RESUMEN
Amyloid-ß (Aß) proteotoxicity is associated with Alzheimer's disease (AD) and is caused by protein aggregation, resulting in neuronal damage in the brain. In the search for novel treatments, Drosophila melanogaster has been extensively used to screen for anti-Aß proteotoxic agents in studies where toxic Aß peptides are expressed in the fly brain. Since drug molecules often are administered orally there is a risk that they fail to reach the brain, due to their inability to cross the brain barrier. To circumvent this problem, we have designed a novel Drosophila model that expresses the Aß peptides in the digestive tract. In addition, a built-in apoptotic sensor provides a fluorescent signal from the green fluorescent protein as a response to caspase activity. We found that expressing different variants of Aß1-42 resulted in proteotoxic phenotypes such as reduced longevity, aggregate deposition, and the presence of apoptotic cells. Taken together, this gut-based Aß-expressing fly model can be used to study the mechanisms behind Aß proteotoxicity and to identify different substances that can modify Aß proteotoxicity.
Asunto(s)
Enfermedad de Alzheimer , Animales , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Péptidos beta-Amiloides/genética , Tracto Gastrointestinal/metabolismo , Modelos Animales de EnfermedadRESUMEN
The prevailing opinion is that prefibrillar ß-amyloid (Aß) species, rather than end-stage amyloid fibrils, cause neuronal dysfunction in Alzheimer's disease, although the mechanisms behind Aß neurotoxicity remain to be elucidated. Luminescent conjugated oligothiophenes (LCOs) exhibit spectral properties upon binding to amyloid proteins and have previously been reported to change the toxicity of Aß1-42 and prion protein. In a previous study, we showed that an LCO, pentamer formyl thiophene acetic acid (p-FTAA), changed the toxicity of Aß1-42. Here we investigated whether an LCO, heptamer formyl thiophene acetic acid (h-FTAA), could change the toxicity of Aß1-42 by comparing its behavior with that of p-FTAA. Moreover, we investigated the effects on toxicity when Aß with the Arctic mutation (AßArc) was aggregated with both LCOs. Cell viability assays on SH-SY5Y neuroblastoma cells demonstrated that h-FTAA has a stronger impact on Aß1-42 toxicity than does p-FTAA. Interestingly, h-FTAA, but not p-FTAA, rescued the AßArc-mediated toxicity. Aggregation kinetics and binding assay experiments with Aß1-42 and AßArc when aggregated with both LCOs showed that h-FTAA and p-FTAA either interact with different species or affect the aggregation in different ways. In conclusion, h-FTAA protects against Aß1-42 and AßArc toxicity, thus showing h-FTAA to be a useful tool for improving our understanding of the process of Aß aggregation linked to cytotoxicity.
Asunto(s)
Acetatos/química , Precursor de Proteína beta-Amiloide/metabolismo , Tiofenos/química , Acetatos/metabolismo , Amiloide/química , Péptidos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/fisiología , Precursor de Proteína beta-Amiloide/toxicidad , Proteínas Amiloidogénicas/química , Colorantes Fluorescentes/química , Humanos , Cinética , Luminiscencia , Fragmentos de Péptidos/metabolismo , Agregado de Proteínas/efectos de los fármacos , Agregado de Proteínas/fisiología , Coloración y Etiquetado/métodos , Tiofenos/metabolismoRESUMEN
Alzheimer's disease is a widespread and devastating neurological disorder associated with proteotoxic events caused by the misfolding and aggregation of the amyloid-ß peptide. To find therapeutic strategies to combat this disease, Drosophila melanogaster has proved to be an excellent model organism that is able to uncover anti-proteotoxic candidates due to its outstanding genetic toolbox and resemblance to human disease genes. In this review, we highlight the use of Drosophila melanogaster to both study the proteotoxicity of the amyloid-ß peptide and to screen for drug candidates. Expanding the knowledge of how the etiology of Alzheimer's disease is related to proteotoxicity and how drugs can be used to block disease progression will hopefully shed further light on the field in the search for disease-modifying treatments.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/genética , Animales , Modelos Animales de Enfermedad , Drosophila melanogaster , HumanosRESUMEN
Aggregation of the amyloid-ß peptide (Aß) in the brain leads to the formation of extracellular amyloid plaques, which is one of the pathological hallmarks of Alzheimer disease (AD). It is a general hypothesis that soluble prefibrillar assemblies of the Aß peptide, rather than mature amyloid fibrils, cause neuronal dysfunction and memory impairment in AD. Thus, reducing the level of these prefibrillar species by using molecules that can interfere with the Aß fibrillation pathway may be a valid approach to reduce Aß cytotoxicity. Luminescent-conjugated oligothiophenes (LCOs) have amyloid binding properties and spectral properties that differ when they bind to protein aggregates with different morphologies and can therefore be used to visualize protein aggregates. In this study, cell toxicity experiments and biophysical studies demonstrated that the LCO p-FTAA was able to reduce the pool of soluble toxic Aß species in favor of the formation of larger insoluble nontoxic amyloid fibrils, there by counteracting Aß-mediated cytotoxicity. Moreover, p-FTAA bound to early formed Aß species and induced a rapid formation of ß-sheet structures. These p-FTAA generated amyloid fibrils were less hydrophobic and more resistant to proteolysis by proteinase K. In summary, our data show that p-FTAA promoted the formation of insoluble and stable Aß species that were nontoxic which indicates that p-FTAA might have therapeutic potential.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/metabolismo , Agregación Patológica de Proteínas/metabolismo , Tiofenos/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Línea Celular Tumoral , Humanos , Agregación Patológica de Proteínas/tratamiento farmacológico , Agregación Patológica de Proteínas/patología , Estabilidad Proteica/efectos de los fármacos , Estructura Secundaria de ProteínaRESUMEN
The hallmarks of Alzheimer disease are amyloid-ß plaques and neurofibrillary tangles accompanied by signs of neuroinflammation. Lysozyme is a major player in the innate immune system and has recently been shown to prevent the aggregation of amyloid-ß1-40 in vitro. In this study we found that patients with Alzheimer disease have increased lysozyme levels in the cerebrospinal fluid and lysozyme co-localized with amyloid-ß in plaques. In Drosophila neuronal co-expression of lysozyme and amyloid-ß1-42 reduced the formation of soluble and insoluble amyloid-ß species, prolonged survival and improved the activity of amyloid-ß1-42 transgenic flies. This suggests that lysozyme levels rise in Alzheimer disease as a compensatory response to amyloid-ß increases and aggregation. In support of this, in vitro aggregation assays revealed that lysozyme associates with amyloid-ß1-42 and alters its aggregation pathway to counteract the formation of toxic amyloid-ß species. Overall, these studies establish a protective role for lysozyme against amyloid-ß associated toxicities and identify increased lysozyme in patients with Alzheimer disease. Therefore, lysozyme has potential as a new biomarker as well as a therapeutic target for Alzheimer disease.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Muramidasa/metabolismo , Fragmentos de Péptidos/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/ultraestructura , Animales , Encéfalo/patología , Muerte Celular , Drosophila melanogaster , Femenino , Humanos , Proteínas de Insectos/metabolismo , Locomoción , Masculino , Persona de Mediana Edad , Muramidasa/sangre , Muramidasa/líquido cefalorraquídeo , Muramidasa/farmacología , Fragmentos de Péptidos/ultraestructura , Placa Amiloide/metabolismo , Placa Amiloide/ultraestructura , Células Tumorales Cultivadas , Proteínas tau/metabolismoRESUMEN
The spreading of pathology through neuronal pathways is likely to be the cause of the progressive cognitive loss observed in Alzheimer's disease (AD) and other neurodegenerative diseases. We have recently shown the propagation of AD pathology via cell-to-cell transfer of oligomeric amyloid beta (Aß) residues 1-42 (oAß1-42) using our donor-acceptor 3-D co-culture model. We now show that different Aß-isoforms (fluorescently labeled 1-42, 3(pE)-40, 1-40 and 11-42 oligomers) can transfer from one cell to another. Thus, transfer is not restricted to a specific Aß-isoform. Although different Aß isoforms can transfer, differences in the capacity to clear and/or degrade these aggregated isoforms result in vast differences in the net amounts ending up in the receiving cells and the net remaining Aß can cause seeding and pathology in the receiving cells. This insufficient clearance and/or degradation by cells creates sizable intracellular accumulations of the aggregation-prone Aß1-42 isoform, which further promotes cell-to-cell transfer; thus, oAß1-42 is a potentially toxic isoform. Furthermore, cell-to-cell transfer is shown to be an early event that is seemingly independent of later appearances of cellular toxicity. This phenomenon could explain how seeds for the AD pathology could pass on to new brain areas and gradually induce AD pathology, even before the first cell starts to deteriorate, and how cell-to-cell transfer can act together with the factors that influence cellular clearance and/or degradation in the development of AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Comunicación Celular/fisiología , Neuritas/metabolismo , Fragmentos de Péptidos/metabolismo , Péptidos beta-Amiloides/ultraestructura , Factor Neurotrófico Derivado del Encéfalo/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Transformada , Técnicas de Cocultivo , Matriz Extracelular/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lisosomas/metabolismo , Lisosomas/ultraestructura , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Factor de Crecimiento Nervioso/farmacología , Neurregulina-1/farmacología , Neuritas/ultraestructura , Neuroblastoma/patología , Fragmentos de Péptidos/ultraestructura , Isoformas de Proteínas , Factores de Tiempo , Tretinoina/farmacologíaRESUMEN
We have created a Drosophila model of lysozyme amyloidosis to investigate the in vivo behavior of disease-associated variants. To achieve this objective, wild-type (WT) protein and the amyloidogenic variants F57I and D67H were expressed in Drosophila melanogaster using the UAS-gal4 system and both the ubiquitous and retinal expression drivers Act5C-gal4 and gmr-gal4. The nontransgenic w(1118) Drosophila line was used as a control throughout. We utilized ELISA experiments to probe lysozyme protein levels, scanning electron microscopy for eye phenotype classification, and immunohistochemistry to detect the unfolded protein response (UPR) activation. We observed that expressing the destabilized F57I and D67H lysozymes triggers UPR activation, resulting in degradation of these variants, whereas the WT lysozyme is secreted into the fly hemolymph. Indeed, the level of WT was up to 17 times more abundant than the variant proteins. In addition, the F57I variant gave rise to a significant disruption of the eye development, and this correlated to pronounced UPR activation. These results support the concept that the onset of familial amyloid disease is linked to an inability of the UPR to degrade completely the amyloidogenic lysozymes prior to secretion, resulting in secretion of these destabilized variants, thereby leading to deposition and associated organ damage.
Asunto(s)
Amiloidosis/enzimología , Anomalías del Ojo/enzimología , Muramidasa/metabolismo , Respuesta de Proteína Desplegada/fisiología , Amiloidosis/patología , Animales , Animales Modificados Genéticamente , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Proteínas de Drosophila/genética , Drosophila melanogaster , Estrés del Retículo Endoplásmico/fisiología , Anomalías del Ojo/genética , Anomalías del Ojo/patología , Femenino , Proteínas Fluorescentes Verdes/genética , Hemolinfa/enzimología , Humanos , Masculino , Metamorfosis Biológica/fisiología , Microscopía Electrónica de Rastreo , Muramidasa/genética , Células Fotorreceptoras de Invertebrados/enzimología , Células Fotorreceptoras de Invertebrados/patología , Células Fotorreceptoras de Invertebrados/ultraestructura , SolubilidadRESUMEN
Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (Abeta) peptide by using a small engineered binding protein (Z(Abeta3)) that binds with nanomolar affinity to Abeta, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z(Abeta3) in the brains of Drosophila melanogaster expressing either Abeta(42) or the aggressive familial associated E22G variant of Abeta(42) abolishes their neurotoxic effects. Biochemical analysis indicates that monomer Abeta binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of Abeta aggregation and reveal that Z(Abeta3) not only inhibits the initial association of Abeta monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation.
Asunto(s)
Péptidos beta-Amiloides , Fragmentos de Péptidos , Amiloide/metabolismo , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/toxicidad , Animales , Animales Modificados Genéticamente , Encéfalo/metabolismo , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Modelos Moleculares , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/ultraestructura , Unión Proteica , Conformación Proteica , Tasa de SupervivenciaRESUMEN
The formation of amyloid-ß peptide (Aß) aggregates at an early stage during the self-assembly process is an important factor in the development of Alzheimer's disease. The toxic effect is believed to be exerted by prefibrillar species of Aß. It is therefore important to identify which prefibrillar species are toxic and characterize their distinct properties. In the present study, we investigated the in vitro aggregation behavior of Aß-derived peptides possessing different levels of neurotoxic activity, using fluorescence spectroscopy in combination with transmission electron microscopy. The toxicity of various Aß aggregates was assessed by using cultures of human neuroblastoma cells. Through combined use of the fluorescence probe 8-anilino-1-napthalenesulfonate (ANS) and the novel luminescent probe pentamer formyl thiophene acetic acid (p-FTAA), we were able to identify those Aß peptide-derived prefibrillar species which exhibited cellular toxicity. In particular, species, which formed early during the aggregation process and showed strong p-FTAA and ANS fluorescence, were the species that possessed toxic activities. Moreover, by manipulating the aggregation conditions, it was possible to change the capacity of the Aß peptide to form nontoxic versus toxic species.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/toxicidad , Fragmentos de Péptidos/química , Fragmentos de Péptidos/toxicidad , Acetatos/química , Naftalenosulfonatos de Anilina/química , Línea Celular , Colorantes Fluorescentes/química , Humanos , Microscopía Electrónica de Transmisión , Tiofenos/químicaRESUMEN
We report the formation of a non-native, folded state of human IgG4-Fc induced by a high temperature at neutral pH and at a physiological salt concentration. This structure is similar to the molten globule state in that it displays a high degree of secondary structure content and surface-exposed hydrophobic residues. However, it is highly resistant to chemical denaturation. The thermally induced state of human IgG4-Fc is thus associated with typical properties of the so-called alternatively folded state previously described for murine IgG, IgG-Fab, and individual antibody domains (V(L), V(H), C(H)1, and C(H)3) under acidic conditions in the presence of anions. Like some of these molecules, human IgG4-Fc in its alternative fold exists as a mixture of different oligomeric structures, dominated by an equilibrium between monomeric and heptameric species. Heating further induces the formation of fibrous structures in the micrometer range.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Temperatura , Anticuerpos/química , Humanos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía Electrónica de Transmisión , Modelos Moleculares , Conformación Proteica , Pliegue de ProteínaRESUMEN
The extent to which proteins aggregate into distinct structures ranging from prefibrillar oligomers to amyloid fibrils is key to the pathogenesis of many age-related degenerative diseases. We describe here for the Alzheimer's disease-related amyloid beta peptide (Abeta) an investigation of the sequence-based determinants of the balance between the formation of prefibrillar aggregates and amyloid fibrils. We show that by introducing single-point mutations, it is possible to convert the normally harmless Abeta40 peptide into a pathogenic species by increasing its relative propensity to form prefibrillar but not fibrillar aggregates, and, conversely, to abolish the pathogenicity of the highly neurotoxic E22G Abeta42 peptide by reducing its relative propensity to form prefibrillar species rather than mature fibrillar ones. This observation can be rationalized by the demonstration that whereas regions of the sequence of high aggregation propensity dominate the overall tendency to aggregate, regions with low intrinsic aggregation propensities exert significant control over the balance of the prefibrillar and fibrillar species formed, and therefore play a major role in determining the neurotoxicity of the Abeta peptide.
Asunto(s)
Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/toxicidad , Neurotoxinas/química , Neurotoxinas/toxicidad , Péptidos beta-Amiloides/ultraestructura , Animales , Benzotiazoles , Drosophila melanogaster/efectos de los fármacos , Cinética , Longevidad/efectos de los fármacos , Proteínas Mutantes/química , Proteínas Mutantes/toxicidad , Proteínas Mutantes/ultraestructura , Mutación/genética , Estructura Cuaternaria de Proteína , Tiazoles/metabolismo , Factores de TiempoRESUMEN
Protein aggregation into amyloid fibrils and protofibrillar aggregates is associated with a number of the most common neurodegenerative diseases. We have established, using a computational approach, that knowledge of the primary sequences of proteins is sufficient to predict their in vitro aggregation propensities. Here we demonstrate, using rational mutagenesis of the Abeta42 peptide based on such computational predictions of aggregation propensity, the existence of a strong correlation between the propensity of Abeta42 to form protofibrils and its effect on neuronal dysfunction and degeneration in a Drosophila model of Alzheimer disease. Our findings provide a quantitative description of the molecular basis for the pathogenicity of Abeta and link directly and systematically the intrinsic properties of biomolecules, predicted in silico and confirmed in vitro, to pathogenic events taking place in a living organism.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Locomoción/fisiología , Longevidad/fisiología , Fragmentos de Péptidos/metabolismo , Péptidos beta-Amiloides/genética , Animales , Modelos Animales de Enfermedad , Drosophila melanogaster , Mutación Missense , Fragmentos de Péptidos/genéticaRESUMEN
Alzheimer's disease (AD) is the most common form of dementia, affecting millions of people and currently lacking available disease-modifying treatments. Appropriate disease models are necessary to investigate disease mechanisms and potential treatments. Drosophila melanogaster models of AD include the Aß fly model and the AßPP-BACE1 fly model. In the Aß fly model, the Aß peptide is fused to a secretion sequence and directly overexpressed. In the AßPP-BACE1 model, human AßPP and human BACE1 are expressed in the fly, resulting in in vivo production of Aß peptides and other AßPP cleavage products. Although these two models have been used for almost two decades, the underlying mechanisms resulting in neurodegeneration are not yet clearly understood. In this study, we have characterized toxic mechanisms in these two AD fly models. We detected neuronal cell death and increased protein carbonylation (indicative of oxidative stress) in both AD fly models. In the Aß fly model, this correlates with high Aß1-42 levels and down-regulation of the levels of mRNA encoding lysosomal-associated membrane protein 1, lamp1 (a lysosomal marker), while in the AßPP-BACE1 fly model, neuronal cell death correlates with low Aß1-42 levels, up-regulation of lamp1 mRNA levels and increased levels of C-terminal fragments. In addition, a significant amount of AßPP/Aß antibody (4G8)-positive species, located close to the endosomal marker rab5, was detected in the AßPP-BACE1 model. Taken together, this study highlights the similarities and differences in the toxic mechanisms which result in neuronal death in two different AD fly models. Such information is important to consider when utilizing these models to study AD pathogenesis or screening for potential treatments.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Animales Modificados Genéticamente , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Proteínas de Drosophila , Drosophila melanogaster , Humanos , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/fisiopatología , Estrés OxidativoRESUMEN
Many conflicting reports about the involvement of serum amyloid P component (SAP) in amyloid diseases have been presented over the years; SAP is known to be a universal component of amyloid aggregates but it has been suggested that it can both induce and suppress amyloid formation. By using our Drosophila model of systemic lysozyme amyloidosis, SAP has previously been shown to reduce the toxicity induced by the expression of the disease-associated lysozyme variant, F57I, in the Drosophila central nervous system. This study further investigates the involvement of SAP in modulating lysozyme toxicity using histochemistry and spectral analyses on the double transgenic WT and F57I lysozyme flies to probe; i) formation of aggregates, ii) morphological differences of the aggregated lysozyme species formed in the presence or absence of SAP, iii) location of lysozyme and iv) co-localisation of lysozyme and SAP in the fly brain. We found that SAP can counteract the toxicity (measured by the reduction in the median survival time) induced by F57I lysozyme by converting toxic F57I species into less toxic amyloid-like structures, as reflected by the spectral changes that p-FTAA undergoes when bound to lysozyme deposits in F57I-F57I-SAP flies as compared to F57I-F57I flies. Indeed, when SAP was introduced to in vitro lysozyme fibril formation, the endpoint fibrils had enhanced ThT fluorescence intensity as compared to lysozyme fibrils alone. This suggests that a general mechanism for SAP's role in amyloid diseases may be to promote the formation of stable, amyloid-like fibrils, thus decreasing the impact of toxic species formed along the aggregation pathway.
Asunto(s)
Amiloidosis/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Muramidasa/metabolismo , Componente Amiloide P Sérico/metabolismo , Amiloide/genética , Amiloide/metabolismo , Amiloide/ultraestructura , Amiloidosis/genética , Amiloidosis/patología , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Humanos , Muramidasa/genética , Agregado de Proteínas , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patologíaRESUMEN
More than 100 distinct mutations in the gene (SOD1) for human copper-zinc superoxide dismutase (CuZnSOD) have been associated with familial amyotrophic lateral sclerosis (fALS). Studies of these mutant proteins, which often have been performed under far from physiological conditions, have indicated effects on protein stabilities, catalytic activity, and metal binding affinities but with no common pattern. Also, with the knowledge that ALS is a late onset disease it is apparent that protein interactions which contribute to the disorder might, in the natural cellular milieu, depend on a delicate balance between intrinsic protein properties. In this study, we have used experimental conditions as near as possible to the in vivo conditions to reduce artifacts emanating from the experimental setup. Using 1H-15N HSQC NMR spectroscopy, we have analyzed hydrogen exchange at the amide groups of wild-type (wt) CuZnSOD and the fALS-associated G93A SOD variant in their fully metalated states. From analyses of the exchange pattern, we have characterized the local dynamics at 64% of all positions in detail in both the wt and G93A protein. The results show that the G93A mutation had no effect on the dynamics at a majority of the investigated positions. However, the mutation results in local destabilization at the site of the mutation and also in stabilization at a few positions that were apparently scattered over the entire protein surface. Most remarkably, the mutation selectively destabilized the remote metal binding region. The results indicate that the metal binding region may affect the intermolecular protein-protein interactions which cause formation of protein aggregates.
Asunto(s)
Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/genética , Cobre/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Zinc/metabolismo , Alanina/genética , Cobre/química , Glicina/genética , Humanos , Unión Proteica/genética , Estabilidad Proteica , Zinc/químicaRESUMEN
BACKGROUND: Oligomeric and fibrillar aggregates of the amyloid beta-peptide (Abeta) have been implicated in the pathogenesis of Alzheimer's disease (AD). The characterization of Abeta assemblies is essential for the elucidation of the mechanisms of Abeta neurotoxicity, but requires large quantities of pure peptide. Here we describe a novel approach to the recombinant production of Abeta. The method is based on the coexpression of the affibody protein ZAbeta3, a selected affinity ligand derived from the Z domain three-helix bundle scaffold. ZAbeta3 binds to the amyloidogenic central and C-terminal part of Abeta with nanomolar affinity and consequently inhibits aggregation. RESULTS: Coexpression of ZAbeta3 affords the overexpression of both major Abeta isoforms, Abeta(1-40) and Abeta(1-42), yielding 4 or 3 mg, respectively, of pure 15N-labeled peptide per liter of culture. The method does not rely on a protein-fusion or -tag and thus does not require a cleavage reaction. The purified peptides were characterized by NMR, circular dichroism, SDS-PAGE and size exclusion chromatography, and their aggregation propensities were assessed by thioflavin T fluorescence and electron microscopy. The data coincide with those reported previously for monomeric, largely unstructured Abeta. ZAbeta3 coexpression moreover permits the recombinant production of Abeta(1-42) carrying the Arctic (E22G) mutation, which causes early onset familial AD. Abeta(1-42)E22G is obtained in predominantly monomeric form and suitable, e.g., for NMR studies. CONCLUSION: The coexpression of an engineered aggregation-inhibiting binding protein offers a novel route to the recombinant production of amyloidogenic Abeta peptides that can be advantageously employed to study the molecular basis of AD. The presented expression system is the first for which expression and purification of the aggregation-prone Arctic variant (E22G) of Abeta(1-42) is reported.
Asunto(s)
Péptidos beta-Amiloides/biosíntesis , Proteínas Recombinantes/biosíntesis , Sustitución de Aminoácidos , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/aislamiento & purificación , Péptidos beta-Amiloides/metabolismo , Ligandos , Espectroscopía de Resonancia Magnética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Péptidos/química , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
We have analysed hydrogen exchange at amide groups to characterise the energy landscape of the 72 amino acid residue protein MerP. From the guanidine hydrochloride (GuHCl) dependence of exchange in the pre-transitional region we have determined free energy values of exchange (DeltaG(HX)) and corresponding m-values for individual amide protons. Detailed analysis of the exchange patterns indicates that for one set of amide protons there is a weak dependence on denaturant, indicating that the exchange is dominated by local fluctuations. For another set of amide protons a linear, but much stronger, denaturant dependence is observed. Notably, the plots of free energy of exchange versus [GuHCl] for 16 amide protons show pronounced upward curvature, and a close inspection of the structure shows that these residues form a well-defined core in the protein. The hydrogen exchange that was measured at various concentrations of NaCl shows an apparent selective stabilisation of this core. Detailed analysis of this exchange pattern indicates that it may originate from selective destabilisation of the unfolded state by guanidinium ions and/or selective stabilisation of the core in the native state by chloride ions.
Asunto(s)
Guanidina/química , Hidrógeno/química , Conformación Proteica , Proteínas/química , Cloruro de Sodio/química , Amidas/química , Secuencia de Aminoácidos , Guanidina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Parasimpaticomiméticos/química , Estructura Secundaria de Proteína , Proteínas/metabolismo , ProtonesRESUMEN
Lysozyme amyloidosis is a hereditary disease in which mutations in the gene coding for lysozyme leads to misfolding and consequently accumulation of amyloid material. To improve understanding of the processes involved we expressed human wild type (WT) lysozyme and the disease-associated variant F57I in the central nervous system (CNS) of a Drosophila melanogaster model of lysozyme amyloidosis, with and without co-expression of serum amyloid p component (SAP). SAP is known to be a universal constituent of amyloid deposits and to associate with lysozyme fibrils. There are clear indications that SAP may play an important role in lysozyme amyloidosis, which requires further elucidation. We found that flies expressing the amyloidogenic variant F57I in the CNS have a shorter lifespan than flies expressing WT lysozyme. We also identified apoptotic cells in the brains of F57I flies demonstrating that the flies' neurological functions are impaired when F57I is expressed in the nerve cells. However, co-expression of SAP in the CNS prevented cell death and restored the F57I flies' lifespan. Thus, SAP has the apparent ability to protect nerve cells from damage caused by F57I. Furthermore, it was found that co-expression of SAP prevented accumulation of insoluble forms of lysozyme in both WT- and F57I-expressing flies. Our findings suggest that the F57I mutation affects the aggregation process of lysozyme resulting in the formation of cytotoxic species and that SAP is able to prevent cell death in the F57I flies by preventing accumulation of toxic F57I structures.
Asunto(s)
Amiloidosis/genética , Sistema Nervioso Central/metabolismo , Muramidasa/genética , Placa Amiloide/genética , Agregación Patológica de Proteínas/genética , Componente Amiloide P Sérico/genética , Amiloidosis/metabolismo , Amiloidosis/patología , Animales , Animales Modificados Genéticamente , Apoptosis , Sistema Nervioso Central/patología , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Expresión Génica , Humanos , Longevidad/genética , Muramidasa/metabolismo , Mutación , Neuronas/metabolismo , Neuronas/patología , Placa Amiloide/metabolismo , Placa Amiloide/patología , Placa Amiloide/prevención & control , Factores Protectores , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Agregación Patológica de Proteínas/prevención & control , Componente Amiloide P Sérico/metabolismo , TransgenesRESUMEN
The aggregation of the amyloid-ß (Aß) peptide into fibrillar deposits has long been considered the key neuropathological hallmark of Alzheimer's disease (AD). Aß peptides are generated from proteolytic processing of the transmembrane Aß precursor protein (AßPP) via sequential proteolysis through the ß-secretase activity of ß-site AßPP-cleaving enzyme (BACE1) and by the intramembranous enzyme γ-secretase. For over a decade, Drosophila melanogaster has been used as a model organism to study AD, and two different approaches have been developed to investigate the toxicity caused by AD-associated gene products in vivo In one model, the Aß peptide is directly over-expressed fused to a signal peptide, allowing secretion of the peptide into the extracellular space. In the other model, human AßPP is co-expressed with human BACE1, resulting in production of the Aß peptide through the processing of AßPP by BACE1 and by endogenous fly γ-secretase. Here, we performed a parallel study of flies that expressed the Aß1-42 peptide alone or that co-expressed AßPP and BACE1. Toxic effects (assessed by eye phenotype, longevity and locomotor assays) and levels of the Aß1-42, Aß1-40 and Aß1-38 peptides were examined. Our data reveal that the toxic effect per amount of detected Aß1-42 peptide was higher in the flies co-expressing AßPP and BACE1 than in the Aß1-42-expressing flies, and that the co-existence of Aß1-42 and Aß1-40 in the flies co-expressing AßPP and BACE1 could be of significant importance to the neurotoxic effect detected in these flies. Thus, the toxicity detected in these two fly models seems to have different modes of action and is highly dependent on how and where the peptide is generated rather than on the actual level of the Aß1-42 peptide in the flies. This is important knowledge that needs to be taken into consideration when using Drosophila models to investigate disease mechanisms or therapeutic strategies in AD research.
RESUMEN
Genetic polymorphisms of immune genes that associate with higher risk to develop Alzheimer's disease (AD) have led to an increased research interest on the involvement of the immune system in AD pathogenesis. A link between amyloid pathology and immune gene expression was suggested in a genome-wide gene expression study of transgenic amyloid mouse models. In this study, the gene expression of lysozyme, a major player in the innate immune system, was found to be increased in a comparable pattern as the amyloid pathology developed in transgenic mouse models of AD. A similar pattern was seen at protein levels of lysozyme in human AD brain and CSF, but this lysozyme pattern was not seen in a tau transgenic mouse model. Lysozyme was demonstrated to be beneficial for different Drosophila melanogaster models of AD. In flies that expressed Aß1-42 or AßPP together with BACE1 in the eyes, the rough eye phenotype indicative of toxicity was completely rescued by coexpression of lysozyme. In Drosophila flies bearing the Aß1-42 variant with the Arctic gene mutation, lysozyme increased the fly survival and decreased locomotor dysfunction dose dependently. An interaction between lysozyme and Aß1-42 in the Drosophila eye was discovered. We propose that the increased levels of lysozyme, seen in mouse models of AD and in human AD cases, were triggered by Aß1-42 and caused a beneficial effect by binding of lysozyme to toxic species of Aß1-42 , which prevented these from exerting their toxic effects. These results emphasize the possibility of lysozyme as biomarker and therapeutic target for AD.