RESUMEN
The over-expression and aggregation of α-synuclein (αSyn) are linked to the onset and pathology of Parkinson's disease. Native monomeric αSyn exists in an intrinsically disordered ensemble of interconverting conformations, which has made its therapeutic targeting by small molecules highly challenging. Nonetheless, here we successfully target the monomeric structural ensemble of αSyn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes. Using a surface plasmon resonance high-throughput screen, in which monomeric αSyn is incubated with microchips arrayed with tethered compounds, we identified novel αSyn interacting drug-like compounds. Because these small molecules could impact a variety of αSyn forms present in the ensemble, we tested representative hits for impact on multiple αSyn malfunctions in vitro and in cells including aggregation and perturbation of vesicular dynamics. We thereby identified a compound that inhibits αSyn misfolding and is neuroprotective, multiple compounds that restore phagocytosis impaired by αSyn overexpression, and a compound blocking cellular transmission of αSyn. Our studies demonstrate that drug-like small molecules that interact with native αSyn can impact a variety of its pathological processes. Thus, targeting the intrinsically disordered ensemble of αSyn offers a unique approach to the development of small molecule research tools and therapeutics for Parkinson's disease.
Asunto(s)
Bibliotecas de Moléculas Pequeñas/farmacología , alfa-Sinucleína/metabolismo , Amiloide/antagonistas & inhibidores , Amiloide/metabolismo , Línea Celular , Transferencia Resonante de Energía de Fluorescencia , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Proteínas Intrínsecamente Desordenadas/metabolismo , Fagocitosis/efectos de los fármacos , Pliegue de Proteína , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/toxicidad , Resonancia por Plasmón de Superficie , alfa-Sinucleína/química , alfa-Sinucleína/efectos de los fármacosRESUMEN
The androgen receptor is a transcription factor that plays a key role in the development of prostate cancer, and its interactions with general transcription regulators are therefore of potential therapeutic interest. The mechanistic basis of these interactions is poorly understood due to the intrinsically disordered nature of the transactivation domain of the androgen receptor and the generally transient nature of the protein-protein interactions that trigger transcription. Here, we identify a motif of the transactivation domain that contributes to transcriptional activity by recruiting the C-terminal domain of subunit 1 of the general transcription regulator TFIIF. These findings provide molecular insights into the regulation of androgen receptor function and suggest strategies for treating castration-resistant prostate cancer.
Asunto(s)
ADN/química , Proteínas Intrínsecamente Desordenadas/química , Receptores Androgénicos/química , Factores de Transcripción TFII/química , Secuencias de Aminoácidos , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Masculino , Modelos Moleculares , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción TFII/genética , Factores de Transcripción TFII/metabolismo , Activación TranscripcionalRESUMEN
Castration-resistant prostate cancer is the lethal condition suffered by prostate cancer patients that become refractory to androgen deprivation therapy. EPI-001 is a recently identified compound active against this condition that modulates the activity of the androgen receptor, a nuclear receptor that is essential for disease progression. The mechanism by which this compound exerts its inhibitory activity is however not yet fully understood. Here we show, by using high resolution solution nuclear magnetic resonance spectroscopy, that EPI-001 selectively interacts with a partially folded region of the transactivation domain of the androgen receptor, known as transactivation unit 5, that is key for the ability of prostate cells to proliferate in the absence of androgens, a distinctive feature of castration-resistant prostate cancer. Our results can contribute to the development of more potent and less toxic novel androgen receptor antagonists for treating this disease.
Asunto(s)
Compuestos de Bencidrilo/farmacología , Clorhidrinas/farmacología , Orquiectomía , Neoplasias de la Próstata/tratamiento farmacológico , Receptores Androgénicos/metabolismo , Compuestos de Bencidrilo/uso terapéutico , Clorhidrinas/uso terapéutico , Humanos , Masculino , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Activación TranscripcionalRESUMEN
Correlated inter-domain motions in proteins can mediate fundamental biochemical processes such as signal transduction and allostery. Here we characterize at structural level the inter-domain coupling in a multidomain enzyme, Adenylate Kinase (AK), using computational methods that exploit the shape information encoded in residual dipolar couplings (RDCs) measured under steric alignment by nuclear magnetic resonance (NMR). We find experimental evidence for a multi-state equilibrium distribution along the opening/closing pathway of Adenylate Kinase, previously proposed from computational work, in which inter-domain interactions disfavour states where only the AMP binding domain is closed. In summary, we provide a robust experimental technique for study of allosteric regulation in AK and other enzymes.
Asunto(s)
Adenilato Quinasa/química , Adenilato Quinasa/metabolismo , Algoritmos , Regulación Alostérica , Biología Computacional , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de ProteínaRESUMEN
Insight into how amyloid ß (Aß) aggregation occurs in vivo is vital for understanding the molecular pathways that underlie Alzheimer's disease and requires new techniques that provide detailed kinetic and mechanistic information. Using noninvasive fluorescence lifetime recordings, we imaged the formation of Aß(1-40) and Aß(1-42) aggregates in live cells. For both peptides, the cellular uptake via endocytosis is rapid and spontaneous. They are then retained in lysosomes, where their accumulation leads to aggregation. The kinetics of Aß(1-42) aggregation are considerably faster than those of Aß(1-40) and, unlike those of the latter peptide, show no detectable lag phase. We used superresolution fluorescence imaging to examine the resulting aggregates and could observe compact amyloid structures, likely because of spatial confinement within cellular compartments. Taken together, these findings provide clues as to how Aß aggregation may occur within neurons.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/metabolismo , Agregado de Proteínas , Agregación Patológica de Proteínas , Péptidos beta-Amiloides/biosíntesis , Supervivencia Celular , Humanos , Cinética , Fragmentos de Péptidos/biosíntesis , Células Tumorales CultivadasRESUMEN
The misfolding of intrinsically disordered proteins such as α-synuclein, tau and the Aß peptide has been associated with many highly debilitating neurodegenerative syndromes including Parkinson's and Alzheimer's diseases. Therapeutic targeting of the monomeric state of such intrinsically disordered proteins by small molecules has, however, been a major challenge because of their heterogeneous conformational properties. We show here that a combination of computational and experimental techniques has led to the identification of a drug-like phenyl-sulfonamide compound (ELN484228), that targets α-synuclein, a key protein in Parkinson's disease. We found that this compound has substantial biological activity in cellular models of α-synuclein-mediated dysfunction, including rescue of α-synuclein-induced disruption of vesicle trafficking and dopaminergic neuronal loss and neurite retraction most likely by reducing the amount of α-synuclein targeted to sites of vesicle mobilization such as the synapse in neurons or the site of bead engulfment in microglial cells. These results indicate that targeting α-synuclein by small molecules represents a promising approach to the development of therapeutic treatments of Parkinson's disease and related conditions.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/antagonistas & inhibidores , Terapia Molecular Dirigida , Enfermedad de Parkinson/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéutico , alfa-Sinucleína/antagonistas & inhibidores , Animales , Sitios de Unión , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Ratones , Modelos Biológicos , Modelos Moleculares , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Enfermedad de Parkinson/patología , Fagocitos/efectos de los fármacos , Fagocitos/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMEN
Structural conversion of the presynaptic, intrinsically disordered protein α-synuclein into amyloid fibrils underlies neurotoxicity in Parkinson's disease. The detailed mechanism by which this conversion occurs is largely unknown. Here, we identify a discrete pattern of transient tertiary interactions in monomeric α-synuclein involving amino acid residues that are, in the fibrillar state, part of ß-strands. Importantly, this pattern of pairwise interactions does not correspond to that found in the amyloid state. A redistribution of this network of fibril-like contacts must precede aggregation into the amyloid structure.
Asunto(s)
Multimerización de Proteína , alfa-Sinucleína/química , Secuencia de Aminoácidos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Solubilidad , alfa-Sinucleína/metabolismoRESUMEN
In the last decade it has become evident that disordered states of proteins play important physiological and pathological roles and that the transient tertiary interactions often present in these systems can play a role in their biological activity. The structural characterization of such states has so far largely relied on ensemble representations, which in principle account for both their local and global structural features. However, these approaches are inherently of low resolution due to the large number of degrees of freedom of conformational ensembles and to the sparse nature of the experimental data used to determine them. Here, we overcome these limitations by showing that tertiary interactions in disordered states can be mapped at high resolution by fitting paramagnetic relaxation enhancement data to a small number of conformations, which can be as low as one. This result opens up the possibility of determining the topology of cooperatively collapsed and hidden folded states when these are present in the vast conformational landscape accessible to disordered states of proteins. As a first application, we study the long-range tertiary interactions of acid-unfolded apomyoglobin from experimentally measured paramagnetic relaxation enhancement data.
Asunto(s)
Apoproteínas/química , Simulación de Dinámica Molecular , Mioglobina/química , Pliegue de Proteína , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
We report observations of an intrinsic fluorescence in the visible range, which develops during the aggregation of a range of polypeptides, including the disease-related human peptides amyloid-ß(1-40) and (1-42), lysozyme and tau. Characteristic fluorescence properties such as the emission lifetime and spectra were determined experimentally. This intrinsic fluorescence is independent of the presence of aromatic side-chain residues within the polypeptide structure. Rather, it appears to result from electronic levels that become available when the polypeptide chain folds into a cross-ß sheet scaffold similar to what has been reported to take place in crystals. We use these findings to quantify protein aggregation in vitro by fluorescence imaging in a label-free manner.
Asunto(s)
Péptidos beta-Amiloides/química , Amiloide/química , Fluorescencia , Muramidasa/química , Fragmentos de Péptidos/química , Estructura Terciaria de Proteína , Proteínas tau/química , Humanos , Estructura Secundaria de ProteínaRESUMEN
Inefficient gene transfer and low virion concentrations are common limitations of retroviral transduction. We and others have previously shown that peptides derived from human semen form amyloid fibrils that boost retroviral gene delivery by promoting virion attachment to the target cells. However, application of these natural fibril-forming peptides is limited by moderate efficiencies, the high costs of peptide synthesis, and variability in fibril size and formation kinetics. Here, we report the development of nanofibrils that self-assemble in aqueous solution from a 12-residue peptide, termed enhancing factor C (EF-C). These artificial nanofibrils enhance retroviral gene transfer substantially more efficiently than semen-derived fibrils or other transduction enhancers. Moreover, EF-C nanofibrils allow the concentration of retroviral vectors by conventional low-speed centrifugation, and are safe and effective, as assessed in an ex vivo gene transfer study. Our results show that EF-C fibrils comprise a highly versatile, convenient and broadly applicable nanomaterial that holds the potential to significantly facilitate retroviral gene transfer in basic research and clinical applications.
Asunto(s)
Nanopartículas/química , Péptidos/química , Retroviridae/genética , Transducción Genética , Virión/química , Amiloide/química , Amiloide/genética , Animales , Centrifugación , Terapia Genética , Vectores Genéticos , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Humanos , Ratones , Microscopía de Fuerza Atómica , Microscopía Confocal , Espectroscopía Infrarroja por Transformada de Fourier , Virión/genética , Virión/aislamiento & purificación , Difracción de Rayos XRESUMEN
Here, we use single-molecule techniques to study the aggregation of α-synuclein, the protein whose misfolding and deposition is associated with Parkinson's disease. We identify a conformational change from the initially formed oligomers to stable, more compact proteinase-K-resistant oligomers as the key step that leads ultimately to fibril formation. The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells. The structural conversion is remarkably slow, indicating a high kinetic barrier for the conversion and suggesting that there is a significant period of time for the cellular protective machinery to operate and potentially for therapeutic intervention, prior to the onset of cellular damage. In the absence of added soluble protein, the assembly process is reversed and fibrils disaggregate to form stable oligomers, hence acting as a source of cytotoxic species.
Asunto(s)
alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Animales , Células Cultivadas , Endopeptidasa K/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Cinética , Modelos Moleculares , Neuronas/metabolismo , Estrés Oxidativo , RatasRESUMEN
Misfolding and aggregation of peptides and proteins is a characteristic of many neurodegenerative disorders, including Alzheimer's disease (AD). In AD the ß-amyloid peptide (Aß) aggregates to form characteristic fibrillar structures, which are the deposits found as plaques in the brains of patients. We have used direct stochastic optical reconstruction microscopy, dSTORM, to probe the process of in situ Aß aggregation and the morphology of the ensuing aggregates with a resolution better than 20 nm. We are able to distinguish different types of structures, including oligomeric assemblies and mature fibrils, and observe a number of morphological differences between the species formed in vitro and in vivo, which may be significant in the context of disease. Our data support the recent view that intracellular Aß could be associated with Aß pathogenicity in AD, although the major deposits are extracellular, and suggest that this approach will be widely applicable to studies of the molecular mechanisms of protein deposition diseases.
Asunto(s)
Amiloide/biosíntesis , Amiloide/química , Microscopía Fluorescente/métodos , Enfermedad de Alzheimer , Amiloide/ultraestructura , Línea Celular , Humanos , Métodos , Microscopía Electrónica de TransmisiónRESUMEN
The fibrillation of amyloidogenic proteins is a critical step in the etiology of neurodegenerative disorders such as Alzheimer and Parkinson diseases. There is major interest in the therapeutic intervention on such aberrant aggregation phenomena, and the utilization of polyaromatic scaffolds has lately received considerable attention. In this regard, the molecular and structural basis of the anti-amyloidogenicity of polyaromatic compounds, required to evolve this molecular scaffold toward therapeutic drugs, is not known in detail. We present here biophysical and biochemical studies that have enabled us to characterize the interaction of metal-substituted, tetrasulfonated phthalocyanines (PcTS) with α-synuclein (AS), the major protein component of amyloid-like deposits in Parkinson disease. The inhibitory activity of the assayed compounds on AS amyloid fibril formation decreases in the order PcTS[Ni(II)] ~ PcTS > PcTS[Zn(II)] >> PcTS[Al(III)] ≈ 0. Using NMR and electronic absorption spectroscopies we demonstrated conclusively that the differences in binding capacity and anti-amyloid activity of phthalocyanines on AS are attributed to their relative ability to self-stack through π-π interactions, modulated by the nature of the metal ion bound at the molecule. Low order stacked aggregates of phthalocyanines were identified as the active amyloid inhibitory species, whose effects are mediated by residue specific interactions. Such sequence-specific anti-amyloid behavior of self-stacked phthalocyanines contrasts strongly with promiscuous amyloid inhibitors with self-association capabilities that act via nonspecific sequestration of AS molecules. The new findings reported here constitute an important contribution for future drug discovery efforts targeting amyloid formation.
Asunto(s)
Amiloide/química , Indoles/química , alfa-Sinucleína/química , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Amiloide/genética , Amiloide/metabolismo , Descubrimiento de Drogas , Humanos , Isoindoles , Resonancia Magnética Nuclear Biomolecular , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Misfolding and aggregation of amyloidogenic polypeptides lie at the root of many neurodegenerative diseases. Whilst protein aggregation can be readily studied in vitro by established biophysical techniques, direct observation of the nature and kinetics of aggregation processes taking place in vivo is much more challenging. We describe here, however, a Förster resonance energy transfer sensor that permits the aggregation kinetics of amyloidogenic proteins to be quantified in living systems by exploiting our observation that amyloid assemblies can act as energy acceptors for variants of fluorescent proteins. The observed lifetime reduction can be attributed to fluorescence energy transfer to intrinsic energy states associated with the growing amyloid species. Indeed, for a-synuclein, a protein whose aggregation is linked to Parkinson's disease, we have used this sensor to follow the kinetics of the self-association reactions taking place in vitro and in vivo and to reveal the nature of the ensuing aggregated species. Experiments were conducted in vitro, in cells in culture and in living Caenorhabditis elegans. For the latter the readout correlates directly with the appearance of a toxic phenotype. The ability to measure the appearance and development of pathogenic amyloid species in a living animal and the ability to relate such data to similar processes observed in vitro provides a powerful new tool in the study of the pathology of the family of misfolding disorders. Our study confirms the importance of the molecular environment in which aggregation reactions take place, highlighting similarities as well as differences between the processes occurring in vitro and in vivo, and their significance for defining the molecular physiology of the diseases with which they are associated.
Asunto(s)
Amiloide/química , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Caenorhabditis elegans/metabolismo , Línea Celular Tumoral , Transferencia Resonante de Energía de Fluorescencia , Humanos , Cinética , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The misfolding and aggregation of amyloidogenic polypeptides are characteristics of many neurodegenerative syndromes including Alzheimer's and Parkinson's disease. There is a major interest in the availability of amyloid-specific probes that exhibit fluorescence properties, for its use as reporters of protein aggregation in spectroscopy and microscopy methodologies. In this review, we intend to provide an overview of novel fluorescence-based probes and procedures applied for addressing fundamental aspects of amyloid self-assembly in vitro and in vivo. We highlight the utilization in vitro of several small-molecule fluorescent probes as extrinsic and site-specific reporters of amyloid formation, including single-molecule determinations. Detection of amyloid self-assembly employing compounds such as JC-1, DCVJ, ANS derivatives and luminescent conjugated polymers, as well as site-specific probes such as pyrene and ESIPT is discussed. We further review novel fluorescent probes developed for the non-invasive optical imaging of protein aggregates in vivo, including BTA-1, Methoxy-X04, NIAD-4 and CRANAD-2. Availability of increasingly versatile amyloid-specific fluorescent probes is having a very positive impact in the drug discovery and diagnostics fields.
Asunto(s)
Amiloide/química , Colorantes Fluorescentes/química , Pliegue de Proteína , Amiloide/biosíntesis , Humanos , Estructura Molecular , Deficiencias en la Proteostasis/diagnóstico , Deficiencias en la Proteostasis/fisiopatologíaRESUMEN
Intrinsically disordered proteins (IDPs) lack well-defined structure but are widely represented in eukaryotic proteomes. Although the functions of most IDPs are not understood, some have been shown to have molecular recognition and/or regulatory roles where their disordered nature might be advantageous. Anhydrin is an uncharacterized IDP induced by dehydration in an anhydrobiotic nematode, Aphelenchus avenae. We show here that anhydrin is a moonlighting protein with two novel, independent functions relating to desiccation tolerance. First, it has a chaperone-like activity that can reduce desiccation-induced enzyme aggregation and inactivation in vitro. When expressed in a human cell line, anhydrin localizes to the nucleus and reduces the propensity of a polyalanine expansion protein associated with oculopharyngeal muscular dystrophy to form aggregates. This in vivo activity is distinguished by a loose association of anhydrin with its client protein, consistent with a role as a molecular shield. In addition, anhydrin exhibits a second function as an endonuclease whose substrates include supercoiled, linear, and chromatin linker DNA. This nuclease activity could be involved in either repair of desiccation-induced DNA damage incurred during anhydrobiosis or in apoptotic or necrotic processes, for example, but it is particularly unexpected for anhydrin because IDP functions defined to date anticorrelate with enzyme activity. Enzymes usually require precise three-dimensional positioning of residues at the active site, but our results suggest this need not be the case. Anhydrin therefore extends the range of IDP functional categories to include catalysis and highlights the potential for the discovery of new functions in disordered proteomes.
Asunto(s)
Biocatálisis , Desecación , Chaperonas Moleculares/química , Tylenchida/química , Secuencia de Aminoácidos , Animales , Línea Celular , ADN/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Tylenchida/metabolismoRESUMEN
Misfolding and aggregation of proteins are characteristics of a range of increasingly prevalent neurodegenerative disorders including Alzheimer's and Parkinson's diseases. In Parkinson's disease and several closely related syndromes, the protein alpha-synuclein (AS) aggregates and forms amyloid-like deposits in specific regions of the brain. Fluorescence microscopy using fluorescent proteins, for instance the yellow fluorescent protein (YFP), is the method of choice to image molecular events such as protein aggregation in living organisms. The presence of a bulky fluorescent protein tag, however, may potentially affect significantly the properties of the protein of interest; for AS in particular, its relative small size and, as an intrinsically unfolded protein, its lack of defined secondary structure could challenge the usefulness of fluorescent-protein-based derivatives. Here, we subject a YFP fusion of AS to exhaustive studies in vitro designed to determine its potential as a means of probing amyloid formation in vivo. By employing a combination of biophysical and biochemical studies, we demonstrate that the conjugation of YFP does not significantly perturb the structure of AS in solution and find that the AS-YFP protein forms amyloid deposits in vitro that are essentially identical with those observed for wild-type AS, except that they are fluorescent. Of the several fluorescent properties of the YFP chimera that were assayed, we find that fluorescence anisotropy is a particularly useful parameter to follow the aggregation of AS-YFP, because of energy migration Förster resonance energy transfer (emFRET or homoFRET) between closely positioned YFP moieties occurring as a result of the high density of the fluorophore within the amyloid species. Fluorescence anisotropy imaging microscopy further demonstrates the ability of homoFRET to distinguish between soluble, pre-fibrillar aggregates and amyloid fibrils of AS-YFP. Our results validate the use of fluorescent protein chimeras of AS as representative models for studying protein aggregation and offer new opportunities for the investigation of amyloid aggregation in vivo using YFP-tagged proteins.
Asunto(s)
Amiloide/biosíntesis , Amiloide/química , Proteínas Bacterianas/química , Proteínas Luminiscentes/química , alfa-Sinucleína/química , Amiloide/ultraestructura , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Encéfalo/metabolismo , Encéfalo/ultraestructura , Polarización de Fluorescencia , Transferencia Resonante de Energía de Fluorescencia , Humanos , Técnicas In Vitro , Cuerpos de Lewy/metabolismo , Cuerpos de Lewy/ultraestructura , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Luminiscentes/ultraestructura , Microscopía Electrónica de Transmisión , Resonancia Magnética Nuclear Biomolecular , Multimerización de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/ultraestructura , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , alfa-Sinucleína/ultraestructuraRESUMEN
The identification of aggregation inhibitors and the investigation of their mechanism of action are fundamental in the quest to mitigate the pathological consequences of amyloid formation. Here, characterization of the structural and mechanistic basis for the antiamyloidogenic effect of phthalocyanine tetrasulfonate (PcTS) on alpha-synuclein (AS) allowed us to demonstrate that specific aromatic interactions are central for ligand-mediated inhibition of amyloid formation. We provide evidence indicating that the mechanism behind the antiamyloidogenic effect of PcTS is correlated with the trapping of prefibrillar AS species during the early stages of the assembly process. By using NMR spectroscopy, we have located the primary binding region for PcTS to a specific site in the N terminus of AS, involving the amino acid Tyr-39 as the anchoring residue. Moreover, the residue-specific structural characterization of the AS-PcTS complex provided the basis for the rational design of nonamyloidogenic species of AS, highlighting the role of aromatic interactions in driving AS amyloid assembly. A comparative analysis with other proteins involved in neurodegenerative disorders reveals that aromatic recognition interfaces might constitute a key structural element to target their aggregation pathways. These findings emphasize the use of aggregation inhibitors as molecular probes to assess structural and toxic mechanisms related to amyloid formation and the potential of small molecules as therapeutics for amyloid-related pathologies.
Asunto(s)
Amiloide/biosíntesis , Indoles/farmacología , alfa-Sinucleína/antagonistas & inhibidores , Sitios de Unión , Humanos , Espectroscopía de Resonancia Magnética , Sondas Moleculares , Multimerización de ProteínaRESUMEN
The ATP-dependent protein chaperone heat-shock protein 70 (Hsp70) displays broad anti-aggregation functions and has a critical function in preventing protein misfolding pathologies. According to in vitro and in vivo models of Parkinson's disease (PD), loss of Hsp70 activity is associated with neurodegeneration and the formation of amyloid deposits of alpha-synuclein (alphaSyn), which constitute the intraneuronal inclusions in PD patients known as Lewy bodies. Here, we show that Hsp70 depletion can be a direct result of the presence of aggregation-prone polypeptides. We show a nucleotide-dependent interaction between Hsp70 and alphaSyn, which leads to the aggregation of Hsp70, in the presence of ADP along with alphaSyn. Such a co-aggregation phenomenon can be prevented in vitro by the co-chaperone Hip (ST13), and the hypothesis that it might do so also in vivo is supported by studies of a Caenorhabditis elegans model of alphaSyn aggregation. Our findings indicate that a decreased expression of Hip could facilitate depletion of Hsp70 by amyloidogenic polypeptides, impairing chaperone proteostasis and stimulating neurodegeneration.
Asunto(s)
Proteínas Portadoras/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Homeostasis/fisiología , Complejos Multiproteicos/metabolismo , Enfermedad de Parkinson/metabolismo , Proteínas Supresoras de Tumor/fisiología , alfa-Sinucleína/metabolismo , Adenosina Trifosfato/fisiología , Amiloide/antagonistas & inhibidores , Amiloide/biosíntesis , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Línea Celular Tumoral , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Humanos , Chaperonas Moleculares , Complejos Multiproteicos/antagonistas & inhibidores , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/prevención & control , Enfermedad de Parkinson/etiología , Péptidos/antagonistas & inhibidores , Péptidos/fisiología , Pliegue de Proteína , Estabilidad Proteica , Ratas , Proteínas Supresoras de Tumor/antagonistas & inhibidores , alfa-Sinucleína/antagonistas & inhibidoresRESUMEN
Increasing evidence links the misfolding and aberrant self-assembly of proteins with the molecular events that underlie a range of neurodegenerative diseases, yet the mechanistical details of these processes are still poorly understood. The fact that many of these proteins are intrinsically unstructured makes it particularly challenging to develop strategies for discovering small molecule inhibitors of their aggregation. We present here a broad biophysical approach that enables us to characterize the mechanisms of interaction between alpha-synuclein, a protein whose aggregation is closely connected with Parkinson's disease, and two small molecules, Congo red and Lacmoid, which inhibit its fibrillization. Both compounds are found to interact with the N-terminal and central regions of the monomeric protein although with different binding mechanisms and affinities. The differences can be attributed to the chemical nature of the compounds as well as their abilities to self-associate. We further show that alpha-synuclein binding and aggregation inhibition are mediated by small oligomeric species of the compounds that interact with distinct regions of the monomeric protein. These findings provide potential explanations of the nonspecific antiamyloid effect observed for these compounds as well as important mechanistical information for future drug discovery efforts targeting the misfolding and aggregation of intrinsically unstructured proteins.