64 research outputs found

    Malalties neurodegeneratives: un problema de plegament proteic (Seminaris de Recerca 2013)

    Get PDF

    Neurodegenerative diseases: A protein misfolding consequence

    Get PDF
    Podeu consultar el llibre complet a: http://hdl.handle.net/2445/63704Protein misfolding and aggregation into amyloid-like structures is related with an increasing number of both non-neuropathic (either localized or systemic) and neurodegenerative human disorders. Decrypting the mechanisms and implications underlying amyloid assemblies has become a central issue in biology and medicine. Compelling evidence show that the formation of amyloid aggregates has a negative impact in cell physiology, entailing the cell dysfunction and finally apoptosis and cell death. The aim of the present review is to illustrate the currently status of the most common and/or debilitating conformational diseases, from Alzheimer to prion diseases

    Characterization of the amyloid bacterial inclusion bodies of the HET-s fungal prion

    Full text link
    The formation of amyloid aggregates is related to the onset of a number of human diseases. Recent studies provide compelling evidence for the existence of related fibrillar structures in bacterial inclusion bodies (IBs). Bacteria might thus provide a biologically relevant and tuneable system to study amyloid aggregation and how to interfere with it. Particularly suited for such studies are protein models for which structural information is available in both IBs and amyloid states. The only high-resolution structure of an infectious amyloid state reported to date is that of the HET-s prion forming domain (PFD). Importantly, recent solid-state NMR data indicates that the structure of HET-s PFD in IBs closely resembles that of the infectious fibrils. Here we present an exhaustive conformational characterization of HET-s IBs in order to establish the aggregation of this prion in bacteria as a consistent cellular model in which the effect of autologous or heterologous protein quality machineries and/or anti-aggregational and anti-prionic drugs can be further studied

    Yeast prions form infectious amyloid inclusion bodies in bacteria

    Full text link
    Background Prions were first identified as infectious proteins associated with fatal brain diseases in mammals. However, fungal prions behave as epigenetic regulators that can alter a range of cellular processes. These proteins propagate as self-perpetuating amyloid aggregates being an example of structural inheritance. The best-characterized examples are the Sup35 and Ure2 yeast proteins, corresponding to [PSI+] and [URE3] phenotypes, respectively. Results Here we show that both the prion domain of Sup35 (Sup35-NM) and the Ure2 protein (Ure2p) form inclusion bodies (IBs) displaying amyloid-like properties when expressed in bacteria. These intracellular aggregates template the conformational change and promote the aggregation of homologous, but not heterologous, soluble prionogenic molecules. Moreover, in the case of Sup35-NM, purified IBs are able to induce different [PSI+] phenotypes in yeast, indicating that at least a fraction of the protein embedded in these deposits adopts an infectious prion fold. Conclusions An important feature of prion inheritance is the existence of strains, which are phenotypic variants encoded by different conformations of the same polypeptide. We show here that the proportion of infected yeast cells displaying strong and weak [PSI+] phenotypes depends on the conditions under which the prionogenic aggregates are formed in E. coli, suggesting that bacterial systems might become useful tools to generate prion strain diversity

    What Makes a Protein Sequence a Prion?

    Get PDF
    Typical amyloid diseases such as Alzheimer's and Parkinson's were thought to exclusively result from de novo aggregation, but recently it was shown that amyloids formed in one cell can cross-seed aggregation in other cells, following a prion-like mechanism. Despite the large experimental effort devoted to understanding the phenomenon of prion transmissibility, it is still poorly understood how this property is encoded in the primary sequence. In many cases, prion structural conversion is driven by the presence of relatively large glutamine/asparagine (Q/N) enriched segments. Several studies suggest that it is the amino acid composition of these regions rather than their specific sequence that accounts for their priogenicity. However, our analysis indicates that it is instead the presence and potency of specific short amyloid-prone sequences that occur within intrinsically disordered Q/N-rich regions that determine their prion behaviour, modulated by the structural and compositional context. This provides a basis for the accurate identification and evaluation of prion candidate sequences in proteomes in the context of a unified framework for amyloid formation and prion propagation

    Using bacterial inclusion bodies to screen for amyloid aggregation inhibitors

    Full text link
    Background: The amyloid-β peptide (Aβ42) is the main component of the inter-neuronal amyloid plaques characteristic of Alzheimer's disease (AD). The mechanism by which Aβ42 and other amyloid peptides assemble into insoluble neurotoxic deposits is still not completely understood and multiple factors have been reported to trigger their formation. In particular, the presence of endogenous metal ions has been linked to the pathogenesis of AD and other neurodegenerative disorders. Results: Here we describe a rapid and high-throughput screening method to identify molecules able to modulate amyloid aggregation. The approach exploits the inclusion bodies (IBs) formed by Aβ42 when expressed in bacteria. We have shown previously that these aggregates retain amyloid structural and functional properties. In the present work, we demonstrate that their in vitro refolding is selectively sensitive to the presence of aggregation-promoting metal ions, allowing the detection of inhibitors of metal-promoted amyloid aggregation with potential therapeutic interest. Conclusions: Because IBs can be produced at high levels and easily purified, the method overcomes one of the main limitations in screens to detect amyloid modulators: the use of expensive and usually highly insoluble synthetic peptides

    Ultra rapid in vivo screening for anti-Alzheimer anti-amyloid drugs

    Get PDF
    More than 46 million people worldwide suffer from Alzheimer's disease. A large number of potential treatments have been proposed; among these, the inhibition of the aggregation of amyloid β-peptide (Aβ), considered one of the main culprits in Alzheimer's disease. Limitations in monitoring the aggregation of Aβ in cells and tissues restrict the screening of anti-amyloid drugs to in vitro studies in most cases. We have developed a simple but powerful method to track Aβ aggregation in vivo in realtime, using bacteria as in vivo amyloid reservoir. We use the specific amyloid dye Thioflavin-S (Th-S) to stain bacterial inclusion bodies (IBs), in this case mainly formed of Aβ in amyloid conformation. Th-S binding to amyloids leads to an increment of fluorescence that can be monitored. The quantification of the Th-S fluorescence along the time allows tracking Aβ aggregation and the effect of potential antiaggregating agents

    On the Binding of Congo Red to Amyloid Fibrils

    Get PDF
    Amyloids are characterized by their capacity to bind Congo red (CR), one of the most used amyloid‐specific dyes. The structural features of CR binding were unknown for years, mainly because of the lack of amyloid structures solved at high resolution. In the last few years, solid‐state NMR spectroscopy enabled the determination of the structural features of amyloids, such as the HET‐s prion forming domain (HET‐s PFD), which also has recently been used to determine the amyloid-CR interface at atomic resolution. Herein, we combine spectroscopic data with molecular docking, molecular dynamics, and excitonic quantum/molecular mechanics calculations to examine and rationalize CR binding to amyloids. In contrast to a previous assumption on the binding mode, our results suggest that CR binding to the HET‐s PFD involves a cooperative process entailing the formation of a complex with 1:1 stoichiometry. This provides a molecular basis to explain the bathochromic shift in the maximal absorbance wavelength when CR is bound to amyloids

    Thioflavin-S staining of bacterial inclusion bodies for the fast, simple, and inexpensive screening of amyloid aggregation inhibitors

    Get PDF
    Amyloid aggregation is linked to a large number of human disorders, from neurodegenerative diseases as Alzheimer"s disease (AD) or spongiform encephalopathies to non-neuropathic localized diseases as type II diabetes and cataracts. Because the formation of insoluble inclusion bodies (IBs) during recombinant protein production in bacteria has been recently shown to share mechanistic features with amyloid self-assembly, bacteria have emerged as a tool to study amyloid aggregation. Herein we present a fast, simple, inexpensive and quantitative method for the screening of potential anti-aggregating drugs. This method is based on monitoring the changes in the binding of thioflavin-S to intracellular IBs in intact Eschericchia coli cells in the presence of small chemical compounds. This in vivo technique fairly recapitulates previous in vitro data. Here we mainly use the Alzheimer"s related beta-amyloid peptide as a model system, but the technique can be easily implemented for screening inhibitors relevant for other conformational diseases simply by changing the recombinant amyloid protein target. Indeed, we show that this methodology can be also applied to the evaluation of inhibitors of the aggregation of tau protein, another amyloidogenic protein with a key role in AD

    Evidence of protein adsorption in pegylated liposomes: Influence of liposomal decoration

    Get PDF
    In order to contribute to a better knowledge of the events involved in the formation of the protein corona when nanoparticles (NPs) come in contact with proteins, we report a study about the changes on the physicochemical properties of pristine, PEGylated and Cyclic Arginine-Glycine-Aspartate peptide (RGD)-functionalized large unilamelar liposomes (LUVs) or magnetoliposomes (MLs) upon incubation with Bovine Serum Albumin (BSA). The main phospholipid component of both LUVs and MLs was L-α-phosphatydylcholine (PC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with 20% of cholesterol. The most obvious indication of the interaction of BSA-nanosystems is given by changes in the hydrodynamic diameter of the particles but other evidence is needed to corroborate the process. Our findings indicate that size modification is a process that is accomplished in few hours and that is strongly dependent not only on the surface decoration but also of the lipid composition of both LUVs and MLs. Fluorescence quenching experiments as well as cryogenic transmission electron microscopy (Cryo-TEM) images assessed these changes and confirmed that although each system has to be studied in a particular way, we can establish three distinctive features that turn into more reactive systems: (a) compositions containing PC compared with their DMPC counterparts; (b) the presence of PEG and/or RGD compared to the pristine counterparts; and (c) the presence of SPIONs: MLs show higher interaction than LUVs of the same lipid composition. Consequently, PEGylation (that is supposed to make stealth NPs) actually fails in preventing complete protein binding
    corecore