RESUMEN
The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.
Asunto(s)
Proteínas de Unión al Calcio , Insuficiencia Cardíaca , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Corazón , Ratones , ARN MensajeroRESUMEN
BACKGROUND: The human L39X phospholamban (PLN) cardiomyopathic mutant has previously been reported as a null mutation but the detailed molecular pathways that lead to the complete lack of detectable protein remain to be clarified. Previous studies have shown the implication between an impaired cellular degradation homeostasis and cardiomyopathy development. Therefore, uncovering the underlying mechanism responsible for the lack of PLN protein has important implications in understanding the patient pathology, chronic human calcium dysregulation and aid the development of potential therapeutics. METHODS: A panel of mutant and wild-type reporter tagged PLN modified mRNA (modRNA) constructs were transfected in human embryonic stem cell-derived cardiomyocytes. Lysosomal and proteasomal chemical inhibitors were used together with cell imaging and protein analysis tools in order to dissect degradation pathways associated with expressed PLN constructs. Transcriptional profiling of the cardiomyocytes transfected by wild-type or L39X mutant PLN modRNA was analysed with bulk RNA sequencing. RESULTS: Our modRNA assay system revealed that transfected L39X mRNA was stable and actively translated in vitro but with only trace amount of protein detectable. Proteasomal inhibition of cardiomyocytes transfected with L39X mutant PLN modRNA showed a fourfold increase in protein expression levels. Additionally, RNA sequencing analysis of protein degradational pathways showed a significant distinct transcriptomic signature between wild-type and L39X mutant PLN modRNA transfected cardiomyocytes. CONCLUSION: Our results demonstrate that the cardiomyopathic PLN null mutant L39X is rapidly, actively and specifically degraded by proteasomal pathways. Herein, and to the best of our knowledge, we report for the first time the usage of modified mRNAs to screen for and illuminate alternative molecular pathways found in genes associated with inherited cardiomyopathies.
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Proteínas de Unión al Calcio/genética , Cardiomiopatías/etiología , Cardiomiopatías/metabolismo , Homocigoto , Mutación , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Mensajero/genética , Alelos , Sustitución de Aminoácidos , Biomarcadores , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Cardiomiopatías/diagnóstico , Línea Celular , Susceptibilidad a Enfermedades , Perfilación de la Expresión Génica , Humanos , Biosíntesis de Proteínas , Estabilidad del ARNRESUMEN
Peptides and proteins have evolved to self-assemble into supramolecular entities through a set of non-covalent interactions. Such structures and materials provide the functional basis of life. Crucially, biomolecular assembly processes can be highly sensitive to and modulated by environmental conditions, including temperature, light, ionic strength and pH, providing the inspiration for the development of new classes of responsive functional materials based on peptide building blocks. Here, it is shown that the stimuli-responsive assembly of amyloidogenic peptide can be used as the basis of environmentally responsive microcapsules which exhibit release characteristics triggered by a change in pH. The microcapsules are biocompatible and biodegradable and may act as vehicles for controlled release of a wide range of biomolecules. Cryo-SEM images reveal the formation of a fibrillar network of the capsule interior with discrete compartments in which cargo molecules can be stored. In addition, the reversible formation of these microcapsules by modulating the solution pH is investigated and their potential application for the controlled release of encapsulated cargo molecules, including antibodies, is shown. These results suggest that the approach described here represents a promising venue for generating pH-responsive functional peptide-based materials for a wide range of potential applications for molecular encapsulation, storage, and release.
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Péptidos , Cápsulas , Concentración de Iones de Hidrógeno , TemperaturaRESUMEN
Therapeutics designed to target α-synuclein (α-syn) aggregation may be critical in halting the progression of pathology in Parkinson's disease (PD) patients. Nanobodies are single-domain antibody fragments that bind with antibody specificity, but allow readier genetic engineering and delivery. When expressed intracellularly as intrabodies, anti-α-syn nanobodies fused to a proteasome-targeting proline, aspartate or glutamate, serine, and threonine (PEST) motif can modulate monomeric concentrations of target proteins. Here we aimed to validate and compare the in vivo therapeutic potential of gene therapy delivery of two proteasome-directed nanobodies selectively targeting α-syn in a synuclein overexpression-based PD model: VH14*PEST (non-amyloid component region) and NbSyn87*PEST (C-terminal region). Stereotaxic injections of adeno-associated viral 5-α-syn (AAV5-α-syn) into the substantia nigra (SN) were performed in Sprague-Dawley rats that were sorted into three cohorts based on pre-operative behavioral testing. Rats were treated with unilateral SN injections of vectors for VH14*PEST, NbSyn87*PEST, or injected with saline 3 weeks post lesion. Post-mortem assessments of the SN showed that both nanobodies markedly reduced the level of phosphorylated Serine-129 α-syn labeling relative to saline-treated animals. VH14*PEST showed considerable maintenance of striatal dopaminergic tone in comparison to saline-treated and NbSyn87*PEST-treated animals as measured by tyrosine hydroxylase immunoreactivity (optical density), DAT immunoreactivity (optical density), and dopamine concentration (high-performance liquid chromatography). Microglial accumulation and inflammatory response, assessed by stereological counts of Iba-1-labeled cells, was modestly increased in NbSyn87*PEST-injected rats but not in VH14*PEST-treated or saline-treated animals. Modest behavioral rescue was also observed, although there was pronounced variability among individual animals. These data validate in vivo therapeutic efficacy of vector-delivered intracellular nanobodies targeting α-syn misfolding and aggregation in synucleinopathies such as PD.
RESUMEN
Intracellular deposits of α-synuclein in the form of Lewy bodies are major hallmarks of Parkinson's disease (PD) and a range of related neurodegenerative disorders. Post-translational modifications (PTMs) of α-synuclein are increasingly thought to be major modulators of its structure, function, degradation and toxicity. Among these PTMs, phosphorylation near the C-terminus at S129 has emerged as a dominant pathogenic modification as it is consistently observed to occur within the brain and cerebrospinal fluid (CSF) of post-mortem PD patients, and its level appears to correlate with disease progression. Phosphorylation at the neighboring tyrosine residue Y125 has also been shown to protect against α-synuclein toxicity in a Drosophila model of PD. In the present study we address the potential roles of C-terminal phosphorylation in modulating the interaction of α-synuclein with other protein partners, using a single domain antibody fragment (NbSyn87) that binds to the C-terminal region of α-synuclein with nanomolar affinity. The results reveal that phosphorylation at S129 has negligible effect on the binding affinity of NbSyn87 to α-synuclein while phosphorylation at Y125, only four residues away, decreases the binding affinity by a factor of 400. These findings show that, despite the fact that α-synuclein is intrinsically disordered in solution, selective phosphorylation can modulate significantly its interactions with other molecules and suggest how this particular form of modification could play a key role in regulating the normal and aberrant function of α-synuclein.
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Procesamiento Proteico-Postraduccional , Anticuerpos de Dominio Único/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Autopsia , Sitios de Unión , Encéfalo/metabolismo , Humanos , Enfermedad de Parkinson/metabolismo , Fosforilación , Unión Proteica , Serina/metabolismo , Tirosina/metabolismo , alfa-Sinucleína/líquido cefalorraquídeoRESUMEN
Neuroinflammation mediated by chronically activated microglia, largely caused by abnormal accumulation of misfolded α-synuclein (αSyn) protein, is known to contribute to the pathophysiology of Parkinson's disease (PD). In this work, based on the immunomodulatory activities displayed by particular heat-shock proteins (HSPs), we tested a novel vaccination strategy that used a combination of αSyn and Grp94 (HSPC4 or Gp96) chaperone and a murine PD model. We used two different procedures, first, the adoptive transfer of splenocytes from αSyn/Grp94-immunized mice to recipient animals, and second, direct immunization with αSyn/Grp94, to study the effects in a chronic mouse MPTP-model of parkinsonism. We found that both approaches promoted a distinct profile in the peripheral system-supported by humoral and cellular immunity-consisting of a Th1-shifted αSyn-specific response accompanied by an immune-regulatory/Th2-skewed general phenotype. Remarkably, this mixed profile sustained by αSyn/Grp94 immunization led to strong suppression of microglial activation in the substantia nigra and striatum, pointing to a newly described positive effect of anti-αSyn Th1-responses in the context of PD. This strategy is the first to target αSyn and report the suppression of PD-associated microgliosis. Overall, we show that the αSyn/Grp94 combination supports a distinct and long-lasting immune profile in the peripheral system, which has an impact at the CNS level by suppressing chronic microglial activation in an MPTP model of PD. Furthermore, our study demonstrates that reshaping peripheral immunity by vaccination with appropriate misfolding protein/HSP combinations could be highly beneficial as a treatment for neurodegenerative misfolding diseases.
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Gliosis/etiología , Gliosis/terapia , Inmunización/métodos , Intoxicación por MPTP , Glicoproteínas de Membrana/inmunología , alfa-Sinucleína/inmunología , Traslado Adoptivo , Análisis de Varianza , Animales , Antígenos CD4/metabolismo , Enfermedad Crónica , Citocinas/metabolismo , Modelos Animales de Enfermedad , Intoxicación por MPTP/inducido químicamente , Intoxicación por MPTP/complicaciones , Intoxicación por MPTP/inmunología , Intoxicación por MPTP/terapia , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/patología , Sustancia Negra/metabolismo , Sustancia Negra/patología , Linfocitos T Reguladores/metabolismoRESUMEN
Populating transient and partially unfolded species is a crucial step in the formation and accumulation of amyloid fibrils formed from pathogenic variants of human lysozyme linked with a rare but fatal hereditary systemic amyloidosis. The partially unfolded species possess an unstructured ß-domain and C-helix with the rest of the α-domain remaining native-like. Here we use paramagnetic relaxation enhancement (PRE) measured by NMR spectroscopy to study the transient intermolecular interactions between such intermediate species. Nitroxide spin labels, introduced specifically at three individual lysine residues, generate distinct PRE profiles, indicating the presence of intermolecular interactions between residues within the unfolded ß-domain. This study describes the applicability to PRE NMR measurements of selective lysine labeling, at different sites within a protein, as an alternative to the introduction of spin labels via engineered cysteine residues. These results reveal the importance of the ß-sheet region of lysozyme for initiating self-assembly into amyloid fibrils.
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Amiloide/metabolismo , Lisina/metabolismo , Muramidasa/metabolismo , Humanos , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Marcadores de SpinRESUMEN
In nature, a wide range of functional materials is based on proteins. Increasing attention is also turning to the use of proteins as artificial biomaterials in the form of films, gels, particles, and fibrils that offer great potential for applications in areas ranging from molecular medicine to materials science. To date, however, most such applications have been limited to single component materials despite the fact that their natural analogues are composed of multiple types of proteins with a variety of functionalities that are coassembled in a highly organized manner on the micrometer scale, a process that is currently challenging to achieve in the laboratory. Here, we demonstrate the fabrication of multicomponent protein microcapsules where the different components are positioned in a controlled manner. We use molecular self-assembly to generate multicomponent structures on the nanometer scale and droplet microfluidics to bring together the different components on the micrometer scale. Using this approach, we synthesize a wide range of multiprotein microcapsules containing three well-characterized proteins: glucagon, insulin, and lysozyme. The localization of each protein component in multishell microcapsules has been detected by labeling protein molecules with different fluorophores, and the final three-dimensional microcapsule structure has been resolved by using confocal microscopy together with image analysis techniques. In addition, we show that these structures can be used to tailor the release of such functional proteins in a sequential manner. Moreover, our observations demonstrate that the protein release mechanism from multishell capsules is driven by the kinetic control of mass transport of the cargo and by the dissolution of the shells. The ability to generate artificial materials that incorporate a variety of different proteins with distinct functionalities increases the breadth of the potential applications of artificial protein-based materials and provides opportunities to design more refined functional protein delivery systems.
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Cápsulas/química , Liberación de Fármacos , Glucagón/química , Insulina/química , Muramidasa/químicaRESUMEN
BACKGROUND: The aggregation of the protein É-synuclein (ÉS) underlies a range of increasingly common neurodegenerative disorders including Parkinson's disease. One widely explored therapeutic strategy for these conditions is the use of antibodies to target aggregated ÉS, although a detailed molecular-level mechanism of the action of such species remains elusive. Here, we characterize ÉS aggregation in vitro in the presence of two ÉS-specific single-domain antibodies (nanobodies), NbSyn2 and NbSyn87, which bind to the highly accessible C-terminal region of ÉS. RESULTS: We show that both nanobodies inhibit the formation of ÉS fibrils. Furthermore, using single-molecule fluorescence techniques, we demonstrate that nanobody binding promotes a rapid conformational conversion from more stable oligomers to less stable oligomers of ÉS, leading to a dramatic reduction in oligomer-induced cellular toxicity. CONCLUSIONS: The results indicate a novel mechanism by which diseases associated with protein aggregation can be inhibited, and suggest that NbSyn2 and NbSyn87 could have significant therapeutic potential.
Asunto(s)
Anticuerpos de Dominio Único/metabolismo , alfa-Sinucleína/metabolismo , Humanos , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/fisiopatología , Unión ProteicaRESUMEN
Naturally spun silks generate fibres with unique properties, including strength, elasticity and biocompatibility. Here we describe a microfluidics-based strategy to spin liquid native silk, obtained directly from the silk gland of Bombyx mori silkworms, into micron-scale capsules with controllable geometry and variable levels of intermolecular ß-sheet content in their protein shells. We demonstrate that such micrococoons can store internally the otherwise highly unstable liquid native silk for several months and without apparent effect on its functionality. We further demonstrate that these native silk micrococoons enable the effective encapsulation, storage and release of other aggregation-prone proteins, such as functional antibodies. These results show that native silk micrococoons are capable of preserving the full activity of sensitive cargo proteins that can aggregate and lose function under conditions of bulk storage, and thus represent an attractive class of materials for the storage and release of active biomolecules.
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Bombyx/metabolismo , Seda/química , Animales , Bombyx/química , Bombyx/crecimiento & desarrollo , Elasticidad , Conformación Proteica en Lámina beta , Estabilidad Proteica , Seda/metabolismoRESUMEN
The aggregation of the amyloid ß peptide (Aß) into amyloid fibrils is a defining characteristic of Alzheimer's disease. Because of the complexity of this aggregation process, effective therapeutic inhibitors will need to target the specific microscopic steps that lead to the production of neurotoxic species. We introduce a strategy for generating fibril-specific antibodies that selectively suppress fibril-dependent secondary nucleation of the 42-residue form of Aß (Aß42). We target this step because it has been shown to produce the majority of neurotoxic species during aggregation of Aß42. Starting from large phage display libraries of single-chain antibody fragments (scFvs), the three-stage approach that we describe includes (i) selection of scFvs with high affinity for Aß42 fibrils after removal of scFvs that bind Aß42 in its monomeric form; (ii) ranking, by surface plasmon resonance affinity measurements, of the resulting candidate scFvs that bind to the Aß42 fibrils; and (iii) kinetic screening and analysis to find the scFvs that inhibit selectively the fibril-catalyzed secondary nucleation process in Aß42 aggregation. By applying this approach, we have identified four scFvs that inhibit specifically the fibril-dependent secondary nucleation process. Our method also makes it possible to discard antibodies that inhibit elongation, an important factor because the suppression of elongation does not target directly the production of toxic oligomers and may even lead to its increase. On the basis of our results, we suggest that the method described here could form the basis for rationally designed immunotherapy strategies to combat Alzheimer's and related neurodegenerative diseases.
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Péptidos beta-Amiloides/metabolismo , Anticuerpos/metabolismo , Bacteriófagos/metabolismo , Enfermedad de Alzheimer/metabolismo , Amiloide/metabolismo , Proteínas Amiloidogénicas/metabolismo , Amiloidosis/metabolismo , Humanos , Cinética , Fragmentos de Péptidos/metabolismo , Biblioteca de PéptidosRESUMEN
In the gas phase, protein ions can adopt a broad range of structures, which have been investigated extensively in the past using ion mobility-mass spectrometry (IM-MS)-based methods. Compact ions with low number of charges undergo a Coulomb-driven transition to partially folded species when the charge increases, and finally form extended structures with presumably little or no defined structure when the charge state is high. However, with respect to the secondary structure, IM-MS methods are essentially blind. Infrared (IR) spectroscopy, on the other hand, is sensitive to such structural details and there is increasing evidence that helices as well as ß-sheet-like structures can exist in the gas phase, especially for ions in low charge states. Very recently, we showed that also the fully extended form of highly charged protein ions can adopt a distinct type of secondary structure that features a characteristic C5-type hydrogen bond pattern. Here we use a combination of IM-MS and IR spectroscopy to further investigate the influence of the initial, native conformation on the formation of these structures. Our results indicate that when intramolecular Coulomb-repulsion is large enough to overcome the stabilization energies of the genuine secondary structure, all proteins, regardless of their sequence or native conformation, form C5-type hydrogen bond structures. Furthermore, our results suggest that in highly charged proteins the positioning of charges along the sequence is only marginally influenced by the basicity of individual residues. Graphical Abstract á .
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Espectrometría de Masas/métodos , Péptidos/química , Ubiquitina/química , alfa-Sinucleína/química , Secuencia de Aminoácidos , Humanos , Enlace de Hidrógeno , Espectrometría de Movilidad Iónica/métodos , Iones/química , Modelos Moleculares , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Espectrofotometría Infrarroja/métodosRESUMEN
The conversion of human lysozyme into amyloid fibrils is associated with a rare but fatal hereditary form of nonneuropathic systemic amyloidosis. The accumulation of large amounts of aggregated protein is thought to be initiated by the formation of transient intermediate species of disease-related lysozyme variants, essentially due to the loss of global cooperativity under physiologically relevant conditions. Interestingly, all five naturally occurring, amyloidogenic, single-point mutations are located in the ß-domain of lysozyme, the region that is predominantly unfolded during the formation of the transient intermediate species. Given the lack of known naturally occurring, amyloidogenic, single-point mutations in the α-domain, we chose three specific mutations to address the effects that location may have on native-state dynamics, as studied by hydrogen-deuterium (HD) exchange experiments analyzed by NMR spectroscopy, and mass spectrometry. We compared the effect of a destabilizing α-domain mutation (I23A) with that of the well-characterized I59T ß-domain variant. We also investigated the effect of a mutation that has minor effects on native-state stability at the domain interface (I56V) and compared it with that of a variant with similar stability within the C-helix (I89V). We show that when variants have similar reduced native-state stabilities, the location of the mutation (I23A versus I59T) is crucial to the native-state dynamics, with the α-domain mutation having a significantly lower ability to populate transient intermediate species under physiologically relevant conditions. Interestingly, the mutation at the interface (I56V) has a greater effect in facilitating the formation of transient intermediate species at elevated temperatures compared with the variants containing α-domain mutations, even though this mutation results in only minor changes to the native-state stability of lysozyme. These findings reveal that the location of specific mutations is an important factor in determining the native-state dynamical properties of human lysozyme in the context of its propensity to populate the aggregation-prone transient intermediate species associated with pathogenic amyloid formation.
Asunto(s)
Muramidasa/química , Muramidasa/genética , Mutación , Amiloide/química , Estabilidad de Enzimas , Humanos , Modelos Moleculares , Dominios Proteicos , Multimerización de Proteína , Estructura Secundaria de ProteínaRESUMEN
Local delivery of amyloid beta oligomers from the tip of a nanopipette, controlled over the cell surface, has been used to deliver physiological picomolar oligomer concentrations to primary astrocytes or neurons. Calcium influx was observed when as few as 2000 oligomers were delivered to the cell surface. When the dosing of oligomers was stopped the intracellular calcium returned to basal levels or below. Calcium influx was prevented by the presence in the pipette of the extracellular chaperone clusterin, which is known to selectively bind oligomers, and by the presence a specific nanobody to amyloid beta. These data are consistent with individual oligomers larger than trimers inducing calcium entry as they cross the cell membrane, a result supported by imaging experiments in bilayers, and suggest that the initial molecular event that leads to neuronal damage does not involve any cellular receptors, in contrast to work performed at much higher oligomer concentrations.
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Péptidos beta-Amiloides/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Fragmentos de Péptidos/metabolismo , Agregado de Proteínas/fisiología , Péptidos beta-Amiloides/síntesis química , Péptidos beta-Amiloides/inmunología , Animales , Astrocitos/citología , Astrocitos/metabolismo , Carbocianinas/química , Células Cultivadas , Clusterina/metabolismo , Microscopía Fluorescente , Neuronas/citología , Neuronas/metabolismo , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Fotoblanqueo , Ratas , Anticuerpos de Dominio Único/inmunologíaRESUMEN
The measurement of molecular interactions with pathological protein aggregates, including amyloid fibrils, is of central importance in the context of the development of diagnostic and therapeutic strategies against protein misfolding disorders. Probing such interactions by conventional methods can, however, be challenging because of the supramolecular nature of protein aggregates, their heterogeneity, and their often dynamic nature. Here we demonstrate that direct measurement of diffusion on a microfluidic platform enables the determination of affinity and kinetics data for ligand binding to amyloid fibrils in solution. This method yields rapid binding information from only microlitres of sample, and is therefore a powerful technique for identifying and characterising molecular species with potential therapeutic or diagnostic application.
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Amiloide/aislamiento & purificación , Técnicas Analíticas Microfluídicas , Deficiencias en la Proteostasis/diagnóstico , Tiazoles/aislamiento & purificación , Amiloide/antagonistas & inhibidores , Amiloide/química , Benzotiazoles , Sitios de Unión/efectos de los fármacos , Difusión , Humanos , Cinética , Ligandos , Nanopartículas/química , Tamaño de la Partícula , Agregado de Proteínas/efectos de los fármacos , Deficiencias en la Proteostasis/tratamiento farmacológico , Tiazoles/química , Tiazoles/farmacologíaRESUMEN
α-Synuclein is an intrinsically disordered protein whose aggregation is associated with Parkinson's disease and other related neurodegenerative disorders. Recently, two single-domain camelid antibodies (nanobodies) were shown to bind α-synuclein with high affinity. Herein, we investigated how these two nanobodies (NbSyn2 and NbSyn87), which are directed to two distinct epitopes within the C-terminal domain of α-synuclein, affect the conformational properties of this protein. Our results suggest that nanobody NbSyn2, which binds to the five C-terminal residues of α-synuclein (residues 136-140), does not disrupt the transient long-range interactions that generate a degree of compaction within the native structural ensemble of α-synuclein. In contrast, the data that we report indicate that NbSyn87, which targets a central region within the C-terminal domain (residues 118-128), has more substantial effects on the fluctuating secondary and tertiary structure of the protein. These results are consistent with the different effects that the two nanobodies have on the aggregation behavior of α-synuclein in vitro. Our findings thus provide new insights into the type of effects that nanobodies can have on the conformational ensemble of α-synuclein.
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Epítopos/metabolismo , Anticuerpos de Dominio Único/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Animales , Camélidos del Nuevo Mundo , Epítopos/inmunología , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Dominios Proteicos , Anticuerpos de Dominio Único/inmunología , alfa-Sinucleína/inmunologíaRESUMEN
We have investigated the potential role of molecular chaperones as modulators of the immune response by using α-synuclein (αSyn) as an aggregation-prone model protein. We first performed an in vitro immunoscreening with 21 preselected candidate chaperones and selected 2 from this set as displaying immunological activity with differential profiles, Grp94/Gp96 and FKBP4/52. We then immunized mice with both chaperone/α-synuclein combinations using monomeric or oligomeric α-synuclein (MαSyn or OαSyn, respectively), and we characterized the immune response generated in each case. We found that Grp94 promoted αSyn-specific T-helper (Th)1/Th17 and IgG1 antibody responses (up to a 3-fold increase) with MαSyn and OαSyn, respectively, coupled to a Th2-type general phenotype (generating 2.5-fold higher IgG1/IgG2 levels). In addition, we observed that FKBP4 favored a Th1-skewed phenotype with MαSyn but strongly supported a Th2-type phenotype with OαSyn (with a 3-fold higher IL-10/IFN-γ serum levels). Importantly, results from adoptive transfer of splenocytes from immunized animals in a Parkinson's disease mouse model indicates that these effects are robust, stable in time, and physiologically relevant. Taken together, Grp94 and FKBP4 are able to generate differential immune responses to α-synuclein-based immunizations, depending both on the nature of the chaperone and on the aggregation state of α-synuclein. Our work reveals that several chaperones are potential modulators of the immune response and suggests that different chaperones could be exploited to redirect the amyloid-elicited immunity both for basic studies of the immunological processes associated with neurodegeneration and for immunotherapy of pathologies associated with protein misfolding and aggregation.
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Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/fisiología , Proteínas de Unión a Tacrolimus/metabolismo , alfa-Sinucleína/metabolismo , Inmunidad Adaptativa , Animales , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Inmunidad Innata , Masculino , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Pliegue de Proteína , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/inmunología , alfa-Sinucleína/genéticaRESUMEN
Characterizing the sizes and interactions of macromolecules under native conditions is a challenging problem in many areas of molecular sciences, which fundamentally arises from the polydisperse nature of biomolecular mixtures. Here, we describe a microfluidic platform for diffusional sizing based on monitoring micron-scale mass transport simultaneously in space and time. We show that the global analysis of such combined space-time data enables the hydrodynamic radii of individual species within mixtures to be determined directly by deconvoluting average signals into the contributions from the individual species. We demonstrate that the ability to perform rapid noninvasive sizing allows this method to be used to characterize interactions between biomolecules under native conditions. We illustrate the potential of the technique by implementing a single-step quantitative immunoassay that operates on a time scale of seconds and detects specific interactions between biomolecules within complex mixtures.
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Inmunoensayo , Técnicas Analíticas Microfluídicas , Microfluídica/métodos , Coloración y Etiquetado/métodos , Animales , Bovinos , Difusión , Colorantes Fluorescentes/química , Glucagón/química , Proteínas HSP70 de Choque Térmico/química , Humanos , Hidrodinámica , Microfluídica/instrumentación , Peso Molecular , Albúmina Sérica Bovina/química , Anticuerpos de Dominio Único/química , Soluciones , Agua/química , alfa-Sinucleína/química , o-Ftalaldehído/químicaRESUMEN
The study of biomolecular interactions is central to an understanding of function, malfunction and therapeutic modulation of biological systems, yet often involves a compromise between sensitivity and accuracy. Many conventional analytical steps and the procedures required to facilitate sensitive detection, such as the incorporation of chemical labels, are prone to perturb the complexes under observation. Here we present a 'latent' analysis approach that uses chemical and microfluidic tools to reveal, through highly sensitive detection of a labelled system, the behaviour of the physiologically relevant unlabelled system. We implement this strategy in a native microfluidic diffusional sizing platform, allowing us to achieve detection sensitivity at the attomole level, determine the hydrodynamic radii of biomolecules that vary by over three orders of magnitude in molecular weight, and study heterogeneous mixtures. We illustrate these key advantages by characterizing a complex of an antibody domain in the solution phase and under physiologically relevant conditions.