Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins. We recently reported the identification of the gene cluster responsible for the biosynthesis of these toxins in cyanobacteria and proposed a biosynthetic scheme starting from L-proline and involving three polyketide synthases which starter would be (S)-1-pyrroline-5-carboxylate bound to an acyl carrier protein, AnaD. We now report the in vitro reconstitution of the first steps of this biosynthesis in Oscillatoria PCC 6506. We identified in PCC 6506, the gene coding for an Sfp-like phosphopantetheinyl transferase and purified the gene product, OsPPT that catalyzed the transfer of the phosphopantetheinyl arm to the serine 41 of AnaD. The pure adenylation protein AnaC loaded L-proline on holo-AnaD and was specific for L-proline (Km = 0.97 mM, kcat = 68 min-1) among the 20 natural amino acids. Among 6 close structural analogs of L-proline, including (S)-1-pyrroline-5-carboxylate, we only found 3,4-dehydro-L-proline to be an alternate substrate for AnaC (Km = 1.5 mM, kcat = 29 min-1). The putative prolyl-AnaD dehydrogenase, AnaB, purified to homogeneity as a histidine-tagged protein, showed an absorption spectrum characteristic of FAD containing proteins. It oxidized prolyl-AnaD to dehydroprolyl-AnaD as shown by tryptic digestion of the protein followed by liquid chromatography coupled to tandem mass spectrometry. Alignment of the amino acid sequence of this dehydrogenase with related enzymes showed that AnaB belongs to the acyl-CoA dehydrogenase superfamily and thus probably catalyzes an a-b-dehydrogenation of the thioester bound proline followed by an aza-allylic isomerization to yield (S)-pyrroline-5-carboxyl-AnaD, the proposed starter for the subsequent polyketide synthase, AnaE.

The Arp2/3 complex generates branched actin networks when activated by Nucleation Promoting Factors (NPFs). Recently, the WASH family of NPFs has been identified, but its cellular role is unclear. Here, we show that WASH generates an actin network on a restricted domain of sorting and recycling endosomes. We found that WASH belongs to a multiprotein complex containing seven subunits, including the heterodimer of capping protein (CP). In vitro, the purified WASH complex activates Arp2/3-mediated actin nucleation and binds directly to liposomes. WASH also interacts with dynamin. WASH depletion gives rise to long membrane tubules pulled out from endosomes along microtubules, as does dynamin inhibition. Accordingly, WASH is required for efficient transferrin recycling. Together, these data suggest that the WASH molecular machine, integrating CP with a NPF, controls the fission of endosomes through an interplay between the forces generated by microtubule motors and actin polymerization.

The Wave proteins activate the Arp2/3 complex at the leading edge of migrating cells. The resulting actin polymerization powers the projection of the plasma membrane in lamellipodia and membrane ruffles. The Wave proteins are always found associated with partner proteins. The canonical Wave complex is a stable complex containing five subunits. Even though it is well admitted that this complex plays an essential regulatory role on Wave function, the mechanisms by which Wave proteins are regulated within the complex are still elusive. Even the constitutive activity or inactivity of the complex is controversial. The major difficulty of these assays resides in the long and difficult purification of the Wave complex by a combination of several chromatography steps, which gives an overall low yield and increases the chance of Wave complex denaturation. Here we report a greatly simplified approach to purify the human Wave complex using a stable cell line expressing a tagged subunit and affinity chromatography. This protocol provided us with sufficient amount of pure Wave complex for functional assays. These assays unambiguously established that the Wave complex in its native conformation is intrinsically inactive, indicating that, like WASP proteins, Wave proteins have a masked C-terminal Arp2/3 binding site at resting state. As a consequence, the Wave complex has to be recruited and activated at the plasma membrane to project migration structures. Importantly, the approach we describe here for multiprotein complex purification is likely applicable to a wide range of human multiprotein complexes. Cell Motil. Cytoskeleton 2009.(c) 2009 Wiley-Liss, Inc.

BACKGROUND: The physiological function of the ubiquitous cellular prion protein, PrP(c), is still under debate. It was essentially studied in nervous system, but poorly investigated in epithelial cells. We previously reported that PrP(c) is targeted to cell-cell junctions of polarized epithelial cells, where it interacts with c-Src. METHODOLOGY/FINDINGS: We show here that, in cultured human enterocytes and in intestine in vivo, the mature PrP(c) is differentially targeted either to the nucleus in dividing cells or to cell-cell contacts in polarized/differentiated cells. By proteomic analysis, we demonstrate that the junctional PrP(c) interacts with cytoskeleton-associated proteins, such as gamma- and beta-actin, alpha-spectrin, annexin A2, and with the desmosome-associated proteins desmoglein, plakoglobin and desmoplakin. In addition, co-immunoprecipitation experiments revealed complexes associating PrP(c), desmoglein and c-Src in raft domains. Through siRNA strategy, we show that PrP(c) is necessary to complete the process of epithelial cell proliferation and for the sub-cellular distribution of proteins involved in cell architecture and junctions. Moreover, analysis of the architecture of the intestinal epithelium of PrP(c) knock-out mice revealed a net decrease in the size of desmosomal junctions and, without change in the amount of BrdU incorporation, a shortening of the length of intestinal villi. CONCLUSIONS/SIGNIFICANCE: From these results, PrP(c) could be considered as a new partner involved in the balance between proliferation and polarization/differentiation in epithelial cells.

Refractile bodies (RB), whose function is still unknown, are specific structures of Eimeriidae parasites. In order to study their proteome, RB were purified from Eimeria tenella sporozoites by a new procedure using a reversible fixation followed by centrifugation. RB proteins were resolved by two-dimensional electrophoresis. Around 76 and 89 spots were detected on RB two-dimensional gels using gradients in the 3-10 and 4-7 range, respectively. RB proteins were located mainly between pH 5 and 7. RB gels were then compared with previously established maps of the entire sporozoite proteome. Proteins appearing in new spots were identified by mass spectrometry. Thirty protein isoforms were located in RB. Added to the already known RB proteins such as Eimepsin and SO7', the new RB proteins were defined as haloacid dehalogenase, hydrolase, subtilase, lactacte dehydrogenase or ubiquitin family proteins. The RB proteome analysis confirmed the hypothesis that this structure is a reservoir for proteins necessary to invasion but also suggests that RB have energetic and metabolic functions.

Nasopharyngeal carcinomas (NPC) are etiologically related to the Epstein-Barr virus (EBV), and malignant NPC cells have consistent although heterogeneous expression of the EBV latent membrane protein 1 (LMP1). LMP1 trafficking and signaling require its incorporation into membrane rafts. Conversely, raft environment is likely to modulate LMP1 activity. In order to investigate NPC-specific raft partners of LMP1, rafts derived from the C15 NPC xenograft were submitted to preparative immunoprecipitation of LMP1 combined with mass spectrometry analysis of coimmunoprecipitated proteins. Through this procedure, galectin 9, a beta-galactoside binding lectin and Hodgkin tumor antigen, was identified as a novel LMP1 partner. LMP1 interaction with galectin 9 was confirmed by coimmunoprecipitation and Western blotting in whole-cell extracts of NPC and EBV-transformed B cells (lymphoblastoid cell lines [LCLs]). Using mutant proteins expressed in HeLa cells, LMP1 was shown to bind galectin 9 in a TRAF3-independent manner. Galectin 9 is abundant in NPC biopsies as well as in LCLs, whereas it is absent in Burkitt lymphoma cells. In subsequent experiments, NPC cells were treated with Simvastatin, a drug reported to dissociate LMP1 from membrane rafts in EBV-transformed B cells. We found no significant effects of Simvastatin on the distribution of LMP1 and galectin 9 in NPC cell rafts. However, Simvastatin was highly cytotoxic for NPC cells, regardless of the presence or absence of LMP1. This suggests that Simvastatin is a potentially useful agent for the treatment of NPCs although it has distinct mechanisms of action in NPC and LCL cells.

INTRODUCTION: The herbal medicinal product kava-kava, used for treating anxiety disorders, was assessed positively by the Cochrane Review. However, it was withdrawn from the market in Switzerland and Germany due to cases of liver failure and 'unproven' efficacy. METHODS: A protocol for the meta-analysis based on patient source data was written, a literature search was done, and six placebo-controlled, randomized trials with the kava extract WS1490 were identified. The endpoints were the change in HAMA during treatment (continuous and binary). RESULTS: WS1490 has an effective success rate of OR=3.3 (95% confidence interval of 2.09-5.22) in patients with non-psychotic anxiety disorders. The continuous outcome supports this result: mean improvement with WS1490 by 5.94 (95% confidence interval -0.86 to 12.8) points on the HAMA scale better than placebo. Kava seems to be more effective in females and in younger patients. DISCUSSION: This meta-analysis has no publication bias, no remarkable heterogeneity and is based on trials with high methodological standards. It is concluded that WS1490, and possibly other kava extracts, are effective. Therefore they remain alternatives to benzodiazepines, selective serotonin re-uptake inhibitors (SSRIs) and other antidepressants in the treatment of non-psychotic anxiety disorders.