Structural Consequences of Copper Binding to the Prion Protein

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

In Vitro Aggregation Assays for the Characterization of \u3b1-synuclein Prion-Like Properties

Aggregation of \u3b1-synuclein plays a crucial role in the pathogenesis of synucleinopathies, a group of neurodegenerative diseases including Parkinson disease (PD), dementia with Lewy bodies (DLB), diffuse Lewy body disease (DLBD) and multiple system atrophy (MSA). The common feature of these diseases is a pathological deposition of protein aggregates, known as Lewy bodies (LBs) in the central nervous system. The major component of these aggregates is \u3b1-synuclein, a natively unfolded protein, which may undergo dramatic structural changes resulting in the formation of \u3b2-sheet rich assemblies. In vitro studies have shown that recombinant \u3b1-synuclein protein may polymerize into amyloidogenic fibrils resembling those found in LBs. These aggregates may be uptaken and propagated between cells in a prion-like manner. Here we present the mechanisms and kinetics of \u3b1-synuclein aggregation in vitro, as well as crucial factors affecting this process. We also describe how PD-linked \u3b1-synuclein mutations and some exogenous factors modulate in vitro aggregation. Furthermore, we present a current knowledge on the mechanisms by which extracellular aggregates may be internalized and propagated between cells, as well as the mechanisms of their toxicity. \ua9 2014 Landes Bioscience

A novel expression system for production of soluble prion proteins in E. coli

Expression of eukaryotic proteins in Escherichia coli is challenging, especially when they contain disulfide bonds. Since the discovery of the prion protein (PrP) and its role in transmissible spongiform encephalopathies, the need to obtain large quantities of the recombinant protein for research purposes has been essential. Currently, production of recombinant PrP is achieved by refolding protocols. Here, we show that the co-expression of two different PrP with the human Quiescin Sulfhydryl OXidase (QSOX), a human chaperone with thiol/disulfide oxidase activity, in the cytoplasm of E. coli produces soluble recombinant PrP. The structural integrity of the soluble PrP has been confirmed by nuclear magnetic resonance spectroscopy, demonstrating that properly folded PrP can be easily expressed in bacteria. Furthermore, the soluble recombinant PrP produced with this method can be used for functional and structural studies

Probing Early Misfolding Events in Prion Protein Mutants by NMR Spectroscopy

The post-translational conversion of the ubiquitously expressed cellular form of the prion protein, PrPC, into its misfolded and pathogenic isoform, known as prion or PrPSc, plays a key role in prion diseases. These maladies are denoted transmissible spongiform encephalopathies (TSEs) and affect both humans and animals. A prerequisite for understanding TSEs is unraveling the molecular mechanism leading to the conversion process whereby most \u3b1-helical motifs are replaced by \u3b2-sheet secondary structures. Importantly, most point mutations linked to inherited prion diseases are clustered in the C-terminal domain region of PrPC and cause spontaneous conversion to PrPSc. Structural studies with PrP variants promise new clues regarding the proposed conversion mechanism and may help identify "hot spots" in PrPC involved in the pathogenic conversion. These investigations may also shed light on the early structural rearrangements occurring in some PrPC epitopes thought to be involved in modulating prion susceptibility. Here we present a detailed overview of our solution-state NMR studies on human prion protein carrying different pathological point mutations and the implications that such findings may have for the future of prion research

Innovative Non-PrP-Targeted Drug Strategy Designed to Enhance Prion Clearance

Prion diseases are a group of neurodegenerative disorders characterized by the accumulation of misfolded prion protein (called PrPSc). Although conversion of the cellular prion protein (PrPC) to PrPSc is still not completely understood, most of the therapies developed until now are based on blocking this process. Here, we propose a new drug strategy aimed at clearing prions without any direct interaction with neither PrPC nor PrPSc. Starting from the recent discovery of SERPINA3/SerpinA3n upregulation during prion diseases, we have identified a small molecule, named compound 5 (ARN1468), inhibiting the function of these serpins and effectively reducing prion load in chronically infected cells. Although the low bioavailability of this compound does not allow in vivo studies in prion-infected mice, our strategy emerges as a novel and effective approach to the treatment of prion disease

The mechanisms of humic substances self-assembly with biological molecules: The case study of the prion protein

Humic substances (HS) are the largest constituent of soil organic matter and are considered as a key component of the terrestrial ecosystem. HS may facilitate the transport of organic and inorganic molecules, as well as the sorption interactions with environmentally relevant proteins such as prions. Prions enter the environment through shedding from live hosts, facilitating a sustained incidence of animal prion diseases such as Chronic Wasting Disease and scrapie in cervid and ovine populations, respectively. Changes in prion structure upon environmental exposure may be significant as they can affect prion infectivity and disease pathology. Despite its relevance, the mechanisms of prion interaction with HS are still not completely understood. The goal of this work is to advance a structural-level picture of the encapsulation of recombinant, non-infectious, prion protein (PrP) into different natural HS. We observed that PrP precipitation upon addition of HS is mainly driven by a mechanism of “salting-out” whereby PrP molecules are rapidly removed from the solution and aggregate in insoluble adducts with humic molecules. Importantly, this process does not alter the protein folding since insoluble PrP retains its α-helical content when in complex with HS. The observed ability of HS to promote PrP insolubilization without altering its secondary structure may have potential relevance in the context of “prion ecology”. These results suggest that soil organic matter interacts with prions possibly without altering the protein structures. This may facilitate prions preservation from biotic and abiotic degradation leading to their accumulation in the environment

Prion protein interaction with soil humic substances: environmental implications

Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders caused by prions. Animal TSE include scrapie in sheep and goats, and chronic wasting disease (CWD) in cervids. Effective management of scrapie in many parts of the world, and of CWD in North American deer population is complicated by the persistence of prions in the environment. After shedding from diseased animals, prions persist in soil, withstanding biotic and abiotic degradation. As soil is a complex, multi-component system of both mineral and organic components, it is important to understand which soil compounds may interact with prions and thus contribute to disease transmission. Several studies have investigated the role of different soil minerals in prion adsorption and infectivity; we focused our attention on the interaction of soil organic components, the humic substances (HS), with recombinant prion protein (recPrP) material. We evaluated the kinetics of recPrP adsorption, providing a structural and biochemical characterization of chemical adducts using different experimental approaches. Here we show that HS act as potent anti-prion agents in prion infected neuronal cells and in the amyloid seeding assays: HS adsorb both recPrP and prions, thus sequestering them from the prion replication process. We interpreted our findings as highly relevant from an environmental point of view, as the adsorption of prions in HS may affect their availability and consequently hinder the environmental transmission of prion diseases in ruminants

NMR Structure of the Human Prion Protein with the Pathological Q212P Mutation Reveals Unique Structural Features

Prion diseases are fatal neurodegenerative disorders caused by an aberrant accumulation of the misfolded cellular prion protein (PrPC) conformer, denoted as infectious scrapie isoform or PrPSc. In inherited human prion diseases, mutations in the open reading frame of the PrP gene (PRNP) are hypothesized to favor spontaneous generation of PrPSc in specific brain regions leading to neuronal cell degeneration and death. Here, we describe the NMR solution structure of the truncated recombinant human PrP from residue 90 to 231 carrying the Q212P mutation, which is believed to cause Gerstmann-Sträussler-Scheinker (GSS) syndrome, a familial prion disease. The secondary structure of the Q212P mutant consists of a flexible disordered tail (residues 90–124) and a globular domain (residues 125–231). The substitution of a glutamine by a proline at the position 212 introduces novel structural differences in comparison to the known wild-type PrP structures. The most remarkable differences involve the C-terminal end of the protein and the β2–α2 loop region. This structure might provide new insights into the early events of conformational transition of PrPC into PrPSc. Indeed, the spontaneous formation of prions in familial cases might be due to the disruptions of the hydrophobic core consisting of β2–α2 loop and α3 helix

Toward the Identification of the Structural Determinants of Prion Conversion

The post-translational conversion of PrPC into the misfolded, pathogenic form PrPSc plays a key role in prion diseases or transmissible spongiform encephalopathies. These disorders are neurodegenerative maladies affecting both human and animals. One of the crucial questions in prion biology is the identification of the regions on PrPC that lead to the conversion process, whereby most alpha-helical motifs are replaced by betasheet secondary structures. In order to gain insights into the structural determinants involved in PrPSc formation we investigated the effect of HuPrPC point mutations linked to genetic form of prion diseases. Pathological point mutations cause spontaneous formation of PrPSc in the brain. Structural studies with HuPrP variants may provide new clues regarding the conversion mechanism, as well as help in the identification of a \u201chot spot\u201d on PrPC side which may be a possible target for pharmacological intervention. We first evaluated by molecular dynamics simulations the structural facets of three HuPrP variants located in the globular domain: two pathological mutations, Q212P and E200K, linked to Gerstmann-Str\ue4ussler\u2013Scheinker (GSS) and familial Creutzfeldt-Jakob disease (fCJD) respectively, and the protective polymorphism E219K. Such simulation-based structural predictions provided preliminary hints on the effects of mutations clustered in the globular HuPrP domain. Subsequently, we carried out structural investigation determining the high-resolution NMR threedimensional (3D) structure of the truncated recHuPrP(90-231) carrying both the fCJD-linked V210I and the GSS-causing Q212P mutations. Moreover, we determined the 3D NMR structure of the E219K polymorphism in order to find the structural basis responsible for its protective effect. Such structural studies led to the preliminary conclusion that the structural disorders of the b2-a2 loop region together with the increased spacing between this loop and the C-terminal part of a3 helix represent key pathological features. This observation raises the possibility that the spontaneous formation of prions might start from the disruption of the hydrophobic core present in the structured HuPrP domain. We then investigated whether the b2-a2 loop region might be exploited to test the effectiveness of drug candidates that would halt prion replication by binding to this epitope. We took advantage of a camel antibody fragment, denoted as nanobody, raised against the b2-a2 loop region of both Mo and HuPrP. We found that the binding of the nanobody to the b2-a2 loop resulted in inhibition of PrPSc replication when the nanobody was added to chronically infected mouse hypothalamic cells and to fibrillization assays. Finally, we have evaluated the effect of the pathological mutations on the N-terminal unstructured domain. We used synchrotron-based X-ray absorption fine structure (XAFS) technique to study the coordination geometries of both Cu2+ and Cu1+ in the Q212P mutant and we compared these findings with the WT. We clearly showed that Q212P mutant causes a dramatic modification on the non-OR copper binding site in the presence of both copper oxidative states (Cu2+ and Cu1+). These findings are a step forward towards showing a structure-function relationship which provides a biological basis for understanding the spontaneous generation of PrPSc in inherited prion diseases

Synthetic prions

Neuropathology of human prion diseases -- The nature of the infectious agents : prp models of resistant species to prion diseases (dog, rabbit and horses) -- Inducing transmissible prion diseases with recombinant fibrils : a new concept on genesis and evolution of infectious prions -- Synthetic prions -- Recent developments in cell biology : role of glycosylation in prion diseases -- Transmissibility of prion diseases in rodent models : atypical strains -- Pre-mortem diagnostic screening for transmissible spongiform encephalopathies by proteomic approaches -- Added value of biochemical tools for the diagnosis of human prion disease -- Anti-prion strategies for in vivo gene therapy assays