconstitution of holo APC/C and its sub-complexes that, when combined with electron microscopy, mass spectrometry and docking of crystallographic and homology-derived coordinates, provides a precise definition of the organization and structure of all essential APC/C subunits, resulting in a pseudo-atomic model for 70% of the APC/C. A lattice-like appearance of the APC/C is generated by multiple repeat motifs of most APC/C subunits. Three conserved tetratricopeptide repeat (TPR) subunits (Cdc16, Cdc23 and Cdc27) share related superhelical homo-dimeric architectures that assemble to generate a quasi-symmetrical structure. Our structure explains how this TPR sub-complex, together with additional scaffolding subunits (Apc1, Apc4 and Apc5), coordinate the juxtaposition of the catalytic and substrate recognition module (Apc2, Apc11 and Apc10 (also known as Doc1)), and TPR-phosphorylation sites, relative to co-activator, regulatory proteins and substrates.     

Spermatid differentiation requires the assembly of a cell polarity complex downstream of junctional adhesion molecule-C

During spermatogenesis in the mammalian testis, stem cells (spermatogonia) differentiate into spermatocytes, which subsequently undergo two consecutive meiotic divisions to give rise to haploid spermatids. These cells are initially round but progressively elongate, condense their nuclei, acquire flagellar and acrosomal structures, and shed a significant amount of their cytoplasm to form spermatozoa (the sperm cells) in a developmental cascade termed spermiogenesis. Defects in these processes will lead to a lack of mature sperm cells (azoospermia), which is a major cause of male infertility in the human population. Here we report that a cell-surface protein of the immunoglobulin superfamily, junctional adhesion molecule-C (JAM-C), is critically required for the differentiation of round spermatids into spermatozoa in mice. We found that Jam-C is essential for the polarization of round spermatids, a function that we attribute to its role in the assembly of a cell polarity complex.     

Cellular peptide composition governed by major histocompatibility complex class I molecules

Major histocompatibility complex (MHC) class I molecules present peptides derived from cellular proteins to cytotoxic T lymphocytes (CTLs), which check these peptides for abnormal features. How such peptides arise in the cell is not known. Here we show that the MHC molecules themselves are substantially involved in determining which peptides occur intracellularly: normal mouse spleen cells identical at all genes but MHC class I express different patterns of peptides derived from cellular non-MHC proteins. We suggest several models to explain this influence of MHC class I molecules on cellular peptide composition.     

A heterodimeric complex that promotes the assembly of mammalian 20S proteasomes

The 26S proteasome is a multisubunit protease responsible for regulated proteolysis in eukaryotic cells. It comprises one catalytic 20S proteasome and two axially positioned 19S regulatory complexes. The 20S proteasome is composed of 28 subunits arranged in a cylindrical particle as four heteroheptameric rings, alpha1-7beta1-7beta1-7alpha1-7 (refs 4, 5), but the mechanism responsible for the assembly of such a complex structure remains elusive. Here we report two chaperones, designated proteasome assembling chaperone-1 (PAC1) and PAC2, that are involved in the maturation of mammalian 20S proteasomes. PAC1 and PAC2 associate as heterodimers with proteasome precursors and are degraded after formation of the 20S proteasome is completed. Overexpression of PAC1 or PAC2 accelerates the formation of precursor proteasomes, whereas knockdown by short interfering RNA impairs it, resulting in poor maturation of 20S proteasomes. Furthermore, the PAC complex provides a scaffold for alpha-ring formation and keeps the alpha-rings competent for the subsequent formation of half-proteasomes. Thus, our results identify a mechanism for the correct assembly of 20S proteasomes.     

Specific low-affinity recognition of major histocompatibility complex plus peptide by soluble T-cell receptor.

The T-cell receptor is necessary and sufficient for recognition of peptides presented by major histocompatibility complex molecules. Other adhesion molecules, like CD4 or CD8, play an auxiliary role in antigen recognition by T cells. Here we analyse T-cell receptor (TCR) binding using a soluble rather than a cell-bound receptor molecule. A TCR-immunoglobulin chimaera is constructed with the variable and the first constant regions of both the TCR alpha- and beta-chains linked to the immunoglobulin light-chain constant regions. This soluble TCR is expressed, assembled and secreted as an alpha beta heterodimer by a myeloma cell line transfected with the recombinant genes. Furthermore, the soluble TCR is biologically active: it specifically inhibits antigen-dependent activation of the relevant T-cell clones and thus discriminates between proper and irrelevant peptides presented by major histocompatibility complex molecules.     

复凝聚法制备玉米多肽微胶囊及释放特性研究

采用复凝聚法制备玉米多肽微胶囊,分析了壁材浓度、明胶和羧甲基纤维素钠(CMC)比例、壁芯比3个因素对微胶囊包埋率的影响.在单因素和正交试验基础上,依据响应面分析法确定了微胶囊最佳制备工艺,并分析其在体外模拟胃肠液环境中的释放特性.微胶囊最优制备工艺:壁材浓度1.2％,明胶︰CMC为7.59︰1,壁芯比为5.97︰1,在...     

具有相互干扰的捕食-食饵系统的定性分析

考虑了捕食者具有相互干扰的Holling-Tanner型捕食-食饵系统,得到了正平衡态存在的条件,进而得到了正平衡态全局稳定的充分条件.     

干扰条件下一个破产模型的改进

在经典风险模型的基础上,讨论了带有干扰项的保费收取次数是一个Poisson过程的破产概率模型,并且将每次收到的保费额看作是服从指数分布的随机变量,分析了获利过程的性质,给出了调节系数方程及相应的破产概率的上限.