19 research outputs found
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. The binding site has been designed by computational modelling and the redesigned peptides have been synthesized and characterized by optical, fluorescence, electron spin resonance and nuclear magnetic resonance spectroscopy. The novel peptides, named Cupricyclin-1 and -2, bind one Cu2+ ion per molecule with nanomolar affinity. Cupricyclins display redox activity and catalyze the dismutation of superoxide anions with an activity comparable to that of non-peptidic superoxide dismutase mimics. We thus propose knottins as a novel scaffold for the design of catalytically-active mini metalloproteins
Heterologous expression and biochemical characterization of a highly active and stable chloroplastic CuZn-superoxide dismutase from Pisum sativum
Espressione della NOS nei neogasteropodi: prime evidenze biochimiche, distribuzione, parziale clonaggio e sequenziamento in Stramonita haemastoma (Muricidae)
Interleukin-1 beta induces ceruloplasmin and ferroportin-1 gene expression via MAP kinases and C/EBP beta, AP-1, and NF-kB activation
Previously, we demonstrated that IL-1 beta was able to increase iron efflux from glial cells through a coordinate induction of both ferroportin-1 (Fpn) and ceruloplasmin (Cp) synthesis. In this study, we have investigated the signaling pathways that are involved in the transcriptional activation of the Cp and Fpn. Our data show that the expression of Cp and Fpn in response to IL-1 beta requires the activation of MAP kinase pathways as a consequence of an IL-1 beta receptor stimulation. Moreover, we have observed that IL-1 beta regulates the expression of Cp and Fpn genes through (i) p38 MAPK-mediated activation of C/EBP transcription factor, (ii) ERK1/2-, JNK1- and partially p38 MAPK-dependent activation of AP-1, and through (iii) activation of NF-kappa B partially mediated by p38 MAPK. (C) 2010 Elsevier Ireland Ltd. All rights reserved
The essential role of Glu185 and Tyr354 residues in the ferroxidase activity of Saccharomyces cerevisiae Fet3
The structural determinants required for ferroxidase activity by the yeast multicopper oxidase Fet3 have been partially clarified by site-directed mutagenesis based on homology modeling. Glu-185 and Tyr-354 mere substituted with Ala and Phe, respectively, Fet3 E185A retained ca, 5% residual ferroxidase catalytic efficiency, and almost 40% oxidase efficiency. On the other hand, Fet3 Y354F exhibited 50% residual efficiency as a ferroxidase and more than 70%, as an oxidase, These results provide new insights in the mechanism of iron binding and oxidation by Fet3, establishing the essential role of Glu-185 and Tyr-354, and allowing to dissect ferroxidase from non-iron oxidase activity
Lactoferrin differently modulates the inflammatory response in epithelial models mimicking human inflammatory and infectious diseases
Conflicting data are reported on pro- or anti-inflammatory activity of bovine lactoferrin (bLf) in different cell models as phagocytes or epithelial cell lines infected by bacteria. Here we evaluated the bLf effect on epithelial models mimicking two human pathologies characterized by inflammation and infection with specific bacterial species. Primary bronchial epithelium from a cystic fibrosis (CF) patient and differentiated intestinal epithelial cells were infected with Pseudomonas aeruginosa LESB58 isolated from a CF patient and Adherent-Invasive Escherichia coli LF82 isolated from a Crohn's disease patient. Surprisingly, bLf significantly reduced the intracellular bacterial survival, but differently modulated the inflammatory response. These data lead us to hypothesize that bLf differentially acts depending on the epithelial model and infecting pathogen. To verify this hypothesis, we explored whether bLf could modulate ferroportin (Fpn), the only known cellular iron exporter from cells, that, by lowering the intracellular iron level, determines a non permissive environment for intracellular pathogens. Here, for the first time, we describe the bLf ability to up-regulate Fpn protein in infected epithelial models. Our data suggest that the mechanism underlying the bLf modulating activity on inflammatory response in epithelial cells is complex and the bLf involvement in modulating cellular iron homeostasis should be taken into account. © 2014 Springer Science+Business Media New York
Dominant mutants of ceruloplasmin impair the copper loading machinery in aceruloplasminemia
Ceruloplasmin impairs endothelium-dependent relaxation of rabbit aorta.
This study evaluated the effects of ceruloplasmin, the copper-containing blue oxidase of vertebrate plasma, on the relaxation of rabbit aortic rings after endothelial release of nitric oxide (NO). Ceruloplasmin at physiological, i.e., micromolar, concentrations inhibited relaxation of rabbit aorta induced by endothelium-dependent agonists hire acetylcholine or ADP, whereas it was ineffective toward vasodilation due to direct stimulation of smooth muscle cells by nitroglycerin. The effect was reversible and specific for native, fully metalated ceruloplasmin, since relaxation was not impaired by the heat-treated or metal-depleted derivatives. A trapping mechanism, involving a direct interaction of NO or other NO-containing species (like nitrosothiols and iron-dinitrosyls) with the copper sites and/or with the free thiol of ceruloplasmin, could be safely excluded on the basis of spectroscopic and chemical analyses of the protein exposed to authentic NO, nitrosothiols, or iron-dinitrosyls. The data presented in this paper constitute the first evidence of impairment of the endothelium-dependent vasodilatation by a plasma protein and may shed some Light on the still uncertain physiological role of ceruloplasmin
Titration of Cupricyclin-1 with CuSO<sub>4</sub> monitored by <sup>1</sup>H NMR.
<p>The molar ratio CuSO<sub>4</sub>/Cupricyclin-1 is reported on the left side of each spectrum.</p
<b>Table 2.</b> Superoxide dismutase activity of Cupriknottins.
<p><b>Table 2.</b> Superoxide dismutase activity of Cupriknottins.</p