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1.
J Exp Med ; 221(7)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38805014

RESUMO

Phenotypic plasticity is a rising cancer hallmark, and lung adeno-to-squamous transition (AST) triggered by LKB1 inactivation is significantly associated with drug resistance. Mechanistic insights into AST are urgently needed to identify therapeutic vulnerability in LKB1-deficient lung cancer. Here, we find that ten-eleven translocation (TET)-mediated DNA demethylation is elevated during AST in KrasLSL-G12D/+; Lkb1L/L (KL) mice, and knockout of individual Tet genes reveals that Tet2 is required for squamous transition. TET2 promotes neutrophil infiltration through STAT3-mediated CXCL5 expression. Targeting the STAT3-CXCL5 nexus effectively inhibits squamous transition through reducing neutrophil infiltration. Interestingly, tumor-infiltrating neutrophils are laden with triglycerides and can transfer the lipid to tumor cells to promote cell proliferation and squamous transition. Pharmacological inhibition of macropinocytosis dramatically inhibits neutrophil-to-cancer cell lipid transfer and blocks squamous transition. These data uncover an epigenetic mechanism orchestrating phenotypic plasticity through regulating immune microenvironment and metabolic communication, and identify therapeutic strategies to inhibit AST.


Assuntos
Quimiocina CXCL5 , Proteínas de Ligação a DNA , Dioxigenases , Neoplasias Pulmonares , Neutrófilos , Proteínas Proto-Oncogênicas , Fator de Transcrição STAT3 , Animais , Neutrófilos/metabolismo , Fator de Transcrição STAT3/metabolismo , Camundongos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Quimiocina CXCL5/metabolismo , Quimiocina CXCL5/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Humanos , Dioxigenases/metabolismo , Pinocitose , Linhagem Celular Tumoral , Infiltração de Neutrófilos , Camundongos Knockout , Camundongos Endogâmicos C57BL , Metabolismo dos Lipídeos
2.
Biol Open ; 13(5)2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38713004

RESUMO

Recent research has shown that membrane trafficking plays an important role in canonical Wnt signaling through sequestration of the ß-catenin destruction complex inside multivesicular bodies (MVBs) and lysosomes. In this study, we introduce Ouabain, an inhibitor of the Na,K-ATPase pump that establishes electric potentials across membranes, as a potent inhibitor of Wnt signaling. We find that Na,K-ATPase levels are elevated in advanced colon carcinoma, that this enzyme is elevated in cancer cells with constitutively activated Wnt pathway and is activated by GSK3 inhibitors that increase macropinocytosis. Ouabain blocks macropinocytosis, which is an essential step in Wnt signaling, probably explaining the strong effects of Ouabain on this pathway. In Xenopus embryos, brief Ouabain treatment at the 32-cell stage, critical for the earliest Wnt signal in development-inhibited brains, could be reversed by treatment with Lithium chloride, a Wnt mimic. Inhibiting membrane trafficking may provide a way of targeting Wnt-driven cancers.


Assuntos
Neoplasias do Colo , Ouabaína , Pinocitose , ATPase Trocadora de Sódio-Potássio , Via de Sinalização Wnt , Neoplasias do Colo/metabolismo , Neoplasias do Colo/etiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Humanos , Ouabaína/farmacologia , Linhagem Celular Tumoral , Xenopus
3.
J Cell Biol ; 223(7)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38748250

RESUMO

Dynamic presynaptic actin remodeling drives structural and functional plasticity at synapses, but the underlying mechanisms remain largely unknown. Previous work has shown that actin regulation via Rac1 guanine exchange factor (GEF) Vav signaling restrains synaptic growth via bone morphogenetic protein (BMP)-induced receptor macropinocytosis and mediates synaptic potentiation via mobilization of reserve pool vesicles in presynaptic boutons. Here, we find that Gef26/PDZ-GEF and small GTPase Rap1 signaling couples the BMP-induced activation of Abelson kinase to this Vav-mediated macropinocytosis. Moreover, we find that adenylate cyclase Rutabaga (Rut) signaling via exchange protein activated by cAMP (Epac) drives the mobilization of reserve pool vesicles during post-tetanic potentiation (PTP). We discover that Rap1 couples activation of Rut-cAMP-Epac signaling to Vav-mediated synaptic potentiation. These findings indicate that Rap1 acts as an essential, convergent node for Abelson kinase and cAMP signaling to mediate BMP-induced structural plasticity and activity-induced functional plasticity via Vav-dependent regulation of the presynaptic actin cytoskeleton.


Assuntos
Plasticidade Neuronal , Terminações Pré-Sinápticas , Transdução de Sinais , Animais , Citoesqueleto de Actina/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Terminações Pré-Sinápticas/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/genética , Complexo Shelterina/metabolismo , Pinocitose , Drosophila
4.
Anticancer Res ; 44(5): 1939-1946, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38677763

RESUMO

BACKGROUND/AIM: Macropinocytosis is a non-selective form of endocytosis that facilitates the uptake of extracellular substances, such as nutrients and macromolecules, into the cells. In KRAS-driven cancers, including pancreatic ductal adenocarcinoma, macropinocytosis and subsequent lysosomal utilization are known to be enhanced to overcome metabolic stress. In this study, we investigated the role of Casein Kinase 2 (CK2) inhibition in macropinocytosis and subsequent metabolic processes in KRAS mutant cholangiocarcinoma (CCA) cell lines. MATERIALS AND METHODS: The bovine serum albumin (BSA) uptake indicating macropinocytosis was performed by flow cytometry using the HuCCT1 KRAS mutant CCA cell line. To validate macropinosome, the Rab7 and LAMP2 were labeled and analyzed via immunocytochemistry and western blot. The CX-4945 (Silmitasertib), CK2 inhibitor, was used to investigate the role of CK2 in macropinocytosis and subsequent lysosomal metabolism. RESULTS: The TFK-1, a KRAS wild-type CCA cell line, showed only apoptotic morphological changes. However, the HuCCT1 cell line showed macropinocytosis. Although CX-4945 induced morphological changes accompanied by the accumulation of intracellular vacuoles and cell death, the level of macropinocytosis did not change. These intracellular vacuoles were identified as late macropinosomes, representing Rab7+ vesicles before fusion with lysosomes. In addition, CX-4945 suppressed LAMP2 expression following the inhibition of the Akt-mTOR signaling pathway, which interrupts mature macropinosome and lysosomal metabolic utilization. CONCLUSION: Macropinocytosis is used as an energy source in the KRAS mutant CCA cell line HuCCT1. The inhibition of CK2 by CX-4945 leads to cell death in HuCCT1 cells through alteration of the lysosome-dependent metabolism.


Assuntos
Neoplasias dos Ductos Biliares , Caseína Quinase II , Colangiocarcinoma , Lisossomos , Mutação , Naftiridinas , Fenazinas , Pinocitose , Piperazinas , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Lisossomos/metabolismo , Linhagem Celular Tumoral , Colangiocarcinoma/patologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/genética , Pinocitose/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Caseína Quinase II/metabolismo , Caseína Quinase II/genética , Caseína Quinase II/antagonistas & inibidores , Piperazinas/farmacologia , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/genética , proteínas de unión al GTP Rab7/metabolismo , Morte Celular/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética
5.
ACS Appl Mater Interfaces ; 16(14): 17069-17079, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38563247

RESUMO

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), transfer bioactive molecules from donor to recipient cells in various pathophysiological settings, thereby mediating intercellular communication. Despite their significant roles in extracellular signaling, the cellular uptake mechanisms of different EV subpopulations remain unknown. In particular, plasma membrane-derived MVs are larger vesicles (100 nm to 1 µm in diameter) and may serve as efficient molecular delivery systems due to their large capacity; however, because of size limitations, receptor-mediated endocytosis is considered an inefficient means for cellular MV uptake. This study demonstrated that macropinocytosis (lamellipodia formation and plasma membrane ruffling, causing the engulfment of large fluid volumes outside cells) can enhance cellular MV uptake. We developed experimental techniques to induce macropinocytosis-mediated MV uptake by modifying MV membranes with arginine-rich cell-penetrating peptides for the intracellular delivery of therapeutic molecules.


Assuntos
Micropartículas Derivadas de Células , Peptídeos Penetradores de Células , Vesículas Extracelulares , Arginina , Pinocitose , Vesículas Extracelulares/metabolismo , Peptídeos Penetradores de Células/química
6.
J Extracell Vesicles ; 13(4): e12426, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38532609

RESUMO

Besides participating in diverse pathological and physiological processes, extracellular vesicles (EVs) are also excellent drug-delivery vehicles. However, clinical drugs modulating EV levels are still lacking. Here, we show that proton pump inhibitors (PPIs) reduce EVs by enhancing macropinocytosis-mediated EV uptake. PPIs accelerate intestinal cell endocytosis of autocrine immunosuppressive EVs through macropinocytosis, thereby aggravating inflammatory bowel disease. PPI-induced macropinocytosis facilitates the clearance of immunosuppressive EVs from tumour cells, improving antitumor immunity. PPI-induced macropinocytosis also increases doxorubicin and antisense oligonucleotides of microRNA-155 delivery efficiency by EVs, leading to enhanced therapeutic effects of drug-loaded EVs on tumours and acute liver failure. Mechanistically, PPIs reduce cytosolic pH, promote ATP6V1A (v-ATPase subunit) disassembly from the vacuolar membrane and enhance the assembly of plasma membrane v-ATPases, thereby inducing macropinocytosis. Altogether, our results reveal a mechanism for macropinocytic regulation and PPIs as potential modulators of EV levels, thus regulating their functions.


Assuntos
Vesículas Extracelulares , Inibidores da Bomba de Prótons , Endocitose , Pinocitose , Adenosina Trifosfatases
7.
Front Immunol ; 15: 1360370, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38533500

RESUMO

Acute respiratory distress syndrome (ARDS) is associated with high morbidity and mortality but lacks specific therapeutic options. Diverse endocytic processes play a key role in all phases of acute lung injury (ALI), including the initial insult, development of respiratory failure due to alveolar flooding, as a consequence of altered alveolar-capillary barrier function, as well as in the resolution or deleterious remodeling after injury. In particular, clathrin-, caveolae-, endophilin- and glycosylphosphatidyl inositol-anchored protein-mediated endocytosis, as well as, macropinocytosis and phagocytosis have been implicated in the setting of acute lung damage. This manuscript reviews our current understanding of these endocytic pathways and subsequent intracellular trafficking in various phases of ALI, and also aims to identify potential therapeutic targets for patients with ARDS.


Assuntos
Lesão Pulmonar Aguda , Síndrome do Desconforto Respiratório , Humanos , Síndrome do Desconforto Respiratório/terapia , Endocitose , Lesão Pulmonar Aguda/terapia , Pinocitose , Fagocitose
8.
Dev Cell ; 59(5): 645-660.e8, 2024 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-38325371

RESUMO

Macropinocytosis, an evolutionarily conserved endocytic pathway, mediates nonselective bulk uptake of extracellular fluid. It is the primary route for axenic Dictyostelium cells to obtain nutrients and has also emerged as a nutrient-scavenging pathway for mammalian cells. How cells adjust macropinocytic activity in various physiological or developmental contexts remains to be elucidated. We discovered that, in Dictyostelium cells, the transcription factors Hbx5 and MybG form a functional complex in the nucleus to maintain macropinocytic activity during the growth stage. In contrast, during starvation-induced multicellular development, the transcription factor complex undergoes nucleocytoplasmic shuttling in response to oscillatory cyclic adenosine 3',5'-monophosphate (cAMP) signals, which leads to increased cytoplasmic retention of the complex and progressive downregulation of macropinocytosis. Therefore, by coupling macropinocytosis-related gene expression to the cAMP oscillation system, which facilitates long-range cell-cell communication, the dynamic translocation of the Hbx5-MybG complex orchestrates a population-level adjustment of macropinocytic activity to adapt to changing environmental conditions.


Assuntos
Dictyostelium , Animais , Dictyostelium/metabolismo , Pinocitose/fisiologia , Citoplasma , Núcleo Celular , Fatores de Transcrição/metabolismo , Mamíferos
9.
Mol Biol Cell ; 35(3): br9, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38265917

RESUMO

Cells rely on a diverse array of engulfment processes to sense, exploit, and adapt to their environments. Among these, macropinocytosis enables indiscriminate and rapid uptake of large volumes of fluid and membrane, rendering it a highly versatile engulfment strategy. Much of the molecular machinery required for macropinocytosis has been well established, yet how this process is regulated in the context of organs and organisms remains poorly understood. Here, we report the discovery of extensive macropinocytosis in the outer epithelium of the cnidarian Hydra vulgaris. Exploiting Hydra's relatively simple body plan, we developed approaches to visualize macropinocytosis over extended periods of time, revealing constitutive engulfment across the entire body axis. We show that the direct application of planar stretch leads to calcium influx and the inhibition of macropinocytosis. Finally, we establish a role for stretch-activated channels in inhibiting this process. Together, our approaches provide a platform for the mechanistic dissection of constitutive macropinocytosis in physiological contexts and highlight a potential role for macropinocytosis in responding to cell surface tension.


Assuntos
Hydra , Animais , Hydra/metabolismo , Pinocitose
10.
Cancer Res Commun ; 4(1): 170-181, 2024 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-38259097

RESUMO

Extracellular vesicles (EV) have emerged as critical effectors in the cross-talk between cancer and normal cells by transferring intracellular materials between adjacent or distant cells. Previous studies have begun to elucidate how cancer cells, by secreting EVs, adapt normal cells at a metastatic site to facilitate cancer cell metastasis. In this study, we utilized a high-content microscopic screening platform to investigate the mechanisms of EV uptake by primary lung fibroblasts. A selected library containing 90 FDA-approved anticancer drugs was screened for the effect on fibroblast uptake of EVs from MDA-MB-231 breast cancer cells. Among the drugs identified to inhibit EV uptake without exerting significant cytotoxicity, we validated the dose-dependent effect of Trametinib (a MEK1/2 inhibitor) and Copanlisib (a PI3K inhibitor). Trametinib suppressed macropinocytosis in lung fibroblasts and inhibited EV uptake with a higher potency comparing with Copanlisib. Gene knockdown and overexpression studies demonstrated that uptake of MDA-MB-231 EVs by lung fibroblasts required MEK2. These findings provide important insights into the mechanisms underlying lung fibroblast uptake of breast cancer cell-derived EVs, which could play a role in breast cancer metastasis to the lungs and suggest potential therapeutic targets for preventing or treating this deadly disease. SIGNIFICANCE: Through a phenotypic screen, we found that MEK inhibitor Trametinib suppressed EV uptake and macropinocytosis in lung fibroblasts, and that EV uptake is mediated by MEK2 in these cells. Our results suggest that MEK2 inhibition could serve as a strategy to block cancer EV uptake by lung fibroblasts.


Assuntos
Neoplasias da Mama , Vesículas Extracelulares , MAP Quinase Quinase 2 , Pinocitose , Transporte Biológico , Fibroblastos , Pulmão , Fosfatidilinositol 3-Quinases , Humanos , Células MDA-MB-231 , MAP Quinase Quinase 2/metabolismo , Neoplasias da Mama/metabolismo
11.
Angew Chem Int Ed Engl ; 63(10): e202318615, 2024 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-38126926

RESUMO

Cell-penetrating peptides (CPPs) play a significant role in the delivery of cargos into human cells. We report the first CPPs based on peptide-bismuth bicycles, which can be readily obtained from commercially available peptide precursors, making them accessible for a wide range of applications. These CPPs enter human cells as demonstrated by live-cell confocal microscopy using fluorescently labelled peptides. We report efficient sequences that demonstrate increased cellular uptake compared to conventional CPPs like the TAT peptide (derived from the transactivating transcriptional activator of human immunodeficiency virus 1) or octaarginine (R8 ), despite requiring only three positive charges. Bicyclization triggered by the presence of bismuth(III) increases cellular uptake by more than one order of magnitude. Through the analysis of cell lysates using inductive coupled plasma mass spectrometry (ICP-MS), we have introduced an alternative approach to examine the cellular uptake of CPPs. This has allowed us to confirm the presence of bismuth in cells after exposure to our CPPs. Mechanistic studies indicated an energy-dependent endocytic cellular uptake sensitive to inhibition by rottlerin, most likely involving macropinocytosis.


Assuntos
Peptídeos Penetradores de Células , Humanos , Peptídeos Penetradores de Células/química , Endocitose/fisiologia , Bismuto , Ciclismo , Pinocitose
12.
MAbs ; 15(1): 2263926, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37824334

RESUMO

In this investigation, we tested the hypothesis that a physiologically based pharmacokinetic (PBPK) model incorporating measured in vitro metrics of off-target binding can largely explain the inter-antibody variability in monoclonal antibody (mAb) pharmacokinetics (PK). A diverse panel of 83 mAbs was evaluated for PK in wild-type mice and subjected to 10 in vitro assays to measure major physiochemical attributes. After excluding for target-mediated elimination and immunogenicity, 56 of the remaining mAbs with an eight-fold variability in the area under the curve (AUC0-672h: 1.74 × 106 -1.38 × 107 ng∙h/mL) and 10-fold difference in clearance (2.55-26.4 mL/day/kg) formed the training set for this investigation. Using a PBPK framework, mAb-dependent coefficients F1 and F2 modulating pinocytosis rate and convective transport, respectively, were estimated for each mAb with mostly good precision (coefficient of variation (CV%) <30%). F1 was estimated to be the mean and standard deviation of 0.961 ± 0.593, and F2 was estimated to be 2.13 ± 2.62. Using principal component analysis to correlate the regressed values of F1/F2 versus the multidimensional dataset composed of our panel of in vitro assays, we found that heparin chromatography retention time emerged as the predictive covariate to the mAb-specific F1, whereas F2 variability cannot be well explained by these assays. A sigmoidal relationship between F1 and the identified covariate was incorporated within the PBPK framework. A sensitivity analysis suggested plasma concentrations to be most sensitive to F1 when F1 > 1. The predictive utility of the developed PBPK model was evaluated against a separate panel of 14 mAbs biased toward high clearance, among which area under the curve of PK data of 12 mAbs was predicted within 2.5-fold error, and the positive and negative predictive values for clearance prediction were 85% and 100%, respectively. MAb heparin chromatography assay output allowed a priori identification of mAb candidates with unfavorable PK.


Assuntos
Anticorpos Monoclonais , Modelos Biológicos , Camundongos , Animais , Pinocitose , Bioensaio , Heparina
13.
Front Immunol ; 14: 1182180, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37545539

RESUMO

Invariant chain (Ii, CD74) is a type II transmembrane glycoprotein that acts as a chaperone and facilitates the folding and transport of MHC II chains. By assisting the assembly and subcellular targeting of MHC II complexes, Ii has a wide impact on the functions of antigen-presenting cells such as antigen processing, endocytic maturation, signal transduction, cell migration, and macropinocytosis. Ii is a multifunctional molecule that can alter endocytic traffic and has several interacting molecules. To understand more about Ii's function and to identify further Ii interactors, a yeast two-hybrid screening was performed. Retinoic Acid-Induced 14 (Rai14) was detected as a putative interaction partner, and the interaction was confirmed by co-immunoprecipitation. Rai14 is a poorly characterized protein, which is believed to have a role in actin cytoskeleton and membrane remodeling. In line with this, we found that Rai14 localizes to membrane ruffles, where it forms macropinosomes. Depletion of Rai14 in antigen-presenting cells delays MHC II internalization, affecting macropinocytic activity. Intriguingly, we demonstrated that, similar to Ii, Rai14 is a positive regulator of macropinocytosis and a negative regulator of cell migration, two antagonistic processes in antigen-presenting cells. This antagonism is known to depend on the interaction between myosin II and Ii. Here, we show that Rai14 also binds to myosin II, suggesting that Ii, myosin II, and Rai14 work together to coordinate macropinocytosis and cell motility.


Assuntos
Antígenos de Histocompatibilidade Classe II , Tretinoína , Pinocitose/fisiologia , Proteínas do Citoesqueleto , Miosina Tipo II
14.
Curr Biol ; 33(15): R812-R814, 2023 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-37552948

RESUMO

Macropinocytosis is a form of endocytosis in which cells engulf relatively large quantities of extracellular fluid through cup-shaped invaginations of the plasma membrane. New work shows that macropinosome closure occurs without a localized constriction of actin filaments, indicating that membrane tension drives cup closure.


Assuntos
Endocitose , Pinocitose , Endossomos , Citoesqueleto de Actina , Membrana Celular
15.
J Neurosci Methods ; 397: 109947, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37574078

RESUMO

BACKGROUND: Macropinocytosis is a pathway utilized for the internalization of extracellular fluid, albumin and dissolved molecules. Assessing macropinocytosis has been challenging in the past because the combination of manual acquisition and visual evaluation of images is laborious, making this type of assessment difficult for high-throughput applications. Therefore, there is a need to develop sensitive and specific macropinocytosis evaluation methods. METHODS: This paper proposed a quantitative and time-saving method for macropinocytosis detection based on high-content analysis (HCA). Additionally, cell proliferation was evaluated using CCK8 test. RESULTS: The term "macropinosome index" was defined to estimate macropinocytosis and allow comparisons between different cell lines and treatments. Furthermore, we demonstrated that macropinocytosis can promote glioblastoma (GBM) cell survival under L-glutamine (L-Gln)-deficient conditions that resemble the tumour microenvironment. CONCLUSIONS: HCA represents a novel, nonsubjective and high-throughput assay for macropinocytosis assessment. In addition, L-Gln deprivation increased the macropinosome index in GBM cells, suggesting that this process may be used to design GBM therapies. AVAILABILITY OF DATA AND MATERIALS: The datasets supporting the conclusions of this article are included within the article and its supplementary materials.


Assuntos
Glioblastoma , Humanos , Glioblastoma/patologia , Pinocitose , Linhagem Celular , Proliferação de Células , Microambiente Tumoral
16.
Vet Microbiol ; 284: 109831, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37480660

RESUMO

Orf virus (ORFV), also known as infectious pustular virus, leads to an acute contagious zoonotic infectious disease. ORFV can directly contact and infect epithelial cells of skin and mucosa, causing damage to tissue cells. So far, the pathway of ORFV entry into cells is unclear. Therefore, finding the internalization pathway of ORFV will help to elucidate the cellular and molecular mechanisms of ORFV infection and invasion, which in turn will provide a certain reference for the prevention and treatment of ORFV. In the present study, chemical inhibitors were used to analyze the mechanism of ORFV entry into target cells. The results showed that the inhibitor of clathrin-mediated endocytosis could inhibit ORFV entry into cells. However, the inhibitor of caveolae-mediated endocytosis cannot inhibit ORFV entry into cells. In addition, inhibition of macropinocytosis pathway also significantly reduced ORFV internalization. Furthermore, the inhibitors of acidification and dynamin also prevented ORFV entry. However, results demonstrated that inhibitors inhibited ORFV entry but did not inhibit ORFV binding. Notably, extracellular trypsin promoted ORFV entry into cells directly, even when the endocytic pathway was inhibited. In conclusion, ORFV enters into its target cells by clathrin-mediated endocytosis and macropinocytosis, while caveolae-dependent endocytosis has little effects on this process. In addition, the entry into target cells by ORFV required an acid environment and the effect of dynamin. Meanwhile, we emphasize that broad-spectrum antiviral inhibitors and extracellular enzyme inhibitors are likely to be effective strategies for the prevention and treatment of ORFV infection.


Assuntos
Ectima Contagioso , Vírus do Orf , Doenças dos Ovinos , Animais , Ovinos , Endocitose , Pinocitose , Internalização do Vírus , Clatrina
17.
Adv Healthc Mater ; 12(27): e2301162, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37449948

RESUMO

Bioactive macromolecules show great promise for the treatment of various diseases. However, the cytosolic delivery of peptide-based drugs remains a challenging task owing to the existence of multiple intracellular barriers and ineffective endosomal escape. To address these issues, herein, programmable self-assembling peptide vectors are reported to amplify cargo internalization into the cytoplasm through receptor-activated macropinocytosis. Programmable self-assembling peptide vector-active human epidermal growth factor receptor-2 (HER2) signaling induces the receptor-activated macropinocytosis pathway, achieving efficient uptake in tumor cells. Shrinking macropinosomes accelerate the process of assembly dynamics and form nanostructures in the cytoplasm to increase peptide-based cargo accumulation and retention. Inductively coupled plasma mass (ICP-MS) spectrometry quantitative analysis indicates that the Gd delivery efficiency in tumor tissue through the macropinocytosis pathway is improved 2.5-fold compared with that through the use of active targeting molecular delivery. Finally, compared with nanoparticles and active targeting delivery, the delivery of bioactive peptide drugs through the self-assembly of peptide vectors maintains high drug activity (the IC50 decreased twofold) in the cytoplasm and achieves effective inhibition of tumor cell growth. Programmable self-assembling peptide vectors represent a promising platform for the intracellular delivery of diverse bioactive drugs, including molecular drugs, peptides, and biologics.


Assuntos
Nanoestruturas , Peptídeos , Humanos , Peptídeos/química , Pinocitose , Citosol/metabolismo , Endossomos/metabolismo , Proteínas de Transporte/metabolismo
18.
Anal Chem ; 95(30): 11410-11419, 2023 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-37468434

RESUMO

A fluorescent sensor that allows simultaneous analysis of environmental factors in a limited cellular space is useful for understanding precise molecular interactions in live cells and their biological responses. Macropinocytosis is a ubiquitous endocytic pathway for massive uptake of extracellular fluids, resulting in the formation of macropinosomes. Although macropinocytosis may impact intracellular delivery and cancer proliferation, information on the intracellular behaviors of macropinosomes is limited. Here, we aimed to develop a macropinoscope, a sensor that simultaneously detects pH and cathepsin B activity in individual macropinosomes. A macropinosome-specific marker, dextran (70 kDa), was employed as a platform, onto which fluorescein, Oregon Green, and tetramethylrhodamine were loaded for ratiometric pH sensing and imaging. A cathepsin-B-cleavable peptide sequence bearing sulfo-Cy5 and the quencher BHQ-3 was also mounted; cleavage of the sequence was detected as an increase in sulfo-Cy5 fluorescence. A steep decrease in pH was observed 5-10 min after macropinosome formation, which was accompanied by an immediate increase in cathepsin B activity. Our design concept will lead to the development of other macropinoscopes for the simultaneous detection of other parameters in individual macropinosomes.


Assuntos
Catepsina B , Endossomos , Catepsina B/metabolismo , Endossomos/metabolismo , Pinocitose/fisiologia , Concentração de Íons de Hidrogênio
19.
J Control Release ; 359: 302-314, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37307923

RESUMO

Macropinocytosis is a widely-observed and evolutionarily-conserved endocytic process found in the eukaryotic cells. In comparison to other endocytic routes, macropinocytosis allows for the internalization of greater quantities of fluid-phase drugs, making it an attractive avenue for drug delivery. Recent evidence showed that various drug delivery systems can be internalized through macropinocytosis. Utilizing macropinocytosis may therefore provide a new avenue for targeted intracellular delivery. In this review, we provide an overview into the origins and distinctive properties of macropinocytosis, summarize the roles of macropinocytosis under healthy and pathological settings. Furthermore, we highlight the biomimetic and synthetic drug delivery systems that employ macropinocytosis as the primary internalization mechanism. To facilitate the clinical applications of these drug delivery systems, additional research can be conducted to enhance the cell-type selectivity of macropinocytosis, the control of drug release at the target, and the prevention of potential toxicity. The rapidly emerging field of macropinocytosis-based targeted drug delivery and therapies holds great potential to drastically increase the efficiency and specificity of drug delivery.


Assuntos
Endocitose , Pinocitose
20.
Methods Mol Biol ; 2692: 375-384, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37365480

RESUMO

All forms of endocytosis involve the incidental uptake of fluid (pinocytosis). Macropinocytosis is a specialized type of endocytosis that results in the bulk ingestion of extracellular fluid via large (>0.2 µm) vacuoles called macropinosomes. The process is a means of immune surveillance, a point of entry for intracellular pathogens, and a source of nutrients for proliferating cancer cells. Macropinocytosis has also recently emerged as a tractable system that can be experimentally exploited to understand fluid handling in the endocytic pathway. In this chapter, we describe how stimulating macropinocytosis in the presence of extracellular fluids of a defined ionic composition can be combined with high-resolution microscopy to understand the role of ion transport in controlling membrane traffic.


Assuntos
Endocitose , Pinocitose , Endossomos , Vacúolos , Transporte Proteico
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