Scavenger receptor BI boosts hepatocyte permissiveness to Plasmodium infection

Infection of hepatocytes by Plasmodium falciparum sporozoites requires the host tetraspanin CD81. CD81 is also predicted to be a coreceptor, along with scavenger receptor BI (SR-BI), for hepatitis C virus. Using SR-BI-knockout, SR-BI-hypomorphic and SR-BI-transgenic primary hepatocytes, as well as specific SR-BI-blocking antibodies, we demonstrate that SR-BI significantly boosts hepatocyte permissiveness to P. falciparum, P. yoelii, and P. berghei entry and promotes parasite development. We show that SR-BI, but not the low-density lipoprotein receptor, acts as a major cholesterol provider that enhances Plasmodium infection. SR-BI regulates the organization of CD81 at the plasma membrane, mediating an arrangement that is highly permissive to penetration by sporozoites. Concomitantly, SR-BI upregulates the expression of the liver fatty-acid carrier L-FABP, a protein implicated in Plasmodium liver-stage maturation. These findings establish the mechanistic basis of the CD81-dependent Plasmodium sporozoite invasion pathway.     

Life-cycle and genome of OtV5, a large DNA virus of the pelagic marine unicellular green alga Ostreococcus tauri

Large DNA viruses are ubiquitous, infecting diverse organisms ranging from algae to man, and have probably evolved from an ancient common ancestor. In aquatic environments, such algal viruses control blooms and shape the evolution of biodiversity in phytoplankton, but little is known about their biological functions. We show that Ostreococcus tauri, the smallest known marine photosynthetic eukaryote, whose genome is completely characterized, is a host for large DNA viruses, and present an analysis of the life-cycle and 186,234 bp long linear genome of OtV5. OtV5 is a lytic phycodnavirus which unexpectedly does not degrade its host chromosomes before the host cell bursts. Analysis of its complete genome sequence confirmed that it lacks expected site-specific endonucleases, and revealed the presence of 16 genes whose predicted functions are novel to this group of viruses. OtV5 carries at least one predicted gene whose protein closely resembles its host counterpart and several other host-like sequences, suggesting that horizontal gene transfers between host and viral genomes may occur frequently on an evolutionary scale. Fifty seven percent of the 268 predicted proteins present no similarities with any known protein in Genbank, underlining the wealth of undiscovered biological diversity present in oceanic viruses, which are estimated to harbour 200Mt of carbon.     

Regulatory signals for endothelial podosome formation

Podosomes are punctate actin-rich adhesion structures which spontaneously form in cells of the myelomonocytic lineage. Their formation is dependent on Src and RhoGTPases. Recently, podosomes have also been described in vascular cells. These podosomes differ from the former by the fact that they are inducible. In endothelial cells, such a signal can be provided by either constitutively active Cdc42, the PKC activator PMA or TGFbeta, depending on the model. Consequently, other regulatory pathways have been reported to contribute to podosome formation. To get more insight into the mechanisms by which podosomes form in endothelial cells, we have explored the respective contribution of signal transducers such as Cdc42-related GTPases, Smads and PKCs in three endothelial cell models. Results presented demonstrate that, in addition to Cdc42, TC10 and TCL GTPases can also promote podosome formation in endothelial cells. We also show that PKCalpha can be either necessary or entirely dispensable, depending on the cell model. In contrast, PKCdelta is essential for podosome formation in endothelial cells but not smooth muscle cells. Finally, although podosomes vary very little in their molecular composition, the signalling pathways involved in their assembly appear very diverse.     

The VLCFA elongase gene family in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>: phylogenetic analysis, 3D modelling and expression profiling

As precursors of wax compounds, very long chain fatty acids participate in the limitation of non-stomatal water loss and the prevention of pathogen attacks. They also serve as energy storage in seeds and as membrane building blocks. Their biosynthesis is catalyzed by the acyl-CoA elongase, a membrane-bound enzymatic complex containing four distinct enzymes (KCS, KCR, HCD and ECR). Twenty-one 3-ketoacyl-CoA synthase (KCS) genes have been identified in Arabidopsis thaliana genome. In this paper we present an overview of the acyl-CoA elongase genes in Arabidopsis focusing on the entire KCS family. We show that the KCS family is made up of 8 distinct subclasses, according to their phylogeny, duplication history, genomic organization, protein topology and 3D modelling. The analysis of the subcellular localization in tobacco cells of the different subunits of the acyl-CoA elongase shows that all these proteins are localized in the endoplasmic reticulum demonstrating that VLCFA production occurs in this compartment. The expression patterns in Arabidopsis of the acyl-CoA elongase genes suggest several levels of regulations at the tissular or organ level but also under stress conditions suggesting a complex organization of this multigenic family.     

Multifaceted roles of human elafin and secretory leukocyte proteinase inhibitor (SLPI), two serine protease inhibitors of the chelonianin family

Elafin and SLPI are low-molecular weight proteins that were first identified as protease inhibitors in mucous fluids including lung secretions, where they help control excessive proteolysis due to neutrophil serine proteases (elastase, proteinase 3 and cathepsin G). Elafin and SLPI are structurally related in that both have a fold with a four-disulfide core or whey acidic protein (WAP) domain responsible for inhibiting proteases. Elafin is derived from a precursor, trappin-2 or pre-elafin, by proteolysis. Trappin-2, which is itself a protease inhibitor, has a unique N-terminal domain that enables it to become cross-linked to extracellular matrix proteins by transglutaminase(s). SLPI and elafin/trappin-2 are attractive candidates as therapeutic molecules for inhibiting neutrophil serine proteases in inflammatory lung diseases. Hence, they have become the WAP proteins most studied over the last decade. This review focuses on recent findings revealing that SLPI and elafin/trappin-2 have many biological functions as diverse as anti-bacterial, anti-fungal, anti-viral, anti-inflammatory and immuno-modulatory functions, in addition to their well-recognized role as protease inhibitors.     

Metal binding and antioxidant properties of chimeric tri- and tetra-domained metallothioneins

An unusual tri-domained (alpha-beta-beta) natural oyster metallothionein (MT) is known, and non-oxidative MT dimers occur in vivo in mollusk species and in mammals. To assess the respective role of the MT domains, two chimeric MTs were constructed: a tetra-domained oyster MT corresponding to the alpha-beta-alpha-beta structure, in order to mimic the natural non-oxidative dimeric form, and a tri-domained alpha-beta-alpha oyster MT. Metal binding and putative antioxidant properties of these two chimeric MTs were investigated using expression of the related genes in the bacteria Escherichia coli. In a wild-type strain these MTs could efficiently bind Cd. In a superoxide dismutase (sodA sodB) null mutant, the tri-domained MT was found to exacerbate Cd toxicity whereas the tetra-domained MT efficiently protected bacteria from Cd. The paradoxical toxicity displayed by the tri-domained MT upon Cd contamination was linked to the generation of superoxide radicals generated by a mechanism which most probably involves a copper-redox cycling reaction, since a Cd-contaminated sodA sodB strain expressing this MT produced 4 times more O2(-) than the control bacteria, and MT toxicity disappeared in the presence of bathocuproine disulfonic acid, a copper chelator. In contrast, the tetra-domained form did not. Interestingly, in bacteria producing superoxide dismutase but hypersensitive to oxidative stress due to either mutations in thioredoxin and glutathione reductase pathways (WM104 mutant) or to a lack of gamma-glutamylcysteine synthetase (gshA mutant), both chimeric MTs were protecting against Cd toxicity. However, an unexpected lack of antioxidant function was observed for both chimeric MTs, which were found to enhance the toxicity of hydrogen peroxide in WM104, or that of menadione in QC1726. Altogether, our results suggest that superoxide dismutase activity counteracts the potential prooxidative effect of the tri-domained MT mediated by Cu ions and that the tetra-domained form is a very efficient protector against metal toxicity in vivo.     

Neutrophil elastase, proteinase 3 and cathepsin G: physicochemical properties, activity and physiopathological functions

Polymorphonuclear neutrophils form a primary line of defense against bacterial infections using complementary oxidative and non-oxidative pathways to destroy phagocytized pathogens. The three serine proteases elastase, proteinase 3 and cathepsin G, are major components of the neutrophil primary granules that participate in the non-oxidative pathway of intracellular pathogen destruction. Neutrophil activation and degranulation results in the release of these proteases into the extracellular medium as proteolytically active enzymes, part of them remaining exposed at the cell surface. Extracellular neutrophil serine proteases also help kill bacteria and are involved in the degradation of extracellular matrix components during acute and chronic inflammation. But they are also important as specific regulators of the immune response, controlling cellular signaling through the processing of chemokines, modulating the cytokine network, and activating specific cell surface receptors. Neutrophil serine proteases are also involved in the pathogenicity of a variety of human diseases. This review focuses on the structural and functional properties of these proteases that may explain their specific biological roles, and facilitate their use as molecular targets for new therapeutic strategies.     

Characterization of oxidized low-density lipoprotein-induced hormesis-like effects in osteoblastic cells

Epidemiological studies indicate that patients suffering from atherosclerosis are predisposed to develop osteoporosis. Atherogenic determinants such as oxidized low-density lipoprotein (oxLDL) particles have been shown both to stimulate the proliferation and promote apoptosis of bone-forming osteoblasts. Given such opposite responses, we characterized the oxLDL-induced hormesis-like effects in osteoblasts. Biphasic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reductive activity responses were induced by oxLDL where low concentrations (10–50 µg/ml) increased and high concentrations (from 150 µg/ml) reduced the MTT activity. Cell proliferation stimulation by oxLDL partially accounted for the increased MTT activity. No alteration of mitochondria mass was noticed, whereas low concentrations of oxLDL induced mitochondria hyperpolarization and increased the cellular levels of reactive oxygen species (ROS). The oxLDL-induced MTT activity was not related to intracellular ROS levels. OxLDL increased NAD(P)H-associated cellular fluorescence and flavoenzyme inhibitor diphenyleneiodonium reduced basal and oxLDL-induced MTT activity, suggesting an enhancement of NAD(P)H-dependent cellular reduction potential. Low concentrations of oxLDL reduced cellular thiol content and increased metallothionein expression, suggesting the induction of compensatory mechanisms for the maintenance of cell redox state. These concentrations of oxLDL reduced osteoblast alkaline phosphatase activity and cell migration. Our results indicate that oxLDL particles cause hormesis-like response with the stimulation of both proliferation and cellular NAD(P)H-dependent reduction potential by low concentrations, whereas high concentrations lead to reduction of MTT activity associated with the cell death. Given the effects of low concentrations of oxLDL on osteoblast functions, oxLDL may contribute to the impairment of bone remodeling equilibrium. osteoblasts; atherosclerosis; oxysterol     

Parkin and CASK/LIN-2 associate via a PDZ-mediated interaction and are co-localized in lipid rafts and postsynaptic densities in brain.

Mutations in the gene encoding parkin cause an autosomal recessive juvenile-onset form of Parkinson's disease. Parkin functions as a RING-type E3 ubiquitin-ligase, coordinating the transfer of ubiquitin to substrate proteins and thereby targeting them for degradation by the proteasome. We now report that the extreme C terminus of parkin, which is selectively truncated by a Parkinson's disease-causing mutation, functions as a class II PDZ-binding motif that binds CASK, the mammalian homolog of Caenorhabditis elegans Lin-2, but not other PDZ proteins in brain extracts. Importantly, parkin co-localizes with CASK at synapses in cultured cortical neurons as well as in postsynaptic densities and lipid rafts in brain. Further, parkin associates not only with CASK but also with other postsynaptic proteins in the N-methyl d-aspartate (NMDA) receptor-signaling complex, in rat brain in vivo. Finally, despite exhibiting E2-dependent ubiquitin ligase activity, rat brain parkin does not ubiquitinate CASK, suggesting that CASK may function in targeting or scaffolding parkin within the postsynaptic complex rather than as a direct substrate for parkin-mediated ubiquitination. These data implicate for the first time a PDZ-mediated interaction between parkin and CASK in neurodegeneration and possibly in ubiquitination of proteins involved in synaptic transmission and plasticity.     

Mechanism of porcine pancreatic alpha-amylase. Inhibition of amylose and maltopentaose hydrolysis by alpha-, beta- and gamma-cyclodextrins.

The effects of alpha-, beta- and gamma-cyclodextrins on the amylose and maltopentaose hydrolysis catalysed by porcine pancreatic alpha-amylase (PPA) were investigated. The results of the statistical analysis performed on the kinetic data using the general initial velocity equation of a one-substrate reaction in the presence of one inhibitor indicate that the type of inhibition involved depends on the substrate used: the inhibition of amylose hydrolysis by alpha-, beta- and gamma-cyclodextrin is of the competitive type, while the inhibition of maltopentaose hydrolysis is of the mixed noncompetitive type. Consistently, the Lineweaver-Burk plots intersect on the vertical axis when amylose is used as the substrate, while in the case of maltopentaose, the intersection occurs at a point located in the second quadrant. The inhibition of the hydrolysis therefore involves only one abortive complex, PPA-cyclodextrin, when amylose is used as the substrate, while two abortive complexes, PPA-cyclodextrin and PPA-maltopentaose-cyclodextrin, are involved with maltopentaose. The mixed noncompetitive inhibition thus shows the existence of one accessory binding site. In any case, only one molecule of inhibitor binds to PPA. In line with these findings, the difference spectra of PPA produced by alpha-, beta- and gamma-cyclodextrin indicate that binding occurs at a tryptophan and a tyrosine residue. The corresponding dissociation constants and the inhibition constants obtained using the kinetic approach are in the same range (1.2-7 mM). The results obtained here on the inhibition of maltopentaose hydrolysis by cyclodextrin are similar to those previously obtained with acarbose as the inhibitor [Alkazaz, M., Desseaux, V., Marchis-Mouren, G., Prodanov, E. & Santimone, M. (1998) Eur. J. Biochem. 252, 100-107], but differ from those obtained with amylose as the substrate and acarbose as inhibitor [Alkazaz, M., Desseaux, V., Marchis-Mouren, G., Payan, F., Forest, E. & Santimone, M. (1996) Eur. J. Biochem. 241, 787-796]. It is concluded that the hydrolysis of both long and short chain substrates requires at least one secondary binding site, including a tryptophan residue.