ABSTRACT
The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrPSc , the aggregated and infectious isoform of the cellular prion protein (PrPC ), with largely unsuccessful results. Conversely, targeting PrPC expression, stability or cell surface localization are poorly explored strategies. We recently characterized the mode of action of chlorpromazine, an anti-psychotic drug known to inhibit prion replication and toxicity by inducing the re-localization of PrPC from the plasma membrane. Unfortunately, chlorpromazine possesses pharmacokinetic properties unsuitable for chronic use in vivo, namely low specificity and high toxicity. Here, we employed HEK293 cells stably expressing EGFP-PrP to carry out a semi-automated high content screening (HCS) of a chemical library directed at identifying non-cytotoxic molecules capable of specifically relocalizing PrPC from the plasma membrane as well as inhibiting prion replication in N2a cell cultures. We identified four candidate hits inducing a significant reduction in cell surface PrPC , one of which also inhibited prion propagation and toxicity in cell cultures in a strain-independent fashion. This study defines a new screening method and novel anti-prion compounds supporting the notion that removing PrPC from the cell surface could represent a viable therapeutic strategy for prion diseases.
Subject(s)
Cell Membrane/chemistry , PrPC Proteins/analysis , Prions/antagonists & inhibitors , Animals , Casein Kinase II/antagonists & inhibitors , Cell Line, Tumor , Cell Survival/drug effects , Drug Evaluation, Preclinical/methods , Fluorescent Dyes , Gene Expression , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , HEK293 Cells , Harmaline/analogs & derivatives , Harmaline/pharmacology , Hematoxylin/analogs & derivatives , Hematoxylin/pharmacology , Humans , Mice , Neuroblastoma , PrPC Proteins/genetics , Prions/biosynthesis , Prions/toxicity , Quinacrine/pharmacology , Tacrolimus/pharmacologyABSTRACT
Peptidylprolyl isomerase A (PPIA), also known as cyclophilin A, is a multifunctional protein with peptidyl-prolyl cis-trans isomerase activity. PPIA is also a translational biomarker for amyotrophic lateral sclerosis, and is enriched in aggregates isolated from amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients. Its normal function in the central nervous system is unknown. Here we show that PPIA is a functional interacting partner of TARDBP (also known as TDP-43). PPIA regulates expression of known TARDBP RNA targets and is necessary for the assembly of TARDBP in heterogeneous nuclear ribonucleoprotein complexes. Our data suggest that perturbation of PPIA/TARDBP interaction causes 'TDP-43' pathology. Consistent with this model, we show that the PPIA/TARDBP interaction is impaired in several pathological conditions. Moreover, PPIA depletion induces TARDBP aggregation, downregulates HDAC6, ATG7 and VCP, and accelerates disease progression in the SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Targeting the PPIA/TARDBP interaction may represent a novel therapeutic avenue for conditions involving TARDBP/TDP-43 pathology, such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration.
Subject(s)
DNA-Binding Proteins/physiology , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Peptidylprolyl Isomerase/metabolism , Adult , Aged , Aged, 80 and over , Animals , Cell Line, Tumor , DNA-Binding Proteins/chemistry , Female , HEK293 Cells , Heterogeneous-Nuclear Ribonucleoproteins/chemistry , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Humans , Male , Mice , Mice, 129 Strain , Mice, Transgenic , Middle Aged , Peptidylprolyl Isomerase/geneticsABSTRACT
Growing evidence suggests that a physiological activity of the cellular prion protein (PrP(C)) plays a crucial role in several neurodegenerative disorders, including prion and Alzheimer's diseases. However, how the functional activity of PrP(C) is subverted to deliver neurotoxic signals remains uncertain. Transgenic (Tg) mice expressing PrP with a deletion of residues 105-125 in the central region (referred to as ΔCR PrP) provide important insights into this problem. Tg(ΔCR) mice exhibit neonatal lethality and massive degeneration of cerebellar granule neurons, a phenotype that is dose dependently suppressed by the presence of wild-type PrP. When expressed in cultured cells, ΔCR PrP induces large, ionic currents that can be detected by patch-clamping techniques. Here, we tested the hypothesis that abnormal ion channel activity underlies the neuronal death seen in Tg(ΔCR) mice. We find that ΔCR PrP induces abnormal ionic currents in neurons in culture and in cerebellar slices and that this activity sensitizes the neurons to glutamate-induced, calcium-mediated death. In combination with ultrastructural and biochemical analyses, these results demonstrate a role for glutamate-induced excitotoxicity in PrP-mediated neurodegeneration. A similar mechanism may operate in other neurodegenerative disorders attributable to toxic, ß-rich oligomers that bind to PrP(C).
Subject(s)
Excitatory Amino Acid Agonists/toxicity , Glutamic Acid/toxicity , Mice, Inbred C57BL/physiology , Mutation/physiology , Neurons/physiology , PrPC Proteins/biosynthesis , Animals , Cells, Cultured , Mice , Mice, Transgenic , Neurons/drug effects , Organ Culture Techniques , PrPC Proteins/geneticsABSTRACT
Prion propagation involves a templating reaction in which the infectious form of the prion protein (PrP(Sc)) binds to the cellular form (PrP(C)), generating additional molecules of PrP(Sc). While several regions of the PrP(C) molecule have been suggested to play a role in PrP(Sc) formation based on in vitro studies, the contribution of these regions in vivo is unclear. Here, we report that mice expressing PrP deleted for a short, polybasic region at the N terminus (residues 23-31) display a dramatically reduced susceptibility to prion infection and accumulate greatly reduced levels of PrP(Sc). These results, in combination with biochemical data, demonstrate that residues 23-31 represent a critical site on PrP(C) that binds to PrP(Sc) and is essential for efficient prion propagation. It may be possible to specifically target this region for treatment of prion diseases as well as other neurodegenerative disorders due to ß-sheet-rich oligomers that bind to PrP(C).
Subject(s)
Brain/metabolism , Peptide Fragments/metabolism , PrPC Proteins/metabolism , PrPSc Proteins/chemistry , PrPSc Proteins/metabolism , Prion Diseases/metabolism , Age Factors , Animals , Brain/pathology , Cell Line, Transformed , Cricetinae , Disease Models, Animal , Endocytosis/genetics , Gene Expression Regulation/genetics , Humans , Immunization/methods , Membrane Microdomains/metabolism , Membrane Microdomains/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neuroblastoma/pathology , Peptide Fragments/genetics , PrPC Proteins/genetics , Prion Diseases/genetics , Prion Diseases/immunology , Prion Diseases/pathology , Protein Binding/genetics , Protein Structure, Secondary/genetics , Scrapie/metabolism , Scrapie/pathology , Sequence Deletion/genetics , Time Factors , TransfectionABSTRACT
There is evidence that alterations in the normal physiological activity of PrP(C) contribute to prion-induced neurotoxicity. This mechanism has been difficult to investigate, however, because the normal function of PrP(C) has remained obscure, and there are no assays available to measure it. We recently reported that cells expressing PrP deleted for residues 105-125 exhibit spontaneous ionic currents and hypersensitivity to certain classes of cationic drugs. Here, we utilize cell culture assays based on these two phenomena to test how changes in PrP sequence and/or cellular localization affect the functional activity of the protein. We report that the toxic activity of Δ105-125 PrP requires localization to the plasma membrane and depends on the presence of a polybasic amino acid segment at the N terminus of PrP. Several different deletions spanning the central region as well as three disease-associated point mutations also confer toxic activity on PrP. The sequence domains identified in our study are also critical for PrP(Sc) formation, suggesting that common structural features may govern both the functional activity of PrP(C) and its conversion to PrP(Sc).
Subject(s)
Cell Membrane/metabolism , Prions/chemistry , Animals , Cell Line , Cytoplasm/metabolism , Detergents/pharmacology , Electrophysiology/methods , Gene Deletion , Humans , Ions/chemistry , Mice , Mutation , Neurodegenerative Diseases/pathology , Prion Diseases/metabolism , Protein Structure, TertiaryABSTRACT
Although a great deal of progress has been made in elucidating the molecular identity of the infectious agent in prion diseases, the mechanisms by which prions kill neurons, and the role of the cellular prion protein (PrP(C)) in this process, remain enigmatic. A window into the normal function of PrP(C), and how it can be corrupted to produce neurotoxic effects, is provided by a PrP deletion mutant called ΔCR, which produces a lethal phenotype when expressed in transgenic mice. In a previous study, we described the unusual observation that cells expressing ΔCR PrP are hyper-sensitive to the toxic effects of two cationic antibiotics (G418 and Zeocin) that are typically used for selection of transfected cell lines. We have used this drug-sensitizing effect to develop a simple Drug-Based Cell Assay (DBCA) that reproduces several features of mutant PrP toxicity observed in vivo, including the rescuing activity of wild-type PrP. In this paper, we present a detailed guide for executing the DBCA in several, different experimental settings, including a new slot blot-based format. This assay provides a unique tool for studying PrP cytotoxic and cytoprotective activities in cell culture.
Subject(s)
Biological Assay/methods , Cytoprotection , Cytotoxins/pharmacology , Prions/pharmacology , Amino Acid Sequence , Cell Survival , Coloring Agents , Formazans/chemistry , HEK293 Cells , Histones/metabolism , Humans , In Situ Nick-End Labeling/methods , Prions/genetics , Sequence Deletion , Tetrazolium Salts/chemistryABSTRACT
The prion protein (PrP) is a glycosylphosphatidylinositol-anchored membrane glycoprotein that plays a vital role in prion diseases, a class of fatal neurodegenerative disorders of humans and animals. Approximately 20% of human prion diseases display autosomal dominant inheritance and are linked to mutations in the PrP gene on chromosome 20. PrP mutations are thought to favor the conformational conversion of PrP into a misfolded isoform that causes disease by an unknown mechanism. The PrP mutation D178N/Met-129 is linked to fatal familial insomnia, which causes severe sleep abnormalities and autonomic dysfunction. We showed by immunoelectron microscopy that this mutant PrP accumulates abnormally in the endoplasmic reticulum and Golgi of transfected neuroblastoma N2a cells. To investigate the impact of intracellular PrP accumulation on cellular homeostasis, we did a two-dimensional gel-based differential proteomics analysis. We used wide range immobilized pH gradient strips, pH 4-7 and 6-11, to analyze a large number of proteins. We found changes in proteins involved in energy metabolism, redox regulation, and vesicular transport. Rab GDP dissociation inhibitor alpha (GDI) was one of the proteins that changed most. GDI regulates vesicular protein trafficking by acting on the activity of several Rab proteins. We found a specific reduction in the level of functional Rab11 in mutant PrP-expressing cells associated with impaired post-Golgi trafficking. Our data are consistent with a model by which mutant PrP induces overexpression of GDI, activating a cytotoxic feedback loop that leads to protein accumulation in the secretory pathway.
Subject(s)
Guanine Nucleotide Dissociation Inhibitors/metabolism , Prions/genetics , Prions/metabolism , rab GTP-Binding Proteins/metabolism , Animals , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Gene Expression/physiology , Golgi Apparatus/drug effects , Golgi Apparatus/metabolism , Guanine Nucleotide Dissociation Inhibitors/genetics , Guanine Nucleotide Dissociation Inhibitors/physiology , Humans , Mice , Mutant Proteins/antagonists & inhibitors , Mutant Proteins/genetics , Mutant Proteins/metabolism , Neurons/metabolism , Prions/antagonists & inhibitors , Prions/physiology , Protein Transport/drug effects , Protein Transport/genetics , Protein Transport/physiology , Proteomics , RNA, Small Interfering/pharmacology , Secretory Pathway/drug effects , Secretory Pathway/genetics , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/physiology , Tumor Cells, Cultured , rab GTP-Binding Proteins/physiologyABSTRACT
In prion diseases, the infectious isoform of the prion protein (PrP(Sc)) may subvert a normal, physiological activity of the cellular isoform (PrP(C)). A deletion mutant of the prion protein (Delta105-125) that produces a neonatal lethal phenotype when expressed in transgenic mice provides a window into the normal function of PrP(C) and how it can be corrupted to produce neurotoxic effects. We report here the surprising and unexpected observation that cells expressing Delta105-125 PrP and related mutants are hypersensitive to the toxic effects of two classes of antibiotics (aminoglycosides and bleomycin analogues) that are commonly used for selection of stably transfected cell lines. This unusual phenomenon mimics several essential features of Delta105-125 PrP toxicity seen in transgenic mice, including rescue by co-expression of wild type PrP. Cells expressing Delta105-125 PrP are susceptible to drug toxicity within minutes, suggesting that the mutant protein enhances cellular accumulation of these cationic compounds. Our results establish a screenable cellular phenotype for the activity of neurotoxic forms of PrP, and they suggest possible mechanisms by which these molecules could produce their pathological effects in vivo.
Subject(s)
Biological Assay/methods , Drug Evaluation, Preclinical/methods , PrPC Proteins , PrPSc Proteins , Prion Diseases/metabolism , Amebicides/pharmacology , Animals , Antibiotics, Antineoplastic/pharmacology , Bleomycin/pharmacology , Cell Line/drug effects , Cell Survival , Cinnamates/pharmacology , GPI-Linked Proteins , Gentamicins/pharmacology , Humans , Hygromycin B/analogs & derivatives , Hygromycin B/pharmacology , Mice , Mice, Transgenic , Phenotype , PrPC Proteins/genetics , PrPC Proteins/metabolism , PrPC Proteins/toxicity , PrPSc Proteins/genetics , PrPSc Proteins/metabolism , PrPSc Proteins/toxicity , Prions/genetics , Prions/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Stem Cells/cytology , Stem Cells/drug effects , Stem Cells/physiologyABSTRACT
Approx. 15% of human prion diseases have a pattern of autosomal dominant inheritance, and are linked to mutations in the gene encoding PrP (prion protein), a GPI (glycosylphosphatidylinositol)-anchored protein whose function is not clear. The cellular mechanisms by which PrP mutations cause disease are also not known. Soon after synthesis in the ER (endoplasmic reticulum), several mutant PrPs misfold and become resistant to phospholipase cleavage of their GPI anchor. The biosynthetic maturation of the misfolded molecules in the ER is delayed and, during transit in the secretory pathway, they form detergent-insoluble and protease-resistant aggregates, suggesting that intracellular PrP aggregation may play a pathogenic role. We have investigated the consequence of deleting residues 114-121 within the hydrophobic core of PrP on the aggregation and cellular localization of two pathogenic mutants that accumulate in the ER and Golgi apparatus. Compared with their full-length counterparts, the deleted molecules formed smaller protease-sensitive aggregates and were more efficiently transported to the cell surface and released by phospholipase cleavage. These results indicate that mutant PrP aggregation and intracellular retention are closely related and depend critically on the integrity of the hydrophobic core. The discovery that Delta114-121 counteracts misfolding and improves the cellular trafficking of mutant PrP provides an unprecedented model for assessing the role of intracellular aggregation in the pathogenesis of prion diseases.
Subject(s)
Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Mutant Proteins/metabolism , Prions/metabolism , Animals , Blotting, Western , Cell Line , Gene Deletion , Glycosylphosphatidylinositols/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Intracellular Space/metabolism , Mice , Microscopy, Confocal , Mutant Proteins/chemistry , Mutant Proteins/genetics , Phospholipases/metabolism , Prions/chemistry , Prions/genetics , Protein Conformation , Protein Folding , Secretory PathwayABSTRACT
The prion protein (PrPC) is a central player in neurodegenerative diseases, such as prion diseases or Alzheimer's disease. In contrast to disease-promoting cell surface PrPC, extracellular fragments act neuroprotective by blocking neurotoxic disease-associated protein conformers. Fittingly, PrPC release by the metalloprotease ADAM10 represents a protective mechanism. We used biochemical, cell biological, morphological, and structural methods to investigate mechanisms stimulating this proteolytic shedding. Shed PrP negatively correlates with prion conversion and is markedly redistributed in murine brain in the presence of prion deposits or amyloid plaques, indicating a sequestrating activity. PrP-directed ligands cause structural changes in PrPC and increased shedding in cells and organotypic brain slice cultures. As an exception, some PrP-directed antibodies targeting repetitive epitopes do not cause shedding but surface clustering, endocytosis, and degradation of PrPC. Both mechanisms may contribute to beneficial actions described for PrP-directed ligands and pave the way for new therapeutic strategies against currently incurable neurodegenerative diseases.
ABSTRACT
Recent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. These techniques allowed us to design a drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein levels by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP and identified four different small molecule ligands for this conformer, all capable of selectively lowering the load of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression.
Subject(s)
Drug Evaluation, Preclinical/methods , Prion Diseases/drug therapy , Prion Proteins/chemistry , Prion Proteins/metabolism , Protein Folding , Animals , Binding Sites , Computer Simulation , Endoplasmic Reticulum/metabolism , Fibroblasts , HEK293 Cells , Humans , Ligands , Lysosomes/drug effects , Lysosomes/metabolism , Mice , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Processing, Post-Translational , Reproducibility of ResultsABSTRACT
IL-7 is required for T cell differentiation and mature T cell homeostasis and promotes pro-B cell proliferation and survival. Tyrosine phosphorylation plays a central role in IL-7 signaling. We identified by two-dimensional electrophoresis followed by anti-phosphotyrosine immunoblotting and mass spectrometry sixteen tyrosine phosphorylated proteins from the IL-7-dependent cell line D1. IL-7 stimulation induced the phosphorylation of the proteins STI1, ATIC and hnRNPH, involved in pathways related to survival, proliferation and gene expression, respectively, and increased the phosphorylation of CrkL, a member of a family of adaptors including the highly homologous Crk isoforms CrkII and CrkI, important in multiple signaling pathways. We observed an increased phosphorylation of CrkL in murine pro-B cells and in murine and human T cells. In addition, IL-7 increased the association of CrkL with the transcription factor Stat5, essential for IL-7 pro-survival activity. The selective tyrosine kinase inhibitor Imatinib. counteracted the IL-7 pro-survival effect in D1 cells and decreased CrkL phosphorylation. These data suggested that CrkL could play a pro-survival role in IL-7-mediated signaling. We observed that pro-B cells also expressed, in addition to CrkL, the Crk isoforms CrkII and CrkI and therefore utilized pro-B cells conditionally deficient in all three to evaluate the role of these proteins. The observation that the IL-7 pro-survival effect was reduced in Crk/CrkL conditionally-deficient pro-B cells further pointed to a pro-survival role of these adaptors. To further evaluate the role of these proteins, gene expression studies were performed in Crk/CrkL conditionally-deficient pro-B cells. IL-7 decreased the transcription of the receptor LAIR1, which inhibits B cell proliferation, in a Crk/CrkL-dependent manner, suggesting that the Crk family of proteins may promote pro-B cell proliferation. Our data contribute to the understanding of IL-7 signaling and suggest the involvement of Crk family proteins in pathways promoting survival and proliferation.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Interleukin-7/pharmacology , Nuclear Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Apoptosis/drug effects , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , Gene Expression Regulation/drug effects , Humans , Imatinib Mesylate/pharmacology , Male , Mice , Mice, Inbred C57BL , Nuclear Proteins/genetics , Phosphorylation/drug effects , Protein Isoforms/genetics , Protein Isoforms/metabolism , STAT5 Transcription Factor/metabolism , Signal Transduction/drug effects , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , T-Lymphocytes/metabolismABSTRACT
Prion diseases are neurodegenerative conditions characterized by the conformational conversion of the cellular prion protein (PrPC), an endogenous membrane glycoprotein of uncertain function, into PrPSc, a pathological isoform that replicates by imposing its abnormal folding onto PrPC molecules. A great deal of evidence supports the notion that PrPC plays at least two roles in prion diseases, by acting as a substrate for PrPSc replication, and as a mediator of its toxicity. This conclusion was recently supported by data suggesting that PrPC may transduce neurotoxic signals elicited by other disease-associated protein aggregates. Thus, PrPC may represent a convenient pharmacological target for prion diseases, and possibly other neurodegenerative conditions. Here, we sought to characterize the activity of chlorpromazine (CPZ), an antipsychotic previously shown to inhibit prion replication by directly binding to PrPC. By employing biochemical and biophysical techniques, we provide direct experimental evidence indicating that CPZ does not bind PrPC at biologically relevant concentrations. Instead, the compound exerts anti-prion effects by inducing the relocalization of PrPC from the plasma membrane. Consistent with these findings, CPZ also inhibits the cytotoxic effects delivered by a PrP mutant. Interestingly, we found that the different pharmacological effects of CPZ could be mimicked by two inhibitors of the GTPase activity of dynamins, a class of proteins involved in the scission of newly formed membrane vesicles, and recently reported as potential pharmacological targets of CPZ. Collectively, our results redefine the mechanism by which CPZ exerts anti-prion effects, and support a primary role for dynamins in the membrane recycling of PrPC, as well as in the propagation of infectious prions.
Subject(s)
Antipsychotic Agents/pharmacology , Chlorpromazine/pharmacology , Prion Proteins/metabolism , Antipsychotic Agents/metabolism , Cell Line , Chlorpromazine/metabolism , Dynamins/antagonists & inhibitors , Humans , Ligands , Mutation , Prion Proteins/genetics , Protein Transport/drug effectsABSTRACT
Into the fold: Prion diseases are neurodegenerative disorders characterized by the accumulation in the brain of a self-replicating, misfolded isoform (PrPSc ) of the cellular prion protein (PrPC ). No therapies are available for these pathologies. We capitalized on previously described cell-based assays to screen a library of small molecules, and identified 55, a compound capable of counteracting both prion replication and toxicity. Compound 55 may represent the starting point for the development of a completely new class of therapeutics for prion diseases.
Subject(s)
Prion Proteins/metabolism , Small Molecule Libraries/metabolism , Animals , Brain/metabolism , Cell Line , Cell Survival/drug effects , HEK293 Cells , Humans , Inhibitory Concentration 50 , Mutagenesis , PrPSc Proteins/antagonists & inhibitors , PrPSc Proteins/genetics , PrPSc Proteins/metabolism , Prion Diseases/metabolism , Prion Diseases/pathology , Prion Proteins/antagonists & inhibitors , Prion Proteins/genetics , Protein Binding , Small Molecule Libraries/chemistry , Small Molecule Libraries/toxicityABSTRACT
Prion diseases are rare neurodegenerative conditions associated with the conformational conversion of the cellular prion protein (PrP(C)) into PrP(Sc), a self-replicating isoform (prion) that accumulates in the central nervous system of affected individuals. The structure of PrP(Sc) is poorly defined, and likely to be heterogeneous, as suggested by the existence of different prion strains. The latter represents a relevant problem for therapy in prion diseases, as some potent anti-prion compounds have shown strain-specificity. Designing therapeutics that target PrP(C) may provide an opportunity to overcome these problems. PrP(C) ligands may theoretically inhibit the replication of multiple prion strains, by acting on the common substrate of any prion replication reaction. Here, we characterized the properties of a cationic tetrapyrrole [Fe(III)-TMPyP], which was previously shown to bind PrP(C), and inhibit the replication of a mouse prion strain. We report that the compound is active against multiple prion strains in vitro and in cells. Interestingly, we also find that Fe(III)-TMPyP inhibits several PrP(C)-related toxic activities, including the channel-forming ability of a PrP mutant, and the PrP(C)-dependent synaptotoxicity of amyloid-ß (Aß) oligomers, which are associated with Alzheimer's Disease. These results demonstrate that molecules binding to PrP(C) may produce a dual effect of blocking prion replication and inhibiting PrP(C)-mediated toxicity.
Subject(s)
Metalloporphyrins/chemistry , PrPC Proteins/metabolism , Prion Proteins/antagonists & inhibitors , Tetrapyrroles/chemistry , Amyloid beta-Peptides/metabolism , Animals , Binding Sites , Cell Line, Tumor , HEK293 Cells , Humans , Metalloporphyrins/pharmacology , Mice, Inbred C57BL , Mice, Knockout , Mutation , Porphyrins , PrPC Proteins/chemistry , PrPC Proteins/genetics , Prion Proteins/chemistry , Protein Binding , Recombinant Proteins/metabolism , Tetrapyrroles/pharmacologyABSTRACT
Transglutaminase 2 (TG2) is a multifunctional protein with Ca(2+)-dependent transamidating and G protein activity. Previously, we reported that tgm2 -/- mice have an impaired insulin secretion and that naturally occurring TG2 mutations associated with familial, early-onset type 2 diabetes, show a defective transamidating activity. Aim of this study was to get a better insight into the role of TG2 in insulin secretion by identifying substrates of TG2 transamidating activity in the pancreatic beta cell line INS-1E. To this end, we labeled INS-1E that are capable of secreting insulin upon glucose stimulation in the physiologic range, with an artificial acyl acceptor (biotinamido-pentylamine) or donor (biotinylated peptide), in basal condition and after stimulus with glucose for 2, 5, and 8 min. Biotinylated proteins were analyzed by two-dimensional electrophoresis and mass spectrometry. In addition, subcellular localization of TG2 in human endocrine pancreas was studied by electron microscopy. Among several TG2's transamidating substrates in INS-1E, mass spectrometry identified cytoplasmic actin (a result confirmed in human pancreatic islet), tropomyosin, and molecules that participate in insulin granule structure (e.g., GAPDH), glucose metabolism, or [Ca(2+)] sensing (e.g., calreticulin). Physical interaction between TG2 and cytoplasmic actin during glucose-stimulated first-phase insulin secretion was confirmed by co-immunoprecipitation. Electron microscopy revealed that TG2 is localized close to insulin and glucagon granules in human pancreatic islet. We propose that TG2's role in insulin secretion may involve cytoplasmic actin remodeling and may have a regulative action on other proteins during granule movement. A similar role of TG2 in glucagon secretion is also suggested.
Subject(s)
GTP-Binding Proteins/metabolism , Insulin-Secreting Cells/metabolism , Insulin/metabolism , Transglutaminases/metabolism , Animals , Cell Line, Tumor , GTP-Binding Proteins/chemistry , Glucose/metabolism , Humans , Insulin Secretion , Insulin-Secreting Cells/chemistry , Mass Spectrometry , Protein Glutamine gamma Glutamyltransferase 2 , Rats , Substrate Specificity , Transglutaminases/chemistryABSTRACT
Insight into the normal function of PrP(C), and how it can be subverted to produce neurotoxic effects, is provided by PrP molecules carrying deletions encompassing the conserved central region. The most neurotoxic of these mutants, Δ105-125 (called ΔCR), produces a spontaneous neurodegenerative illness when expressed in transgenic mice, and this phenotype can be dose-dependently suppressed by co-expression of wild-type PrP. Whether the toxic activity of ΔCR PrP and the protective activity or wild-type PrP are cell-autonomous, or can be exerted on neighboring cells, is unknown. To investigate this question, we have utilized co-cultures of differentiated neural stem cells derived from mice expressing ΔCR or wild-type PrP. Cells from the two kinds of mice, which are marked by the presence or absence of GFP, are differentiated together to yield neurons, astrocytes, and oligodendrocytes. As a surrogate read-out of ΔCR PrP toxicity, we assayed sensitivity of the cells to the cationic antibiotic, Zeocin. In a previous study, we reported that cells expressing ΔCR PrP are hypersensitive to the toxic effects of several cationic antibiotics, an effect that is suppressed by co-expression of wild type PrP, similar to the rescue of the neurodegenerative phenotype observed in transgenic mice. Using this system, we find that while ΔCR-dependent toxicity is cell-autonomous, the rescuing activity of wild-type PrP can be exerted in trans from nearby cells. These results provide important insights into how ΔCR PrP subverts a normal physiological function of PrP(C), and the cellular mechanisms underlying the rescuing process.
Subject(s)
Cell Communication/physiology , Membrane Proteins/metabolism , Neural Stem Cells/metabolism , PrPC Proteins/genetics , PrPC Proteins/metabolism , Animals , Bleomycin/toxicity , Blotting, Western , Cells, Cultured , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , In Situ Nick-End Labeling , Mice , Microscopy, Fluorescence , Mutation/genetics , Neural Stem Cells/drug effects , PrPC Proteins/toxicity , Time-Lapse ImagingABSTRACT
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease, and protein aggregation has been proposed as a possible pathogenetic mechanism. However, the aggregate protein constituents are poorly characterized so knowledge on the role of aggregation in pathogenesis is limited. METHODOLOGY/PRINCIPAL FINDINGS: We carried out a proteomic analysis of the protein composition of the insoluble fraction, as a model of protein aggregates, from familial ALS (fALS) mouse model at different disease stages. We identified several proteins enriched in the detergent-insoluble fraction already at a preclinical stage, including intermediate filaments, chaperones and mitochondrial proteins. Aconitase, HSC70 and cyclophilin A were also significantly enriched in the insoluble fraction of spinal cords of ALS patients. Moreover, we found that the majority of proteins in mice and HSP90 in patients were tyrosine-nitrated. We therefore investigated the role of nitrative stress in aggregate formation in fALS-like murine motor neuron-neuroblastoma (NSC-34) cell lines. By inhibiting nitric oxide synthesis the amount of insoluble proteins, particularly aconitase, HSC70, cyclophilin A and SOD1 can be substantially reduced. CONCLUSION/SIGNIFICANCE: Analysis of the insoluble fractions from cellular/mouse models and human tissues revealed novel aggregation-prone proteins and suggests that nitrative stress contribute to protein aggregate formation in ALS.
Subject(s)
Amyotrophic Lateral Sclerosis/pathology , Detergents/pharmacology , Proteins/chemistry , Proteins/metabolism , Stress, Physiological/drug effects , Tyrosine/analogs & derivatives , Aconitate Hydratase/metabolism , Aconitate Hydratase/ultrastructure , Amino Acid Substitution/genetics , Amyotrophic Lateral Sclerosis/enzymology , Animals , Disease Models, Animal , Disease Progression , Electrophoresis, Gel, Two-Dimensional , Humans , Immunohistochemistry , Mice , NG-Nitroarginine Methyl Ester/pharmacology , Protein Structure, Quaternary , Proteomics , Reproducibility of Results , Solubility/drug effects , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spinal Cord/drug effects , Spinal Cord/ultrastructure , Superoxide Dismutase/metabolism , Superoxide Dismutase-1 , Tyrosine/metabolismABSTRACT
Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, whose primary mechanisms or causes are still not defined and for which no effective treatment is available. We have recently reported that before disease onset the level of tyrosine nitrated proteins is increased in the G93A SOD1 transgenic mouse model of ALS. In the present investigation, we carried out a proteomic analysis of spinal cord extracts from G93A SOD1 mice at the presymptomatic stage of the disease to further unravel primary events in the pathogenesis and tentatively screen for potential pharmacological targets. Using a robust two-dimensional gel electrophoresis-based proteomic approach, we detected a number of proteins differentially represented in presymptomatic mice in comparison with controls. Alterations of these proteins correlate with mitochondrial dysfunction, aggregation, and stress response. Moreover, we found a variation in the isoform pattern of cyclophilin A, a molecular chaperone that protects cells from the oxidative stress.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Proteomics , Superoxide Dismutase/genetics , Animals , Cyclophilin A/genetics , Disease Models, Animal , Electrophoresis, Gel, Two-Dimensional , Female , Mice , Mice, Transgenic , Nerve Tissue Proteins/metabolism , Phosphorylation , Protein Isoforms/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spinal Cord/metabolismABSTRACT
Inherited prion diseases are linked to insertional and point mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. Transgenic (Tg) (PG14) mice express a mouse PrP homolog of a nine-octapeptide insertion associated with an inherited prion disorder. These mice develop a progressive neurological syndrome characterized by ataxia and cerebellar atrophy due to synaptic degeneration in the molecular layer and massive apoptosis of granule neurons. To investigate the molecular events that may contribute to neurological dysfunction, we carried out a differential proteomic analysis of cerebella from Tg(PG14) mice at the preclinical, onset, and symptomatic phases of their neurological illness. 2-D maps of cerebellar proteins from Tg(PG14) mice were compared to those obtained from age-matched Tg(WT) mice that express wild-type PrP and remain healthy. Proteins whose levels were significantly modified in at least one stage of the Tg(PG14) disease were identified by PMF. Analysis detected a preclinical decrease of the calcium/calmodulin-dependent phosphatase calcineurin (CaN) in granule neurons, suggesting that dysregulation of CaN activity induced by mutant PrP may be responsible for the cerebellar dysfunction in Tg(PG14) mice.