New insights into structural determinants of prion protein folding and stability

Prions are the etiological agent of fatal neurodegenerative diseases called prion diseases or transmissible spongiform encephalopathies. These maladies can be sporadic, genetic or infectious disorders. Prions are due to post-translational modifications of the cellular prion protein leading to the formation of a \u3b2-sheet enriched conformer with altered biochemical properties. The molecular events causing prion formation in sporadic prion diseases are still elusive. Recently, we published a research elucidating the contribution of major structural determinants and environmental factors in prion protein folding and stability. Our study highlighted the crucial role of octarepeats in stabilizing prion protein; the presence of a highly enthalpically stable intermediate state in prion-susceptible species; and the role of disulfide bridge in preserving native fold thus avoiding the misfolding to a \u3b2-sheet enriched isoform. Taking advantage from these findings, in this work we present new insights into structural determinants of prion protein folding and stability

A rapid high-performance liquid chromatography-tandem mass spectrometry assay for unambiguous detection of different milk species employed in cheese manufacturing

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Prion protein and copper cooperatively protect neurons by modulating NMDA receptor through S-nitrosylation

AIMS: Several neurodegenerative disorders show alterations in glutamatergic synapses and increased susceptibility to excitotoxicity. Mounting evidence suggests a central role for the cellular prion protein (PrP(C)) in neuroprotection. Therefore, the loss of PrP(C) function occurring in prion disorders may contribute to the disease progression and neurodegeneration. Indeed, PrP(C) modulates N-methyl-d-aspartate receptors (NMDAR), thus preventing cell death. In this study, we show that PrP(C) and copper cooperatively inhibit NMDAR through S-nitrosylation, a post-translational modification resulting from the chemical reaction of nitric oxide (NO) with cysteines. RESULTS: Comparing wild-type Prnp (Prnp(+/+)) and PrP(C) knockout (Prnp(0/0)) mouse hippocampi, we found that GluN1 and GluN2A S-nitrosylation decrease in Prnp(0/0). Using organotypic hippocampal cultures, we found that copper chelation decreases NMDAR S-nitrosylation in Prnp(+/+) but not in Prnp(0/0). This suggests that PrP(C) requires copper to support the chemical reaction between NO and thiols. We explored PrP(C)-Cu neuroprotective role by evaluating neuron susceptibility to excitotoxicity in Prnp(+/+) and Prnp(0/0) cultures. We found that (i) PrP(C)-Cu modulates GluN2A-containing NMDAR, those inhibited by S-nitrosylation; (ii) PrP(C) and copper are interdependent to protect neurons from insults; (iii) neuronal NO synthase inhibition affects susceptibility in wild-type but not in Prnp(0/0), while (iv) the addition of a NO donor enhances Prnp(0/0) neurons survival. INNOVATION AND CONCLUSIONS: Our results show that PrP(C) and copper support NMDAR S-nitrosylation and cooperatively exert neuroprotection. In addition to NMDAR, PrP(C) may also favor the S-nitrosylation of other proteins. Therefore, this mechanism may be investigated in the context of the different cellular processes in which PrP(C) is involved

Modulation of Alpha-Synuclein Aggregation by Dopamine Analogs

The action of dopamine on the aggregation of the unstructured alpha-synuclein (α-syn) protein may be linked to the pathogenesis of Parkinson's disease. Dopamine and its oxidation derivatives may inhibit α-syn aggregation by non-covalent binding. Exploiting this fact, we applied an integrated computational and experimental approach to find alternative ligands that might modulate the fibrillization of α-syn. Ligands structurally and electrostatically similar to dopamine were screened from an established library. Five analogs were selected for in vitro experimentation from the similarity ranked list of analogs. Molecular dynamics simulations showed they were, like dopamine, binding non-covalently to α-syn and, although much weaker than dopamine, they shared some of its binding properties. In vitro fibrillization assays were performed on these five dopamine analogs. Consistent with our predictions, analyses by atomic force and transmission electron microscopy revealed that all of the selected ligands affected the aggregation process, albeit to a varying and lesser extent than dopamine, used as the control ligand. The in silico/in vitro approach presented here emerges as a possible strategy for identifying ligands interfering with such a complex process as the fibrillization of an unstructured protein

In Absence of the Cellular Prion Protein, Alterations in Copper Metabolism and Copper-Dependent Oxidase Activity Affect Iron Distribution

Essential elements as copper and iron modulate a wide range of physiological functions. Their metabolism is strictly regulated by cellular pathways, since dysregulation of metal homeostasis is responsible for many detrimental effects. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and prion diseases are characterized by alterations of metal ions. These neurodegenerative maladies involve proteins that bind metals and mediate their metabolism through not well-defined mechanisms. Prion protein, for instance, interacts with divalent cations via multiple metal-binding sites and it modulates several metal-dependent physiological functions, such as S-nitrosylation of NMDA receptors. In this work we focused on the effect of prion protein absence on copper and iron metabolism during development and adulthood. In particular, we investigated copper and iron functional values in serum and several organs such as liver, spleen, total brain and isolated hippocampus. Our results show that iron content is diminished in prion protein-null mouse serum, while it accumulates in liver and spleen. Our data suggest that these alterations can be due to impairments in copper-dependent cerulopalsmin activity which is known to affect iron mobilization. In prion protein-null mouse total brain and hippocampus, metal ion content shows a fluctuating trend, suggesting the presence of homeostatic compensatory mechanisms. However, copper and iron functional values are likely altered also in these two organs, as indicated by the modulation of metal-binding protein expression levels. Altogether, these results reveal that the absence of the cellular prion protein impairs copper metabolism and copper-dependent oxidase activity, with ensuing alteration of iron mobilization from cellular storage compartments

A Path to the Stars: The Evolution of the Species

During the last years, a number of telescopes have been dedicated to the followup of the GRBs. But after the Swift launch, the average observed intensity of the GRBs showed to be lower than thought before. Our experience with the robotic 60 cm REM telescope confirmed this evidence, with a large number oflostGRBs. Then, we proposed to study the feasibility of a 4 m fast pointing class telescope, equipped with a multichannel imagers, from Visible to Near Infrared. In this paper, we present the main result of the feasibility study we performed so far