Academic literature on the topic 'ARTIFICIAL CELL, NMR, PROTEIN, FABP'

Human epidermal-type fatty acid-binding protein (E-FABP) belongs to a family of intracellular 14–15kDa lipid-binding proteins, whose functions have been associated with fatty acid signalling, cell growth, regulation and differentiation. As a contribution to understanding the structure—function relationship, we report in the present study features of its solution structure and backbone dynamics determined by NMR spectroscopy. Applying multi-dimensional high-resolution NMR techniques on unlabelled and 15N-enriched recombinant human E-FABP, the 1H and 15N resonance assignments were completed. On the basis of 2008 distance restraints, the three-dimensional solution structure of human E-FABP was subsequently obtained (backbone atom root-mean-square deviation of 0.92±0.11Å; where 1Å = 0.1nm), consisting mainly of 10 anti-parallel β-strands that form a β-barrel structure. 15N relaxation experiments (T1, T2 and heteronuclear nuclear Overhauser effects) at 500, 600 and 800MHz provided information on the internal dynamics of the protein backbone. Nearly all non-terminal backbone amide groups showed order parameters S2>0.8, with an average value of 0.88±0.04, suggesting a uniformly low backbone mobility in the nanosecond-to-picosecond time range. Moreover, hydrogen/deuterium exchange experiments indicated a direct correlation between the stability of the hydrogen-bonding network in the β-sheet structure and the conformational exchange in the millisecond-to-microsecond time range. The features of E-FABP backbone dynamics elaborated in the present study differ markedly from those of the phylogenetically closely related heart-type FABP and the more distantly related ileal lipid-binding protein, implying a strong interdependence with the overall protein stability and possibly also with the ligand-binding affinity for members of the lipid-binding protein family.

Nanometer scale Ca-deficient hydroxyapatite (nanoapatite) is a potential candidate as artificial bone substitute materials owing to its similarity to the bone with respect to composition, morphology and osteoclastic degradation or adsorbent materials for blood purification therapy to remove pathogenic substances. The initial biodegradation behaviors, the initial cell-material interaction and the protein adsorption properties of nanoapatite must depend on the microstructure. The purpose of this study is the preparation of nanoapatite particles and their structural characterization by using X-ray diffraction (XRD) and solid-state NMR spectroscopy. The nanoapatite particles were prepared by precipitation processing method, and the effects of magnesium ions on the precipitation of calcium phosphate were examined, because Mg ions are well-known to play a role of inhibition of crystal growth. The addition of Mg ions led to the precipitation of nanometer scale Ca-deficient apatite crystals having 1.33-1.63 of the molar ratio (Mg+Ca)/P. NMR analyses showed that the microstructure of Mg•HAp particles can be explained by a crystalline HAp core covered with a thin amorphous hydrated calcium phosphate layer.

Treatment options for infections caused by antimicrobial-resistant bacteria are rendered ineffective, and drug alternatives are needed—either from new chemical classes or drugs with new modes of action. Historically, natural products have been important contributors to drug discovery. In a recent study, the dimeric naphthopyrone lulworthinone produced by an obligate marine fungus in the family Lulworthiaceae was discovered. The observed potent antibacterial activity against Gram-positive bacteria, including several clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates, prompted this follow-up mode of action investigation. This paper aimed to characterize the antibacterial mode of action (MOA) of lulworthinone by combining in vitro assays, NMR experiments and microscopy. The results point to a MOA targeting the bacterial membrane, leading to improper cell division. Treatment with lulworthinone induced an upregulation of genes responding to cell envelope stress in Bacillus subtilis. Analysis of the membrane integrity and membrane potential indicated that lulworthinone targets the bacterial membrane without destroying it. This was supported by NMR experiments using artificial lipid bilayers. Fluorescence microscopy revealed that lulworthinone affects cell morphology and impedes the localization of the cell division protein FtsZ. Surface plasmon resonance and dynamic light scattering assays showed that this activity is linked with the compound‘s ability to form colloidal aggregates. Antibacterial agents acting at cell membranes are of special interest, as the development of bacterial resistance to such compounds is deemed more difficult to occur.

AbstractThe gut is of importance in the pathology of COVID-19 both as a route of infection, and gut dysfunction influencing the severity of disease. Systemic changes caused by SARS-CoV-2 gut infection include alterations in circulating levels of metabolites, nutrients and microbial products which alter immune and inflammatory responses. Circulating plasma markers for gut inflammation and damage such as zonulin, lipopolysaccharide and β-glycan increase in plasma along with severity of disease. However, Intestinal Fatty Acid Binding Protein / Fatty Acid Binding Protein 2 (I-FABP/FABP2), a widely used biomarker for gut cell death, has paradoxically been shown to be reduced in moderate to severe COVID-19. We also found this pattern in a pilot cohort of mild (n = 18) and moderately severe (n = 19) COVID-19 patients in Milan from March to June 2020. These patients were part of the first phase of COVID-19 in Europe and were therefore all unvaccinated. After exclusion of outliers, patients with more severe vs milder disease showed reduced FABP2 levels (median [IQR]) (124 [368] vs. 274 [558] pg/mL, P < 0.01). A reduction in NMR measured plasma relative lipid-CH3 levels approached significance (median [IQR]) (0.081 [0.011] vs. 0.073 [0.024], P = 0.06). Changes in circulating lipid levels are another feature commonly observed in severe COVID-19 and a weak positive correlation was observed in the more severe group between reduced FABP2 and reduced relative lipid-CH3 and lipid-CH2 levels. FABP2 is a key regulator of enterocyte lipid import, a process which is inhibited by gut SARS-CoV-2 infection. We propose that the reduced circulating FABP2 in moderate to severe COVID-19 is a marker of infected enterocyte functional change rather than gut damage, which could also contribute to the development of hypolipidemia in patients with more severe disease.

La biologia sintetica di solito sviluppa il disegno cellulare utilizzando strategie "bottom-up", dal basso verso l’alto, inserendo e/o cancellando geni provenienti da organismi esistenti. Invece, gli approcci "top-down", dall’alto verso il basso, potrebbero consentire il controllo di cellule viventi in un modo che non richiede manipolazione genetica diretta. Ad esempio, le cellule artificiali (cellule costruite in laboratorio, ovvero, cellule sintetiche che mimano le cellule naturali), possono essere utilizzate per "tradurre" segnali esterni che le cellule naturali possono "comprendere" senza modificarle geneticamente. Così, abbiamo sviluppato sistemi cellulari artificiali che inviano messaggi chimici specifici per le cellule naturali. Queste cellule artificiali ci hanno ispirato un'altra applicazione finalizzata alla comprensione della chimica delle proteine nei sistemi cito-mimetici. Il complesso ambiente cellulare modula significativamente il comportamento delle macromolecole, agendo sulla loro struttura, dinamica e stabilità. Studi basati su sistemi cellulari semplificati che mimano le cellule naturali potrebbero fornire importanti informazioni sulla chimica delle proteine in ambienti che imitano quello nativo. Una caratteristica distintiva dei sistemi cellulari è che il citoplasma è profondamente affollato con concentrazioni significative di macromolecole (50-400 g / L) che incidono su diverse proprietà delle proteine (per esempio la capacità di legare, le interazioni proteina-proteina, il folding, ecc). In questa tesi di dottorato, due proteine citosoliche piccole ma dinamiche, sono state studiate all'interno di ambienti cellulari artificiali mediante spettroscopia NMR: la proteina del fegato umano che lega gli acidi grassi (LFABP) e la proteina dell’intestino umano che lega gli acidi biliari (IBABP), entrambe appartenenti alla famiglia di proteine intracellulari leganti acidi grassi (iLBPs). L'ambiente affollato è stato imitato utilizzando agenti sintetici (PEG, Ficoll o Destrano) e bio-macromolecule (BSA, Lisozima o Ubiquitina) nel range di concentrazioni 50-300 g/L. È stato osservato che Ficoll e/o Destrano sono relativamente inerti. Invece, è stato dimostrato che BSA e Lisozima causano delle interazioni non specifiche e specifiche, rispettivamente, con le proteine oggetto di studio. Le interazioni causate da PEG con le proteine non possono essere descritte solo quantitativamente in termini di volume escluso infatti i nostri risultati confermano la precedente constatazione che il PEG ha tendenza a interagire con le proteine. Inoltre, anche i lisati di E. coli e i sistemi cellulari artificiali come lo sono le emulsioni acqua-in-olio e i liposomi, sono stati utilizzati per simulare l'ambiente cellulare complesso e ristretto dato che le membrane lipidiche delimitano l’ambiente citosolico. Oltre alle proprietà strutturali e dinamiche, abbiamo studiato il meccanismo di legame di LFABP agli acidi grassi, in condizioni di affollamento macromolecolari.
Synthetic biology approaches usually develop cellular design using “bottom-up” strategies, inserting and deleting genes from existing organisms. Instead, “top-down” approaches could allow controlling living cells in a manner that does not require direct genetic modification. For example, artificial cells (laboratory-built cells, namely cell-like systems), can be used to “translate” external signals that the natural cells can “understand” without genetic modifications. Thus, we have developed artificial cellular mimics that send specific chemical messages to natural cells. These artificial cells inspired us another application aimed at understanding protein chemistry in cytomimetic systems. The complex cellular environment significantly modulates the behavior of macromolecules, affecting their structure, dynamics, and stability. Studies based on simplified cell mimics could provide important insights into protein chemistry in native-like environments. A distinctive feature of cellular systems is that the cytoplasmic medium is deeply crowded with significant concentrations of macromolecules (50-400 g/L) which affect several protein attributes (i.e. ligand binding, protein-protein interaction, folding, etc.). In this PhD thesis, two cytosolic, small, dynamic proteins were studied within an artificial cell environment by NMR Spectroscopy: human liver fatty acid binding protein (LFABP) and human ileal bile acid binding protein (IBABP), belonging to the intracellular lipid binding proteins (iLBPs). The crowded environment was mimicked by use of synthetic crowding agents (PEG, Ficoll or Dextran) and biomacromolecules (BSA, Lysozyme or Ubiquitin) in the range of concentrations 50-300 g/L. It was observed that Ficoll and/or Dextran are relatively inert. Instead, BSA and Lysozyme engaged in non-specific and specific interactions, respectively, with the test proteins. PEG interactions with proteins cannot be described quantitatively in terms of excluded volume alone and our results confirm the previous finding that PEG has tendency to interact with proteins. Moreover, also E. coli lysates and artificial cell systems such as water-in-oil emulsions and liposomes, were used to mimic the complex cellular environment and the restricted, lipid-bounded cytosolic milieu. In addition to structural and dynamic attributes, we investigated the lipid binding mechanism of LFABP under macromolecular crowding conditions.