N-terminal nesprin-2 variants regulate ß-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. Immunofluorescence microscopy revealed that nesprin-2 N-terminal variants colocalised with ß-catenin at cell-cell junctions in U2OS cells. Calcium switch assays demonstrated that nesprin-2 and ß-catenin are lost from cell-cell junctions in low calcium conditions whereas emerin localisation at the NE remained unaltered, furthermore, an N-terminal fragment of nesprin-2 was sufficient for cell-cell junction localisation and interacted with ß-catenin. Disruption of these N-terminal nesprin-2 variants, using siRNA depletion resulted in loss of ß-catenin from cell-cell junctions, nuclear accumulation of active ß-catenin and augmented ß-catenin transcriptional activity. Importantly, we show that U2OS cells lack nesprin-2 giant, suggesting that the N-terminal nesprin-2 variants regulate ß-catenin signalling independently of the NE. Together, these data identify N-terminal nesprin-2 variants as novel regulators of ß-catenin signalling that tether ß-catenin to cell-cell contacts to inhibit ß-catenin transcriptional activity.

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. Using yeast two-hybrid assays, co-immunoprecipitation, and chemical crosslinking, we show that tyrosine-124 and tryptophan-127 within the enzymatically inactive N-terminal CDA domain mediate A3G oligomerization, and this coincides with packaging into HIV-1 virions. In addition to the importance of specific residues in A3G, oligomerization is also shown to be RNA-dependent. Homology modelling of A3G onto the A2 template structure indicates an accumulation of positive charge in a pocket formed by a putative dimer interface. Substitution of arginine residues at positions 24, 30, and 136 within this pocket resulted in reduced virus inhibition, virion packaging, and oligomerization. Consistent with RNA serving a central role in all these activities, the oligomerization-deficient A3G proteins associated less efficiently with several cellular RNA molecules. Accordingly, we propose that occupation of the positively charged pocket by RNA promotes A3G oligomerization, packaging into virions and antiviral function.

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.

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). This active conformation is accessible to the cytoplasmic tail of p22(phox) and initiates the formation of the membrane-bound functional enzyme complex. Molecular dynamics simulations reveal insights in the mechanism of activation of the autoinhibited form of p47(phox) by in silico phosphorylation, of the three serine residues, Ser(303), Ser(304), and Ser(328). The simulations highlight the major collective coordinates generating the opening and the closing of the two SH3 domains and the residues that cause the unmasking of the p22(phox) binding site.

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 structure-based homology modelling revealed subtle differences between ASPP2 and ASPP1 and together with the experimental data provide novel mechanistic insights.

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. Analysis of the P. ostreatus laccase gene family allowed the identification of a "laccase subfamily" consisting of three genes. A peculiar intron-exon structure was revealed for the gene of one of the new laccases, along with a high instability of the recombinant enzyme due to lability of its copper ligand. This study allowed enlarging the assortment of P. ostreatus laccases and increasing knowledge to improve laccase production.

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. The availability of established recombinant expression systems has allowed the construction of mutated, "better performing" enzymes through molecular evolution techniques. In the present work, random mutagenesis experiments on poxc and poxa1b cDNAs, using error prone PCR (EP-PCR) have been performed. By screening a library of 1100 clones the mutant 1M9B was selected, it shows a single mutation (L112F) leading to an enzyme more active but less stable with respect to the wild-type enzyme (POXA1b) in all the analyzed conditions. This mutant has been used as a template for a second round of EP-PCR. From this second generation library of 1200 clones, three mutants have been selected. Properties of the four mutants, 1M9B screened from the first library, and 1L2B, 1M10B, and 3M7C from the second library, were analyzed. The better performing mutant 3M7C presents, besides L112F, only one substitution (P494T) responsible both for the significantly increased stability and for the exhibited higher activity of this mutant. Molecular dynamics simulations have been performed on three-dimensional models of POXA1b, 1M9B, and 3M7C, and hypotheses on the structure-function relationships of these proteins have been formulated.

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. This counterintuitive synergy between inhibitor and growth factor depends on their ability to promote atypical HER2-HER3 heterodimerisation. By stabilising a particular HER2 conformer, lapatinib drives HER2-HER3 kinase domain heterocomplex formation. This dimer exists in a head-to-head orientation distinct from the canonical asymmetric active dimer. The associated clustering observed for these dimers predisposes to neuregulin responses, affording a proliferative outcome. Our findings provide mechanistic insights into the liabilities involved in targeting kinases with ATP-competitive inhibitors and highlight the complex role of protein conformation in acquired resistance.

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). To gain greater insight into the inhibitory process, the authors selected two strongly inhibitory antibodies (designated 12.8 and 12.10) and modeled their structures by homology. Computational docking was used to generate antigen-antibody complexes and a selection filter based on NMR data was applied to obtain plausible models. Molecular Dynamics simulations of the selected complexes were performed to evaluate the role of specific side chains in the binding. Favorable complexes were obtained that complement the NMR data in defining specific binding sites. These models can provide valuable guidelines for future experimental work that is devoted to the understanding of the action mechanism of invasion-inhibitory antibodies.

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. In order to distinguish genuine nesprin variants and eliminate confusion with degradation products of larger nesprin isoforms, in this chapter we discuss methods including 5' and 3' Rapid Amplification of cDNA Ends (RACE) and RT-PCR in combination with EST database searching, for identifying and validating putative nesprin isoforms. This information is essential to allow a better understanding of nesprin functions in different cell types.

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. We present a bioinformatical approach to accurate definition of the boundaries of each SR by comparison with canonical SR structures, allowing for a large-scale homology modelling of the 74 nesprin-1 and 56 nesprin-2 SRs. The exposed and evolutionary conserved residues identify important pbs for protein-protein interactions that can guide tailored binding experiments. Most importantly, the bioinformatics analyses and the 3D models have been central to the design of selected constructs for protein expression. 1D NMR and CD spectra have been performed of the expressed SRs, showing a folded, stable, high content α-helical structure, typical of SRs. Molecular Dynamics simulations have been performed to study the structural and elastic properties of consecutive SRs, revealing insights in the mechanical properties adopted by these modules in the cell.

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. The POPS program for the SASA computation on single molecules and complex interfaces is described in detail. Explicit solvent behaviour is described here in the form of solvent density maps at the protein surface. We highlight the usefulness of that approach in defining the organisation of specific water molecules at functional sites and in determining hydrophobicity scores for the identification of potential interaction patches.

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. RESULTS: In this study we set out to identify novel alternatively transcribed nesprin variants by screening the EST database and by using RACE analysis to identify cDNA ends. These two methods provided potential hits for alternative start and termination sites that were validated by PCR and DNA sequencing. We show that these alternative sites are not only expressed in a tissue specific manner but by combining different sites together it is possible to create a wide array of nesprin variants. By cloning and expressing small novel nesprin variants into human fibroblasts and U2OS cells we show localization to actin stress-fibres, focal adhesions, microtubules, the nucleolus, nuclear matrix and the nuclear envelope (NE). Furthermore we show that the sub-cellular localization of individual nesprin variants can vary depending on the cell type, suggesting any single nesprin variant may have different functions in different cell types. CONCLUSIONS: These studies suggest nesprins act as highly versatile tissue specific intracellular protein scaffolds and identify potential novel functions for nesprins beyond cytoplasmic-nuclear coupling. These alternate functions may also account for the diverse range of disease phenotypes observed when these genes are mutated.

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. In the course of these simulations, we observed a general trend towards increased definition of the beta2 strand for those structures that have a distorted starting conformation of beta2. Solvent density maps around the protein as calculated from MD simulations indicated that this distortion is dependent on preferential hydration of residues within the beta2 strand. We also demonstrate that the identification of a pre-defined DNA binding site is prevented by the inherent flexibility of loops that determine access to the deaminase catalytic core. We discuss the implications of our analyses for the as yet unresolved structure of the full-length A3G protein and its biological functions with regard to hypermutation of DNA.

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. The mutation results in loss of RAD51 focus formation in response to DNA damage and in increased cellular sensitivity to the DNA interstrand cross-linking agent mitomycin C and the topoisomerase-1 inhibitor camptothecin. Thus, biallelic germline mutations in a RAD51 paralog are associated with an FA-like syndrome.

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. haloplanktis TAC125, and the recombinant product (after named PhTAC125 Lip1) was purified. PhTAC125 Lip1 was found to be associated to the outer membrane and exhibited higher enzymatic activity towards synthetic substrates with long acyl chains. A structural model was constructed using the structure of carboxylesterase Est30 from Geobacillus stearothermophilus as template. The model covered the central part of the protein with the exceptions of PhTAC125 Lip1 N- and C-terminal regions, where the psychrophilic protein displays extra-domains. The constructed model showed a typical alpha/beta-hydrolase fold, and confirmed the presence of a canonical catalytic triad consisting of Ser, Asp and His. The sequence analysis showed that PhTAC125 Lip1 is distantly related to other lipolytic enzymes, but closely related to other putative psychrophilic esterases/lipases. The aligned proteins share common features, such as: (1) a conserved new active-site pentapeptide motif (LGG(F/L/Y)STG); (2) the likely extra-cytoplasmic localization, (3) the absence of a typical calcium-binding pocket, and (4) the absence of a canonical lid. These observations strongly suggest that aligned proteins constitute a novel lipase family, typical of psychrophilic marine gamma-proteobacteria, and PhTAC125 Lip1 could be considered the first characterised member of this family.

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. Two genes encoding POXA3a and POXA3b small subunits have been identified, and the corresponding amino acid sequences show only two amino acid substitutions. The 18- and 16-kDa subunits of both POXA3a and POXA3b differ for N-glycosylation at Asn150 of the 16-kDa subunit. The POXA3 large subunit 3D model allows us to highlight peculiarities of this molecule with respect to the laccases whose 3D structures are known.

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. We found distinctive features respect mesophilic counterparts that highlighted for the first time the occurrence of two adjacent chromosomal DsbA genes organised in a functional operon. The sophisticated transcriptional regulation mechanism that controls the expression of these two genes was also defined. The two DsbA proteins, named PhDsbA and PhDsbA2, respectively, were expressed in Escherichia coli and characterized. Results reported in this paper provide some insights into disulfide bonds formation in a micro organism isolated in the Antarctic sea water.