Mycobacterium tuberculosis Glucosyl-3-Phosphoglycerate Synthase: Structure of a Key Enzyme in Methylglucose Lipopolysaccharide Biosynthesis

Tuberculosis constitutes today a serious threat to human health worldwide, aggravated by the increasing number of identified multi-resistant strains of Mycobacterium tuberculosis, its causative agent, as well as by the lack of development of novel mycobactericidal compounds for the last few decades. The increased resilience of this pathogen is due, to a great extent, to its complex, polysaccharide-rich, and unusually impermeable cell wall. The synthesis of this essential structure is still poorly understood despite the fact that enzymes involved in glycosidic bond synthesis represent more than 1% of all M. tuberculosis ORFs identified to date. One of them is GpgS, a retaining glycosyltransferase (GT) with low sequence homology to any other GTs of known structure, which has been identified in two species of mycobacteria and shown to be essential for the survival of M. tuberculosis. To further understand the biochemical properties of M. tuberculosis GpgS, we determined the three-dimensional structure of the apo enzyme, as well as of its ternary complex with UDP and 3-phosphoglycerate, by X-ray crystallography, to a resolution of 2.5 and 2.7 Å, respectively. GpgS, the first enzyme from the newly established GT-81 family to be structurally characterized, displays a dimeric architecture with an overall fold similar to that of other GT-A-type glycosyltransferases. These three-dimensional structures provide a molecular explanation for the enzyme''s preference for UDP-containing donor substrates, as well as for its glucose versus mannose discrimination, and uncover the structural determinants for acceptor substrate selectivity. Glycosyltransferases constitute a growing family of enzymes for which structural and mechanistic data urges. The three-dimensional structures of M. tuberculosis GpgS now determined provide such data for a novel enzyme family, clearly establishing the molecular determinants for substrate recognition and catalysis, while providing an experimental scaffold for the structure-based rational design of specific inhibitors, which lay the foundation for the development of novel anti-tuberculosis therapies.     

Microglia at the Centre of Brain Research: Accomplishments and Challenges for the Future

Neurochemical Research - Microglia are the immune guardians of the central nervous system (CNS), with critical functions in development, maintenance of homeostatic tissue balance, injury and...     

ORIGINAL ARTICLE: Mass extinctions in the Azores during the last glaciation: fact or myth?

Aim The influence of the last glaciation on the shallow‐water marine malacofauna of the Azores Islands is reviewed. We test, for this fauna, the ‘Pleistocene temperature theory’ of J.C. Briggs, which hypothesizes that a (supposed) lack of endemics in the older (Azorean endemic) fauna resulted from extinctions caused by a severe drop in sea surface temperatures during the Pleistocene. Location Santa Maria Island, Azores, Portugal. Methods We compare the fossil mollusc fauna of Prainha, Praia do Calhau and Lagoinhas Pleistocene outcrops with the recent mollusc fauna of the Azores Islands. We dated the fossil fauna, using shells of Patella aspera Röding, 1798, by standard U/Th methodology at the GEOTOP laboratory (Université du Québec à Montreal, Canada). Results Dating of the shells of P. aspera indicates that the deposition of the lower unit of the Prainha outcrop corresponded to Marine Oxygen Isotope Substage 5e (MISS 5e). Not a single endemic Azorean species of mollusc that is present in the Pleistocene fossil record has since become extinct, and we found no signs of ‘mass extinctions’ in the littoral marine molluscs of the Azores. The only species that were extirpated from these islands were thermophilic molluscs and littoral bivalves living in fine sand. Main conclusions Our results do not support Briggs’‘Pleistocene temperature theory’. Nor did we find evidence supporting the hypothesis that most of the marine organisms now present in the Azores recolonized the islands after the last glacial maximum.     

Azurin interaction with the lipid raft components ganglioside GM-1 and caveolin-1 increases membrane fluidity and sensitivity to anti-cancer drugs

Membrane lipid rafts are highly ordered microdomains and essential components of plasma membranes. In this work, we demonstrate that azurin uptake by cancer cells is, in part, mediated by caveolin-1 and GM-1, lipid rafts’ markers. This recognition is mediated by a surface exposed hydrophobic core displayed by azurin since the substitution of a phenylalanine residue in position 114 facing the hydrophobic cavity by alanine impacts such interactions, debilitating the uptake of azurin by cancer cells. Treating of cancer cells with azurin leads to a sequence of events: alters the lipid raft exposure at plasma membranes, causes a decrease in the plasma membrane order as examined by Laurdan two-photon imaging and leads to a decrease in the levels of caveolin-1. Caveolae, a subset of lipid rafts characterized by the presence of caveolin-1, are gaining increasing recognition as mediators in tumor progression and resistance to standard therapies. We show that azurin inhibits growth of cancer cells expressing caveolin-1, and this inhibition is only partially observed with mutant azurin. Finally, the simultaneous administration of azurin with anticancer therapeutic drugs (paclitaxel and doxorubicin) results in an enhancement in their activity, contrary to the mutated protein.     

Omiganan interaction with bacterial membranes and cell wall models. Assigning a biological role to saturation

Omiganan pentahydrochloride (ILRWPWWPWRRK-NH₂·5Cl) is an antimicrobial peptide currently in phase III clinical trials. This study aims to unravel the mechanism of action of this drug at the membrane level and address the eventual protective role of peptidoglycan in cell walls. The interaction of omiganan pentahydrochloride with bacterial and mammalian membrane models - large unilamellar vesicles of different POPC:POPG proportions - was characterized by UV-Vis fluorescence spectroscopy. The molar ratio partition constants obtained for the two anionic bacterial membrane models were very high ((18.9 ± 1.3) × 10³ and (43.5 ± 8.7) × 10³) and about one order of magnitude greater than for the neutral mammalian models ((3.7 ± 0.4) × 10³ for 100% POPC bilayers). At low lipid:peptide ratios there were significant deviations from the usual hyperbolic-like partition behavior of peptide vesicle titration curves, especially for the most anionic systems. Membrane saturation can account for such observations and mathematical models were derived to further characterize the peptide-lipid interaction under those conditions; a possible relation between saturation and MIC was deduced; this was supported by differential quenching studies of peptide internalization. Interaction with the bacterial cell wall was assessed using Staphylococcus aureus peptidoglycan extracts as a model. A strong partition towards the peptidoglycan mesh was observed, but not as large as for the membrane models.     

The Polycomb group protein MEDEA controls cell proliferation and embryonic patterning in Arabidopsis

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