AliasServer: a web server to handle multiple aliases used to refer to proteins

SUMMARY: AliasServer provides services that facilitate the assembly of data or datasets that make use of different identifiers for refering to the same protein. This resource relies on a database which contains, for a given organism, a non-redundant list of protein sequences associated with a set of aliases. AVAILABILITY: AliasServer is available as an interactive Web server at http://cbi.labri.fr/outils/alias/ and as a web service using a SOAP interface. The complete tool, including sources and data, is available for local installations upon request. SUPPLEMENTARY INFORMATION: Technical documentation is available at http://cbi.labri.fr/outils/alias/asdoc.pdf     

Functional characterization of the postulated intramolecular sphingolipid activator protein domain of human acid sphingomyelinase

Degradation of membrane-bound sphingomyelin to phosphorylcholine and ceramide is catalyzed by the water-soluble lysosomal acid sphingomyelinase (A-SMase). The presence of sphingolipid activator proteins (Saps: saposins A-D; GM2 activator) is not essential to mediate this reaction at the water-lipid interface in vivo . A hypothesis based on amino acid sequence alignments suggests that the enzyme possesses an N-terminal saposin-homologous domain, which may facilitate the enzymatic reaction at the interface. We mutated one homologous and three conserved amino acid residues of this domain and studied the activity of the variant enzymes using different sphingomyelin degradation assays. A variant with an exchange of a conserved amino acid residue, Pro153Ala, still exhibited enzyme activity of approximately 52% of normal in a detergent-containing micellar assay, but only 13% of normal in a detergent-free liposomal assay system, which suggests that the Sap-homologous domain fulfills membrane-disturbing functions. Addition of saposin C to the liposomal assay mixtures increased the Pro153Ala variant sphingomyelinase activity to 46% of normal, indicating that the variant saposin-like domain can be substituted by the presence of the sphingolipid activator protein. On the other hand, the addition of saposin C did not result in complete restoration of the variant activity. Thus, the Sap-like domain may also have another role, e.g., to stabilize the fold of acid sphingomyelinase, which cannot be compensated by the presence of saposin C or a detergent. Such an essential second function of the saposin-like domain as an integral part of acid sphingomyelinase is confirmed by our observation that the Lys118Glu, Cys120Ser and Cys131Ser variants were almost completely devoid of activity in the detergent-containing micellar assay system as well as in the liposomal assay system in the presence of saposin C.     

Non-muscle alpha-actinin-4 interacts with plasminogen activator inhibitor type-1 (PAI-1)

PAI-1 modulates many biological processes involving fibrinolysis, cell migration or tissue remodelling. In addition to inhibiting serine proteases (mainly tPA and uPA), PAI-1 interacts with vitronectin (Vn), fibrin or alpha(1)-acid glycoprotein, interactions which are important for PAI-1-mediated effects in inflammation, tumor invasion and metastasis. To further identify proteins interacting with PAI-1, the yeast two-hybrid strategy was employed. Screening of a human placenta cDNA library identified--in addition to the C-terminal region of cytokeratin 18 (CK18(182-430))--a large C-terminal fragment of alpha-actinin-4 (Act-4) as a binding partner for PAI-1. Two different cDNA clones encoding Act-4(287-911) and Act-4(330-911) respectively, were isolated. An Act-4(330-911)/GST-fusion protein, but not GST alone, was immunoprecipitated together with active PAI-1. In solid phase binding assays, active wild-type PAI-1 as well as the PAI-1 variant Q123K (which does not interact with multimeric Vn) was found to bind to Act-4(330-911)/GST. Latent PAI-1, latent Q123K, and the inactive PAI-1 variant Q55P did not display any binding activity. Act-4 is mainly present intracellularly and is involved in cellular motility via interaction with the actin cytoskeleton, thus probably affecting the metastatic potential of tumor cells. However, an extracellular Act-4-derived fragment (mactinin) has previously been identified, which (i) is generated by proteolytic action of uPA, (ii) displays significant chemotactic activity for monocytes, and (iii) promotes monocyte/macrophage maturation. We suggest that PAI-1, via interaction with both Act-4 and uPA, may function as a modulator of this mononuclear phagocyte response, not only in inflammation but also in tumor invasion and metastasis.     

Effect of Vibrio parahaemolyticus haemolysin on human erythrocytes

Haemolysin Kanagawa, a toxin from Vibrio parahaemolyticus, is known to trigger haemolysis. Flux studies indicated that haemolysin forms a cation channel. In the present study, channel properties were elucidated by patch clamp and functional significance of ion fluxes by fluorescence-activated cell sorting (FACS) analysis. Treatment of human erythrocytes with 1 U ml-1 haemolysin within minutes induces a non-selective cation permeability. Moreover, haemolysin activates clotrimazole-sensitive K+ channels, pointing to stimulation of Ca2+-sensitive Gardos channels. Haemolysin (1 U ml-1) leads within 5 min to slight cell shrinkage, which is reversed in Ca2+-free saline. Erythrocytes treated with haemolysin (0.1 U ml-1) do not undergo significant haemolysis within the first 60 min. Replacement of extracellular Na+ with NMDG+ leads to slight cell shrinkage, which is potentiated by 0.1 U ml-1 haemolysin. According to annexin binding, treatment of erythrocytes with 0.1 U ml-1 haemolysin leads within 30 min to breakdown of phosphatidylserine asymmetry of the cell membrane, a typical feature of erythrocyte apoptosis. The annexin binding is significantly blunted at increased extracellular K+ concentrations and by K+ channel blocker clotrimazole. In conclusion, haemolysin Kanagawa induces cation permeability and activates endogenous Gardos K+ channels. Consequences include breakdown of phosphatidylserine asymmetry, which depends at least partially on cellular loss of K+.     

Evidence for a size-sensing mechanism in animal cells

Continuously proliferating cells exactly double their mass during each cell cycle. Here we have addressed the controversial question of if and how cell size is sensed and regulated. We used erythroblasts that proliferate under the control of a constitutively active oncogene (v-ErbB) or under the control of physiological cytokines (stem cell factor, erythropoietin and v-ErbB inhibitor). The oncogene-driven cells proliferated 1.7 times faster and showed a 1.5-fold increase in cell volume. The two phenotypes could be converted into each other 24 h after altering growth factor signalling. The large cells had a higher rate of protein synthesis, together with a shortened G1 phase. Additional experiments with chicken erythroblasts and mouse fibroblasts, synchronized by centrifugal elutriation, provided further evidence that vertebrate cells can respond to cell size alterations (induced either through different growth factor signalling or DNA synthesis inhibitors) by compensatory shortening of the subsequent G1 phase. Taken together, these data suggest that an active size threshold mechanism exists in G1, which induces adjustment of cell-cycle length in the next cycle, thus ensuring maintenance of a proper balance between growth and proliferation rates in vertebrates.     

ARF6-dependent interaction of the TWIK1 K+ channel with EFA6, a GDP/GTP exchange factor for ARF6

TWIK1 belongs to a family of K(+) channels involved in neuronal excitability and cell volume regulation. Its tissue distribution suggests a role in epithelial potassium transport. Here we show that TWIK1 is expressed in a subapical compartment in renal proximal tubules and in polarized MDCK cells. In nonpolarized cells, this compartment corresponds to pericentriolar recycling endosomes. We identified EFA6, an exchange factor for the small G protein ADP-ribosylation factor 6 (ARF6), as a protein binding to TWIK1. EFA6 interacts with TWIK1 only when it is bound to ARF6. Because ARF6 modulates endocytosis at the apical surface of epithelial cells, the ARF6/EFA6/TWIK1 association is probably important for channel internalization and recycling.     

Characterization of the microheterogeneity of transthyretin in plasma and urine using SELDI-TOF-MS immunoassay

Background  

It has been shown that transthyretin (TTR) exists in different molecular variants. Besides point mutations associated with different diseases such as amyloidosis, other posttranslational modifications occur that might be of diagnostic interest.     

Involvement of putative SNF2 chromatin remodeling protein DRD1 in RNA-directed DNA methylation

In plants, the mechanism by which RNA can induce de novo cytosine methylation of homologous DNA is poorly understood. Cytosines in all sequence contexts become modified in response to RNA signals. Recent work has implicated the de novo DNA methyltransferases (DMTases), DRM1 and DRM2, in establishing RNA-directed methylation of the constitutive nopaline synthase promoter, as well as the DMTase MET1 and the putative histone deacetylase HDA6 in maintaining or enhancing CpG methylation induced by RNA. Despite the identification of enzymes that catalyze epigenetic modifications in response to RNA signals, it is unclear how RNA targets DNA for methylation. A screen for mutants defective in RNA-directed DNA methylation identified a novel putative chromatin-remodeling protein, DRD1. This protein belongs to a previously undefined, plant-specific subfamily of SWI2/SNF2-like proteins most similar to the RAD54/ATRX subfamily. In drd1 mutants, RNA-induced non-CpG methylation is almost eliminated at a target promoter, resulting in reactivation, whereas methylation of centromeric and rDNA repeats is unaffected. Thus, unlike the SNF2-like proteins DDM1/Lsh1 and ATRX, which regulate methylation of repetitive sequences, DRD1 is not a global regulator of cytosine methylation. DRD1 is the first SNF2-like protein implicated in an RNA-guided, epigenetic modification of the genome.     

Pollinator attractiveness increases with distance from flowering orchids

Orchids are extraordinary among plants because many species are pollinated through sexual duplicity by producing flowers that mimic female insects to lure unsuspecting males. Previous work showed that sexual deception by the orchid Chiloglottis trapeziformis can have a negative impact on its wasp pollinator Neozeleboria cryptoides. We report that female wasps may be capable of mitigating the cost of the orchids' deception. Although male wasps quickly habituated to areas planted with unrewarding flower decoys, we found that the effectiveness of the chemical cue used by the wingless females to attract males increases with increasing distance from an orchid patch. The apparent specificity of the males' site-based avoidance strategy means that females emerging in areas occupied by flowering orchids could, potentially, leave the orchid colony by walking to increase their attractiveness.     

Increased luminal pH in the epididymis of infertile c-ros knockout mice and the expression of sodium-hydrogen exchangers and vacuolar proton pump H+-ATPase

Transgenic mice targeted for the c-ros gene, which are fertile when heterozygous (HET), but infertile when homozygous (knockout, KO) and associated with failure in pubertal differentiation of the epididymal initial segment, provide a model for studying the role of the epididymal luminal environment in sperm development. Luminal fluid from the cauda epididymidis was measured by both ion-selective microelectrodes and pH strips to be 0.3 pH units higher in the KO than HET. Of the genes responsible for luminal acidification, expression of mRNA of vacuolar H(+)-ATPase was found in all epididymal regions, but with no difference between KO and HET. Immunohistochemistry showed its presence in epithelial apical cells and clear cells. The Na(+)-hydrogen exchanger NHE2 was expressed at mRNA and protein levels in the caput but only marginally detectable if at all in the distal epididymis. This was compensated for by NHE3 which was expressed strongest in the cauda region, in agreement with immunohistochemical staining. Quantification of Western blot data revealed slight, but significant, decreases of NHE2 in the caput and of NHE3 in the cauda in the KO mice. The increase in luminal fluid pH in the KO mice could also be contributed to by other epithelial regulating factors including the Na(+)-dependent glutamate transporter EAAC1 formerly reported to be down regulated in the KO.