Maria Cristina De Rosa

Haemoglobin polymorphisms affect the oxygen- binding properties in Atlantic cod populations

A major challenge in evolutionary biology is to identify the genes underlying adaptation. The oxygen-transporting haemoglobins directly link external conditions with metabolic needs and therefore represent a unique system for studying environmental effects on molecular evolution. We have discovered two haemoglobin polymorphisms in Atlantic cod populations inhabiting varying temperature and oxygen regimes in the North Atlantic. Three-dimensional modelling of the tetrameric haemoglobin structure demonstrated that the two amino acid replacements Met55β1Val and Lys62β1Ala are located at crucial positions of the α1β1 subunit interface and haem pocket, respectively. The replacements are proposed to affect the oxygen-binding properties by modifying the haemoglobin quaternary structure and electrostatic feature. Intriguingly, the same molecular mechanism for facilitating oxygen binding is found in avian species adapted to high altitudes, illustrating convergent evolution in water- and air-breathing vertebrates to reduction in environmental oxygen availability. Cod populations inhabiting the cold Arctic waters and the low-oxygen Baltic Sea seem well adapted to these conditions by possessing the high oxygen affinity Val55–Ala62 haplotype, while the temperature-insensitive Met55–Lys62 haplotype predominates in the southern populations. The distinct distributions of the functionally different haemoglobin variants indicate that the present biogeography of this ecologically and economically important species might be seriously affected by global warming.

Modeling the ternary complex TCR-Vβ/collagenii(261-273)/HLA-DR4 associated with rheumatoid arthritis

Background It is known that genetic predisposition to rheumatoid arthritis (RA) is associated with the MHC class II allele HLA-DR4 and that residues 261–273 of type II collagen (huCollp261) represent an immunodominant T cell epitope restricted by the DR4 molecule. Despite recent advances in characterization of MHC and T cell receptor (TCR) contacts to this epitope, the atomic details of TCR/huCollp261/HLA-DR4 ternary complex are not known. Methodology/Principal Findings Here we have used computational modeling to get insight into this interaction. A three-dimensional model of the TCR Vβ domain from a DR4+ patient affected by RA has been derived by homology modeling techniques. Subsequently, the structure of the TCR Vβ domain in complex with huCollp261/HLA-DR4 was obtained from a docking approach in conjunction with a filtering procedure based on biochemical information. The best complex from the docking experiments was then refined by 20 ns of molecular dynamics simulation in explicit water. The predicted model is consistent with available experimental data. Our results indicate that residues 97–101 of CDR3β are critical for recognition of huCollp261/HLA-DR4 by TCR. We also show that TCR contacts on p/MHC surface affect the conformation of the shared epitope expressed by DR alleles associated with RA susceptibility. Conclusions/Significance This work presents a three-dimensional model for the ternary complex TCR-Vβ/collagenII(261–273)/HLA-DR4 associated with rheumatoid arthritis that can provide insights into the molecular mechanisms of self reactivity.

Investigations on the 4-Quinolone-3-carboxylic Acid Motif. 4. Identification of New Potent and Selective Ligands for the Cannabinoid Type 2 Receptor with Diverse Substitution Patterns and Antihyperalgesic Effects in Mice

Experimental evidence suggests that selective CB2 receptor modulators may provide access to antihyperalgesic agents devoid of psychotropic effects. Taking advantage of previous findings on structure-activity/selectivity relationships for a class of 4-quinolone-3-carboxamides, further structural modifications of the heterocyclic scaffold were explored, leading to the discovery of the 8-methoxy derivative 4a endowed with the highest affinity and selectivity ever reported for a CB2 ligand. The compound, evaluated in vivo in the formalin test, behaved as an inverse agonist by reducing at a dose of 6 mg/kg the second phase of the formalin-induced nocifensive response in mice.

In vitro assembly of nucleocapsid-like particles from purified recombinant capsid protein of dengue-2 virus

The capsid protein is one of the three structural proteins of flaviviruses and is the building block of the nucleocapsid. It has also a predominant role in the replication of dengue virus. To obtain nucleocapsid-like particles from recombinant dengue-2 capsid protein produced in E. coli, a purification process using cation exchange chromatography was established. The purified protein exhibited a molecular mass corresponding to a dimer; therefore, similar to that reported for alphaviruses, an in vitro assembly reaction using single-stranded DNA was performed. In all cases, particles were obtained independently of the specificity and the length of the oligonucleotides used. The present work is the first report of in vitro assembly of the recombinant dengue capsid protein, which could constitute a powerful tool in the development of vaccine candidates.

Investigations on the 4-Quinolone-3-carboxylic Acid Motif. 3. Synthesis, Structure—Affinity Relationships, and Pharmacological Characterization of 6-Substituted 4-Quinolone-3-carboxamides as Highly Selective Cannabinoid-2 Receptor Ligands

A set of quinolone-3-carboxamides 2 bearing diverse substituents at position 1, 3, and 6 of the bicyclic nucleus was prepared. Except for six compounds exhibiting Ki>100 nM, all the quinolone-3-carboxamides 2 proved to be high affinity CB2 ligands, with Ki values ranging from 73.2 to 0.7 nM and selectivity [SI=Ki(CB1)/Ki(CB2)] varying from >14285 to 1.9, with only 2ah exhibiting a reverse selectivity (SI<1). In the formalin test of peripheral acute and inflammatory pain in mice, 2ae showed analgesic activity that was antagonized by a selective CB2 antagonist. By contrast, 2e was inactive per se and antagonized the effect of a selective CB2 agonist. Finally, 2g and 2p exhibited CB2 inverse agonist-like behavior in this in vivo test. However, two different functional assays carried out in vitro on 2e and 2g indicated for both compounds an overall inverse agonist activity at CB2 receptors.

Alignment of flexible molecules at their receptor site using 3D descriptors and Hi-PCA.

Three categories of molecular flexibility are defined. A novel method ofaligning partly flexible molecules with each other is described. The bindingmode of one of these molecules to its receptor site was already well knownfrom previous crystallographic studies, and this known binding mode was usedto predict the binding mode of the other molecules at their receptor. Thepredictions were checked by comparison with previous observations, and werecorrect. Two novel methods were combined in this research. It was necessaryto take account of the conformational changes which occur when each ligandmolecule binds to the protein, and a new release of programme Grid was usedfor this. It was also necessary to analyse the Grid results in order todistinguish the role of each chemical group at the receptor site. This wasdone by applying hierarchical principal component analysis (Hi-PCA) methodsto the descriptors obtained from Grid.

A gain-of-glycosylation mutation associated with myoclonus-dystonia syndrome affects trafficking and processing of mouse ε-sarcoglycan in the late secretory pathway†

Missense mutations in the SGCE gene encoding ε‐sarcoglycan account for approximately 15% of SGCE‐positive cases of myoclonus‐dystonia syndrome (MDS) in humans. In this study, we show that while the majority of MDS‐associated missense mutants modeled with a murine ε‐sarcoglycan cDNA are substrates for endoplasmic reticulum‐associated degradation, one mutant, M68T (analogous to human c.275T>C, p.M92T), located in the Ig‐like domain of ε‐sarcoglycan, results in a gain‐of‐glycosylation mutation producing a protein that is targeted to the plasma membrane, albeit at reduced levels compared to wild‐type ε‐sarcoglycan. Removal of the ectopic N‐linked glycan failed to restore efficient plasma membrane targeting of M68T demonstrating that the substitution rather than the glycan was responsible for the trafficking defect of this mutant. M68T also colocalized with CD63‐positive vesicles in the endosomal–lysosomal system and was found to be more susceptible to lysosomal proteolysis than wild‐type ε‐sarcoglycan. Finally, we demonstrate impaired ectodomain shedding of M68T, a process that occurs physiologically for ε‐sarcoglycan resulting in the lysosomal trafficking of the intracellular C‐terminal domain of the protein. Our findings show that functional analysis of rare missense mutations can provide a mechanistic insight into the pathogenesis of MDS and the physiological role of ε‐sarcoglycan. Hum Mutat 32:1246–1258, 2011. ©2011 Wiley Periodicals, Inc.

Allosteric properties of hemoglobin and the plasma membrane of the erythrocyte: new insights in gas transport and metabolic modulation.

Within the red blood cell the hemoglobin molecule is subjected to modulation mechanisms, namely homo‐ and heterotropic interactions, which optimize its functional behavior to the specific physiological requirements. At the cellular level, these modulation mechanisms are utilized to perform a number of other functions that are not minor with respect to the basic function of oxygen transport. Here we report some key examples concerning: (i) the interaction of hemoglobin with band 3 and its influence on glucose metabolism; (ii) the role of the ligand‐linked quaternary transition of hemoglobin in the control of “NO bioactivity” and of gas diffusion; (iii) the interaction of plasma membrane with the various oxidative derivatives of the hemoglobin molecule.

A second Ig-like domain identified in dystroglycan by molecular modelling and dynamics

Dystroglycan (DG) is a cell surface receptor which is composed of two subunits that interact noncovalently, namely α- and β-DG. In skeletal muscle, DG is the central component of the dystrophin-glycoprotein complex (DGC) that anchors the actin cytoskeleton to the extracellular matrix. To date only the three-dimensional structure of the N-terminal region of α-DG has been solved by X-ray crystallography. To expand such a structural analysis, a theoretical molecular model of the murine α-DG C-terminal region was built based on folding recognition/threading techniques. Although there is no a significant (<30%) sequence homology with the N-terminal region of α-DG, protein fold recognition methods found a significant resemblance to the α-DG N-terminal crystallographic structure. Our in silico structural prediction identified two subdomains in this region. Amino acid residues ∼ 500-600 of α-DG were predicted to adopt an immunoglobulin-like (Ig-like) β-sandwich fold. Such modeled domain includes the β-DG binding epitope of α-DG and, confirming our previous experimental results, suggests that the linear epitope (residues 550-565) assumes a β-strand conformation. The remaining segment of the α-DG C-terminal region (residues 601-653) is organized in a coil-helix-coil motif. A 20-ns molecular dynamics simulation in explicit water solvent provided support to the predicted Ig-like model structure. The identification of a second Ig-like domain in DG represents another important step towards a full structural and functional description of the α/β DG interface. Preliminary characterization of a novel recombinant peptide (505-600) encompassing this second Ig-like domain demonstrates that it is soluble and stable, further corroborating our in silico analysis.

Polymorphism, selection and tandem duplication of transferrin genes in Atlantic cod (Gadus morhua) - Conserved synteny between fish monolobal and tetrapod bilobal transferrin loci

BackgroundThe two homologous iron-binding lobes of transferrins are thought to have evolved by gene duplication of an ancestral monolobal form, but any conserved synteny between bilobal and monolobal transferrin loci remains unexplored. The important role played by transferrin in the resistance to invading pathogens makes this polymorphic gene a highly valuable candidate for studying adaptive divergence among local populations.ResultsThe Atlantic cod genome was shown to harbour two tandem duplicated serum transferrin genes (Tf1, Tf2), a melanotransferrin gene (MTf), and a monolobal transferrin gene (Omp). Whereas Tf1 and Tf2 were differentially expressed in liver and brain, the Omp transcript was restricted to the otoliths. Fish, chicken and mammals showed highly conserved syntenic regions in which monolobal and bilobal transferrins reside, but contrasting with tetrapods, the fish transferrin genes are positioned on three different linkage groups. Sequence alignment of cod Tf1 cDNAs from Northeast (NE) and Northwest (NW) Atlantic populations revealed 22 single nucleotide polymorphisms (SNP) causing the replacement of 16 amino acids, including eight surface residues revealed by the modelled 3D-structures, that might influence the binding of pathogens for removal of iron. SNP analysis of a total of 375 individuals from 14 trans-Atlantic populations showed that the Tf1-NE variant was almost fixed in the Baltic cod and predominated in the other NE Atlantic populations, whereas the NW Atlantic populations were more heterozygous and showed high frequencies of the Tf- NW SNP alleles.ConclusionsThe highly conserved synteny between fish and tetrapod transferrin loci infers that the fusion of tandem duplicated Omp-like genes gave rise to the modern transferrins. The multiple nonsynonymous substitutions in cod Tf1 with putative structural effects, together with highly divergent allele frequencies among different cod populations, strongly suggest evidence for positive selection and local adaptation in trans-Atlantic cod populations.