RESUMO
The perpetuation of inflammation is an important pathophysiological contributor to the global medical burden. Chronic inflammation is promoted by non-programmed cell death1,2; however, how inflammation is instigated, its cellular and molecular mediators, and its therapeutic value are poorly defined. Here we use mouse models of atherosclerosis-a major underlying cause of mortality worldwide-to demonstrate that extracellular histone H4-mediated membrane lysis of smooth muscle cells (SMCs) triggers arterial tissue damage and inflammation. We show that activated lesional SMCs attract neutrophils, triggering the ejection of neutrophil extracellular traps that contain nuclear proteins. Among them, histone H4 binds to and lyses SMCs, leading to the destabilization of plaques; conversely, the neutralization of histone H4 prevents cell death of SMCs and stabilizes atherosclerotic lesions. Our data identify a form of cell death found at the core of chronic vascular disease that is instigated by leukocytes and can be targeted therapeutically.
Assuntos
Aterosclerose/patologia , Morte Celular , Membrana Celular/metabolismo , Histonas/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Porosidade , Animais , Artérias/patologia , Membrana Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Histonas/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/patologia , Neutrófilos/citologia , Ligação Proteica/efeitos dos fármacosRESUMO
Imbalances in the amounts of amyloid-ß peptides (Aß) generated by the membrane proteases ß- and γ-secretase are considered as a trigger of Alzheimer's disease (AD). Cell-free studies of γ-secretase have shown that increasing membrane thickness modulates Aß generation but it has remained unclear if these effects are translatable to cells. Here we show that the very long-chain fatty acid erucic acid (EA) triggers acyl chain remodeling in AD cell models, resulting in substantial lipidome alterations which included increased esterification of EA in membrane lipids. Membrane remodeling enhanced γ-secretase processivity, resulting in the increased production of the potentially beneficial Aß37 and/or Aß38 species in multiple cell lines. Unexpectedly, we found that the membrane remodeling stimulated total Aß secretion by cells expressing WT γ-secretase but lowered it for cells expressing an aggressive familial AD mutant γ-secretase. We conclude that EA-mediated modulation of membrane composition is accompanied by complex lipid homeostatic changes that can impact amyloidogenic processing in different ways and elicit distinct γ-secretase responses, providing critical implications for lipid-based AD treatment strategies.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Humanos , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Lipídeos de Membrana/metabolismo , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Linhagem Celular , Precursor de Proteína beta-Amiloide/metabolismo , Presenilina-1/metabolismoRESUMO
GGGGCC (G4C2) repeat expansion in the C9orf72 gene has been shown to cause frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Dipeptide repeat proteins produced through repeat-associated non-AUG (RAN) translation are recognized as potential drivers for neurodegeneration. Therefore, selective inhibition of RAN translation could be a therapeutic avenue to treat these neurodegenerative diseases. It was previously known that the porphyrin TMPyP4 binds to G4C2 repeat RNA. However, the consequences of this interaction have not been well characterized. Here, we confirmed that TMPyP4 inhibits C9orf72 G4C2 repeat translation in cellular and in in vitro translation systems. An artificial insertion of an AUG codon failed to cancel the translation inhibition, suggesting that TMPyP4 acts downstream of non-AUG translation initiation. Polysome profiling assays also revealed polysome retention on G4C2 repeat RNA, along with inhibition of translation, indicating that elongating ribosomes stall on G4C2 repeat RNA. Urea-resistant interaction between G4C2 repeat RNA and TMPyP4 likely contributes to this ribosome stalling and thus to selective inhibition of RAN translation. Taken together, our data reveal a novel mode of action of TMPyP4 as an inhibitor of G4C2 repeat translation elongation.
Assuntos
Proteína C9orf72/biossíntese , Expansão das Repetições de DNA , Modelos Biológicos , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Porfirinas/farmacologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Proteína C9orf72/genética , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/metabolismo , Células HeLa , Humanos , Polirribossomos/metabolismoRESUMO
BACKGROUND: Acute infection is a well-established risk factor of cardiovascular inflammation increasing the risk for a cardiovascular complication within the first weeks after infection. However, the nature of the processes underlying such aggravation remains unclear. Lipopolysaccharide derived from Gram-negative bacteria is a potent activator of circulating immune cells including neutrophils, which foster inflammation through discharge of neutrophil extracellular traps (NETs). Here, we use a model of endotoxinemia to link acute infection and subsequent neutrophil activation with acceleration of vascular inflammation Methods: Acute infection was mimicked by injection of a single dose of lipopolysaccharide into hypercholesterolemic mice. Atherosclerosis burden was studied by histomorphometric analysis of the aortic root. Arterial myeloid cell adhesion was quantified by intravital microscopy. RESULTS: Lipopolysaccharide treatment rapidly enhanced atherosclerotic lesion size by expansion of the lesional myeloid cell accumulation. Lipopolysaccharide treatment led to the deposition of NETs along the arterial lumen, and inhibition of NET release annulled lesion expansion during endotoxinemia, thus suggesting that NETs regulate myeloid cell recruitment. To study the mechanism of monocyte adhesion to NETs, we used in vitro adhesion assays and biophysical approaches. In these experiments, NET-resident histone H2a attracted monocytes in a receptor-independent, surface charge-dependent fashion. Therapeutic neutralization of histone H2a by antibodies or by in silico designed cyclic peptides enables us to reduce luminal monocyte adhesion and lesion expansion during endotoxinemia. CONCLUSIONS: Our study shows that NET-associated histone H2a mediates charge-dependent monocyte adhesion to NETs and accelerates atherosclerosis during endotoxinemia.
Assuntos
Aterosclerose/metabolismo , Adesão Celular/fisiologia , Endotoxemia/metabolismo , Monócitos/metabolismo , Eletricidade Estática , Animais , Aterosclerose/induzido quimicamente , Aterosclerose/patologia , Adesão Celular/efeitos dos fármacos , Endotoxemia/induzido quimicamente , Endotoxemia/patologia , Armadilhas Extracelulares/metabolismo , Humanos , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monócitos/efeitos dos fármacos , Monócitos/patologiaRESUMO
Repeat expansion in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Expanded sense and antisense repeat RNA transcripts in C9orf72 are translated into five dipeptide-repeat proteins (DPRs) in an AUG-independent manner. We previously identified the heterogeneous ribonucleoprotein (hnRNP) A3 as an interactor of the sense repeat RNA that reduces its translation into DPRs. Furthermore, we found that hnRNPA3 is depleted from the nucleus and partially mislocalized to cytoplasmic poly-GA inclusions in C9orf72 patients, suggesting that poly-GA sequesters hnRNPA3 within the cytoplasm. We now demonstrate that hnRNPA3 also binds to the antisense repeat RNA. Both DPR production and deposition from sense and antisense RNA repeats are increased upon hnRNPA3 reduction. All DPRs induced DNA double strand breaks (DSB), which was further enhanced upon reduction of hnRNPA3. Poly-glycine-arginine and poly-proline-arginine increased foci formed by phosphorylated Ataxia Telangiectasia Mutated (pATM), a major sensor of DSBs, whereas poly-glycine-alanine (poly-GA) evoked a reduction of pATM foci. In dentate gyri of C9orf72 patients, lower nuclear hnRNPA3 levels were associated with increased DNA damage. Moreover, enhanced poly-GA deposition correlated with reduced pATM foci. Since cytoplasmic pATM deposits partially colocalized with poly-GA deposits, these results suggest that poly-GA, the most frequent DPR observed in C9orf72 patients, differentially causes DNA damage and that poly-GA selectively sequesters pATM in the cytoplasm inhibiting its recruitment to sites of DNA damage. Thus, mislocalization of nuclear hnRNPA3 caused by poly-GA leads to increased poly-GA production, which partially depletes pATM, and consequently enhances DSB.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína C9orf72/genética , Repetições de Dinucleotídeos/fisiologia , Degeneração Lobar Frontotemporal/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Idoso , Esclerose Lateral Amiotrófica/metabolismo , Dano ao DNA/genética , Feminino , Degeneração Lobar Frontotemporal/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , FosforilaçãoRESUMO
Intramembrane cleavage of the ß-amyloid precursor protein C99 substrate by γ-secretase is implicated in Alzheimer's disease pathogenesis. Biophysical data have suggested that the N-terminal part of the C99 transmembrane domain (TMD) is separated from the C-terminal cleavage domain by a di-glycine hinge. Because the flexibility of this hinge might be critical for γ-secretase cleavage, we mutated one of the glycine residues, G38, to a helix-stabilizing leucine and to a helix-distorting proline. Both mutants impaired γ-secretase cleavage and also altered its cleavage specificity. Circular dichroism, NMR, and backbone amide hydrogen/deuterium exchange measurements as well as molecular dynamics simulations showed that the mutations distinctly altered the intrinsic structural and dynamical properties of the substrate TMD. Although helix destabilization and/or unfolding was not observed at the initial ε-cleavage sites of C99, subtle changes in hinge flexibility were identified that substantially affected helix bending and twisting motions in the entire TMD. These resulted in altered orientation of the distal cleavage domain relative to the N-terminal TMD part. Our data suggest that both enhancing and reducing local helix flexibility of the di-glycine hinge may decrease the occurrence of enzyme-substrate complex conformations required for normal catalysis and that hinge mobility can thus be conducive for productive substrate-enzyme interactions.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Membrana Celular/metabolismo , Simulação de Dinâmica Molecular , Proteólise , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/genética , Mutação , Domínios ProteicosRESUMO
OBJECTIVE: Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) occur in a proportion of patients with inflammatory demyelinating diseases of the central nervous system (CNS). We analyzed their pathogenic activity by affinity-purifying these antibodies (Abs) from patients and transferring them to experimental animals. METHODS: Patients with Abs to MOG were identified by cell-based assay. We determined the cross-reactivity to rodent MOG and the recognized MOG epitopes. We produced the correctly folded extracellular domain of MOG and affinity-purified MOG-specific Abs from the blood of patients. These purified Abs were used to stain CNS tissue and transferred in 2 models of experimental autoimmune encephalomyelitis. Animals were analyzed histopathologically. RESULTS: We identified 17 patients with MOG Abs from our outpatient clinic and selected 2 with a cross-reactivity to rodent MOG; both had recurrent optic neuritis. Affinity-purified Abs recognized MOG on transfected cells and stained myelin in tissue sections. The Abs from the 2 patients recognized different epitopes on MOG, the CC' and the FG loop. In both patients, these Abs persisted during our observation period of 2 to 3 years. The anti-MOG Abs from both patients were pathogenic upon intrathecal injection in 2 different rat models. Together with cognate MOG-specific T cells, these Abs enhanced T-cell infiltration; together with myelin basic protein-specific T cells, they induced demyelination associated with deposition of C9neo, resembling a multiple sclerosis type II pathology. INTERPRETATION: MOG-specific Abs affinity purified from patients with inflammatory demyelinating disease induce pathological changes in vivo upon cotransfer with myelin-reactive T cells, suggesting that these Abs are similarly pathogenic in patients. Ann Neurol 2018;84:315-328.
Assuntos
Autoanticorpos/sangue , Encéfalo/metabolismo , Encéfalo/patologia , Glicoproteína Mielina-Oligodendrócito/sangue , Adulto , Idoso , Animais , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Cobaias , Humanos , Inflamação/sangue , Inflamação/diagnóstico , Masculino , Pessoa de Meia-Idade , Ratos , Ratos Endogâmicos Lew , Adulto JovemRESUMO
Sulfoconjugates of sterols play important roles as neurosteroids, neurotransmitters, and ion channel ligands in health and disease. In most cases, sterol conjugate analysis is performed with liquid chromatography-mass spectrometry. This is a valuable tool for routine analytics with the advantage of direct sterol sulfates analysis without previous cleavage and/or derivatization. The complementary technique gas chromatography-mass spectrometry (GC-MS) is a preeminent discovery tool in the field of sterolomics, but the analysis of sterol sulfates is hampered by mandatory deconjugation and derivatization. Despite the difficulties in sample workup, GC-MS is an indispensable tool for untargeted analysis and steroid profiling. There are no general sample preparation protocols for sterol sulfate analysis using GC-MS. In this study we present a reinvestigation and evaluation of different deconjugation and derivatization procedures with a set of representative sterol sulfates. The advantages and disadvantages of trimethylsilyl (TMS), methyloxime-trimethylsilyl (MO-TMS), and trifluoroacetyl (TFA) derivatives were examined. Different published procedures of sterol sulfate deconjugation, including enzymatic and chemical cleavage, were reinvestigated and examined for diverse sterol sulfates. Finally, we present a new protocol for the chemical cleavage of sterol sulfates, allowing for simultaneous deconjugation and derivatization, simplifying GC-MS based sterol sulfate analysis.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas , Esteroides/química , Esteróis/química , Sulfatos/química , Humanos , Estrutura Molecular , Solventes , Esteroides/análise , Esteróis/análise , Sulfatos/análiseRESUMO
The uncoupling protein (UCP1) is a proton (H+) transporter in the mitochondrial inner membrane. By dissipating the electrochemical H+ gradient, UCP1 uncouples respiration from ATP synthesis, which drives an increase in substrate oxidation via the TCA cycle flux that generates more heat. The mitochondrial uncoupling-mediated non-shivering thermogenesis in brown adipose tissue is vital primarily to mammals, such as rodents and new-born humans, but more recently additional functions in adult humans have been described. UCP1 is regulated by ß-adrenergic receptors through the sympathetic nervous system and at the molecular activity level by nucleotides and fatty acid to meet thermogenesis needs. The discovery of novel UCP homologs has greatly contributed to the understanding of human diseases, such as obesity and diabetes. In this article, we review the progress made towards the molecular mechanism and function of the UCPs, in particular focusing on the influential contributions from Martin Klingenberg's laboratory. Because all members of the UCP family are potentially promising drug targets, we also present and discuss possible approaches and methods for UCP-related drug discovery.
Assuntos
Proteínas de Desacoplamento Mitocondrial/química , Proteínas de Desacoplamento Mitocondrial/metabolismo , Trifosfato de Adenosina/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Sítios de Ligação , Ácidos Graxos não Esterificados/metabolismo , Humanos , Ligação Proteica , Termogênese/fisiologiaRESUMO
Intronic hexanucleotide (G4C2) repeat expansions in C9orf72 are genetically associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat RNA accumulates within RNA foci but is also translated into disease characterizing dipeptide repeat proteins (DPR). Repeat-dependent toxicity may affect nuclear import. hnRNPA3 is a heterogeneous nuclear ribonucleoprotein, which specifically binds to the G4C2 repeat RNA We now report that a reduction of nuclear hnRNPA3 leads to an increase of the repeat RNA as well as DPR production and deposition in primary neurons and a novel tissue culture model that reproduces features of the C9orf72 pathology. In fibroblasts derived from patients carrying extended C9orf72 repeats, nuclear RNA foci accumulated upon reduction of hnRNPA3. Neurons in the hippocampus of C9orf72 patients are frequently devoid of hnRNPA3. Reduced nuclear hnRNPA3 in the hippocampus of patients with extended C9orf72 repeats correlates with increased DPR deposition. Thus, reduced hnRNPA3 expression in C9orf72 cases leads to increased levels of the repeat RNA as well as enhanced production and deposition of DPR proteins and RNA foci.
Assuntos
Dipeptídeos/metabolismo , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Proteínas/genética , RNA Mensageiro/genética , Animais , Encéfalo/metabolismo , Proteína C9orf72 , Fibroblastos , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Neurônios/metabolismo , Ligação Proteica , Transporte Proteico , Células Piramidais/metabolismo , Transporte de RNA , RNA Interferente Pequeno/genética , RatosRESUMO
Intramembrane proteolysis has emerged as a key mechanism required for membrane proteostasis and cellular signaling. One of the intramembrane-cleaving proteases (I-CLiPs), γ-secretase, is also intimately implicated in Alzheimer's disease, a major neurodegenerative disease and leading cause of dementia. High-resolution crystal structural analyses have revealed that I-CLiPs harbor their active sites buried deeply in the membrane bilayer. Surprisingly, however, the key kinetic constants of these proteases, turnover number kcat and catalytic efficiency kcat/KM, are largely unknown. By investigating the kinetics of intramembrane cleavage of the Alzheimer's disease-associated ß-amyloid precursor protein in vitro and in human embryonic kidney cells, we show that γ-secretase is a very slow protease with a kcat value similar to those determined recently for rhomboid-type I-CLiPs. Our results indicate that low turnover numbers may be a general feature of I-CLiPs.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Proteólise , Células HEK293 , Humanos , Membranas Intracelulares/química , CinéticaRESUMO
Aggregation of α-synuclein (αS) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of αS is largely unknown. We demonstrate with in vitro vesicle fusion experiments that αS has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, αS binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age-dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous αS. In contrast, siRNA-mediated downregulation of αS results in elongated mitochondria in cell culture. αS can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, αS prevents fusion of differently labelled mitochondrial populations. Thus, αS inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of αS is rescued by coexpression of PINK1, parkin or DJ-1 but not the PD-associated mutations PINK1 G309D and parkin Δ1-79 or by DJ-1 C106A.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fusão de Membrana/fisiologia , Mitocôndrias/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , alfa-Sinucleína/metabolismo , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Linhagem Celular , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitocôndrias/ultraestrutura , Proteínas Oncogênicas/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteína Desglicase DJ-1 , Proteínas Quinases/genética , Ubiquitina-Proteína Ligases/genética , alfa-Sinucleína/genéticaRESUMO
Amino acid scales are crucial for protein prediction tasks, many of them being curated in the AAindex database. Despite various clustering attempts to organize them and to better understand their relationships, these approaches lack the fine-grained classification necessary for satisfactory interpretability in many protein prediction problems. To address this issue, we developed AAontology-a two-level classification for 586 amino acid scales (mainly from AAindex) together with an in-depth analysis of their relations-using bag-of-word-based classification, clustering, and manual refinement over multiple iterations. AAontology organizes physicochemical scales into 8 categories and 67 subcategories, enhancing the interpretability of scale-based machine learning methods in protein bioinformatics. Thereby it enables researchers to gain a deeper biological insight. We anticipate that AAontology will be a building block to link amino acid properties with protein function and dysfunctions as well as aid informed decision-making in mutation analysis or protein drug design.
RESUMO
Pathogenic generation of amyloid ß-peptide (Aß) by sequential cleavage of ß-amyloid precursor protein (APP) by ß- and γ-secretases is widely believed to causally underlie Alzheimer disease (AD). ß-Secretase initially cleaves APP thereby generating a membrane-bound APP C-terminal fragment, from which γ-secretase subsequently liberates 37-43-amino acid long Aß species. Although the latter cleavages are intramembranous and although lipid alterations have been implicated in AD, little is known of how the γ-secretase-mediated release of the various Aß species, in particular that of the pathogenic longer variants Aß(42) and Aß(43), is affected by the lipid environment. Using a cell-free system, we have directly and systematically investigated the activity of γ-secretase reconstituted in defined model membranes of different thicknesses. We found that bilayer thickness is a critical parameter affecting both total activity as well as cleavage specificity of γ-secretase. Whereas the generation of the pathogenic Aß(42/43) species was markedly attenuated in thick membranes, that of the major and rather benign Aß(40) species was enhanced. Moreover, the increased production of Aß(42/43) by familial AD mutants of presenilin 1, the catalytic subunit of γ-secretase, could be substantially lowered in thick membranes. Our data demonstrate an effective modulation of γ-secretase activity by membrane thickness, which may provide an approach to lower the generation of the pathogenic Aß(42/43) species.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Membrana Celular/metabolismo , Lipídeos de Membrana/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/genética , Membrana Celular/patologia , Células HEK293 , Humanos , Lipídeos de Membrana/genética , MutaçãoRESUMO
Cumulative evidence now suggests that the abnormal aggregation of the protein α-synuclein (αS) is a critical factor in triggering neurodegeneration in Parkinson's disease (PD). In particular, a fundamental pathogenetic mechanism appears to involve targeting of neuronal membranes by soluble oligomeric intermediates of αS, leading to their disruption or permeabilisation. Therefore, a model assay was developed in which fluorophore-loaded unilamellar vesicles were permeabilised by soluble oligomers, the latter formed by aggregation of human recombinant αS protein. The αS oligomers induced an impairment of membrane integrity similar to that of the pore-forming bacterial peptide gramicidin. The lipid vesicle permeabilisation assay was then utilised to screen 11 natural polyphenolic compounds, 8 synthetic N'-benzylidene-benzohydrazide compounds and black tea extract for protection against membrane damage by wild-type and mutant (A30P, A53T) synuclein aggregates. A select group of potent inhibitory compounds included apigenin, baicalein, morin, nordihydroguaiaretic acid, and black tea extract. Structure-activity analysis further suggests that a 5,7-dihydroxy-chromen-4-one moiety appears to be favourable for the inhibition reaction. In conclusion, we have identified a group of polyphenols that can effectively hinder membrane damage by αS aggregates. These may serve as a viable source of lead compounds for the development and design of novel therapeutic agents in PD.
Assuntos
Lipídeos de Membrana/metabolismo , Polifenóis/farmacologia , alfa-Sinucleína/toxicidade , Permeabilidade da Membrana Celular , Técnicas In Vitro , Cinética , Lipossomos , Espectrometria de Fluorescência , Relação Estrutura-AtividadeRESUMO
Aggregation of α-synuclein is involved in the pathogenesis of Parkinson's disease (PD). Studies of in vitro aggregation of α-synuclein are rendered complex because of the formation of a heterogeneous population of oligomers. With the use of confocal single-molecule fluorescence techniques, we demonstrate that small aggregates (oligomers) of α-synuclein formed from unbound monomeric species in the presence of organic solvent (DMSO) and iron (Fe(3+)) ions have a high affinity to bind to model membranes, regardless of the lipid-composition or membrane curvature. This binding mode contrasts with the well-established membrane binding of α-synuclein monomers, which is accompanied with α-helix formation and requires membranes with high curvature, defects in the lipid packing, and/or negatively charged lipids. Additionally, we demonstrate that membrane-bound α-synuclein monomers are protected from aggregation. Finally, we identified compounds that potently dissolved vesicle-bound α-synuclein oligomers into monomers, leaving the lipid vesicles intact. As it is commonly believed that formation of oligomers is related PD progression, such compounds may provide a promising strategy for the design of novel therapeutic drugs in Parkinson's disease.
Assuntos
Multimerização Proteica , Espectrometria de Fluorescência/métodos , Lipossomas Unilamelares/metabolismo , alfa-Sinucleína/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Dimetil Sulfóxido/química , Transferência Ressonante de Energia de Fluorescência , Doença de Parkinson/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Lipossomas Unilamelares/química , alfa-Sinucleína/químicaRESUMO
Anti-amyloidogenic processing of the amyloid precursor protein APP by α-secretase prevents formation of the amyloid-ß peptide, which accumulates in senile plaques of Alzheimer disease patients. α-Secretase belongs to the family of a disintegrin and metalloproteases (ADAMs), and ADAM10 is the primary candidate for this anti-amyloidogenic activity. We recently demonstrated that ADAM10 translation is repressed by its 5'-UTR and that in particular the first half of ADAM10 5'-UTR is responsible for translational repression. Here, we asked whether specific sequence motifs exist in the ADAM10 5'-UTR that are able to form complex secondary structures and thus potentially inhibit ADAM10 translation. Using circular dichroism spectroscopy, we demonstrate that a G-rich region between nucleotides 66 and 94 of the ADAM10 5'-UTR forms a highly stable, intramolecular, parallel G-quadruplex secondary structure under physiological conditions. Mutation of guanines in this sequence abrogates the formation of the G-quadruplex structure. Although the G-quadruplex structure efficiently inhibits translation of a luciferase reporter in in vitro translation assays and in living cells, inhibition of G-quadruplex formation fails to do so. Moreover, expression of ADAM10 was similarly repressed by the G-quadruplex. Mutation of the G-quadruplex motif results in a significant increase of ADAM10 levels and consequently APPsα secretion. Thus, we identified a critical RNA secondary structure within the 5'-UTR, which contributes to the translational repression of ADAM10.
Assuntos
Regiões 5' não Traduzidas/fisiologia , Proteínas ADAM/biossíntese , Secretases da Proteína Precursora do Amiloide/biossíntese , Proteínas de Membrana/biossíntese , Conformação de Ácido Nucleico , Biossíntese de Proteínas/fisiologia , Proteínas ADAM/genética , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , MutaçãoRESUMO
The two proteases beta-secretase and gamma-secretase generate the amyloid beta peptide and are drug targets for Alzheimer's disease. Here we tested the possibility of targeting the cellular environment of beta-secretase cleavage instead of the beta-secretase enzyme itself. beta-Secretase has an acidic pH optimum and cleaves the amyloid precursor protein in the acidic endosomes. We identified two drugs, bepridil and amiodarone, that are weak bases and are in clinical use as calcium antagonists. Independently of their calcium-blocking activity, both compounds mildly raised the membrane-proximal, endosomal pH and inhibited beta-secretase cleavage at therapeutically achievable concentrations in cultured cells, in primary neurons, and in vivo in guinea pigs. This shows that an alkalinization of the cellular environment could be a novel therapeutic strategy to inhibit beta-secretase. Surprisingly, bepridil and amiodarone also modulated gamma-secretase cleavage independently of endosomal alkalinization. Thus, both compounds act as dual modulators that simultaneously target beta- and gamma-secretase through distinct molecular mechanisms. In addition to Alzheimer's disease, compounds with dual properties may also be useful for drug development targeting other membrane proteins.
Assuntos
Amiodarona/farmacologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Bepridil/farmacologia , Inibidores Enzimáticos/farmacologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Amiodarona/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Bepridil/química , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/metabolismo , Linhagem Celular , Células Cultivadas , Inibidores Enzimáticos/química , Feminino , Cobaias , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/metabolismo , Nexinas de Proteases , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismoRESUMO
Nicastrin is a type I transmembrane glycoprotein, which is part of the high molecular weight γ-secretase complex. γ-Secretase is one of the key players associated with the generation of Alzheimer's disease pathology, since it liberates the neurotoxic amyloid ß-peptide. Four proteins Nicastrin, anterior pharynx-defective-1 (Aph-1), presenilin enhancer-2 (Pen-2) and Presenilin are essential to form the active γ-secretase complex. Recently it has been shown, that Nicastrin has a key function in stabilizing the mature γ-secretase complex and may also be involved in substrate recognition. So far no structural data for the Nicastrin ectodomain or any other γ-secretase component are available. We therefore used Circular Dichroism (CD) spectroscopy to demonstrate that Nicastrin, similar to its homologues, the Streptomyces griseus aminopeptidase (SGAP) and the transferrin receptor (TfR), adopts a thermostable secondary structure. Furthermore, the Nicastrin ectodomain has an exceptionally high propensity to refold after thermal denaturation. These findings provide evidence to further support the hypothesis that Nicastrin may share evolutionary conserved properties with the aminopeptidase and the transferrin receptor family.
Assuntos
Secretases da Proteína Precursora do Amiloide/química , Glicoproteínas de Membrana/química , Doença de Alzheimer/metabolismo , Aminopeptidases/química , Linhagem Celular , Dicroísmo Circular , Humanos , Redobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores da Transferrina/química , Streptomyces griseus/enzimologia , TemperaturaRESUMO
Steroids play an important role in cell regulation and homeostasis. Many diseases like Alzheimer's disease or Smith-Lemli-Opitz syndrome are known to be associated with deviations in the steroid profile. Most published methods only allow the analysis of small subgroups of steroids and cannot give an overview of the total steroid profile. We developed and validated a method that allows the analysis of free neutral steroids, including intermediates of cholesterol biosynthesis, free oxysterols, C19 and C21 steroids, free steroid acids, including bile acids, and sterol sulfates using gas chromatography-mass spectrometry. Samples were analyzed in scan mode for screening purposes and in dynamic multiple reaction monitoring mode for highly sensitive quantitative analysis. The method was validated for mouse brain and liver tissue and consists of sample homogenization, lipid extraction, steroid group separation, deconjugation, derivatization and gas chromatography-mass spectrometry analysis. We applied the method on brain and liver samples of mice (10 months and 3 weeks old) and cultured N2a cells and report the endogenous concentrations of 29 physiological steroids.