Research Activity

The natural complement to the characterization of cloned genes is their structural characterization. The knowledge of the three-dimensional structure of macromolecules involved in biological processes can help rationalize the data deriving from functional studies, as well as reveal unexpected novel activities. Our main interest is the understanding of the structure-function relationship in biological macromolecules whose functions range from protein/DNA recognition to enymatic catalysis, protein-protein interaction in receptor-ligand systems, and transport across membranes. We carry out all the processes required to go from the DNA to the purified protein, through the crystallization and finally to three-dimensional structure using X-ray crystallography. We also use a variety of biophysical methods to structurally and functionally characterize macromolecules, such as circular dichroism and dynamic light scattering. Here we outline the main research lines in the lab.

1. Structural Immunology

T lymphocytes discriminate between self and foreign antigens bound to antigen presenting molecules via the interaction of a clonotypic T cell receptor (TCR). TCRs mainly interact with antigens in the form of peptides bound to either class I or class II major histocompatibility complex molecules (MHC). The structures of class I- and class II-MHC molecules highlighted the structural framework for the binding of peptide antigens, and defined the structural constraints for peptide length and side chain properties. The structure of unliganded TCRs showed the similarities and differences with Fab portions of antibodies, and ultimately the TCR/MHC/peptide complex structures showed how TCRs recognize antigens.

Figure 1 X-ray structure of a TCR/MHC/peptide complex. The class I-MHC molecule (in green) is non-covalently associated with the soluble beta2-microglobulin (dark green), and binds 8 or 9 residue long peptides (in yellow) in the alpha1 and alpha2 domains. The heterodimeric alphabeta TCR (pink and light blue) is positioned over the bound peptide to optimally "read-out" the contents of the antigen-binding groove. The structure helps understanding how mutations at peptide residues can change the antigen potency from agonist to antagonist.

However, recent data clearly demonstrated that TCRs can also recognize different antigens, such as lipids and glycolipids, bound to non-classical MHC-like molecules, such as CD1. Glycolipids such as alpha-Galactosyl-Ceramide bound to CD1 is able to activate a subpopulation of T cells (NKT cells) linked to the innate immune response that are suggested to play a protective function against autoimmune diseases. We are currently pursuing the structure of CD1 in complex with different glycolipid antigens in order to shed light on the structural requirements for lipid presentation by MHC-like molecules.

2. Nucleotide Metabolism and Cancer

Cancerous cells have increased requirements for nucleotides to supplement the rapid division process. Interfering with nucleotide biosynthesis with specific compounds has long been the therapeutic approach of choice in chemotherapy, providing increasing success rates. 5-fluorouridines are currently used, for instance, in the colorectal and breast cancer therapies. These nitrogenous bases act as prodrugs, and are processed by cellular enzymes to a 5-fluorouridine nucleotide that inhibits thymidylate synthase. This conversion involves two separate steps, a deamination followed by the transfer of phosphoribosyl pyrophosphate. Human enzymes are poorly efficient in these two steps, thus requiring high doses of the prodrug that in turn leads to high systemic toxicity and unwanted side effects. We identified, cloned and characterized several bacterial and eukariotic enzymes with nucleoside hydrolase activity (NHs) that convert with high efficiency lowly toxic, bioavailable 5-fluoro nucleosides to the corresponding base. These enzymes were long thought to belong only to protozoan parasites, which lack purine biosynthetic enzymes and rely on the host for uptake of purine bases.

Figure 2. The ribbon diagram of the pyrimidine specific NH from E. coli, an open a/b structure with a helical ‘cap’ that provides substrate specificity. Highlighted are the residues involved in the binding of the active site Ca2+ ion, the substrate, and water nucleophile.

Using a genomic approach, we are investigating the structure and specificity of these enzymes. The high resolution structures of pyrimidine nucleosidases guides the engineering of mutated proteins with enhanced specificity towards 5-fluoropyrimidine nucleosides. The mutated enzymes are candidates for use in suiceide gene therapy approaches to dramatically enhance the efficiency of these compounds against solid tumors.

3. uPAR

Urokinase (uPA) is a serine protease involved in the processes of fibrinolysis. uPA binds with high affinity to its receptor, a GPI-anchored glycoprotein termed the urokinase plasminogen activator receptor (uPAR). uPAR is composed of three disulfide-rich homologous domains, D1 D2 and D3. Upon binding of urokinase, uPAR is cleaved to the D2D3 domains, and becomes endowed with high chemotactic activity. The uPA/uPAR complex plays important roles in cell migration and proliferation. The role of uPAR in metastatic processes has been established in several model systems. In collaboration with the group of Francesco Blasi (http://www.sanraffaele.org/research/blasi) we are pursuing the determination of the three-dimensional structure of uPAR in its intact or activated form, and its complex with uPA. Knowledge of the structure of uPAR in its different forms will help understand at the molecular level the events that trigger cancer invasiveness. Moreover, the structures will provide a template for the development of novel antagonists to fight metastatic processes.

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