RESUMEN
Oligomeric species populated during α-synuclein aggregation are considered key drivers of neurodegeneration in Parkinson's disease. However, the development of oligomer-targeting therapeutics is constrained by our limited knowledge of their structure and the molecular determinants driving their conversion to fibrils. Phenol-soluble modulin α3 (PSMα3) is a nanomolar peptide binder of α-synuclein oligomers that inhibits aggregation by blocking oligomer-to-fibril conversion. Here, we investigate the binding of PSMα3 to α-synuclein oligomers to discover the mechanistic basis of this protective activity. We find that PSMα3 selectively targets an α-synuclein N-terminal motif (residues 36-61) that populates a distinct conformation in the mono- and oligomeric states. This α-synuclein region plays a pivotal role in oligomer-to-fibril conversion as its absence renders the central NAC domain insufficient to prompt this structural transition. The hereditary mutation G51D, associated with early onset Parkinson's disease, causes a conformational fluctuation in this region, leading to delayed oligomer-to-fibril conversion and an accumulation of oligomers that are resistant to remodeling by molecular chaperones. Overall, our findings unveil a new targetable region in α-synuclein oligomers, advance our comprehension of oligomer-to-amyloid fibril conversion, and reveal a new facet of α-synuclein pathogenic mutations.
Asunto(s)
alfa-Sinucleína , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Humanos , Enfermedad de Parkinson/metabolismo , Secuencias de AminoácidosRESUMEN
BACKGROUND: α-Synuclein (αS) aggregation is the main neurological hallmark of a group of neurodegenerative disorders, collectively referred to as synucleinopathies, of which Parkinson's disease (PD) is the most prevalent. αS oligomers are elevated in the cerebrospinal fluid (CSF) of PD patients, standing as a biomarker for disease diagnosis. However, methods for early PD detection are still lacking. We have recently identified the amphipathic 22-residue peptide PSMα3 as a high-affinity binder of αS toxic oligomers. PSMα3 displayed excellent selectivity and reproducibility, binding to αS toxic oligomers with affinities in the low nanomolar range and without detectable cross-reactivity with functional monomeric αS. RESULTS: In this work, we leveraged these PSMα3 unique properties to design a plasmonic-based biosensor for the direct detection of toxic oligomers under label-free conditions. SIGNIFICANCE AND NOVELTY: We describe the integration of the peptide in a lab-on-a-chip plasmonic platform suitable for point-of-care measurements of αS toxic oligomers in CSF samples in real-time and at an affordable cost, providing an innovative biosensor for PD early diagnosis in the clinic.
Asunto(s)
Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Humanos , alfa-Sinucleína , Reproducibilidad de los Resultados , Enfermedad de Parkinson/diagnóstico , PéptidosRESUMEN
α-Synuclein (αS) aggregation is the main neurological hallmark of a group of debilitating neurodegenerative disorders, collectively referred to as synucleinopathies, of which Parkinson's disease is the most prevalent. αS oligomers formed during the initial stages of aggregation are considered key pathogenic drivers of disease onset and progression, standing as privileged targets for therapeutic intervention and diagnosis. However, the structure of αS oligomers and the mechanistic basis of oligomer to fibril conversion are yet poorly understood, thereby precluding the rational formulation of strategies aimed at targeting oligomeric species. In this review, we delve into the recent advances in the structural and mechanistic characterization of αS oligomers. We also discuss how these advances are transforming our understanding of these elusive species and paving the way for oligomer-targeting therapeutics and diagnosis.
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Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder, yet effective treatments able to stop or delay disease progression remain elusive. The aggregation of a presynaptic protein, α-synuclein (aSyn), is the primary neurological hallmark of PD and, thus, a promising target for therapeutic intervention. However, the lack of consensus on the molecular properties required to specifically bind the toxic species formed during aSyn aggregation has hindered the development of therapeutic molecules. Recently, we defined and experimentally validated a peptide architecture that demonstrated high affinity and selectivity in binding to aSyn toxic oligomers and fibrils, effectively preventing aSyn pathogenic aggregation. Human peptides with such properties may have neuroprotective activities and hold a huge therapeutic interest. Driven by this idea, here, we developed a discriminative algorithm for the screening of human endogenous neuropeptides, antimicrobial peptides and diet-derived bioactive peptides with the potential to inhibit aSyn aggregation. We identified over 100 unique biogenic peptide candidates and ensembled a comprehensive database (aSynPEP-DB) that collects their physicochemical features, source datasets and additional therapeutic-relevant information, including their sites of expression and associated pathways. Besides, we provide access to the discriminative algorithm to extend its application to the screening of artificial peptides or new peptide datasets. aSynPEP-DB is a unique repository of peptides with the potential to modulate aSyn aggregation, serving as a platform for the identification of previously unexplored therapeutic agents. Database URL: https://asynpepdb.ppmclab.com/.
Asunto(s)
Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Humanos , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , PéptidosRESUMEN
The aggregation of wild-type transthyretin (TTR) and over 130 genetic TTR variants underlies a group of lethal disorders named TTR amyloidosis (ATTR). TTR chemical chaperones are molecules that hold great promise to modify the course of ATTR progression. In previous studies, we combined rational design and molecular dynamics simulations to generate a series of TTR selective kinetic stabilizers displaying exceptionally high affinities. In an effort to endorse the previously developed molecules with optimal pharmacokinetic properties, we conducted structural design optimization, leading to the development of PITB. PITB binds with high affinity to TTR, effectively inhibiting tetramer dissociation and aggregation of both the wild-type protein and the two most prevalent disease-associated TTR variants. Importantly, PITB selectively binds and stabilizes TTR in plasma, outperforming tolcapone, a drug currently undergoing clinical trials for ATTR. Pharmacokinetic studies conducted on mice confirmed that PITB exhibits encouraging pharmacokinetic properties, as originally intended. Furthermore, PITB demonstrates excellent oral bioavailability and lack of toxicity. These combined attributes position PITB as a lead compound for future clinical trials as a disease-modifying therapy for ATTR.
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Neuropatías Amiloides Familiares , Prealbúmina , Ratones , Animales , Prealbúmina/metabolismo , Neuropatías Amiloides Familiares/tratamiento farmacológico , Neuropatías Amiloides Familiares/metabolismo , Tolcapona/uso terapéutico , Simulación de Dinámica MolecularRESUMEN
Stress granules (SGs) are highly conserved cytoplasmic condensates that assemble in response to stress and contribute to maintaining protein homeostasis. These membraneless organelles are dynamic, disassembling once the stress is no longer present. Persistence of SGs due to mutations or chronic stress has been often related to age-dependent protein-misfolding diseases in animals. Here, we find that the metacaspase MC1 is dynamically recruited into SGs upon proteotoxic stress in Arabidopsis (Arabidopsis thaliana). Two predicted disordered regions, the prodomain and the 360 loop, mediate MC1 recruitment to and release from SGs. Importantly, we show that MC1 has the capacity to clear toxic protein aggregates in vivo and in vitro, acting as a disaggregase. Finally, we demonstrate that overexpressing MC1 delays senescence and this phenotype is dependent on the presence of the 360 loop and an intact catalytic domain. Together, our data indicate that MC1 regulates senescence through its recruitment into SGs and this function could potentially be linked to its remarkable protein aggregate-clearing activity.
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Arabidopsis , Animales , Arabidopsis/genética , Arabidopsis/metabolismo , Agregado de Proteínas , Gránulos de Estrés , Gránulos Citoplasmáticos/metabolismo , Estrés FisiológicoRESUMEN
Transthyretin amyloidosis (ATTR) is a group of fatal diseases described by the misfolding and amyloid deposition of transthyretin (TTR). Discovering small molecules that bind and stabilize the TTR tetramer, preventing its dissociation and subsequent aggregation, is a therapeutic strategy for these pathologies. Departing from the crystal structure of TTR in complex with tolcapone, a potent binder in clinical trials for ATTR, we combined rational design and molecular dynamics (MD) simulations to generate a series of novel halogenated kinetic stabilizers. Among them, M-23 displays one of the highest affinities for TTR described so far. The TTR/M-23 crystal structure confirmed the formation of unprecedented protein-ligand contacts, as predicted by MD simulations, leading to an enhanced tetramer stability both in vitro and in whole serum. We demonstrate that MD-assisted design of TTR ligands constitutes a new avenue for discovering molecules that, like M-23, hold the potential to become highly potent drugs to treat ATTR.
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Neuropatías Amiloides Familiares , Prealbúmina , Humanos , Prealbúmina/química , Amiloide/metabolismo , Neuropatías Amiloides Familiares/tratamiento farmacológico , Neuropatías Amiloides Familiares/metabolismo , Tolcapona/uso terapéutico , CinéticaRESUMEN
Proteome-wide analyses suggest that most globular proteins contain at least one amyloidogenic region, whereas these aggregation-prone segments are thought to be underrepresented in intrinsically disordered proteins (IDPs). In recent work, we reported that intrinsically disordered regions (IDRs) indeed sustain a significant amyloid load in the form of cryptic amyloidogenic regions (CARs). CARs are widespread in IDRs, but they are necessarily exposed to solvent, and thus they should be more polar and have a milder aggregation potential than conventional amyloid regions protected inside globular proteins. CARs are connected with IDPs function and, in particular, with the establishment of protein-protein interactions through their IDRs. However, their presence also appears associated with pathologies like cancer or Alzheimer's disease. Given the relevance of CARs for both IDPs function and malfunction, we developed CARs-DB, a database containing precomputed predictions for all CARs present in the IDPs deposited in the DisProt database. This web tool allows for the fast and comprehensive exploration of previously unnoticed amyloidogenic regions embedded within IDRs sequences and might turn helpful in identifying disordered interacting regions. It contains >8,900 unique CARs identified in a total of 1711 IDRs. CARs-DB is freely available for users and can be accessed at http://carsdb.ppmclab.com. To validate CARs-DB, we demonstrate that two previously undescribed CARs selected from the database display full amyloidogenic potential. Overall, CARs-DB allows easy access to a previously unexplored amyloid sequence space.
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α-Synuclein (a-syn) oligomers and fibrils are behind neurodegeneration in Parkinson's disease (PD), but therapeutically targeting them is challenging. Amphipathic and cationic helical peptides inhibit amyloid formation and suppress neurotoxicity by selectively binding the solvent-accessible regions in these toxic species. Can endogenous peptides, like LL-37, constitute a new therapeutic paradigm in PD?
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Enfermedad de Parkinson , Amiloide , Humanos , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
Prion-like domains (PrLDs) are intrinsically disordered regions (IDRs) of low sequence complexity with a similar composition to yeast prion domains. PrLDs-containing proteins have been involved in different organisms' regulatory processes. Regions of moderate amyloid propensity within IDRs have been shown to assemble autonomously into amyloid fibrils. These sequences tend to be rich in polar amino acids and often escape from the detection of classical bioinformatics screenings that look for highly aggregation-prone hydrophobic sequence stretches. We defined them as cryptic amyloidogenic regions (CARs) and recently developed an integrated database that collects thousands of predicted CARs in IDRs. CARs seem to be evolutionary conserved among disordered regions because of their potential to stablish functional contacts with other biomolecules. Here we have focused on identifying and characterizing CARs in prion-like proteins (pCARs) from plants, a lineage that has been poorly studied in comparison with other prionomes. We confirmed the intrinsic amyloid potential for a selected pCAR from Arabidopsis thaliana and explored functional enrichments and compositional bias of pCARs in plant prion-like proteins.
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The treatment of Parkinson's disease (PD), the second most common neurodegenerative human disorder, continues to be symptomatic. Development of drugs able to stop or at least slowdown PD progression would benefit several million people worldwide. SynuClean-D is a low molecular weight 2-pyridone-based promising drug candidate that inhibits the aggregation of α-synuclein in human cultured cells and prevents degeneration of dopaminergic neurons in a Caenorhabditis elegans model of PD. Improving SynuClean-D pharmacokinetic/pharmacodynamic properties, performing structure/activity studies and testing its efficacy in mammalian models of PD requires the use of gr-amounts of the compound. However, not enough compound is on sale, and no synthetic route has been reported until now, which hampers the molecule progress towards clinical trials. To circumvent those problems, we describe here an efficient and economical route that enables the synthesis of SynuClean-D with good yields as well as the synthesis of SynuClean-D derivatives. Structure-activity comparison of the new compounds with SynuClean-D reveals the functional groups of the molecule that can be disposed of without activity loss and those that are crucial to interfere with α-synuclein aggregation. Several of the derivatives obtained retain the parent's compound excellent in vitro anti-aggregative activity, without compromising its low toxicity. Computational predictions and preliminary testing indicate that the blood brain barrier (BBB) permeability of SynuClean-D is low. Importantly, several of the newly designed and obtained active derivatives are predicted to display good BBB permeability. The synthetic route developed here will facilitate their synthesis for BBB permeability determination and for efficacy testing in mammalian models of PD.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Diseño de Fármacos , Enfermedad de Parkinson/tratamiento farmacológico , Piridonas/farmacología , alfa-Sinucleína/antagonistas & inhibidores , Animales , Barrera Hematoencefálica/metabolismo , Caenorhabditis elegans , Relación Dosis-Respuesta a Droga , Estructura Molecular , Enfermedad de Parkinson/metabolismo , Agregado de Proteínas/efectos de los fármacos , Piridonas/síntesis química , Piridonas/química , Relación Estructura-Actividad , alfa-Sinucleína/metabolismoRESUMEN
The amyloid conformation is considered a fundamental state of proteins and the propensity to populate it a generic property of polypeptides. Multiple proteome-wide analyses addressed the presence of amyloidogenic regions in proteins, nurturing our understanding of their nature and biological implications. However, these analyses focused on highly aggregation-prone and hydrophobic stretches that are only marginally found in intrinsically disordered regions (IDRs). Here, we explore the prevalence of cryptic amyloidogenic regions (CARs) of polar nature in IDRs. CARs are widespread in IDRs and associated with IDPs function, with particular involvement in protein-protein interactions, but their presence is also connected to a risk of malfunction. By exploring this function/malfunction dichotomy, we speculate that ancestral CARs might have evolved into functional interacting regions playing a significant role in protein evolution at the origins of life.
RESUMEN
α-Synuclein aggregation is a key driver of neurodegeneration in Parkinson's disease and related syndromes. Accordingly, obtaining a molecule that targets α-synuclein toxic assemblies with high affinity is a long-pursued objective. Here, we exploit the biophysical properties of toxic oligomers and amyloid fibrils to identify a family of α-helical peptides that bind to these α-synuclein species with low nanomolar affinity, without interfering with the monomeric functional protein. This activity is translated into a high anti-aggregation potency and the ability to abrogate oligomer-induced cell damage. Using a structure-guided search we identify a human peptide expressed in the brain and the gastrointestinal tract with analogous binding, anti-aggregation, and detoxifying properties. The chemical entities we describe here may represent a therapeutic avenue for the synucleinopathies and are promising tools to assist diagnosis by discriminating between native and toxic α-synuclein species.
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Amiloide/metabolismo , Enfermedad de Parkinson/patología , Agregación Patológica de Proteínas/patología , alfa-Sinucleína/metabolismo , Encéfalo/metabolismo , Tracto Gastrointestinal/metabolismo , HumanosRESUMEN
Prions are self-perpetuating proteins able to switch between a soluble state and an aggregated-and-transmissible conformation. These proteinaceous entities have been widely studied in yeast, where they are involved in hereditable phenotypic adaptations. The notion that such proteins could play functional roles and be positively selected by evolution has triggered the development of computational tools to identify prion-like proteins in different kingdoms of life. These algorithms have succeeded in screening multiple proteomes, allowing the identification of prion-like proteins in a diversity of unrelated organisms, evidencing that the prion phenomenon is well conserved among species. Interestingly enough, prion-like proteins are not only connected with the formation of functional membraneless protein-nucleic acid coacervates, but are also linked to human diseases. This review addresses state-of-the-art computational approaches to identify prion-like proteins, describes proteome-wide analysis efforts, discusses these unique proteins' functional role, and illustrates recently validated examples in different domains of life.
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Biología Computacional , Priones/química , Priones/metabolismo , Proteoma , Proteómica , Algoritmos , Animales , Biología Computacional/métodos , Humanos , Proteínas de Plantas , Proteínas Priónicas/química , Proteínas Priónicas/metabolismo , Proteómica/métodos , Reproducibilidad de los ResultadosRESUMEN
Hereditary transthyretin amyloidosis (ATTR) is a disease characterized by the extracellular deposition of transthyretin (TTR) amyloid fibrils. Highly destabilizing TTR mutations cause leptomeningeal amyloidosis, a rare, but fatal, disorder in which TTR aggregates in the brain. The disease remains intractable, since liver transplantation, the reference therapy for systemic ATTR, does not stop mutant TTR production in the brain. In addition, despite current pharmacological strategies have shown to be effective against in vivo TTR aggregation by stabilizing the tetramer native structure and precluding its dissociation, they display low brain permeability. Recently, we have repurposed tolcapone as a molecule to treat systemic ATTR. Crystal structures and biophysical analysis converge to demonstrate that tolcapone binds with high affinity and specificity to three unstable leptomeningeal TTR variants, stabilizing them and, consequently, inhibiting their aggregation. Because tolcapone is an FDA-approved drug that crosses the blood-brain barrier, our results suggest that it can translate into a first disease-modifying therapy for leptomeningeal amyloidosis. DATABASES: PDB codes for A25T-TTR, V30G-TTR, and Y114C-TTR bound to tolcapone are 6TXV, 6TXW, and 6XTK, respectively.
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Amiloide/antagonistas & inhibidores , Antiparkinsonianos/química , Fármacos Neuroprotectores/química , Prealbúmina/química , Agregado de Proteínas/efectos de los fármacos , Tolcapona/química , Amiloide/química , Neuropatías Amiloides Familiares/tratamiento farmacológico , Neuropatías Amiloides Familiares/genética , Neuropatías Amiloides Familiares/metabolismo , Neuropatías Amiloides Familiares/patología , Antiparkinsonianos/farmacología , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Reposicionamiento de Medicamentos , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutación , Fármacos Neuroprotectores/farmacología , Prealbúmina/genética , Prealbúmina/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Desnaturalización Proteica , Pliegue de Proteína/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tolcapona/farmacología , Urea/químicaRESUMEN
Synucleinopathies are a group of disorders characterized by the accumulation of α-Synuclein amyloid inclusions in the brain. Preventing α-Synuclein aggregation is challenging because of the disordered nature of the protein and the stochastic nature of fibrillogenesis, but, at the same time, it is a promising approach for therapeutic intervention in these pathologies. A high-throughput screening initiative allowed us to discover ZPDm, the smallest active molecule in a library of more than 14.000 compounds. Although the ZPDm structure is highly related to that of the previously described ZPD-2 aggregation inhibitor, we show here that their mechanisms of action are entirely different. ZPDm inhibits the aggregation of wild-type, A30P, and H50Q α-Synuclein variants in vitro and interferes with α-Synuclein seeded aggregation in protein misfolding cyclic amplification assays. However, ZPDm distinctive feature is its strong potency to dismantle preformed α-Synuclein amyloid fibrils. Studies in a Caenorhabditis elegans model of Parkinson's Disease, prove that these in vitro properties are translated into a significant reduction in the accumulation of α-Synuclein inclusions in ZPDm treated animals. Together with previous data, the present work illustrates how different chemical groups on top of a common molecular scaffold can result in divergent but complementary anti-amyloid activities.
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Protein aggregation is a widespread phenomenon that stems from the establishment of non-native intermolecular contacts resulting in protein precipitation. Despite its deleterious impact on fitness, protein aggregation is a generic property of polypeptide chains, indissociable from protein structure and function. Protein aggregation is behind the onset of neurodegenerative disorders and one of the serious obstacles in the production of protein-based therapeutics. The development of computational tools opened a new avenue to rationalize this phenomenon, enabling prediction of the aggregation propensity of individual proteins as well as proteome-wide analysis. These studies spotted aggregation as a major force driving protein evolution. Actual algorithms work on both protein sequences and structures, some of them accounting also for conformational fluctuations around the native state and the protein microenvironment. This toolbox allows to delineate conformation-specific routines to assist in the identification of aggregation-prone regions and to guide the optimization of more soluble and stable biotherapeutics. Here we review how the advent of predictive tools has change the way we think and address protein aggregation.
RESUMEN
Parkinson's disease (PD) is characterized by progressive loss of dopaminergic neurons and the accumulation of deposits of α-synuclein (α-syn) in the brain. The pivotal role of α-syn aggregation in PD makes it an attractive target for potential disease-modifying therapies. However, the disordered nature of the protein, its multistep aggregation mechanism, and the lack of structural information on intermediate species complicate the discovery of modulators of α-syn amyloid deposition. Despite these difficulties, small molecules have been shown to block the misfolding and aggregation of α-syn, and can even disentangle mature α-syn amyloid fibrils. In this review we provide an updated overview of these leading small compounds and discuss how these chemical chaperones hold great promise to alter the course of PD progression.