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1.
J Hazard Mater ; 421: 126802, 2022 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-34396977

RESUMO

The coexistence of hazardous substances enhances their toxicities to plants, but its mechanism is still unclear due to the unknown cytochemical behavior of hazardous substance in plants. In this study, by using interdisciplinary methods, we observed the cytochemical behavior of coexisting hazardous substances {terbium [Tb(III)], benzo(a)pyrene (BaP) and cadmium [Cd(II)] in environments} in plants and thus identified a new mechanism by which coexisting hazardous substances in environments enhance their toxicities to plants. First, Tb(III) at environmental exposure level (1.70 × 10-10 g/L) breaks the inert rule of clathrin-mediated endocytosis (CME) in leaf cells. Specifically, Tb(III) binds to its receptor [FASCICLIN-like arabinogalactan protein 17 (FLA17)] on the plasma membrane of leaf cells and then docks to an intracellular adaptor protein [adaptor protein 2 (AP2)] to form ternary complex [Tb(III)-FLA17-AP2], which finally initiates CME pathway in leaf cells. Second, coexisting Tb(III), BaP and Cd(II) in environments are simultaneously transported into leaf cells via Tb(III)-initiated CME pathway, leading to the accumulation of them in leaf cells. Finally, these accumulated hazardous substances simultaneously poison plant leaf cells. These results provide theoretical and experimental bases for elucidating the mechanisms of hazardous substances in environments poisoning plants, evaluating their risks, and protecting ecosystems.


Assuntos
Clatrina , Substâncias Perigosas , Ecossistema , Endocitose , Substâncias Perigosas/toxicidade , Plantas
2.
Environ Pollut ; 292(Pt A): 118308, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34626705

RESUMO

Aggravating the pollution of microcystins (MCs) in freshwater environments is detrimental to aquatic living organisms and humans, and thus threatens the stability of ecosystems. Some environmental factors have been verified to promote the production of MCs in Microcystis aeruginosa, thereby aggravating the pollution of MCs. However, the effects of cerium (Ce), the most abundant rare earth element in global water environments, on the production of MCs in M. aeruginosa are unknown. Here, Lake Taihu water was selected as a representative of freshwater environments. By using interdisciplinary methods, it was found that: (1) the exposure level of Ce [Ce(III) and Ce(IV)] in Lake Taihu water is in the range of 0.271-0.282 µg/L; (2) Ce exposure in Lake Taihu water promoted the contents of three main MCs (MC-LR, MC-LW and MC-YR) in M. aeruginosa and water; (3) a cellular mechanism of Ce promoting the production of MCs in M. aeruginosa in Lake Taihu water was suggested: Ce enhanced endocytosis in cells of M. aeruginosa to promote the essential element uptake by M. aeruginosa for MC synthesis. Thus, Ce exposure in Lake Taihu water aggravates the pollution of MCs via enhancing endocytosis in cells of M. aeruginosa. The results provide reference for assessing the environmental risk of Ce in water environments, investigating the mechanism of the pollution of MCs induced by environmental factors, and developing strategies aimed at preventing and controlling the pollution of MCs.


Assuntos
Cério , Microcystis , Cério/toxicidade , China , Ecossistema , Endocitose , Humanos , Lagos , Microcistinas , Água
3.
Parasitol Int ; 86: 102444, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34464754

RESUMO

Trypanosoma cruzi proliferative forms perform endocytosis through a specialized structure named the cytostome-cytopharynx complex (SPC). The SPC is a specialized invagination of the cell membrane that extends through the cell body towards the posterior regions, with its aperture close to the flagellar pocket. Recently, diverse proteins were found along the cytopharynx, including two myosin motors. One of these is the orphan myosin MyoF, that was proved to be essential for endocytosis in epimastigotes. However, the dynamics of MyoF localization along the endocytic pathway and through the T. cruzi life cycle remain unclear. Using CRISPR-Cas9 genome editing, we generated epimastigotes expressing MyoF fused to mNeonGreen from its endogenous locus. Using these cells, we observed that during the epimastigote cell cycle MyoF signal disappeared during G2, reappearing at early cytokinesis. Additionally, we show that MyoF localization during metacyclogenesis is compatible with the progressive disappearance of the SPC, being absent in metacyclic trypomastigotes. Detergent fractionation showed that MyoF was predominantly present in the insoluble fraction and immunolocalized at the SPC microtubules in whole-mount cytoskeleton preparations. Moreover, during tracer uptake through the SPC, MyoF followed the tracer along the endocytic pathway and was found in posterior compartments after 30 min. Taken together, the data suggest that MyoF may play a role not only at the cargo entry site but also along the endocytic pathway.


Assuntos
Endocitose , Miosinas/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/fisiologia , Miosinas/metabolismo , Proteínas de Protozoários/metabolismo
4.
Int J Nanomedicine ; 16: 7269-7281, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34737564

RESUMO

Purpose: Small molecule modified antitumor drug conjugate nanoparticles have the advantages of high drug loading, simple synthesis and preparation, and better biocompatibility. Due to the large demand for exogenous α-linolenic acid (ALA) by tumor cells, we synthesized α-linolenic acid-paclitaxel conjugate (ALA-PTX) and prepared α-linolenic acid-paclitaxel conjugate nanoparticles (ALA-PTX NPs), in order to obtain better tumor cellular uptake and antitumor activity in vitro and in vivo. Methods: We synthesized and characterized ALA-PTX, and then prepared and characterized ALA-PTX NPs. The cellular uptake, uptake pathways, intracellular behavior, in vitro and in vivo antitumor activity of ALA-PTX NPs were evaluated. Results: The size of ALA-PTX NPs was approximately 110.7±1.7 nm. The drug loading was approximately 90% (w/w) with CrEL-free and organic solvent-free characteristics. The cellular uptake of ALA-PTX NPs was significantly higher than that of PTX injection by MCF-7, MCF-7/ADR and HepG2 cells. In these three cell lines, the cellular uptake of ALA-PTX NPs at 6h was approximately 1.5-2.6 times higher than that of PTX injection. ALA-PTX NPs were ingested through clathrin-mediated endocytosis, then transferred to lysosomes, and could dissolve in cells to play an antitumor activity. The in vitro and in vivo antitumor activity of ALA-PTX NPs was confirmed in MCF-7/ADR and HepG2 cell models and tumor-bearing nude mouse models. Conclusion: ALA-PTX NPs developed in our study could provide a new method for the preparation of nano-delivery systems suitable for antitumor therapy that could increase tumor cellular uptake and enhance antitumor activity.


Assuntos
Antineoplásicos Fitogênicos , Antineoplásicos , Nanopartículas , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Endocitose , Camundongos , Paclitaxel , Ácido alfa-Linoleico
5.
Int J Mol Sci ; 22(19)2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34639129

RESUMO

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system that finally leads to demyelination. Demyelinating optic neuritis is a frequent symptom in MS. Recent studies also revealed synapse dysfunctions in MS patients and MS mouse models. We previously reported alterations of photoreceptor ribbon synapses in the experimental auto-immune encephalomyelitis (EAE) mouse model of MS. In the present study, we found that the previously observed decreased imunosignals of photoreceptor ribbons in early EAE resulted from a decrease in synaptic ribbon size, whereas the number/density of ribbons in photoreceptor synapses remained unchanged. Smaller photoreceptor ribbons are associated with fewer docked and ribbon-associated vesicles. At a functional level, depolarization-evoked exocytosis as monitored by optical recording was diminished even as early as on day 7 after EAE induction. Moreover compensatory, post-depolarization endocytosis was decreased. Decreased post-depolarization endocytosis in early EAE correlated with diminished synaptic enrichment of dynamin3. In contrast, basal endocytosis in photoreceptor synapses of resting non-depolarized retinal slices was increased in early EAE. Increased basal endocytosis correlated with increased de-phosphorylation of dynamin1. Thus, multiple endocytic pathways in photoreceptor synapse are differentially affected in early EAE and likely contribute to the observed synapse pathology in early EAE.


Assuntos
Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Endocitose , Exocitose , Esclerose Múltipla/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Sinapses/patologia , Animais , Dinaminas/metabolismo , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/etiologia , Esclerose Múltipla/metabolismo , Fosforilação , Retina/metabolismo , Retina/patologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/patologia
6.
Nat Commun ; 12(1): 5812, 2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34608164

RESUMO

The advantage of locally applied anesthetics is that they are not associated with the many adverse effects, including addiction liability, of systemically administered analgesics. This therapeutic approach has two inherent pitfalls: specificity and a short duration of action. Here, we identified nociceptor endocytosis as a promising target for local, specific, and long-lasting treatment of inflammatory pain. We observed preferential expression of AP2α2, an α-subunit isoform of the AP2 complex, within CGRP+/IB4- nociceptors in rodents and in CGRP+ dorsal root ganglion neurons from a human donor. We utilized genetic and pharmacological approaches to inhibit nociceptor endocytosis demonstrating its role in the development and maintenance of acute and chronic inflammatory pain. One-time injection of an AP2 inhibitor peptide significantly reduced acute and chronic pain-like behaviors and provided prolonged analgesia. We evidenced sexually dimorphic recovery responses to this pharmacological approach highlighting the importance of sex differences in pain development and response to analgesics.


Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Dor Crônica/tratamento farmacológico , Endocitose/efeitos dos fármacos , Nociceptores/efeitos dos fármacos , Complexo 2 de Proteínas Adaptadoras/antagonistas & inibidores , Complexo 2 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Subunidades alfa do Complexo de Proteínas Adaptadoras/antagonistas & inibidores , Subunidades alfa do Complexo de Proteínas Adaptadoras/genética , Subunidades alfa do Complexo de Proteínas Adaptadoras/metabolismo , Animais , Dor Crônica/metabolismo , Dor Crônica/fisiopatologia , Epiderme/inervação , Feminino , Gânglios Espinais/metabolismo , Humanos , Inflamação , Masculino , Camundongos , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/metabolismo , Nociceptores/metabolismo , Nociceptores/fisiologia , Peptídeos/administração & dosagem , Peptídeos/metabolismo , Peptídeos/farmacologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia
7.
Int J Mol Sci ; 22(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34638549

RESUMO

Selective endocytosis followed by degradation is a major mechanism for downregulating plasma membrane transporters in response to specific environmental cues. In Saccharomyces cerevisiae, this endocytosis is promoted by ubiquitylation catalyzed by the Rsp5 ubiquitin-ligase, targeted to transporters via adaptors of the alpha-arrestin family. However, the molecular mechanisms of this targeting and their control according to conditions remain incompletely understood. In this work, we dissect the molecular mechanisms eliciting the endocytosis of Can1, the arginine permease, in response to cycloheximide-induced TORC1 hyperactivation. We show that cycloheximide promotes Rsp5-dependent Can1 ubiquitylation and endocytosis in a manner dependent on the Bul1/2 alpha-arrestins. Also crucial for this downregulation is a short acidic patch sequence in the N-terminus of Can1 likely acting as a binding site for Bul1/2. The previously reported inhibition by cycloheximide of transporter recycling, from the trans-Golgi network to the plasma membrane, seems to additionally contribute to efficient Can1 downregulation. Our results also indicate that, contrary to the previously described substrate-transport elicited Can1 endocytosis mediated by the Art1 alpha-arrestin, Bul1/2-mediated Can1 ubiquitylation occurs independently of the conformation of the transporter. This study provides further insights into how distinct alpha-arrestins control the ubiquitin-dependent downregulation of a specific amino acid transporter under different conditions.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Antifúngicos/farmacologia , Cicloeximida/farmacologia , Endocitose/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Transporte Proteico/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitinação/efeitos dos fármacos
8.
Nat Cell Biol ; 23(10): 1052-1053, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34616023
9.
Int J Pharm ; 609: 121200, 2021 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-34662643

RESUMO

The administration of plasmid DNA (pDNA) using a pyro-drive jet injector allows gene expression in cells of the treated tissue; however, the detailed plasmid uptake process remains to be determined. A recent theory suggests that shear stress enhances the endocytosis pathway and pDNA internalization. Here, we investigated the process of pDNA uptake in the context of a pyro-drive jet injector-based administration as a way to optimize gene transfer efficiency via the increase in DNA uptake. The gene expression was significantly improved when the shear stress caused by the jet was generated where the pDNA was retained. Contrarily, heparin, an inhibitor of the spontaneous uptake of injected DNA, inhibited the gene expression in jet injection. In addition, treatment with typical endocytosis inhibitors (chlorpromazine, methyl-ß-cyclodextrin, dimethyl amiloride, rottlerin, and NSC23766) also reduced plasmid expression efficiency in the context of jet injection; conversely, endosome escape in the context of chloroquine treatment increased the expression efficiency. Altogether, our results not only clarify the mechanism of pDNA uptake in the context of jet injection but also highlight the key role of endosomes on the intracellular trafficking of pDNA. Importantly, such findings may impact other studies on gene transfer and endocytosis and boost further efforts to improve the efficiency and safety of jet injection in the context of both basic and translational applications.


Assuntos
DNA , Endocitose , Terapia Genética , Injeções a Jato , Plasmídeos/genética
10.
Int J Mol Sci ; 22(19)2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34638976

RESUMO

Lysosomal degradation, the common destination of autophagy and endocytosis, is one of the most important elements of eukaryotic metabolism. The small GTPases Rab39A and B are potential new effectors of this pathway, as their malfunction is implicated in severe human diseases like cancer and neurodegeneration. In this study, the lysosomal regulatory role of the single Drosophila Rab39 ortholog was characterized, providing valuable insight into the potential cell biological mechanisms mediated by these proteins. Using a de novo CRISPR-generated rab39 mutant, we found no failure in the early steps of endocytosis and autophagy. On the contrary, we found that Rab39 mutant nephrocytes internalize and degrade endocytic cargo at a higher rate compared to control cells. In addition, Rab39 mutant fat body cells contain small yet functional autolysosomes without lysosomal fusion defect. Our data identify Drosophila Rab39 as a negative regulator of lysosomal clearance during both endocytosis and autophagy.


Assuntos
Autofagia/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Endocitose/genética , Lisossomos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Proteínas de Drosophila/genética , Larva/enzimologia , Larva/genética , Fenótipo , Proteínas rab de Ligação ao GTP/genética
11.
Colloids Surf B Biointerfaces ; 208: 112140, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34597939

RESUMO

Semiconductor quantum dots (QDs) have been extensively explored for extensive bioapplications, yet their cellular fate, especially exocytosis, has not been thoroughly investigated. Herein, we systematically investigated the whole cellular process from the endocytosis, intercellular trafficking, to the exocytosis of a typical QD, core/shell CdSe/ZnS QD. Using confocal laser scanning microscopy and flow cytometry, and after carefully eliminating the effect of cell division, we found that the QDs were internalized by HeLa cells with a time-, dose-, and serum-dependent manner. The cellular uptake was inhibited by serum, but eventually peaked after 4-6 h incubation with or without serum. The primary endocytosis pathway was clathrin-mediated, and actin- and microtubule-dependent in the medium with serum, while the caveolae-mediated endocytosis and macropinocytosis were more important for the QDs in the serum-free medium. Inside cells, most QDs distributed in lysosomes, and some entered mitochondria, endoplasmic reticulum, and Golgi apparatus. The translocation of the QDs from other organelles to Golgi apparatus was observed. The exocytosis of QDs was faster than the endocytosis, reaching the maximum in about one hour after cultured in fresh culture medium, with around 60% of the internalized QDs remained undischarged. The exocytosis process was energy- and actin-dependent, and the lysosome exocytosis and endoplasmic reticulum/Golgi pathway were the main routes. This study provides a full picture of behavior and fate of QDs in cells, which may facilitate the design of ideal QDs applied in biomedical and other fields.


Assuntos
Compostos de Cádmio , Pontos Quânticos , Compostos de Selênio , Endocitose , Exocitose , Células HeLa , Humanos , Sulfetos , Compostos de Zinco
12.
Nat Cell Biol ; 23(10): 1073-1084, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34616024

RESUMO

Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis and cancer invasion. Unlike cargo-specific clathrin-mediated endocytosis, the clathrin- and dynamin-independent endocytic pathway (CLIC-GEEC, CG pathway) is considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids. While the core molecular players of CG-endocytosis have been recently defined, evidence of cargo-specific adaptors or selective uptake of proteins for the pathway are lacking. Here we identify the actin-binding protein Swiprosin-1 (Swip1, EFHD2) as a cargo-specific adaptor for CG-endocytosis. Swip1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery-Arf1, IRSp53 and actin-and is critical for integrin endocytosis. Through this function, Swip1 supports integrin-dependent cancer-cell migration and invasion, and is a negative prognostic marker in breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG pathway and a role for specific adaptors in recruitment into this endocytic route.


Assuntos
Neoplasias da Mama/patologia , Clatrina/metabolismo , Dinaminas/metabolismo , Endocitose , Integrina beta1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Actinas/metabolismo , Transporte Biológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Movimento Celular , Clatrina/genética , Dinaminas/genética , Feminino , Humanos , Integrina beta1/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas rab de Ligação ao GTP/genética
13.
mBio ; 12(5): e0254221, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34634931

RESUMO

Damage in COVID-19 results from both the SARS-CoV-2 virus and its triggered overactive host immune responses. Therapeutic agents that focus solely on reducing viral load or hyperinflammation fail to provide satisfying outcomes in all cases. Although viral and cellular factors have been extensively profiled to identify potential anti-COVID-19 targets, new drugs with significant efficacy remain to be developed. Here, we report the potent preclinical efficacy of ALD-R491, a vimentin-targeting small molecule compound, in treating COVID-19 through its host-directed antiviral and anti-inflammatory actions. We found that by altering the physical properties of vimentin filaments, ALD-491 affected general cellular processes as well as specific cellular functions relevant to SARS-CoV-2 infection. Specifically, ALD-R491 reduced endocytosis, endosomal trafficking, and exosomal release, thus impeding the entry and egress of the virus; increased the microcidal capacity of macrophages, thus facilitating the pathogen clearance; and enhanced the activity of regulatory T cells, therefore suppressing the overactive immune responses. In cultured cells, ALD-R491 potently inhibited the SARS-CoV-2 spike protein and human ACE2-mediated pseudoviral infection. In aged mice with ongoing, productive SARS-CoV-2 infection, ALD-R491 reduced disease symptoms as well as lung damage. In rats, ALD-R491 also reduced bleomycin-induced lung injury and fibrosis. Our results indicate a unique mechanism and significant therapeutic potential for ALD-R491 against COVID-19. We anticipate that ALD-R491, an oral, fast-acting, and non-cytotoxic agent targeting the cellular protein with multipart actions, will be convenient, safe, and broadly effective, regardless of viral mutations, for patients with early- or late-stage disease, post-COVID-19 complications, and other related diseases. IMPORTANCE With the Delta variant currently fueling a resurgence of new infections in the fully vaccinated population, developing an effective therapeutic drug is especially critical and urgent in fighting COVID-19. In contrast to the many efforts to repurpose existing drugs or address only one aspect of COVID-19, we are developing a novel agent with first-in-class mechanisms of action that address both the viral infection and the overactive immune system in the pathogenesis of the disease. Unlike virus-directed therapeutics that may lose efficacy due to viral mutations, and immunosuppressants that require ideal timing to be effective, this agent, with its unique host-directed antiviral and anti-inflammatory actions, can work against all variants of the virus, be effective during all stages of the disease, and even resolve post-disease damage and complications. Further development of the compound will provide an important tool in the fight against COVID-19 and its complications, as well as future outbreaks of new viruses.


Assuntos
Anti-Inflamatórios/uso terapêutico , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/metabolismo , Compostos Orgânicos/uso terapêutico , Glicoproteína da Espícula de Coronavírus/metabolismo , Vimentina/metabolismo , Animais , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Células HEK293 , Humanos , Camundongos , Células RAW 264.7
14.
Nat Commun ; 12(1): 6065, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34663803

RESUMO

Different types of cellular membranes have unique lipid compositions that are important for their functional identity. PI(4,5)P2 is enriched in the plasma membrane where it contributes to local activation of key cellular events, including actomyosin contraction and cytokinesis. However, how cells prevent PI(4,5)P2 from accumulating in intracellular membrane compartments, despite constant intermixing and exchange of lipid membranes, is poorly understood. Using the C. elegans early embryo as our model system, we show that the evolutionarily conserved lipid transfer proteins, PDZD-8 and TEX-2, act together with the PI(4,5)P2 phosphatases, OCRL-1 and UNC-26/synaptojanin, to prevent the build-up of PI(4,5)P2 on endosomal membranes. In the absence of these four proteins, large amounts of PI(4,5)P2 accumulate on endosomes, leading to embryonic lethality due to ectopic recruitment of proteins involved in actomyosin contractility. PDZD-8 localizes to the endoplasmic reticulum and regulates endosomal PI(4,5)P2 levels via its lipid harboring SMP domain. Accumulation of PI(4,5)P2 on endosomes is accompanied by impairment of their degradative capacity. Thus, cells use multiple redundant systems to maintain endosomal PI(4,5)P2 homeostasis.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Actomiosina/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Membrana Celular/metabolismo , Citocinese , Desenvolvimento Embrionário , Retículo Endoplasmático/metabolismo , Homeostase , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso , Fosfatidilinositóis , Monoéster Fosfórico Hidrolases
15.
Nat Commun ; 12(1): 5726, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34593813

RESUMO

Cell membrane coated nanoparticles (NPs) have recently been recognized as attractive nanomedical tools because of their unique properties such as immune escape, long blood circulation time, specific molecular recognition and cell targeting. However, the integrity of the cell membrane coating on NPs, a key metrics related to the quality of these biomimetic-systems and their resulting biomedical function, has remained largely unexplored. Here, we report a fluorescence quenching assay to probe the integrity of cell membrane coating. In contradiction to the common assumption of perfect coating, we uncover that up to 90% of the biomimetic NPs are only partially coated. Using in vitro homologous targeting studies, we demonstrate that partially coated NPs could still be internalized by the target cells. By combining molecular simulations with experimental analysis, we further identify an endocytic entry mechanism for these NPs. We unravel that NPs with a high coating degree (≥50%) enter the cells individually, whereas the NPs with a low coating degree (<50%) need to aggregate together before internalization. This quantitative method and the fundamental understanding of how cell membrane coated NPs enter the cells will enhance the rational designing of biomimetic nanosystems and pave the way for more effective cancer nanomedicine.


Assuntos
Antineoplásicos Alquilantes/administração & dosagem , Materiais Biomiméticos/química , Membrana Celular/química , Portadores de Fármacos/química , Neoplasias/tratamento farmacológico , Animais , Composição de Medicamentos/métodos , Endocitose , Células HeLa , Humanos , Masculino , Camundongos , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Nanopartículas/ultraestrutura , Porosidade , Células RAW 264.7 , Propriedades de Superfície
16.
Nat Commun ; 12(1): 5739, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34667166

RESUMO

Protein aggregates associated with neurodegenerative diseases have the ability to transmit to unaffected cells, thereby templating their own aberrant conformation onto soluble homotypic proteins. Proteopathic seeds can be released into the extracellular space, secreted in association with extracellular vesicles (EV) or exchanged by direct cell-to-cell contact. The extent to which each of these pathways contribute to the prion-like spreading of protein misfolding is unclear. Exchange of cellular cargo by both direct cell contact or via EV depends on receptor-ligand interactions. We hypothesized that enabling these interactions through viral ligands enhances intercellular proteopathic seed transmission. Using different cellular models propagating prions or pathogenic Tau aggregates, we demonstrate that vesicular stomatitis virus glycoprotein and SARS-CoV-2 spike S increase aggregate induction by cell contact or ligand-decorated EV. Thus, receptor-ligand interactions are important determinants of intercellular aggregate dissemination. Our data raise the possibility that viral infections contribute to proteopathic seed spreading by facilitating intercellular cargo transfer.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Vesículas Extracelulares/metabolismo , Glicoproteínas de Membrana/metabolismo , Agregação Patológica de Proteínas/virologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas do Envelope Viral/metabolismo , Adulto , Idoso , Encéfalo/patologia , Estudos de Casos e Controles , Linhagem Celular , Endocitose , Feminino , Humanos , Microscopia Intravital , Masculino , Pessoa de Meia-Idade , Príons/metabolismo , Agregação Patológica de Proteínas/patologia , Dobramento de Proteína , Proteínas tau/metabolismo
17.
Int J Mol Sci ; 22(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34638858

RESUMO

TMEM175 (transmembrane protein 175) coding sequence variants are associated with increased risk of Parkinson's disease. TMEM175 is the ubiquitous lysosomal K+ channel regulated by growth factor receptor signaling and direct interaction with protein kinase B (PKB/Akt). In the present study, we show that the expression of mouse TMEM175 results in very small K+ currents through the plasma membrane in Xenopus laevis oocytes, in good accordance with the previously reported intracellular localization of the channel. However, the application of the dynamin inhibitor compounds, dynasore or dyngo-4a, substantially increased TMEM175 currents measured by the two-electrode voltage clamp method. TMEM175 was more permeable to cesium than potassium ions, voltage-dependently blocked by 4-aminopyridine (4-AP), and slightly inhibited by extracellular acidification. Immunocytochemistry experiments indicated that dyngo-4a increased the amount of epitope-tagged TMEM175 channel on the cell surface. The coexpression of dominant-negative dynamin, and the inhibition of clathrin- or caveolin-dependent endocytosis increased TMEM175 current much less than dynasore. Therefore, dynamin-independent pharmacological effects of dynasore may also contribute to the action on the channel. TMEM175 current rapidly decays after the withdrawal of dynasore, raising the possibility that an efficient internalization mechanism removes the channel from the plasma membrane. Dyngo-4a induced about 20-fold larger TMEM175 currents than the PKB activator SC79, or the coexpression of a constitutively active mutant PKB with the channel. In contrast, the allosteric PKB inhibitor MK2206 diminished the TMEM175 current in the presence of dyngo-4a. These data suggest that, in addition to the lysosomes, PKB-dependent regulation also influences TMEM175 current in the plasma membrane.


Assuntos
Membrana Celular/metabolismo , Hidrazonas/farmacologia , Lisossomos/metabolismo , Naftóis/farmacologia , Canais de Potássio/metabolismo , 4-Aminopiridina/farmacologia , Animais , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Microscopia Confocal/métodos , Oócitos/citologia , Oócitos/metabolismo , Oócitos/fisiologia , Técnicas de Patch-Clamp/métodos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/genética , Transporte Proteico/efeitos dos fármacos , Xenopus laevis
18.
Nanomedicine (Lond) ; 16(23): 2075-2094, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34523349

RESUMO

Background: Poly(lactic-co-glycolic) acid (PLGA) nanoparticles can be prepared by emulsion-solvent-evaporation from o/w and w1/o/w2 emulsions. Aims: To elaborate similarities and differences regarding mechanical, morphological and physicochemical properties, as well as endocytosis and dose-dependent immune responses by primary human leukocytes between nanoparticles prepared by these two methods. Methods: Fluorescently labeled as well as TLR agonist (R848)-loaded PLGA nanoparticles were prepared via both single- and double-emulsion solvent evaporation. Results: Particles prepared by both methods were similar in chemical composition and surface charge but exhibited slight differences in size and morphology. Pronounced differences were found for loading, dissolution and mechanical properties. The particles were differently endocytosed by monocytes and induced qualitatively and quantitatively different immune responses. Conclusions: Variations in nanoparticle preparation can affect particle-derived immunological characteristics.


Assuntos
Nanopartículas , Ácido Poliglicólico , Portadores de Fármacos , Emulsões , Endocitose , Glicóis , Humanos , Ácido Láctico , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico
19.
Int J Med Sci ; 18(15): 3533-3543, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34522180

RESUMO

Importance: Despite the availability of a vaccine against the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), humans will have to live with this virus and the after-effects of the coronavirus disease 2019 (COVID-19) infection for a long time. Cholesterol plays an important role in the infection and prognosis of SARS-CoV-2, and the study of its mechanism is of great significance not only for the treatment of COVID-19 but also for research on generic antiviral drugs. Observations: Cholesterol promotes the development of atherosclerosis by activating NLR family pyrin domain containing 3 (NLRP3), and the resulting inflammatory environment indirectly contributes to COVID-19 infection and subsequent deterioration. In in vitro studies, membrane cholesterol increased the number of viral entry sites on the host cell membrane and the number of angiotensin-converting enzyme 2 (ACE2) receptors in the membrane fusion site. Previous studies have shown that the fusion protein of the virus interacts with cholesterol, and the spike protein of SARS-CoV-2 also requires cholesterol to enter the host cells. Cholesterol in blood interacts with the spike protein to promote the entry of spike cells, wherein the scavenger receptor class B type 1 (SR-B1) plays an important role. Because of the cardiovascular protective effects of lipid-lowering therapy and the additional anti-inflammatory effects of lipid-lowering drugs, it is currently recommended to continue lipid-lowering therapy for patients with COVID-19, but the safety of extremely low LDL-C is questionable. Conclusions and Relevance: Cholesterol can indirectly increase the susceptibility of patients to SARS-CoV-2 and increase the risk of death from COVID-19, which are mediated by NLRP3 and atherosclerotic plaques, respectively. Cholesterol present in the host cell membrane, virus, and blood may also directly participate in the virus cell entry process, but the specific mechanism still needs further study. Patients with COVID-19 are recommended to continue lipid-lowering therapy.


Assuntos
COVID-19/complicações , Hipercolesterolemia/complicações , Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/uso terapêutico , Aterosclerose/fisiopatologia , COVID-19/diagnóstico , COVID-19/tratamento farmacológico , COVID-19/terapia , Membrana Celular/metabolismo , HDL-Colesterol/metabolismo , LDL-Colesterol/metabolismo , Endocitose , Humanos , Hipercolesterolemia/diagnóstico , Hipercolesterolemia/terapia , Inflamação , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/sangue , Prognóstico , SARS-CoV-2 , Receptores Depuradores Classe B/metabolismo
20.
Front Immunol ; 12: 636966, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34557180

RESUMO

Since 2003, the world has been confronted with three new betacoronaviruses that cause human respiratory infections: SARS-CoV, which causes severe acute respiratory syndrome (SARS), MERS-CoV, which causes Middle East respiratory syndrome (MERS), and SARS-CoV-2, which causes Coronavirus Disease 2019 (COVID-19). The mechanisms of coronavirus transmission and dissemination in the human body determine the diagnostic and therapeutic strategies. An important problem is the possibility that viral particles overcome tissue barriers such as the intestine, respiratory tract, blood-brain barrier, and placenta. In this work, we will 1) consider the issue of endocytosis and the possibility of transcytosis and paracellular trafficking of coronaviruses across tissue barriers with an emphasis on the intestinal epithelium; 2) discuss the possibility of antibody-mediated transcytosis of opsonized viruses due to complexes of immunoglobulins with their receptors; 3) assess the possibility of the virus transfer into extracellular vesicles during intracellular transport; and 4) describe the clinical significance of these processes. Models of the intestinal epithelium and other barrier tissues for in vitro transcytosis studies will also be briefly characterized.


Assuntos
Endocitose , Mucosa Intestinal/virologia , SARS-CoV-2/metabolismo , Anticorpos Antivirais/metabolismo , Antivirais/farmacologia , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/transmissão , COVID-19/virologia , Ensaios Clínicos como Assunto , Endocitose/efeitos dos fármacos , Humanos , Mucosa Intestinal/metabolismo , Modelos Biológicos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , Junções Íntimas/metabolismo , Junções Íntimas/virologia , Transcitose/efeitos dos fármacos , Ligação Viral
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