Over-expression of Rififylin, a new RING finger and FYVE-like domain-containing protein, inhibits recycling from the endocytic recycling compartment

Endocytosed membrane components are recycled to the cell surface either directly from early/sorting endosomes or after going through the endocytic recycling compartment (ERC). Studying recycling mechanisms is difficult, in part due to the fact that specific tools to inhibit this process are scarce. In this study, we have characterized a novel widely expressed protein, named Rififylin (Rffl) for RING Finger and FYVE-like domain-containing protein, that, when overexpressed in HeLa cells, induced the condensation of transferrin receptor-, Rab5-, and Rab11-positive recycling tubulovesicular membranes in the perinuclear region. Internalized transferrin was able to access these condensed endosomes but its exit from this compartment was delayed. Using deletion mutants, we show that the carboxy-terminal RING finger of Rffl is dispensable for its action. In contrast, the amino-terminal domain of Rffl, which shows similarities with the phosphatidylinositol-3-phosphate-binding FYVE finger, is critical for the recruitment of Rffl to recycling endocytic membranes and for the inhibition of recycling, albeit in a manner that is independent of PtdIns(3)-kinase activity. Rffl overexpression represents a novel means to inhibit recycling that will help to understand the mechanisms involved in recycling from the ERC to the plasma membrane.     

Dendrimeric coating of glass slides for sensitive DNA microarrays analysis

Successful use and reliability of microarray technology is highly dependent on several factors, including surface chemistry parameters and accessibility of cDNA targets to the DNA probes fixed onto the surface. Here, we show that functionalisation of glass slides with homemade dendrimers allow production of more sensitive and reliable DNA microarrays. The dendrimers are nanometric structures of size-controlled diameter with aldehyde function at their periphery. Covalent attachment of these spherical reactive chemical structures on amino-silanised glass slides generates a reactive ~100 Å layer onto which amino-modified DNA probes are covalently bound. This new grafting chemistry leads to the formation of uniform and homogenous spots. More over, probe concentration before spotting could be reduced from 0.2 to 0.02 mg/ml with PCR products and from 20 to 5 µM with 70mer oligonucleotides without affecting signal intensities after hybridisation with Cy3- and Cy5-labelled targets. More interestingly, while the binding capacity of captured probes on dendrimer-activated glass surface (named dendrislides) is roughly similar to other functionalised glass slides from commercial sources, detection sensitivity was 2-fold higher than with other available DNA microarrays. This detection limit was estimated to 0.1 pM of cDNA targets. Altogether, these features make dendrimer-activated slides ideal for manufacturing cost-effective DNA arrays applicable for gene expression and detection of mutations.     

The Freter model: A simple model of biofilm formation

A simple, conceptual model of biofilm formation, due to R. Freter et al. (1983), is studied analytically and numerically in both CSTR and PFR. Two steady state regimes are identified, namely, the complete washout of the microbes from the reactor and the successful colonization of both the wall and bulk fluid. One of these is stable for any particular set of parameter values and sharp and explicit conditions are given for the stability of each. The effects of adding an anti-microbial agent to the CSTR are examined.Supported by NSF Grant DMS 0107439 and UTA Grant REP 14748717Supported by NSF Grant DMS 0107160     

The EMBL Nucleotide Sequence Database: major new developments

The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/) incorporates, organizes and distributes nucleotide sequences from all available public sources. The database is located and maintained at the European Bioinformatics Institute (EBI) near Cambridge, UK. In an international collaboration with DDBJ (Japan) and GenBank (USA), data are exchanged amongst the collaborating databases on a daily basis to achieve optimal synchronization. Webin is the preferred web-based submission system for individual submitters, while automatic procedures allow incorporation of sequence data from large-scale genome sequencing centres and from the European Patent Office (EPO). Database releases are produced quarterly. Network services allow free access to the most up-to-date data collection via FTP, Email and World Wide Web interfaces. EBI's Sequence Retrieval System (SRS) integrates and links the main nucleotide and protein databases plus many other specialized molecular biology databases. For sequence similarity searching, a variety of tools (e.g. Fasta, BLAST) are available which allow external users to compare their own sequences against the latest data in the EMBL Nucleotide Sequence Database and SWISS-PROT. All resources can be accessed via the EBI home page at http://www.ebi.ac.uk.     

CCK-A receptor activates RhoA through G alpha 12/13 in NIH3T3 cells

Cholecystokinin (CCK) is a major regulator of pancreatic acinar cells and was shown previously to be capable of inducing cytoskeletal changes in these cells. In the present study, using NIH3T3 cells stably transfected with CCK-A receptors as a model cell, we demonstrate that CCK can induce actin stress fibers through a G₁₃- and RhoA-dependent mechanism. CCK induced stress fibers within minutes similar to those induced by lysophosphatidic acid (LPA), the active component of serum. The effects of CCK were mimicked by active RhoV14 and blocked by dominant-negative RhoN19, Clostridium botulinum C3 transferase, and the Rho-kinase inhibitor Y-27632. CCK rapidly induced active Rho in cells as shown with a pull-down assay using the Rho binding domain of rhotekin and by a serum response element (SRE)-luciferase reporter assay. To evaluate the G protein mediating the action of CCK, cells were transfected with active -subunits; G₁₃ and G₁₂ but not G_q induced stress fibers and in some cases cell rounding. A p115 Rho guanine nucleotide exchange factor (GEF) regulator of G protein signaling (RGS) domain known to interact with G_12/13 inhibited active _12/13-and CCK-induced stress fibers, whereas RGS2 and RGS4, which are known to inhibit G_q, had no effect. Cotransfection with plasmids coding for the G protein -subunit carboxy-terminal peptide from ₁₃ and, to a lesser extent ₁₂, also inhibited the effect of CCK, whereas the peptide from _q did not. These results show that in NIH3T3 cells bearing CCK-A receptors, CCK activates Rho primarily through G₁₃, leading to rearrangement of the actin cytoskeleton. actin; cholecystokinin; Rho; Rho-kinase; stress fibers     

Motions and structural variability within toxins: implication for their use as scaffolds for protein engineering

Animal toxins are small proteins built on the basis of a few disulfide bonded frameworks. Because of their high variability in sequence and biologic function, these proteins are now used as templates for protein engineering. Here we report the extensive characterization of the structure and dynamics of two toxin folds, the "three-finger" fold and the short alpha/beta scorpion fold found in snake and scorpion venoms, respectively. These two folds have a very different architecture; the short alpha/beta scorpion fold is highly compact, whereas the "three-finger" fold is a beta structure presenting large flexible loops. First, the crystal structure of the snake toxin alpha was solved at 1.8-A resolution. Then, long molecular dynamics simulations (10 ns) in water boxes of the snake toxin alpha and the scorpion charybdotoxin were performed, starting either from the crystal or the solution structure. For both proteins, the crystal structure is stabilized by more hydrogen bonds than the solution structure, and the trajectory starting from the X-ray structure is more stable than the trajectory started from the NMR structure. The trajectories started from the X-ray structure are in agreement with the experimental NMR and X-ray data about the protein dynamics. Both proteins exhibit fast motions with an amplitude correlated to their secondary structure. In contrast, slower motions are essentially only observed in toxin alpha. The regions submitted to rare motions during the simulations are those that exhibit millisecond time-scale motions. Lastly, the structural variations within each fold family are described. The localization and the amplitude of these variations suggest that the regions presenting large-scale motions should be those tolerant to large insertions or deletions.     

Parallel liquid synthesis of N,N'-Disubstituted 3-amino azepin-2-ones as potent and specific farnesyl transferase inhibitors

A rapid structure-activity study was performed by parallel liquid synthesis on N,N'-disubstitution of 3-amino azepin-2-one to afford potent and specific farnesyl transferase inhibitors with low nM enzymatic and cellular activities. The activities of the selected compounds were validated in vivo, and compounds 41a and 44a presented significant antitumour activity.     

基于支持向量机的蛋白质同源寡聚体分类研究

基于支持向量机和贝叶斯方法,从蛋白质一级序列出发对蛋白质同源二聚体、同源三聚体、同源四聚体、同源六聚体进行分类研究,结果表明：基于支持向量机, 采用“一对多”和“一对一”策略, 其分类总精度分别为77.36%和93.43%, 分别比基于贝叶斯协方差判别法的分类总精度50.64%提高26.72和42.79个百分点.从而说明支持向量机可用于蛋白质同源寡聚体分类,且是一种非常有效的方法.对于多类蛋白质同源寡聚体分类,基于相同的机器学习方法（如支持向量机）,采用“一对一”策略比“一对多”效果好.同时亦表明蛋白质同源寡聚体一级序列包含四级结构信息.     

Crystal structure of tabtoxin resistance protein complexed with acetyl coenzyme A reveals the mechanism for beta-lactam acetylation

Tabtoxin resistance protein (TTR) is an enzyme that renders tabtoxin-producing pathogens, such as Pseudomonas syringae, tolerant to their own phytotoxins. Here, we report the crystal structure of TTR complexed with its natural cofactor, acetyl coenzyme A (AcCoA), to 1.55A resolution. The binary complex forms a characteristic "V" shape for substrate binding and contains the four motifs conserved in the GCN5-related N-acetyltransferase (GNAT) superfamily, which also includes the histone acetyltransferases (HATs). A single-step mechanism is proposed to explain the function of three conserved residues, Glu92, Asp130 and Tyr141, in catalyzing the acetyl group transfer to its substrate. We also report that TTR possesses HAT activity and suggest an evolutionary relationship between TTR and other GNAT members.