3,291 research outputs found

    EPR Methods for Biological Cu(II): L-Band CW and NARS

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    Abstract: Copper has many roles in biology that involve the change of coordination sphere and/or oxidation state of the copper ion. Consequently, the study of copper in heterogeneous environments is an important area in biophysics. EPR is a primary technique for the investigation of paramagnetic copper, which is usually the isolated Cu(II) ion, but sometimes as Cu(II) in different oxidation states of multitransition ion clusters. The gross geometry of the coordination environment of Cu(II) can often be determined from a simple inspection of the EPR spectrum, recorded in the traditional X-band frequency range (9–10 GHz). Identification and quantitation of the coordinating ligand atoms, however, is not so straightforward. In particular, analysis of the superhyperfine structure on the EPR spectrum, to determine the number of coordinated nitrogen atoms, is fraught with difficulty at X-band, despite the observation that the overwhelming number of EPR studies of Cu(II) in the literature have been carried out at X-band. Greater reliability has been demonstrated at S-band (3–4 GHz), using the low-field parallel (gz) features. However, analysis relies on clear identification of the outermost superhyperfine line, which has the lowest intensity of all the spectral features. Computer simulations have subsequently indicated that the much more intense perpendicular region of the spectrum can be reliably interpreted at L-band (2 GHz). The present work describes the development of L-band EPR of Cu(II) into a routine method that is applicable to biological samples

    A survey and a molecular dynamics study on the (central) hydrophobic region of prion proteins

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    Prion diseases are invariably fatal neurodegenerative diseases that affect humans and animals. Unlike most other amyloid forming neurodegenerative diseases, these can be highly infectious. Prion diseases occur in a variety of species. They include the fatal human neurodegenerative diseases Creutzfeldt-Jakob Disease (CJD), Fatal Familial Insomnia (FFI), Gerstmann-Straussler-Scheinker syndrome (GSS), Kuru, the bovine spongiform encephalopathy (BSE or 'mad-cow' disease) in cattle, the chronic wasting disease (CWD) in deer and elk, and scrapie in sheep and goats, etc. Transmission across the species barrier to humans, especially in the case of BSE in Europe, CWD in North America, and variant CJDs (vCJDs) in young people of UK, is a major public health concern. Fortunately, scientists reported that the (central) hydrophobic region of prion proteins (PrP) controls the formation of diseased prions. This article gives a detailed survey on PrP hydrophobic region and does molecular dynamics studies of human PrP(110-136) to confirm some findings from the survey. The structural bioinformatics presented in this article can be helpful as a reference in three-dimensional images for laboratory experimental works to study PrP hydrophobic region

    The prion protein regulates glutamate-mediated Ca2+ entry and mitochondrial Ca2+ accumulation in neurons

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    The cellular prion protein (PrPC) whose conformational misfolding leads to the production of deadly prions, has a still-unclarified cellular function despite decades of intensive research. Following our recent finding that PrPC limits Ca2+ entry via store-operated Ca2+ channels in neurons, we investigated whether the protein could also control the activity of ionotropic glutamate receptors (iGluRs). To this end, we compared local Ca2+ movements in primary cerebellar granule neurons and cortical neurons transduced with genetically encoded Ca2+ probes and expressing, or not expressing, PrPC. Our investigation demonstrated that PrPC downregulates Ca2+ entry through each specific agonist-stimulated iGluR and after stimulation by glutamate. We found that, although PrP-knockout (KO) mitochondria were displaced from the plasma membrane, glutamate addition resulted in a higher mitochondrial Ca2+ uptake in PrP-KO neurons than in their PrPC-expressing counterpart. This was because the increased Ca2+ entry through iGluRs in PrP-KO neurons led to a parallel increase in Ca2+-induced Ca2+ release via ryanodine receptor channels. These data thus suggest that PrPC takes part in the cell apparatus controlling Ca2+ homeostasis, and that PrPC is involved in protecting neurons from toxic Ca2+ overloads

    Molecular Dynamics Studies on 3D Structures of the Hydrophobic Region PrP(109-136)

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    Prion diseases caused by the conversion from a soluble normal cellular prion protein into insoluble abnormally folded infectious prions, are invariably fatal and highly infectious degenerative diseases that affect a wide variety of mammalian species. The palindrome and the Glycine-rich conserved segment in the hydrophobic region 109-136 control the conversion from normal prion protein to form into diseased prions. This paper gives detailed reviews on the 109-136 region and presents the studies of its 3D structures and structural dynamics.Comment: This paper was accepted on 18-02-2013 by the journal Acta Biochimica et Biophysica Sinica, in press in Vol 45 No 4, Apr 201

    Synthesis and structural characterization of a mimetic membrane-anchored prion protein

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    During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrPSc) of the host encoded prion protein (PrPC) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrPC and PrPSc have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrPC to PrPSc, but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C-terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP-GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane-anchored PrP. We show that the structure of PrP-GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor-free forms of PrP represent the structure of cellular, membrane-anchored PrP. The availability of a lipid-anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP

    Gene expression profiling en association with prion-related lesions in the medulla oblongata of symptomatic natural scrapie animals.

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    The pathogenesis of natural scrapie and other prion diseases remains unclear. Examining transcriptome variations in infected versus control animals may highlight new genes potentially involved in some of the molecular mechanisms of prion-induced pathology. The aim of this work was to identify disease-associated alterations in the gene expression profiles of the caudal medulla oblongata (MO) in sheep presenting the symptomatic phase of natural scrapie. The gene expression patterns in the MO from 7 sheep that had been naturally infected with scrapie were compared with 6 controls using a Central Veterinary Institute (CVI) custom designed 4×44K microarray. The microarray consisted of a probe set on the previously sequenced ovine tissue library by CVI and was supplemented with all of the Ovis aries transcripts that are currently publicly available. Over 350 probe sets displayed greater than 2-fold changes in expression. We identified 148 genes from these probes, many of which encode proteins that are involved in the immune response, ion transport, cell adhesion, and transcription. Our results confirm previously published gene expression changes that were observed in murine models with induced scrapie. Moreover, we have identified new genes that exhibit differential expression in scrapie and could be involved in prion neuropathology. Finally, we have investigated the relationship between gene expression profiles and the appearance of the main scrapie-related lesions, including prion protein deposition, gliosis and spongiosis. In this context, the potential impacts of these gene expression changes in the MO on scrapie development are discussed

    Characterizing the role of the PrPC N-terminal domain in protein and metal binding: NMR and XAS studies

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    The conversion of the cellular prion protein PrPC into the infectious isoform (PrPSc) is the key event in prion diseases. The physiological role of PrPC remains one of main challenges in prion biology, and it is an absolute requirement also for understanding prion diseases. Putative roles for PrPC are based on its localization in the central and peripheral nervous systems and on PrPC- interacting molecules or metal ions through its unstructured N-terminal domain. We analysed the function of the cellular prion protein using structural biology techniques aimed to analyze the interaction between PrPC and NCAM and PrPC with copper ions. We first focused on the structural determinants responsible for human PrPC (HuPrP) and NCAM interaction using Stimulated Emission Depletion (STED) nanoscopy, surface plasma resonance (SPR) and NMR spectroscopy approaches. Such structural and biological investigations revealed surface interacting epitopes governing the interaction between HuPrP N-terminus and the second module of NCAM Fibronectin type-3 domain, providing molecular details about the interaction between HuPrP and NCAM Fibronectin domain, and revealed a new role of PrPC N- terminus as a dynamic and functional element responsible for protein-protein interaction. Subsequently, we have investigated the role of copper in prion conversion and susceptibility with a special focus on the non-OR copper binding site. The molecular mechanisms of prion conversion are still debated. NMR-based studies on HuPrP and MoPrP globular domains have identified the \u3b22-\u3b12 loop as important element able to modulate the susceptibility of a given species to prion disease. However, recent studies have highlighted also the importance of the N- terminal region in promoting structural rearrangements to PrPSc. We studied copper coordination in the non-OR region of different species including human, sheep, bank vole and opossum. By using in vitro approaches, cell-based and computational techniques, we propose two types of copper coordination geometries, where the type-1 Cu(II) coordination displays a closed non-OR region conformation associated with less-susceptible species, while in type-2 a less structured and solvent exposed non-OR region might render the overall PrPC structure more flexible, therefore we correlate this with higher susceptibility to prion diseases. Our data highlighted how copper coordination in the non-OR copper binding site may explain the different susceptibility to prion diseases observed in these mammalian species. Ultimately, in the present thesis we expanded our knowledge on how the N-terminus of PrPC regulates the physiological functions of PrPC and how it is involved in the prion conversion

    Structural Consequences of Copper Binding to the Prion Protein

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    Prion, or PrPSc, is the pathological isoform of the cellular prion protein (PrPC) and it is the etiological agent of transmissible spongiform encephalopathies (TSE) affecting humans and animal species. The most relevant function of PrPC is its ability to bind copper ions through its flexible N-terminal moiety. This review includes an overview of the structure and function of PrPC with a focus on its ability to bind copper ions. The state-of-the-art of the role of copper in both PrPC physiology and in prion pathogenesis is also discussed. Finally, we describe the structural consequences of copper binding to the PrPC structure

    Metal Ion Regulation in the Central Nervous System and in Glutamatergic Synapses: Role of the Cellular Prion Protein

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    Despite many efforts, the molecular mechanisms underlying the pathophysiology of neurodegenerative disorders have not been fully understood. Results published in literature highlight that different neurodegenerative diseases share common features: protein aggregation in neuronal tissue; oxidation of neuronal tissue mediated by redox-active metal ions interaction with a target protein; and functional demise. So, unveiling the physiological function of protein that aggregate in neurodegenerating tissues, as well as their interplay with metal ions, becomes a prominent issue, in order to understand the etiological trigger and to define a possible therapeutic strategy. Metal ions are essential elements for cellular processes, but at the same they are potentially dangerous since they can give rise to Fenton reaction and oxidative/nitrosative stress. So, their homeostasis is strictly regulated in each district of the organism, but in particular in the brain. The brain, having the highest metabolic rate and depending predominantly on oxidative metabolism for its energy, has developed fine mechanisms to compartmentalize, distribute, uptake and excrete the different ionic species. Alterations in one of these mechanisms can lead to great neuronal damages, and maybe neurodegenerative disorders. This work has been focused on the cellular prion protein (PrPC), whose conformational isoform, the scrapie prion protein (PrPSc) is the causative agent of prion disordes and whose function has not been clearly defined, yet. Metal ions are a common denominator to all the cellular pathways in which PrPC seems to be actively involved. In particular, metal ions homeostasis maintainance, neuroprotection in excitotoxic condition and ionotropic receptor modulation have been studied. In the first part of the project, PrPC role in metal ion homeostais maintainance has been investigated. To this aim, copper, manganese, zinc and iron content, as well as metal binding proteins expression have been measured in a PrP knockout murine model, compared to wild-type. The results describe the global rearrangement occurring in the expression of metal binding proteins to maintain trace metals homeostasis, trying to compensate PrPC absence. At the same time, a pronounced decrease in Ceruloplasmin ferroxidase activity has been detected in PrP null mouse serum, pointing out a global impairment in copper metabolism in PrPC absence. In the second part of the project, the importance of the interaction between PrPC and copper ions in excitotoxic conditions and in synapses functionality has been studied. It has been published that PrP null mice show higher levels of neuronal cell death in stressful conditions and when subjected to toxic treatment with glutamate receptor agonists. Moreover, these mice show altered kinetics of N-methyl-D-aspartate (NMDA) receptor current. These alterations appears to be due to an inhibitory regulation that PrPC exerts on NMDA receptors via copper ions, lacking in PrP null hippocampi. First, the enhanced suceptibility to excitotoxicity of PrP knockout mice has been verified and characterised in organotypic hippocampal cultures upon treatment with NMDA. Higher neuronal cell death levels have been detected in all the investigated hippocampal regions. To identify which cellular regulatory mechanism is alterd in PrPC absence, the expression of the proteins mainly involved in excitotoxicity has been compared between PrP knockout and wild-type hippocampi. Among other minor differences, a different modulation of calcium transporters expression has been identified in PrP knockout hippocampi and brains. This global alteration appears to be necessary to maintain calcium homeostasis, since calcium content measurements did not reveal any strong difference between PrP null and wild-type samples. NMDA receptors can be S-nitrosylated on extracellular cysteines and this reaction is always inhibitory. S-nytrosilation requires an electron acceptor to occur, for this reason copper ions are often involved in these kind of reactions. Moreover, copper ions are known to modulate NMDA receptor activity, but the precise mechanism has not been described, yet. Since PrPC is known to support the S-nitrosylation of other membrane proteins, the S-nitrosylation levels of NMDA receptor subunits GluN1 and GluN2A have been measured in PrP knockout hippocampi from adult mice and compared to wild-type ones. Results show that the S-nitrosylated fractions of both GluN1 and GluN2A are reduced in PrP absence. So, this reveals that PrPC modulates NMDA receptor activity providing the copper ions necessary to support their inhibitory S-nitrosylation reaction. Through this mechanism, PrPC contributes to inhibit NMDA receptor currents, as well as to protect neurons in excitotoxic conditions

    pH MODULATION OF FIBRIL DISSOCIATION AND COPPER BINDING PROPERTIES OF THE PRION PROTEIN

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    The cellular form of prion protein (PrPC) is a cell-surface glycoprotein attached to lipid rafts via its glycosylphosphatidylinositol anchor. Conversion of PrPC to its scrapie conformer (PrPSc, the fibrillar form) constitutes the key event of the etiology of prion diseases. Fibril dissociation is necessary for efficient conversion and continued propagation of the disease state. Recent studies have revealed that conversion occurs along the endocytic pathway. To better understand the dissociation process, we have investigated the effect of low pH on the stability of recombinant prion fibrils. We show that under conditions that mimic the endocytic environment, amyloid fibrils made from full-length recombinant prion protein dissociate both laterally and axially to form protofilaments. About 5% of the protofilaments are short enough to be considered soluble and contain ~100-300 monomers per structure; these also retain the biophysical characteristics of the filaments. We propose that protonation of His residues and charge repulsion in the N-terminal domain trigger fibril dissociation. Our data suggest that lysosomes and late endosomes are competent milieus for propagating the misfolded state not only by destabilizing the normal prion protein, but by accelerating fibril dissociation into smaller structures that may act as seeds for further fibril formation. PrPC binds four Cu(II) in its octarepeat region and another at the fifth binding site. Previous work has demonstrated detailed structural information on copper binding to these sites at neutral pH. Both types of binding sites contain ionizable groups, thus the effect of pH on copper binding needs to be clarified. Moreover, much less attention has been devoted to understanding copper binding in PrPSc, which is more pathologically relevant. These two aspects are investigated here using isothermal titration calorimetry and X-band electron paramagnetic spectroscopy. Our results confirm that copper binding to both the octarepeats and the fifth binding site is pH-dependent. We show that both sites bind copper in the fibrillar form with coordination modes similar to their monomeric counterparts. However, the ratios of the different coordination modes have changed in the fibril, which might suggest changes in their affinities after conversion and have potential effects on the redox properties of fibrils
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