Example PagesOur recent research interests can be divided, with some overlap, into two main directions: development and/or improvements of computational biology methods and their application to the study of problems of biomedical interest.
To the first area, belong the development of methods for the prediction of the structure and function of proteins and nucleic acid.
Access to the tools can be obtained following the link on the left. They include:
Maistas: A publicly available server (http://www.bioinformatica.crs4.org/maistas/) that, given a gene or a protein of an organism for which the genome is available and included in the Ensembl datasets, inspects the genome, collects all putative isoforms of the input gene, builds, whenever possible, three-dimensional models for all of them using state of the art methodologies and evaluates their structural feasibility. The main aim of the work is to provide users with comparative models of the protein(s) of their interest together with valuable practical information about which isoforms are likely to correspond to a functional protein.
Meps and Mepmaps: a method able to find the surface region of a protein that can be effectively mimicked by a peptide, given the structure of the protein and the maximum number of side chains deemed to be required for recognition. The method is implemented as a publicly available server. It can also find and report all peptide sequences of a specified length that can mimic the surface of a given protein and store them in a database. (In collaboration with Tiziana Castrignano')
Pigs: a web server for the automatic prediction of immunoglobulin variable domains based on the canonical structure model. The server is user friendly and flexible. It allows the user to select the templates for the frameworks and the loops using different strategies. The final output is a full-fledged three-dimensional model of the variable domains of the target immunoglobulin.
Movin: a publicly available server that allows the user to investigate protein interaction data in light of other biological information, such as their sequences, presence of specific domains, process and component ontologies. The server can be effectively used to construct a highconfidence set of mutually exclusive interactions by identifying similar features in groups of proteins sharing a common interaction partner.
The Protein Model Database (PMDB) is a public resource aimed at storing manually built 3D models
of proteins. The database is designed to provide access tomodels published in the scientific literature,
together with validating experimental data. It is a relational database. The system is accessible at http://www.caspur.it/PMDB and allows predictors to submit models along with related supporting evidence and users to download them through a simple and intuitive interface. Users can navigate in the database and retrieve models referring to the same target protein or to different regions of the same protein. Each model is assigned a unique identifier. All our models are deposited there. (In collaboration with Tiziana Castrignano')
The other aspect of our research has been the investigation of important biomedical problems related to pathologies induced by foreign agent such as Hepatitis C and malaria and genetic diseases as well as the study of biotechnologically important molecules. In the following we only briefly describe some examples:
Malaria is caused by protozoan parasites of the genus Plasmodium. Four species of Plasmodium can infect humans: P. falciparum, P. malariae, P. vivax, and P. ovale. P. falciparum is the only able to
cytoadhere to the surface of postcapillary endothelial cells. A key role in cytoadherence is played by the interaction between the PfEMP1 P. falciparum protein and the human intracellular adhesion molecule (ICAM-1) although very little is known about the molecular details of this complex. Here we propose a model for this interaction on the basis of a homology model of the functional domain of PfEMP1 and of the ICAM-1 three dimensional structures. Our model is consistent with the results of many experimental observations, provides a rational explanation for the different binding abilities of different strains of P. falciparum and explains the reduced binding affinity of the A4 strain of P. falciparum for the ICAM-1Kilifi polymorphism. On the basis of our model, we can also explain why the murine ICAM-1, although sharing 70% sequence similarity with its human homologue, does not bind PfEMP1, and why the binding of fibrinogen and PfEMP1 to ICAM-1 is mutually exclusive. The model of the complex can serve as a useful tool for the design and interpretation of biochemical and immunological experimental results.
Down syndrome (DS) is the most frequent form of mental retardation and is caused by chromosome 21 (HSA21) trisomy. Despite the number of known genes involved in DS and its high therapeutic interest, biological mechanisms leading to the DS phenotype are not fully clear.We presented a functional hypothesis based on fold recognition and hidden Markov model techniques for four HSA21 genes located in the DS Candidate Region (DSCR). More specifically, we propose that they are members of a novel mitogen-activated protein kinase pathway with DYRK1A, SNF1LK and RIPK4 gene products being elements of the kinase cascade and the DSCR3 acting as structural scaffold for their interaction. This hypothesis finds support in various biochemical studies concerning the biological behavior and features of the involved HSA21 proteins. Our analysis calls for specifically designed experiments to validate our prediction and establish its relevance in terms of therapeutic approaches to the disease.
Alternative splicing enables genes to generate more than one gene product. Splicing events that occur within protein coding regions have the potential to alter the biological function of the expressed protein and even to create new protein functions. Alternative splicing has been suggested as one explanation for the discrepancy between the number of human genes and functional complexity. In collaboration with several groups, we carried out a detailed study of the alternatively spliced gene products annotated in the ENCODE pilot project. We find that alternative splicing in human genes is more frequent than has commonly been suggested, and we demonstrate that many of the potential alternative gene products will have markedly different structure and function from their constitutively spliced counterparts. For the vast majority of these alternative isoforms, little evidence exists to suggest they have a role as functional proteins, and it seems unlikely that the spectrum of conventional enzymatic or structural functions can be substantially extended through alternative splicing. (In collaboration with groups from the Biosapiens NoE)
Studies of B cell antigen receptors (BCRs) expressed by leukemic lymphocytes from patients with B cell chronic lymphocytic leukemia (B-CLL) suggest that B lymphocytes with some level of BCR structural restriction become transformed. While analyzing rearranged VHDJH and VLJL genes of 25 non–IgM-producing B-CLL cases, we found five IgG+ cases that display strikingly similar BCRs (use of the same H- and L-chain V gene segments with unique, shared heavy chain third complementarity-determining region [HCDR3] and light chain third complementarity-determining region [LCDR3] motifs). These H- and L-chain characteristics were not identified in other B-CLL cases or in normal B lymphocytes whose sequences are available in the public databases. Three-dimensional modeling studies suggest that these BCRs could bind the same antigenic epitope. The structural features of the B-CLL BCRs resemble those of mAb’s reactive with carbohydrate determinants of bacterial capsules or viral coats and with certain autoantigens. These findings suggest that the B lymphocytes that gave rise to these IgG+ B-CLL cells were selected for this unique BCR structure. This selection could have occurred because the precursors of the B-CLL cells were chosen for their antigen-binding capabilities by antigen(s) of restricted nature and structure, or because the precursors derived from a B cell subpopulation with limited BCR heterogeneity, or both. (In collaboration with Fabio Ghiotto)
UL18 is a glycoprotein encoded by the human cytomegalovirus genome and is thought to play a pivotal role during human cytomegalovirus infection, although its exact function is still a matter of debate. UL18 shares structural similarity with MHC classI and binds the receptor CD85j on immune cells. Besides UL18, CD85j binds MHC class I molecules. The binding properties of CD85j to MHC class I molecules have been thoroughly studied. Conversely, very little information is available on the CD85j/UL18 complex, namely that UL18 binds CD85j through its alpha3 domain with an affinity that is about 1000-fold higher than the MHC class I affinity for CD85j. Deeper knowledge of features of the UL18/CD85j complex would help to disclose the function of UL18 when it binds to CD85j. We first demonstrated that the UL18alpha3 domain is not sufficient per se for binding and that beta2-microglobulin is necessary for UL18–CD85j interaction. We then dissected structural determinants of binding UL18 to CD85j. To this end, we constructed a three-dimensional model of the complex. The model was used to design mutants in selected regions of the putative interaction interface, the effects of which were measured on binding. Six regions in both the alpha2 and alpha3 domains and specific amino acids within them were identified that are potentially involved in the UL18–CD85j interaction. The higher affinity of UL18 to CD85j, compared with MHC class I, seems to be due not to additional interaction regions but to an overall better fit of the two molecules. (In collaboration with Fabio Ghiotto)
Itch, a member of the E6AP carboxy terminus (HECT) domain-containing family of ubiquitin E3 ligases, acts in concert with the ubiquitin activating enzyme (E1) and the ubiquitin conjugating enzyme (E2) to catalyze ubiquitylation of protein targets. This sub-family of E3s shares a 350 residue C-terminal HECT domain having a strictly conserved catalytic Cys, and recruiting its cognate ubiquitin-loaded E2. HECT domains possess intrinsic enzymatic activity, by accepting ubiquitin from an E2, forming a ubiquitin thiolester intermediate, and directly catalyzing ubiquitylation of the target protein. Several hypotheses have been proposed for the biochemical mechanism underlying the structural relationship of the HECT-E2 association and subsequent ubiquitin transfer. We used molecular dynamic simulations, free energy calculations, protein modelling techniques and normal modes analysis to get a deeper characterization of the static and dynamical properties of this interaction mechanism. We hypothesize a correlated slow-frequency motion that involves two different hinge regions of the HECT domain. The identification of the amino acid residues responsible for the HECT-E2 interaction, and for the dynamical properties of the ubiquitin transfer process, may be of relevant interest for pharmacological and therapeutical purposes. (In collaboration with Gerry Melino)
We studied alterations in mitochondrial proteins in a patient suffering from recurrent profound muscle weakness, associated with ethylmalonic-adipic aciduria, who had benefited from high dose of riboflavin treatment. Morphological and biochemical alterations included muscle lipid accumulation, low muscle carnitine content, reduction in fatty acid b-oxidation and reduced activity of complexes I and II of the respiratory chain. Riboflavin therapy partially or totally reversed these symptoms and increased the level of muscle flavin adenine dinucleotide, suggesting that aberrant flavin cofactor metabolism accounted for the disease. Proteomic investigation of muscle mitochondria revealed decrease or absence of several flavoenzymes, enzymes related to flavin cofactor-dependent mitochondrial pathways and mitochondrial or mitochondria-associated calcium-binding proteins. All these deficiencies were completely rescued after riboflavin treatment. This study indicates for the first time a profound involvement of riboflavin/flavin cofactors in modulating the level of a number of functionally coordinated polypeptides involved in fatty acyl-CoA and amino acid metabolism, extending the number of enzymatic pathways altered in riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency. (In collaboration with Elisabetta Gianazza)
The environmental sequencing of the Sargasso Sea has introduced a huge new resource of genomic information. Unlike the protein sequences held in the current searchable databases, the Sargasso Sea sequences originate from a single marine environment and have been sequenced from species that are not easily obtainable by laboratory cultivation. The resource also contains very many fragments of whole protein sequences, a side effect of the shotgun sequencing method. These sequences form a significant addendum to the current searchable databases but also present us with some intrinsic difficulties. While it is important to know whether it is possible to assign function to these sequences with the current methods and whether they will increase our capacity to explore sequence space, it is also interesting to know how current bioinformatics techniques will deal with the new sequences in the resource. We found that the Sargasso Sea sequences seem to introduce a bias that decreases the potential of current methods to propose structure and function for new proteins. In particular the high proportion of sequence fragments in the resource seems to result in poor quality multiple alignments. These observations suggest that the new sequences should be used with care, especially if the information is to be used in large scale analyses. On a positive note, the results may just spark improvements in computational and experimental methods to take into account the fragments generated by environmental sequencing techniques. (In collaboration with Alfonso Valencia)
More info on our projects will be added in these pages soon
