RESUMEN
The dynamic processes operating on genomic DNA, such as gene expression and cellular division, lead inexorably to topological challenges in the form of entanglements, catenanes, knots, "bubbles", R-loops, and other outcomes of supercoiling and helical disruption. The resolution of toxic topological stress is the function attributed to DNA topoisomerases. A prominent example is the negative supercoiling (nsc) trailing processive enzymes such as DNA and RNA polymerases. The multiple equilibrium states that nscDNA can adopt by redistribution of helical twist and writhe include the left-handed double-helical conformation known as Z-DNA. Thirty years ago, one of our labs isolated a protein from Drosophila cells and embryos with a 100-fold greater affinity for Z-DNA than for B-DNA, and identified it as topoisomerase II (gene Top2, orthologous to the human UniProt proteins TOP2A and TOP2B). GTP increased the affinity and selectivity for Z-DNA even further and also led to inhibition of the isomerase enzymatic activity. An allosteric mechanism was proposed, in which topoII acts as a Z-DNA-binding protein (ZBP) to stabilize given states of topological (sub)domains and associated multiprotein complexes. We have now explored this possibility by comprehensive bioinformatic analyses of the available protein sequences of topoII representing organisms covering the whole tree of life. Multiple alignment of these sequences revealed an extremely high level of evolutionary conservation, including a winged-helix protein segment, here denoted as Zτ, constituting the putative structural homolog of Zα, the canonical Z-DNA/Z-RNA binding domain previously identified in the interferon-inducible RNA Adenosine-to-Inosine-editing deaminase, ADAR1p150. In contrast to Zα, which is separate from the protein segment responsible for catalysis, Zτ encompasses the active site tyrosine of topoII; a GTP-binding site and a GxxG sequence motif are in close proximity. Quantitative Zτ-Zα similarity comparisons and molecular docking with interaction scoring further supported the "B-Z-topoII hypothesis" and has led to an expanded mechanism for topoII function incorporating the recognition of Z-DNA segments ("Z-flipons") as an inherent and essential element. We further propose that the two Zτ domains of the topoII homodimer exhibit a single-turnover "conformase" activity on given G(ate) B-DNA segments ("Z-flipins"), inducing their transition to the left-handed Z-conformation. Inasmuch as the topoII-Z-DNA complexes are isomerase inactive, we infer that they fulfill important structural roles in key processes such as mitosis. Topoisomerases are preeminent targets of anti-cancer drug discovery, and we anticipate that detailed elucidation of their structural-functional interactions with Z-DNA and GTP will facilitate the design of novel, more potent and selective anti-cancer chemotherapeutic agents.
Asunto(s)
ADN Forma B , ADN de Forma Z , Humanos , Simulación del Acoplamiento Molecular , ADN/química , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Guanosina Trifosfato , Adenosina Desaminasa/metabolismoRESUMEN
Förster resonance energy transfer (FRET) imaging methods provide unique insight into the spatial distribution of energy transfer and (bio)molecular interaction events, though they deliver average information for an ensemble of events included in a diffraction-limited volume. Coupling super-resolution fluorescence microscopy and FRET has been a challenging and elusive task. Here, we present STED-FRET, a method of general applicability to obtain super-resolved energy transfer images. In addition to higher spatial resolution, STED-FRET provides a more accurate quantification of interaction and has the capacity of suppressing contributions of noninteracting partners, which are otherwise masked by averaging in conventional imaging. The method capabilities were first demonstrated on DNA-origami model systems, verified on uniformly double-labeled microtubules, and then utilized to image biomolecular interactions in the membrane-associated periodic skeleton (MPS) of neurons.
RESUMEN
The defining feature of the extensive family of amyloid diseases is the formation of networks of entangled elongated protein fibrils and amorphous aggregates exhibiting crossed ß-sheet secondary structure. The time course of amyloid conversion has been studied extensively in vitro with the proteins involved in the neurodegenerative pathology of Parkinson's disease (α-synuclein), Alzheimer's disease (Tau) and Huntington's disease (Huntingtin). Although much is known about the thermodynamics and kinetics of the transition from a soluble, intrinsically disordered monomer to the fibrillar end state, the putative oligomeric intermediates, currently considered to be the major initiators of cellular toxicity, are as yet poorly defined. We have detected and characterized amyloid precursors by monitoring AS aggregation with ESIPT (excited state intramolecular protein transfer) probes, one of which, 7MFE [7-(3-maleimido-N-propanamide)-2-(4-diethyaminophenyl)-3-hydroxychromone], is introduced here and compared with a related compound, 6MFC, used previously. A series of 140 spectra for sparsely labeled AS was acquired during the course of aggregation, and resolved into the relative contributions (spectra, intensities) of discrete molecular species including the monomeric, fibrillar, and ensemble of intermediate forms. Based on these findings, a kinetic scheme was devised to simulate progress curves as a function of key parameters. An essential feature of the model, one not previously invoked in schemes of amyloid aggregation, is the catalysis of molecular fuzziness by discrete colloidal nanoparticles arising spontaneously via monomer condensation upon exposure of AS to ≥ 37 °C.
Asunto(s)
Cromonas/química , Maleimidas/química , Agregado de Proteínas , Protones , alfa-Sinucleína/química , Enlace de Hidrógeno , CinéticaRESUMEN
α-Synuclein misfolding and aggregation is a hallmark in Parkinson's disease and in several other neurodegenerative diseases known as synucleinopathies. The toxic properties of α-synuclein are conserved from yeast to man, but the precise underpinnings of the cellular pathologies associated are still elusive, complicating the development of effective therapeutic strategies. Combining molecular genetics with target-based approaches, we established that glycation, an unavoidable age-associated post-translational modification, enhanced α-synuclein toxicity in vitro and in vivo, in Drosophila and in mice. Glycation affected primarily the N-terminal region of α-synuclein, reducing membrane binding, impaired the clearance of α-synuclein, and promoted the accumulation of toxic oligomers that impaired neuronal synaptic transmission. Strikingly, using glycation inhibitors, we demonstrated that normal clearance of α-synuclein was re-established, aggregation was reduced, and motor phenotypes in Drosophila were alleviated. Altogether, our study demonstrates glycation constitutes a novel drug target that can be explored in synucleinopathies as well as in other neurodegenerative conditions.
Asunto(s)
Enfermedades Neurodegenerativas/metabolismo , Agregación Patológica de Proteínas/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidad , Envejecimiento/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Drosophila , Inhibidores Enzimáticos/farmacología , Femenino , Glicosilación/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/fisiología , Masculino , Ratones , Ratones Transgénicos , Procesamiento Proteico-Postraduccional , Piruvaldehído/farmacología , Ratas , Levaduras/efectos de los fármacos , Levaduras/fisiología , alfa-Sinucleína/efectos de los fármacos , alfa-Sinucleína/fisiologíaRESUMEN
Photoswitchable probes are of great utility in fluorescence microscopy, permitting numerous determinations, including molecular localization for super-resolution, based on their modifiable emission intensity and spectra. We have coated a blue-emitting (425â nm) quantum dot (QD) with a diheteroarylethene photochrome (PCf), the closed form isomer of which has absorption and emission maxima at 440 and 520-530â nm, respectively, and thus functions as a fluorescent acceptor for the QD donor in Förster resonance energy transfer (FRET). The transition from the non-absorbing, non-fluorescent open state to the fluorescent closed state is achieved by irradiation in the near-UV and reversed by visible light. The PCf is coupled to an amphiphilic polymer that stably coats the QD, thereby creating a water-soluble color-switching QD (csQD) emitting in the blue after visible light irradiation and in the green after UV irradiation. Thus, csQDs photomodulate between two observable states, without the "off" state of previous constructs. The resulting change in the emission ratios of the QD and PCf is up to 180 %, and the csQD can undergo multiple photocycles with minimal fatigue.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Indenos/química , Puntos Cuánticos/química , Isomerismo , Luz , Solubilidad , Rayos Ultravioleta , Agua/químicaRESUMEN
Among the deoxyribozymes catalyzing the ligation of two RNA substrates, 7S11 generates a branched RNA containing a 2',5'-linkage. We have attached the small fluorogenic probe Bimane to the triphosphate terminated RNA substrate and utilized emission intensity and anisotropy to follow structural rearrangements leading to a catalytically active complex upon addition of Mg(2+). Bimane coupled to synthetic oligonucleotides is quenched by nearby guanines via photoinduced electron transfer. The degree of quenching is sensitive to changes in the base pairing of the residues involved and in their distances to the probe. These phenomena permit the characterization of various sequential processes in the assembly and function of 7S11: binding of Mg(2+) to the triphosphate moiety, release of quenching of the probe by the 5'-terminal G residues of R-RNA as they engage in secondary base-pair interactions, local rearrangement into a distinct active conformation, and continuous release of the Bimane-labeled pyrophosphate during the course of reaction at 37°C. It was possible to assign equilibrium and rate constants and structural interpretations to the sequence of conformational transitions and catalysis, information useful for optimizing the design of next generation deoxyribozymes. The fluorescent signatures, thermodynamic equilibria and catalytic function of numerous mutated (base/substituted) molecules were examined.
Asunto(s)
Compuestos Bicíclicos con Puentes/química , ADN Catalítico/química , Colorantes Fluorescentes/química , Magnesio/química , ARN/química , ADN Catalítico/metabolismo , Cinética , Mutación , ARN/metabolismoRESUMEN
Extracellular α-Synuclein has been implicated in interneuronal propagation of disease pathology in Parkinson's Disease. How α-Synuclein is released into the extracellular space is still unclear. Here, we show that α-Synuclein is present in extracellular vesicles in the central nervous system. We find that sorting of α-Synuclein in extracellular vesicles is regulated by sumoylation and that sumoylation acts as a sorting factor for targeting of both, cytosolic and transmembrane proteins, to extracellular vesicles. We provide evidence that the SUMO-dependent sorting utilizes the endosomal sorting complex required for transport (ESCRT) by interaction with phosphoinositols. Ubiquitination of cargo proteins is so far the only known determinant for ESCRT-dependent sorting into the extracellular vesicle pathway. Our study reveals a function of SUMO protein modification as a Ubiquitin-independent ESCRT sorting signal, regulating the extracellular vesicle release of α-Synuclein. We deciphered in detail the molecular mechanism which directs α-Synuclein into extracellular vesicles which is of highest relevance for the understanding of Parkinson's disease pathogenesis and progression at the molecular level. We furthermore propose that sumo-dependent sorting constitutes a mechanism with more general implications for cell biology.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Vesículas Extracelulares/metabolismo , Oligodendroglía/citología , Proteína SUMO-1/metabolismo , Sumoilación/fisiología , alfa-Sinucleína/metabolismo , Animales , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Vesículas Extracelulares/genética , Ratones , Oligodendroglía/metabolismo , Proteína SUMO-1/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , alfa-Sinucleína/genéticaRESUMEN
Photoswitchable fluorescent diheteroarylethenes are promising candidates for applications in super-resolution molecular localization fluorescence microscopy thanks to their high quantum yields and fatigue-resistant photoswitching characteristics. We have studied the effect of varying substituents on the photophysical properties of six sulfone derivatives of diheteroarylethenes, which display fluorescence in one (closed form) of two thermally stable photochromic states. Electron-donating substituents displace the absorption and emission spectra towards the red without substantially affecting the fluorescence quantum yields. Furthermore, ethoxybromo, a very electron-donating substituent, stabilizes the excited state of the closed isomer to the extent of almost entirely inhibiting its cycloreversion. Multi-parameter Hammett correlations indicate a relationship between the emission maxima and electron-donating character, providing a useful tool in the design of future photochromic molecules. Most of the synthesized compounds exhibit small bathochromic shifts and shorter fluorescence lifetimes with an increase in solvent polarity. However, the ethoxybromo-substituted fluorescent photochrome is unique in its strong solvatochromic behaviour, constituting a photoactivatable (photochromic), fluorescent and highly solvatochromic small organic compound. The Catalán formalism identified solvent dipolarity as the principal basis of the solvatochromism, reflecting the highly polarized nature of this molecule.
Asunto(s)
Colorantes Fluorescentes/química , Absorción , Acetatos/química , Dioxanos/química , Electrones , Fluorescencia , Colorantes Fluorescentes/síntesis química , Heptanos/química , Isomerismo , Modelos Lineales , Metanol/química , Estructura Molecular , Procesos Fotoquímicos , Solventes/química , Espectrometría de FluorescenciaRESUMEN
α-synuclein (AS) is a small (140 amino acids), abundant presynaptic protein, which lacks a unique secondary structure in aqueous solution. Amyloid aggregates of AS in dopaminergic neurons of the midbrain are the hallmark of Parkinson's disease (PD). The process of aggregation involves a series of complex structural transitions from innocuous monomeric AS to oligomeric, presumably neurotoxic, forms and finally to fibril formation. Despite its potential importance for understanding PD pathobiology and devising rational, targeted therapeutic strategies, the details of the aggregation process remain largely unknown. Methodologies and reagents capable of controlling the aggregation kinetics are essential tools for the investigation of the molecular mechanisms of amyloid diseases. In this work, we investigated the influence of citrate-capped gold nanoparticles on the aggregation kinetics of AS using a fluorescent probe (MFC) sensitive to the polarity of the molecular microenvironment via excited state intramolecular proton transfer (ESIPT). The particular effects on the half time, nucleation time, and growth rate were ascertained. Gold nanoparticles produced a strong acceleration of protein aggregation with an influence on both the nucleation and growth phases of the overall mechanism. The effects were dependent on the size and concentration of the nanoparticles, being strongest for nanoparticles 10 nm in diameter, which produced a 3-fold increase in the overall aggregation rate at concentrations as low as 20 nM.
Asunto(s)
Amiloide/química , Oro/química , Enfermedad de Parkinson/patología , alfa-Sinucleína/química , Humanos , Cinética , Nanopartículas del Metal/química , Enfermedad de Parkinson/etiología , Estructura Secundaria de ProteínaRESUMEN
A photomodulatable amphiphilic polymer has been synthesized with a backbone of poly[isobutylene-alt-maleic anhydride] and pendant dodecyl alkyl chains, Lucifer Yellow (LY) fluorescent probes, and diheteroarylethenes photochromic (PC) groups. The latter serve as reversible UV-activated FRET acceptors for the LY donors. We characterized the spectral and switching properties of the polymer in an organic solvent (CHCl(3)). In an aqueous medium the polymer forms polymersomes, constituting fluorescence probes ~75 nm in diameter. Self-assembly of the polymer on the surface of a quantum dot (QD) serving as a template creates a dual-color photoswitchable nanoparticle (psNP) with improved properties due to the increase in polymer density and efficiency of PC photoconversion. The psNP exhibits a second QD red emission band that functions as an internal standard requiring only a single excitation wavelength, and is much reduced in size (<20 nm diameter) compared to the polymersomes. The QD template also greatly increases the depth of modulation by photochromic FRET of the LY emission monitored by both steady-state and time-resolved (lifetime) fluorescence (from 20%â70%, and from 12%â55%, respectively).
Asunto(s)
Color , Nanopartículas , Polímeros/química , Puntos Cuánticos , Transferencia Resonante de Energía de Fluorescencia , Cinética , Solventes/química , Espectrofotometría UltravioletaRESUMEN
Insulin signaling comprises a complex cascade of events, playing a key role in the regulation of glucose metabolism and cellular growth. Impaired response to insulin is the hallmark of diabetes, whereas upregulated insulin activity occurs in many cancers. Two splice variants of the insulin receptor (IR) exist in mammals: IR-A, lacking exon 11, and full-length IR-B. Although considerable biochemical data exist on insulin binding and downstream signaling, little is known about the dynamics of the IR itself. We created functional IR transgenes fused with visible fluorescent proteins for use in combination with biotinamido-caproyl insulin and streptavidin quantum dots. Using confocal and structured illumination microscopy, we visualized the endocytosis of both isoforms in living and fixed cells and demonstrated a higher rate of endocytosis of IR-A than IR-B. These differences correlated with higher and sustained activation of IR-A in response to insulin and with distinctive ERK1/2 activation profiles and gene transcription regulation. In addition, cells expressing IR-B showed higher AKT phosphorylation after insulin stimulation than cells expressing IR-A. Taken together, these results suggest that IR signaling is dependent on localization; internalized IRs regulate mitogenic activity, whereas metabolic balance signaling occurs at the cell membrane.
Asunto(s)
Endocitosis/fisiología , Isoformas de Proteínas/metabolismo , Receptor de Insulina/metabolismo , Transducción de Señal/fisiología , Empalme Alternativo , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HeLa , Humanos , Insulina/química , Insulina/metabolismo , Isoformas de Proteínas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Seudópodos/metabolismo , Seudópodos/ultraestructura , Puntos Cuánticos , Receptor de Insulina/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , TransgenesRESUMEN
We have revealed a reorientation of ectodomain I of the epidermal growth factor receptor (EGFR; ErbB1; Her1) in living CHO cells expressing the receptor, upon binding of the native ligand EGF. The state of the unliganded, nonactivated EGFR was compared to that exhibited after ligand addition in the presence of a kinase inhibitor that prevents endocytosis but does not interfere with binding or the ensuing conformational rearrangements. To perform these experiments, we constructed a transgene EGFR with an acyl carrier protein sequence between the signal peptide and the EGFR mature protein sequence. This protein, which behaves similarly to wild-type EGFR with respect to EGF binding, activation, and internalization, can be labeled at a specific serine in the acyl carrier tag with a fluorophore incorporated into a 4'-phosphopantetheine (P-pant) conjugate transferred enzymatically from the corresponding CoA derivative. By measuring Förster resonance energy transfer between a molecule of Atto390 covalently attached to EGFR in this manner and a novel lipid probe NR12S distributed exclusively in the outer leaflet of the plasma membrane, we determined the apparent relative separation of ectodomain I from the membrane under nonactivating and activating conditions. The data indicate that the unliganded domain I of the EGFR receptor is situated much closer to the membrane before EGF addition, supporting the model of a self-inhibited configuration of the inactive receptor in quiescent cells.
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Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/química , Transferencia Resonante de Energía de Fluorescencia/métodos , Microscopía Fluorescente/métodos , Animales , Células CHO , Membrana Celular/metabolismo , Cricetulus , Endocitosis/efectos de los fármacos , Factor de Crecimiento Epidérmico/química , Receptores ErbB/antagonistas & inhibidores , Colorantes Fluorescentes/metabolismo , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Quinazolinas/farmacología , Proteínas Recombinantes/análisisRESUMEN
Ligand-driven dimerizations of ErbB receptor subunits fulfill a fundamental role in their activation. We have used the number and brightness analysis technique to investigate the existence of preformed ligand-independent dimers and clusters and to characterize the initial steps in the activation of ErbB1 and ErbB2. In cells expressing 50,000-200,000 receptors, ErbB1 was monomeric in the absence of ligand stimulation, whereas in CHO cells with receptor levels >500,000 as much as 30% of ErbB1 was present as preformed dimers. EGF induced the formation of ErbB1 dimers as well as larger clusters (up to pentamers) that colocalized with clathrin-coated pits. The distribution of unstimulated ErbB2 in cells expressing 3·10(5)-10(6) receptors was fundamentally different, in that this receptor was present in preformed homoassociated aggregates containing 5-10 molecules. These constitutive ErbB2 homoclusters colocalized with caveolae, increased in size at subphysiological temperatures, but decreased in size upon EGF stimulation. We conclude that these ErbB2 clusters are promoted primarily by membrane-mediated interactions and are dispersed upon ligand stimulation.
Asunto(s)
Receptores ErbB/metabolismo , Receptor ErbB-2/metabolismo , Animales , Secuencia de Bases , Células CHO , Cricetinae , Cricetulus , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/química , Receptores ErbB/genética , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Ligandos , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Microscopía Fluorescente , Plásmidos/genética , Multimerización de Proteína/efectos de los fármacos , Estructura Cuaternaria de Proteína/efectos de los fármacos , Receptor ErbB-2/química , Receptor ErbB-2/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , TransfecciónRESUMEN
Photoswitchable semiconductor nanoparticles, quantum dots (QDs), couple the advantages of conventional QDs with the ability to reversibly modulate the QD emission, thereby improving signal detection by rejection of background signals. Using a simple coating methodology with polymers incorporating a diheteroarylethene photochromic FRET acceptor as well as a spectrally distinct organic fluorophore, photoswitchable QDs were prepared that are small, biocompatible, and feature ratiometric dual emission. With programmed irradiation, the fluorescence intensity ratio can be modified by up to â¼100%.
RESUMEN
The discovery of a reversible transition in the helical sense of a double-helical DNA was initiated by the first synthesis in 1967 of the alternating sequence poly[d(G-C)]. In 1968, exposure to high salt concentration led to a cooperative isomerization of the double helix manifested by an inversion in the CD spectrum in the 240-310 nm range and in an altered absorption spectrum. The tentative interpretation, reported in 1970 and then in detailed form in a 1972 publication by Pohl and Jovin, was that the conventional right-handed B-DNA structure (R) of poly[d(G-C)] transforms at high salt concentration into a novel, alternative left-handed (L) conformation. The historical course of this development and its aftermath, culminating in the first crystal structure of left-handed Z-DNA in 1979, is described in detail. The research conducted by Pohl and Jovin after 1979 is summarized, ending with an assessment of "unfinished business": condensed Z*-DNA; topoisomerase IIα (TOP2A) as an allosteric ZBP (Z-DNA-binding protein); B-Z transitions of phosphorothioate-modified DNAs; and parallel-stranded poly[d(G-A)], a double helix with high stability under physiological conditions and potentially also left-handed.
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ADN Forma B , ADN , ADN/genética , ADN/metabolismo , Conformación de Ácido Nucleico , PolirribonucleótidosRESUMEN
The morphological features of α-synuclein (AS) amyloid aggregation in vitro and in cells were elucidated at the nanoscale by far-field subdiffraction fluorescence localization microscopy. Labeling AS with rhodamine spiroamide probes allowed us to image AS fibrillar structures by fluorescence stochastic nanoscopy with an enhanced resolution at least 10-fold higher than that achieved with conventional, diffraction-limited techniques. The implementation of dual-color detection, combined with atomic force microscopy, revealed the propagation of individual fibrils in vitro. In cells, labeled protein appeared as amyloid aggregates of spheroidal morphology and subdiffraction sizes compatible with in vitro supramolecular intermediates perceived independently by atomic force microscopy and cryo-electron tomography. We estimated the number of monomeric protein units present in these minute structures. This approach is ideally suited for the investigation of the molecular mechanisms of amyloid formation both in vitro and in the cellular milieu.
Asunto(s)
Microscopía Fluorescente/métodos , Imagen Molecular/métodos , Nanoestructuras/química , Multimerización de Proteína , alfa-Sinucleína/química , Color , Células HeLa , Humanos , Espacio Intracelular/metabolismo , Estructura Secundaria de Proteína , Rodaminas/química , alfa-Sinucleína/metabolismoRESUMEN
The aggregation of α-synuclein is associated with progression of Parkinson's disease. We have identified submicrometer supramolecular structures that mediate the early stages of the overall mechanism. The sequence of structural transformations between metastable intermediates were captured and characterized by atomic force microscopy guided by a fluorescent probe sensitive to preamyloid species. A novel ~0.3-0.6 µm molecular assembly, denoted the acuna, nucleates, expands, and liberates fibers with distinctive segmentation and a filamentous fuzzy fringe. These fuzzy fibers serve as precursors of mature amyloid fibrils. Cryo-electron tomography resolved the acuna inner structure as a scaffold of highly condensed colloidal masses interlinked by thin beaded threads, which were perceived as fuzziness by atomic force microscopy. On the basis of the combined data, we propose a sequential mechanism comprising molecular, colloidal, and fibrillar stages linked by reactions with disparate temperature dependencies and distinct supramolecular forms. We anticipate novel diagnostic and therapeutic approaches to Parkinson's and related neurodegenerative diseases based on these new insights into the aggregation mechanism of α-synuclein and intermediates, some of which may act to cause and/or reinforce neurotoxicity.
Asunto(s)
Multimerización de Proteína , alfa-Sinucleína/química , Microscopía por Crioelectrón , Microscopía de Fuerza Atómica , Modelos Moleculares , Estructura Secundaria de Proteína , Protones , Factores de TiempoRESUMEN
Parkinson disease is characterized cytopathologically by the deposition in the midbrain of aggregates composed primarily of the presynaptic neuronal protein α-synuclein (AS). Neurotoxicity is currently attributed to oligomeric microaggregates subjected to oxidative modification and promoting mitochondrial and proteasomal dysfunction. Unphysiological binding to membranes of these and other organelles is presumably involved. In this study, we performed a systematic determination of the influence of charge, phase, curvature, defects, and lipid unsaturation on AS binding to model membranes using a new sensitive solvatochromic fluorescent probe. The interaction of AS with vesicular membranes is fast and reversible. The protein dissociates from neutral membranes upon thermal transition to the liquid disordered phase and transfers to vesicles with higher affinity. The binding of AS to neutral and negatively charged membranes occurs by apparently different mechanisms. Interaction with neutral bilayers requires the presence of membrane defects; binding increases with membrane curvature and rigidity and decreases in the presence of cholesterol. The association with negatively charged membranes is much stronger and much less sensitive to membrane curvature, phase, and cholesterol content. The presence of unsaturated lipids increases binding in all cases. These findings provide insight into the relation between membrane physical properties and AS binding affinity and dynamics that presumably define protein localization in vivo and, thereby, the role of AS in the physiopathology of Parkinson disease.