Unfolding of beta-lactoglobulin on the surface of polystyrene nanoparticles: experimental and computational approaches.

Structural changes ensuing from the non-covalent absorption of bovine beta-lactoglobulin (BLG) on the surface of polystyrene nanoparticles were investigated by using spectroscopic approaches, by assessing the reactivity of specific residues, and by limited proteolysis/mass spectrometry. Also, the immunoreactivity of absorbed and free BLG was compared. All these approaches indicated substantial rearrangements of the protein structure in the absorbed state, in spite of the reported structural rigidity of BLG. Changes made evident by experimental measurements were confirmed by computational approaches. These indicate that adsorption-related changes are most marked in the area between the main C-terminal alpha helix and the beta-barrel, and lead to full exposure of the thiol on Cys121 , consistent with experimental measurements. In the computational model of bound BLG, both Trp61 and Trp19 also move away from their neighboring quenchers and become solvent-exposed, as indicated by fluorescence measurement. Upon binding, the beta-barrel also loosens, with a substantial increase in immunoreactivity and with noticeable changes in the trypsinolytic pattern. The possible general significance of the structural changes reported here for non-covalently adsorbed BLG is discussed with respect to recognition events involving surface-bound proteins, as are aspects related to the carrier function(s) of BLG, and to its use as a common ingredient in many food systems.

Recombinant S. cerevisiae expressing Old Yellow Enzymes from non-conventional yeasts: an easy system for selective reduction of activated alkenes.

BACKGROUND: Old Yellow Enzymes (OYEs) are flavin-dependent enoate reductases (EC 1.6.99.1) that catalyze the stereoselective hydrogenation of electron-poor alkenes. Their ability to generate up to two stereocenters by the trans-hydrogenation of the C = C double bond is highly demanded in asymmetric synthesis. Isolated redox enzymes utilization require the addition of cofactors and systems for their regeneration. Microbial whole-cells may represent a valid alternative combining desired enzymatic activity and efficient cofactor regeneration. Considerable efforts were addressed at developing novel whole-cell OYE biocatalysts, based on recombinant Saccharomyces cerevisiae expressing OYE genes. RESULTS: Recombinant S. cerevisiae BY4741∆Oye2 strains, lacking endogenous OYE and expressing nine separate OYE genes from non-conventional yeasts, were used as whole-cell biocatalysts to reduce substrates with an electron-poor double bond activated by different electron-withdrawing groups. Ketoisophorone, α-methyl-trans-cinnamaldehyde, and trans-ß-methyl-ß-nitrostyrene were successfully reduced with high rates and selectivity. A series of four alkyl-substituted cyclohex-2-enones was tested to check the versatility and efficiency of the biocatalysts. Reduction of double bond occurred with high rates and enantioselectivity, except for 3,5,5-trimethyl-2-cyclohexenone. DFT (density functional theory) computational studies were performed to investigate whether the steric hindrance and/or the electronic properties of the substrates were crucial for reactivity. The three-dimensional structure of enoate reductases from Kluyveromyces lodderae and Candida castellii, predicted through comparative modeling, resulted similar to that of S. cerevisiae OYE2 and revealed the key role of Trp116 both in substrate specificity and stereocontrol. All the modeling studies indicate that steric hindrance was a major determinant in the enzyme reactivity. CONCLUSIONS: The OYE biocatalysts, based on recombinant S. cerevisiae expressing OYE genes from non-conventional yeasts, were able to differently reduce the activated double bond of enones, enals and nitro-olefins, exhibiting a wide range of substrate specificity. Moreover whole-cells biocatalysts bypassed the necessity of the cofactor recycling and, tuning reaction parameters, allowed the synthetic exploitation of endogenous carbonyl reductases. Molecular modeling studies highlighted key structural features for further improvement of catalytic properties of OYE enzymes.

Inflammatory serum proteome pattern in mice fed a high-fat diet.

To investigate the influence of diet on serum protein pattern, mice were fed for 8 weeks either control chow or a high-fat diet (containing 21 % w/w milk fat and 0.2 % w/w cholesterol); sera were collected and analyzed by 2-DE. The main positive acute-phase reactant proteins, haptoglobin and hemopexin, were significantly up-regulated in animals receiving the high-fat diet. Data on all other proteins also pointed to an inflammatory condition in these animals. The largest change in concentration was observed for carboxylesterase N, a circulating enzyme seldom connected with lipid metabolism in earlier reports. These observations agree with the notion of a link between diet-induced hyperlipidemia and the inflammatory component of its cardiovascular sequels in humans, but the effects in the experimental animals are massive and obviously affect most of the major serum proteins.

Neglected markers: altered serum proteome in murine models of disease.

More than a decade ago our groups pioneered the analysis of serum proteins of laboratory animals with up-to-date proteomic techniques. We were, and still are, convinced that conforming animal procedures to the minimally invasive approaches typical of clinical biochemistry focuses attention on the actual conditions under which any finding arrived at on animal models of disease may eventually be applied to human patients for screening/diagnosis. We are also convinced that, besides the proteins present in trace level as a result of tissue leakage during disorders affecting specific peripheral organs, changes in the concentration of some of the major serum proteins as part of an acute-phase response may be taken as biological end-points during a number of experimental procedures. When reviewing literature data about proteomic investigations on plasma or serum of mice, we realized that not much work has been done in the direction we favor. In addition, we noticed that sometimes information about serum proteome has been coarsely treated and in a few cases even misunderstood/misused. In the following, we present current findings on serum/plasma proteome of the laboratory mouse not only under control conditions and during an experimentally induced acute-phase reaction, but also in a number of models of disease, mainly related to cancer and to metabolic disorders.

Electrostatics of folded and unfolded bovine ß-lactoglobulin.

We report on electrophoretic, spectroscopic, and computational studies aimed at clarifying, at atomic resolution, the electrostatics of folded and unfolded bovine ß-lactoglobulin (BLG) with a detailed characterization of the specific aminoacids involved. The procedures we used involved denaturant gradient gel electrophoresis, isoelectric focusing, electrophoretic titration curves, circular dichroism and fluorescence spectra in the presence of increasing concentrations of urea (up to 8 M), electrostatics computations and low-mode molecular dynamics. Discrepancy between electrophoretic and spectroscopic evidence suggests that changes in mobility induced by urea are not just the result of changes in gyration radius upon unfolding. Electrophoretic titration curves run across a pH range of 3.5-9 in the presence of urea suggest that more than one aminoacid residue may have anomalous pKa value in native BLG. Detailed computational studies indicate a shift in pKa of Glu44, Glu89, and Glu114, mainly due to changes in global and local desolvation. For His161, the formation of hydrogen bond(s) could add up to desolvation contributions. However, since His161 is at the C terminus, the end-effect associated to the solvated form strongly influences its pKa value with extreme variation between crystal structures on one side and NMR or low-mode molecular dynamics structures on the other. The urea concentration effective in BLG unfolding depends on pH, with higher stability of the protein at lower pH.

Wards in the keyway: amino acids with anomalous pK(a)s in calycins.

As a follow-up to our recent analysis of the electrostatics of bovine ß-lactoglobulin (Eberini et al. in Amino Acids 42:2019-2030, 2011), we investigated whether the occurrence in the native structure of calycins-the superfamily to which ß-lactoglobulin belongs-of amino acids with anomalous pK (a)s is an infrequent or, on the contrary, a common occurrence, and whether or not a general pattern may be recognized. To this aim, we randomly selected four calycins we had either purified from natural sources or prepared with recombinant DNA technologies during our previous and current structural and functional studies on this family. Their pIs vary over several pH units and their known functions are as diverse as carriers, enzymes, immunomodulators and/or extracellular chaperones. In our survey, we used both in silico prediction methods and in vitro procedures, such as isoelectric focusing, electrophoretic titration curves and spectroscopic techniques. By comparing the results under native conditions (no exposure of the proteins to chaotropic agents) to those after protein unfolding (in the presence of 8 M urea), a shift is observed in the pK (a) of at least one amino acid per protein, which results in a measurable change in pI. Three types of amino acids are involved: Cys, Glu, and His, their position varies along the calycin sequence. Although no common mechanism may thus be recognized, we hypothesize that the 'normalization' of anomalous pK (a)s may be the phenomenon that accompanies, and favors, structural rearrangements such as those involved in ligand binding by these proteins. An interesting, if anecdotal, validation to this view comes from the behavior of human retinol binding protein, for which the pI of the folded and liganded protein is intermediate between those of the folded and unliganded and of the unfolded protein forms. Likewise, both solid (from crystallography) and solution state (from CD spectroscopy) data confirm that the protein undergoes structural rearrangement upon retinol binding.

Energy matters: mitochondrial proteomics for biomedicine.

This review compiles results of medical relevance from mitochondrial proteomics, grouped either according to the type of disease - genetic or degenerative - or to the involved mechanism - oxidative stress or apoptosis. The findings are commented in the light of our current understanding of uniformity/variability in cell responses to different stimuli. Specificities in the conceptual and technical approaches to human mitochondrial proteomics are also outlined.

In silico identification of new ligands for GPR17: a promising therapeutic target for neurodegenerative diseases.

GPR17, a previously orphan receptor responding to both uracil nucleotides and cysteinyl-leukotrienes, has been proposed as a novel promising target for human neurodegenerative diseases. Here, in order to specifically identify novel potent ligands of GPR17, we first modeled in silico the receptor by using a multiple template approach, in which extracellular loops of the receptor, quite complex to treat, were modeled making reference to the most similar parts of all the class-A GPCRs crystallized so far. A high-throughput virtual screening exploration of GPR17 binding site with more than 130,000 lead-like compounds was then applied, followed by the wet functional and pharmacological validation of the top-scoring chemical structures. This approach revealed successful for the proposed aim, and allowed us to identify five agonists or partial agonists with very diverse chemical structure. None of these compounds could have been expected 'a priori' to act on a GPCR, and all of them behaved as much more potent ligands than GPR17 endogenous activators.

Identification of small molecules uncoupling the Notch::Jagged interaction through an integrated high-throughput screening.

Notch signaling plays an important role in several cellular functions including growth, differentiation, cell fate determination and stemness. Increased Notch activity has been linked to several types of cancers. Activation of Notch signaling is triggered by the interaction of Notch receptors (Notch1-4) with 5 different ligands (Jagged1-2 and Dll1-3-4) expressed on the neighbouring cells. Currently, indirect approaches to inhibit Notch signalling are based on the inhibition of the key step of Notch activation catalyzed by the γ-Secretase and thereby affect several different γ-Secretase substrates; conversely direct strategies get advantage of antibody-based drugs. The evidence that Jagged-mediated Notch activation plays a key role in cancer cell biology and the interplay with the surrounding microenvironment prompted us to develop a strategy to directly inhibit Notch activation by uncoupling its interaction with the Jagged, using an unprecedented approach based on small molecules. We set-up a screening strategy based on: protein::protein docking of crystallographic structures of Notch1 with Jagged1; comparative modelling of the Notch2:Jagged2 complex, based on the Notch1::Jagged1 complex; in silico high-throughput screening directed to Notch2 interaction surface of a virtual chemical library containing a large variety of molecules commercially available. The predicted pharmacological activity of the selected compounds was validated in vitro by a gene reporter and a viability assay. This approach led to the successful identification of two candidates with different anti-proliferative potency and efficacy. This represents the first step towards the rational identification of candidate molecules for the development of entirely novel drugs directed to inhibit Notch signaling in cancer.

Targeting Toxoplasma gondii CPSF3 as a new approach to control toxoplasmosis.

Toxoplasma gondii is an important food and waterborne pathogen causing toxoplasmosis, a potentially severe disease in immunocompromised or congenitally infected humans. Available therapeutic agents are limited by suboptimal efficacy and frequent side effects that can lead to treatment discontinuation. Here we report that the benzoxaborole AN3661 had potent in vitro activity against T. gondii Parasites selected to be resistant to AN3661 had mutations in TgCPSF3, which encodes a homologue of cleavage and polyadenylation specificity factor subunit 3 (CPSF-73 or CPSF3), an endonuclease involved in mRNA processing in eukaryotes. Point mutations in TgCPSF3 introduced into wild-type parasites using the CRISPR/Cas9 system recapitulated the resistance phenotype. Importantly, mice infected with T. gondii and treated orally with AN3661 did not develop any apparent illness, while untreated controls had lethal infections. Therefore, TgCPSF3 is a promising novel target of T. gondii that provides an opportunity for the development of anti-parasitic drugs.

All-Purpose Containers? Lipid-Binding Protein - Drug Interactions.

The combined use of in vitro (19F-NMR) and in silico (molecular docking) procedures demonstrates the affinity of a number of human calycins (lipid-binding proteins from ileum, liver, heart, adipose tissue and epidermis, and retinol-binding protein from intestine) for different drugs (mainly steroids and vastatins). Comparative evaluations on the complexes outline some of the features relevant for interaction (non-polar character of the drugs; amino acids and water molecules in the protein calyx most often involved in binding). Dissociation constants (Ki) for drugs typically lie in the same range as Ki for natural ligands; in most instances (different proteins and docking conditions), vastatins are the strongest interactors, with atorvastatin ranking top in half of the cases. The affinity of some calycins for some of the vastatins is in the order of magnitude of the drug Cmax after systemic administration in humans. The possible biological implications of this feature are discussed in connection with drug delivery parameters (route of administration, binding to carrier proteins, distribution to, and accumulation in, human tissues).

A computational approach to evaluate the androgenic affinity of iprodione, procymidone, vinclozolin and their metabolites.

Our research is aimed at devising and assessing a computational approach to evaluate the affinity of endocrine active substances (EASs) and their metabolites towards the ligand binding domain (LBD) of the androgen receptor (AR) in three distantly related species: human, rat, and zebrafish. We computed the affinity for all the selected molecules following a computational approach based on molecular modelling and docking. Three different classes of molecules with well-known endocrine activity (iprodione, procymidone, vinclozolin, and a selection of their metabolites) were evaluated. Our approach was demonstrated useful as the first step of chemical safety evaluation since ligand-target interaction is a necessary condition for exerting any biological effect. Moreover, a different sensitivity concerning AR LBD was computed for the tested species (rat being the least sensitive of the three). This evidence suggests that, in order not to over-/under-estimate the risks connected with the use of a chemical entity, further in vitro and/or in vivo tests should be carried out only after an accurate evaluation of the most suitable cellular system or animal species. The introduction of in silico approaches to evaluate hazard can accelerate discovery and innovation with a lower economic effort than with a fully wet strategy.

Nimesulide binding site in the B0AT1 (SLC6A19) amino acid transporter. Mechanism of inhibition revealed by proteoliposome transport assay and molecular modelling.

The effect of pharmaceutical compounds on the rat kidney B0AT1 transporter in proteoliposomes has been screened. To this aim, inhibition of the transport activity by the different compounds was measured on Na(+)-[(3)H]glutamine co-transport in the presence of membrane potential positive outside. Most of the tested drugs had no effect on the transport activity. Some compounds exhibited inhibitory effects from 5 to 88% at concentration of 300µM. Among the tested compounds, only the anti-inflammatory drug nimesulide exerted potent inhibition on B0AT1. From dose response analysis, an IC50 value of 23µM was found. Inhibition kinetic analysis was performed: noncompetitive inhibition of the glutamine transport was observed while competitive behaviour was found when the inhibition was analyzed with respect to the Na(+) concentration. Several molecules harbouring functional groups of nimesulide (analogues) were tested as inhibitors. None among the tested molecules has the capacity to inhibit the transport with the exception of the compound NS-398, whose chemical structure is very close to that of whole nimesulide. The IC50 for this compound was 131µM. Inhibition kinetics showed behaviour of NS-398 identical to that of nimesulide, i.e., noncompetitive inhibition respect to glutamine and competitive inhibition respect to Na(+). Molecular docking of nimesulide suggested that this drug is able to bind B0AT1 in an external dedicated binding site and that its binding produces a steric hindrance effect of the protein translocation path abolishing the transporter activity.

Distant homology modeling of LCAT and its validation through in silico targeting and in vitro and in vivo assays.

LCAT (lecithin:cholesterol acyltransferase) catalyzes the transacylation of a fatty acid of lecithin to cholesterol, generating a cholesteryl ester and lysolecithin. The knowledge of LCAT atomic structure and the identification of the amino acids relevant in controlling its structure and function are expected to be very helpful to understand the enzyme catalytic mechanism, as involved in HDL cholesterol metabolism. However - after an early report in the late '90 s - no recent advance has been made about LCAT three-dimensional structure. In this paper, we propose an LCAT atomistic model, built following the most up-to-date molecular modeling approaches, and exploiting newly solved crystallographic structures. LCAT shows the typical folding of the α/ß hydrolase superfamily, and its topology is characterized by a combination of α-helices covering a central 7-strand ß-sheet. LCAT presents a Ser/Asp/His catalytic triad with a peculiar geometry, which is shared with such other enzyme classes as lipases, proteases and esterases. Our proposed model was validated through different approaches. We evaluated the impact on LCAT structure of some point mutations close to the enzyme active site (Lys218Asn, Thr274Ala, Thr274Ile) and explained, at a molecular level, their phenotypic effects. Furthermore, we devised some LCAT modulators either designed through a de novo strategy or identified through a virtual high-throughput screening pipeline. The tested compounds were proven to be potent inhibitors of the enzyme activity.

Oxysterols act as promiscuous ligands of class-A GPCRs: in silico molecular modeling and in vitro validation.

According to classical pharmacology, each neurotransmitter/hormonal receptor, including GPCRs, is exclusively activated by highly specific ligands. However, recent evidence challenges this dogma. Oxysterols are produced at inflammatory sites and can surprisingly potently activate the Epstein Barr virus induced gene receptor-2 (EBI2), a GPCR involved in adaptive immune responses. Similarly, oxysterols promiscuously operate CXCR2, a chemokine receptor participating to immune reactions and cancer development. Both EBI2 and CXCR2 are phylogenetically related to GPR17, another GPCR implicated in inflammatory/immune neurodegenerative events. Here, we used an integrated approach combining comparative modeling, molecular docking and in vitro experiments to investigate their potential interactions with oxysterols. All three receptors share the binding site to allocate oxysterols with different local arrangements, higher sensitivity to specific oxysterols and different activation thresholds. Such differences may dictate the diverse biological effects induced by oxysterols, depending on production site, concentration, specific spatiotemporal features and receptor expression on targeted cells. Thus, EBI2, CXCR2 and GPR17 are promiscuously operated by oxysterols making this class of ligands a 'fil rouge' linking oxidative stress, inflammation and neurodegeneration. Such a transversal role may represent a conserved, "unspecific" (but selective) signaling mode, by which emergency molecules activate multiple receptors involved in inflammatory/immune responses.

In between - Proteomics of dog biological fluids.

Dogs are relevant to biomedical research in connection both to veterinary medicine for their role as pets and to basic investigations for their use as animal models in pathology, pharmacology and toxicology studies. Proteomic analysis of biological fluids is less advanced for dogs than for other animal species but a wealth of information has already been gathered, which we summarize in this review. As a remarkable feature, we also assemble here for due reference a number of 2-DE serum/plasma or urine patterns in health and disease; some of them correspond to unpublished data from the University of Veterinary Medicine Vienna.

A proteomic portrait of atherosclerosis.

We have arranged in this review the main evidence about proteome alterations in different cell and body fluid compartments along the progression of atherosclerosis. With time the description of the molecular phenomena is becoming more and more detailed yet the complex interrelationships among different factors are still elusive and previously neglected aspects (such as size for lipoprotein particles) emerge as not less relevant than the absolute abundance of individual proteins. Physiological limits to the kinetics of protein distribution through the biological fluids seem to hinder the early diagnosis of acute conditions through plasma analysis but suggest urine analysis as a workable alternative for the assessment of chronic conditions. The survey of literature data is complemented with a few unpublished results from our laboratories, featuring 2DE maps of the proteins extracted from human thrombi.

The oxysterol-CXCR2 axis plays a key role in the recruitment of tumor-promoting neutrophils.

Tumor-infiltrating immune cells can be conditioned by molecules released within the microenvironment to thwart antitumor immune responses, thereby facilitating tumor growth. Among immune cells, neutrophils play an important protumorigenic role by favoring neoangiogenesis and/or by suppressing antitumor immune responses. Tumor-derived oxysterols have recently been shown to favor tumor growth by inhibiting dendritic cell migration toward lymphoid organs. We report that tumor-derived oxysterols recruit protumor neutrophils in a liver X receptor (LXR)-independent, CXCR2-dependent manner, thus favoring tumor growth by promoting neoangiogenesis and immunosuppression. We demonstrate that interfering with the oxysterol-CXCR2 axis delays tumor growth and prolongs the overall survival of tumor-bearing mice. These results identify an unanticipated protumor function of the oxysterol-CXCR2 axis and a possible target for cancer therapy.

Proteomics of rat biological fluids--the tenth anniversary update.

In addition to a remarkable sexual dimorphism of serum and urine proteomes, the rat is exceptional for the wide difference between the serum patterns during an acute phase reaction vs baseline conditions. This feature allows monitoring with high sensitivity onset and progression of any pathological state that involves an inflammatory component as well as assessing the outcome of any therapeutic intervention. Reference maps have been defined for the proteomes of serum, urine, cerebrospinal fluid and bronchoalveolar lavage fluid. For both serum and urine most of the proteomic investigations have dealt with toxicological testing, for BALF with allergic or irritative reactions, whereas with CSF the main aim was the characterization of rat models of neurological disorders. When surveying more than ten years of literature on rat biological fluid proteomics, it is puzzling to see how seldom a consistent analytical plan has been set up for the comparative investigation on two or more types of sample, whether to fully characterize a disease model or to evaluate pharmacological/toxicological effects of a drug. It is also regrettable that in several cases only a negligible part of the results is discussed at length whereas most data are not even made known to the scientific community.