Maria Teresa Cambria

Curcumin and the cellular stress response in free radical-related diseases

Free radicals play a main pathogenic role in several human diseases such as neurodegenerative disorders, diabetes, and cancer. Although there has been progress in treatment of these diseases, the development of important side effects may complicate the therapeutic course. Curcumin, a well known spice commonly used in India to make foods colored and flavored, is also used in traditional medicine to treat mild or moderate human diseases. In the recent years, a growing body of literature has unraveled the antioxidant, anticarcinogenic, and antinfectious activity of curcumin based on the ability of this compound to regulate a number of cellular signal transduction pathways. These promising data obtained in vitro are now being translated to the clinic and more than ten clinical trials are currently ongoing worldwide. This review outlines the biological activities of curcumin and discusses its potential use in the prevention and treatment of human diseases.

The hormetic role of dietary antioxidants in free radical-related diseases.

Regular consumption of cruciferous vegetables or spices is associated with a reduced incidence of cancer and reduction of markers for neurodegenerative damage. Furthermore, greater health benefit may be obtained from raw as opposed to cooked vegetables. Nutritional interventions, by increasing dietary intake of fruits and vegetables, can retard and even reverse age-related declines in brain function and cognitive performance. The mechanisms through which dietary supplementation with antioxidants may be useful to prevent free radical-related diseases is related to their ability to counteract toxic production of both reactive oxygen and nitrogen species, along with the up-regulation of vitagenes, such as members of the heat shock protein (Hsp) family heme oxygenase-1 and Hsp70. The most prominent dietary factor that affects the risk of many different chronic diseases is energy intake - excessive calorie intake increases the risk. Reducing energy intake by controlled caloric restriction or intermittent fasting increases lifespan and protects various tissues against diseases, in part, by hormetic mechanisms that increase cellular stress resistance. This biphasic dose-response relationship, referred to here as hormesis, display low-dose stimulation and a high-dose inhibition. Despite the current interest in hormesis by the toxicology community, quantitatively similar U-shaped dose responses have long been recognized by researchers to be involved with factors affecting memory, learning, and performance, as well as nutritional antioxidants and oxidative stress-mediated degenerative reactions. Dietary polyphenols present strong cytoprotective effects, however under uncontrolled nutritional supplementation gene induction effects and the interaction with detoxification responses can have negative consequences through the generation of more reactive and harmful intermediates.

Docking simulation and competitive experiments validate the interaction between the 2,5-xylidine inhibitor and Rigidoporus lignosus laccase.

Abstract Laccases are polyphenol oxidases which oxidize a broad range of reducing substrates, preferably phenolic compounds, and their use in biotechnological applications is increasing. Recently, the first X-ray structure of active laccase from white rot fungus Rigidoporus lignosus has been reported containing a full complement of copper ions. Comparison among selected fungal laccases of known 3D structure has shown that the Rigidoporus lignosus laccase has a very high similarity with the Trametes versicolor laccase that, being co-crystallized with 2,5-xylidine, shows a well defined binding pocket for the substrate. Global sequence alignment between Rigidoporus lignosus and Trametes versicolor laccases shows 73% of identity but, surprisingly, there is no identity and neither conservative substitutions between the residues composing the loops directly contacting the 2,5-xylidine. Moreover the structural alignment of these two enzymes identifies in these loops a striking structural similarity proposing the question if 2,5-xylidine may bind in same enzyme pocket. Here we report the protein-ligand docking simulation of 3D structure of Rigidoporus lignosus laccase and 2,5-xylidine. Docking simulation analyses show that spatial conformation of the two 2,5-xylidine binding pockets, despite differences in the residues directly contacting the ligand, may arrange a similar pocket that allows a comparable accommodation of the inhibitor. To validate these results the binding of 2,5-xylidine in the substrate cavity has been confirmed by kinetic competitive experiments.

Redox regulation of cellular stress response in multiple sclerosis

Multiple sclerosis (MS) is an autoimmune-mediated neurodegenerative disease with characteristic foci of inflammatory demyelination in the brain, spinal cord, and optic nerves. Recent studies have demonstrated not only that axonal damage and neuronal loss are significant pathologic components of MS, but that this neuronal damage is thought to cause the permanent neurologic disability often seen in MS patients. Emerging finding suggests that altered redox homeostasis and increased oxidative stress, primarily implicated in the pathogenesis of MS, are a trigger for activation of a brain stress response. Relevant to maintenance of redox homeostasis, integrated mechanisms controlled by vitagenes operate in brain in preserving neuronal survival during stressful conditions. Vitagenes encode for heat shock proteins (Hsp) Hsp32, Hsp70, the thioredoxin and the sirtuin protein systems. In the present study we assess stress response mechanisms in the CSF, plasma and lymphocytes of control patients compared to MS patients. We found that the levels of vitagenes Hsp72, Hsc70, HO-1, as well as oxidative stress markers carbonyls and hydroxynonenals were significantly higher in the blood and CSF of MS patients than in control patients. In addition, an increased expression of Trx and sirtuin 1, together with a decrease in the expression of TrxR were observed. Our data strongly support a pivotal role for redox homeostasis disruption in the pathogenesis of MS and, consistently with the notion that new therapies that prevent neurodegeneration through nonimmunomodulatory mechanisms can have a tremendous potential to work synergistically with current MS therapies, unravel important targets for new cytoprotective strategies.

Cellular stress response, sirtuins and UCP proteins in Alzheimer disease: role of vitagenes

Alzheimer’s Disease (AD) is a neurodegenerative disorder affecting up to one third of individuals reaching the age of 80. Different integrated responses exist in the brain to detect oxidative stress which is controlled by several genes termed Vitagenes. Vitagenes encode for cytoprotective heat shock proteins (Hsp), as well as thioredoxin, sirtuins and uncouple proteins (UCPs). In the present study we evaluate stress response mechanisms in plasma and lymphocytes of AD patients, as compared to controls, in order to provide evidence of an imbalance of oxidant/antioxidant mechanisms and oxidative damage in AD patients and the possible protective role of vitagenes.We found that the levels of Sirt-1 and Sirt-2 in AD lymphocytes were significantly higher than in control subjects. Interestingly, analysis of plasma showed in AD patients increased expression of Trx, a finding associated with reduced expression of UCP1, as compared to control group.This finding can open up new neuroprotective strategies, as molecules inducing this defense mechanisms can represent a therapeutic target to minimize the deleterious consequences associated to oxidative stress, such as in brain aging and neurodegenerative disorders.

Enhanced Laccase Production in White-Rot Fungus Rigidoporus lignosus by the Addition of Selected Phenolic and Aromatic Compounds

The white rot fungus Rigidoporus lignosus produces substantial amounts of extracellular laccase, a multicopper blue oxidase which is capable of oxidizing a wide range of organic substrates. Laccase production can be greatly enhanced in liquid cultures supplemented with various aromatic and phenolic compounds. The maximum enzyme activity was reached at the 21st or 24th day of fungal cultivation after the addition of inducers. The zymograms of extracellular fluid of culture preparation in the presence of inducers, at maximum activity day, revealed two bands with enzymatic activity, called Lac1 and Lac2, having different intensities. Lac2 band shows the higher intensity which changed with the different inducers. Laccase induction can be also obtained by adding to the culture medium olive mill wastewaters, which shows a high content of phenolic compounds

STRUCTURE–ACTIVITY RELATIONSHIP ON FUNGAL LACCASE FROM RIGIDOPORUS LIGNOSUS: A FOURIER-TRANSFORM INFRARED SPECTROSCOPIC STUDY

The structure and thermal stability of a laccase from Rigidoporus lignosus (Rl) was analysed by Fourier-transform infrared (FT-IR) spectroscopy. The enzyme was depleted of copper atoms, then part of the apoenzyme was re-metalled and these two forms of the protein were analysed as well. The enzymatic activity, lost by the removal of copper atoms, was restored in the re-metalled apoenzyme and resulted similar to that of native protein. The infrared data indicated that the enzyme contains a large amount of beta-sheets and a small content of alpha-helices, and it displayed a marked thermostability showing the T(m) at 92.5 degrees C. The apoenzyme and the re-metalled apoenzyme did not show remarkable differences in the secondary structure with respect to the native protein, but the thermal stability of the apoenzyme was dramatically reduced showing a T(m) close to 72 degrees C, while the re-metalled protein displayed the T(m) at 90 degrees C. These data indicate that copper atoms, beside their role in catalytic activity, play also an important role on the stabilisation of the structure of Rl laccase. About 35% of the polypeptide chain is buried and/or constitutes a particular compact structure, which, beside copper atoms, is probably involved in the high thermal stability of the protein. Another small part of the structure is particularly sensitive to high temperatures and it could be the cause of the loss of enzymatic activity when the temperature is raised above 45-50 degrees C.

Structure and Stability of the Insulin Dimer Investigated by Molecular Dynamics Simulation

Abstract Molecular dynamics simulation indicates that the dynamical behaviour of the insulin dimer is asymmetric. Atomic level knowledge of the interaction modes and protein conformation in the solvation state identifies dynamical structures, held by hydrogen bonds that stabilize, mainly in one monomer, the interaction between the chains. Dynamic cross-correlation analysis shows that the two insulin monomers behave asymmetrically and are almost independent. Solvation energy, calculated to evaluate the contribute of each interface residue to the dimer association pattern, well compares with the experimental association state found in protein mutants indicating that this parameter is an important factor to explain the association properties of mutated insulin dimers.

Spectroscopic and molecular dynamics simulation studies of the interaction of insulin with glucose

The interaction between monomeric insulin and monosaccharides has been investigated through circular dichroism, fluorescence spectroscopy and two dimensional nuclear magnetic resonance. CD spectra indicate that D-glucose interacts with monomeric insulin whereas D-galactose, D-mannose and 2-deoxy-D-glucose have a lower effect. Fluorescence emission was quenched at sugar concentrations of 5-10 mM. Titration with the different sugars produces a quenching of the tyrosine spectrum from which a binding free energy value for the insulin-sugar complexes has been evaluated. Transfer nuclear Overhauser enhancement NMR experiments indicate the existence of dipolar interactions at short interatomic distances between C-1 proton of D-glucose in the beta form and the monomeric insulin. Further, NMR total correlation spectra experiments revealed that the hormone is in the monomeric form and that upon addition of glucose no aggregation occurs. The interaction does not involve relevant changes in the secondary structure of insulin suggesting that the interaction occur at the side chain level. Molecular dynamics simulations and modeling studies, based on the dynamic fluctuations of potential binding moiety sidechains, argued from results of NMR spectroscopy, provide additional informations to locate the putative binding sites of D-glucose to insulin.

Silibinin Regulates Lipid Metabolism and Differentiation in Functional Human Adipocytes.

Silibinin, a natural plant flavonolignan is the main active constituent found in milk thistle (Silybum marianum). It is known to have hepatoprotective, anti-neoplastic effect, and suppresses lipid accumulation in adipocytes. Objective of this study was to investigate the effect of silibinin on adipogenic differentiation and thermogenic capacity of human adipose tissue derived mesenchymal stem cells. Silibinin (10 μM) treatment, either at the beginning or at the end of adipogenic differentiation, resulted in an increase of SIRT-1, PPARα, Pgc-1α, and UCPs gene expression. Moreover, silibinin administration resulted in a decrease of PPARγ, FABP4, FAS, and MEST/PEG1 gene expression during the differentiation, confirming that this compound is able to reduce fatty acid accumulation and adipocyte size. Our data showed that silibinin regulated adipocyte lipid metabolism, inducing thermogenesis and promoting a brown remodeling in adipocyte. Taken together, our findings suggest that silibinin increases UCPs expression by stimulation of SIRT1, PPARα, and Pgc-1α, improved metabolic parameters, decreased lipid mass leading to the formation of functional adipocytes.