42 research outputs found

    NMR Structural and interaction studies of bile acid binding proteinscomplexed with physiological ligands and bile acid-derived contrastagents

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    Il progetto svolto durante il mio Dottorato di Ricerca riguarda essenzialmente la cartterizzazione strutturale di complessi proteinalegante tramite spettroscopia di Risonanza Magnetica Nucleare (NMR). Studiare le interazioni proteiche a livello molecolare \ue8 di fondamentale importanza per acquisire informazioni su molti processi biologici, quali lo sviluppo di malattie nell\u2019uomo o il relativo disegno di farmaci. La Risonanza Magnetica Nucleare \ue8 una tecnica particolarmente adatta per la caratterizzazione, a livello atomico, delle interazioni tra proteine e altre molecole in soluzione. La Risonanza Magnetica Nucleare pu\uf2 essere utilizzata per valutare gli aspetti strutturali, termodinamici e cinetici di una reazione di interazione proteina-legante e pu\uf2 rilevare anche le interazioni pi\uf9 deboli. In questo contesto, durante il mio Dottorato di Ricerca ho affrontato l'indagine strutturale e lo studio dei determinanti molecolari dell\u2019interazione di differenti proteine, le bile acid binding proteins (BABPs) in complesso sia con i loro leganti fisiologici sia legate a farmaci sintetitici. Le BABPs sono piccole proteine citosoliche che svolgono la loro funzione negli epatociti ed enterociti, dove agiscono come trasportatori intracellulari degli acidi biliari, permettendo il loro ricircolo tramite la circolazione enteroepatica. Nella prima prima parte di questa tesi viene descritta la determinazione strutturale tramite NMR della BABP di fegato di pollo (cL-BABP) in complesso con l\u2019addotto acido biliare-Gd-DTPA, un potenziale agente di contrasto epatospecifo per la Risonanza Magnetica per Immagine (MRI). Il razionale di questo studio deriva da una ricerca di nuovi agenti di contrasto epatospecifici per la discriminazione e la diagnosi di lesioni focali o tumori nel fegato, tramite la Risonanza Magnetica per Immagine. La seconda parte di questa tesi \ue8 focalizzata sulla caratterizzazione delle interazioni della BABP ileale di pollo (cI-BABP) con acidi biliari. Questo studio \ue8 stato svolto utilizzando diversi approcci, cos\uec come la spettroscopia NMR e misure calorimetriche. La terza parte del mio lavoro di tesi si basa sulla determinazione strutturale tramite NMR del complesso ternario tra la CI-BABP e due molecole di acido glicochenodeossicolico (GCDA). Infine, un singolo e doppio mutante della proteina CI-BABP (A101S e H99Q/A101S) sono stati prodotti per studiare i determinanti molecolari della cooperativit\ue0 di legame. I dati ottenuti permettono di capire le basi chimiche del trasporto intracellulare degli acidi biliari. Questa conoscenza pu\uf2 aprire nuove vie per l'esplorazione di strategie di prevenzione e trattamento delle malattie metaboliche.The research project of my PhD concerns essentially the Nuclear Magnetic Resonance (NMR) structural studies of protein-ligand complexes. Studying protein interactions at the molecular level is crucial to the understanding of many biological processes, such as human diseases and drug design. NMR spectroscopy is particularly well suited to the investigation, at the atomic level, of the interactions between proteins and other molecules in solution. NMR can be used to evaluate the structural, thermodynamic and kinetic aspects of a binding reaction, even for weak protein-ligand interactions. In this contest, during my PhD I addressed the structural investigation and the study of the molecular determinants of binding of different bile acid binding proteins (BABPs) and their native ligands or synthetic drugs. BABPs are small cytosolic proteins that display their function in the hepatocytes and enterocytes where they act as bile acids transporters participating to the enterohepatic circulation. The first part of this thesis is related to the structural determination of chicken liver-BABP (cL-BABP) in complex with a bile acid-based gadolinium(III)-chelate, a potential hepatospecific contrast agent for magnetic resonance imaging (MRI). The rationale of this study derived from a search for new hepatospecific MRI contrast agents for the discrimination and diagnosis of focal lesions or hepatic malignancies. The second part of this thesis is focused on the characterization of the interactions of chicken ileal BABP (cI-BABP) with bile acids. This study has been performed using different approaches, such as NMR spectroscopy and calorimetric measurements. The third part of the my thesis work is based on the NMR structural determination of the ternary complex between cI-BABP and two molecules of glycochenodeoxycholic acid (GCDA). Finally, a single and double mutant of cI-BABP (A101S and H99Q/A101S) were produced to investigate the molecular determinants of binding cooperativity. The obtained data allow shedding light on the chemical basis of intracellular bile acid transport. This notion may open new avenues for exploration of strategies for prevention and treatment of metabolic diseases

    Integrated microscopy and metabolomics to test an innovative fluid dynamic system for skin explants in vitro

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    The in vitro models are receiving growing attention in studies on skin permeation, penetration, and irritancy, especially for the preclinical development of new transcutaneous drugs. However, synthetic membranes or cell cultures are unable to effectively mimic the permeability and absorption features of the cutaneous barrier. The use of explanted skin samples maintained in a fluid dynamic environment would make it possible for an in vitro experimentation closer to in vivo physiological conditions. To this aim, in the present study, we have modified a bioreactor designed for cell culture to host explanted skin samples. The preservation of the skin was evaluated by combining light, transmission, and scanning electron microscopy, for the histo/cytological characterization, with nuclear magnetic resonance spectroscopy, for the identification in the culture medium of metabolites indicative of the functional state of the explants. Our morphological and metabolomics results demonstrated that fluid dynamic conditions ameliorate significantly the structural and functional preservation of skin explants in comparison with conventional culture conditions. Our in vitro system is, therefore, reliable to test novel therapeutic agents intended for transdermal administration in skin samples from biopsies or surgical materials, providing predictive information suitable for focused in vivo research and reducing animal experimentation

    Molecular properties of human guanylate cyclase-activating protein 2 (GCAP2) and its retinal dystrophy-associated variant G157R

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    In murine and bovine photoreceptors, guanylate cyclase activating-protein 2 (GCAP2) activates retinal guanylate cyclases (GC) at low Ca2+ levels, thus contributing to the Ca2+/cGMP negative feedback on the cyclase together with its paralog GCAP1, which has the same function but different Ca2+ sensitivity. In humans, a GCAP2 missense mutation (G157R) has been associated with inherited-retinal degeneration (IRD) via an unknown molecular mechanism. Here, we characterized the biochemical properties of human GCAP2 and the G157R variant, focusing on its dimerization and the Ca2+/Mg2+-binding processes in the presence or absence of N-terminal myristoylation. We found that human GCAP2 and its bovine/murine orthologs significantly differ in terms of oligomeric properties, cation binding, and GC regulation. Myristoylated GCAP2 endothermically binds up to three Mg2+ ions with high affinity and forms a compact dimer that may reversibly dissociate in the presence of Ca2+. Conversely, non-myristoylated GCAP2 does not bind Mg2+ over the physiological range, and remains as a monomer in the absence of Ca2+. Both myristoylated and non-myristoylated GCAP2 bind Ca2+ with high affinity. At odds with GCAP1 and independently of myristoylation, human GCAP2 does not significantly activate retinal GC1 in a Ca2+-dependent fashion. The IRD-associated G157R variant is characterized by a partly misfolded, molten globule-like conformation with reduced affinity for cations, and is prone to form aggregates, likely mediated by hydrophobic interactions. Our findings suggest that GCAP2 in human photoreceptors might be mostly implicated in processes other than phototransduction, and suggest a possible molecular mechanism for G157R-associated IRD

    Chemical Characterization of a Collagen-Derived Protein Hydrolysate and Biostimulant Activity Assessment of Its Peptidic Components

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    Protein hydrolysates (PHs) are plant biostimulants consisting of oligopeptides and free amino acids exploited in agriculture to increase crop productivity. This work aimed to fractionate a commercial collagen-derived protein hydrolysate (CDPH) according to the molecular mass of the peptides and evaluate the bioactivity of different components. First, the CDPH was dialyzed and/or filtrated and analyzed on maize, showing that smaller compounds were particularly active in stimulating lateral root growth. The CDPH was then fractionated through fast protein liquid chromatography and tested on in vitro grown tomatoes proving that all the fractions were bioactive. Furthermore, these fractions were characterized by liquid chromatography-electrospray ionization- tandem mass spectrometry revealing a consensus sequence shared among the identified peptides. Based on this sequence, a synthetic peptide was produced. We assessed its structural similarity with the CDPH, the collagen, and polyproline type II helix by comparing the respective circular dichroism spectra and for the first time, we proved that a signature peptide was as bioactive as the whole CDPH

    Ozone activates the Nrf2 pathway and improves preservation of explanted adipose tissue in vitro

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    In clinical practice, administration of low ozone (O3) dosages is a complementary therapy for many diseases, due to the capability of O3 to elicit an antioxidant response through the Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2)-dependent pathway. Nrf2 is also involved in the adipogenic differentiation of mesenchymal stem cells, and low O3 concentrations have been shown to stimulate lipid accumulation in human adipose-derived adult stem cells in vitro. Thus, O3 treatment is a promising procedure to improve the survival of explanted adipose tissue, whose reabsorption after fat grafting is a major problem in regenerative medicine. In this context, we carried out a pilot study to explore the potential of mild O3 treatment in preserving explanted murine adipose tissue in vitro. Scanning and transmission electron microscopy, Western blot, real-time polymerase chain reaction and nuclear magnetic resonance spectroscopy were used. Exposure to low O3 concentrations down in the degradation of the explanted adipose tissue and induced a concomitant increase in the protein abundance of Nrf2 and in the expression of its target gene Hmox1. These findings provide a promising background for further studies aimed at the clinical application of O3 as an adjuvant treatment to improve fat engraftment

    Pentosan Polysulfate Inhibits Attachment and Infection by SARS-CoV-2 In Vitro: Insights into Structural Requirements for Binding

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    Two years since the outbreak of the novel coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic, there remain few clinically effective drugs to complement vaccines. One is the anticoagulant, heparin, which in 2004 was found able to inhibit invasion of SARS-CoV (CoV-1) and which has been employed during the current pandemic to prevent thromboembolic complications and moderate potentially damaging inflammation. Heparin has also been shown experimentally to inhibit SARS-CoV-2 attachment and infection in susceptible cells. At high therapeutic doses however, heparin increases the risk of bleeding and prolonged use can cause heparin-induced thrombocytopenia, a serious side effect. One alternative, with structural similarities to heparin, is the plant-derived, semi-synthetic polysaccharide, pentosan polysulfate (PPS). PPS is an established drug for the oral treatment of interstitial cystitis, is well-tolerated, and exhibits weaker anticoagulant effects than heparin. In an established Vero cell model, PPS and its fractions of varying molecular weights inhibited invasion by SARS-CoV-2. Intact PPS and its size-defined fractions were characterized by molecular weight distribution and chemical structure using nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry, then employed to explore the structural basis of interactions with SARS-CoV-2 spike protein receptor-binding domain (S1 RBD) and the inhibition of Vero cell invasion. PPS was as effective as unfractionated heparin, but more effective in inhibiting cell infection than low-molecular-weight heparin (on a weight/volume basis). Isothermal titration calorimetry and viral plaque-forming assays demonstrated size-dependent binding to S1 RBD and inhibition of Vero cell invasion, suggesting the potential application of PPS as a novel inhibitor of SARS-CoV-2 infection

    Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy

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    The use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications. In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxation rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products

    New Eu(III)-based complex with a C1 symmetric chiral ligand: A spectroscopic study

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    The synthesis of the new chiral ligand N, N'-bis[(2-quinolylmethyl]-cyclohexanediamine-N-tert-butylacetate N'-acetic acid and its Eu complex is presented. The spectroscopic investigation of the complex has been performed both in the solid state and in solution of ethanol. Thanks to the heteroaromatic quinoline ring, an efficient ligand to europium energy transfer (antenna effect) is possible. The low symmetry of the ligand (C-1 point symmetry) guarantees a strongly distorted Eu(III) geometric environment and the predominance of the D-5(0)-> F-7(2) hypersensitive band, in the Eu(III) luminescence emission spectrum. As for the majority of the Eu(III)-based coordination compounds, the observed lifetime of the D-5(0) excited state of the complex under investigation falls in the range of ms. Further, its good solubility in polar protic solvent (ethanol) and the excitation around 320 nm, which minimizes interfering excitation of chromophores in biological media, candidates this molecule for applications in the biomedical field

    The long variant of human ileal bile acid-binding protein associated with colorectal cancer exhibits sub-cellular localization and lipid binding behaviour distinct from those of the common isoform

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    Ileal bile acid-binding protein, IBABP, participates in the intracellular trafficking of bile salts and influences their signaling activities. The recently discovered variant, IBABP-L, bearing an N-terminal 49-amino acid extension, was found to be associated with colorectal cancer and to protect cancer cells from the cytotoxic effects of deoxycholate. However, the precise function and the molecular properties of this variant are currently unknown
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