RESUMEN
Protein misfolding and aggregation processes involve local polarity and viscosity fluctuation. Herein we modulated the polarity and viscosity sensitivities of merocyanine dyes to detect protein aggregation. We demonstrated how structural modulation balanced these two fluorescence sensitivities and affected the detection of misfolded and aggregated proteins.
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Benzopiranos/química , Indoles/química , Agregación Patológica de Proteínas/diagnóstico , Deficiencias en la Proteostasis/diagnóstico , Colorantes Fluorescentes , Humanos , Estructura MolecularRESUMEN
BACKGROUND: Syringes containing anti-vascular endothelial growth factor drugs to treat retinal diseases are prepared in different ways by various parties with syringe selection, preparation, and storage conditions affecting the risk of injecting particles into the vitreous. This study examines particle loads from various syringes over time. METHODS: Four syringes were studied: two plastic transfer syringes lubricated with silicone oil or oleamide, a glass syringe with baked-on silicone, and a lubricant-free polymer syringe. Syringes were rinsed with water or filled with buffer and analyzed over time; particles were quantified by flow imaging. Particle formation in a bevacizumab formulation was also characterized. RESULTS: Insulin syringes consistently showed very high particle counts. Oleamide-lubricated syringes had substantially fewer particles, but showed appreciable increases over time (leading to visible particles). Baked-on silicone glass syringes and lubricant-free polymer syringes both showed low particle levels ≥10 µm. Lubricant-free syringes showed the lowest particle levels ≥1 µm and the lowest particle levels with bevacizumab agitation. CONCLUSION: Syringes have different intrinsic particle loads which can contribute to particle loads in the delivered drug. Oleamide-lubricated transfer syringes, commonly used for bevacizumab repackaging, have time-dependent particle loads and are associated with the formation of visible particles beyond 30 days of storage.
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Inhibidores de la Angiogénesis/metabolismo , Bevacizumab/metabolismo , Composición de Medicamentos/métodos , Material Particulado/metabolismo , Agregación Patológica de Proteínas/etiología , Jeringas , Embalaje de Medicamentos , Inyecciones Intravítreas , Lubricantes , Agregación Patológica de Proteínas/diagnóstico , Agregación Patológica de Proteínas/metabolismo , Aceites de Silicona/química , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
The accumulation of amyloid protein aggregates in tissues is the basis for the onset of diseases known as amyloidoses. Intriguingly, many amyloidoses impact the central nervous system (CNS) and usually are devastating diseases. It is increasingly apparent that neurotoxic soluble oligomers formed by amyloidogenic proteins are the primary molecular drivers of these diseases, making them lucrative diagnostic and therapeutic targets. One promising diagnostic/therapeutic strategy has been the development of antibody fragments against amyloid oligomers. Antibody fragments, such as fragment antigen-binding (Fab), scFv (single chain variable fragments), and VHH (heavy chain variable domain or single-domain antibodies) are an alternative to full-length IgGs as diagnostics and therapeutics for a variety of diseases, mainly because of their increased tissue penetration (lower MW compared to IgG), decreased inflammatory potential (lack of Fc domain), and facile production (low structural complexity). Furthermore, through the use of in vitro-based ligand selection, it has been possible to identify antibody fragments presenting marked conformational selectivity. In this review, we summarize significant reports on antibody fragments selective for oligomers associated with prevalent CNS amyloidoses. We discuss promising results obtained using antibody fragments as both diagnostic and therapeutic agents against these diseases. In addition, the use of antibody fragments, particularly scFv and VHH, in the isolation of unique oligomeric assemblies is discussed as a strategy to unravel conformational moieties responsible for neurotoxicity. We envision that advances in this field may lead to the development of novel oligomer-selective antibody fragments with superior selectivity and, hopefully, good clinical outcomes.
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Amiloide/inmunología , Amiloidosis/diagnóstico , Síndromes de Neurotoxicidad/diagnóstico , Agregación Patológica de Proteínas/diagnóstico , Amiloide/antagonistas & inhibidores , Amiloidosis/inmunología , Amiloidosis/patología , Animales , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/patología , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Síndromes de Neurotoxicidad/inmunología , Síndromes de Neurotoxicidad/patología , Fragmentos de Péptidos/inmunología , Agregación Patológica de Proteínas/inmunología , Anticuerpos de Dominio Único , Relación Estructura-ActividadRESUMEN
Understanding of the structural changes during their aggregation and interaction is a prerequisite for establishing the precise clinical relevance of human islet amyloid polypeptide (hIAPP) (involved in Type-II Diabetes Mellitus) in the treatment of Alzheimer's disease stemmed from beta-amyloid (Aß). Herein, we show that the steady-state emission spectra obtained from photoluminescence (PL) simultaneously capture both the tyrosine derivative (tyrosinate) and the structure-specific intrinsic fluorescence during the aggregation of Aß and hIAPP. We observe multiple peaks in the emission spectra which exist for structure-specific intrinsic fluorescence, and use the second derivative UV-Vis spectra and the shift in the tyrosine peak as a quantitative measure of the dissimilitude in the electronic states and the fibril growth. We further applied these techniques to detect the static electric field (0, 40, 120, 200â V/cm) induced promotion and inhibition of fibrillation in Aß, hIAPP and their electric field dependent role in the fibrillation of Aß : hIAPP(1 : 1). The results were corroborated by field-emission scanning electron microscopy (FESEM), and the determinations of secondary structures by Fourier transform infrared spectroscopy (FTIR). The results indicate that the emission spectrum can be used as a sensor to detect the presence of fibrils; hence for screening potential inhibitors of amyloid fibrillation.
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Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/análisis , Fluorescencia , Polipéptido Amiloide de los Islotes Pancreáticos/análisis , Agregación Patológica de Proteínas/diagnóstico , Tirosina/análisis , Humanos , Microscopía Electrónica de Rastreo , Agregado de Proteínas , Estructura Secundaria de Proteína , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja por Transformada de Fourier , Electricidad EstáticaRESUMEN
Several neurodegenerative diseases, like Alzheimer's and Parkinson's are linked with protein aggregation into amyloid fibrils. Conformational changes of native protein into the ß-sheet structure are associated with a significant change in the vibrational spectrum. This is especially true for amide bands which are inherently sensitive to the secondary structure of a protein. Raman amide bands are greatly intensified under resonance conditions, in the UV spectral range, allowing for the selective probing of the peptide backbone. In this work, we examine parallel ß-sheet forming GGVVIA, the C-terminus segment of amyloid-ß peptide, using UV-Vis, FTIR, and multiwavelength Raman spectroscopy. We find that amide bands are enhanced far from the expected UV range, i.e., at 442 nm. A reasonable two-fold relative intensity increase is observed for amide II mode (normalized according to the δCH2/δCH3 vibration) while comparing 442 and 633 nm excitations; an increase in relative intensity of other amide bands was also visible. The observed relative intensification of amide II, amide S, and amide III modes in the Raman spectrum recorded at 442 nm comparing with longer wavelength (633/785/830 nm) excited spectra allows unambiguous identification of amide bands in the complex Raman spectra of peptides and proteins containing the ß-sheet structure.
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Amidas/química , Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/química , Espectrometría Raman/métodos , Secuencia de Aminoácidos , Amiloide/química , Humanos , Microscopía de Fuerza Atómica/métodos , Estructura Molecular , Agregado de Proteínas , Agregación Patológica de Proteínas/diagnóstico , Estructura Secundaria de ProteínaRESUMEN
Tissue accumulation of abnormal aggregates of amyloidogenic proteins such as prion protein, α-synuclein, and tau represents the hallmark of most common neurodegenerative disorders and precedes the onset of symptoms by years. As a consequence, the sensitive and specific detection of abnormal forms of these proteins in patients' accessible tissues or fluids as biomarkers may have a significant impact on the clinical diagnosis of these disorders. By exploiting seeded polymerization propagation mechanisms to obtain cell-free reactions that allow highly amplified detection of these amyloid proteins, novel emerging in vitro techniques, such as the real-time quaking-induced conversion assay (RT-QuIC) have paved the way towards this important goal. Given its high accuracy in identifying misfolded forms of prion protein from Creutzfeldt-Jakob disease (CJD) CSF, RT-QuIC has already been included in the diagnostic criteria for the clinical diagnosis of sporadic CJD, the most common human prion disease. By showing that this assay may also accurately discriminate between Lewy body disorders and other forms of parkinsonisms or dementias, more recent studies strongly suggested that CSF RT-QuIC can also be successfully applied to synucleinopathies. Finally, preliminary encouraging data also suggested that CSF RT-QuIC might also work for tau protein, and accurately distinguish between 3R- and 4R tauopathies, including Pick's disease, progressive supranuclear palsy, and corticobasal degeneration. Here we will review the state of the art of cell-free aggregation assays, their current diagnostic value and putative limitations, and the future perspectives for their expanded use in clinical practice.
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Proteínas Amiloidogénicas/análisis , Diagnóstico Precoz , Enfermedades Neurodegenerativas/diagnóstico , Animales , Bioensayo/métodos , Sistema Libre de Células , Humanos , Técnicas In Vitro , Agregación Patológica de Proteínas/diagnósticoRESUMEN
Alzheimer's disease (AD) is progressive brain amyloidosis that damages brain regions associated with memory, thinking, behavioral and social skills. Neuropathologically, AD is characterized by intraneuronal hyperphosphorylated tau inclusions as neurofibrillary tangles (NFTs), and buildup of extracellular amyloid-beta (Aß) peptide as senile plaques. Several biomarker tests capturing these pathologies have been developed. However, for the full clinical expression of the neurodegenerative events of AD, there exist other central molecular pathways. In terms of understanding the unidentified underlying processes for the progression and development of AD, a complete comprehension of the structure and composition of atypical aggregation of proteins is essential. Presently, to aid the prognosis, diagnosis, detection, and development of drug targets in AD, neuroproteomics is elected as one of the leading essential tools for the efficient exploratory discovery of prospective biomarker candidates estimated to play a crucial role. Therefore, the aim of this review is to present the role of neuroproteomics to analyze the complexity of AD.
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Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/metabolismo , Agregado de Proteínas , Agregación Patológica de Proteínas/diagnóstico , Agregación Patológica de Proteínas/metabolismo , Proteómica , Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/metabolismo , Biomarcadores/análisis , Biomarcadores/metabolismo , HumanosRESUMEN
Multiple neurodegenerative diseases are characterized by aggregation of tau molecules. Adult humans express six isoforms of tau that contain either 3 or 4 microtubule binding repeats (3R or 4R tau). Different diseases involve preferential aggregation of 3R (e.g Pick disease), 4R (e.g. progressive supranuclear palsy), or both 3R and 4R tau molecules [e.g. Alzheimer disease and chronic traumatic encephalopathy]. Three ultrasensitive cell-free seed amplification assays [called tau real-time quaking induced conversion (tau RT-QuIC) assays] have been developed that preferentially detect 3R, 4R, or 3R/4R tau aggregates in biospecimens. In these reactions, low-fg amounts of a given self-propagating protein aggregate (the seed) are incubated with a vast excess of recombinant tau monomers (the substrate) in multi-well plates. Over time, the seeds incorporate the substrate to grow into amyloids that can then be detected using thioflavin T fluorescence. Here we describe a tau RT-QuIC assay (K12 RT-QuIC) that, using a C-terminally extended recombinant 3R tau substrate (K12CFh), enables sensitive detection of Pick disease, Alzheimer disease, and chronic traumatic encephalopathy seeds in brain homogenates. The discrimination of Pick disease from Alzheimer disease and chronic traumatic encephalopathy cases is then achieved through the quantitative differences in K12 RT-QuIC assay thioflavin T responses, which correlate with structural properties of the reaction products. In particular, Fourier transform infrared spectroscopy analysis of the respective K12CFh amyloids showed distinct ß-sheet conformations, suggesting at least partial propagation of the original seed conformations in vitro. Thus, K12 RT-QuIC provides a single assay for ultrasensitive detection and discrimination of tau aggregates comprised mainly of 3R, or both 3R and 4R, tau isoforms.
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Enfermedad de Alzheimer/diagnóstico , Bioensayo/métodos , Encéfalo/metabolismo , Enfermedad de Pick/diagnóstico , Agregación Patológica de Proteínas/diagnóstico , Proteínas tau/análisis , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Biomarcadores/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedad de Pick/metabolismo , Agregación Patológica de Proteínas/metabolismoRESUMEN
INTRODUCTION: Light chain deposition disease is a rare anatomo-clinical disorder, which rarely leads to cystic lung destruction. CASE REPORT: We report the case of a 62years old female patient with a history of a monoclonal gammopathy of unknown significance who developed progressive dyspnea. Thoracic CT-scan demonstrated a diffuse pulmonary cystic disorder with predominance in the right lower lobe. Thoracoscopic surgical resection of that lobe led to a diagnosis of non-amyloid kappa light chain deposits. Surgery also resulted in a lung volume reduction effect with clinical and functional benefits related to improved ventilation of adjacent segments. CONCLUSION: This report of pulmonary cystic disorder related to a light chain deposition disease highlights the potential clinical and functional benefits observed after lung volume reduction surgery.
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Quistes/cirugía , Cadenas Ligeras de Inmunoglobulina/metabolismo , Enfermedades Pulmonares/cirugía , Gammopatía Monoclonal de Relevancia Indeterminada/cirugía , Agregación Patológica de Proteínas/patología , Quistes/metabolismo , Quistes/patología , Diagnóstico Diferencial , Femenino , Humanos , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/diagnóstico , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares/patología , Persona de Mediana Edad , Gammopatía Monoclonal de Relevancia Indeterminada/complicaciones , Gammopatía Monoclonal de Relevancia Indeterminada/diagnóstico , Gammopatía Monoclonal de Relevancia Indeterminada/patología , Neumonectomía , Agregación Patológica de Proteínas/complicaciones , Agregación Patológica de Proteínas/diagnóstico , Agregación Patológica de Proteínas/metabolismo , Resultado del TratamientoRESUMEN
Although the causative role of the accumulation of amyloid ß 1-42 (Aß42) deposits in the pathogenesis of Alzheimer's disease (AD) has been under debate for many years, it is supposed that the toxicity soluble oligomers of Tau protein (TauOs) might be also the pathogenic factor acting on the initial stages of this disease. Therefore, we performed a thorough search for literature pertaining to our investigation via the MEDLINE/PubMed database. It was shown that soluble TauOs, especially granular forms, may be the most toxic form of this protein. Hyperphosphorylated TauOs can reduce the number of synapses by missorting into axonal compartments of neurons other than axon. Furthermore, soluble TauOs may be also responsible for seeding Tau pathology within AD brains, with probable link to AßOs toxicity. Additionally, the concentrations of TauOs in the cerebrospinal fluid (CSF) and plasma of AD patients were higher than in non-demented controls, and revealed a negative correlation with mini-mental state examination (MMSE) scores. It was postulated that adding the measurements of TauOs to the panel of CSF biomarkers could improve the diagnosis of AD.
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Enfermedad de Alzheimer/metabolismo , Agregación Patológica de Proteínas/metabolismo , Pliegue de Proteína , Deficiencias en la Proteostasis/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/patología , Animales , Humanos , Agregación Patológica de Proteínas/diagnóstico , Agregación Patológica de Proteínas/patología , Deficiencias en la Proteostasis/diagnóstico , Deficiencias en la Proteostasis/patologíaRESUMEN
The variation of amyloid ß peptide (Aß) concentration and Aß aggregation are closely associated with the etiology of Alzheimer's diseases (AD). The interaction of Aß with the monosialoganglioside-rich neuronal cell membrane has been suggested to influence Aß aggregation. Therefore, studies on the mechanism of Aß and sialic acids (SA) interaction would greatly contribute to better understanding the pathogenesis of AD. Herein, we report a novel approach for Aß-SA interaction analysis and highly sensitive Aß detection by mimicing the cell surface presentation of SA clusters through engineering of SA-modified peptide nanofiber (SANF). The SANF displayed well-ordered 1D nanostructure with high density of SA on surface. Using FAM-labeled Aß fragments of Aß1-16, Aß16-23, and Aß24-40, the interaction between Aß and SA was evaluated by the fluorescence titration experiments. It was found that the order of the SA-binding affinity was Aß1-16 > Aß24-40 > Aß16-23. Importantly, the presence of full-length Aß1-40 monomer triggered a significant fluorescence enhancement due to the multivalent binding of Aß1-40 to the nanofiber. This fluorescent turn-on response showed high selectivity and sensitivity for Aß1-40 detection and the method was further used for Aß aggregation process monitoring and inhibitor screening. The results suggest the proposed strategy is promising to serve as a tool for mechanism study and the early diagnosis of Alzheimer's disease.
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Péptidos beta-Amiloides/análisis , Glicopéptidos/química , Ácido N-Acetilneuramínico/química , Nanofibras/química , Fragmentos de Péptidos/análisis , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/líquido cefalorraquídeo , Animales , Bovinos , Humanos , Fragmentos de Péptidos/líquido cefalorraquídeo , Agregado de Proteínas , Agregación Patológica de Proteínas/líquido cefalorraquídeo , Agregación Patológica de Proteínas/diagnóstico , Espectrometría de FluorescenciaRESUMEN
Systemic light chain amyloidosis (AL) is a life-threatening disease caused by aggregation and deposition of monoclonal immunoglobulin light chains (LC) in target organs. Severity of heart involvement is the most important factor determining prognosis. Here, we report the 4.0 Å resolution cryo-electron microscopy map and molecular model of amyloid fibrils extracted from the heart of an AL amyloidosis patient with severe amyloid cardiomyopathy. The helical fibrils are composed of a single protofilament, showing typical 4.9 Å stacking and cross-ß architecture. Two distinct polypeptide stretches (total of 77 residues) from the LC variable domain (Vl) fit the fibril density. Despite Vl high sequence variability, residues stabilizing the fibril core are conserved through different cardiotoxic Vl, highlighting structural motifs that may be common to misfolding-prone LCs. Our data shed light on the architecture of LC amyloids, correlate amino acid sequences with fibril assembly, providing the grounds for development of innovative medicines.
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Amiloide/ultraestructura , Cadenas Ligeras de Inmunoglobulina/ultraestructura , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/patología , Miocardio/ultraestructura , Agregación Patológica de Proteínas/patología , Anciano , Secuencia de Aminoácidos , Amiloide/inmunología , Amiloide/metabolismo , Autopsia , Microscopía por Crioelectrón , Humanos , Cadenas Ligeras de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/metabolismo , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/diagnóstico , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/inmunología , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/metabolismo , Masculino , Miocardio/inmunología , Miocardio/metabolismo , Miocardio/patología , Agregación Patológica de Proteínas/diagnóstico , Agregación Patológica de Proteínas/inmunología , Agregación Patológica de Proteínas/metabolismo , Conformación Proteica en Lámina beta , Pliegue de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Índice de Severidad de la EnfermedadRESUMEN
BACKGROUND: Congenital cataract is the leading cause of blindness in children worldwide. Approximately half of all congenital cataracts have a genetic basis. Protein aggregation is the single most important factor in cataract formation. METHODS: A four-generation Chinese family diagnosed with autosomal dominant congenital cataracts and microphthalmia was recruited at the Shengjing Hospital of China Medical University. Genomic DNA was extracted from the peripheral blood of the participants. All coding exons and flanking regions of seven candidate genes (CRYAA, CRYBA4, CRYBB2, CRYGC, GJA8, MAF, and PITX3) were amplified and sequenced. Restriction fragment length polymorphism (RFLP) assays were performed to confirm the candidate causative variant, c.35G > T in the CRYAA gene. We constructed pcDNA3.1(+)-CRYAA expression plasmids containing either the wild-type or the R12L mutant alleles and respectively transfected them into HEK293T cells and into HeLa cells. Western blotting was performed to determine protein expression levels and protein solubility. Immunofluorescence was performed to determine protein sub-cellular localization. RESULTS: A heterozygous variant c.35G > T was identified in exon 1 of CRYAA, which resulted in a substitution of arginine to leucine at codon 12 (p.R12L). The nucleotide substitution c.35G > T was co-segregated with the disease phenotype in the family. The mutant R12L-CRYAA in HEK293T cells showed a significant increase in the expression level of the CRYAA protein compared with the wild-type cells. Moreover, a large amount of the mutant protein aggregated in the precipitate where the wild-type protein was not detected. Immunofluorescence studies showed that the overexpressed mutant CRYAA in HeLa cells formed large cytoplasmic aggregates and aggresomes. CONCLUSIONS: In summary, we described a case of human congenital cataract and microphthalmia caused by a novel mutation in the CRYAA gene, which substituted an arginine at position 12 in the N-terminal region of αA-crystallin. The molecular mechanisms that underlie the pathogenesis of human congenital cataract may be characterized by the prominent effects of the p.R12L mutation on αA-crystallin aggregation and solubility. Our study also expands the spectrum of known CRYAA mutations.
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Catarata/genética , Trastornos de los Cromosomas/genética , Cristalinas/genética , Microftalmía/genética , Mutación Missense , Agregación Patológica de Proteínas/genética , Adulto , Pueblo Asiatico , Secuencia de Bases , Catarata/diagnóstico , Catarata/etnología , Catarata/patología , Niño , China , Trastornos de los Cromosomas/diagnóstico , Trastornos de los Cromosomas/etnología , Trastornos de los Cromosomas/patología , Exones , Femenino , Expresión Génica , Pruebas Genéticas , Células HEK293 , Células HeLa , Heterocigoto , Humanos , Masculino , Microftalmía/diagnóstico , Microftalmía/etnología , Microftalmía/patología , Linaje , Polimorfismo de Longitud del Fragmento de Restricción , Agregación Patológica de Proteínas/diagnóstico , Agregación Patológica de Proteínas/etnología , Agregación Patológica de Proteínas/patologíaRESUMEN
The accumulation of mutant aggregate-prone proteins is a hallmark of the majority of neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases. Autophagy, a cytosolic bulk degradation system, is the major clearance pathway for several aggregate-prone proteins, such as mutant huntingtin. The autophagosome-associated protein LC3-II is a specific marker of autophagic flux within cells, whereas aggregate formation of mutant huntingtin represents a good readout for studying autophagy modulation. Here we describe the method of assessing autophagic flux using LC3-II western blotting and substrate clearance by expressing the N-terminal fragment of huntingtin (htt exon 1) containing an expanded polyglutamine tract in mammalian cells.
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Autofagia/genética , Western Blotting/métodos , Proteína Huntingtina/metabolismo , Proteínas Asociadas a Microtúbulos/análisis , Enfermedades Neurodegenerativas/diagnóstico , Agregación Patológica de Proteínas/diagnóstico , Western Blotting/instrumentación , Exones/genética , Células HeLa , Humanos , Proteína Huntingtina/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patología , ProteolisisRESUMEN
N-terminal mutant huntingtin (mHTT) fragments with pathogenic polyglutamine (polyQ) tracts spontaneously form stable, amyloidogenic protein aggregates with a fibrillar morphology. Such structures are detectable in brains of Huntington's disease (HD) patients and various model organisms, suggesting that they play a critical role in pathogenesis. Heat-stable, fibrillar mHTT aggregates can be detected and quantified in cells and tissues using a denaturing filter retardation assay (FRA). Here, we describe step-by-step protocols and experimental procedures for the investigation of mHTT aggregates in complex biosamples using FRAs. The methods are illustrated with examples from studies in cellular, transgenic fly, and mouse models of HD, but can be adapted for any disease-relevant protein with amyloidogenic polyQ tracts.
Asunto(s)
Encéfalo/patología , Proteína Huntingtina/análisis , Enfermedad de Huntington/diagnóstico , Immunoblotting/métodos , Agregación Patológica de Proteínas/diagnóstico , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Drosophila melanogaster , Calor , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Immunoblotting/instrumentación , Ratones , Mutación , Agregado de Proteínas , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patologíaRESUMEN
Dominant mutations in the gene encoding copper and zinc-binding superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS). Abnormal accumulation of misfolded SOD1 proteins in spinal motoneurons is a major pathological hallmark in SOD1-related ALS. Dissociation of copper and/or zinc ions from SOD1 has been shown to trigger the protein aggregation/oligomerization in vitro, but the pathological contribution of such metal dissociation to the SOD1 misfolding still remains obscure. Here, we tested the relevance of the metal-deficient SOD1 in the misfolding in vivo by developing a novel antibody (anti-apoSOD), which exclusively recognized mutant SOD1 deficient in metal ions at its copper-binding site. Notably, anti-apoSOD-reactive species were detected specifically in the spinal cords of the ALS model mice only at their early pre-symptomatic stages but not at the end stage of the disease. The cerebrospinal fluid as well as the spinal cord homogenate of one SOD1-ALS patient also contained the anti-apoSOD-reactive species. Our results thus suggest that metal-deficiency in mutant SOD1 at its copper-binding site is one of the earliest pathological features in SOD1-ALS.
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Esclerosis Amiotrófica Lateral/diagnóstico , Cobre/metabolismo , Agregación Patológica de Proteínas/diagnóstico , Superóxido Dismutasa-1/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/inmunología , Esclerosis Amiotrófica Lateral/patología , Animales , Anticuerpos/inmunología , Enfermedades Asintomáticas , Sitios de Unión/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Neuronas Motoras/patología , Mutación , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/inmunología , Agregación Patológica de Proteínas/patología , Unión Proteica/genética , Pliegue de Proteína , Sensibilidad y Especificidad , Médula Espinal/citología , Médula Espinal/patología , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/inmunología , Zinc/metabolismoRESUMEN
Multiple sclerosis (MS) is a disease which manifests demyelination of neuronal cells in the brain. Despite extensive research on the mechanisms of disease development and progression, the exact mechanism is not elucidated yet, which has hampered drug development and subsequent treatment of the disease. We have recently shown that the serum levels of arsenic and malondialdehyde, a lipid peroxidation marker, are high in MS patients. In this article, we would like to formulate the hypothesis that arsenic may cause MS by induction of inflammation, degeneration, and apoptosis in neuronal cells. The induction of ROS generation in cells upon exposure to arsenic as a heavy metal may be involved in the pathogenesis of MS. Tau protein, a member of the family of microtubule-associated proteins, is mainly expressed in neurons and contribute to the assembly of neuronal microtubules network. Arsenic may affect the hyperphosphorylation and aggregation of tau proteins and may be involved in the cascade leading to deregulation of tau function associated with neurodegeneration. For validation of this hypothesis, studies might be conducted to evaluate the association of arsenic levels and tau protein levels in MS patients. Further studies might also focus on the trafficking along microtubules in neurons of MS patient with regard to hyperphosphorylation of tau protein. This hypothesis may add a new dimension to the understanding of MS etiology and help to design novel therapeutic agents against potential targets that might be discovered. If this hypothesis proves to be true, tau phosphorylation inhibitors can be potential candidates for MS drug development.
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Arsénico/toxicidad , Inflamación/diagnóstico , Esclerosis Múltiple/diagnóstico , Agregación Patológica de Proteínas/diagnóstico , Proteínas tau/metabolismo , Animales , Apoptosis , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Línea Celular , Progresión de la Enfermedad , Humanos , Inflamación/inducido químicamente , Peroxidación de Lípido , Malondialdehído/sangre , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Esclerosis Múltiple/inducido químicamente , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosforilación , Agregación Patológica de Proteínas/inducido químicamente , Proteínas tau/genéticaRESUMEN
An increasing amount of findings suggests that the aggregation of soluble peptides and proteins into amyloid fibrils is a relevant upstream process in the complex cascade of events leading to the pathology of Alzheimer's disease and several other neurodegenerative disorders. Nevertheless, several aspects of the correlation between the aggregation process and the onset and development of the pathology remain largely elusive. In this context, biophysical and biochemical studies in test tubes have proven extremely powerful in providing quantitative information about the structure and the reactivity of amyloids at the molecular level. In this review we use selected recent examples to illustrate the importance of such biophysical research to complement phenomenological studies based on cellular and molecular biology, and we discuss the implications for pharmaceutical applications associated with Alzheimer's disease and other neurodegenerative disorders in both academic and industrial contexts.
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Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Amiloide/metabolismo , Descubrimiento de Drogas , Enfermedad de Alzheimer/diagnóstico , Animales , Descubrimiento de Drogas/métodos , Humanos , Agregación Patológica de Proteínas/diagnóstico , Agregación Patológica de Proteínas/tratamiento farmacológico , Agregación Patológica de Proteínas/metabolismo , Pliegue de ProteínaRESUMEN
A boronic acid-based fluorescent probe was developed for diagnosis of amyloid-ß (Aß) plaques from Alzheimer's disease (AD). Probe 4c, which included boronic acid as a functional group, exhibited a significant increase (64.37-fold, FAß/F0) in fluorescence intensity as a response to Aß aggregates, with a blue shift (105nm) in the maximum emission wavelength. We found that boronic acid as a functional group improved the binding affinity (KD value=0.79±0.05µM for 4c) for Aß aggregates and confirmed that 4c selectively stained Aß plaques in brain sections from APP/PS1 mice. Ex vivo fluorescence imaging using mice (normal and APP/PS1) also revealed that 4c was able to penetrate the blood-brain barrier (BBB) and to stain Aß plaques in the brain. From these results, we believe that 4c will be useful as a fluorescent probe in preclinical research related to AD. Furthermore, we believe that our results with boronic acid also provide valuable information for the development of a probe for Aß plaques.
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Enfermedad de Alzheimer/diagnóstico , Precursor de Proteína beta-Amiloide/análisis , Ácidos Borónicos/química , Encéfalo/patología , Colorantes Fluorescentes/química , Placa Amiloide/diagnóstico , Enfermedad de Alzheimer/patología , Animales , Ácidos Borónicos/farmacocinética , Colorantes Fluorescentes/farmacocinética , Ratones , Ratones Transgénicos , Imagen Óptica/métodos , Placa Amiloide/patología , Agregación Patológica de Proteínas/diagnóstico , Agregación Patológica de Proteínas/patologíaRESUMEN
The intracellular and extracellular accumulation of disordered proteins and aggregated proteins occurs in many protein conformational diseases, such as aging-related neurodegeneration and alcoholic liver diseases. However, the conventional methods to study protein structural changes are limited for the rapid detection and monitoring of protein aggregation because of long incubation times (i.e., usually several days), complicated sample pretreatment steps, and expensive instrumentation. Here, we describe an ultrafast colorimetric method for the real-time monitoring of protein structure evolution and the determination of predominant structures via nanoparticle-assisted protein aggregation. During the aggregation process, nanoparticles act as nucleation cores, which form networks depending on the structures of the protein aggregates, and accelerate the kinetics of the protein aggregation. Simultaneously, these nanoparticles exhibit colorimetric responses according to their embedded shapes (e.g., fibrillar and amorphous) on the protein aggregates. We observed distinct spectral shifts and concomitant colorimetric responses of concentration- and type-dependent protein aggregation with the naked eye within a few minutes (<2 min) under acidic conditions. Moreover, the morphological transitions from small aggregates to larger aggregates of nanoparticle-assisted protein aggregates were visualized with dark-field microscope imaging, which show a similar trend with that of protein aggregates formed without the aid of nanoparticles. Finally we show that our proposed method can be utilized to screen the protein aggregation propensity under a variety of conditions such as different pH levels, high temperature, and chemicals. These findings suggest that the proposed method is an easy way to study the molecular biophysics of protein aggregation and to rapidly screen anti-aggregation drugs for protein conformational diseases.