RESUMO
The assembly of amyloidogenic proteins into highly-structured fibrillar aggregates is related to the onset and progression of several amyloidoses, including neurodegenerative Alzheimer's or Parkinson's diseases. Despite years of research and a general understanding of the process of such aggregate formation, there are currently still very few drugs and treatment modalities available. One of the factors that is relatively insufficiently understood is the cross-interaction between different amyloid-forming proteins. In recent years, it has been shown that several of these proteins or their aggregates can alter each other's fibrillization properties, however, there are still many unknowns in the amyloid interactome. In this work, we examine the interaction between amyloid disease-related prion protein and superoxide dismutase-1. We show that not only does superoxide dismutase-1 increase the lag time of prion protein fibril formation, but it also changes the conformation of the resulting aggregates.
Assuntos
Fragmentos de Peptídeos/metabolismo , Proteínas Priônicas/metabolismo , Agregados Proteicos/efeitos dos fármacos , Superóxido Dismutase-1/metabolismo , Animais , Ligação de Hidrogênio , Camundongos , Fragmentos de Peptídeos/química , Proteínas Priônicas/química , Conformação Proteica em Folha beta/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacosRESUMO
Lymphatic filariasis and its associated health hazards have taken enormous tolls especially in the tropical and sub-tropical countries round the globe. Our present work contemplates the immunomodulatory role of filarial Thioredoxin reductase (TrxR) for the survival of the parasite inside the human host. For this, the protein TrxR was purified from the filarial parasite Setaria cervi and further substantiated through specific anti-TrxR antibody raised in mice. Both commercially available anti-TrxR antibody and laboratory raised antibody produced a single band with a molecular mass of ~80 kDa on western blot. The protein is optimally active at pH 7.0 and at temperature 37 °C. This protein contains both alpha helix and beta pleated sheet with selenocysteine at its active site. The Km was found to be 2.75 ± 0.49 mM. TrxR was found to downregulate lipopolysaccharide (LPS)-induced inflammation in macrophages due to inhibition of TLR4-NF-κB pathway. The result was further supported by the downregulation of inflammasome pathway and activation of alternatively activated macrophages upon TrxR treatment. Hence this study projects insights into the importance of filarial TrxR in host-parasite interface as well as it illustrates novel therapeutic strategy towards anti-filarial drug development.
Assuntos
Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Macrófagos/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxina Dissulfeto Redutase/farmacologia , Animais , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Proteínas de Helminto/metabolismo , Inflamação/induzido quimicamente , Lipopolissacarídeos/farmacologia , Camundongos , NF-kappa B/metabolismo , Conformação Proteica em alfa-Hélice/efeitos dos fármacos , Conformação Proteica em Folha beta/efeitos dos fármacos , Células RAW 264.7 , Setaria (Nematoide)/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismoRESUMO
Although silk proteins are considered promising in building a scaffold for tissue engineering, one of the silk proteins, Bombyx mori silk sericin (BS), has limited processability in producing nanofibrous scaffolds because its surface charge anisotropy promotes gelation instead. To overcome this daunting challenge, we developed a mild and simple procedure for assembling BS into nanofibers and nanofibrous scaffolds. Briefly, arginine was added to the aqueous BS solution to reduce the negative charge of BS, thereby inducing BS to self-assemble into nanofibers in the solution. Circular dichroism (CD) and Fourier transform infrared (FT-IR) spectra showed that arginine promoted the formation of ß-sheet conformation in BS and increased its thermal stability. Furthermore, the arginine-induced BS nanofiber solution could be casted into scaffolds made of abundant network-like nanofibrous structures. The BS scaffolds promoted cell adhesion and growth and stimulated osteogenic differentiation of the bone marrow mesenchymal stem cells (BMSCs) in the absence of differentiation inducers in culture media. Our study presents a new strategy for assembling proteins into osteogenic nanofibrous scaffolds for inducing stem cell differentiation in regenerative medicine.
Assuntos
Arginina/química , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Nanofibras/química , Sericinas/farmacologia , Alicerces Teciduais/química , Animais , Bombyx/química , Membranas Artificiais , Conformação Proteica em Folha beta/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Sericinas/químicaRESUMO
Molecules inhibiting the amyloid beta (Aß) peptide aggregation and/or disaggregating mature fibrils are a promising approach for the Alzheimer's disease (AD) therapy, as the Aß fibrillation is one of the key triggers of the disease. Gallic acid (GA) is a phenolic acid with anti-amyloidogenic activity against Aß in buffered solutions. However, there is still no evidence of these properties in vivo. Given the rate of failures of AD drug development, there is a huge demand of replicating the in vivo environment in in vitro studies, thus allowing to stop earlier the study of molecules with no effect in vivo. Thus, this study aims to evaluate the effect of in vitro neuronal membranes on the GA's ability in preventing Aß1-42 aggregation and disrupting preformed fibrils. To this end, liposomes were employed to mimic the cell membrane environment. The results reveal that the lipid membranes did not affect the GA's ability in inhibiting Aß1-42 fibrillation. However, in vitro neuronal membranes modulate the GA-induced Aß fibrils disaggregation, which may be related with the moderate affinity of the compound for the lipid membrane. Even so, GA presented strong anti-amyloidogenic properties in the cell membrane-like environment. This work highlights the promising value of GA on preventing and treating AD, thus justifying its study in animal models.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Ácido Gálico/química , Lipossomos/química , Fragmentos de Peptídeos/metabolismo , Multimerização Proteica/efeitos dos fármacos , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/química , Humanos , Cinética , Fragmentos de Peptídeos/química , Conformação Proteica em alfa-Hélice/efeitos dos fármacos , Conformação Proteica em Folha beta/efeitos dos fármacosRESUMO
Macromolecular associates, such as membraneless organelles or lipid-protein assemblies, provide a hydrophobic environment, i.e., a liquid protein phase (LP), where folding preferences can be drastically altered. LP as well as the associated phase change from water (W) is an intriguing phenomenon related to numerous biological processes and also possesses potential in nanotechnological applications. However, the energetic effects of a hydrophobic yet water-containing environment on protein folding are poorly understood. Here, we focus on small ß-sheets, the key motifs of proteins, undergoing structural changes in liquid-liquid phase separation (LLPS) and also model the mechanism of energy-coupled unfolding, e.g., in proteases, during W â LP transition. Due to the importance of the accurate description for hydrogen bonding patterns, the employed models were studied by using quantum mechanical calculations. The results demonstrate that unfolding is energetically less favored in LP by ~0.3-0.5 kcal·mol-1 per residue in which the difference further increased by the presence of explicit structural water molecules, where the folded state was preferred by ~1.2-2.3 kcal·mol-1 per residue relative to that in W. Energetics at the LP/W interfaces was also addressed by theoretical isodesmic reactions. While the models predict folded state preference in LP, the unfolding from LP to W renders the process highly favorable since the unfolded end state has >1 kcal·mol-1 per residue excess stabilization.
Assuntos
Transição de Fase/efeitos dos fármacos , Conformação Proteica em Folha beta/efeitos dos fármacos , Água/farmacologia , Motivos de Aminoácidos/efeitos dos fármacos , Fracionamento Químico/métodos , Simulação por Computador , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Cinética , Substâncias Macromoleculares/química , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Teoria Quântica , Viscosidade , Água/químicaRESUMO
Studies have established that congenital cataract is the major cause of blindness in children across the globe. The ß-crystallin protein family is the richest and most soluble structural protein in the lens. Their solubility and stability are essential in maintaining lens transparency. In this study, we identified a novel ßB2 mutation W151R in a rare progressive cortical congenital cataract family and explored its pathogenesis using purified protein and mutant related cataract-cell models. Due to its low solubility and poor structural stability, the ßB2 W151R mutation was prone to aggregation. Moreover, the W151R mutation enhanced the exposure of the hydrophobic side chains in the fourth Greek Key motif, which were readily degraded by trypsin. However, upon the administration of lanosterol, the negative effect of the W151R mutation was reversed. Therefore, lanosterol is a potential therapeutic option for cataracts.
Assuntos
Catarata/congênito , Lanosterol/uso terapêutico , Cristalino/patologia , Agregação Patológica de Proteínas/genética , Cadeia B de beta-Cristalina/genética , Catarata/tratamento farmacológico , Catarata/genética , Catarata/patologia , Pré-Escolar , Análise Mutacional de DNA , Feminino , Células HEK293 , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Lanosterol/farmacologia , Cristalino/efeitos dos fármacos , Masculino , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Linhagem , Agregação Patológica de Proteínas/congênito , Agregação Patológica de Proteínas/tratamento farmacológico , Conformação Proteica em Folha beta/efeitos dos fármacos , Conformação Proteica em Folha beta/genética , Proteólise/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Tripsina/metabolismo , Cadeia B de beta-Cristalina/química , Cadeia B de beta-Cristalina/isolamento & purificação , Cadeia B de beta-Cristalina/metabolismoRESUMO
An in vitro effect of (+)MK-801 (dizocilpine), an inhibitor of the glutamate/NMDA and nicotinic acetylcholine receptors, on the Aß[1-42] and Aß[1-40] peptides is described and compared to that of memantine. Memantine has been approved by the U.S. Food and Drug Administration for the treatment of mild-moderate Alzheimer's disease. Both compounds accelerated the formation of a ß-sheet structure by Aß[1-42], (+)MK-801 more rapidly than memantine, as observed in a thioflavin T fluorescence assay. The acceleration was followed by a decrease in the fluorescence signal that was not observed when the ligand was absent. Nuclear magnetic resonance spectra of the soluble peptides in the presence and absence of (+)MK-801 demonstrated that the monomeric form did not bind (+)MK-801 and that in the presence of (+)MK-801 the concentration of the monomeric form progressively decreased. Small angle X-ray scattering confirmed that the presence of (+)MK-801 resulted in a more rapid and characteristic transition to an insoluble form. These results suggest that (+)MK-801 and memantine accelerate the transition of Aß[1-42] and Aß[1-40] to ThT-negative insoluble forms.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/efeitos dos fármacos , Maleato de Dizocilpina/farmacologia , Memantina/farmacologia , Benzotiazóis , Antagonistas de Aminoácidos Excitatórios/farmacologia , Corantes Fluorescentes , Humanos , Técnicas In Vitro , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/efeitos dos fármacos , Conformação Proteica em Folha beta/efeitos dos fármacos , Espectrometria de FluorescênciaRESUMO
Fibrillar aggregates of amyloid-ß (Aß) are the main component of plaques lining the cerebrovasculature in cerebral amyloid angiopathy. As the predominant Aß isoform in vascular deposits, Aß40 is a valuable target in cerebral amyloid angiopathy research. However, the slow process of Aß40 aggregation in vitro is a bottleneck in the search for Aß-targeting molecules. In this study, we sought a method to accelerate the aggregation of Aß40 in vitro, to improve experimental screening procedures. We evaluated the aggregating ability of bicine, a biological buffer, using various in vitro methods. Our data suggest that bicine promotes the aggregation of Aß40 with high speed and reproducibility, yielding a mixture of aggregates with significant ß-sheet-rich fibril formation and toxicity.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Angiopatia Amiloide Cerebral/patologia , Glicina/análogos & derivados , Fragmentos de Peptídeos/metabolismo , Agregados Proteicos/efeitos dos fármacos , Peptídeos beta-Amiloides/toxicidade , Animais , Linhagem Celular , Sobrevivência Celular , Glicina/farmacologia , Humanos , Camundongos , Neurônios , Fragmentos de Peptídeos/toxicidade , Conformação Proteica em Folha beta/efeitos dos fármacosRESUMO
Rheumatoid arthritis is a rheumatic disease for which a healing treatment does not presently exist. Silk fibroin has been extensively studied for use in drug delivery systems due to its uniqueness, versatility and strong clinical track record in medicine. However, in general, natural polymeric materials are not mechanically stable enough, and have high rates of biodegradation. Thus, synthetic materials such as gellan gum can be used to produce composite structures with biological signals to promote tissue-specific interactions while providing the desired mechanical properties. In this work, we aimed to produce hydrogels of tyramine-modified gellan gum with silk fibroin (Ty-GG/SF) via horseradish peroxidase (HRP), with encapsulated betamethasone, to improve the biocompatibility and mechanical properties, and further increase therapeutic efficacy to treat rheumatoid arthritis (RA). The Ty-GG/SF hydrogels presented a ß-sheet secondary structure, with gelation time around 2-5 min, good resistance to enzymatic degradation, a suitable injectability profile, viscoelastic capacity with a significant solid component and a betamethasone-controlled release profile over time. In vitro studies showed that Ty-GG/SF hydrogels did not produce a deleterious effect on cellular metabolic activity, morphology or proliferation. Furthermore, Ty-GG/SF hydrogels with encapsulated betamethasone revealed greater therapeutic efficacy than the drug applied alone. Therefore, this strategy can provide an improvement in therapeutic efficacy when compared to the traditional use of drugs for the treatment of rheumatoid arthritis.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Betametasona/farmacologia , Fibroínas/farmacologia , Hidrogéis/farmacologia , Inflamação/tratamento farmacológico , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Artrite Reumatoide/patologia , Betametasona/química , Técnicas de Cultura de Células , Sistemas de Liberação de Medicamentos/métodos , Fibroínas/química , Humanos , Hidrogéis/química , Inflamação/patologia , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/farmacologia , Conformação Proteica em Folha beta/efeitos dos fármacos , Coelhos , Engenharia Tecidual , Tiramina/química , Tiramina/farmacologiaRESUMO
In this study, inhibitory kinetics of Nuciferine and Methyl Ganoderate extrated from Lotus Leaves and Ganoderma lucidum on Mucor miehei Lipase were studied first. The molecular structure of Nuciferine and Methyl Ganoderate were determined. The inhibitory effects of two extracts on lipase were reversible, with the IC50 values of 0.194 and 0.332 mg/mL, respectively. The inhibition kinetic analysis by Lineweaver-Burk plots showed that they were a mixed-type inhibitor of lipase, with inhibition constants KI of 0.16 and 0.29 mg/mL, and KIS of 0.36 and 0.49 mg/mL, respectively. Results of spectral analysis showed that the UV absorption and the molecule fluorescence spectrum of the lipase hydrolyzate were significantly decreased after the inhibitor was added. The molecular docking further suggested that the interaction site between the active substance and inhibitor was located in an α-helix and a ß-sheet of the lipase, and the lipase active site was interfered by the inhibitor near the cap structure. In addition, the proliferation and differentiation of 3 T3-L1 preadipocytes were inhibited by two extracts. Total triglycerides and cholesterol were significantly reduced in the cells. The results confirmed that Nuciferine and Methyl Ganoderate can be used as potential obesity treatment drugs.
Assuntos
Aporfinas/farmacologia , Lipase/metabolismo , Rhizomucor/efeitos dos fármacos , Triterpenos/farmacologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Animais , Fármacos Antiobesidade/farmacologia , Domínio Catalítico/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colesterol/metabolismo , Cinética , Camundongos , Simulação de Acoplamento Molecular/métodos , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Conformação Proteica em alfa-Hélice/efeitos dos fármacos , Conformação Proteica em Folha beta/efeitos dos fármacos , Reishi/química , Triglicerídeos/metabolismoRESUMO
Irreversible aggregation can extremely limit the bioavailability and therapeutic activity of peptide-based drugs. There is therefore an urgent demand of effective strategy to control peptide aggregation. Recently, we found that tyrosine nitration at certain sites of peptide can effectively inhibit its aggregation. This minor modification may be an ideal strategy to the rational design of peptide-based drugs with low aggregation propensity yet without loss of bioactivity. Human calcitonin (hCT) is such a peptide hormone known for its hypocalcaemic effect but has limited pharmaceutical potential due to a high tendency to aggregate. In this study, by using multiple techniques including Fluorescence, TEM, Nu-PAGE and CD, we demonstrated that Y12 nitration of hCT would significantly inhibit its self-assembles, and we also found that this modification would not only reduce the cytotoxicity induced by peptide aggregation, but also had little effect on its potency. This finding may provide a novel strategy for clinically application of hCT instead of sCT.
Assuntos
Calcitonina/farmacologia , Nitrobenzenos/química , Multimerização Proteica/efeitos dos fármacos , Tirosina/química , Sequência de Aminoácidos , Animais , Calcitonina/química , Calcitonina/fisiologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Conformação Proteica em Folha beta/efeitos dos fármacosRESUMO
Our recent publication illustrates the critical role of phenylalanine-mediated aromatic-aromatic interactions in determining the assembly of peptidic ß-sheets. However, the effect of phenylalanine number on regulating the assembly efficacy of peptidic ß-sheets remains poorly understood. We herein evaluate the assembly efficacy of ß-sheets of a series of oligopeptides which contain 0, 1, 2, or 3 phenylalanine in their molecular backbones. In our assembly system, two phenylalanine (2F) is the minimum number for driving the assembly of ß-sheets of oligopeptides. Oligopeptides with three phenylalanine (3F) show significantly increased assembly efficacy of ß-sheets compared to that with 2F. These results suggest a positive correlation between the phenylalanine number and assembly efficacy of ß-sheets. By improving the assembly efficacy of ß-sheets, we further develop a highly sensitive HIV analytical system in which the specific binding of ß-sheets with Congo Red induces enhanced fluorescence. For HIV p24 detection, the 3F-based analytical system (0.61 pg/mL) shows a significantly lower limit of detection (LOD) than the 2F-based analytical system (2.44 pg/mL), both of which are more sensitive than commercial ELISA (5 pg/mL) used in the clinic. This work not only illustrates the effect of phenylalanine number on regulating the assembly efficacy of ß-sheets but also provides a guideline for the construction of a highly sensitive analytical system of disease diagnosis.
Assuntos
Proteína do Núcleo p24 do HIV/sangue , HIV/química , Conformação Proteica em Folha beta/efeitos dos fármacos , Sangue/virologia , Vermelho Congo/química , Vermelho Congo/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Proteína do Núcleo p24 do HIV/química , Proteína do Núcleo p24 do HIV/metabolismo , Humanos , Limite de Detecção , Fenilalanina/química , Ligação ProteicaRESUMO
One innate immune response in insects is the proteolytic activation of hemolymph prophenoloxidase (proPO), regulated by protease inhibitors called serpins. In the inhibition reaction of serpins, a protease cleaves a peptide bond in a solvent-exposed reactive center loop (RCL) of the serpin, and the serpin undergoes a conformational change, incorporating the amino-terminal segment of the RCL into serpin ß-sheet A as a new strand. This results in an irreversible inhibitory complex of the serpin with the protease. We synthesized four peptides with sequences from the hinge region in the RCL of Manduca sexta serpin-3 and found they were able to block serpin-3 inhibitory activity, resulting in suppression of inhibitory protease-serpin complex formation. An RCL-derived peptide with the sequence Ser-Val-Ala-Phe-Ser (SVAFS) displayed robust blocking activity against serpin-3. Addition of acetyl-SVAFS-amide to hemolymph led to unregulated proPO activation. Serpin-3 associated with Ac-SVAFS-COO- had an altered circular dichroism spectrum and enhanced thermal resistance to change in secondary structure, indicating that these two molecules formed a binary complex, most likely by insertion of the peptide into ß-sheet A. The interference of RCL-derived peptides with serpin activity may lead to new possibilities of "silencing" arthropod serpins with unknown functions for investigation of their physiological roles.
Assuntos
Catecol Oxidase/química , Precursores Enzimáticos/química , Manduca/química , Peptídeos/farmacologia , Serpinas/química , Animais , Catecol Oxidase/antagonistas & inibidores , Catecol Oxidase/ultraestrutura , Precursores Enzimáticos/antagonistas & inibidores , Precursores Enzimáticos/ultraestrutura , Hemolinfa/enzimologia , Imunidade Inata/efeitos dos fármacos , Peptídeo Hidrolases/química , Peptídeo Hidrolases/ultraestrutura , Peptídeos/síntese química , Peptídeos/química , Conformação Proteica em Folha beta/efeitos dos fármacos , Serpinas/ultraestruturaRESUMO
The effect of adding ethylammonium nitrate (EAN), which is an ionic liquid (IL), on the aggregate formation of α-synuclein (α-Syn) in aqueous solution has been investigated. FTIR and Raman spectroscopy were used to investigate changes in the secondary structure of α-Syn and in the states of water molecules and EAN. The results presented here show that the addition of EAN to α-Syn causes the formation of an intermolecular ß-sheet structure in the following manner: native disordered state â polyproline II (PPII)-helix â intermolecular ß-sheet (α-Syn amyloid-like aggregates: α-SynA). Although cations and anions of EAN play roles in masking the charged side chains and PPII-helix-forming ability involved in the formation of α-SynA, water molecules are not directly related to its formation. We conclude that EAN-induced α-Syn amyloid-like aggregates form at hydrophobic associations in the middle of the molecules after masking the charged side chains at the N- and C-terminals of α-Syn.
Assuntos
Agregados Proteicos , Compostos de Amônio Quaternário/química , alfa-Sinucleína/química , Amiloide/síntese química , Amiloide/química , Precipitação Química/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Agregados Proteicos/efeitos dos fármacos , Conformação Proteica em Folha beta/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Compostos de Amônio Quaternário/farmacologia , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Água/químicaRESUMO
Cellulose nanocrystals (CNC) were successfully prepared from wheat bran, and their effects on the gelling properties and microstructure of heat-induced whey protein isolate (WPI) gels were investigated. The results showed that the water holding capacity, gel strength, viscoelasticity, and thermal stability of the composite gels were improved by increasing the CNC concentration from 0 to 1.0 % (w/v). The incorporation of CNC restricted water mobility and facilitated conformation conversion of the secondary structure from α-helix to ß-sheet. CNC has good compatibility with the protein matrixes at relatively low concentrations. At higher CNC concentrations, the agglomerated CNC can serve as an active dehydrating agent to absorb moisture in the protein matrixes, which promotes unfolding and cross-linking of the protein molecules. Moreover, the active filling effects of CNC contributed to the formation of a compact and homogeneous gel structure. Therefore, naturally sourced CNC is suggested as a potential gel modifier in food industry.
Assuntos
Celulose/química , Nanopartículas/química , Conformação Proteica/efeitos dos fármacos , Proteínas do Soro do Leite/química , Fibras na Dieta , Indústria Alimentícia , Géis/química , Temperatura Alta , Humanos , Conformação Proteica em alfa-Hélice/efeitos dos fármacos , Conformação Proteica em Folha beta/efeitos dos fármacos , Água/química , Proteínas do Soro do Leite/ultraestruturaRESUMO
Tauopathy belongs to a various class of neurodegenerative diseases in which insoluble Tau aggregates are formed, and cellular function is lost, leading to neuronal death. Various therapeutic strategies have been investigated, and many drugs have been proposed as a cure for these diseases but their toxicity and adverse side effects, limit their consumption by humans. Alternatively, the use of non-toxic medicine without any adverse or undesirable secondary effects like nutrients, vitamins, as well as herbal and mineral supplements is common and continues to increase each year. Folic acid is a form of vitamin B and plays a vital role in the synthesis of nucleic acid, methionine regeneration, and in shuttling one-carbon units essential for normal cell division and growth. We investigated the interaction between folic acid and Tau protein, then experimental and theoretical evidence were provided for the suppressing effects of folic acid on Tau fibril formation. The obtained results showed that folic acid could interact with Tau through a spontaneous binding process, mainly by hydrophobic forces, and this interaction leads to a decline in protein-protein interactions through stabilizing its native state, limiting successful Tau-seed oligomerization and consequently decelerating polymerization of Tau amyloid aggregates.
Assuntos
Amiloide/química , Ácido Fólico/farmacologia , Agregados Proteicos/efeitos dos fármacos , Proteínas tau/química , Simulação de Acoplamento Molecular , Conformação Proteica em Folha beta/efeitos dos fármacosRESUMO
Neurofibrillary tangles of the Tau protein and plaques of the amyloid ß peptide are hallmarks of Alzheimer's disease (AD), which is characterized by the conversion of monomeric proteins/peptides into misfolded ß-sheet rich fibrils. Halting the fibrillation process and disrupting the existing aggregates are key challenges for AD drug development. Previously, we performed in vitro high-throughput screening for the identification of potent inhibitors of Tau aggregation using a proxy model, a highly aggregation-prone hexapeptide fragment 306VQIVYK311 (termed PHF6) derived from Tau. Here we have characterized a hit molecule from that screen as a modulator of Tau aggregation using in vitro, in silico, and in vivo techniques. This molecule, an anthraquinone derivative named Purpurin, inhibited ~ 50% of PHF6 fibrillization in vitro at equimolar concentration and disassembled pre-formed PHF6 fibrils. In silico studies showed that Purpurin interacted with key residues of PHF6, which are responsible for maintaining its ß-sheets conformation. Isothermal titration calorimetry and surface plasmon resonance experiments with PHF6 and full-length Tau (FL-Tau), respectively, indicated that Purpurin interacted with PHF6 predominantly via hydrophobic contacts and displayed a dose-dependent complexation with FL-Tau. Purpurin was non-toxic when fed to Drosophila and it significantly ameliorated the AD-related neurotoxic symptoms of transgenic flies expressing WT-FL human Tau (hTau) plausibly by inhibiting Tau accumulation and reducing Tau phosphorylation. Purpurin also reduced hTau accumulation in cell culture overexpressing hTau. Importantly, Purpurin efficiently crossed an in vitro human blood-brain barrier model. Our findings suggest that Purpurin could be a potential lead molecule for AD therapeutics.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Antraquinonas/farmacologia , Oligopeptídeos/genética , Agregados Proteicos/efeitos dos fármacos , Proteínas tau/genética , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/efeitos dos fármacos , Animais , Animais Geneticamente Modificados/genética , Barreira Hematoencefálica/efeitos dos fármacos , Modelos Animais de Doenças , Drosophila melanogaster/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/genética , Fosforilação/efeitos dos fármacos , Conformação Proteica em Folha beta/efeitos dos fármacos , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genéticaRESUMO
Globular proteins are typically unfolded by SDS to form protein-decorated micelle-like structures. Several proteins have been shown subsequently to refold by addition of the nonionic surfactant octaethylene glycol monododecyl ether (C12E8). Thus SDS converts ß-lactoglobulin, which has mainly ß-sheet secondary structure, into a state rich in α-helicality, while addition of C12E8 leads to refolding and recovery of the original ß-sheet structure. Here we extend these studies to the large ß-sheet-rich cellulase Cel7b from Humicola insolens whose enzymatic activity provides a very sensitive refolding parameter. The enzymes widespread usage in the detergent industry makes it an obvious model system for protein-surfactant interactions. SDS-unfolding and subsequent refolding using C12E8 were investigated at pHâ¯4.2 using near- and far-UV circular dichroism (CD), small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), size-exclusion chromatography (SEC) and activity measurements. The Cel7b:SDS complex can be described as a random configuration of 3-4 connected core-shell structures in which the protein is converted to a mainly α-helical secondary structure. Addition of C12E8 recovers almost all the secondary structure, part of the tertiary structure, about 50% of the activity and dissociates part of the protein population completely from detergent micelles. The lack of complete refolding may be due to charge neutralisation of Cel7b by SDS, kinetically trapping the enzyme into aggregated structures. In support of this, aggregates did not form when C12E8 was first mixed with Cel7b followed by addition of SDS. Formation of such aggregates may be a general phenomenon hampering quantitative refolding from the SDS-denatured state.
Assuntos
Celulase/química , Desdobramento de Proteína/efeitos dos fármacos , Dodecilsulfato de Sódio/farmacologia , Tensoativos/farmacologia , Calorimetria , Celulase/efeitos dos fármacos , Dicroísmo Circular , Cinética , Polietilenoglicóis/farmacologia , Conformação Proteica/efeitos dos fármacos , Conformação Proteica em alfa-Hélice/efeitos dos fármacos , Conformação Proteica em Folha beta/efeitos dos fármacos , Desnaturação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Espalhamento a Baixo Ângulo , Sordariales/enzimologia , Tensoativos/química , Difração de Raios XRESUMO
Coordination complexes have emerged as prominent modulators of amyloid aggregation via their interaction with the N-terminal histidine residues of amyloid-ß (Aß). Herein, we report the synthesis and characterization of a novel cobalt(III) Schiff base complex with methylamine axial ligands, and we present both computational and experimental data demonstrating the reduction of ß-sheet formation by this complex. The computations include molecular dynamics simulations of both monomeric and pentameric Aß, which demonstrate decreased formation of ß-sheet structures, destabilization of preformed ß-sheets, and suppression of aggregation. These results are consistent with a dose dependence in experimental bulk aggregation data using thioflavin T fluorescence, and overall this study demonstrates useful drug activity of the cobalt complex.
Assuntos
Amiloide/química , Cobalto/química , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Agregados Proteicos/efeitos dos fármacos , Bases de Schiff/química , Modelos Moleculares , Conformação Proteica em Folha beta/efeitos dos fármacosRESUMO
Anthrax is an ancient and deadly disease caused by the spore-forming bacterial pathogen Bacillus anthracis. At present, anthrax mostly affects wildlife and livestock, although it remains a concern for human public health-primarily for people who handle contaminated animal products and as a bioterrorism threat due to the high resilience of spores, a high fatality rate of cases and the lack of a civilian vaccination programme1,2. The cell surface of B. anthracis is covered by a protective paracrystalline monolayer-known as surface layer or S-layer-that is composed of the S-layer proteins Sap or EA1. Here, we generate nanobodies to inhibit the self-assembly of Sap, determine the structure of the Sap S-layer assembly domain (SapAD) and show that the disintegration of the S-layer attenuates the growth of B. anthracis and the pathology of anthrax in vivo. SapAD comprises six ß-sandwich domains that fold and support the formation of S-layers independently of calcium. Sap-inhibitory nanobodies prevented the assembly of Sap and depolymerized existing Sap S-layers in vitro. In vivo, nanobody-mediated disruption of the Sap S-layer resulted in severe morphological defects and attenuated bacterial growth. Subcutaneous delivery of Sap inhibitory nanobodies cleared B. anthracis infection and prevented lethality in a mouse model of anthrax disease. These findings highlight disruption of S-layer integrity as a mechanism that has therapeutic potential in S-layer-carrying pathogens.