Obiettivi e caratteristiche della conferenza

Il Programma per la Cooperazione transfrontaliera Italia-Slovenia 2007-2013 ha finanziato sei progetti di ricerca e innovazione in ambito biomedico. In totale, sono stati concessi circa 8,5 milioni di \u20ac a 44 beneficiari, distribuiti nell\u2019Area Programma. La durata dei progetti va da due anni e mezzo a tre anni e mezzo, a seconda dell\u2019ambiziosit\ue0 degli obiettivi e del livello di coinvolgimento dei partner. Il finanziamento rappresenta il 6% dell\u2019intero budget di Programma (137,6 MLN \u20ac) e il 23% del budget assegnato all\u2019asse prioritario n. 2 - Competitivit\ue0 e societ\ue0 basata sulla conoscenza. I coordinatori dei sei progetti si sono incontrati per la prima volta in occasione della tavola rotonda organizzata in occasione del Convegno sulla sanit\ue0 transfrontaliera di Gorizia il 4 dicembre 2013. Gi\ue0 in quell\u2019occasione sono bastati alcuni rapidi contatti tra i Team Manager per decidere di ripetere l\u2019incontro ed approfondire la conoscenza reciproca. L\u2019Universit\ue0 di Trieste, Lead partner del progetto strategico Trans2Care, ha messo a disposizione i suoi spazi in edificio H3 per ospitare la Conferenza. Il sito web del progetto \ue8 stato messo a disposizione per promuovere l\u2019evento e fornire tutte le informazioni necessarie ai partecipanti unitamente agli obiettivi della Conferenza

LE CATELICIDINE, UNA NUOVA FAMIGLIA DI PRECURSORI DI PEPTIDI ANTIMICROBICI DEI NEUTROFILI. CARATTERIZZAZIONE MOLECOLARE E STRUTTURALE

1994/1995VII Ciclo1966Versione digitalizzata della tesi di dottorato cartacea

The cDNA of the neutrophil antibiotic Bac5 predicts a pro-sequence homologous to a cysteine proteinase inhibitor that is common to other neutrophil antibiotics.

Bac5 is a 5-kDa proline- and arginine-rich antibiotic, stored as inactive precursor (proBac5) in the large granules of bovine neutrophils. A full-length cDNA encoding the precursor form of Bac5 has been cloned. The encoded protein (pre-proBac5) has a calculated mass of 20,031 Da and a pI of 9.21. This comprises a putative signal peptide of 29 amino acid residues and a 101-residue pro-sequence that precede the mature antibiotic. The pro-sequence is acidic and may neutralize the highly cationic Bac5, thus accounting for the inactivation of the antibiotic activity observed in in vitro experiments. The structure of mature Bac5 agrees closely with the amino acid sequence previously determined, with an additional tripeptide tail predicting carboxyl-terminal amidation. A valyl residue is deduced at the cleavage site for the proteolytic maturation of proBac5, consistent with a previous observation showing elastase as the enzyme involved in this processing step. The region upstream of Bac5 reveals high identity to corresponding regions of two neutrophil antimicrobial polypeptides, CAP18 from rabbit and bovine indolicidin. The COOH-terminal sequences of these antibiotics are completely unrelated. The proregion also exhibits remarkable similarity to pig cathelin, an inhibitor of cathepsin L, indicating a common evolutionary origin

Bovine Neutrophil Antibiotic Peptides and Their Precursors: Structure and Role in Innate Immunity

Four peptides were characterized in extracts of bovine neutrophil granules: an Arg-rich dodecapeptide, maintained in a cyclic structure by a disulfide bridge; a Trp-rich tridecapeptide named indo- licidin; and two 43- and 59 amino acids long peptides, named Bac5 and Bac7, with frequent repeats of the triplets Arg-Pro-Pro and Pro-Arg-Pro, respectively. The full length cDNA of the first three of these peptides was characterized recently. Sequence analysis showed that the prosequences of the predicted precursors of all the three peptides are highly identical and exhibited also a remarkable similarity to cathelin, a porcine inhibitor of cathepsin L. Purified proBacö actually proved in in vitro assays to inhibit cathepsin L, but not other cysteine proteinases such as cathepsin B. Unlike proBacö, proBac7 is selectively chemotactic to monocytes. Several fragments of Bac5 and Bac7 (from 6 to 35 residues) were synthesized by the Fmoc method. The results of antibacterial assays show that the N-terminal portion, the most cationic one in both Bac5 and Bac7, is essential for the antimicrobial activity and that the minimal length necessary to arrest the growth of susceptible bacteria is 18-20 residues

Insight into GEBR\u201032a: Chiral Resolution, Absolute Configuration and Enantiopreference in PDE4D Inhibition

Alzheimer\u2019s disease is the most common type of dementia, affecting millions of people worldwide. One of its main consequences is memory loss, which is related to downstream effectors of cyclic adenosine monophosphate (cAMP). A well\u2010established strategy to avoid cAMP degradation is the inhibition of phosphodiesterase (PDE). In recent years, GEBR\u201032a has been shown to possess selective inhibitory properties against PDE type 4 family members, resulting in an improvement in spatial memory processes without the typical side effects that are usually correlated with this mechanism of action. In this work, we performed the HPLC chiral resolution and absolute configuration assignment of GEBR\u201032a. We developed an efficient analytical and semipreparative chromatographic method exploiting an amylose\u2010based stationary phase, we studied the chiroptical properties of both enantiomers and we assigned their absolute configuration by 1H\u2010NMR (nuclear magnetic resonance). Lastly, we measured the IC50 values of both enantiomers against both the PDE4D catalytic domain and the long PDE4D3 isoform. Results strongly support the notion that GEBR\u201032a inhibits the PDE4D enzyme by interacting with both the catalytic pocket and the regulatory domains

engineering methionine γ lyase from citrobacter freundii for anticancer activity

Abstract Methionine deprivation of cancer cells, which are deficient in methionine biosynthesis, has been envisioned as a therapeutic strategy to reduce cancer cell viability. Methionine γ-lyase (MGL), an enzyme that degrades methionine, has been exploited to selectively remove the amino acid from cancer cell environment. In order to increase MGL catalytic activity, we performed sequence and structure conservation analysis of MGLs from various microorganisms. Whereas most of the residues in the active site and at the dimer interface were found to be conserved, residues located in the C-terminal flexible loop, forming a wall of the active site entry channel, were found to be variable. Therefore, we carried out site-saturation mutagenesis at four independent positions of the C-terminal flexible loop, P357, V358, P360 and A366 of MGL from Citrobacter freundii, generating libraries that were screened for activity. Among the active variants, V358Y exhibits a 1.9-fold increase in the catalytic rate and a 3-fold increase in KM, resulting in a catalytic efficiency similar to wild type MGL. V358Y cytotoxic activity was assessed towards a panel of cancer and nonmalignant cell lines and found to exhibit IC50 lower than the wild type. The comparison of the 3D-structure of V358Y MGL with other MGL available structures indicates that the C-terminal loop is either in an open or closed conformation that does not depend on the amino acid at position 358. Nevertheless, mutations at this position allosterically affects catalysis

Broad-spectrum coronavirus 3C-like protease peptidomimetic inhibitors effectively block SARS-CoV-2 replication in cells: Design, synthesis, biological evaluation, and X-ray structure determination

Despite the approval of vaccines, monoclonal antibodies and restrictions during the pandemic, the demand for new efficacious and safe antivirals is compelling to boost the therapeutic arsenal against the COVID-19. The viral 3-chymotrypsin-like protease (3CLpro) is an essential enzyme for replication with high homology in the active site across CoVs and variants showing an almost unique specificity for Leu-Gln as P2–P1 residues, allowing the development of broad-spectrum inhibitors. The design, synthesis, biological activity, and cocrystal structural information of newly conceived peptidomimetic covalent reversible inhibitors are herein described. The inhibitors display an aldehyde warhead, a Gln mimetic at P1 and modified P2–P3 residues. Particularly, functionalized proline residues were inserted at P2 to stabilize the β-turn like bioactive conformation, modulating the affinity. The most potent compounds displayed low/sub-nM potency against the 3CLpro of SARS-CoV-2 and MERS-CoV and inhibited viral replication of three human CoVs, i.e. SARS-CoV-2, MERS-CoV, and HCoV 229 in different cell lines. Particularly, derivative 12 exhibited nM-low μM antiviral activity depending on the virus, and the highest selectivity index. Some compounds were co-crystallized with SARS-CoV-2 3CLpro validating our design. Altogether, these results foster future work toward broad-spectrum 3CLpro inhibitors to challenge CoVs related pandemics

p53 Transactivation and the Impact of Mutations, Cofactors and Small Molecules Using a Simplified Yeast-Based Screening System

The p53 tumor suppressor, which is altered in most cancers, is a sequence-specific transcription factor that is able to modulate the expression of many target genes and influence a variety of cellular pathways. Inactivation of the p53 pathway in cancer frequently occurs through the expression of mutant p53 protein. In tumors that retain wild type p53, the pathway can be altered by upstream modulators, particularly the p53 negative regulators MDM2 and MDM4. promoter, ii) single copy, chromosomally located p53-responsive and control luminescence reporters, iii) enhanced chemical uptake using modified ABC-transporters, iv) small-volume formats for treatment and dual-luciferase assays, and v) opportunities to co-express p53 with other cofactor proteins. This robust system can distinguish different levels of expression of WT and mutant p53 as well as interactions with MDM2 or 53BP1.We found that the small molecules Nutlin and RITA could both relieve the MDM2-dependent inhibition of WT p53 transactivation function, while only RITA could impact p53/53BP1 functional interactions. PRIMA-1 was ineffective in modifying the transactivation capacity of WT p53 and missense p53 mutations. This dual-luciferase assay can, therefore, provide a high-throughput assessment tool for investigating a matrix of factors that can influence the p53 network, including the effectiveness of newly developed small molecules, on WT and tumor-associated p53 mutants as well as interacting proteins

Topoisomerase II\u3b2 mediates the resistance of glioblastoma stem cells to replication stress-inducing drugs

The mesenchymal state in cancer is usually associated with poor prognosis due to the metastatic predisposition and the hyper-activated metabolism. Exploiting cell glucose metabolism we propose a new method to detect mesenchymal-like cancer cells. We demonstrate that the uptake of glucose-coated magnetic nanoparticles (MNPs) by mesenchymal-like cells remains constant when the glucose in the medium is increased from low (5.5 mM) to high (25 mM) concentration, while the MNPs uptake by epithelial-like cells is significantly reduced. These findings reveal that the glucose-shell of MNPs plays a major role in recognition of cells with high-metabolic activity. By selectively blocking the glucose transporter 1 channels we showed its involvement in the internalization process of glucose-coated MNPs. Our results suggest that glucose-coated MNPs can be used for metabolic-based assays aimed at detecting cancer cells and that can be used to selectively target cancer cells taking advantage, for instance, of the magnetic-thermotherapy