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We present the state of the art on the study of surfaces and tenuous atmospheres of the icy Galilean satellites Ganymede, Europa and Callisto, from past and ongoing space exploration conducted with several spacecraft to recent telescopic observations, and we show how the ESA JUICE mission plans to explore these surfaces and atmospheres in detail with its scientific payload. The surface geology of the moons is the main evidence of their evolution and reflects the internal heating provided by tidal interactions. Surface composition is the result of endogenous and exogenous processes, with the former providing valuable information about the potential composition of shallow subsurface liquid pockets, possibly connected to deeper oceans. Finally, the icy Galilean moons have tenuous atmospheres that arise from charged particle sputtering affecting their surfaces. In the case of Europa, plumes of water vapour have also been reported, whose phenomenology at present is poorly understood and requires future close exploration. In the three main sections of the article, we discuss these topics, highlighting the key scientific objectives and investigations to be achieved by JUICE. Based on a recent predicted trajectory, we also show potential coverage maps and other examples of reference measurements. The scientific discussion and observation planning presented here are the outcome of the JUICE Working Group 2 (WG2): "Surfaces and Near-surface Exospheres of the Satellites, dust and rings".
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Saturn's moon Titan was explored by the Cassini spacecraft from 2004 to 2017. While Cassini revealed a lot about this Earth-like world, its radar observations could only provide limited information about Titan's liquid hydrocarbons seas Kraken, Ligeia and Punga Mare. Here, we show the results of the analysis of the Cassini mission bistatic radar experiments data of Titan's polar seas. The dual-polarized nature of bistatic radar observations allow independent estimates of effective relative dielectric constant and small-scale roughness of sea surface, which were not possible via monostatic radar data. We find statistically significant variations in effective dielectric constant (i.e., liquid composition), consistent with a latitudinal dependence in the methane-ethane mixing-ratio. The results on estuaries suggest lower values than the open seas, compatible with methane-rich rivers entering seas with higher ethane content. We estimate small-scale roughness of a few millimeters from the almost purely coherent scattering from the sea surface, hinting at the presence of capillary waves. This roughness is concentrated near estuaries and inter-basin straits, perhaps indicating active tidal currents.
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Vaults are eukaryotic ribonucleoproteins consisting of 78 copies of the major vault protein (MVP), which assemble into a nanoparticle with an about 60 nm volume-based size, enclosing other proteins and RNAs. Regardless of their physiological role(s), vaults represent ideal, natural hollow nanoparticles, which are produced by the assembly of the sole MVP. Here, we have expressed in Komagataella phaffi and purified an MVP variant carrying a C-terminal Z peptide (vault-Z), which can tightly bind an antibody's Fc portion, in view of targeted delivery. Via surface plasmon resonance analysis, we could determine a 2.5 nM affinity to the monoclonal antibody Trastuzumab (Tz)/vault-Z 1:1 interaction. Then, we characterized the in-solution interaction via co-incubation, ultracentrifugation, and analysis of the pelleted proteins. This showed virtually irreversible binding up to an at least 10:1 Tz/vault-Z ratio. As a proof of concept, we labeled the Fc portion of Tz with a fluorophore and conjugated it with the nanoparticle, along with either Tz or Cetuximab, another monoclonal antibody. Thus, we could demonstrate antibody-dependent, selective uptake by the SKBR3 and MDA-MB 231 breast cancer cell lines. These investigations provide a novel, flexible technological platform that significantly extends vault-Z's applications, in that it can be stably conjugated with finely adjusted amounts of antibodies as well as of other molecules, such as fluorophores, cell-targeting peptides, or drugs, using the Fc portion as a scaffold.
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Nanopartículas , Trastuzumab , Partículas Ribonucleoproteicas en Bóveda , Humanos , Partículas Ribonucleoproteicas en Bóveda/metabolismo , Partículas Ribonucleoproteicas en Bóveda/química , Nanopartículas/química , Trastuzumab/química , Línea Celular Tumoral , Cetuximab/química , Anticuerpos Monoclonales/química , Inmunoconjugados/químicaRESUMEN
Amyloid aggregates are diverse proteinaceous assemblies, including one or more protein species, wherein the molecules interact according to characteristic patterns [...].
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Proteínas AmiloidogénicasRESUMEN
The vault nanoparticle is a eukaryotic assembly consisting of 78 copies of the 99-kDa major vault protein. They generate two cup-shaped symmetrical halves, which in vivo enclose protein and RNA molecules. Overall, this assembly is mainly involved in pro-survival and cytoprotective functions. It also holds a remarkable biotechnological potential for drug/gene delivery, thanks to its huge internal cavity and the absence of toxicity/immunogenicity. The available purification protocols are complex, partly because they use higher eukaryotes as expression systems. Here, we report a simplified procedure that combines human vault expression in the yeast Komagataella phaffii, as described in a recent report, and a purification process we have developed. This consists of RNase pretreatment followed by size-exclusion chromatography, which is far simpler than any other reported to date. Protein identity and purity was confirmed by SDS-PAGE, Western blot and transmission electron microscopy. We also found that the protein displayed a significant propensity to aggregate. We thus investigated this phenomenon and the related structural changes by Fourier-transform spectroscopy and dynamic light scattering, which led us to determine the most suitable storage conditions. In particular, the addition of either trehalose or Tween-20 ensured the best preservation of the protein in native, soluble form.
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Nanopartículas , Humanos , Nanopartículas/química , Microscopía Electrónica de TransmisiónRESUMEN
Ribosome-inactivating proteins, including Saporin toxin, have found application in the search for innovative alternative cancer therapies to conventional chemo- and radiotherapy. Saporin's main mechanism of action involves the inhibition of cytoplasmic protein synthesis. Its strong theoretical efficacy is counterbalanced by negligible cell uptake and diffusion into the cytosol. In this work, we demonstrate that by immobilizing Saporin on iron oxide nanoparticles coated with an amphiphilic polymer, which promotes nanoconjugate endosomal escape, a strong cytotoxic effect mediated by ribosomal functional inactivation can be achieved. Cancer cell death was mediated by apoptosis dependent on nanoparticle concentration but independent of surface ligand density. The cytotoxic activity of Saporin-conjugated colloidal nanoparticles proved to be selective against three different cancer cell lines in comparison with healthy fibroblasts.
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Assessing the toxic effect in living organisms remains a major issue for the development of safe nanomedicines and exposure of researchers involved in the synthesis, handling and manipulation of nanoparticles. In this study, we demonstrate that Caenorhabditis elegans could represent an in vivo model alternative to superior mammalians for the collection of several physiological functionality parameters associated to both short-term and long-term effects of colloidally stable nanoparticles even in absence of microbial feeding, usually reported to be necessary to ensure appropriate intake. Contextually, we investigated the impact of surface charge on toxicity of superparamagnetic iron oxide coated with a wrapping polymeric envelop that confers them optimal colloidal stability. By finely tuning the functional group composition of this shallow polymer-obtaining totally anionic, partially pegylated, partially anionic and partially cationic, respectively-we showed that the ideal surface charge organization to optimize safety of colloidal nanoparticles is the one containing both cationic and anionic groups. Our results are in accordance with previous evidence that zwitterionic nanoparticles allow long circulation, favorable distribution in the tumor area and optimal tumor penetration and thus support the hypothesis that zwitterionic iron oxide nanoparticles could be an excellent solution for diagnostic imaging and therapeutic applications in nanooncology.
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The vault nanoparticle is a eukaryotic ribonucleoprotein complex consisting of 78 individual 97 kDa-"major vault protein" (MVP) molecules that form two symmetrical, cup-shaped, hollow halves. It has a huge size (72.5 × 41 × 41 nm) and an internal cavity, wherein the vault poly(ADP-ribose) polymerase (vPARP), telomerase-associated protein-1 (TEP1), and some small untranslated RNAs are accommodated. Plenty of literature reports on the biological role(s) of this nanocomplex, as well as its involvement in diseases, mostly oncological ones. Nevertheless, much has still to be understood as to how vault participates in normal and pathological mechanisms. In this comprehensive review, current understanding of its biological roles is discussed. By different mechanisms, vault's individual components are involved in major cellular phenomena, which result in protection against cellular stresses, such as DNA-damaging agents, irradiation, hypoxia, hyperosmotic, and oxidative conditions. These diverse cellular functions are accomplished by different mechanisms, mainly gene expression reprogramming, activation of proliferative/prosurvival signaling pathways, export from the nucleus of DNA-damaging drugs, and import of specific proteins. The cellular functions of this nanocomplex may also result in the onset of pathological conditions, mainly (but not exclusively) tumor proliferation and multidrug resistance. The current understanding of its biological roles in physiological and pathological processes should also provide new hints to extend the scope of its exploitation as a nanocarrier for drug delivery.
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Amyloid aggregation of human ataxin-3 (ATX3) is responsible for spinocerebellar ataxia type 3, which belongs to the class of polyglutamine neurodegenerative disorders. It is widely accepted that the formation of toxic oligomeric species is primarily involved in the onset of the disease. For this reason, to understand the mechanisms underlying toxicity, we expressed both a physiological (ATX3-Q24) and a pathological ATX3 variant (ATX3-Q55) in a simplified cellular model, Escherichia coli. It has been observed that ATX3-Q55 expression induces a higher reduction of the cell growth compared to ATX3-Q24, due to the bacteriostatic effect of the toxic oligomeric species. Furthermore, the Fourier transform infrared microspectroscopy investigation, supported by multivariate analysis, made it possible to monitor protein aggregation and the induced cell perturbations in intact cells. In particular, it has been found that the toxic oligomeric species associated with the expression of ATX3-Q55 are responsible for the main spectral changes, ascribable mainly to the cell envelope modifications. A structural alteration of the membrane detected through electron microscopy analysis in the strain expressing the pathological form supports the spectroscopic results.
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Amiloide/genética , Proteínas Amiloidogénicas/genética , Ataxina-3/genética , Enfermedad de Machado-Joseph/genética , Membrana Celular/genética , Proliferación Celular/genética , Escherichia coli/genética , Regulación de la Expresión Génica/genética , Humanos , Enfermedad de Machado-Joseph/metabolismo , Enfermedad de Machado-Joseph/patología , Proteínas del Tejido Nervioso/genética , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patologíaRESUMEN
BACKGROUND: We have previously demonstrated the neuroprotective activity of tetracycline on a Spinocerebellar Ataxia 3 nematode model. Here, we present the screening of a small library of tetracycline congeners in order to identify the most effective compound in preventing ataxin-3 aggregation. METHODS: We performed the assays on the Josephin Domain as it is directly involved in the onset of fibrillation. We used thioflavin T and solubility assays to spot out the most effective tetracycline congeners; Fourier transform infrared and NMR spectroscopies to characterize their mode of action. We employed an ataxic Caenorhabditis elegans model to evaluate the pharmacological efficacy of tetracycline congeners. RESULTS: Methacycline was identified as the most effective compound. Like tetracycline, methacycline neither significantly affected the aggregation kinetics nor did it change the secondary structures of the final aggregates but increased the solubility of the aggregated species. Saturation transfer NMR experiments demonstrated methacycline capability to only bind the oligomeric species of Josephin Domain. Competition assays also showed that methacycline binds to the Josephin Domain more tightly than tetracycline. The treatment with methacycline induced a significant improvement in motility and locomotion of the transgenic C. elegans without changing its lifespan. The efficacy was distinctly stronger than that of tetracycline. Noteworthy, unlike tetracycline, methacycline was able to retard aging-related decline in motility of even the healthy worms used. CONCLUSIONS: The apparent absence of toxic effects displayed by methacycline, along with its stronger efficacy in contrasting expanded ataxin-3 toxicity, makes it a possible candidate for a chronic treatment of the disease.
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Antibacterianos/farmacología , Ataxina-3/antagonistas & inhibidores , Caenorhabditis elegans/efectos de los fármacos , Metaciclina/farmacología , Modelos Biológicos , Animales , Ataxina-3/metabolismo , Caenorhabditis elegans/metabolismo , Cinética , Agregado de Proteínas/efectos de los fármacos , Estructura Secundaria de ProteínaRESUMEN
Amyloids result from the aggregation of a set of diverse proteins, due to either specific mutations or promoting intra- or extra-cellular conditions. Structurally, they are rich in intermolecular ß-sheets and are the causative agents of several diseases, both neurodegenerative and systemic. It is believed that the most toxic species are small aggregates, referred to as oligomers, rather than the final fibrillar assemblies. Their mechanisms of toxicity are mostly mediated by aberrant interactions with the cell membranes, with resulting derangement of membrane-related functions. Much effort is being exerted in the search for natural antiamyloid agents, and/or in the development of synthetic molecules. Actually, it is well documented that the prevention of amyloid aggregation results in several cytoprotective effects. Here, we portray the state of the art in the field. Several natural compounds are effective antiamyloid agents, notably tetracyclines and polyphenols. They are generally non-specific, as documented by their partially overlapping mechanisms and the capability to interfere with the aggregation of several unrelated proteins. Among rationally designed molecules, we mention the prominent examples of ß-breakers peptides, whole antibodies and fragments thereof, and the special case of drugs with contrasting transthyretin aggregation. In this framework, we stress the pivotal role of the computational approaches. When combined with biophysical methods, in several cases they have helped clarify in detail the protein/drug modes of interaction, which makes it plausible that more effective drugs will be developed in the future.
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Amiloide/metabolismo , Agregado de Proteínas , Agregación Patológica de Proteínas/metabolismo , Amiloidosis/tratamiento farmacológico , Amiloidosis/etiología , Amiloidosis/metabolismo , Amiloidosis/patología , Animales , Diseño de Fármacos , Desarrollo de Medicamentos , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Terapia Molecular Dirigida , Agregado de Proteínas/efectos de los fármacos , Agregación Patológica de Proteínas/tratamiento farmacológicoRESUMEN
BACKGROUND: Vaults are eukaryotic ribonucleoprotein particles composed of up 78 copies of the 97â¯kDa major vault protein that assembles into a barrel-like, "nanocapsule" enclosing poly(ADP-ribose) polymerase, telomerase-associated protein-1 and small untranslated RNAs. Overall, the molecular mass of vault particles amounts to about 13â¯MDa. Although it has been implicated in several cellular functions, its physiological roles remain poorly understood. Also, the possibility to exploit it as a nanovector for drug delivery is currently being explored in several laboratories. METHODS: Using the baculovirus expression system, vaults were expressed and purified by a dialysis step using a 1â¯MDa molecular weight cutoff membrane and a subsequent size exclusion chromatography. Purity was assessed by SDS-PAGE, transmission electron microscopy and dynamic light scattering. Particle's endocytic uptake was monitored by flow cytometry and confocal microscopy. RESULTS: The purification protocol here reported is far simpler and faster than those currently available and lead to the production of authentic vault. We then demonstrated its clathrin-mediated endocytic uptake by normal fibroblast and glioblastoma, but not carcinoma cell lines. In contrast, no significant caveolin-mediated endocytosis was detected. CONCLUSIONS: These results provide the first evidence for an intrinsic propensity of the vault complex to undergo endocytic uptake cultured eukaryotic cells. GENERAL SIGNIFICANCE: The newly developed purification procedure will greatly facilitate any investigation based on the use of the vault particle as a natural nanocarrier. Its clathrin-mediated endocytic uptake observed in normal and in some tumor cell lines sheds light on its physiological role.
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Endocitosis/fisiología , Fibroblastos/citología , Glioblastoma/metabolismo , Nanopartículas/administración & dosificación , Partículas Ribonucleoproteicas en Bóveda/química , Partículas Ribonucleoproteicas en Bóveda/metabolismo , Animales , Células Cultivadas , Sistemas de Liberación de Medicamentos , Endocitosis/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Glioblastoma/patología , Humanos , Nanopartículas/química , Transducción de Señal , SpodopteraRESUMEN
The protein ataxin-3 contains a polyglutamine stretch that triggers amyloid aggregation when it is expanded beyond a critical threshold. This results in the onset of the spinocerebellar ataxia type 3. The protein consists of the globular N-terminal Josephin domain and a disordered C-terminal tail where the polyglutamine stretch is located. Expanded ataxin-3 aggregates via a two-stage mechanism: first, Josephin domain self-association, then polyQ fibrillation. This highlights the intrinsic amyloidogenic potential of Josephin domain. Therefore, much effort has been put into investigating its aggregation mechanism(s). A key issue regards the conformational requirements for triggering amyloid aggregation, as it is believed that, generally, misfolding should precede aggregation. Here, we have assayed the effect of 2,2,2-trifluoroethanol, a co-solvent capable of stabilizing secondary structures, especially α-helices. By combining biophysical methods and molecular dynamics, we demonstrated that both secondary and tertiary JD structures are virtually unchanged in the presence of up to 5% 2,2,2-trifluoroethanol. Despite the preservation of JD structure, 1% of 2,2,2-trifluoroethanol suffices to exacerbate the intrinsic aggregation propensity of this domain, by slightly decreasing its conformational stability. These results indicate that in the case of JD, conformational fluctuations might suffice to promote a transition towards an aggregated state without the need for extensive unfolding, and highlights the important role played by the environment on the aggregation of this globular domain.
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Amiloide/efectos de los fármacos , Ataxina-3/metabolismo , Agregado de Proteínas/efectos de los fármacos , Proteínas Represoras/metabolismo , Trifluoroetanol/farmacología , Ataxina-3/química , Dicroismo Circular , Humanos , Conformación Molecular , Simulación de Dinámica Molecular , Péptidos/metabolismo , Conformación Proteica/efectos de los fármacos , Dominios Proteicos/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Proteínas Represoras/químicaRESUMEN
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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A novel surface plasmon resonance (SPR) optical fiber biosensor, able to bind perfluorooctanoate and perfluorooctanesulfonate compounds, is presented. In the first step, an ad hoc antibody compound has been designed, produced and tested by ELISA, then, in the second step, the gold surface of a plastic optical fiber sensor has been derivatizated and functionalized with this new bio-receptor, able to bind target analytes with high affinity and selectivity. The experimental data have shown that the developed SPR optical fiber biosensor makes it possible to detect these compounds. One advantage of this approach stems from the possibility to monitor the perfluorinated compounds in the environment exploiting the remote sensing capability offered by the optical fibers. The measurements were performed in laboratory, also exploiting matrices mimicking the real environment. The limit of detection of the assay was 0.21ppb, a value that is lower than the maximum residue limit fixed by the European Union regulations.
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The protein ataxin-3 carries a polyglutamine stretch close to the C-terminus that triggers a neurodegenerative disease in humans when its length exceeds a critical threshold. A role as a transcriptional regulator but also as a ubiquitin hydrolase has been proposed for this protein. Here, we report that, when expressed in the yeast Pichia pastoris, full-length ataxin-3 enabled almost normal growth at 37 °C, well above the physiological optimum of 30 °C. The N-terminal Josephin domain (JD) was also effective but significantly less, whereas catalytically inactive JD was completely ineffective. Based on MudPIT proteomic analysis, we observed that the strain expressing full-length, functional ataxin-3 displayed persistent upregulation of enzymes involved in mitochondrial energy metabolism during growth at 37 °C compared with the strain transformed with the empty vector. Concurrently, in the transformed strain intracellular ATP levels at 37 °C were even higher than normal ones at 30 °C. Elevated ATP was also paralleled by upregulation of enzymes involved in both protein biosynthesis and biosynthetic pathways, as well as of several stress-induced proteins. A similar pattern was observed when comparing a strain expressing JD with another expressing its catalytically inactive counterpart. We suggest that such effects mostly result from mechanisms of transcriptional regulation.
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Ataxina-3/genética , Proteínas Fúngicas/genética , Respuesta al Choque Térmico , Pichia/metabolismo , Adenosina Trifosfato/metabolismo , Ataxina-3/química , Ataxina-3/metabolismo , Metabolismo Energético , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Pichia/genéticaRESUMEN
The protein ataxin-3 (ATX3) triggers an amyloid-related neurodegenerative disease when its polyglutamine stretch is expanded beyond a critical threshold. We formerly demonstrated that the polyphenol epigallocatechin-3-gallate (EGCG) could redirect amyloid aggregation of a full-length, expanded ATX3 (ATX3-Q55) towards non-toxic, soluble, SDS-resistant aggregates. Here, we have characterized other related phenol compounds, although smaller in size, i.e. (-)-epigallocatechin gallate (EGC), and gallic acid (GA). We analysed the aggregation pattern of ATX3-Q55 and of the N-terminal globular Josephin domain (JD) by assessing the time course of the soluble protein, as well its structural features by FTIR and AFM, in the presence and the absence of the mentioned compounds. All of them redirected the aggregation pattern towards soluble, SDS-resistant aggregates. They also prevented the appearance of ordered side-chain hydrogen bonding in ATX3-Q55, which is the hallmark of polyQ-related amyloids. Molecular docking analyses on the JD highlighted three interacting regions, including the central, aggregation-prone one. All three compounds bound to each of them, although with different patterns. This might account for their capability to prevent amyloidogenesis. Saturation transfer difference NMR experiments also confirmed EGCG and EGC binding to monomeric JD. ATX3-Q55 pre-incubation with any of the three compounds prevented its calcium-influx-mediated cytotoxicity towards neural cells. Finally, all the phenols significantly reduced toxicity in a transgenic Caenorhabditis elegans strain expressing an expanded ATX3. Overall, our results show that the three polyphenols act in a substantially similar manner. GA, however, might be more suitable for antiamyloid treatments due to its simpler structure and higher chemical stability.
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Ataxina-3/metabolismo , Catequina/análogos & derivados , Amiloide/metabolismo , Proteínas Amiloidogénicas , Animales , Caenorhabditis elegans/metabolismo , Catequina/química , Catequina/metabolismo , Modelos Animales de Enfermedad , Humanos , Enlace de Hidrógeno , Simulación del Acoplamiento Molecular , Proteínas del Tejido Nervioso/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Péptidos , Fenoles/química , Fenoles/metabolismoRESUMEN
BACKGROUND: The discovery of new solutions with antibacterial activity as efficient and safe alternatives to common preservatives (such as parabens) and to combat emerging infections and drug-resistant bacterial pathogens is highly expected in cosmetics and pharmaceutics. Colloidal silver nanoparticles (NPs) are attracting interest as novel effective antimicrobial agents for the prevention of several infectious diseases. METHODS: Water-soluble, negatively charged silver nanoparticles (AgNPs) were synthesized by reduction with citric and tannic acid and characterized by transmission electron microscopy, dynamic light scattering, zeta potential, differential centrifuge sedimentation, and ultraviolet-visible spectroscopy. AgNPs were tested with model Gram-negative and Gram-positive bacteria in comparison to two different kinds of commercially available AgNPs. RESULTS: In this work, AgNPs with higher antibacterial activity compared to the commercially available colloidal silver solutions were prepared and investigated. Bacteria were plated and the antibacterial activity was tested at the same concentration of silver ions in all samples. The AgNPs did not show any significant reduction in the antibacterial activity for an acceptable time period. In addition, AgNPs were transferred to organic phase and retained their antibacterial efficacy in both aqueous and nonaqueous media and exhibited no toxicity in eukaryotic cells. CONCLUSION: We developed AgNPs with a 20 nm diameter and negative zeta potential with powerful antibacterial activity and low toxicity compared to currently available colloidal silver, suitable for cosmetic preservatives and pharmaceutical preparations administrable to humans and/or animals as needed.
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Antibacterianos/farmacología , Nanopartículas del Metal/química , Plata/farmacología , Células 3T3-L1/efectos de los fármacos , Animales , Antibacterianos/efectos adversos , Antibacterianos/química , Ácido Cítrico/química , Coloides/química , Evaluación Preclínica de Medicamentos/métodos , Dispersión Dinámica de Luz , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Nanopartículas del Metal/efectos adversos , Ratones , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Transmisión , Plata/química , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja por Transformada de Fourier , Taninos/químicaRESUMEN
The Hsp70 family of chaperones plays an essential role in suppressing protein aggregation in the cell. Here we investigate the factors controlling the intrinsic ability of human Hsp70 to inhibit the elongation of amyloid fibrils formed by the Parkinson's disease-related protein α-synuclein. Using kinetic analysis, we show that Hsp70 binds preferentially to α-synuclein fibrils as a consequence of variations in the association and dissociation rate constants of binding to the different aggregated states of the protein. Our findings illustrate the importance of the kinetics of binding of molecular chaperones, and also of potential therapeutic molecules, in the efficient suppression of specific pathogenic events linked to neurodegeneration.
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Unión Competitiva , Proteínas HSP70 de Choque Térmico/metabolismo , Multimerización de Proteína , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Humanos , Cinética , Estructura Secundaria de Proteína , Especificidad por SustratoRESUMEN
Epigallocatechin-3-gallate (EGCG) and tetracycline are two known inhibitors of amyloid aggregation able to counteract the fibrillation of most of the proteins involved in neurodegenerative diseases. We have recently investigated their effect on ataxin-3 (AT3), the polyglutamine-containing protein responsible for spinocerebellar ataxia typeâ 3. We previously showed that EGCG and tetracycline can contrast the aggregation process and toxicity of expanded AT3, although by different mechanisms. Here, we have performed further experiments by using the sole Josephin domain (JD) to further elucidate the mechanism of action of the two compounds. By protein solubility assays and FTIR spectroscopy we have first observed that EGCG and tetracycline affect the JD aggregation essentially in the same way displayed when acting on the full-length expanded AT3. Then, by saturation transfer difference (STD) NMR experiments, we have shown that EGCG binds both the monomeric and the oligomeric JD form, whereas tetracycline can only interact with the oligomeric one. Surface plasmon resonance (SPR) analysis has confirmed the capability of the sole EGCG to bind monomeric JD, although with a KD value suggestive for a non-specific interaction. Our investigations provide new details on the JD interaction with EGCG and tetracycline, which could explain the different mechanisms by which the two compounds reduce the toxicity of AT3.