RESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease affecting motor neurons and characterized by microglia-mediated neurotoxic inflammation whose underlying mechanisms remain incompletely understood. In this work, we reveal that MAPK/MAK/MRK overlapping kinase (MOK), with an unknown physiological substrate, displays an immune function by controlling inflammatory and type-I interferon (IFN) responses in microglia which are detrimental to primary motor neurons. Moreover, we uncover the epigenetic reader bromodomain-containing protein 4 (Brd4) as an effector protein regulated by MOK, by promoting Ser492-phospho-Brd4 levels. We further demonstrate that MOK regulates Brd4 functions by supporting its binding to cytokine gene promoters, therefore enabling innate immune responses. Remarkably, we show that MOK levels are increased in the ALS spinal cord, particularly in microglial cells, and that administration of a chemical MOK inhibitor to ALS model mice can modulate Ser492-phospho-Brd4 levels, suppress microglial activation, and modify the disease course, indicating a pathophysiological role of MOK kinase in ALS and neuroinflammation.
Assuntos
Esclerose Lateral Amiotrófica , Proteínas que Contêm Bromodomínio , Proteínas Quinases Ativadas por Mitógeno , Doenças Neurodegenerativas , Animais , Camundongos , Esclerose Lateral Amiotrófica/metabolismo , Modelos Animais de Doenças , Microglia/metabolismo , Doenças Neurodegenerativas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas que Contêm Bromodomínio/genética , Proteínas que Contêm Bromodomínio/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismoRESUMO
Parkinson's disease (PD) is characterised neuropathologically by the degeneration of dopaminergic neurons in the ventral midbrain, the accumulation of α-synuclein (α-syn) aggregates in neurons, and chronic neuroinflammation. In the past two decades, in vitro, ex vivo and in vivo studies have consistently shown the involvement of inflammatory responses mediated by microglia and astrocytes, which may be elicited by pathological α-syn or signals from affected neurons and other cell types, and are directly linked to neurodegeneration and disease development. Besides the prominent immune alterations seen in the central nervous system (CNS), including the infiltration of T-cells into the brain, more recent studies have demonstrated important changes in the peripheral immune profile within both the innate and adaptive compartments, particularly involving monocytes, CD4+ and CD8+ T-cells. This review aims to integrate the consolidated understanding of immune-related processes underlying the pathogenesis of PD, focusing on both central and peripheral immune cells, neuron-glia crosstalk as well as the central-peripheral immune interaction during the development of PD. Our analysis seeks to provide a comprehensive view of the emerging knowledge of the mechanisms of immunity in PD and the implications of this for better understanding the overall pathogenesis of this disease.
RESUMO
Increasing evidence indicates that peripheral immune cells play a prominent role in neurodegeneration connected to protein misfolding, which are associated with formation of aberrant aggregates, including soluble protein misfolded oligomers. The precise links, however, between the physicochemical features of diverse oligomers and their effects on the immune system, particularly on adaptive immunity, remain currently unexplored, due partly to the transient and heterogeneous nature of the oligomers themselves. To overcome these limitations, we took advantage of two stable and well-characterized types of model oligomers (A and B), formed by HypF-N bacterial protein, type B oligomers displaying lower solvent-exposed hydrophobicity. Exposure to oligomers of human peripheral blood mononuclear cells (PBMCs) revealed differential effects, with type B, but not type A, oligomers leading to a reduction in CD4+ cells. Type A oligomers promoted enhanced differentiation towards CD4+ CD25High FoxP3+ Tregs and displayed a higher suppressive effect on lymphocyte proliferation than Tregs treated with oligomers B or untreated cells. Moreover, our results reveal Th1 and Th17 lymphocyte differentiation mediated by type A oligomers and a differential balance of TGF-ß, IL-6, IL-23, IFN-γ and IL-10 mediators. These results indicate that type B oligomers recapitulate some of the biological responses associated with Parkinson's disease in peripheral immunocompetent cells, while type A oligomers resemble responses associated with Alzheimer's disease. We anticipate that further studies characterizing the differential effects of protein misfolded oligomers on the peripheral immune system may lead to the development of blood-based diagnostics, which could report on the type and properties of oligomers present in patients.
Assuntos
Leucócitos Mononucleares/metabolismo , Deficiências na Proteostase/metabolismo , Adulto , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Citocinas/metabolismo , Humanos , Ativação Linfocitária/fisiologia , Pessoa de Meia-Idade , Dobramento de Proteína , Linfócitos T Reguladores/metabolismo , Células Th1/metabolismo , Células Th17/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is the most common neurodegenerative disorder of motor neurons in adults, with a median survival of 3-5 years after appearance of symptoms, and with no curative treatment currently available. Frontotemporal dementia (FTD) is also an adult-onset neurodegenerative disease, displaying not only clinical overlap with ALS, but also significant similarities at genetic and pathologic levels. Apart from the progressive loss of neurons and the accumulation of protein inclusions in certain cells and tissues, both disorders are characterized by chronic inflammation mediated by activated microglia and astrocytes, with an early and critical impact of neurodegeneration along the disease course. Despite the progress made in the last two decades in our knowledge around these disorders, the underlying molecular mechanisms of such non-cell autonomous neuronal loss still need to be clarified. In particular, immune signaling kinases are currently thought to have a key role in determining the neuroprotective or neurodegenerative nature of the central and peripheral immune states in health and disease. This review provides a comprehensive and updated view of the proposed mechanisms, therapeutic potential, and ongoing clinical trials of immune-related kinases that have been linked to ALS and/or FTD, by covering the more established TBK1, RIPK1/3, RACK I, and EPHA4 kinases, as well as other emerging players in ALS and FTD immune signaling.
Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Demência Frontotemporal/enzimologia , Sistema Imunitário/enzimologia , Inflamação , Fosfotransferases/metabolismo , Transdução de Sinais , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/imunologia , Esclerose Lateral Amiotrófica/metabolismo , Demência Frontotemporal/tratamento farmacológico , Demência Frontotemporal/imunologia , Demência Frontotemporal/metabolismo , Humanos , Sistema Imunitário/metabolismo , Fosfotransferases/antagonistas & inibidoresRESUMO
Neuroinflammation mediated by chronically activated microglia, largely caused by abnormal accumulation of misfolded α-synuclein (αSyn) protein, is known to contribute to the pathophysiology of Parkinson's disease (PD). In this work, based on the immunomodulatory activities displayed by particular heat-shock proteins (HSPs), we tested a novel vaccination strategy that used a combination of αSyn and Grp94 (HSPC4 or Gp96) chaperone and a murine PD model. We used two different procedures, first, the adoptive transfer of splenocytes from αSyn/Grp94-immunized mice to recipient animals, and second, direct immunization with αSyn/Grp94, to study the effects in a chronic mouse MPTP-model of parkinsonism. We found that both approaches promoted a distinct profile in the peripheral system-supported by humoral and cellular immunity-consisting of a Th1-shifted αSyn-specific response accompanied by an immune-regulatory/Th2-skewed general phenotype. Remarkably, this mixed profile sustained by αSyn/Grp94 immunization led to strong suppression of microglial activation in the substantia nigra and striatum, pointing to a newly described positive effect of anti-αSyn Th1-responses in the context of PD. This strategy is the first to target αSyn and report the suppression of PD-associated microgliosis. Overall, we show that the αSyn/Grp94 combination supports a distinct and long-lasting immune profile in the peripheral system, which has an impact at the CNS level by suppressing chronic microglial activation in an MPTP model of PD. Furthermore, our study demonstrates that reshaping peripheral immunity by vaccination with appropriate misfolding protein/HSP combinations could be highly beneficial as a treatment for neurodegenerative misfolding diseases.
Assuntos
Gliose/etiologia , Gliose/terapia , Imunização/métodos , Intoxicação por MPTP , Glicoproteínas de Membrana/imunologia , alfa-Sinucleína/imunologia , Transferência Adotiva , Análise de Variância , Animais , Antígenos CD4/metabolismo , Doença Crônica , Citocinas/metabolismo , Modelos Animais de Doenças , Intoxicação por MPTP/induzido quimicamente , Intoxicação por MPTP/complicações , Intoxicação por MPTP/imunologia , Intoxicação por MPTP/terapia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/patologia , Substância Negra/metabolismo , Substância Negra/patologia , Linfócitos T Reguladores/metabolismoRESUMO
Dysregulated microglial responses are central in neurodegenerative proteinopathies, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar disease (FTLD). Pathologic TDP-43, which is typically found in intracellular inclusions, is a misfolding protein with emerging roles in ALS and FTLD. Recently, TDP-43 species have been found in extracellular fluids of patients; however, the overall implications of TDP-43-mediated signaling linked to neuroinflammation are poorly understood. Our work-the first, to our knowledge, to focus on innate immunity responses to TDP-43 aggregates-shows that such species are internalized by microglia and cause abnormal mobilization of endogenous TDP-43. Exposure to TDP-43 aggregates elicited not only IL-1ß, but also NLRP3-dependent and noncanonical IL-18 processing. Moreover, we report a link between TDP-43 and neuronal loss via the apoptosis-independent emerging roles of caspase-3 in neurotoxic inflammation. Our results further support the view of noncell autonomous neurodegenerative mechanisms in ALS. Remarkably, we demonstrate that TDP-43 aggregates bind to and colocalize with MAPK/MAK/MRK overlapping kinase (MOK) and show that its phosphorylation status is disrupted. Finally, we show that this TDP-43-caused activation state can be altered by exogenous Hsp27 and Hsp70 chaperones. Our study provides new insight into the immune phenotype, mechanisms, and signaling pathways that operate in microglial neurotoxic activation in ALS.-Leal-Lasarte, M. M., Franco, J. M., Labrador-Garrido, A., Pozo, D., Roodveldt, C. Extracellular TDP-43 aggregates target MAPK/MAK/MRK overlapping kinase (MOK) and trigger caspase-3/IL-18 signaling in microglia.
Assuntos
Caspase 3/metabolismo , Proteínas de Ligação a DNA/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Interleucina-18/metabolismo , Microglia/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Caspase 3/genética , Sobrevivência Celular , Células Cultivadas , Proteínas de Ligação a DNA/administração & dosagem , Regulação Enzimológica da Expressão Gênica/fisiologia , Inflamassomos/metabolismo , Inflamação/metabolismo , Interleucina-18/genética , Masculino , Camundongos , Proteínas Quinases Ativadas por Mitógeno/genéticaRESUMO
We describe the isolation and detailed structural characterization of stable toxic oligomers of α-synuclein that have accumulated during the process of amyloid formation. Our approach has allowed us to identify distinct subgroups of oligomers and to probe their molecular architectures by using cryo-electron microscopy (cryoEM) image reconstruction techniques. Although the oligomers exist in a range of sizes, with different extents and nature of ß-sheet content and exposed hydrophobicity, they all possess a hollow cylindrical architecture with similarities to certain types of amyloid fibril, suggesting that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their ß-sheet structures. Our findings reveal the inherent multiplicity of the process of protein misfolding and the key role the ß-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the kinetic stability and the pathological nature of individual oligomeric species.
Assuntos
Amiloide/química , Multimerização Proteica , alfa-Sinucleína/química , alfa-Sinucleína/toxicidade , Microscopia Crioeletrônica , Interações Hidrofóbicas e Hidrofílicas , Imageamento Tridimensional , Modelos Moleculares , Peso Molecular , Estrutura Secundária de Proteína , alfa-Sinucleína/ultraestruturaRESUMO
We have investigated the potential role of molecular chaperones as modulators of the immune response by using α-synuclein (αSyn) as an aggregation-prone model protein. We first performed an in vitro immunoscreening with 21 preselected candidate chaperones and selected 2 from this set as displaying immunological activity with differential profiles, Grp94/Gp96 and FKBP4/52. We then immunized mice with both chaperone/α-synuclein combinations using monomeric or oligomeric α-synuclein (MαSyn or OαSyn, respectively), and we characterized the immune response generated in each case. We found that Grp94 promoted αSyn-specific T-helper (Th)1/Th17 and IgG1 antibody responses (up to a 3-fold increase) with MαSyn and OαSyn, respectively, coupled to a Th2-type general phenotype (generating 2.5-fold higher IgG1/IgG2 levels). In addition, we observed that FKBP4 favored a Th1-skewed phenotype with MαSyn but strongly supported a Th2-type phenotype with OαSyn (with a 3-fold higher IL-10/IFN-γ serum levels). Importantly, results from adoptive transfer of splenocytes from immunized animals in a Parkinson's disease mouse model indicates that these effects are robust, stable in time, and physiologically relevant. Taken together, Grp94 and FKBP4 are able to generate differential immune responses to α-synuclein-based immunizations, depending both on the nature of the chaperone and on the aggregation state of α-synuclein. Our work reveals that several chaperones are potential modulators of the immune response and suggests that different chaperones could be exploited to redirect the amyloid-elicited immunity both for basic studies of the immunological processes associated with neurodegeneration and for immunotherapy of pathologies associated with protein misfolding and aggregation.
Assuntos
Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/fisiologia , Proteínas de Ligação a Tacrolimo/metabolismo , alfa-Sinucleína/metabolismo , Imunidade Adaptativa , Animais , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Imunidade Inata , Masculino , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Dobramento de Proteína , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/imunologia , alfa-Sinucleína/genéticaRESUMO
Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded oligomers preformed from three different proteins added extracellularly to cultured cells. All the chaperones were found to decrease oligomer toxicity significantly, even at very low chaperone/protein molar ratios, provided that they were added extracellularly rather than being overexpressed in the cytosol. Infrared spectroscopy and site-directed labeling experiments using pyrene ruled out structural reorganizations within the discrete oligomers. Rather, confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and chaperones. Moreover, atomic force microscopy imaging indicated that larger assemblies of oligomers are formed in the presence of the chaperones. This suggests that the chaperones bind to the oligomers and promote their assembly into larger species, with consequent shielding of the reactive surfaces and a decrease in their diffusional mobility. Overall, the data indicate a generic ability of chaperones to neutralize extracellular misfolded oligomers efficiently and reveal that further assembly of protein oligomers into larger species can be an effective strategy to neutralize such extracellular species.
Assuntos
Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Multimerização Proteica , Linhagem Celular Tumoral , Humanos , Chaperonas Moleculares/genéticaRESUMO
How proteins with new functions (e.g., drug or antibiotic resistance or degradation of man-made chemicals) evolve in a matter of months or years is still unclear. This ability is dependent on the induction of new phenotypic traits by a small number of mutations (plasticity). But mutations often have deleterious effects on functions that are essential for survival. How are these seemingly conflicting demands met at the single-protein level? Results from directed laboratory evolution experiments indicate that the evolution of a new function is driven by mutations that have little effect on the native function but large effects on the promiscuous functions that serve as starting point. Thus, an evolving protein can initially acquire increased fitness for a new function without losing its original function. Gene duplication and the divergence of a completely new protein may then follow.
Assuntos
Arildialquilfosfatase/genética , Anidrase Carbônica II/genética , Evolução Molecular , Hidrolases de Triester Fosfórico/genética , Arildialquilfosfatase/fisiologia , Bactérias/enzimologia , Bactérias/genética , Anidrase Carbônica II/fisiologia , Variação Genética , Humanos , Hidrolases de Triester Fosfórico/fisiologia , Reação em Cadeia da Polimerase , Estrutura Terciária de ProteínaRESUMO
The ATP-dependent protein chaperone heat-shock protein 70 (Hsp70) displays broad anti-aggregation functions and has a critical function in preventing protein misfolding pathologies. According to in vitro and in vivo models of Parkinson's disease (PD), loss of Hsp70 activity is associated with neurodegeneration and the formation of amyloid deposits of alpha-synuclein (alphaSyn), which constitute the intraneuronal inclusions in PD patients known as Lewy bodies. Here, we show that Hsp70 depletion can be a direct result of the presence of aggregation-prone polypeptides. We show a nucleotide-dependent interaction between Hsp70 and alphaSyn, which leads to the aggregation of Hsp70, in the presence of ADP along with alphaSyn. Such a co-aggregation phenomenon can be prevented in vitro by the co-chaperone Hip (ST13), and the hypothesis that it might do so also in vivo is supported by studies of a Caenorhabditis elegans model of alphaSyn aggregation. Our findings indicate that a decreased expression of Hip could facilitate depletion of Hsp70 by amyloidogenic polypeptides, impairing chaperone proteostasis and stimulating neurodegeneration.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Homeostase/fisiologia , Complexos Multiproteicos/metabolismo , Doença de Parkinson/metabolismo , Proteínas Supressoras de Tumor/fisiologia , alfa-Sinucleína/metabolismo , Trifosfato de Adenosina/fisiologia , Amiloide/antagonistas & inibidores , Amiloide/biossíntese , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Humanos , Chaperonas Moleculares , Complexos Multiproteicos/antagonistas & inibidores , Degeneração Neural/metabolismo , Degeneração Neural/prevenção & controle , Doença de Parkinson/etiologia , Peptídeos/antagonistas & inibidores , Peptídeos/fisiologia , Dobramento de Proteína , Estabilidade Proteica , Ratos , Proteínas Supressoras de Tumor/antagonistas & inibidores , alfa-Sinucleína/antagonistas & inibidoresRESUMO
The aggregation process of α-synuclein, a protein closely associated with Parkinson's disease, is highly sensitive to sequence variations. It is therefore of great importance to understand the factors that define the aggregation propensity of specific mutational variants as well as their toxic behavior in the cellular environment. In this context, we investigated the extent to which the aggregation behavior of α-synuclein can be altered to resemble that of ß-synuclein, an aggregation-resistant homologue of α-synuclein not associated with disease, by swapping residues between the two proteins. Because of the vast number of possible swaps, we have applied a rational design procedure to single out a mutational variant, called α2ß, in which two short stretches of the sequence in the NAC region have been replaced in α-synuclein from ß-synuclein. We find not only that the aggregation rate of α2ß is close to that of ß-synuclein, being much lower than that of α-synuclein, but also that α2ß effectively changes the cellular toxicity of α-synuclein to a value similar to that of ß-synuclein upon exposure of SH-SY5Y cells to preformed oligomers. Remarkably, control experiments on the corresponding mutational variant of ß-synuclein, called ß2α, confirmed that the mutations that we have identified also shift the aggregation behavior of this protein toward that of α-synuclein. These results demonstrate that it is becoming possible to control in quantitative detail the sequence code that defines the aggregation behavior and toxicity of α-synuclein.
Assuntos
Estrutura Quaternária de Proteína , alfa-Sinucleína/química , beta-Sinucleína/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Sobrevivência Celular , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , Células Tumorais Cultivadas , alfa-Sinucleína/genética , alfa-Sinucleína/toxicidade , beta-Sinucleína/toxicidadeRESUMO
The formation of misfolded protein oligomers during early stages of amyloid aggregation and the activation of neuroinflammatory responses are two key events associated with neurodegenerative diseases. Although it has been established that misfolded oligomers are involved in the neuroinflammatory process, the links between their structural features and their functional effects on the immune response remain unknown. To explore such links, we took advantage of two structurally distinct soluble oligomers (type A and B) of protein HypF-N and compared the elicited microglial inflammatory responses. By using confocal microscopy, protein pull-down, and high-throughput mass spectrometry, we found that, even though both types bound to a common pool of microglial proteins, type B oligomers-with a lower solvent-exposed hydrophobicity-showed enhanced protein binding, correlating with the observed inflammatory response. Furthermore, the interactome associated with inflammatory-mediated neurodegeneration revealed previously unidentified receptors and signaling molecules likely to be involved in the oligomer-elicited innate immune response.
Assuntos
Carboxil e Carbamoil Transferases/imunologia , Proteínas de Escherichia coli/imunologia , Imunidade Inata/imunologia , Microglia/imunologia , Agregação Patológica de Proteínas/imunologia , Animais , Linhagem Celular , Cricetinae , Humanos , Camundongos , Microglia/patologia , Agregação Patológica de Proteínas/patologia , Ligação ProteicaRESUMO
Recent developments have been made in the application of directed evolution to achieve the efficient heterologous expression of proteins in Escherichia coli and yeast by increasing the stability and solubility of the protein in the host environment. One interesting conclusion that emerges is that the evolutionary process often improves the stability and solubility of an intermediate (apoprotein, proprotein or folding intermediate) that otherwise constitutes a bottleneck to functional expression, rather than altering the protein's final state.
Assuntos
Evolução Molecular Direcionada/métodos , Escherichia coli/fisiologia , Regulação da Expressão Gênica/fisiologia , Engenharia de Proteínas/métodos , Proteínas/genética , Proteínas/metabolismo , Saccharomyces cerevisiae/fisiologia , Clonagem Molecular/métodos , Evolução Molecular , Melhoramento Genético/métodos , Proteínas/química , Proteínas Recombinantes/biossíntese , SolubilidadeRESUMO
The past few years have seen significant advances in research related to the 'latent skills' of enzymes - namely, their capacity to promiscuously catalyze reactions other than the ones they evolved for. These advances regard (i) the mechanism of catalytic promiscuity - how enzymes, that generally exert exquisite specificity, promiscuously catalyze other, and sometimes barely related, reactions; (ii) the evolvability of promiscuous functions - namely, how latent activities evolve further, and in particular, how promiscuous activities can firstly evolve without severely compromising the original activity. These findings have interesting implications on our understanding of how new enzymes evolve. They support the key role of catalytic promiscuity in the natural history of enzymes, and suggest that today's enzymes diverged from ancestral proteins catalyzing a whole range of activities at low levels, to create families and superfamilies of potent and highly specialized enzymes.
Assuntos
Enzimas/química , Evolução Molecular , Animais , Catálise , Ativação Enzimática/fisiologia , Enzimas/fisiologia , Humanos , Especificidade por SubstratoRESUMO
The eukaryotic chaperonin CCT (chaperonin containing TCP-1) uses cavities built into its double-ring structure to encapsulate and to assist folding of a large subset of proteins. CCT can inhibit amyloid fibre assembly and toxicity of the polyQ extended mutant of huntingtin, the protein responsible for Huntington's disease. This raises the possibility that CCT modulates other amyloidopathies, a still-unaddressed question. We show here that CCT inhibits amyloid fibre assembly of α-synuclein A53T, one of the mutants responsible for Parkinson's disease. We evaluated fibrillation blockade in α-synuclein A53T deletion mutants and CCT interactions of full-length A53T in distinct oligomeric states to define an inhibition mechanism specific for α-synuclein. CCT interferes with fibre assembly by interaction of its CCTζ and CCTγ subunits with the A53T central hydrophobic region (NAC). This interaction is specific to NAC conformation, as it is produced once soluble α-synuclein A53T oligomers form and blocks the reaction before fibres begin to grow. Finally, we show that this association inhibits α-synuclein A53T oligomer toxicity in neuroblastoma cells. In summary, our results and those for huntingtin suggest that CCT is a general modulator of amyloidogenesis via a specific mechanism.
Assuntos
Amiloide/metabolismo , Chaperonina com TCP-1/metabolismo , alfa-Sinucleína/metabolismo , Amiloide/toxicidade , Linhagem Celular Tumoral , Chaperonina com TCP-1/química , Humanos , Microscopia Eletrônica de Transmissão , Mutagênese Sítio-Dirigida , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMO
Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disease that primarily affects motor neurons and is accompanied by sustained unregulated immune responses, but without clear indications of the ultimate causative mechanisms. The identification of a diverse array of ALS phenotypes, a series of recently discovered mutations, and the links between ALS and frontotemporal degeneration have significantly increased our knowledge of the disease. In this review we discuss the main features involved in ALS pathophysiology in the context of recent advances in 'omics' approaches, including genomics, proteomics, and others. We emphasize the pressing need to combine clinical imaging with various different parameters taken from omics fields to facilitate early, accurate diagnosis and rational drug design in the treatment of ALS.
Assuntos
Esclerose Lateral Amiotrófica/fisiopatologia , Esclerose Lateral Amiotrófica/terapia , Feminino , Genômica , Humanos , Masculino , Medicina de Precisão , ProteômicaRESUMO
In the present work we propose a simple method for affinity purification of the 67-kDa lectin-like glycoprotein (LLGP-67) from Trypanosoma cruzi, the causative agent of Chagas' disease. The LLGP-67, which presents galactose binding activity and participates in the host cell recognition process, was previously purified by methods based on its interaction with galactose residues on erythrocytic membranes. We describe herein results showing that this protein can be purified from T. cruzi in a direct way using non-derivatized agarose as a chromatographic ligand. We also demonstrate the relevance of LLGP-67 as an antigen for human diagnosis of chagasic infection. Sensitivity and specificity for this antigen were calculated, being 98 and 98.11% respectively.
Assuntos
Antígenos de Protozoários/isolamento & purificação , Doença de Chagas/diagnóstico , Lectinas/isolamento & purificação , Proteínas de Protozoários/isolamento & purificação , Trypanosoma cruzi/química , Animais , Antígenos de Protozoários/análise , Antígenos de Protozoários/imunologia , Doença de Chagas/parasitologia , Cromatografia de Afinidade , Cromatografia em Agarose , Ensaio de Imunoadsorção Enzimática , Feminino , Galactose/metabolismo , Humanos , Soros Imunes , Lectinas/análise , Lectinas/imunologia , Proteínas de Protozoários/análise , Proteínas de Protozoários/imunologia , Coelhos , Trypanosoma cruzi/imunologiaRESUMO
α-Synuclein (αSyn) is a 140-residue amyloid-forming protein whose aggregation is linked to Parkinson's disease (PD). It has also been found to play a critical role in the immune imbalance that accompanies disease progression, a characteristic that has prompted the search for an effective αSyn-based immunotherapy. In this study, we have simultaneously exploited two important features of certain heat-shock proteins (HSPs): their classical "chaperone" activities and their recently discovered and diverse "immunoactive" properties. In particular, we have explored the immune response elicited by immunization of C57BL/6 mice with an αSyn/Hsp70 protein combination in the absence of added adjuvant. Our results show differential effects for mice immunized with the αSyn/Hsp70 complex, including a restrained αSyn-specific (IgM and IgG) humoral response as well as minimized alterations in the Treg (CD4(+)CD25(+)Foxp3(+)) and Teff (CD4(+)Foxp3(-)) cell populations, as opposed to significant changes in mice immunized with αSyn and Hsp70 alone. Furthermore, in vitro-stimulated splenocytes from immunized mice showed the lowest relative response against αSyn challenge for the "αSyn/Hsp70" experimental group as measured by IFN-γ and IL-17 secretion, and higher IL-10 levels when stimulated with LPS. Finally, serum levels of Th1-cytokine IFN-γ and immunomodulatory IL-10 indicated a unique shift toward an immunomodulatory/immunoprotective phenotype in mice immunized with the αSyn/Hsp70 complex. Overall, we propose the use of functional "HSP-chaperoned amyloid/aggregating proteins" generated with appropriate HSP-substrate protein combinations, such as the αSyn/Hsp70 complex, as a novel strategy for immune-based intervention against synucleinopathies and other amyloid or "misfolding" neurodegenerative disorders.
RESUMO
In recent years, it has become accepted that α-synuclein (αSyn) has a key role in the microglia-mediated neuroinflammation, which accompanies the development of Parkinson's disease and other related disorders, such as Dementia with Lewy Bodies and Alzheimer's disease. Nevertheless, the cellular and molecular mechanisms underlying its pathological actions, especially in the sporadic forms of the diseases, are not completely understood. Intriguingly, several epidemiological and animal model studies have revealed a link between certain microbial infections and the onset or progression of sporadic forms of these neurodegenerative disorders. In this work, we have characterized the effect of toll-like receptor (TLR) stimulation on primary murine microglial cultures and analysed the impact of priming cells with extracellular wild-type (Wt) αSyn on the subsequent TLR stimulation of cells with a set of TLR ligands. By assaying key interleukins and chemokines we report that specific stimuli, in particular Pam3Csk4 (Pam3) and single-stranded RNA40 (ssRNA), can differentially affect the TLR2/1- and TLR7-mediated responses of microglia when pre-conditioned with αSyn by augmenting IL-6, MCP-1/CCL2 or IP-10/CXCL10 secretion levels. Furthermore, we report a skewing of αSyn-primed microglia stimulated with ssRNA (TLR7) or Pam3 (TLR2/1) towards intermediate but at the same time differential, M1/M2 phenotypes. Finally, we show that the levels and intracellular location of activated caspase-3 protein change significantly in αSyn-primed microglia after stimulation with these particular TLR agonists. Overall, we report a remarkable impact of non-aggregated αSyn pre-sensitization of microglia on TLR-mediated immunity, a phenomenon that could contribute to triggering the onset of sporadic α-synuclein-related neuropathologies.