RESUMO
Transmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurodegenerative diseases with no approved therapeutics. TSE pathology is characterized by abnormal accumulation of amyloidogenic and infectious prion protein conformers (PrPSc) in the central nervous system. Herein, we examined the role of gallate group in green tea catechins in modulating the aggregation of human prion protein (HuPrP) using two green tea constituents i.e., epicatechin 3-gallate (EC3G; with intact gallate ring) and epigallocatechin (EGC; without gallate ring). Molecular docking indicated distinct differences in hydrogen bonding and hydrophobic interactions of EC3G and EGC at the ß2-α2 loop of HuPrP. These differences were substantiated by 44-fold higher KD for EC3G as compared to EGC with the former significantly reducing Thioflavin T (ThT) binding aggregates of HuPrP. Conformational alterations in HuPrP aggregates were validated by particle sizing, AFM analysis and A11 and OC conformational antibodies. As compared to EGC, EC3G showed relatively higher reduction in toxicity and cellular internalization of HuPrP oligomers in Neuro-2a cells. Additionally, EC3G also displayed higher fibril disaggregating properties as observed by ThT kinetics and electron microscopy. Our observations were supported by molecular dynamics (MD) simulations that showed markedly reduced α2-α3 and ß2-α2 loop mobilities in presence of EC3G that may lead to constriction of HuPrP conformational space with lowered ß-sheet conversion. In totality, gallate moiety of catechins play key role in modulating HuPrP aggregation, and toxicity and could be a new structural motif for designing therapeutics against prion diseases and other neurodegenerative disorders.
Assuntos
Catequina , Doenças Priônicas , Príons , Humanos , Príons/química , Proteínas Priônicas/química , Chá , Simulação de Acoplamento Molecular , Catequina/farmacologiaRESUMO
Anticancer drugs, such as fluorouracil (5-FU), oxaliplatin, and doxorubicin (Dox) are commonly used to treat colorectal cancer (CRC); however, owing to their low response rate and adverse effects, the development of efficient drug delivery systems (DDSs) is required. The cellular prion protein PrPC, which is a cell surface glycoprotein, has been demonstrated to be overexpressed in CRC, however, there has been no research on the development of PrPC-targeting DDSs for targeted drug delivery to CRC. In this study, PrPC aptamer (Apt)-conjugated gold nanoparticles (AuNPs) were synthesized for targeted delivery of Dox to CRC. Thiol-terminated PrPC-Apt was conjugated to AuNPs, followed by hybridization of its complementary DNA for drug loading. Finally, Dox was loaded onto the AuNPs to synthesize PrPC-Apt-functionalized doxorubicin-oligomer-AuNPs (PrPC-Apt DOA). The PrPC-Apt DOA were spherical nanoparticles with an average diameter of 20 nm. Treatment of CRC cells with PrPC-Apt DOA induced reactive oxygen species generation by decreasing catalase and superoxide dismutase activities. In addition, treatment with PrPC-Apt DOA inhibited mitochondrial functions by decreasing the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha, complex 4 activity, and oxygen consumption rates. Compared to free Dox, PrPC-Apt DOA decreased proliferation and increased apoptosis of CRC cells to a greater degree. In this study, we demonstrated that PrPC-Apt DOA targeting could effectively deliver Dox to CRC cells. PrPC-Apt DOA can be used as a treatment for CRC, and have the potential to replace existing anticancer drugs, such as 5-FU, oxaliplatin, and Dox.
Assuntos
Aptâmeros de Nucleotídeos/química , Neoplasias Colorretais/tratamento farmacológico , Doxorrubicina/administração & dosagem , Doxorrubicina/uso terapêutico , Sistemas de Liberação de Medicamentos , Ouro/química , Nanopartículas Metálicas/química , Proteínas Priônicas/química , Apoptose/efeitos dos fármacos , Catalase/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/farmacologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Recent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. These techniques allowed us to design a drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein levels by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP and identified four different small molecule ligands for this conformer, all capable of selectively lowering the load of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Doenças Priônicas/tratamento farmacológico , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Dobramento de Proteína , Animais , Sítios de Ligação , Simulação por Computador , Retículo Endoplasmático/metabolismo , Fibroblastos , Células HEK293 , Humanos , Ligantes , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Reprodutibilidade dos TestesRESUMO
Endoplasmic reticulum (ER) stress is a common threat to photoreceptors during the pathogenesis of chronic retinopathies and often results in irreversible visual impairment. 2,3,5,6-Tetramethylpyrazine (TMP), which possesses many beneficial pharmacological activities, is a potential drug that could be used to protect photoreceptors. In the present study, we found that the cellular growth rate of 661 W cells cultured under low glucose conditions was lower than that of control cells, while the G2/M phase of the cell cycle was longer. We further found that the mitochondrial membrane potential (ΔΨm) was lower and that ER stress factor expression was increased in 661 W cells cultured under low glucose conditions. TMP reversed these trends. Visual function and cell counts in the outer nuclear layer (ONL) were low and the TUNEL-positive rate in the ONL was high in a C3H mouse model of spontaneous retinal degeneration. Similarly, visual function was decreased, and the TUNEL-positive rate in the ONL was increased in fasted C57/BL6j mice compared with control mice. On the other hand, ER stress factor expression was found to be increased in the retinas of both mouse models, as shown by reverse transcription real-time PCR (RT-qPCR) and western blotting. TMP reversed the physiological and molecular biological variations observed in both mouse models, and ATF4 expression was enhanced again. Further investigation by using western blotting illustrated that the proportion of insoluble prion protein (PRP) versus soluble PRP was reduced both in vitro and in vivo. Taken together, these results suggest that TMP increased the functions of photoreceptors by alleviating ER stress in vitro and in vivo, and the intrinsic mechanism was the ATF4-mediated inhibition of PRP aggregation. TMP may potentially be used clinically as a therapeutic agent to attenuate the functional loss of photoreceptors during the pathogenesis of chronic retinopathies. KEY MESSAGES: ⢠Already known: TMP is a beneficial drug mainly used in clinic to enhance organ functions, and the intrinsic mechanism is still worthy of exploring. ⢠New in the study: We discovered that TMP ameliorated retinal photoreceptors function via ER stress alleviation, which was promoted by ATF4-mediated inhibition of PRP aggregation. ⢠Application prospect: In prospective clinical practices, TMP may potentially be used in the clinic as a therapeutic agent to attenuate the photoreceptors functional reduction in chronic retinopathies.
Assuntos
Fator 4 Ativador da Transcrição/fisiologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas Priônicas/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/prevenção & controle , Pirazinas/farmacologia , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Degeneração Retiniana/prevenção & controle , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Eletrorretinografia , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Jejum , Feminino , Glucose/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Proteínas Priônicas/química , Agregação Patológica de Proteínas/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/fisiopatologia , Método Simples-Cego , Solubilidade , Organismos Livres de Patógenos Específicos , Transcrição Gênica/efeitos dos fármacosRESUMO
In Creutzfeldt Jakob, Alzheimer and Parkinson diseases, copper metalloproteins such as prion, amyloid protein precursor and α-synuclein are able to protect against free radicals by reduction from cupric Cu+2 to cupreous Cu+. In these pathologies, a regional copper (Cu) brain decrease correlated with an iron, zinc or manganese (Mn) increase has previously been observed, leading to local neuronal death and abnormal deposition of these metalloproteins in ß-sheet structures. In this study we demonstrate the protective effect of Cu metalloproteins against deleterious free-radical effects. With neuroblastoma SH-SY5Y cell cultures, we show that bovine brain prion protein in Cu but not Mn form prevents free radical-induced neuronal death. The survival ratio of SH-SY5Y cells has been measured after UV irradiation (free radical production), when the incubating medium is supplemented with bovine brain homogenate in native, Cu or Mn forms. This ratio, about 28% without any addition or with bovine brain protein added in Mn form, increases by as much as 54.73% with addition to the culture medium of native bovine brain protein and by as much as 95.95% if the addition is carried out in cupric form. This protective effect of brain copper protein against free radical-induced neuronal death has been confirmed with Inductively Coupled Plasma Mass Spectrometry Mn and Cu measurement in bovine brain homogenates: respectively lower than detection limit and 9.01µg/g dry weight for native form; lower than detection limit and 825.85µg/g dry weight for Cu-supplemented form and 1.75 and 68.1µg/g dry weight in Mn-supplemented brain homogenate.
Assuntos
Cobre/metabolismo , Neuroblastoma/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Animais , Bovinos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cobre/farmacologia , Radicais Livres/antagonistas & inibidores , Radicais Livres/química , Radicais Livres/farmacologia , Humanos , Manganês/metabolismo , Manganês/farmacologia , Proteínas Priônicas/farmacologia , Células Tumorais CultivadasRESUMO
Copper plays a critical role in prion protein (PrP) physiology. Cu(2+) binds with high affinity to the PrP N-terminal octarepeat (OR) domain, and intracellular copper promotes PrP expression. The molecular details of copper coordination within the OR are now well characterized. Here we examine how Cu(2+) influences the interaction between the PrP N-terminal domain and the C-terminal globular domain. Using nuclear magnetic resonance and copper-nitroxide pulsed double electron-electron resonance, with molecular dynamics refinement, we localize the position of Cu(2+) in its high-affinity OR-bound state. Our results reveal an interdomain cis interaction that is stabilized by a conserved, negatively charged pocket of the globular domain. Interestingly, this interaction surface overlaps an epitope recognized by the POM1 antibody, the binding of which drives rapid cerebellar degeneration mediated by the PrP N terminus. The resulting structure suggests that the globular domain regulates the N-terminal domain by binding the Cu(2+)-occupied OR within a complementary pocket.