| Selection of single chain antibodies (scFv) against amyloid aggregates top page Amyloid is a key topic in modern research. It essentially derives from a particular and highly ordered protein aggregation mechanism based on the concept that partially or completely unfolded proteins may gain a strong stability when complexed with other proteins in a beta cross linked 3-D structure. The resulting amyloid fibrils are insoluble wire-shaped bodies that aggregate inside and outside the cells and form plaques which compromise the functionality at various levels. Such fibrils (and all the route of aggregation that brings to them) are involved in many human pathologies (called amyloidosis) including Alzheimer's, Parkinson's, prion-based diseases and many others. scFv are "recombinant antibodies" genereted by in-vitro extensive mutagenesis of the variable fragments of Ig light chains. When placed in an appropriate expression vector (a phagemid, which uses the phages ability of replication and infection for propagation) these variable regions can be modeled to form what is usually considered the "antigen specific" part of an immuno globulin, the upper extremity of the double arm of the famous "Y". As in the immunitary sustem the variability of amino acid composition of such region is able to generate over 10 billions different combinations, thanks to random mutagenesis that number can also be increased. scFv libraries can be screened searching for a clone over the entire population which specifically and strongly recognizes virtually any physiological or non-physiological antigen: in brief, we can produce specific "antibodies" whith a complete in vitro approach, totally animal and cell-free. Our research is intended to obtain scFv clones which can limit, prevent or reverse protein amyloid aggregation working on different model proteins which have proved a good propensity to form aggregates.  |
| Characterization of a novel Escherichia coli acylphosphatase top page Acylphosphatase activity in Escherichia coli has been attributed for long time to non-specific acid phosphatases. However, the sequencing of the whole genome of Escherichia coli has shown the presence of a gene (b0968) containing an open reading frame (ORF) for a putative acylphosphatase protein (P75877). The effective expression of such an ORF was checked in E. coli cells by specific RT-PCR. We have then cloned and expressed it into the same cells. The purified enzyme named EcoAcP, is the first acylphosphatase from eubacteria to be characterized. It is a fully structured and active acylphosphatase displaying the same substrate affinity but a very reduced specific activity with respect to other members of the same family. When compared to the well known eukaryotic acylphosphatases, EcoAcP displays some peculiar enzymatic and structural features; indeed it appears to have a disulphide bridge, to gain catalytic efficiency in mild destabilising conditions (small urea concentrations and high temperature) and to possess a conformational stability higher than that of eukariotic acylphosphatases. At the moment we are collaborating in the determination of the enzyme structure through Nuclear Magnetic Resonance and we are investigating its catalytic peculiarities.  see Corazza A. et al. 2006 for EcoAcP structure determined by NMR see Ramazzotti M. et al. 2006 for EcoAcP kinetic and thermodynamic characterization. |
| Protein extraction from refined oils and allergenic potency top page Severe allergic reactions (anaphylaxis) can be triggered by a wide range of foods. Theoretically almost any food may be implicated, but by far the most common culprits are peanut, soy, sunflower and tree nuts. It is sensible to view any allergic reaction to these foods as potentially serious. Even apparently mild symptoms should not be ignored because it is possible that future reactions may be more severe. Medical advice should be sought and the food causing the problem must be avoided. Refined vegetable oil are used in a wide variety of food and as additive in preparation. Commercially refined oils are derived from plants which are recognized as potent allergens (peanut, soy, sun flower etc.). The refining of the oil results almost incomplete in the removal of proteins. This leads to a debate about the safety of refined oils and whether to label each oil because of the potential risk of allergenicity. The prevalence of food anaphylaxis due to masked allergens has increased within the last 10 years. Contamination of manufactured products by food allergens is a key concern for food industry. Moreover several severe reactions to oil after parenteral medical application have been reported. The term allergy refers to a condition in which an immunological reaction to a substance produces IgE-mediated clinical symptoms. This implies that some component of the food, almost always proteins, is recognized by cell of the immune system and an immune response occurs. We are now trying to identify a general pattern of protein which characterize each oil and to detect the most allergenic elements.  |
| Analysis of codon usage in protein families top page Codon usage is for many organism a driving force in high levels of protein expression. The levels of each tRNA in the cells is not equal and therefore not all amino acid are available in the same way. A strong promoter seems to induce a sort of selection in the codons of the gene downstream in order to avoid amino acid deprivation or misincorporation. We developed a package of softwares (named CAI Analyser Package) for the analysis of codon usage in whole genomes whose aim is to infer the levels of gene expression by observing the codon usage and by scoring the genes with the Codon Adaptation Index. The software optimization and the statistics on the results are still in refinement, towards the mapping of the importance of the different methabolic pathways in different organisms.   see Ramazzotti M. et al. 2007 for the software we developed (CAIAP) for this project or visit the CAIAP server |
| Tracing the evolution of acylphosphatase proteins top page Acylphosphatases (EC 3.6.1.7) are a small cytosolic enzyme. The main activity is to catalyse the hydrolysis of the carboxyl phosphate bonds some basic compounds such as the b-aspartyl phosphate intermediates formed during the action of Na+/K+ and Ca++-membrane ATPase. This action on the phosphointermediate led us to the proposal that the physiological funcion of AcP consists in controlling the ion transport across the membrane. The physiological role of AcP is, however, still a matter of debate. During the years and thanks to the increasing number of sequenced genomes available, we found out that acylphosphatases possess an unexpected high evolutionary conservation from archea to bacteria, from invertebrates to mammalians and in nearly all organism so far sequenced. We are collecting all the acylphosphatases available and we are now trying to trace the evolutionary pathway of this enzyme by computational techniques such as multiple sequence alignment, positional scoring algorithms and predictive tools in order to understand the real role of this enzyme in lower organisms and the reason for which evolution has reserved for it a special place during the millennia.  see Ramazzotti M. et al. 2004 for the program we developed for analysing AcP hystory |
| Anti-amyloidogenic effect of some vegetal compounds top page Amyloidosis encompasses a large group of diseases characterized by tissue deposition of proteins assembled in regular -strands. Human lysozyme is a bacteriolytic protein present in external secretions and in polymorphs and macrophages. 4 of the 5 variants of this enzyme have been associated with non-neuropathic systemic amyloidosis. HEWL (hen egg white lysozyme) is a good model for studies on amyloid fibril formation. Similar to the human form, it can be easily fragmented in peptides prone to amyloid aggregation. We tested the effect of some polyphenols commonly present in vegetal foods and often used in industry as antioxidants (caffeoil derivatives, catechins, gallic acid derivatives, anthocyanins) on amyloid aggregation of HEWL or its slightly modified 49-64 peptide (S in 64, instead of C). The kinetics of HEWL amyloid aggregation resembled a sigmoidal curve with a lag phase of 2-3 days, corresponding to enzyme fragmentation. The process resulted complete in about 7 days, when tested with ThT assay. Lyzozyme peptide showed an exponential kinetics of about 7 hours. Several tested polyphenols reduced the ThT signal, although the first part of kinetic curve was conserved. The highest effects on the aggregation process were obtained using rosmarinic acid, cichoric acid, epigallocatechin gallate, tannic acid and keracyanin. In polyphenols the presence of 2 distinct phenolic rings, particularly with 2 hydroxyl groups in orto positions, resulted essential for the interference with the aggregation process.   |
Used techniques
| Electrophoresis: for proteins and nucleic acids, with direct staining or Western blotting |
| Spectrophotometry, spectropolarimetry (CD) and spectrofluorimetry |
| Mass spectrometry: with MALDI-TOF spectrometer |
| Chromatography: Gravity, FPLC and HPLC, affinity, reverse-phase, gel-filtration |
| PCR-based gene cloning and recombinant protein expression |
| Phage display |
| Immuno-assays: ELISA and immuno-blotting |
| Cell culture |