3-O-Methyltolcapone and Its Lipophilic Analogues Are Potent Inhibitors of Transthyretin Amyloidogenesis with High Permeability and Low Toxicity.

Transthyretin (TTR) is an amyloidogenic homotetramer involved in the transport of thyroxine in blood and cerebrospinal fluid. To date, more than 130 TTR point mutations are known to destabilise the TTR tetramer, leading to its extracellular pathological aggregation accumulating in several organs, such as heart, peripheral and autonomic nerves, and leptomeninges. Tolcapone is an FDA-approved drug for Parkinson's disease that has been repurposed as a TTR stabiliser.

A Comparative Analysis of Punicalagin Interaction with PDIA1 and PDIA3 by Biochemical and Computational Approaches.

In a previous work, it was shown that punicalagin, an active ingredient of pomegranate, is able to bind to PDIA3 and inhibit its disulfide reductase activity. Here we provide evidence that punicalagin can also bind to PDIA1, the main expressed form of protein disulfide isomerase (PDI). In this comparative study, the affinity and the effect of punicalagin binding on each protein were evaluated, and a computational approach was used to identify putative binding sites.

Halting hyaluronidase activity with hyaluronan-based nanohydrogels: development of versatile injectable formulations.

Hyaluronan (HA) is among the most used biopolymers for viscosupplementation and dermocosmetics. However, the current injectable HA-based formulations present relevant limitations: I) unmodified HA is quickly degraded by endogenous hyaluronidases (HAase), resulting in short lasting properties; II) cross-linked HA, although shows enhanced stability against HAase, often contains toxic chemical cross-linkers. As such, herein, we present biocompatible self-assembled hyaluronan-cholesterol nanohydrogels (HA-CH NHs) able to bind to HAase and inhibit the enzyme activity in vitro, more efficiently than currently marketed HA-based cross-linked formulations (e.

Insights into the GTP-dependent allosteric control of c-di-GMP hydrolysis from the crystal structure of PA0575 protein from Pseudomonas aeruginosa.

Bis-(3'-5')-cyclic diguanylic acid (c-di-GMP) belongs to the class of cyclic dinucleotides, key carriers of cellular information in prokaryotic and eukaryotic signal transduction pathways. In bacteria, the intracellular levels of c-di-GMP and their complex physiological outputs are dynamically regulated by environmental and internal stimuli, which control the antagonistic activities of diguanylate cyclases (DGCs) and c-di-GMP specific phosphodiesterases (PDEs). Allostery is one of the major modulators of the c-di-GMP-dependent response.

Punicalagin, an active pomegranate component, is a new inhibitor of PDIA3 reductase activity.

Background: Polyphenolic compounds isolated from pomegranate fruit possess several pharmacological activities including anti-inflammatory, hepatoprotective, antigenotoxic and anticoagulant activities. The present work focuses the attention on PDIA3 interaction with punicalagin and ellagic acid, the most predominant components of pomegranate extracts. PDIA3, a member of the protein disulfide isomerase family involved in several cellular functions, is associated with different human diseases and it has the potential to be a pharmacological target.

Spectroscopic and calorimetric characterization of spermine oxidase and its association forms.

Spermine oxidase (SMOX) is a flavin-containing enzyme that oxidizes spermine to produce spermidine, 3-aminopropanaldehyde, and hydrogen peroxide. SMOX has been shown to play key roles in inflammation and carcinogenesis; indeed, it is differentially expressed in several human cancer types. Our previous investigation has revealed that SMOX purified after heterologous expression in actually consists of monomers, covalent homodimers, and other higher-order forms.

Behind Resveratrol Stabilization by Carboxymethylated (1,3/1,6)-β-d-Glucan: Does the Polyphenol Play a Role in Polymer Structural Organization?

Resveratrol stability in solution can be improved by combining the polyphenol with carboxymethylated (1,3/1,6)-β-d-glucan (CM-glucan), a carbohydrate polymer widely used in the food and pharmaceutical industries. The present work was undertaken to elucidate the mechanism behind this stabilizing effect. The supramolecular structural, physico-chemical and morphological features of the CM-glucan/resveratrol complex have been studied under different physical and chemical stimuli by means of spectroscopic techniques, microscopy and physical methods such as UV-Visible spectroscopy (UV-Vis), spectrofluorimetry, Circular Dichroism (CD), Infrared spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC), Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM).

Analysis of the interaction of calcitriol with the disulfide isomerase ERp57.

Calcitriol, the active form of vitamin D, can regulate the gene expression through the binding to the nuclear receptor VDR, but it can also display nongenomic actions, acting through a membrane-associated receptor, which has been discovered as the disulfide isomerase ERp57. The aim of our research is to identify the binding sites for calcitriol in ERp57 and to analyze their interaction. We first studied the interaction through bioinformatics and fluorimetric analyses.

Structural basis for PPAR partial or full activation revealed by a novel ligand binding mode.

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in the regulation of the metabolic homeostasis and therefore represent valuable therapeutic targets for the treatment of metabolic diseases. The development of more balanced drugs interacting with PPARs, devoid of the side-effects showed by the currently marketed PPARγ full agonists, is considered the major challenge for the pharmaceutical companies. Here we present a structure-based virtual screening approach that let us identify a novel PPAR pan-agonist with a very attractive activity profile and its crystal structure in the complex with PPARα and PPARγ, respectively.

Stability of spermine oxidase to thermal and chemical denaturation: comparison with bovine serum amine oxidase.

Spermine oxidase (SMOX) is a flavin-containing enzyme that specifically oxidizes spermine to produce spermidine, 3-aminopropanaldehyde and hydrogen peroxide. While no crystal structure is available for any mammalian SMOX, X-ray crystallography showed that the yeast Fms1 polyamine oxidase has a dimeric structure. Based on this scenario, we have investigated the quaternary structure of the SMOX protein by native gel electrophoresis, which revealed a composite gel band pattern, suggesting the formation of protein complexes.

Structural basis of functional diversification of the HD-GYP domain revealed by the Pseudomonas aeruginosa PA4781 protein, which displays an unselective bimetallic binding site.

Unlabelled: The intracellular level of the bacterial secondary messenger cyclic di-3',5'-GMP (c-di-GMP) is determined by a balance between its biosynthesis and degradation, the latter achieved via dedicated phosphodiesterases (PDEs) bearing a characteristic EAL or HD-GYP domain. We here report the crystal structure of PA4781, one of the three Pseudomonas aeruginosa HD-GYP proteins, which we have previously characterized in vitro. The structure shows a bimetallic active site whose metal binding mode is different from those of both HD-GYP PDEs characterized so far.

Structural basis of the transactivation deficiency of the human PPARγ F360L mutant associated with familial partial lipodystrophy.

The peroxisome proliferator-activated receptors (PPARs) are transcription factors that regulate glucose and lipid metabolism. The role of PPARs in several chronic diseases such as type 2 diabetes, obesity and atherosclerosis is well known and, for this reason, they are the targets of antidiabetic and hypolipidaemic drugs. In the last decade, some rare mutations in human PPARγ that might be associated with partial lipodystrophy, dyslipidaemia, insulin resistance and colon cancer have emerged.

The binding of silibinin to ERp57.

The flavonoid silibinin is known to intervene in many cellular processes involved in a variety of pathologies, thus appearing a promising therapeutic tool. The molecular mechanisms responsible for these activities, however, have not been clearly defined, and although some of its interactions with proteins have been identified, the relative affinities are often too low to appear relevant in vivo. Here we describe the interaction of silibinin with the protein disulfide isomerase ERp57, characterized by a submicromolar dissociation constant.

The phytotoxin fusicoccin differently regulates 14-3-3 proteins association to mode III targets.

Modulation of the interaction of regulatory 14-3-3 proteins to their physiological partners through small cell-permeant molecules is a promising strategy to control cellular processes where 14-3-3s are engaged. Here, we show that the fungal phytotoxin fusicoccin (FC), known to stabilize 14-3-3 association to the plant plasma membrane H(+) -ATPase, is able to stabilize 14-3-3 interaction to several client proteins with a mode III binding motif. Isothermal titration calorimetry analysis of the interaction between 14-3-3s and different peptides reproducing a mode III binding site demonstrated the FC ability to stimulate 14-3-3 the association.

Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of Mycobacterium tuberculosis.

Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing.

Open tubular columns containing the immobilized ligand binding domain of peroxisome proliferator-activated receptors α and γ for dual agonists characterization by frontal affinity chromatography with mass spectrometry detection.

The peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily. In the last years novel PPARs ligands have been identified and these include PPARα/γ dual agonists. To rapidly identify novel PPARs dual ligands, a robust binding assay amenable to high-throughput screening toward PPAR isoforms would be desirable.

Cupricyclins, novel redox-active metallopeptides based on conotoxins scaffold.

Highly stable natural scaffolds which tolerate multiple amino acid substitutions represent the ideal starting point for the application of rational redesign strategies to develop new catalysts of potential biomedical and biotechnological interest. The knottins family of disulphide-constrained peptides display the desired characteristics, being highly stable and characterized by hypervariability of the inter-cysteine loops. The potential of knottins as scaffolds for the design of novel copper-based biocatalysts has been tested by engineering a metal binding site on two different variants of an ω-conotoxin, a neurotoxic peptide belonging to the knottins family.

Synthesis, characterization and biological evaluation of ureidofibrate-like derivatives endowed with peroxisome proliferator-activated receptor activity.

A series of ureidofibrate-like derivatives was prepared and assayed for their PPAR functional activity. A calorimetric approach was used to characterize PPARγ-ligand interactions, and docking experiments and X-ray studies were performed to explain the observed potency and efficacy. R-1 and S-1 were selected to evaluate several aspects of their biological activity.

pH-, temperature- and ion-dependent oligomerization of Sulfolobus solfataricus recombinant amidase: a study with site-specific mutants.

Recombinant amidase from Sulfolobus solfataricus occurred as a dimer of 110 kDa comprising identical subunits. Only dimers were present at pHs above 7.0, but with decreasing pH, dimers associated into octamers, with complete oligomerization occurring at pH 3.

Thermal stability and redox properties of M. tuberculosis CuSOD.

The superoxide dismutase from Mycobacterium tuberculosis is the only Cu-containing superoxide dismutase that lacks zinc in the active site. To explore the structural properties of this unusual enzyme, we have investigated its stability by differential scanning calorimetry. We have found that the holo-enzyme is significantly more stable than the apo-protein or the partially metallated enzyme, but that its melting temperature is markedly lower than that of all the other characterized eukaryotic and prokaryotic Cu,Zn superoxide dismutases.

IFI16 and NM23 bind to a common DNA fragment both in the P53 and the cMYC gene promoters.

In the melanoma M14 cell line, we found that the antimetastatic protein NM23/nucleoside diphosphate kinase binds to the promoters of the oncogene cMYC and of P53, a gene often mutated in human cancer (Cervoni et al. [2006] J. Cell.

Regulatory and structural properties differentiating the chromosomal and the bacteriophage-associated Escherichia coli O157:H7 Cu, Zn superoxide dismutases.

Background: Highly virulent enterohemorrhagic Escherichia coli O157:H7 strains possess three sodC genes encoding for periplasmic Cu, Zn superoxide dismutases: sodC, which is identical to the gene present in non-pathogenic E. coli strains, and sodC-F1 and sodC-F2, two nearly identical genes located within lambdoid prophage sequences. The significance of this apparent sodC redundancy in E.

Protein phosphorylation corrects the folding defect of the neuroblastoma (S120G) mutant of human nucleoside diphosphate kinase A/Nm23-H1.

Human nucleoside diphosphate (NDP) kinase A is a 'house-keeping' enzyme essential for the synthesis of nonadenine nucleoside (and deoxynucleoside) 5'-triphosphate. It is involved in complex cellular regulatory functions including the control of metastatic tumour dissemination. The mutation S120G has been identified in high-grade neuroblastomas.

DNA sequences acting as binding sites for NM23/NDPK proteins in melanoma M14 cells.

We isolated and analyzed by chromatin immunoprecipitation (ChIP) in viable M14 cells DNA sequences bound to the antimetastatic protein nucleoside diphosphate kinase (NM23/NDPK) to shed some light on the nuclear functions of this protein and on the mechanism by which it acts in development and cancer. We assessed the presence of selected sequences from promoters of platelet-derived growth factor A (PDGF-A), c-myc, myeloperoxidase (MPO), CD11b, p53, WT1, CCR5, ING1, and NM23-H1 genes in the cross-linked complexes. Quantitative PCR (Q-PCR) showed a substantial enrichment of the correlated oncosuppressor genes p53, WT1, ING1, and NM23-H1 in the immunoprecipitated (IP) DNA.