Advances in Computational Techniques to Study GPCR-Ligand Recognition

G-protein-coupled receptors (GPCRs) are among the most intensely investigated drug targets. The recent revolutions in protein engineering and molecular modeling algorithms have overturned the research paradigm in the GPCR field. While the numerous ligand-bound X-ray structures determined have provided invaluable insights into GPCR structure and function, the development of algorithms exploiting graphics processing units (GPUs) has made the simulation of GPCRs in explicit lipid-water environments feasible within reasonable computation times. In this review we present a survey of the recent advances in structure-based drug design approaches with a particular emphasis on the elucidation of the ligand recognition process in class A GPCRs by means of membrane molecular dynamics (MD) simulations

Lytic Polysaccharide Monooxygenases as Chitin-Specific Virulence Factors in Crayfish Plague

he oomycete pathogen Aphanomyces astaci, also known as "crayfish plague", is an obligate fungal-like parasite of freshwater crustaceans and is considered responsible for the ongoing decline of native European crayfish populations. A. astaci is thought to secrete a wide array of effectors and enzymes that facilitate infection, however their molecular mechanisms have been poorly characterized. Here, we report the identification of AA15 lytic polysaccharide monooxygenases (LPMOs) as a new group of secreted virulence factors in A. astaci. We show that this enzyme family has greatly expanded in A. astaci compared to all other oomycetes, and that it may facilitate infection through oxidative degradation of crystalline chitin, the most abundant polysaccharide found in the crustacean exoskeleton. These findings reveal new roles for LPMOs in animal-pathogen interactions, and could help inform future strategies for the protection of farmed and endangered species

PsAA9A, a C1-specific AA9 lytic polysaccharide monooxigenase from the white-rot basidiomycete Pycnoporus sanguineus

Woody biomass represents an important source of carbon on earth, and its global recycling is highly dependent on Agaricomycetes fungi. White-rot Basidiomycetes are a very important group in this regard, as they possess a large and diverse enzymatic repertoire for biomass decomposition. Among these enzymes, the recently discovered lytic polysaccharide monooxygenases (LPMOs) have revolutionized biomass processing with their novel oxidative mechanism of action. The strikingly high representation of LPMOs in fungal genomes raises the question of their functional versatility. In this work, we studied an AA9 LPMO from the white-rot basidiomycete Pycnoporus sanguineus, PsAA9A. Successfully produced as a recombinant secreted protein in Pichia pastoris, PsAA9A was found to be a C1-specific LPMO active on cellulosic substrates, generating native and oxidized cello-oligosaccharides in the presence of an external electron donor. PsAA9A boosted cellulolytic activity of glysoside hydrolases from families GH1, GH5, and GH6.This study serves as a starting point towards understanding the functional versatility and biotechnological potential of this enzymatic family, highly represented in wood decay fungi, in Pycnoporus genus.Instituto de BiotecnologíaFil: Garrido, Mercedes Maria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Garrido, Mercedes Maria. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Landoni, Malena. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigación en Hidratos de Carbono; ArgentinaFil: Sabbadin, Federico. University of York. Department of Biology. Centre for Novel Agricultural Products (CNAP); Reino UnidoFil: Valacco, Maria Pia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Centro de Estudios Químicos y Biológicos por Espectrometría de Masa; ArgentinaFil: Valacco, Maria Pia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Couto, Alicia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigación en Hidratos de Carbono; ArgentinaFil: Bruce, Neil Charles. University of York. Department of Biology. Centre for Novel Agricultural Products (CNAP); Reino UnidoFil: Wirth, Sonia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada; ArgentinaFil: Wirth, Sonia Alejandra. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; ArgentinaFil: Campos, Eleonora. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Campos, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Characterisation of the enzyme transport path between shipworms and their bacterial symbionts.

BACKGROUND: Shipworms are marine xylophagus bivalve molluscs, which can live on a diet solely of wood due to their ability to produce plant cell wall-degrading enzymes. Bacterial carbohydrate-active enzymes (CAZymes), synthesised by endosymbionts living in specialised shipworm cells called bacteriocytes and located in the animal's gills, play an important role in wood digestion in shipworms. However, the main site of lignocellulose digestion within these wood-boring molluscs, which contains both endogenous lignocellulolytic enzymes and prokaryotic enzymes, is the caecum, and the mechanism by which bacterial enzymes reach the distant caecum lumen has remained so far mysterious. Here, we provide a characterisation of the path through which bacterial CAZymes produced in the gills of the shipworm Lyrodus pedicellatus reach the distant caecum to contribute to the digestion of wood. RESULTS: Through a combination of transcriptomics, proteomics, X-ray microtomography, electron microscopy studies and in vitro biochemical characterisation, we show that wood-digesting enzymes produced by symbiotic bacteria are localised not only in the gills, but also in the lumen of the food groove, a stream of mucus secreted by gill cells that carries food particles trapped by filter feeding to the mouth. Bacterial CAZymes are also present in the crystalline style and in the caecum of their shipworm host, suggesting a unique pathway by which enzymes involved in a symbiotic interaction are transported to their site of action. Finally, we characterise in vitro four new bacterial glycosyl hydrolases and a lytic polysaccharide monooxygenase identified in our transcriptomic and proteomic analyses as some of the major bacterial enzymes involved in this unusual biological system. CONCLUSION: Based on our data, we propose that bacteria and their enzymes are transported from the gills along the food groove to the shipworm's mouth and digestive tract, where they aid in wood digestion

Uncovering the molecular mechanisms of lignocellulose digestion in shipworms

Abstract Lignocellulose forms the structural framework of woody plant biomass and represents the most abundant carbon source in the biosphere. Turnover of woody biomass is a critical component of the global carbon cycle, and the enzymes involved are of increasing industrial importance as industry moves away from fossil fuels to renewable carbon resources. Shipworms are marine bivalve molluscs that digest wood and play a key role in global carbon cycling by processing plant biomass in the oceans. Previous studies suggest that wood digestion in shipworms is dominated by enzymes produced by endosymbiotic bacteria found in the animal’s gills, while little is known about the identity and function of endogenous enzymes produced by shipworms. Using a combination of meta-transcriptomic, proteomic, imaging and biochemical analyses, we reveal a complex digestive system dominated by uncharacterized enzymes that are secreted by a specialized digestive gland and that accumulate in the cecum, where wood digestion occurs. Using a combination of transcriptomics, proteomics, and microscopy, we show that the digestive proteome of the shipworm Lyrodus pedicellatus is mostly composed of enzymes produced by the animal itself, with a small but significant contribution from symbiotic bacteria. The digestive proteome is dominated by a novel 300 kDa multi-domain glycoside hydrolase that functions in the hydrolysis of β-1,4-glucans, the most abundant polymers in wood. These studies allow an unprecedented level of insight into an unusual and ecologically important process for wood recycling in the marine environment, and open up new biotechnological opportunities in the mobilization of sugars from lignocellulosic biomass

An ancient family of lytic polysaccharide monooxygenases with roles in arthropod development and biomass digestion.

Thermobia domestica belongs to an ancient group of insects and has a remarkable ability to digest crystalline cellulose without microbial assistance. By investigating the digestive proteome of Thermobia, we have identified over 20 members of an uncharacterized family of lytic polysaccharide monooxygenases (LPMOs). We show that this LPMO family spans across several clades of the Tree of Life, is of ancient origin, and was recruited by early arthropods with possible roles in remodeling endogenous chitin scaffolds during development and metamorphosis. Based on our in-depth characterization of Thermobia's LPMOs, we propose that diversification of these enzymes toward cellulose digestion might have endowed ancestral insects with an effective biochemical apparatus for biomass degradation, allowing the early colonization of land during the Paleozoic Era. The vital role of LPMOs in modern agricultural pests and disease vectors offers new opportunities to help tackle global challenges in food security and the control of infectious diseases

Hemocyanin facilitates lignocellulose digestion by wood-boring marine crustaceans

Woody (lignocellulosic) plant biomass is an abundant renewable feedstock, rich in polysaccharides that are bound into an insoluble fiber composite with lignin. Marine crustacean woodborers of the genus Limnoria are among the few animals that can survive on a diet of this recalcitrant material without relying on gut resident microbiota. Analysis of fecal pellets revealed that Limnoria targets hexose-containing polysaccharides (mainly cellulose, and also glucomannans), corresponding with the abundance of cellulases in their digestive system, but xylans and lignin are largely unconsumed. We show that the limnoriid respiratory protein, hemocyanin, is abundant in the hindgut where wood is digested, that incubation of wood with hemocyanin markedly enhances its digestibility by cellulases, and that it modifies lignin. We propose that this activity of hemocyanins is instrumental to the ability of Limnoria to feed on wood in the absence of gut symbionts. These findings may hold potential for innovations in lignocellulose biorefining

Engineering cytochromes P450 for biocatalysis and bioremediation

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Allorecognition in Botryllus schlosseri: ultrastructural study of fusion between genetically compatible colonies.

When colonies of the ascidian Botryllus schlosseri contact each other, they can either fuse and anastomise their circulatory systems if genetically compatible, or reject and produce a series of cytotoxic spots along the contact border in the case of incompatibility. Although many studies have been devoted to the analysis of rejection, few data on fusion are available. In order to fill this gap, we started a preliminary study on the morphology of the various steps of the fusion reaction at both light and electron microscope. We were able to distinguish at least five different stages in the fusion process. In stage 1 the tunics of the facing colonies contact each other and epithelial cells of the ampullar tips appear cylindrical in shape with a cytoplasm rich in RER with enlarged cisternae containing homogeneous, finely dispersed material. In stage 2, the tunics are strictly juxtaposed and the cuticular papillae are tightly intermingled, but the two tunics are still distinguishable. In this stage, cells of the ampullar tips contain numerous membrane bound granules, with homogeneous electron-dense material, in the supranuclear (\u201cpad\u201d) region. Stage 3 is marked by the dissolution of the two cuticles and the local fusion of the tunics in front of the facing ampullae. Less granules are now present in the ampullar pad and some haemocytes leak out from the circulation through the ampullar tips. In stage 4, the pads of the two facing epithelial adhere and new junctional complexes are formed. Basal lamina still delimitate the ampullar lumen and appear highly folded. In stage 5 the juxtaposed epithelia open, thus permitting the communication between the vessels of the two colonies. Cell pads are progressively resorbed and cells of the ampullar tips, now lining a new vessel, return to a cubic shape. Future studies will investigate the occurrence of apoptotic events in the process of ampullar fusion in B. schlosseri

Advances in Computational Techniques to Study GPCR-Ligand Recognition

G-protein-coupled receptors (GPCRs) are among the most intensely investigated drug targets. The recent revolutions in protein engineering and molecular modeling algorithms have overturned the research paradigm in the GPCR field. While the numerous ligand-bound X-ray structures determined have provided invaluable insights into GPCR structure and function, the development of algorithms exploiting graphics processing units (GPUs) has made the simulation of GPCRs in explicit lipid-water environments feasible within reasonable computation times. In this review we present a survey of the recent advances in structure-based drug design approaches with a particular emphasis on the elucidation of the ligand recognition process in class A GPCRs by means of membrane molecular dynamics (MD) simulations