RESUMO
Protein post-translational modifications (PTMs) play a crucial role in countless biological processes, profoundly modulating protein properties on both spatial and temporal scales. Protein PTMs have also emerged as reliable biomarkers for several diseases. However, only a handful of techniques are available to accurately measure their levels, capture their complexity at a single molecule level, and characterize their multifaceted roles in health and disease. Nanopore sensing provides high sensitivity for the detection of low-abundance proteins, holding the potential to impact single-molecule proteomics and PTM detection, in particular. Here, we demonstrate the ability of a biological nanopore, the pore-forming toxin aerolysin, to detect and distinguish α-synuclein-derived peptides bearing single or multiple PTMs, namely, phosphorylation, nitration, and oxidation occurring at different positions and in various combinations. The characteristic current signatures of the α-synuclein peptide and its PTM variants could be confidently identified by using a deep learning model for signal processing. We further demonstrate that this framework can quantify α-synuclein peptides at picomolar concentrations and detect the C-terminal peptides generated by digestion of full-length α-synuclein. Collectively, our work highlights the advantage of using nanopores as a tool for simultaneous detection of multiple PTMs and facilitates their use in biomarker discovery and diagnostics.
Assuntos
Aprendizado Profundo , Nanoporos , alfa-Sinucleína/química , Processamento de Proteína Pós-Traducional , Peptídeos/químicaRESUMO
AIMS: Synaptic dysfunction in Parkinson's disease is caused by propagation of pathogenic α-synuclein between neurons. Previously, in multiple system atrophy (MSA), pathologically characterised by ectopic deposition of abnormal α-synuclein predominantly in oligodendrocytes, we demonstrated that the occurrence of memory impairment was associated with the number of α-synuclein-positive neuronal cytoplasmic inclusions (NCIs) in the hippocampus. In the present study, we aimed to investigate how abnormal α-synuclein in the hippocampus can lead to memory impairment. METHODS: We performed pathological and biochemical analyses using a mouse model of adult-onset MSA and human cases (MSA, N = 25; Parkinson's disease, N = 3; Alzheimer's disease, N = 2; normal controls, N = 11). In addition, the MSA model mice were examined behaviourally and physiologically. RESULTS: In the MSA model, inducible human α-synuclein was first expressed in oligodendrocytes and subsequently accumulated in the cytoplasm of excitatory hippocampal neurons (NCI-like structures) and their presynaptic nerve terminals with the development of memory impairment. α-Synuclein oligomers increased simultaneously in the hippocampus of the MSA model. Hippocampal dendritic spines also decreased in number, followed by suppression of long-term potentiation. Consistent with these findings obtained in the MSA model, post-mortem analysis of human MSA brain tissues showed that cases of MSA with memory impairment developed more NCIs in excitatory hippocampal neurons along with α-synuclein oligomers than those without. CONCLUSIONS: Our results provide new insights into the role of α-synuclein oligomers as a possible pathological cause of memory impairment in MSA.
Assuntos
Atrofia de Múltiplos Sistemas , Doença de Parkinson , Humanos , Atrofia de Múltiplos Sistemas/patologia , alfa-Sinucleína/metabolismo , Doença de Parkinson/patologia , Corpos de Inclusão/patologia , Neurônios/patologia , Encéfalo/patologiaRESUMO
Despite the strong evidence linking the aggregation of the Huntingtin protein (Htt) to the pathogenesis of Huntington's disease (HD), the mechanisms underlying Htt aggregation and neurodegeneration remain poorly understood. Herein, we investigated the ultrastructural properties and protein composition of Htt cytoplasmic and nuclear inclusions in mammalian cells and primary neurons overexpressing mutant exon1 of the Htt protein. Our findings provide unique insight into the ultrastructural properties of cytoplasmic and nuclear Htt inclusions and their mechanisms of formation. We show that Htt inclusion formation and maturation are complex processes that, although initially driven by polyQ-dependent Htt aggregation, also involve the polyQ and PRD domain-dependent sequestration of lipids and cytoplasmic and cytoskeletal proteins related to HD dysregulated pathways; the recruitment and accumulation of remodeled or dysfunctional membranous organelles, and the impairment of the protein quality control and degradation machinery. We also show that nuclear and cytoplasmic Htt inclusions exhibit distinct biochemical compositions and ultrastructural properties, suggesting different mechanisms of aggregation and toxicity.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteína Huntingtina/química , Proteína Huntingtina/metabolismo , Neurônios/metabolismo , Animais , Células HEK293 , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/ultraestrutura , Doença de Huntington/metabolismo , Corpos de Inclusão Intranuclear/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/química , Agregação Patológica de Proteínas , ProteomaRESUMO
Huntington's disease is a neurodegenerative disorder caused by the expansion of a polyglutamine repeat (>36Q) in the N-terminal domain of the huntingtin protein (Htt), which renders the protein or fragments thereof more prone to aggregate and form inclusions. Although several Htt N-terminal fragments of different lengths have been identified within Htt inclusions, most studies on the mechanisms, sequence, and structural determinants of Htt aggregation have focused on the Httexon1 (Httex1). Herein, we investigated the aggregation properties of mutant N-terminal Htt fragments of various lengths (Htt171, Htt140, and Htt104) in comparison to mutant Httex1 (mHttex1). We also present a new chemoenzymatic semisynthetic strategy that enables site-specific phosphorylation of Htt beyond Httex1. These advances yielded insights into how post-translational modifications (PTMs) and structured domains beyond Httex1 influence aggregation mechanisms, kinetics, and fibril morphology of longer N-terminal Htt fragments. We demonstrate that phosphorylation at T107 significantly slows the aggregation of mHtt171, whereas phosphorylation at T107 and S116 accelerates the aggregation, underscoring the importance of crosstalk between different PTMs. The mHtt171 proteins aggregate via a different mechanism and form oligomers and fibrillar aggregates with morphological properties that are distinct from that of mHttex1. These observations suggest that different N-terminal fragments could have distinct aggregation mechanisms and that a single polyQ-targeting antiaggregation strategy may not effectively inhibit the aggregation of all N-terminal Htt fragments. Finally, our results underscore the need for further studies to investigate the aggregation mechanisms of Htt fragments and how the various fragments interact with each other and influence Htt toxicity and disease progression.
Assuntos
Proteína Huntingtina/síntese química , Peptídeos/química , Éxons , Humanos , Doença de Huntington/metabolismo , Cinética , Fosforilação , Agregados Proteicos , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
Alteration to endoplasmic reticulum (ER) proteostasis is observed in a variety of neurodegenerative diseases associated with abnormal protein aggregation. Activation of the unfolded protein response (UPR) enables an adaptive reaction to recover ER proteostasis and cell function. The UPR is initiated by specialized stress sensors that engage gene expression programs through the concerted action of the transcription factors ATF4, ATF6f, and XBP1s. Although UPR signaling is generally studied as unique linear signaling branches, correlative evidence suggests that ATF6f and XBP1s may physically interact to regulate a subset of UPR target genes. In this study, we designed an ATF6f/XBP1s fusion protein termed UPRplus that behaves as a heterodimer in terms of its selective transcriptional activity. Cell-based studies demonstrated that UPRplus has a stronger effect in reducing the abnormal aggregation of mutant huntingtin and α-synuclein when compared to XBP1s or ATF6 alone. We developed a gene transfer approach to deliver UPRplus into the brain using adeno-associated viruses (AAVs) and demonstrated potent neuroprotection in vivo in preclinical models of Parkinson's disease and Huntington's disease. These results support the concept in which directing UPR-mediated gene expression toward specific adaptive programs may serve as a possible strategy to optimize the beneficial effects of the pathway in different disease conditions.
Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Doenças Neurodegenerativas/prevenção & controle , Resposta a Proteínas não Dobradas , Proteína 1 de Ligação a X-Box/metabolismo , Fator 6 Ativador da Transcrição/genética , Animais , Modelos Animais de Doenças , Células HEK293 , Humanos , Proteína Huntingtina/genética , Masculino , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Proteína 1 de Ligação a X-Box/genética , alfa-Sinucleína/genéticaRESUMO
Amyloid diseases are global epidemics with profound health, social and economic implications and yet remain without a cure. This dire situation calls for research into the origin and pathological manifestations of amyloidosis to stimulate continued development of new therapeutics. In basic science and engineering, the cross-ß architecture has been a constant thread underlying the structural characteristics of pathological and functional amyloids, and realizing that amyloid structures can be both pathological and functional in nature has fuelled innovations in artificial amyloids, whose use today ranges from water purification to 3D printing. At the conclusion of a half century since Eanes and Glenner's seminal study of amyloids in humans, this review commemorates the occasion by documenting the major milestones in amyloid research to date, from the perspectives of structural biology, biophysics, medicine, microbiology, engineering and nanotechnology. We also discuss new challenges and opportunities to drive this interdisciplinary field moving forward.
Assuntos
Doença de Alzheimer/metabolismo , Amiloide/química , Amiloide/metabolismo , Amiloidose , Cátions Bivalentes/química , Reagentes de Ligações Cruzadas/química , Humanos , Modelos Moleculares , Conformação Molecular , Impressão Tridimensional , Dobramento de Proteína , Processamento de Proteína Pós-TraducionalRESUMO
Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicities and pathology-spreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the development of 11-mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) that efficiently label the edges of synthetic, recombinant, and native amyloid fibrils derived from different amyloidogenic proteins. We demonstrate that these NPs represent powerful tools for assessing amyloid morphological polymorphism, using cryogenic transmission electron microscopy (cryo-EM). The NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including transmission electron microscopy with use of the negative stain or cryo-EM imaging. The use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of a patient with Alzheimer's disease, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain amyloidosis, revealed a high degree of homogeneity across the fibrils derived from human tissue in comparison with fibrils aggregated in vitro. These findings are consistent with, and strongly support, the emerging view that the physiologic milieu is a key determinant of amyloid fibril strains. Together, these advances should not only facilitate the profiling and characterization of amyloids for structural studies by cryo-EM, but also pave the way to elucidate the structural basis of amyloid strains and toxicity, and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.
Assuntos
Amiloide/genética , Amiloide/metabolismo , Encéfalo/metabolismo , Microscopia Crioeletrônica/métodos , Ouro/química , Nanopartículas Metálicas/química , Polimorfismo Genético , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amiloide/química , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Amiloidose de Cadeia Leve de Imunoglobulina/metabolismo , Amiloidose de Cadeia Leve de Imunoglobulina/patologia , Emaranhados NeurofibrilaresRESUMO
A multitude of biological processes are enabled by complex interactions between lipid membranes and proteins. To understand such dynamic processes, it is crucial to differentiate the constituent biomolecular species and track their individual time evolution without invasive labels. Here, we present a label-free mid-infrared biosensor capable of distinguishing multiple analytes in heterogeneous biological samples with high sensitivity. Our technology leverages a multi-resonant metasurface to simultaneously enhance the different vibrational fingerprints of multiple biomolecules. By providing up to 1000-fold near-field intensity enhancement over both amide and methylene bands, our sensor resolves the interactions of lipid membranes with different polypeptides in real time. Significantly, we demonstrate that our label-free chemically specific sensor can analyze peptide-induced neurotransmitter cargo release from synaptic vesicle mimics. Our sensor opens up exciting possibilities for gaining new insights into biological processes such as signaling or transport in basic research as well as provides a valuable toolkit for bioanalytical and pharmaceutical applications.
Assuntos
Técnicas Biossensoriais/métodos , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Espectroscopia de Infravermelho com Transformada de Fourier , Dicroísmo Circular , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Reprodutibilidade dos TestesRESUMO
The formation and spreading of amyloid aggregates from the presynaptic protein α-synuclein in the brain play central roles in the pathogenesis of Parkinson's disease. Here, we use high-resolution atomic force microscopy to investigate the early oligomerization events of α-synuclein with single monomer angstrom resolution. We identify, visualize, and characterize directly the smallest elementary unit in the hierarchical assembly of amyloid fibrils, termed here single-strand protofilaments. We show that protofilaments form from the direct molecular assembly of unfolded monomeric α-synuclein polypeptide chains. To unravel protofilaments' internal structure and elastic properties, we manipulated nanomechanically these species by atomic force spectroscopy. The single-molecule scale identification and characterization of the fundamental unit of amyloid assemblies provide insights into early events underlying their formation and shed light on opportunities for therapeutic intervention at the early stages of aberrant protein self-assembly.
Assuntos
Amiloide/química , Desdobramento de Proteína , alfa-Sinucleína/química , Amiloide/metabolismo , Amiloide/ultraestrutura , Humanos , Microscopia de Força Atômica , Doença de Parkinson/metabolismo , Doença de Parkinson/terapia , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , alfa-Sinucleína/metabolismoRESUMO
Huntington's disease is caused by expansion of a polyglutamine (polyQ) domain within exon 1 of the huntingtin gene (Httex1). The prevailing hypothesis is that the monomeric Httex1 protein undergoes sharp conformational changes as the polyQ length exceeds a threshold of 36-37 residues. Here, we test this hypothesis by combining novel semi-synthesis strategies with state-of-the-art single-molecule Förster resonance energy transfer measurements on biologically relevant, monomeric Httex1 proteins of five different polyQ lengths. Our results, integrated with atomistic simulations, negate the hypothesis of a sharp, polyQ length-dependent change in the structure of monomeric Httex1. Instead, they support a continuous global compaction with increasing polyQ length that derives from increased prominence of the globular polyQ domain. Importantly, we show that monomeric Httex1 adopts tadpole-like architectures for polyQ lengths below and above the pathological threshold. Our results suggest that higher order homotypic and/or heterotypic interactions within distinct sub-populations of neurons, which are inevitable at finite cellular concentrations, are likely to be the main source of sharp polyQ length dependencies of HD.
Assuntos
Éxons/genética , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Doença de Huntington/genética , Prolina/metabolismo , Reprodutibilidade dos TestesRESUMO
Cells are extremely complex systems able to actively modify their metabolism and behavior in response to environmental conditions and stimuli such as pathogenic agents or drugs. The comprehension of these responses is central to understand the molecular bases of human pathologies, including amyloid misfolding diseases. Conventional bulk biological assays are limited by intrinsic cellular heterogeneity in gene, protein and metabolite expression, and can investigate only indirectly cellular reactions in non-physiological conditions. Here we employ a label-free nanomotion sensor to study single neuroblastoma cells exposed to extracellular monomeric and amyloid α-synuclein species in real-time and in physiological conditions. Combining this technique with fluorescence microscopy, we demonstrate multispecies cooperative cytotoxic effect of amyloids and aggregate-induced loss of cellular membrane integrity. Notably, the method can study cellular reactions and cytotoxicity an order of magnitude faster, and using 100-fold smaller volume of reagents when compared to conventional bulk analyses. This rapidity and sensitivity will allow testing novel pharmacological approaches to stop or delay a wide range of human diseases.
RESUMO
Conformational changes in disease-associated or mutant proteins represent a key pathological aspect of Huntington's disease (HD) and other protein misfolding diseases. Using immunoassays and biophysical approaches, we and others have recently reported that polyglutamine expansion in purified or recombinantly expressed huntingtin (HTT) proteins affects their conformational properties in a manner dependent on both polyglutamine repeat length and temperature but independent of HTT protein fragment length. These findings are consistent with the HD mutation affecting structural aspects of the amino-terminal region of the protein, and support the concept that modulating mutant HTT conformation might provide novel therapeutic and diagnostic opportunities. We now report that the same conformational TR-FRET based immunoassay detects polyglutamine- and temperature-dependent changes on the endogenously expressed HTT protein in peripheral tissues and post-mortem HD brain tissue, as well as in tissues from HD animal models. We also find that these temperature- and polyglutamine-dependent conformational changes are sensitive to bona-fide phosphorylation on S13 and S16 within the N17 domain of HTT. These findings provide key clinical and preclinical relevance to the conformational immunoassay, and provide supportive evidence for its application in the development of therapeutics aimed at correcting the conformation of polyglutamine-expanded proteins as well as the pharmacodynamics readouts to monitor their efficacy in preclinical models and in HD patients.
Assuntos
Proteína Huntingtina/química , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Peptídeos/metabolismo , Expansão das Repetições de Trinucleotídeos , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Drosophila/metabolismo , Éxons/genética , Fibroblastos/metabolismo , Células HEK293 , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosforilação , Fosfosserina/metabolismo , Conformação ProteicaRESUMO
The first exon of the Huntingtin protein (Httex1) is one of the most actively studied Htt fragments because its overexpression in R6/2 transgenic mice has been shown to recapitulate several key features of Huntington disease. However, the majority of biophysical studies of Httex1 are based on assessing the structure and aggregation of fusion constructs where Httex1 is fused to large proteins, such as glutathione S-transferase, maltose-binding protein, or thioredoxin, or released in solution upon in situ cleavage of these proteins. Herein, we report an intein-based strategy that allows, for the first time, the rapid and efficient production of native tag-free Httex1 with polyQ repeats ranging from 7Q to 49Q. Aggregation studies on these proteins enabled us to identify interesting polyQ-length-dependent effects on Httex1 oligomer and fibril formation that were previously not observed using Httex1 fusion proteins or Httex1 proteins produced by in situ cleavage of fusion proteins. Our studies revealed the inability of Httex1-7Q/15Q to undergo amyloid fibril formation and an inverse correlation between fibril length and polyQ repeat length, suggesting possible polyQ length-dependent differences in the structural properties of the Httex1 aggregates. Altogether, our findings underscore the importance of working with tag-free Httex1 proteins and indicate that model systems based on non-native Httex1 sequences may not accurately reproduce the effect of polyQ repeat length and solution conditions on Httex1 aggregation kinetics and structural properties.
Assuntos
Amiloide/química , Doença de Huntington/metabolismo , Inteínas , Proteínas do Tecido Nervoso/química , Peptídeos/metabolismo , Motivos de Aminoácidos , Amiloide/genética , Amiloide/metabolismo , Animais , Éxons , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Cinética , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Agregados ProteicosRESUMO
New technologies launch novel materials; besides their performances in products, their health hazards must be tested. This applies to the lead halide perovskite CH3NH3PbI3 as well, which offers fulgurate applications in photovoltaic devices. We report the effects of CH3NH3PbI3 photovoltaic perovskites in human lung adenocarcinoma epithelial cells (A549), human dopaminergic neuroblastoma cells (SH-SY5Y) and murine primary hippocampal neurons by using multiple assays and electron microscopy studies. In cell culture media the major part of the dissolved CH3NH3PbI3 has a strong cell-type dependent effect. Hippocampal primary neurons and neuroblastoma cells suffer a massive apoptotic cell death, whereas exposure to lung epithelial cells dramatically alters the kinetics of proliferation, metabolic activity and cellular morphology without inducing noticeable cell death. Our findings underscore the critical importance of conducting further studies to investigate the effect of short and long-term exposure to CH3NH3PbI3 on health and environment.
RESUMO
Converging lines of evidence indicate that near-infrared light treatment, also known as photobiomodulation (PBM), may exert beneficial effects and protect against cellular toxicity and degeneration in several animal models of human pathologies, including neurodegenerative disorders. In the present study, we report that chronic PMB treatment mitigates dopaminergic loss induced by unilateral overexpression of human α-synuclein (α-syn) in the substantia nigra of an AAV-based rat genetic model of Parkinson's disease (PD). In this model, daily exposure of both sides of the rat's head to 808-nm near-infrared light for 28 consecutive days alleviated α-syn-induced motor impairment, as assessed using the cylinder test. This treatment also significantly reduced dopaminergic neuronal loss in the injected substantia nigra and preserved dopaminergic fibers in the ipsilateral striatum. These beneficial effects were sustained for at least 6 weeks after discontinuing the treatment. Together, our data point to PBM as a possible therapeutic strategy for the treatment of PD and other related synucleinopathies.
Assuntos
Neurônios Dopaminérgicos/efeitos da radiação , Terapia com Luz de Baixa Intensidade , Doença de Parkinson/radioterapia , Substância Negra/efeitos da radiação , Animais , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Corpo Estriado/efeitos da radiação , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Feminino , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Ratos , Ratos Sprague-Dawley , Substância Negra/metabolismo , Substância Negra/patologiaRESUMO
A novel mutation in the α-Synuclein (α-Syn) gene "G51D" was recently identified in two familial cases exhibiting features of Parkinson's disease (PD) and multiple system atrophy (MSA). In this study, we explored the impact of this novel mutation on the aggregation, cellular and biophysical properties of α-Syn, in an attempt to unravel how this mutant contributes to PD/MSA. Our results show that the G51D mutation significantly attenuates α-Syn aggregation in vitro. Moreover, it disrupts local helix formation in the presence of SDS, decreases binding to lipid vesicles C-terminal to the site of mutation and severely inhibits helical folding in the presence of acidic vesicles. When expressed in yeast, α-Syn(G51D) behaves similarly to α-Syn(A30P), as both exhibit impaired membrane association, form few inclusions and are non-toxic. In contrast, enhanced secreted and nuclear levels of the G51D mutant were observed in mammalian cells, as well as in primary neurons, where α-Syn(G51D) was enriched in the nuclear compartment, was hyper-phosphorylated at S129 and exacerbated α-Syn-induced mitochondrial fragmentation. Finally, post-mortem human brain tissues of α-Syn(G51D) cases were examined, and revealed only partial colocalization with nuclear membrane markers, probably due to post-mortem tissue delay and fixation. These findings suggest that the PD-linked mutations may cause neurodegeneration via different mechanisms, some of which may be independent of α-Syn aggregation.
Assuntos
Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Mutação/genética , Doença de Parkinson/genética , Agregação Patológica de Proteínas/genética , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Soluções Tampão , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Corpos de Inclusão/efeitos dos fármacos , Corpos de Inclusão/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neuroblastoma/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Membrana Nuclear/efeitos dos fármacos , Membrana Nuclear/metabolismo , Doença de Parkinson/patologia , Fosforilação/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Transporte Proteico/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Dodecilsulfato de Sódio/farmacologia , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Lipossomas Unilamelares/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/ultraestruturaRESUMO
Increasing evidence suggests that the c-Abl protein tyrosine kinase could play a role in the pathogenesis of Parkinson's disease (PD) and other neurodegenerative disorders. c-Abl has been shown to regulate the degradation of two proteins implicated in the pathogenesis of PD, parkin and α-synuclein (α-syn). The inhibition of parkin's neuroprotective functions is regulated by c-Abl-mediated phosphorylation of parkin. However, the molecular mechanisms by which c-Abl activity regulates α-syn toxicity and clearance remain unknown. Herein, using NMR spectroscopy, mass spectrometry, in vitro enzymatic assays and cell-based studies, we established that α-syn is a bona fide substrate for c-Abl. In vitro studies demonstrate that c-Abl directly interacts with α-syn and catalyzes its phosphorylation mainly at tyrosine 39 (pY39) and to a lesser extent at tyrosine 125 (pY125). Analysis of human brain tissues showed that pY39 α-syn is detected in the brains of healthy individuals and those with PD. However, only c-Abl protein levels were found to be upregulated in PD brains. Interestingly, nilotinib, a specific inhibitor of c-Abl kinase activity, induces α-syn protein degradation via the autophagy and proteasome pathways, whereas the overexpression of α-syn in the rat midbrains enhances c-Abl expression. Together, these data suggest that changes in c-Abl expression, activation and/or c-Abl-mediated phosphorylation of Y39 play a role in regulating α-syn clearance and contribute to the pathogenesis of PD.
Assuntos
Doença de Parkinson/enzimologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/enzimologia , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fosforilação , Proteólise , Proteínas Proto-Oncogênicas c-abl/genética , alfa-Sinucleína/genéticaRESUMO
An increase in α-synuclein levels due to gene duplications/triplications or impaired degradation is sufficient to trigger its aggregation and cause familial Parkinson disease (PD). Therefore, lowering α-synuclein levels represents a viable therapeutic strategy for the treatment of PD and related synucleinopathies. Here, we report that Polo-like kinase 2 (PLK2), an enzyme up-regulated in synucleinopathy-diseased brains, interacts with, phosphorylates and enhances α-synuclein autophagic degradation in a kinase activity-dependent manner. PLK2-mediated degradation of α-synuclein requires both phosphorylation at S129 and PLK2/α-synuclein complex formation. In a rat genetic model of PD, PLK2 overexpression reduces intraneuronal human α-synuclein accumulation, suppresses dopaminergic neurodegeneration, and reverses hemiparkinsonian motor impairments induced by α-synuclein overexpression. This PLK2-mediated neuroprotective effect is also dependent on PLK2 activity and α-synuclein phosphorylation. Collectively, our findings demonstrate that PLK2 is a previously undescribed regulator of α-synuclein turnover and that modulating its kinase activity could be a viable target for the treatment of synucleinopathies.
Assuntos
Regulação da Expressão Gênica/fisiologia , Doença de Parkinson/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , alfa-Sinucleína/metabolismo , Análise de Variância , Animais , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Humanos , Imuno-Histoquímica , Imunoprecipitação , Fosforilação , RatosRESUMO
Increasing evidence implicates Aß peptides self-assembly and fibril formation as crucial events in the pathogenesis of Alzheimer disease. Thus, inhibiting Aß aggregation, among others, has emerged as a potential therapeutic intervention for this disorder. Herein, we employed 3-aminopyrazole as a key fragment in our design of non-dye compounds capable of interacting with Aß42 via a donor-acceptor-donor hydrogen bond pattern complementary to that of the ß-sheet conformation of Aß42. The initial design of the compounds was based on connecting two 3-aminopyrazole moieties via a linker to identify suitable scaffold molecules. Additional aryl substitutions on the two 3-aminopyrazole moieties were also explored to enhance π-π stacking/hydrophobic interactions with amino acids of Aß42. The efficacy of these compounds on inhibiting Aß fibril formation and toxicity in vitro was assessed using a combination of biophysical techniques and viability assays. Using structure activity relationship data from the in vitro assays, we identified compounds capable of preventing pathological self-assembly of Aß42 leading to decreased cell toxicity.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Pirazóis/química , Linhagem Celular Tumoral , Citotoxinas/antagonistas & inibidores , Citotoxinas/química , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
A polyubiquitin chain anchored to the substrate has been the hallmark of proteasomal recognition. However, the degradation signal appears to be more complex and to contain also a substrate's unstructured region. Recent reports have shown that the proteasome can degrade also monoubiquitylated proteins, which adds an additional layer of complexity to the signal. Here, we demonstrate that the size of the substrate is an important determinant in its extent of ubiquitylation: a single ubiquitin moiety fused to a tail of up to â¼150 residues derived from either short artificial repeats or from naturally occurring proteins, is sufficient to target them for proteasomal degradation. Importantly, chemically synthesized adducts, where ubiquitin is attached to the substrate via a naturally occurring isopeptide bond, display similar characteristics. Taken together, these findings suggest that the ubiquitin proteasomal signal is adaptive, and is not always made of a long polyubiquitin chain.