Characterisation of inverse agonism of the orphan-G protein-coupled receptor GPR52 by cannabinoid ligands Cannabidiol and O-1918.

The identification of cannabinoid ligands Cannabidiol and O-1918 as inverse agonists of the orphan receptor GPR52 is reported. Detailed characterisation of GPR52 pharmacology and modelling of the proposed receptor interaction is described. The identification of a novel and further CNS pharmacology for the polypharmacological agent and marketed drug Cannabidiol is noteworthy.

Inhibitor-induced HER2-HER3 heterodimerisation promotes proliferation through a novel dimer interface.

While targeted therapy against HER2 is an effective first-line treatment in HER2 breast cancer, acquired resistance remains a clinical challenge. The pseudokinase HER3, heterodimerisation partner of HER2, is widely implicated in the resistance to HER2-mediated therapy. Here, we show that lapatinib, an ATP-competitive inhibitor of HER2, is able to induce proliferation cooperatively with the HER3 ligand neuregulin.

N-terminal nesprin-2 variants regulate β-catenin signalling.

The spatial compartmentalisation of biochemical signalling pathways is essential for cell function. Nesprins are a multi-isomeric family of proteins that have emerged as signalling scaffolds, herein, we investigate the localisation and function of novel nesprin-2 N-terminal variants. We show that these nesprin-2 variants display cell specific distribution and reside in both the cytoplasm and nucleus.

Identification and Validation of Putative Nesprin Variants.

Nesprins are a family of multi-isomeric scaffolding proteins that were originally identified at the nuclear envelope (NE), where they bind to lamin A/C, emerin, and SUN-domain containing proteins, to form the LInker of Nucleoskeleton-and-Cytoskeleton (LINC) complex that connects the NE to the actin cytoskeleton. However, nesprin genes also give rise to a variety of tissue-specific variants of different sizes with potential roles beyond the NE. These variants are generated through alternative initiation, termination, and splicing, which makes nesprin biology very complex to study due to the difficulty in generating specific antibodies and/or short interfering RNAs (siRNA) to particular isoforms.

Large-scale modelling of the divergent spectrin repeats in nesprins: giant modular proteins.

Nesprin-1 and nesprin-2 are nuclear envelope (NE) proteins characterized by a common structure of an SR (spectrin repeat) rod domain and a C-terminal transmembrane KASH [Klarsicht-ANC-Syne-homology] domain and display N-terminal actin-binding CH (calponin homology) domains. Mutations in these proteins have been described in Emery-Dreifuss muscular dystrophy and attributed to disruptions of interactions at the NE with nesprins binding partners, lamin A/C and emerin. Evolutionary analysis of the rod domains of the nesprins has shown that they are almost entirely composed of unbroken SR-like structures.

Multiple novel nesprin-1 and nesprin-2 variants act as versatile tissue-specific intracellular scaffolds.

Background: Nesprins (Nuclear envelope spectrin-repeat proteins) are a novel family of giant spectrin-repeat containing proteins. The nesprin-1 and nesprin-2 genes consist of 146 and 116 exons which encode proteins of ∼1mDa and ∼800 kDa is size respectively when all the exons are utilised in translation. However emerging data suggests that the nesprins have multiple alternative start and termination sites throughout their genes allowing the generation of smaller isoforms.

Protein-water interactions in MD simulations: POPS/POPSCOMP solvent accessibility analysis, solvation forces and hydration sites.

The effects of solvation on molecular recognition are investigated from different perspectives, ranging from methods to analyse explicit solvent dynamical behaviour at the protein surface to methods for the implicit treatment of solvent effects associated with the conformational behaviour of biomolecules. The here presented implicit solvation method is based on an analytical approximation of the Solvent Accessible Surface Area (SASA) of solute molecules, which is computationally efficient and easy to parametrise. The parametrised SASA solvation method is discussed in the light of protein design and ligand binding studies.

In silico phosphorylation of the autoinhibited form of p47(phox): insights into the mechanism of activation.

Activation of the multicomponent enzyme NADPH oxidase requires the interaction between the tandem SH3 domain of the cytosolic subunit p47(phox) and the cytoplasmic tail of membrane-bound p22(phox). In the resting state, p47(phox) exists in an autoinhibited conformation stabilized by intramolecular contacts between the SH3 domains and an adjacent polybasic region. Phosphorylation of three serine residues, Ser(303), Ser(304), and Ser(328) within this polybasic region has been shown to be sufficient for the disruption of the intramolecular interactions thereby inducing an active state of p47(phox).

Rationalisation of the differences between APOBEC3G structures from crystallography and NMR studies by molecular dynamics simulations.

The human APOBEC3G (A3G) protein is a cellular polynucleotide cytidine deaminase that acts as a host restriction factor of retroviruses, including HIV-1 and various transposable elements. Recently, three NMR and two crystal structures of the catalytic deaminase domain of A3G have been reported, but these are in disagreement over the conformation of a terminal beta-strand, beta2, as well as the identification of a putative DNA binding site. We here report molecular dynamics simulations with all of the solved A3G catalytic domain structures, taking into account solubility enhancing mutations that were introduced during derivation of three out of the five structures.

Mutation of the RAD51C gene in a Fanconi anemia-like disorder.

Fanconi anemia (FA) is a rare chromosomal-instability disorder associated with a variety of developmental abnormalities, bone marrow failure and predisposition to leukemia and other cancers. We have identified a homozygous missense mutation in the RAD51C gene in a consanguineous family with multiple severe congenital abnormalities characteristic of FA. RAD51C is a member of the RAD51-like gene family involved in homologous recombination-mediated DNA repair.

RNA-dependent oligomerization of APOBEC3G is required for restriction of HIV-1.

The human cytidine deaminase APOBEC3G (A3G) is a potent inhibitor of retroviruses and transposable elements and is able to deaminate cytidines to uridines in single-stranded DNA replication intermediates. A3G contains two canonical cytidine deaminase domains (CDAs), of which only the C-terminal one is known to mediate cytidine deamination. By exploiting the crystal structure of the related tetrameric APOBEC2 (A2) protein, we identified residues within A3G that have the potential to mediate oligomerization of the protein.

The Pleurotus ostreatus laccase multi-gene family: isolation and heterologous expression of new family members.

This work was aimed at identifying and at characterizing new Pleurotus ostreatus laccases, in order to individuate the most suitable biocatalysts for specific applications. The existence of a laccase gene clustering was demonstrated in this basidiomycete fungus, and three new laccase genes were cloned, taking advantage of their closely related spatial organization on the fungus genome. cDNAs coding for two of the new laccases were isolated and expressed in the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis, in order to optimize their production and to characterize the recombinant proteins.

Molecular interactions of ASPP1 and ASPP2 with the p53 protein family and the apoptotic promoters PUMA and Bax.

The apoptosis stimulating p53 proteins, ASPP1 and ASPP2, are the first two common activators of the p53 protein family that selectively enable the latter to regulate specific apoptotic target genes, which facilitates yes yet unknown mechanisms for discrimination between cell cycle arrest and apoptosis. To better understand the interplay between ASPP- and p53-family of proteins we investigated the molecular interactions between them using biochemical methods and structure-based homology modelling. The data demonstrate that: (i) the binding of ASPP1 and ASPP2 to p53, p63 and p73 is direct; (ii) the C-termini of ASPP1 and ASPP2 interact with the DNA-binding domains of p53 protein family with dissociation constants, K(d), in the lower micro-molar range; (iii) the stoichiometry of binding is 1:1; (iv) the DNA-binding domains of p53 family members are sufficient for these protein-protein interactions; (v) EMSA titrations revealed that while tri-complex formation between ASPPs, p53 family of proteins and PUMA/Bax is mutually exclusive, ASPP2 (but not ASPP1) formed a complex with PUMA (but not Bax) and displaced p53 and p73.

The cold-active Lip1 lipase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 is a member of a new bacterial lipolytic enzyme family.

The genome of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 was searched for the presence of genes encoding ester-hydrolysing enzymes. Amongst the others, the gene PSHAa0051 coding for a putative secreted esterase/lipase was selected. The psychrophilic gene was cloned, functionally over-expressed in P.

Development of new laccases by directed evolution: functional and computational analyses.

Laccases are blue multicopper oxidases that couple the four-electron reduction of oxygen with the oxidation of a broad range of aromatic substrates. These fungal enzymes can be used for many applications such as bleaching, organic synthesis, bioremediation, and in laundry detergents. Laccases from Pleurotus ostreatus have been successfully heterologously expressed in yeasts.

Structural characterization of heterodimeric laccases from Pleurotus ostreatus.

The subfamily of POXA3 laccase isoenzymes produced by the fungus Pleurotus ostreatus has been characterized as an example of the complexity and heterogeneity of fungal isoenzyme patterns. Two isoenzymes, POXA3a and POXA3b, were previously purified, exhibiting an unusual heterodimeric structure composed of a large (67 kDa) and a small (18 or 16 kDa) subunit. A unique gene encodes the large subunit of both POXA3a and POXA3b, but alternative splicing produces two variants--differing for an insertion of four amino acids--for each isoenzyme.

Interaction of malaria parasite-inhibitory antibodies with the merozoite surface protein MSP1(19) by computational docking.

Merozoite surface protein 1 (MSP1) of the malaria parasite Plasmodium falciparum is an important vaccine candidate antigen. Antibodies specific for the C-terminal maturation product, MSP1(19), have been shown to inhibit erythrocyte invasion and parasite growth. Specific monoclonal antibodies react with conformational epitopes contained within the two EGF-like domains that constitute the antigen MSP1(19).

The thiol-disulfide oxidoreductase system in the cold-adapted bacterium Pseudoalteromonas haloplanktis TAC 125: discovery of a novel disulfide oxidoreductase enzyme.

In prokaryotes, protein disulfide bond oxidation, reduction and isomerization are catalyzed by members of the thioredoxin superfamily, characterized by the conserved C-X-X-C motif in their active site. Thioredoxins and glutaredoxins contribute to the reducing power in the cytoplasm, while the Dsb system catalyzes disulfide bonds formation in the periplasmic space. This paper addresses the question of disulfide bonds formation in a cold-adapted micro-organism, Pseudoalteromonas haloplanktis TAC 125 (PhTAC125) by characterizing the DsbA system.