ABSTRACT
Prion diseases are transmissible neurodegenerative disorders that affect mammals, including humans. The central molecular event is the conversion of cellular prion glycoprotein, PrPC, into a plethora of assemblies, PrPSc, associated with disease. Distinct phenotypes of disease led to the concept of prion strains, which are associated with distinct PrPSc structures. However, the degree to which intra- and inter-strain PrPSc heterogeneity contributes to disease pathogenesis remains unclear. Addressing this question requires the precise isolation and characterization of all PrPSc subpopulations from the prion-infected brains. Until now, this has been challenging. We used asymmetric-flow field-flow fractionation (AF4) to isolate all PrPSc subpopulations from brains of hamsters infected with three prion strains: Hyper (HY) and 263K, which produce almost identical phenotypes, and Drowsy (DY), a strain with a distinct presentation. In-line dynamic and multi-angle light scattering (DLS/MALS) data provided accurate measurements of particle sizes and estimation of the shape and number of PrPSc particles. We found that each strain had a continuum of PrPSc assemblies, with strong correlation between PrPSc quaternary structure and phenotype. HY and 263K were enriched with large, protease-resistant PrPSc aggregates, whereas DY consisted primarily of smaller, more protease-sensitive aggregates. For all strains, a transition from protease-sensitive to protease-resistant PrPSc took place at a hydrodynamic radius (Rh) of 15 nm and was accompanied by a change in glycosylation and seeding activity. Our results show that the combination of AF4 with in-line MALS/DLS is a powerful tool for analyzing PrPSc subpopulations and demonstrate that while PrPSc quaternary structure is a major contributor to PrPSc structural heterogeneity, a fundamental change, likely in secondary/tertiary structure, prevents PrPSc particles from maintaining proteinase K resistance below an Rh of 15 nm, regardless of strain. This results in two biochemically distinctive subpopulations, the proportion, seeding activity, and stability of which correlate with prion strain phenotype.
Subject(s)
Dynamic Light Scattering/methods , Photometry/methods , PrPSc Proteins/analysis , PrPSc Proteins/chemistry , Animals , Cricetinae , Hydrodynamics , Mice , Protein Structure, QuaternaryABSTRACT
A distinctive signature of the prion diseases is the accumulation of the pathogenic isoform of the prion protein, PrPSc, in the central nervous system of prion-affected humans and animals. PrPSc is also found in peripheral tissues, raising concerns about the potential transmission of pathogenic prions through human food supplies and posing a significant risk to public health. Although muscle tissues are considered to contain levels of low prion infectivity, it has been shown that myotubes in culture efficiently propagate PrPSc. Given the high consumption of muscle tissue, it is important to understand what factors could influence the establishment of a prion infection in muscle tissue. Here we used in vitro myotube cultures, differentiated from the C2C12 myoblast cell line (dC2C12), to identify factors affecting prion replication. A range of experimental conditions revealed that PrPSc is tightly associated with proteins found in the systemic extracellular matrix, mostly fibronectin (FN). The interaction of PrPSc with FN decreased prion infectivity, as determined by standard scrapie cell assay. Interestingly, the prion-resistant reserve cells in dC2C12 cultures displayed a FN-rich extracellular matrix while the prion-susceptible myotubes expressed FN at a low level. In agreement with the in vitro results, immunohistopathological analyses of tissues from sheep infected with natural scrapie demonstrated a prion susceptibility phenotype linked to an extracellular matrix with undetectable levels of FN. Conversely, PrPSc deposits were not observed in tissues expressing FN. These data indicate that extracellular FN may act as a natural barrier against prion replication and that the extracellular matrix composition may be a crucial feature determining prion tropism in different tissues.
Subject(s)
Fibronectins , Prion Diseases , Prions , Scrapie , Animals , Humans , Cell Line , Fibronectins/therapeutic use , Prion Diseases/drug therapy , Prion Diseases/prevention & control , Prions/metabolism , Scrapie/metabolism , SheepABSTRACT
Prion diseases are chronic and fatal neurodegenerative diseases characterized by the accumulation of disease-specific prion protein (PrPSc), spongiform changes, neuronal loss, and gliosis. Growing evidence shows that the neuroinflammatory response is a key component of prion diseases and contributes to neurodegeneration. Toll-like receptors (TLRs) have been proposed as important mediators of innate immune responses triggered in the central nervous system in other human neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. However, little is known about the role of TLRs in prion diseases, and their involvement in the neuropathology of natural scrapie has not been studied. We assessed the gene expression of ovine TLRs in four anatomically distinct brain regions in natural scrapie-infected sheep and evaluated the possible correlations between gene expression and the pathological hallmarks of prion disease. We observed significant changes in TLR expression in scrapie-infected sheep that correlate with the degree of spongiosis, PrPSc deposition, and gliosis in each of the regions studied. Remarkably, TLR4 was the only gene upregulated in all regions, regardless of the severity of neuropathology. In the hippocampus, we observed milder neuropathology associated with a distinct TLR gene expression profile and the presence of a peculiar microglial morphology, called rod microglia, described here for the first time in the brain of scrapie-infected sheep. The concurrence of these features suggests partial neuroprotection of the hippocampus. Finally, a comparison of the findings in naturallyinfected sheep versus an ovinized mouse model (tg338 mice) revealed distinct patterns of TLRgene expression.
Subject(s)
Neurodegenerative Diseases , Prion Diseases , Scrapie , Animals , Brain/metabolism , Gliosis/pathology , Mice , Neurodegenerative Diseases/metabolism , Prion Diseases/metabolism , Scrapie/metabolism , Sheep , Toll-Like Receptors/genetics , Toll-Like Receptors/metabolism , TranscriptomeABSTRACT
Prions are lipidated proteins that interact with endogenous lipids and metal ions. They also assemble into multimers and propagate into the infectious scrapie form known as PrPSc The high-resolution structure of the infectious PrPSc state remains unknown, and its analysis largely relies on detergent-based preparations devoid of endogenous ligands. Here we designed polymers that allow isolation of endogenous membrane:protein assemblies in native nanodiscs without exposure to conventional detergents that destabilize protein structures and induce fibrillization. A set of styrene-maleic acid (SMA) polymers including a methylamine derivative facilitated gentle release of the infectious complexes for resolution of multimers, and a thiol-containing version promoted crystallization. Polymer extraction from brain homogenates from Syrian hamsters infected with Hyper prions and WT mice infected with Rocky Mountain Laboratories prions yielded infectious prion nanoparticles including oligomers and microfilaments bound to lipid vesicles. Lipid analysis revealed the brain phospholipids that associate with prion protofilaments, as well as those that are specifically enriched in prion assemblies captured by the methylamine-modified copolymer. A comparison of the infectivity of PrPSc attached to SMA lipid particles in mice and hamsters indicated that these amphipathic polymers offer a valuable tool for high-yield production of intact, detergent-free prions that retain in vivo activity. This native prion isolation method provides an avenue for producing relevant prion:lipid targets and potentially other proteins that form multimeric assemblies and fibrils on membranes.
Subject(s)
Brain/metabolism , Lipids/chemistry , Maleates/chemistry , Nanostructures/chemistry , Polystyrenes/chemistry , Prion Proteins/metabolism , Animals , Cricetinae , Maleates/chemical synthesis , Maleates/metabolism , Methylamines/chemistry , Mice , Phospholipids/chemistry , Phospholipids/metabolism , Polystyrenes/chemical synthesis , Polystyrenes/metabolism , Prion Proteins/chemistry , Prion Proteins/isolation & purification , Sulfhydryl Compounds/chemistryABSTRACT
Prion diseases are progressive neurodegenerative disorders affecting humans and various mammals. The prominent neuropathological change in prion diseases is neuroinflammation characterized by activation of neuroglia surrounding prion deposition. The cause and effect of this cellular response, however, is unclear. We investigated innate immune defenses against prion infection using primary mixed neuronal and glial cultures. Conditional prion propagation occurred in glial cultures depending on their immune status. Preconditioning of the cells with the toll-like receptor (TLR) ligand, lipopolysaccharide, resulted in a reduction in prion propagation, whereas suppression of the immune responses with the synthetic glucocorticoid, dexamethasone, increased prion propagation. In response to recombinant prion fibrils, glial cells up-regulated TLRs (TLR1 and TLR2) expression and secreted cytokines (tumor necrosis factor-α, interleukin-1ß, interleukin-6, granulocyte-macrophage colony-stimulating factor, and interferon-ß). Preconditioning of neuronal and glial cultures with recombinant prion fibrils inhibited prion replication and altered microglial and astrocytic populations. Our results provide evidence that, in early stages of prion infection, glial cells respond to prion infection through TLR-mediated innate immunity.
Subject(s)
Immunity, Innate/immunology , Neuroglia/metabolism , Prions/metabolism , Toll-Like Receptors/metabolism , Animals , Cells, Cultured , Cytokines/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Interleukin-1beta/metabolism , Mice , Neuroglia/immunology , Prions/immunology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
UNLABELLED: Prion diseases are fatal neurodegenerative disorders associated with the conversion of cellular prion protein (PrPC) into its aberrant infectious form (PrPSc). There is no treatment available for these diseases. The bile acids tauroursodeoxycholic acid(TUDCA) and ursodeoxycholic acid (UDCA) have been recently shown to be neuroprotective in other protein misfolding disease models, including Parkinson's, Huntington's and Alzheimer's diseases, and also in humans with amyotrophic lateral sclerosis.Here, we studied the therapeutic efficacy of these compounds in prion disease. We demonstrated that TUDCA and UDCA substantially reduced PrP conversion in cell-free aggregation assays, as well as in chronically and acutely infected cell cultures. This effect was mediated through reduction of PrPSc seeding ability, rather than an effect on PrPC. We also demonstrated the ability of TUDCA and UDCA to reduce neuronal loss in prion-infected cerebellar slice cultures. UDCA treatment reduced astrocytosis and prolonged survival in RML prion-infected mice. Interestingly, these effects were limited to the males, implying a gender-specific difference in drug metabolism. Beyond effects on PrPSc, we found that levels of phosphorylated eIF2 were increased at early time points, with correlated reductions in postsynaptic density protein 95. As demonstrated for other neurodegenerative diseases, we now show that TUDCA and UDCA may have a therapeutic role in prion diseases, with effects on both prion conversion and neuroprotection. Our findings, together with the fact that these natural compounds are orally bioavailable, permeable to the blood-brain barrier, and U.S. Food and Drug Administration-approved for use in humans, make these compounds promising alternatives for the treatment of prion diseases. IMPORTANCE: Prion diseases are fatal neurodegenerative diseases that are transmissible to humans and other mammals. There are no disease-modifying therapies available, despite decades of research. Treatment targets have included inhibition of protein accumulation,clearance of toxic aggregates, and prevention of downstream neurodegeneration. No one target may be sufficient; rather, compounds which have a multimodal mechanism, acting on different targets, would be ideal. TUDCA and UDCA are bile acids that may fulfill this dual role. Previous studies have demonstrated their neuroprotective effects in several neurodegenerative disease models, and we now demonstrate that this effect occurs in prion disease, with an added mechanistic target of upstream prion seeding. Importantly, these are natural compounds which are orally bioavailable, permeable to the blood-brain barrier, and U.S.Food and Drug Administration-approved for use in humans with primary biliary cirrhosis. They have recently been proven efficacious in human amyotrophic lateral sclerosis. Therefore, these compounds are promising options for the treatment of prion diseases.
Subject(s)
Neurons/metabolism , PrPC Proteins/metabolism , PrPSc Proteins/metabolism , Prion Diseases/metabolism , Prion Diseases/physiopathology , Taurochenodeoxycholic Acid/metabolism , Ursodeoxycholic Acid/metabolism , Animals , Bile Acids and Salts/metabolism , Cell Death , Cell Survival , Female , Humans , Male , Mice , Mice, Inbred C57BL , Neurons/cytology , Species SpecificityABSTRACT
Prion diseases are fatal neurodegenerative disorders associated with the polymerization of the cellular form of prion protein (PrP(C)) into an amyloidogenic ß-sheet infectious form (PrP(Sc)). The sequence of host PrP is the major determinant of host prion disease susceptibility. In mice, the presence of allele a (Prnp(a), encoding the polymorphism Leu-108/Thr-189) or b (Prnp(b), Phe-108/Val-189) is associated with short or long incubation times, respectively, following infection with PrP(Sc). The molecular bases linking PrP sequence, infection susceptibility, and convertibility of PrP(C) into PrP(Sc) remain unclear. Here we show that recombinant PrP(a) and PrP(b) aggregate and respond to seeding differently in vitro. Our kinetic studies reveal differences during the nucleation phase of the aggregation process, where PrP(b) exhibits a longer lag phase that cannot be completely eliminated by seeding the reaction with preformed fibrils. Additionally, PrP(b) is more prone to propagate features of the seeds, as demonstrated by conformational stability and electron microscopy studies of the formed fibrils. We propose a model of polymerization to explain how the polymorphisms at positions 108 and 189 produce the phenotypes seen in vivo. This model also provides insight into phenomena such as species barrier and prion strain generation, two phenomena also influenced by the primary structure of PrP.
Subject(s)
Phenylalanine/genetics , Polymorphism, Genetic , Prion Diseases/metabolism , Prions/genetics , Valine/genetics , Alleles , Animals , Benzothiazoles , Circular Dichroism , Dose-Response Relationship, Drug , Kinetics , Mice , Microscopy, Electron/methods , Polymers/chemistry , Prion Diseases/genetics , Protein Conformation , Recombinant Proteins/metabolism , Thiazoles/chemistry , Time FactorsABSTRACT
Synucleinopathies, including Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB), are neurodegenerative disorders caused by the accumulation of misfolded alpha-synuclein protein. Developing effective vaccines against synucleinopathies is challenging due to the difficulty of stimulating an immune-specific response against alpha-synuclein without causing harmful autoimmune reactions, selectively targeting only pathological forms of alpha-synuclein. Previous attempts using linear peptides and epitopes without control of the antigen structure failed in clinical trials. The immune system was unable to distinguish between native alpha-synuclein and its amyloid form. The prion domain of the fungal HET-s protein was selected as a scaffold to introduce select epitopes from the surface of alpha-synuclein fibrils. Four vaccine candidates were generated by introducing specific amino acid substitutions onto the surface of the scaffold protein. The approach successfully mimicked the stacking of the parallel in-register beta-sheet structure seen in alpha-synuclein fibrils. All vaccine candidates induced substantial levels of IgG antibodies that recognized pathological alpha-synuclein fibrils derived from a synucleinopathy mouse model. Furthermore, the antisera recognized pathological alpha-synuclein aggregates in brain lysates from patients who died from DLB, MSA, or PD, but did not recognize linear alpha-synuclein peptides. Our approach, based on the rational design of vaccines using the structure of alpha-synuclein amyloid fibrils and strict control over the exposed antigen structure used for immunization, as well as the ability to mimic aggregated alpha-synuclein, provides a promising avenue toward developing effective vaccines against alpha-synuclein fibrils.
ABSTRACT
Microglia play diverse pathophysiological roles in Alzheimer's disease (AD), with genetic susceptibility factors skewing microglial cell function to influence AD risk. CD33 is an immunomodulatory receptor associated with AD susceptibility through a single nucleotide polymorphism that modulates mRNA splicing, skewing protein expression from a long protein isoform (CD33M) to a short isoform (CD33m). Understanding how human CD33 isoforms differentially impact microglial cell function in vivo has been challenging due to functional divergence of CD33 between mice and humans. We address this challenge by studying transgenic mice expressing either of the human CD33 isoforms crossed with the 5XFAD mouse model of amyloidosis and find that human CD33 isoforms have opposing effects on the response of microglia to amyloid-ß (Aß) deposition. Mice expressing CD33M have increased Aß levels, more diffuse plaques, fewer disease-associated microglia, and more dystrophic neurites compared to 5XFAD control mice. Conversely, CD33m promotes plaque compaction and microglia-plaque contacts, and minimizes neuritic plaque pathology, highlighting an AD protective role for this isoform. Protective phenotypes driven by CD33m are detected at an earlier timepoint compared to the more aggressive pathology in CD33M mice that appears at a later timepoint, suggesting that CD33m has a more prominent impact on microglia cell function at earlier stages of disease progression. In addition to divergent roles in modulating phagocytosis, scRNAseq and proteomics analyses demonstrate that CD33m+ microglia upregulate nestin, an intermediate filament involved in cell migration, at plaque contact sites. Overall, our work provides new functional insights into how CD33, as a top genetic susceptibility factor for AD, modulates microglial cell function.
Subject(s)
Alzheimer Disease , Disease Models, Animal , Mice, Transgenic , Microglia , Protein Isoforms , Sialic Acid Binding Ig-like Lectin 3 , Animals , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Microglia/metabolism , Sialic Acid Binding Ig-like Lectin 3/metabolism , Humans , Mice , Protein Isoforms/metabolism , Amyloid beta-Peptides/metabolism , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathologyABSTRACT
Lewy bodies and Lewy neurites, neuropathological hallmarks of several neurological diseases, are mainly made of filamentous assemblies of α-synuclein. However, other macromolecules including Tau, ubiquitin, glyceraldehyde-3-phosphate dehydrogenase, and glycosaminoglycans are routinely found associated with these amyloid deposits. Glyceraldehyde-3-phosphate dehydrogenase is a glycolytic enzyme that can form fibrillar aggregates in the presence of acidic membranes, but its role in Parkinson disease is still unknown. In this work, the ability of heparin to trigger the amyloid aggregation of this protein at physiological conditions of pH and temperature is demonstrated by infrared and fluorescence spectroscopy, dynamic light scattering, small angle x-ray scattering, circular dichroism, and fluorescence microscopy. Aggregation proceeds through the formation of short rod-like oligomers, which elongates in one dimension. Heparan sulfate was also capable of inducing glyceraldehyde-3-phosphate dehydrogenase aggregation, but chondroitin sulfates A, B, and C together with dextran sulfate had a negligible effect. Aided with molecular docking simulations, a putative binding site on the protein is proposed providing a rational explanation for the structural specificity of heparin and heparan sulfate. Finally, it is demonstrated that in vitro the early oligomers present in the glyceraldehyde-3-phosphate dehydrogenase fibrillation pathway promote α-synuclein aggregation. Taking into account the toxicity of α-synuclein prefibrillar species, the heparin-induced glyceraldehyde-3-phosphate dehydrogenase early oligomers might come in useful as a novel therapeutic strategy in Parkinson disease and other synucleinopathies.
Subject(s)
Amyloid/chemistry , Glyceraldehyde-3-Phosphate Dehydrogenases/chemistry , Heparin/chemistry , Protein Multimerization , alpha-Synuclein/chemistry , Amyloid/metabolism , Animals , Chondroitin Sulfates/chemistry , Chondroitin Sulfates/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Heparitin Sulfate/chemistry , Heparitin Sulfate/metabolism , Humans , Hydrogen-Ion Concentration , Parkinson Disease/metabolism , Rabbits , alpha-Synuclein/metabolismABSTRACT
Prion diseases are fatal neurodegenerative diseases caused by pathogenic misfolding of the prion protein, PrP. They are transmissible between hosts, and sometimes between different species, as with transmission of bovine spongiform encephalopathy to humans. Although PrP is found in a wide range of vertebrates, prion diseases are seen only in certain mammals, suggesting that infectious misfolding was a recent evolutionary development. To explore when PrP acquired the ability to misfold infectiously, we reconstructed the sequences of ancestral versions of PrP from the last common primate, primate-rodent, artiodactyl, placental, bird, and amniote. Recombinant ancestral PrPs were then tested for their ability to form ß-sheet aggregates, either spontaneously or when seeded with infectious prion strains from human, cervid, or rodent species. The ability to aggregate developed after the oldest ancestor (last common amniote), and aggregation capabilities diverged along evolutionary pathways consistent with modern-day susceptibilities. Ancestral bird PrP could not be seeded with modern-day prions, just as modern-day birds are resistant to prion disease. Computational modeling of structures suggested that differences in helix 2 could account for the resistance of ancestral bird PrP to seeding. Interestingly, ancestral primate PrP could be converted by all prion seeds, including both human and cervid prions, raising the possibility that species descended from an ancestral primate have retained the susceptibility to conversion by cervid prions. More generally, the results suggest that susceptibility to prion disease emerged prior to ~100 million years ago, with placental mammals possibly being generally susceptible to disease.
Subject(s)
Prion Diseases , Prions , Pregnancy , Animals , Cattle , Female , Humans , Prion Proteins/chemistry , Placenta/metabolism , Prions/metabolism , Prion Diseases/genetics , Prion Diseases/metabolism , MammalsABSTRACT
Prion diseases are fatal neurodegenerative diseases in mammals with the unique characteristics of misfolding and aggregation of the cellular prion protein (PrPC) to the scrapie prion (PrPSc). Although neuroinflammation and neuronal loss feature within the disease process, the details of PrPC/PrPSc molecular transition to generate different aggregated species, and the correlation between each species and sequence of cellular events in disease pathogenesis are not fully understood. In this study, using mice inoculated with the RML isolate of mouse-adapted scrapie as a model, we applied asymmetric flow field-flow fractionation to monitor PrPC and PrPSc particle sizes and we also measured seeding activity and resistance to proteases. For cellular analysis in brain tissue, we measured inflammatory markers and synaptic damage, and used the isotropic fractionator to measure neuronal loss; these techniques were applied at different timepoints in a cross-sectional study of disease progression. Our analyses align with previous reports defining significant decreases in PrPC levels at pre-clinical stages of the disease and demonstrate that these decreases become significant before neuronal loss. We also identified the earliest PrPSc assemblies at a timepoint equivalent to 40% elapsed time for the disease incubation period; we propose that these assemblies, mostly composed of proteinase K (PK)-sensitive species, play an important role in triggering disease pathogenesis. Lastly, we show that the PK-resistant assemblies of PrPSc that appear at timepoints close to the terminal stage have similar biophysical characteristics, and hence that preparative use of PK-digestion selects for this specific subpopulation. In sum, our data argue that qualitative, as well as quantitative, changes in PrP conformers occur at the midpoint of subclinical phase; these changes affect quaternary structure and may occur at the threshold where adaptive responses become inadequate to deal with pathogenic processes.
Subject(s)
Disease Progression , Down-Regulation , PrPC Proteins/metabolism , PrPSc Proteins/chemistry , Scrapie/pathology , Animals , Biomarkers/metabolism , Brain/pathology , Cell Death , Endopeptidase K/metabolism , Glial Fibrillary Acidic Protein/metabolism , Inflammation/pathology , Mice , Molecular Weight , PrPSc Proteins/metabolism , Protein Structure, Quaternary , Solubility , Synapses/pathology , Time FactorsABSTRACT
Inhibition or reversion of protein self-aggregation has been suggested as a possible preventive mechanism against amyloid diseases, and many efforts are underway to found out molecules capable to restrain the protein aggregation process. In this paper, the inhibitory effects of thyroid hormone analogues on heat-induced fibrillation process of serum albumin are reported. Among the analogues tested, 3,5,3',5'-tetraiodothyroacetic and 3,5,3'-triiodothyroacetic acid showed the most important inhibitory effects on amyloid formation. Thyroxine exhibits a lesser protective effect, while 3,5,3'-triiodothyronine showed no significant inhibition. The gaining of a negative charge together with a size reduction of the hormone molecule could play an essential role in the inhibition of fibrils formation. According to infrared spectroscopy results, the thyroid hormones analogues protective effects proceed via the stabilization of the protein native structure. The current work demonstrates the effectiveness of naturally occurring molecules in the inhibition of albumin fibril formation.
Subject(s)
Serum Albumin/metabolism , Thyroxine/analogs & derivatives , Triiodothyronine/analogs & derivatives , Amyloid/metabolism , Animals , Cattle , Dose-Response Relationship, Drug , Drug Design , Hot Temperature , Kinetics , Protein Binding/drug effects , Protein Structure, Secondary/drug effects , Serum Albumin/chemistry , Spectroscopy, Fourier Transform Infrared , Thyroxine/pharmacology , Triiodothyronine/pharmacologyABSTRACT
α-Synuclein is a protein that aggregates as amyloid fibrils in the brains of patients with Parkinson's disease and dementia with Lewy bodies. Small oligomers of α-synuclein are neurotoxic and are thought to be closely associated with disease. Whereas α-synuclein fibrillization and fibril morphologies have been studied extensively with various methods, the earliest stages of aggregation and the properties of oligomeric intermediates are less well understood because few methods are able to detect and characterize early-stage aggregates. We used fluorescence spectroscopy to investigate the early stages of aggregation by studying pairwise interactions between α-synuclein monomers, as well as between engineered tandem oligomers of various sizes (dimers, tetramers, and octamers). The hydrodynamic radii of these engineered α-synuclein species were first determined by fluorescence correlation spectroscopy and dynamic light scattering. The rate of pairwise aggregation between different species was then monitored using dual-color fluorescence cross-correlation spectroscopy, measuring the extent of association between species labelled with different dyes at various time points during the early aggregation process. The aggregation rate and extent increased with tandem oligomer size. Self-association of the tandem oligomers was found to be the preferred pathway to form larger aggregates: interactions between oligomers occurred faster and to a greater extent than interactions between oligomers and monomers, indicating that the oligomers were not as efficient in seeding further aggregation by addition of monomers. These results suggest that oligomer-oligomer interactions may play an important role in driving aggregation during its early stages.
Subject(s)
Protein Aggregates , Protein Aggregation, Pathological/metabolism , Protein Multimerization , Recombinant Proteins , alpha-Synuclein/chemistry , alpha-Synuclein/genetics , Genetic Engineering , Humans , Kinetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Solubility , alpha-Synuclein/metabolismABSTRACT
The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation.
Subject(s)
Metalloporphyrins/pharmacology , Molecular Chaperones/pharmacology , Peptide Fragments/chemistry , Prions/chemistry , Protein Folding , Pyrroles/pharmacology , Animals , Cricetinae , Mesocricetus , Metalloporphyrins/chemistry , Models, Molecular , Molecular Chaperones/chemistry , Protein Conformation , Pyrroles/chemistryABSTRACT
The sequence of a host's prion protein (PrP) can affect that host's susceptibility to prion disease and is the primary basis for the species barrier to transmission. Yet within many species, polymorphisms of the prion protein gene (Prnp) exist, each of which can further affect susceptibility or influence incubation period, pathology and phenotype. As strains are defined by these features (incubation period, pathology, phenotype), polymorphisms may also lead to the preferential propagation or generation of certain strains. In our recent study of the mouse Prnp(a) and Prnp(b) polymorphisms (which produced the proteins PrP(a) and PrP(b), respectively), we found differences in aggregation tendency, strain adaptability and conformational variability. Comparing our in vitro data with that of in vivo studies, we found that differing incubation periods between Prnp(a) and Prnp(b) mice can primarily be explained on the basis of faster or more efficient aggregation of PrP(a). In addition, and more importantly, we found that the faithful propagation of strains in Prnp(b) mice can be explained by the ability of PrP(b) to adopt a wider range of conformations. This adaptability allows PrP(b) to successfully propagate the structural features of a seed. In contrast, Prnp(a) mice revert PrP(b) strains into PrP(a) -type strains, and overall they have a narrower distribution of incubation periods. This can be explained by PrP(a) having fewer preferred conformations. We propose that Prnp polymorphisms are one route by which certain prion strains may preferentially propagate. This has significant implications for prion disease, chronic wasting disease (CWD) in particular, as it is spreading through North America. Deer which are susceptible to CWD also carry polymorphisms which influence their susceptibility. If these polymorphisms also preferentially allow strain diversification and propagation, this may accelerate the crossing of species barriers and propagation of the disease up the food chain.
Subject(s)
Phenylalanine/genetics , Polymorphism, Genetic , Prion Diseases/metabolism , Prions/genetics , Valine/genetics , AnimalsABSTRACT
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme related with Huntington's, Parkinson's and Alzheimer's diseases. The ability of negatively charged membranes to induce a rapid formation of GAPDH amyloid fibrils has been demonstrated, but the mechanisms by which GAPDH reaches the fibrillar state remains unclear. In this report, we describe the structural changes undergone by GAPDH at physiological pH and temperature conditions right from its interaction with acidic membranes until the amyloid fibril is formed. According to our results, the GAPDH-membrane binding induces a beta-structuring process along with a loss of quaternary structure in the enzyme. In this way, experimental evidences on the initial steps of GAPDH amyloid fibrils formation pathway are provided.
Subject(s)
Amyloid/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Membranes, Artificial , Amyloid/ultrastructure , Glyceraldehyde-3-Phosphate Dehydrogenases/ultrastructure , Humans , Microscopy, Electron, Transmission , Protein Multimerization , Spectroscopy, Fourier Transform InfraredABSTRACT
The muscle-type nicotinic receptor has two pharmacologically distinguishable acetylcholine binding sites at the alpha-gamma and alpha-delta subunit interfaces; alpha-conotoxins can bind them selectively. As reported, alpha-conotoxin MI has greater affinity for the site near the alpha-delta interface of the BC(3)H1 cell receptor but, in the case of the Torpedo californica receptor, displays greater affinity for that near the alpha-gamma interface. To further investigate ligand selectivity, we study the conotoxin MI-Torpedo marmorata receptor interaction. In this work, we show the binding of alpha-conotoxin MI to the T. marmorata receptor and the influence of the antagonist alpha-Bungarotoxin and the agonist carbamylcholine on such binding; in addition, and contrasting with the results for the Torpedo californica receptor, we identify the alpha-delta subunit interface as the high affinity binding site. This is the first work describing different characteristics of the interaction between alpha-conotoxin MI and receptors from different species of the same genus.