960 research outputs found
NMR Interaction studies of Human Liver Fatty Acid Binding Protein with putative ligands and associated proteins
No full textLe molecole lipidiche, come acidi grassi, eicosanoidi e acidi biliari (BAs) sono essenziali per la sopravvivenza cellulare, in quanto possono fungere da fonti di energia, da substrati per la formazione di membrane oppure da molecole segnale per la regolazione del metabolismo cellulare. A causa della loro scarsa solubilità e della loro potenziale citotossicità necessitano di chaperon intracellulari che, legandole, aumentano la loro solubilità in solventi acquosi.
Le proteine che legano gli acidi grassi, FABPs (Fatty Acid Binding Proteins), appartengono alla famiglia delle iLBPs (intracellular lipid binding proteins), una classe di piccole proteine citoplasmatiche (di circa 14-15KDa) evolutivamente correlate fra loro, probabilmente implicate nel trasporto lipidico trans-cellulare. Le iLBP sono proteine versatili che partecipano ai processi nucleari e al mantenimento dell’omeostasi e del metabolismo lipidico; per questo motivo sono state scelte come bersagli di farmaci contro lo sviluppo di dislipidemie.
La FABP di fegato (LFABP), appartenente alla sotto-famiglia II delle FABP, è forse la più peculiare; a differenza degli altri membri può infatti accomodare, nella sua cavità idrofobica, due molecole di acidi grassi a lunga catena, ma anche una grande varietà di molecole idrofobiche come gli esteri dell’acil-CoA, fosfolipidi e acidi biliari.
Per le sue particolari caratteristiche e l'alta concentrazione che può raggiungere all'interno degli epatociti (1-5% delle proteine solubili totali), si è ipotizzato che la LFABP umana (HLFABP) sia implicata nello sviluppo dei parassiti malarici, durante la fase epatica della malattia. Questa fase è asintomatica e potrebbe fornire nuove strategie per arrestare l'infezione.
UIS3 è una piccola proteina trans-membrana, presumibilmente localizzata nella membrana vacuolare parassitofora (PVM), specificamente espressa negli sporozoiti infettivi ed essenziale per il loro sviluppo nella fase iniziale della malaria all’interno del fegato. Un saggio di tipo yeast two hybrid, basato sulla proteina UIS3 derivante dal parassita malarico dei roditori P. yoelii (Py-UIS3), ha permesso di identificare LFABP come possibile partner di interazione. Inoltre, in un lavoro del 2008 di Ashwani e collaboratori, venne riportata un’interazione diretta fra HLFABP e il dominio solubile di UIS3 di Plasmodium falciparum ( PfUIS3(130-229)).
Al fine di ottenere informazioni vincolanti ad un livello atomico di risoluzione, l'interazione tra HLFABP e PfUIS3(130-229) è stata analizzata in dettaglio tramite spettroscopia di Risonanza Magnetica Nucleare(NMR). Inoltre, anche l'associazione con fosfolipidi e acidi grassi è stata analizzata attraverso NMR. I nostri dati, tuttavia, non hanno evidenziato alcuna interazione di PfUIS3(130-229) con HLFABP e/o molecole lipidiche, indicando la necessità di ridefinire il modello attuale di importo di lipidi nei parassiti malarici mediato da LFABP.
Nella seconda parte di questo progetto di ricerca è stata analizzata in dettaglio l’interazione fra HLFABP e gli acidi grassi. L’NMR e la spettroscopia di massa (MS) sono state utilizzate per la prima volta insieme per caratterizzare questa associazione. Campioni di HLFABP in complesso con oleato (OA) e palmitato (PA) sono stati preparati in acqua e successivamente analizzati utilizzando la tecnologia ESI-MS (Electron Spray Ionization Mass Spectroscopy) per determinarne la specificità, la stechiometria e l'affinità relativa. I nostri dati sono concordi con la presenza di due siti di legame distinti con una diversa affinità per gli acidi grassi. Sono poi stati allestiti degli esperimenti di competizione, titolando la proteina sia con OA che con PA; i campioni sono stati analizzati tramite ESI-MS ed i dati indicano che OA e PA competono effettivamente per lo stesso sito di associazione all'interno della proteina e che HLFABP ha una maggiore affinità per gli acidi grassi instauri.
Successivamente abbiamo sfruttato la potenza delle titolazioni 13C-NMR per indagare sia l'interazione fra HLFABP e acidi grassi marcati in 13C, che lo stato di ionizzazione dei ligandi legati nella tasca idrofobica della proteina.
A livello globale, abbiamo sviluppato un metodo adatto per lo studio di altri membri della famiglia FABP, che, nonostante le loro dimensioni molto favorevoli, sono sistemi complessi per essere caratterizzati da una singola tecnica biofisica. Inoltre, questo metodo potrà essere applicato anche per lo studio di HLFABP in complesso con altri ligandi idrofobici.
Nell'ultima parte di questo lavoro ci siamo concentrati sull'interazione tra HLFABP e gli acidi biliari, molecole anfipatiche, che facilitano l'assorbimento dei lipidi, del colesterolo e di vitamine liposolubili nell'intestino tenue. Gli acidi biliari subiscono un riciclaggio tra l'intestino ed il fegato, chiamato "Circolazione enteroepatica", che consente il recupero di quasi il 95% di queste preziose molecole. Dal momento che un trasportatore di acidi biliari non è stato ancora stato identificato negli epatociti dei mammiferi, abbiamo esplorato l’associazione tra HLFABP e gli acidi biliari utilizzando una vasta gamma di tecniche biofisiche, coinvolgendo l'NMR, la spettroscopia di fluorescenza e la spettrometria di massa.
L'interazione tra HLFABP e l’acido glicocolico (GCA), il sale biliare più abbondante presente nel
fegato umano, è stato ampiamente esplorato mediante NMR.
L’NMR è una fra le spettroscopie più potenti e versatili per l'analisi molecolare, poiché
permette di caratterizzare la struttura delle macromolecole biologiche ed i loro complessi ad un livello atomico di risoluzione. Inoltre, l'NMR fornisce informazioni sulla dinamica proteica su una vasta gamma di scale dei tempi . Le dinamiche possono influenzare la velocità e la via di ripiegamento delle proteine, così come l’aggregazione, la catalisi e l’associazione ad un ligando attraverso l’adattamento indotto o la selezione conformazionale. Così, la determinazione delle dinamiche proteiche in soluzione è importante per comprendere l'intero spettro di funzioni macromolecolari svolte dalle proteine.
Esperimenti di titolazione NMR ed esperimenti omonucleari 1H-1H NOESY, eseguiti con diversi schemi di marcatura isotopica, suggeriscono che HLFABP è in grado di legare una sola molecola di GCA. Inoltre, per complementare i dati NMR, è stata eseguita un’analisi computazionale per calcolare la struttura del complesso, utilizzando il programma di docking HADDOCK.
Successivamente, per meglio definire il legame, sono stati acquisiti esperimenti di rilassamento 15N sul backbone di HLFABP nella sua forma apo ed in complesso sia con GCA che con OA e sono state ottenute dinamiche residuo-specifiche su una vasta scala di tempi, che va dai ns ai ms. I nostri dati indicano chiaramente chele dinamiche veloci (ps-ns) non vengono influenzate particolarmente dal binding, mentre le dinamiche lente (μs-ms) sono mantenute o accentuate dopo il legame. Infine sono stati eseguiti esperimenti di scambio idrogeno/deuterio e CLEANEX per monitorare le zone più protette della proteina dallo scambio col solvente in presenza ed in assenza dei ligandi. In presenza di GCA è stato poi osservato un aumento della stabilità proteica.
La spettroscopia di fluorescenza e l'NMR sono state anche utilizzate per caratterizzare l'interazione fra HLFABP e un pool di acidi biliari con diverse modalità di coniugazione e idrossilazione.
I dati NMR mostrano che HLFABP può interagire con un’ampia gamma di acidi biliari in modo complesso, attraverso la formazione di almeno uno stato attivato. In aggiunta, i nostri dati NMR suggeriscono che la struttura della proteina sia preformata per il legame delle diverse molecole idrofobiche. Infatti la rete di legami idrogeno non viene perturbata in modo significativo dall'aggiunta dei vari ligandi. Attraverso la spettroscopia NMR abbiamo poi dimostrato che HLFABP esiste come un insieme di conformeri in scambio veloce fra loro su una scala di tempi NMR.
La spettroscopia di fluorescenza è stata impiegata per calcolare l’affinità di HLFABP verso i vari acidi biliari utilizzati nello screening e attraverso un saggio di competizione, utilizzando il DAUDA come composto fluorescente, è stata calcolata un’affinità μM (da 0.6-7.5 μM) in accordo con quella calcolata tramite spettroscopia NMR. L’affinità maggiore è stata ottenuta per quegli acidi biliari con caratteristiche di idrofobicità maggiori.
Infine, la presenza di complessi eterotipici, formati da HLFABP in complesso sia con acidi biliari che con acidi grassi, è stata analizzata tramite NMR e ESI-MS.Lipidic molecules such as fatty acids (FAs), eicosanoids and bile salts (BAs) are essential for cell survival because they serve as metabolic energy sources, substrates for membranes and signaling molecules for metabolic regulation. Due to their low solubility and in some case cytotoxicity they necessitate intracellular chaperons, which bind them, thus increasing their aqueous solubility.
Fatty acid binding proteins (FABPs), belong to the Intracellular lipid binding proteins (iLBPs) family, a class of evolutionarily related small (14-15 KDa) cytoplasmic proteins, which have been proposed to be implicated in the transcellular transport of lipophilic ligands. Due to their participation in nuclear processes and lipid metabolism and homeostasis, they have recently been proposed as drug targets against the development of lipid related disorders.
Among the other family members, liver fatty acid binding protein (LFABP), belonging to subfamily II of FABPs, is the most unique. Differently from the other FABPs, LFABP is able to accommodate two long chain FAs (LCFAs) molecules, but also a wide range of hydrophobic ligands, such as BAs, eicosanoids, Acyl-CoA esters and phospholipids.
Due to its peculiar characteristics and the high concentration that it could reach within the hepatocytes (1-5% of total soluble proteins), human LFABP (HLFABP) has been hypothesized to be implicated in malaria parasites development, during the hepatic stage of the disease. The hepatic stage is asymptomatic and it would provide novel strategies for arresting the infection.
UIS3 is a small transmembrane protein, presumably localized to the parasitophorous vacuolar membrane (PVM), specifically expressed in infective sporozoites, and essential for early-stage liver development. A yeast two-hybrid screen based on UIS3 of the rodent malaria parasite P. yoelii (Py-UIS3) identified mouse LFABP as an interacting host protein. In addition, a work of 2008 of Ashwani and collaborators reports a direct interaction between HLFABP and the soluble domain of UIS3 from Plasmodium falciparum (PfUIS3(130-229)). In order to gain binding information at an atomic level of resolution, the interaction between HLFABP and PfUIS3(130-229) was analyzed in detail exploiting Nuclear Mgnetic Resonance (NMR) spectroscopy. Furthermore, the direct binding of phospholipids and FAs to UIS3 was also analyzed by NMR. However, our data did not show any interaction of PfUIS3(130-229) with HLFABP and lipid molecules, calling for a redefinition of the current model of FABP-mediated lipid import by human malaria parasites.
In the second part of this research project, we investigated in detail the interaction between HLFABP and fatty acids. For the first time NMR and MS spectroscopy were used in combination to characterize the binding between HLFABP and FAs. Samples of HLFABP in complex with palmitate (PA) or oleate (OA) were prepared in water and analyzed through Electron Spray Ionization mass spectroscopy (ESI-MS) to asses specificity, stoichiometry and relative affinity. Our data are in agreement with the presence of two distinct binding sites with different affinities for FAs. Competition experiments were also performed, titrating the protein with both PA and OA; OA and PA effectively compete for the same binding site within the protein binding pocket. Our results show that HLFABP has an higher affinity for unsaturated FAs. Successively, we exploited the power of 13C NMR titration data to investigate the interaction between HLFABP and 13C FAs and to get information about the ionization state of the bound ligands. Globally, we developed a method suitable for the study of other FABP family members, which, despite their favorable size are really challenging systems to be characterized by only a singular biophysical technique. In addition, this method could be also applied to the study of HLFABP in complex with other hydrophobic ligands.
In the last part of this work we focused on the interaction between HLFABP and BAs, amphipathic molecules, which in the small intestine facilitate the absorption of dietary lipids, cholesterol, and fatsoluble vitamins. BAs undergo a recycling pathway between the intestine and the liver, called “enterohepatic circulation”, which allows the recovery of almost the 95% of these precious molecules. Since a BA carrier within the hepatocytes has not been identified yet, we explored the interaction between HLFABP and BAs using a wide range of biophysical techniques, involving NMR, florescence and mass spectroscopy.
The interaction between HLFABP and glycocholic acid (GCA), the most abundant bile salt present in human liver, was extensively explored using NMR spectroscopy technique.
NMR is one of the most powerful and versatile spectroscopic technique for molecular analysis, since it allows to characterize biological macromolecules and their complexes at an atomic level of resolution. In addition, NMR provides information about protein dynamics on a wide range of time scales. Dynamics can affect the rate and pathway of protein folding, as well as misfolding and aggregation, catalysis and also binding via induced fit or conformational selection. Thus, the determination of protein dynamics in solution is important for realizing the full spectrum of macromolecular functions and for predicting and engineering protein behavior.
NMR titration experiments and 1H-1H homonuclear NOESY filtered experiments, performed with different labeling schemes, suggested that HLFABP is able to accommodate only one molecule of GCA. To complement NMR data, a model of the complex was obtained through a computational analysis, using the docking program HADDOCK.
To better characterize the binding, 15N backbone relaxation experiments on HLFABP in its apo form and in complex with either GCA or OA were recorded and residue specific dynamics, on a time scale ranging from ps to ms, were obtained. Fast time scale dynamics are not significantly perturbed upon OA/GCA addition, while slow motions are retained or enhanced upon binding. Hydrogen/deuterium exchange and CLEANEX experiments were also performed to get information on solvent accessibility to individual sites and to detect protein dynamics occurring on a much slower time scale. An increase in protein stability upon GCA/OA binding was observed.
For the first time NMR and fluorescence spectroscopy were combined on a BA pool, with different pattern of conjugation and hydroxylation.
The NMR data show that HLFABP can interact with a wide range of bile salts, through a complex pathway, involving at least one activated state. In addition the hydrogen bond network was not significantly perturbed upon ligand addition, indicating that the scaffold of the protein is preformed to bind such kind of ligands. Through NMR spectroscopy, we demonstrated also that HLFABP exists as an ensemble of conformers in fast exchange on an NMR time scale.
Fluorescence spectroscopy was used to calculate the affinity of HLFABP toward the different BAs employed in the study. An affinity in the μM range (spanning form 0.6-7.5μM) was obtained through DAUDA displacement assay, in close agreement with the ones calculated by NMR. The higher affinity was obtained for those BAs displaying high hydrophobic properties.
Finally we analyzed both by NMR and mass spectroscopy the existence of an heterotypic complex constituted by HLFABP in complex with both GCA and FAs, which is likely the conformation assumed by the protein in vivo
Evidence from NMR interaction studies challenges the hypothesis of direct lipid transfer from L-FABP to malaria sporozoite protein UIS3.
No full textUIS3 is a malaria parasite protein essential for liver stage development of Plasmodium species, presumably localized to the membrane of the parasitophorous vacuole formed in infected cells. It has been recently proposed that the soluble domain of UIS3 interacts with the host liver fatty acid binding protein (L-FABP), providing the parasite with a pathway for importing exogenous lipids required for its rapid growth. This finding may suggest novel strategies for arresting parasite development. In this study, we have investigated the interaction between human L-FABP and the soluble domain of Plasmodium falciparum UIS3 by NMR spectroscopy. The amino acid residuespecific analysis of 1H,15N-2D NMR spectra excluded the occurrence of a direct interaction between L-FABP (in its unbound and oleate-loaded forms) and Pf-UIS3. Furthermore, the spectrum of Pf-UIS3 was unchanged when oleate or phospholipids were added. The present investigation entails a reformulation of the current model of host-pathogen lipid transfer, possibly redirecting research for early intervention against malaria
A Florentine family in crisis: the Strozzi in the fifteenth century.
No full textPhDIn 1434 the Strozzi lineage had held a leading position in
Florentine society and government for at least one hundred and fifty
years, and was one of the largest and wealthiest of the city's
patrician lineages. The records of the catasto of 1427 and of the
scrutiny of 1433 are used to give a profile of the dominant social,
economic and political position of the Strozzi before the advent of
Medicean dominance. Their record of electoral success, and the
political and cultural leadership of influential and respected men
such as Palla di Nofri and Matteo di Simone, with other factors, put
the Strozzi amongst the greatest enemies of the victorious Medicean
regime of late 1434. The effects of political opposition and exile
on the lineage are examined both directly, through records of office-holding,
and indirectly through such indicators as marriage alliances
and household wealth. The two most prominent lines of the Strozzi
were exiled after 1434. Palla di Nofri's life and preoccupations in
his Paduan exile are examined, together with the lives of his sons;
none of these Strozzi ever returned to Florence, pursued as they were
by the enmity of the Medicean regime. The very different careers of
Filippo di Matteo and his brother Lorenzo are also examined: how they
succeeded in founding a lucrative bank in Naples, and in returning to
Florence to 'rebuild' (rifare) the position of the Strozzi lineage
there. The final decades of the century saw the Strozzi in an
economically more secure position, due substantially to the efforts
of Filippo. Except for a very small number of its members admitted
into the regime, most of the lineage is here shown to have remained
excluded from significant political office until after the fall of
the Medici regime in 1494
Peptidyl Prolyl Isomerase A Modulates the Liquid–Liquid Phase Separation of Proline-Rich IDPs
No full text[Image: see text] Liquid–liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs) and the action of molecular chaperones are tightly connected. An important class of molecular chaperones are peptidyl prolyl isomerases, which enhance the cis/trans-isomerization of proline. However, little is known about the impact of peptidyl prolyl isomerases on the LLPS of IDPs, which often contain many prolines. Here, we demonstrate that the most ubiquitous peptidyl prolyl isomerase, peptidyl prolyl isomerase A (PPIA), concentrates inside liquid-like droplets formed by the Alzheimer’s disease-associated protein tau, as well as inside RNA-induced coacervates of a proline–arginine dipeptide repeat protein. We further show that the recruitment of PPIA into the IDP droplets triggers their dissolution and return to a single mixed phase. NMR-based binding and proline isomerization studies provide insights into the mechanism of LLPS modulation. Together, the results establish a regulatory role of proline isomerases on the liquid–liquid phase separation of proline-rich IDPs
Occupational Asthma Due to Subtilisin: The Power of Specific Inhalation Challenge
No full textWe report the first confirmed case in Italy of occupational asthma caused by subtilisin in a healthcare worker involved in cleaning surgical instruments. The diagnosis was confirmed through a specific inhalation challenge (SIC) per-formed one year after the last exposure and after stopping inhaled corticosteroid therapy. An immediate reaction was observed after three minutes of exposure to diluted Neogiozym TM, with a 30% decrease in FEV1. This case highlights the diagnostic importance of SIC even for high-molecular-weight (HMW ) agents and emphasizes the need to re-consider occupational asthma as a complex, evolving disease influenced by both host and environmental factors. The patient exhibited a Type-2-high phenotype despite negative skin prick tests and normal IgE levels, reinforcing the value of dynamic, multi-marker assessment in occupational endotyping. This case supports the broader use of SIC in occupational settings where allergen-specific IgE testing is limited
Vues présentes sur le celtique cisalpin
No full textPresent views on Cisalpine Celtic.
During the last twenty years, the knowledge of Lepontic and of Italian Gaulish has been notably enriched by very important finds, and also by numerous studies. The author proposes a synthesis of all new linguistical and cultural data provided by epigraphy in this particular resort.Dans les vingt dernières années la connaissance du lépontique et du gaulois d’Italie a été notablement enrichie par certaines trouvailles fort importantes ainsi que par de nombreuses études. Tableau synthétique des nouvelles données linguistiques et culturelles apportées par l’épigraphie de ce domaine.Motta Filippo. Vues présentes sur le celtique cisalpin. In: Etudes Celtiques, vol. 29, 1992. Actes du IXe congrès international d'études celtiques. Paris, 7-12 juillet 1991. Deuxième partie : Linguistique, littératures. pp. 311-318
Key substrate recognition residues in the active site of cystathionine beta-synthase from Toxoplasma gondii
No full text: Cystathionine β-synthase (CBS) catalyzes the condensation of l-serine and l-homocysteine to give l-cystathionine in the transsulfuration pathway. Recently, a few O-acetylserine (l-OAS)-dependent CBSs (OCBSs) have been found in bacteria that can exclusively function with l-OAS. CBS from Toxoplasma gondii (TgCBS) can efficiently use both l-serine and l-OAS to form l-cystathionine. In this work, a series of site-specific variants substituting S84, Y160, and Y246 with hydrophobic residues found at the same positions in OCBSs was generated to explore the roles of the hydroxyl moieties of these residues as determinants of l-serine/l-OAS preference in TgCBS. We found that the S84A/Y160F/Y246V triple mutant behaved like an OCBS in terms of both substrate requirements, showing β-replacement activity only with l-OAS, and pH optimum, which is decreased by ~1 pH unit. Formation of a stable aminoacrylate upon reaction with l-serine is prevented by the triple mutation, indicating the importance of the H-bonds between the hydroxyl groups of Y160, Y246, and S84 with l-serine in formation of the intermediate. Analysis of the independent effect of each mutation on TgCBS activity and investigation of the protein-aminoacrylate complex structure allowed for the conclusion that the hydroxyl group of Y246 has a major, but not exclusive, role in controlling the l-serine preference by efficiently stabilizing its leaving group. These studies demonstrate that differences in substrate specificity of CBSs are controlled by natural variations in as few as three residue positions. A better understanding of substrate specificity in TgCBS will facilitate the design of new antimicrobial compounds
Conformational Plasticity of Centrin 1 from Toxoplasma gondii in Binding to the Centrosomal Protein SFI1
No full textCentrins are calcium (Ca(2+))-binding proteins that are involved in many cellular functions including centrosome regulation. A known cellular target of centrins is SFI1, a large centrosomal protein containing multiple repeats that represent centrin-binding motifs. Recently, a protein homologous to yeast and mammalian SFI1, denominated TgSFI1, which shares SFI1-repeat organization, was shown to colocalize at centrosomes with centrin 1 from Toxoplasma gondii (TgCEN1). However, the molecular details of the interaction between TgCEN1 and TgSFI1 remain largely unknown. Herein, combining different biophysical methods, including isothermal titration calorimetry, nuclear magnetic resonance, circular dichroism, and fluorescence spectroscopy, we determined the binding properties of TgCEN1 and its individual N- and C-terminal domains to synthetic peptides derived from distinct repeats of TgSFI1. Overall, our data indicate that the repeats in TgSFI1 constitute binding sites for TgCEN1, but the binding modes of TgCEN1 to the repeats differ appreciably in terms of binding affinity, Ca(2+) sensitivity, and lobe-specific interaction. These results suggest that TgCEN1 displays remarkable conformational plasticity, allowing for the distinct repeats in TgSFI1 to possess precise modes of TgCEN1 binding and regulation during Ca(2+) sensing, which appears to be crucial for the dynamic association of TgCEN1 with TgSFI1 in the centrosome architecture
Structural Basis for the Functional Diversity of Centrins: A Focus on Calcium Sensing Properties and Target Recognition
No full textCentrins are a family of small, EF hand-containing proteins that are found in all eukaryotes and are often complexed with centrosome-related structures. Since their discovery, centrins have attracted increasing interest due to their multiple, diverse cellular functions. Centrins are similar to calmodulin (CaM) in size, structure and domain organization, although in contrast to CaM, the majority of centrins possess at least one calcium (Ca2+) binding site that is non-functional, thus displaying large variance in Ca2+ sensing abilities that could support their functional versatility. In this review, we summarize current knowledge on centrins from both biophysical and structural perspectives with an emphasis on centrin-target interactions. In-depth analysis of the Ca2+ sensing properties of centrins and structures of centrins complexed with target proteins can provide useful insight into the mechanisms of the different functions of centrins and how these proteins contribute to the complexity of the Ca2+ signaling cascade. Moreover, it can help to better understand the functional redundancy of centrin isoforms and centrin-binding proteins
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