| Name | Pietro |
| Surname | Pucci |
| Institution | Università degli Studi della Campania Luigi Vanvitelli |
| Telephone | +39 081 674 318 (UniNa) |
| Telephone 2 | +39 081 373 7896 (Ceinge) |
| pucci@unina.it | |
| Address | Department of Chemical Sciences, Federico II University, Via Cintia 6, 80126, Naples, Italy |
| Resume | Download |
Myoglobin is an α-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues impairs the productive folding of the protein making the polypeptide chain highly prone to aggregate forming amyloid fibrils at physiological pH and room temperature. The role played by tryptophanyl residues in driving the productive folding process was investigated by providing structural details at low resolution of compact intermediate of three mutated apomyoglobins, i.e., W7F, W14F and the amyloid forming mutant W7FW14F. In particular, we followed the hydrogen/deuterium exchange rate of protein segments using proteolysis with pepsin followed by mass spectrometry analysis. The results revealed significant differences in the N-terminal region, consisting in an alteration of the physico-chemical properties of the 7-11 segment for W7F and in an increase of local flexibility of the 12-29 segment for W14F. In the double trypthophanyl substituted mutant, these effects are additive and impair the formation of native-like contacts and favour inter-chain interactions leading to protein aggregation and amyloid formation at physiological pH.
A protocol for a simple and reliable dot-blot immunoassay was developed and optimized to test work of art samples for the presence of specific proteinaceus material (i.e. ovalbumin-based). The analytical protocol has been extensively set up with respect, among the other, to protein extraction conditions, to densitometric analysis and to the colorimetric reaction conditions. Feasibility evaluation demonstrated that a commercial scanner and a free image analysis software can be used for the data acquisition and elaboration, thus facilitating the application of the proposed protocol to commonly equipped laboratories and to laboratories of museums and conservation centres. The introduction of method of standard additions in the analysis of fresh and artificially aged laboratory-prepared samples, containing egg white and various pigments, allowed us to evaluate the matrix effect and the effect of sample aging and to generate threshold density values useful for the detection of ovalbumin in samples from ancient works of art. The efficacy of the developed dot-blot immunoassay was proved testing microsamples from 13th-16th century mural paintings of Saint Francesco Church in Lodi (Italy). Despite the aging, the altered conditions of conservation, the complex matrix, and the micro-size of samples, the presence of ovalbumin was detected in all those mural painting samples where mass-spectrometry-based proteomic analysis unambiguously detected ovalbumin peptides.
Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibits insulin signaling and action. Understanding the mechanisms underlying ENPP1 expression may help unravel molecular mechanisms of insulin resistance. Recent data suggest a role of ENPP1-3'untraslated region (UTR), in controlling ENPP1 expression. We sought to identify trans-acting ENPP1-3'UTR binding proteins, and investigate their role on insulin signaling. By RNA pull-down, 49 proteins bound to ENPP1-3'UTR RNA were identified by mass spectrometry (MS). Among these, in silico analysis of genome wide association studies and expression profile datasets pointed to N-acetylgalactosaminyltransferase 2 gene (GALNT2) for subsequent investigations. Gene expression levels were evaluated by RT-PCR. Protein expression levels, IRS-1 and Akt phosphorylation were evaluated by Western blot. Insulin receptor (IR) autophosphorylation was evaluated by ELISA. GALNT2 down-regulation increased while GALNT2 over-expression reduced ENPP1 expression levels. In addition, GALNT2 down-regulation reduced insulin stimulation of IR, IRS-1 and Akt phosphorylation and insulin inhibition of phosphoenolpyruvate carboxykinase (PEPCK) expression, a key neoglucogenetic enzyme. Our data point to GALNT2 as a novel factor involved in the modulation of ENPP1 expression as well as insulin signaling and action in human liver HepG2 cells.
Patients with cystic fibrosis (CF) manifest a multisystemic disease due to mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR); despite extensive testing of coding regions, a proportion of CF alleles remains unidentified. We studied 118 patients with CF and CFTR-related disorders, most with one or both unknown mutations after the scanning of CFTR coding regions, and a non-CF control group (n = 75) by sequencing the 6000-bp region at the 5' of the CFTR gene. We identified 23 mutations, of which 9 were novel. We expressed such mutations in vitro using four cell systems to explore their functional effect, relating the data to the clinical expression of each patient. Some mutations reduced expression of the gene reporter firefly luciferase in various cell lines and may act as disease-causing mutations. Other mutations caused an increase in luciferase expression in some cell lines. One mutation had a different effect in different cells. For other mutations, the expression assay excluded a functional role. Gene variants in the large 5' region may cause altered regulation of CFTR gene expression, acting as disease-causing mutations or modifiers of its clinical phenotype. Studies of in vitro expression in different cell systems may help reveal the effect of such mutations.
Gastrokine-1 (GKN1), a protein expressed in normal gastric tissue, but absent in gastric cancer tissues and derived cell lines, has recently emerged as a potential biomarker for gastric cancer. To better establish the molecular properties of GKN1, the first protocol for the production of mature human GKN1 in the expression system of Pichia pastoris was settled. The recombinant protein showed anti-proliferative properties specifically on gastric cancer cell lines thus indicating that it was properly folded. Characterization of structural and biochemical properties of recombinant GKN1 was achieved by limited proteolysis analysis, circular dichroism and fluorescence spectroscopy. The analysis of GKN1 primary structure coupled to proteolytic experiments highlighted that GKN1 was essentially resistant to proteolytic enzymes and showed the presence of at least a disulphide bond between Cys61 and one of the other three Cys (Cys122, Cys145 and Cys159) of the molecule. The secondary structure analysis revealed a prevailing β-structure. Spectroscopic and calorimetric investigations on GKN1 thermal denaturation pointed out its high thermal stability and suggested a more complex than a two-state unfolding process. The resulting protein was endowed with a globular structure characterized by domains showing different stabilities toward chemical and physical denaturants. These results are in agreement with the prediction of GKN1 secondary structure and a three-dimensional structure model. Our findings provide the basis for the development of new pharmaceutical compounds of potential use for gastric cancer therapy.
Lipopolysaccharides (LPSs) are ubiquitous and vital components of the cell surface of Gram-negative bacteria that have been shown to play a relevant role in the induction of the immune-system response. In animal and plant cells, innate immune defenses toward microorganisms are triggered by the perception of pathogen associated molecular patterns. These are conserved and generally indispensable microbial structures such as LPSs that are fundamental in the Gram-negative immunity recognition. This paper reports the development of an integrated strategy based on lipopolysaccharide affinity methodology that represents a new starting point to elucidate the molecular mechanisms elicited by bacterial LPS and involved in the different steps of innate immunity response. Biotin-tagged LPS was immobilized on streptavidin column and used as a bait in an affinity capture procedure to identify protein partners from human serum specifically interacting with this effector. The complex proteins/lipopolysaccharide was isolated and the protein partners were fractionated by gel electrophoresis and identified by mass spectrometry. This procedure proved to be very effective in specifically binding proteins functionally correlated with the biological role of LPS. Proteins specifically bound to LPS essentially gathered within two functional groups, regulation of the complement system (factor H, C4b, C4BP, and alpha 2 macroglobulin) and inhibition of LPS-induced inflammation (HRG and Apolipoproteins). The reported strategy might have important applications in the elucidation of biological mechanisms involved in the LPSs-mediated molecular recognition and anti-infection responses.
Homing of colorectal cancer (CRC) cells to the liver is a non-random process driven by a crosstalk between tumour cells and components of the host tissue. Here we report the isolation of a liver metastasis-specific peptide ligand (CGIYRLRSC) that binds a complex of E-cadherin and α(6) integrin on the surface of CRC cells. We identify angiopoietin-like 6 protein as a peptide-mimicked natural ligand enriched in hepatic blood vessels of CRC patients. We demonstrate that an interaction between hepatic angiopoietin-like 6 and tumoural α(6) integrin/E-cadherin drives liver homing and colonization by CRC cells, and that CGIYRLRSC inhibits liver metastasis through interference with this ligand/receptor system. Our results indicate a mechanism for metastasis whereby a soluble factor accumulated in normal vessels functions as a specific ligand for circulating cancer cells. Consistently, we show that high amounts of coexpressed α(6) integrin and E-cadherin in primary tumours represent a poor prognostic factor for patients with advanced CRC.
Myoglobin is an alpha-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues increases the susceptibility of the polypeptide chain to misfold, causing amyloid aggregation under physiological condition, i.e., neutral pH and room temperature. The role played by tryptophanyl residues in driving the folding process has been investigated by examining three mutated apomyoglobins, i.e., W7F, W14F, and the amyloid-forming mutant W7FW14F, by an integrated approach based on far-ultraviolet (UV) circular dichroism (CD) analysis, fluorescence spectroscopy, and complementary proteolysis. Particular attention has been devoted to examine the conformational and dynamic properties of the equilibrium intermediate formed at pH 4.0, since it represents the early organized structure from which the native fold originates. The results show that the W → F substitutions at position 7 and 14 differently affect the structural organization of the AGH subdomain of apomyoglobin. The combined effect of the two substitutions in the double mutant impairs the formation of native-like contacts and favors interchain interactions, leading to protein aggregation and amyloid formation.
The innate immune system of insects consists of humoural and cellular responses that provide protection against invading pathogens and parasites. Defence reactions against these latter include encapsulation by immune cells and targeted melanin deposition, which is usually restricted to the surface of the foreign invader, to prevent systemic damage. Here we show that a protein produced by haemocytes of Heliothis virescens (Lepidoptera, Noctuidae) larvae, belonging to XendoU family, generates amyloid fibrils, which accumulate in large cisternae of the rough endoplasmic reticulum and are released upon immune challenge, to form a layer coating non-self objects entering the haemocoel. This amyloid layer acts as a molecular scaffold that promotes localised melanin synthesis and the adhesion of immune cells around the non-self intruder during encapsulation response. Our results demonstrate a new functional role for these protein aggregates that are commonly associated with severe human diseases. We predict that insects will offer new powerful experimental systems for studying inducible amyloidogenesis, which will likely provide fresh perspectives for its prevention.
In this study we report that, in response to proteasome inhibition, the E3-Ubiquitin ligase TRIM50 localizes to and promotes the recruitment and aggregation of polyubiquitinated proteins to the aggresome. Using Hdac6-deficient mouse embryo fibroblasts (MEF) we show that this localization is mediated by the histone deacetylase 6, HDAC6. Whereas Trim50-deficient MEFs allow pinpointing that the TRIM50 ubiquitin-ligase regulates the clearance of polyubiquitinated proteins localized to the aggresome. Finally we demonstrate that TRIM50 colocalizes, interacts with and increases the level of p62, a multifunctional adaptor protein implicated in various cellular processes including the autophagy clearance of polyubiquitinated protein aggregates. We speculate that when the proteasome activity is impaired, TRIM50 fails to drive its substrates to the proteasome-mediated degradation, and promotes their storage in the aggresome for successive clearance.