Superoxide Dismutase (SOD)-mimetic M40403 is protective in cell and fly models of paraquat toxicity: Implications for Parkinson disease

Parkinson disease is a debilitating and incurable neurodegenerative disorder affecting 3c1-2% of people over 65 years of age. Oxidative damage is considered to play a central role in the progression of Parkinson disease and strong evidence links chronic exposure to the pesticide paraquat with the incidence of the disease, most probably through the generation of oxidative damage. In this work, we demonstrated in human SH-SY5Y neuroblastoma cells the beneficial role of superoxide dismutase (SOD) enzymes against paraquat-induced toxicity, as well as the therapeutic potential of the SOD-mimetic compound M40403. Having verified the beneficial effects of superoxide dismutation in cells, we then evaluated the effects using Drosophila melanogaster as an in vivo model. Besides protecting against the oxidative damage induced by paraquat treatment, our data demonstrated that in Drosophila M40403 was able to compensate for the loss of endogenous SOD enzymes, acting both at a cytosolic and mitochondrial level. Because previous clinical trials have indicated that the M40403 molecule is well tolerated in humans, this study may have important implication for the treatment of Parkinson disease

Superoxide dismutating molecules rescue the toxic effects of PINK1 and parkin loss.

Reactive oxygen species exert important functions in regulating several cellular signalling pathways. However, an excessive accumulation of reactive oxygen species can perturb the redox homeostasis leading to oxidative stress, a condition which has been associated to many neurodegenerative disorders. Accordingly, alterations in the redox state of cells and mitochondrial homeostasis are established hallmarks in both familial and sporadic Parkinson's disease cases. PINK1 and Parkin are two genes which account for a large fraction of autosomal recessive early-onset forms of Parkinson's disease and are now firmly associated to both mitochondria and redox homeostasis. In this study we explored the hypothesis that superoxide anions participate in the generation of the Parkin and PINK1 associated phenotypic effect by testing the capacity of endogenous and exogenous superoxide dismutating molecules to rescue the toxic effects induced by loss of PINK1 or Parkin, in both cellular and fly models. Our results demonstrate the positive effect of an increased level of superoxide dismutase proteins on the pathological phenotypes, both in vitro and in vivo. A more pronounced effectiveness for mitochondrial SOD2 activity points to the superoxide radicals generated in the mitochondrial matrix as the prime suspect in the definition of the observed phenotypes. Moreover, we also demonstrate the efficacy of a SOD-mimetic compound, M40403, to partially ameliorate PINK1/Parkin phenotypes in vitro and in vivo. These results support the further exploration of SOD-mimetic compounds as a therapeutic strategy against Parkinson's disease

Primary structure and spectroscopic studies of Neurospora copper metallothionein.

When Neurospora crassa is grown in the presence of Cu(II) ions, it accumulates the metal with the concomitant synthesis of a low molecular weight copper-binding protein. The molecule binds 6 g-atom of copper per mole protein (Mr = 2200) and shows a striking sequence homology to the zinc- and cadmium-binding vertebrate metallothioneins. Absorption, circular dichroism, and electron paramagnetic resonance spectroscopy of Neurospora metallothionein indicate the copper to be bound to cysteinyl residues as a Cu(I)-thiolate complex of the polymeric mu-thiolate structure [Cu(I)6RS7]-. This metal-binding mode is also in agreement with the unusual luminescence of the protein. Spectral perturbation studies with HgCl2 and p-(chloromercuri)benzoate suggest that the 6 Cu(I)ions are coordinated to the seven cysteinyl residues in the form of a single metal cluster. Neurospora apometallothionein is also capable of binding in vivo group IIB metal ions [Zn(II), Cd(II), and Hg(II)] as well as paramagnetic Co(II) ions with an overall metal-to-protein stoichiometry of 3. The spectroscopic properties of the fully substituted forms are indicative of a distorted tetrahedral coordination. However, metal titration of the apoprotein shows the third metal ion to be differently coordinated than the other two metal ions. This difference can be explained by the presence of only seven cysteine residues in Neurospora metallothionein as opposed to nine cysteine residues in the three-metal cluster of the mammalian metallothioneins

Characterization of tungstophosphoric acid supported on MCM-41 mesoporous silica by using n-hexane cracking, benzene adsorption, and xray diffraction.

MCM-41 with all-silica composition was synthesized with unit cell parameters a=b=60Å. The material was characterized by X-ray powder diffraction and benzene adsorption. Combinations of unit cell parameter, benzene adsorption and crystal density determination have given values for the pore diameter and wall thickness in the ab-plane of the unit cell. The unit cell size shrank upon impregnation of the material with tungstophosphoric acid.The catalytic activity of tungstophosphoric acid (HPW) supported on the all-silica mesoporous MCM-41 material has been evaluated for the cracking of n-hexane. The catalyst showed a very high activity of n-hexane cracking at a temperature as low as 200oC, at very low space velocity. However, the catalyst deactivated rapidly due to coke deposition on the acid sites. At higher temperature the activity declined, most likely because of the heteropoly-acid thermal instability. Both X-ray powder diffraction and infra-red spectroscopy have shown evidence for the heteropolyacid instability starting at 300oC.The type of the solvent used in the impregnation affected the dispersion of HPW on the MCM-41 material as indicated by benzene adsorption measurement, but it did not influence the catalytic performance for nhexane cracking. In the case of aqueous solution, the benzene adsorption was lower than that of the parent MCM-41 material. The reduced adsorption capacity was probably due to the presence of the HPW at the pore mouth of the cylindrical channels, causing narrowing and blocking of some sections of the channels. Moreover, the adsorption isotherm showed a micropore characteristic supporting the pore narrowing proposal. In the case of using methanol solvent for the impregnation, the full adsorption capacity was retained, indicating a much higher dispersion of the HPW throughout the channels of the MCM-41 material. The benzene adsorption isotherm also showed the characteristic of mesoporosity and the adsorption capacity was not appreciably reduced.Characterization of the spent catalyst after n-hexane cracking test showed additional reduction in the benzene adsorption capacity for the aqueous impregnated catalyst. Again the methanol-impregnated catalyst had almost a full adsorption capacity

Characterization of tungstophosphoric acid supported on MCM-41 mesoporous silica by using n-hexane cracking, benzene adsorption, and xray diffraction.

MCM-41 with all-silica composition was synthesized with unit cell parameters a=b=60Å. The material was characterized by X-ray powder diffraction and benzene adsorption. Combinations of unit cell parameter, benzene adsorption and crystal density determination have given values for the pore diameter and wall thickness in the ab-plane of the unit cell. The unit cell size shrank upon impregnation of the material with tungstophosphoric acid.The catalytic activity of tungstophosphoric acid (HPW) supported on the all-silica mesoporous MCM-41 material has been evaluated for the cracking of n-hexane. The catalyst showed a very high activity of n-hexane cracking at a temperature as low as 200oC, at very low space velocity. However, the catalyst deactivated rapidly due to coke deposition on the acid sites. At higher temperature the activity declined, most likely because of the heteropoly-acid thermal instability. Both X-ray powder diffraction and infra-red spectroscopy have shown evidence for the heteropolyacid instability starting at 300oC.The type of the solvent used in the impregnation affected the dispersion of HPW on the MCM-41 material as indicated by benzene adsorption measurement, but it did not influence the catalytic performance for nhexane cracking. In the case of aqueous solution, the benzene adsorption was lower than that of the parent MCM-41 material. The reduced adsorption capacity was probably due to the presence of the HPW at the pore mouth of the cylindrical channels, causing narrowing and blocking of some sections of the channels. Moreover, the adsorption isotherm showed a micropore characteristic supporting the pore narrowing proposal. In the case of using methanol solvent for the impregnation, the full adsorption capacity was retained, indicating a much higher dispersion of the HPW throughout the channels of the MCM-41 material. The benzene adsorption isotherm also showed the characteristic of mesoporosity and the adsorption capacity was not appreciably reduced.Characterization of the spent catalyst after n-hexane cracking test showed additional reduction in the benzene adsorption capacity for the aqueous impregnated catalyst. Again the methanol-impregnated catalyst had almost a full adsorption capacity

Unveiling the binding and orientation of the antimicrobial peptide Plantaricin 149 in zwitterionic and negatively charged membranes

Antimicrobial peptides are a large group of natural compounds which present promising properties for the pharmaceutical and food industries, such as broad-spectrum activity, potential for use as natural preservatives, and reduced propensity for development of bacterial resistance. Plantaricin 149 (Pln149), isolated from Lactobacillus plantarum NRIC 149, is a peptide with the ability to inhibit bacteria from the Listeria and Staphylococcus genera, which is capable of promoting inhibition and disruption of yeast cells. In this study, the interactions of Pln149 with model membranes composed of zwitterionic and/or anionic phospholipids were investigated using a range of biophysical techniques, including isothermal titration calorimetry, surface tension measurements, synchrotron radiation circular dichroism spectroscopy, oriented circular dichroism spectroscopy, and optical microscopy, in order to elucidate their mode of interactions and provide insight into their functional roles. In anionic model membranes, the binding of Pln149 to lipid bilayers is an endothermic process and induces a helical secondary structure in the peptide. The helices bind parallel to the surfaces of lipid bilayers and can promote vesicle disruption, depending on peptide concentration. Although Pln149 has relatively low affinity for zwitterionic liposomes, it is able to adsorb at their lipid interfaces, disturbing the lipid packing, assuming a similar parallel helix structure with a surface-bound orientation, and promoting an increase in the membrane surface area. Such findings can explain the intriguing inhibitory action of Pln149 in yeast cells whose cell membranes have a significant zwitterionic lipid composition

Physico-chemical and antifungal properties of protease inhibitors from Acacia plumosa

This study was aimed at investigating the purification, biological activity, and some structural properties of three serine protease inhibitors isoforms, denoted ApTIA, ApTIB, and ApTIC from Acacia plumosa Lowe seeds. They were purified from the saline extract of the seeds, using Superdex-75 gel filtration and Mono-S ion exchange chromatography. They were further investigated by mass spectrometry, spectroscopic measurements, surface plasmon resonance, and inhibition assays with proteases and phytopathogenic fungi. The molecular mass of each isoform was estimated at ca. 20 kDa. Each contained two polypeptide chains linked by a disulfide bridge, with different isoelectric points that are acidic in nature. The N-terminal sequences of both chains indicated that they were Kunitz-type inhibitors. Circular dichroism (CD) analyses suggested the predominance of both disordered and beta-strands on ApTI isoforms secondary structure, as expected for β-II proteins. In addition, it was observed that the proteins were very stable, even at either extreme pH values or at high temperature, with denaturation midpoints close to 75 °C. The isoinhibitors could delay, up to 10 times, the blood coagulation time in vitro and inhibited action of trypsin (Ki 1.8 nM), α-chymotrypsin (Ki 10.3 nM) and kallikrein (Ki 0.58 μM). The binding of ApTIA, ApTIB, and ApTIC to trypsin and α-chymotrypsin, was investigated by surface plasmon resonance (SPR), this giving dissociation constants of 0.39, 0.56 and 0.56 nM with trypsin and 7.5, 6.9 and 3.5 nM with α-chymotrypsin, respectively. The growth profiles of Aspergillus niger, Thielaviopsis paradoxa and Colletotrichum sp. P10 were also inhibited by each isoforms. These three potent inhibitors from A. plumosa may therefore be of great interest as specific inhibitors to regulate proteolytic processes.FAPESPCNP

Deconstructing the DGAT1 enzyme: membrane interactions at substrate binding sites

Diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates