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1.
Adv Exp Med Biol ; 1131: 93-129, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646508

RESUMO

Plasma membrane Ca2+ transport ATPases (PMCA1-4, ATP2B1-4) are responsible for removing excess Ca2+ from the cell in order to keep the cytosolic Ca2+ ion concentration at the low level essential for normal cell function. While these pumps take care of cellular Ca2+ homeostasis they also change the duration and amplitude of the Ca2+ signal and can create Ca2+ gradients across the cell. This is accomplished by generating more than twenty PMCA variants each having the character - fast or slow response, long or short memory, distinct interaction partners and localization signals - that meets the specific needs of the particular cell-type in which they are expressed. It has become apparent that these pumps are essential to normal tissue development and their malfunctioning can be linked to different pathological conditions such as certain types of neurodegenerative and heart diseases, hearing loss and cancer. In this chapter we summarize the complexity of PMCA regulation and function under normal and pathological conditions with particular attention to recent developments of the field.


Assuntos
Membrana Celular , ATPases Transportadoras de Cálcio da Membrana Plasmática , Animais , Membrana Celular/enzimologia , Membrana Celular/patologia , Citosol/metabolismo , Homeostase/fisiologia , Humanos , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo
2.
Adv Exp Med Biol ; 1131: 547-604, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646526

RESUMO

Ca2+ release activated Ca2+ (CRAC) channels represent a primary pathway for Ca2+ to enter non-excitable cells. The two key players in this process are the stromal interaction molecule (STIM), a Ca2+ sensor embedded in the membrane of the endoplasmic reticulum, and Orai, a highly Ca2+ selective ion channel located in the plasma membrane. Upon depletion of the internal Ca2+ stores, STIM is activated, oligomerizes, couples to and activates Orai. This review provides an overview of novel findings about the CRAC channel activation mechanisms, structure and gating. In addition, it highlights, among diverse STIM and Orai mutants, also the disease-related mutants and their implications.


Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio , Membrana Celular , Animais , Cálcio/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/sangue , Canais de Cálcio Ativados pela Liberação de Cálcio/genética , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Mutação , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo
3.
Adv Exp Med Biol ; 1131: 699-718, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646531

RESUMO

Calcium exchanges and homeostasis are finely regulated between cellular organelles and in response to physiological signals. Besides ionophores, including voltage-gated Ca2+ channels, ionotropic neurotransmitter receptors, or Store-operated Ca2+ entry, activity of regulatory intracellular proteins finely tune Calcium homeostasis. One of the most intriguing, by its unique nature but also most promising by the therapeutic opportunities it bears, is the sigma-1 receptor (Sig-1R). The Sig-1R is a chaperone protein residing at mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), where it interacts with several partners involved in ER stress response, or in Ca2+ exchange between the ER and mitochondria. Small molecules have been identified that specifically and selectively activate Sig-1R (Sig-1R agonists or positive modulators) at the cellular level and that also allow effective pharmacological actions in several pre-clinical models of pathologies. The present review will summarize the recent data on the mechanism of action of Sig-1R in regulating Ca2+ exchanges and protein interactions at MAMs and the ER. As MAMs alterations and ER stress now appear as a common track in most neurodegenerative diseases, the intracellular action of Sig-1R will be discussed in the context of the recently reported efficacy of Sig-1R drugs in pathologies like Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis.


Assuntos
Membrana Celular , Estresse do Retículo Endoplasmático , Doenças Neurodegenerativas , Receptores sigma , Membrana Celular/metabolismo , Membrana Celular/patologia , Humanos , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Receptores sigma/metabolismo
4.
Adv Exp Med Biol ; 1141: 1-12, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31571163

RESUMO

Absorption, distribution, and excretion of drugs are involved in drug transport across plasma membrane, most of which are mediated by drug transporters. These drug transporters are generally divided into solute carrier (SLC) family and ATP-binding cassette (ABC) family. These transporters not only mediate transport of therapeutic drugs across membrane but also transport various kinds of endogenous compounds. Thus besides being participated in disposal of drug and its clinical efficacy/toxicity, these transporters also play vital roles in maintaining cell homeostasis via regulating transport of endogenous compounds. This chapter will outline classification of drug transporters, their roles in drug disposal/drug response, and remote communication between tissues/organs.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas Carreadoras de Solutos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Comunicação Celular , Membrana Celular/metabolismo , Humanos , Preparações Farmacêuticas/metabolismo , Proteínas Carreadoras de Solutos/metabolismo
5.
Phys Chem Chem Phys ; 21(41): 22679-22694, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31595905

RESUMO

Peptide misfolding and aberrant assembly in membranous micro-environments have been associated with numerous neurodegenerative diseases. The biomolecular mechanisms and biophysical implications of these amyloid membrane interactions have been under extensive research and can assist in understanding disease pathogenesis and potential development of rational therapeutics. But, the complex nature and diversity of biomolecular interactions, structural transitions, and dependence on local environmental conditions have made accurate microscopic characterization challenging. In this review, using cases of Alzheimer's disease (amyloid-beta peptide), Parkinson's disease (alpha-synuclein peptide) and Huntington's disease (huntingtin protein), we illustrate existing challenges in experimental investigations and summarize recent relevant numerical simulation studies into amyloidogenic peptide-membrane interactions. In addition we project directions for future in silico studies and discuss shortcomings of current computational approaches.


Assuntos
Biologia Computacional , Lipídeos/química , Doenças Neurodegenerativas , Dobramento de Proteína , Proteínas Amiloidogênicas/metabolismo , Membrana Celular/metabolismo , Simulação por Computador , Humanos , Metabolismo dos Lipídeos , Doenças Neurodegenerativas/fisiopatologia , Peptídeos/metabolismo
6.
Phys Chem Chem Phys ; 21(36): 20211-20218, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31486459

RESUMO

In spite of their well-known side effects, the nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most commonly prescribed medications for their antipyretic and anti-inflammatory actions. Interaction of NSAIDs with the plasma membrane plays a vital role in their therapeutic actions and defines many of their side effects. In the present study, we investigate the effects of three NSAIDs, aspirin, ibuprofen, and indomethacin, on the structure and dynamics of a model plasma membrane using a combination of small angle neutron scattering (SANS) and neutron spin echo (NSE) techniques. The SANS and NSE measurements were carried out on a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membrane, with and without NSAIDs, at two different temperatures, 11 °C and 37 °C, where the DMPC membrane is in the gel and fluid phase, respectively. SANS data analysis shows that incorporation of NSAIDs leads to bilayer thinning of the membrane in both the phases. The dynamic properties of the membrane are represented by the intermediate scattering functions for NSE data, which are successfully described by the Zilman and Granek model. NSE data analysis shows that in both gel and fluid phases, addition of NSAIDs results in a decrease in the bending rigidity and compressibility modulus of the membrane, which is more prominent when the membrane is in the gel phase. The magnitude of the effect of NSAIDs on the bending rigidity and compressibility modulus of the membrane in the gel phase follows an order of ibuprofen > aspirin > indomethacin, whereas in the fluid phase, it is in the order of aspirin > ibuprofen > indomethacin. We find that the interaction between NSAIDs and phospholipid membranes is strongly dependent on the chemical structure of the drugs and physical state of the membrane. Mechanical properties of the membrane can be quantified by the membrane's bending rigidity. Hence, the present study reveals that incorporation of NSAIDs modulates the mechanical properties of the membrane, which may affect several physiological processes, particularly those linked to the membrane curvature.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Estruturas da Membrana Celular/química , Estruturas da Membrana Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Nêutrons , Espalhamento a Baixo Ângulo
7.
Phys Chem Chem Phys ; 21(36): 20239-20251, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31490518

RESUMO

The amyloid formation of human islet amyloid polypeptide (hIAPP)-an intrinsically disordered peptide, is associated with type II diabetes. Cellular membranes, especially those composed of negatively-charged lipids, accelerate the hIAPP amyloid fibrillation, and their integrity is disrupted during the aggregation process, leading to cell apoptosis. However, the underlying molecular mechanism is not well understood. Herein, we investigated the conformational dynamics during the interactions of hIAPP monomer with POPG membrane bilayer, by carrying out µs-long all-atom molecular dynamics simulations. Starting from the metastable coiled conformations in water, hIAPP monomers tend to adopt transient α-helical and ß-sheet structures when adsorbing to the membrane surface. The amphiphilic N-terminal region further inserts into the membrane interior and is located at the lipid head-tail interface, mainly in turn and α-helical structures. In contrast, the ß-hairpin structures reside on the membrane surface without insertion, and expand laterally with the hydrophobic residues exposed to the solvent. Moreover, the adsorption and insertion of hIAPP monomers induce two distinct local membrane deformations: (1) the hIAPP adsorption on the membrane surface mainly causes membrane bending; (2) the insertion of both turns and α-helices synchronizes with the formation of hydrophobic defects on the POPG membrane, leading to stronger membrane stretching and a longer coherence length of membrane thinning. Based on the structural and dynamical results, we propose that ß-hairpin structures may be a precursor for the fibrillation on the membrane surface due to the flat geometry and hydrophobic regions exposed to solvent, while N-terminal amphiphilic α-helices would facilitate hIAPP assembling into toxic oligomers inside the membrane.


Assuntos
Membrana Celular/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo
8.
Adv Exp Med Biol ; 1159: 5-31, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31502197

RESUMO

Mitochondria and bacteria share a myriad of properties since it is believed that the powerhouses of the eukaryotic cell have evolved from a prokaryotic origin. Ribosomal RNA sequences, DNA architecture and metabolism are strikingly similar in these two entities. Proteins and nucleic acids have been a hallmark for comparison between mitochondria and prokaryotes. In this chapter, similarities (and differences) between mitochondrial and prokaryotic membranes are addressed with a focus on structure-function relationship of different lipid classes. In order to be suitable for the theme of the book, a special emphasis is reserved to the effects of bioactive sphingolipids, mainly ceramide, on mitochondrial membranes and their roles in initiating programmed cell death.


Assuntos
Evolução Biológica , Membrana Celular/química , Lipídeos/química , Mitocôndrias/química , Células Procarióticas/química , Ceramidas , Esfingolipídeos
9.
Adv Exp Med Biol ; 1159: 33-48, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31502198

RESUMO

Are ceramide molecules capable of self-assembling in biological and phospholipid membranes to form ceramide channels: membrane channels capable to translocating proteins through said membranes? A number of papers have been published which support the conclusion that ceramide forms these large channels in membranes. The evidence is extensive and consisting of: flux studies using isolated mitochondria, liposomes and planar membranes; visualization by electron microscopy; elastic deformation studies; and regulation by Bcl-2 family proteins. The evidence supports a structural model of the channel shown to be stable by molecular dynamic simulations and having structural and mechanical properties consistent with multiple experiments. Yet the novelty of this claim raises legitimate questions. Indeed, a recent report questions the existence of ceramide channels based on liposome experiments. This review presents both a comprehensive description of the major observations supporting the case that ceramide channels do exist and addresses the issues raised in the skeptical report.


Assuntos
Membrana Celular/química , Ceramidas/química , Lipossomos/química , Mitocôndrias/química , Fosfolipídeos , Proteínas Proto-Oncogênicas c-bcl-2
10.
Adv Exp Med Biol ; 1159: 79-108, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31502201

RESUMO

Ceramides are the central molecules in sphingolipid metabolism. In addition, they are recognized as important modulators of cell function, playing key roles in several cellular processes that range from cell proliferation to cell death. Moreover, ceramides were implicated in multiple diseases, including cancer, neurodegenerative and metabolic diseases, and also in infection by different pathogens. The mechanisms underlying the diverse biological and pathological actions of ceramides are yet to be fully elucidated. Several lines of evidence suggest that the structural features of ceramides, namely their high hydrophobicity and ability to establish strong H-bond network, are responsible for changes in the biophysical properties of biological membranes that can affect the activity of proteins and activate signaling pathways. Ceramide-induced alterations in membrane biophysical properties might also influence the internalization, trafficking and sorting of lipids, proteins, drugs and even pathogens contributing to cell pathophysiology. In this chapter, we critically discuss the ability of ceramides to form lipid domains with atypical biophysical properties and how these domains can be involved in those processes.


Assuntos
Membrana Celular , Ceramidas/fisiologia , Transdução de Sinais , Humanos , Lipídeos
11.
Adv Exp Med Biol ; 1159: 109-138, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31502202

RESUMO

Despite the advancements in modern medicine, there are still difficulties in diagnosing common illnesses. The invasiveness and price of the tests used to follow up certain diseases can be a barrier to proper patient follow-up. Sphingolipids are a diverse category of lipids. They are structural molecules in cell membranes and signaling molecules involved in the regulation of crucial cell functions, including cell growth, differentiation, proliferation and apoptosis. Recent research has shown that abnormal sphingolipid metabolism is associated with genetic and metabolic disease processes. Given their crucial role to maintain homeostasis within the body, sphingolipids have been investigated as potential biomarkers to predict disease in the population. Here we discuss how sphingolipids levels are altered in different diseases, thus illustrating their possible use as diagnostic and prognostic biomarkers for disease.


Assuntos
Biomarcadores , Transdução de Sinais , Esfingolipídeos , Ciclo Celular , Membrana Celular , Homeostase , Humanos
12.
Chem Commun (Camb) ; 55(82): 12288-12291, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31524898

RESUMO

Here we present a set of fluorescent cages prepared by tethering fluorescent dyes to a photolabile group. The developed molecules enable caging of signalling lipids, their delivery to specific cellular membranes, with further imaging, quantification, and controlled photorelease of active lipids in living cells.


Assuntos
Corantes Fluorescentes/química , Metabolismo dos Lipídeos , Lipídeos/química , Nitrobenzenos/química , Transdução de Sinais , Membrana Celular/química , Células HeLa , Humanos
13.
J Agric Food Chem ; 67(40): 11044-11052, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31545599

RESUMO

Persimmon condensed tannins (PT) are highly polymerized (mDP = 26) and highly galloylated (72%) proanthocyanidins. Its pleiotropic effects in oxidation resistance, neuroprotection, hypolipidemia, and cardio-protection both in vitro and in vivo were widely reported. Because large proanthocyanidins are unlikely to be absorbed in the gastrointestinal tract, it is believed that the interaction of PT with biological membranes may play a crucial role in its biological activities. In the present study, the capacities of PT adsorbing to membrane, partitioning into membrane, and its influence on the membrane fluidity were investigated by fluorescence quenching, isothermal titration calorimetry (ITC) and fluorescence anisotropy measurements in a biomembrane-mimetic system composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), sphingomyelin (SPM), and cholesterol (CHOL). Besides, the effects of PT on the morphology and integrity of the cell membrane were studied by scanning electron microscopy (SEM) and fluorescence staining in the 3T3-L1 cell model. The results suggested that PT could affect cell membrane rafts domains, destroy the cell membrane morphology, and regulate cell membrane fluidity, which might contribute to its biological effects.


Assuntos
Membrana Celular/química , Diospyros/química , Extratos Vegetais/química , Proantocianidinas/química , Animais , Fenômenos Biofísicos , Membrana Celular/metabolismo , Colesterol/química , Colesterol/metabolismo , Frutas/química , Fluidez de Membrana , Camundongos , Células NIH 3T3 , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Extratos Vegetais/metabolismo , Polimerização , Proantocianidinas/metabolismo , Esfingomielinas/química , Esfingomielinas/metabolismo
14.
Chemosphere ; 235: 1116-1124, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31561302

RESUMO

The porous graphitic carbon nitride nanosheets (PCNSs) with high yields were synthesized by using one-step chemical exfoliation method. PCNSs accelerated separation efficiency of photo-generated electron-hole pairs in comparison to bulk graphitic carbon nitride. The PCNS5 (exfoliation for 5 h) exhibited optimal photocatalytic disinfection capability towards Escherichia coli K-12 under simulated solar light irradiation with complete disinfection of 6.5 log10 cfu/mL of E. coil K-12 within 2 h. The enhanced photocatalytic activity of PCNS5 originated from mesoporous nanosheet structure. The possible mechanism of photocatalytic disinfection has proposed that intracellular reactive oxygen species levels and the activities of antioxidant enzymes (e.g., catalase and superoxide dismutase) were enhanced. Transmission electron microscope images observed during photocatalytic disinfection process suggested that the cell membrane was regarded as the first target for oxidation, resulting in a faster leakage of cytoplasmic content and finally degradation of DNA leading to bacterial death. Furthermore, the trapping experiment showed that superoxide radical (•O2-) and holes (h+) were responsible for E. coli K-12 disinfection by PCNS5.


Assuntos
Desinfecção/métodos , Escherichia coli K12/efeitos dos fármacos , Nanoestruturas/química , Nitrilos/toxicidade , Fotólise , Bactérias/efeitos dos fármacos , Membrana Celular/metabolismo , Dano ao DNA , Oxirredução , Porosidade , Espécies Reativas de Oxigênio
16.
Sheng Wu Gong Cheng Xue Bao ; 35(8): 1463-1468, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31441617

RESUMO

We studied the construction of fusion protein TAT-RIG-I-GFP prokaryotic expression vector and verified the function of TAT in transmembrane delivery. First, four pairs of specific primers were designed, and the RIG-I gene of Mallard Duck (Anas platyrhynchos) was cloned. Then, the pET-TAT-RIG-I-GFP and pET-RIG-I-GFP prokaryotic expression vectors were constructed. Meanwhile, they were converted to E. coli BL21 (DE3), which were induced to be expressed after culture. After the purification of His-60 nickel affinity chromatography column and the identification of SDS-PAGE, the purified TAT-RIG-I-GFP and RIG-I-GFP proteins were incubated to DF-1 cells. Finally, fluorescence microscopy was used to observe whether the corresponding fluorescence was produced in DF-1 cells. The results showed that pET-TAT-RIG-I-GFP fusion with TAT showed obvious green fluorescence in DF-1 cells. However, the pET-RIG-I-GFP without TAT cannot display green fluorescence. This shows that TAT-fused protein have successfully delivered DF-1 cells and play a key role in transmembrane delivery. In conclusion, these results provide a solid material basis for further study of antiviral drugs in poultry.


Assuntos
Produtos do Gene tat , Membrana Celular , Primers do DNA , Escherichia coli , Expressão Gênica , Vetores Genéticos , Proteínas Recombinantes de Fusão
17.
Adv Exp Med Biol ; 1155: 977-985, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468461

RESUMO

Taurine (2-aminoethanesulfonic acid) is a sulfur-containing amino acid. It is one of the most abundant free amino acids in many excitable tissues, including the brain, skeletal and cardiac muscles. Physiological actions of taurine are widespread and include regulation of plasma glucose levels, bile acid conjugation, detoxification, membrane stabilization, blood pressure regulation, osmoregulation, neurotransmission, and modulation of mitochondria function and cellular calcium levels. Taurine plays an important role in modulating glutamate and GABA neurotransmission and prevents excitotoxicity in vitro primarily through modulation of intracellular calcium homeostasis. Taurine supplementation prevents age-dependent decline of cognitive functions. Because of the wide spread actions of taurine, its levels are highly regulated through enzymatic biosynthesis or dietary intake. Furthermore, depletion of endogenous or dietary supplementation of exogenous taurine have been shown to induce wide spread actions on multiple organs. Cysteine sulfonic acid decarboxylase (CSAD) was first identified in the liver and is thought to be the rate-limiting enzyme in taurine biosynthesis. CSAD mRNA is expressed in the brain in astrocytes. Homozygous knockout mice lacking CSAD (CSAD-KO) have very reduced taurine content and show severe functional histopathology in the visual system, skeletal system, heart, pancreas and brain. Conversely, dietary supplementation of taurine results in significant health benefits acting through the same organ systems. Fluctuation of taurine bioavailability lead to changes in the expression levels of taurine transporters in neuronal plasma membranes, endothelial cells forming the blood-brain barrier and proximal cells of the kidneys. Suggesting a highly regulated mechanism for maintaining taurine homeostasis and organ systems function. Here we show how alterations in taurine levels directly affect the function of one organ system and through functional interaction and compensatory adaptation; these effects extend to another organ systems with focus on the nervous system.


Assuntos
Sistemas Neurossecretores/fisiologia , Taurina/farmacologia , Animais , Barreira Hematoencefálica , Membrana Celular , Células Endoteliais , Homeostase , Rim , Camundongos , Camundongos Knockout
18.
Int J Nanomedicine ; 14: 5061-5071, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31371947

RESUMO

Background: Photodynamic therapy (PDT) is widely recognized as a promising way to cure cancer. However, the limited tumor homing property of currently available drug delivery systems (DDSs) is the bottleneck for the delivery of photodynamic agents. Purpose: In our study, we decorated silica nanoparticles (SLN) with cell membrane (CM) derived from SGC7901 cells to construct carrier (CM/SLN) which was able to to specifically target the homogenous SGC7901 cells. Materials and methods: Furthermore, the decent drug loading capability of CM/SLN was adopted to load photodynamic agent chlorins e6 (Ce6) to finally construct aDDS suitable for tumor-targeted PDT of gastric cancer. Results: The experimental results suggested that CM/SLN/Ce6 was nano-sized particles with good dispersion and stability in physiological conditions. Moreover, due to the modification of CM,CM/SLN/Ce6 could specifically target the homogenous SGC7901 cells both in vitro and in vivo. Most importantly, further in vivo results demonstrated that the CM/SLN/Ce6 showed a better anticancer outcome compared to SLN/Ce6. Conclusion: CM/SLN/Ce6 might be a promising platform for effective tumor targeted PDT of gastric cancer.


Assuntos
Membrana Celular/patologia , Nanopartículas/química , Fotoquimioterapia , Porfirinas/uso terapêutico , Dióxido de Silício/química , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Peso Corporal/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Coloides , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Humanos , Camundongos , Nanopartículas/ultraestrutura , Tamanho da Partícula , Porfirinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Eletricidade Estática , Distribuição Tecidual/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos
19.
Cell Physiol Biochem ; 53(2): 400-412, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31403270

RESUMO

BACKGROUND/AIMS: Mutations in ABCA4 cause Stargardt macular degeneration, which invariably ends in legal blindness. We studied two common mutants, A1038V (in NBD1) and G1961E (in NBD2), with the purpose of exploring how they interact with the cell's quality control mechanism. The study was designed to determine how these mutants can be rescued. METHODS: We expressed wt and mutant ABCA4 in HEK293 cells and studied the effect of the mutations on trafficking and processing and the ability of correctors to rescue them. We used a combination of western blotting, confocal microscopy and surface biotinylation coupled with pulldown of plasma membrane proteins. RESULTS: G1961E is sensitive to inhibitors of the aggresome, tubacin and the lysosome, bafilomycin A. Both mutants cause a reduction in heat shock protein, Hsp27. Incubation of HEK293 cells expressing the mutants with VX-809, an FDA approved drug for the treatment of cystic fibrosis, increased the levels of A1038V and G1961E by 2- to 3-fold. Importantly, VX-809 increased the levels of both mutants at the plasma membrane suggesting that trafficking had been restored. Transfecting additional Hsp27 to the cells also increased the steady state levels of both mutants. However, in combination with VX-809 the addition of Hsp27 caused a dramatic increase in the protein expression particularly in the G1961 mutant which increased approximately 5-fold. CONCLUSION: Our results provide a new mechanism for the rescue of ABCA4 trafficking mutants based on the restoration of Hsp27. Our results provide a pathway for the treatment of Stargardt disease.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Transportadores de Cassetes de Ligação de ATP/genética , Aminopiridinas/uso terapêutico , Anilidas/farmacologia , Benzodioxóis/uso terapêutico , Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Leupeptinas/farmacologia , Lisossomos/metabolismo , Degeneração Macular/congênito , Degeneração Macular/tratamento farmacológico , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Mutação , Transporte Proteico/efeitos dos fármacos
20.
Dokl Biochem Biophys ; 486(1): 216-219, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31367825

RESUMO

The direct action of singlet oxygen on the bacteriochlorophyll (BChl) of light-harvesting complexes in the membranes of four species of purple non-sulfur and sulfur photosynthesizing bacteria with and without carotenoids was studied. It was found that BChl in carotenoidless samples is generally more resistant to the action of singlet oxygen compared to the control. It is assumed that carotenoids are not required to protect BChl of bacterial light-harvesting complexes from singlet oxygen, and in the classic work by Griffith et al. [1] the apoptosis process in carotenoidless mutant cells, which involves the destruction of complexes, the appearance of monomeric BChl, and the generation of singlet oxygen caused by BChl, followed by BChl oxidation, was mistakenly attributed to the protective function of carotenoids.


Assuntos
Bactérias/citologia , Bactérias/metabolismo , Bacterioclorofilas/metabolismo , Carotenoides/metabolismo , Membrana Celular/metabolismo , Fotossíntese , Oxigênio Singlete/metabolismo , Estresse Oxidativo
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