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1.
J Cell Biol ; 223(4)2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38319250

RESUMO

Endosomes are specialized organelles that function in the secretory and endocytic protein sorting pathways. Endocytosed cell surface receptors and transporters destined for lysosomal degradation are sorted into intraluminal vesicles (ILVs) at endosomes by endosomal sorting complexes required for transport (ESCRT) proteins. The endosomes (multivesicular bodies, MVBs) then fuse with the lysosome. During endosomal maturation, the number of ILVs increases, but the size of endosomes does not decrease despite the consumption of the limiting membrane during ILV formation. Vesicle-mediated trafficking is thought to provide lipids to support MVB biogenesis. However, we have uncovered an unexpected contribution of a large bridge-like lipid transfer protein, Vps13, in this process. Here, we reveal that Vps13-mediated lipid transfer at ER-endosome contact sites is required for the ESCRT pathway. We propose that Vps13 may play a critical role in supplying lipids to the endosome, ensuring continuous ESCRT-mediated sorting during MVB biogenesis.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/genética , Lipídeos , Corpos Multivesiculares , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transporte Proteico
2.
J Mater Chem B ; 12(7): 1892-1904, 2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-38305086

RESUMO

In recent years, a number of initially approved magnetic iron oxide nanoparticle (IONP)-based nano-medicines have been withdrawn due to the obscure nano-bio effects. Therefore, there is an urgent need to study the cellular effects triggered by IONPs on cells. In this study, we investigate the time-course cellular effects on the response of RAW 264.7 cells caused by Si-IONPs via pharmacological and mass spectrometry-based proteomics techniques. Our results revealed that Si-IONPs were internalized by clathrin-mediated endocytosis within 1 hour, and gradually degraded in endolysosomes over time, which might influence autophagy, oxidative stress, innate immune response, and inflammatory response after 12 hours. Our research provides a necessary assessment of Si-IONPs for further clinical treatment.


Assuntos
Endocitose , Proteômica , Lisossomos/metabolismo , Endossomos , Nanopartículas Magnéticas de Óxido de Ferro
3.
Front Immunol ; 15: 1328453, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38343548

RESUMO

Lung infection by influenza A virus (IAV) is a major cause of global mortality from lung injury, a disease defined by widespread dysfunction of the lung's air-blood barrier. Endocytosis of IAV virions by the alveolar epithelium - the cells that determine barrier function - is central to barrier loss mechanisms. Here, we address the current understanding of the mechanistic steps that lead to endocytosis in the alveolar epithelium, with an eye to how the unique structure of lung alveoli shapes endocytic mechanisms. We highlight where future studies of alveolar interactions with IAV virions may lead to new therapeutic approaches for IAV-induced lung injury.


Assuntos
Vírus da Influenza A , Influenza Humana , Lesão Pulmonar , Humanos , Barreira Alveolocapilar , Influenza Humana/patologia , Lesão Pulmonar/patologia , Pulmão/patologia , Endocitose
4.
Sci Rep ; 14(1): 291, 2024 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-38168911

RESUMO

Phosphatidylinositol 4,5-bisphosphate (PIP2) has been shown to be critical for the endocytosis of G protein-coupled receptors (GPCRs). We have previously demonstrated that depletion of PIP2 by chemically induced plasma membrane (PM) recruitment of a 5-phosphatase domain prevents the internalization of the ß2 adrenergic receptor (ß2AR) from the PM to early endosomes. In this study, we tested the effect of hormone-induced PM PIP2 depletion on ß2AR internalization using type-1 angiotensin receptor (AT1R) or M3 muscarinic acetylcholine receptor (M3R). We followed the endocytic route of ß2ARs in HEK 293T cells using bioluminescence resonance energy transfer between the receptor and endosome marker Rab5. To compare the effect of lipid depletion by different means, we created and tested an AT1R fusion protein that is capable of both recruitment-based and hormone-induced depletion methods. The rate of PM PIP2 depletion was measured using a biosensor based on the PH domain of phospholipase Cδ1. As expected, ß2AR internalization was inhibited when PIP2 depletion was evoked by recruiting 5-phosphatase to PM-anchored AT1R. A similar inhibition occurred when wild-type AT1R was activated by adding angiotensin II. However, stimulation of the desensitization/internalization-impaired mutant AT1R (TSTS/4A) caused very little inhibition of ß2AR internalization, despite the higher rate of measurable PIP2 depletion. Interestingly, inhibition of PIP2 resynthesis with the selective PI4KA inhibitor GSK-A1 had little effect on the change in PH-domain-measured PM PIP2 levels but did significantly decrease ß2AR internalization upon either AT1R or M3R activation, indicating the importance of a locally synthetized phosphoinositide pool in the regulation of this process.


Assuntos
Endocitose , Fosfatidilinositóis , Fosfatidilinositóis/metabolismo , Membrana Celular/metabolismo , Receptores de Angiotensina/metabolismo , Hormônios/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo
5.
Adv Exp Med Biol ; 1435: 315-327, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38175481

RESUMO

Membrane vesicles are secreted by growing bacterial cells and are important components of the bacterial secretome, with a role in delivering effector molecules that ultimately enable bacterial survival. Membrane vesicles of Clostridioides difficile likely contribute to pathogenicity and is a new area of research on which there is currently very limited information. This chapter summarizes the current knowledge on membrane vesicle formation, content, methods of characterization and functions in Clostridia and model Gram-positive species.


Assuntos
Clostridioides difficile , Clostridioides , Transporte Biológico , Endocitose , Conhecimento
6.
Int J Nanomedicine ; 19: 633-650, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38269255

RESUMO

Introduction: Most current anti-cancer therapies are associated with major side effects due to a lack of tumor specificity. Appropriate vectorization of drugs using engineered nanovectors is known to increase local concentration of therapeutic molecules in tumors while minimizing their side effects. Mesothelin (MSLN) is a well-known tumor associated antigen overexpressed in many malignancies, in particular in malignant pleural mesothelioma (MPM), and various MSLN-targeting anticancer therapies are currently evaluated in preclinical and clinical assays. In this study, we described, for the first time, the functionalization of fluorescent organic nanoassemblies (NA) with a nanobody (Nb) targeting MSLN for the specific targeting of MSLN expressing MPM cancer cells. Methods: Cell lines from different cancer origin expressing or not MSLN were used. An Nb directed against MSLN was coupled to fluorescent NA using click chemistry. A panel of endocytosis inhibitors was used to study targeted NA internalization by cells. Cancer cells were grown in 2D or 3D and under a flow to evaluate the specificity of the targeted NA. Binding and internalization of the targeted NA were studied using flow cytometry, confocal microscopy and transmission electron microscopy. Results: We show that the targeted NA specifically bind to MSLN-expressing tumor cells. Moreover, such functionalized NA appear to be internalized more rapidly and in significantly larger proportions compared to naked ones in MSLN+ MPM cells, thereby demonstrating both the functionality and interest of the active targeting strategy. We demonstrated that targeted NA are mainly internalized through a clathrin-independent/dynamin-dependent endocytosis pathway and are directed to lysosomes for degradation. A 3D cell culture model based on MSLN-expressing multicellular tumor spheroids reveals NA penetration in the first superficial layers. Conclusion: Altogether, these results open the path to novel anticancer strategies based on MSLN-activated internalization of NA incorporating drugs to promote specific accumulation of active treatments in tumors.


Assuntos
Bioensaio , Mesotelina , Linhagem Celular , Corantes , Endocitose
7.
PLoS Biol ; 22(1): e3002470, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38206965

RESUMO

The bridging integrator 1 (BIN1) gene is an important risk locus for late-onset Alzheimer's disease (AD). BIN1 protein has been reported to mediate tau pathology, but the underlying molecular mechanisms remain elusive. Here, we show that neuronal BIN1 is cleaved by the cysteine protease legumain at residues N277 and N288. The legumain-generated BIN1 (1-277) fragment is detected in brain tissues from AD patients and tau P301S transgenic mice. This fragment interacts with tau and accelerates its aggregation. Furthermore, the BIN1 (1-277) fragment promotes the propagation of tau aggregates by enhancing clathrin-mediated endocytosis (CME). Overexpression of the BIN1 (1-277) fragment in tau P301S mice facilitates the propagation of tau pathology, inducing cognitive deficits, while overexpression of mutant BIN1 that blocks its cleavage by legumain halts tau propagation. Furthermore, blocking the cleavage of endogenous BIN1 using the CRISPR/Cas9 gene-editing tool ameliorates tau pathology and behavioral deficits. Our results demonstrate that the legumain-mediated cleavage of BIN1 plays a key role in the progression of tau pathology. Inhibition of legumain-mediated BIN1 cleavage may be a promising therapeutic strategy for treating AD.


Assuntos
Doença de Alzheimer , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/patologia , Encéfalo/metabolismo , Clatrina/metabolismo , Endocitose , Camundongos Transgênicos , Proteínas tau/genética , Proteínas tau/metabolismo
8.
Soft Matter ; 20(3): 651-660, 2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-38164011

RESUMO

Endocytosis is a fundamental cellular process in eukaryotic cells that facilitates the transport of molecules into the cell. With the help of fluorescence microscopy and electron tomography, researchers have accumulated extensive geometric data of membrane shapes during endocytosis. These data contain rich information about the mechanical properties of membranes, which are hard to access via experiments due to the small dimensions of the endocytic patch. In this study, we propose an approach that combines machine learning with the Helfrich theory of membranes to infer the mechanical properties of membranes during endocytosis from a dataset of membrane shapes extracted from electron tomography. Our results demonstrate that machine learning can output solutions that both match the experimental profile and satisfy the membrane shape equations derived from Helfrich theory. The learning results show that during the early stage of endocytosis, the inferred membrane tension is negative, indicating the presence of strong compressive forces at the boundary of the endocytic invagination. Our method presents a generic framework for extracting membrane information from super-resolution imaging.


Assuntos
Endocitose , Células Eucarióticas , Membrana Celular/metabolismo , Membranas , Aprendizado de Máquina
9.
Nat Commun ; 15(1): 454, 2024 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-38212623

RESUMO

Emerging evidence indicates that the activation of ferroptosis by glutathione peroxidase 4 (GPX4) inhibitors may be a prominent therapeutic strategy for tumor suppression. However, the wide application of GPX4 inhibitors in tumor therapy is hampered due to poor tumor delivery efficacy and the nonspecific activation of ferroptosis. Taking advantage of in vivo self-assembly, we develop a peptide-ferriporphyrin conjugate with tumor microenvironment specific activation to improve tumor penetration, endocytosis and GPX4 inhibition, ultimately enhancing its anticancer activity via ferroptosis. Briefly, a GPX4 inhibitory peptide is conjugated with an assembled peptide linker decorated with a pH-sensitive moiety and ferriporphyrin to produce the peptide-ferriporphyrin conjugate (Gi-F-CAA). Under the acidic microenvironment of the tumor, the Gi-F-CAA self-assembles into large nanoparticles (Gi-F) due to enhanced hydrophobic interaction after hydrolysis of CAA, improving tumor endocytosis efficiency. Importantly, Gi-F exhibits substantial inhibition of GPX4 activity by assembly enhanced binding (AEB) effect, augmenting the oxidative stress of ferriporphyrin-based Fenton reaction, ultimately enabling antitumor properties in multiple tumor models. Our findings suggest that this peptide-ferriporphyrin conjugate design with AEB effect can improve the therapeutic effect via induction of ferroptosis, providing an alternative strategy for overcoming chemoresistance.


Assuntos
Ferroptose , Neoplasias , Humanos , Endocitose , Hemina , Hidrólise , Peptídeos/farmacologia , Linhagem Celular Tumoral , Neoplasias/tratamento farmacológico , Microambiente Tumoral
10.
Cell Death Dis ; 15(1): 60, 2024 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-38233383

RESUMO

The Western diet, characterized by its high content of long-chain fatty acids (LCFAs), is widely recognized as a significant triggering factor for inflammatory bowel disease (IBD). While the link between a high-fat diet and colitis has been observed, the specific effects and mechanisms remain incompletely understood. Our study provides evidence that the diet rich in LCFAs can disrupt the integrity of the intestinal barrier and exacerbate experimental colitis in mice. Mechanistically, LCFAs upregulate the signal transducer and activator of transcription-3 (STAT3) pathway in the inflammatory model, and STAT3 knockout effectively counters the pro-inflammatory effects of LCFAs on colitis. Specifically, palmitic acid (PA), a representative LCFA, enters intestinal epithelial cells via the cluster of differentiation 36 (CD36) pathway and participates in the palmitoylation cycle of STAT3. Inhibiting this cycle using pharmacological inhibitors like 2-Bromopalmitate (2-BP) and ML349, as well as DHHC7 knockdown, has the ability to alleviate inflammation induced by PA. These findings highlight the significant role of dietary LCFAs, especially PA, in the development and progression of IBD. Diet adjustments and targeted modulation offer potential therapeutic strategies for managing this condition. Model of LCFAs involvement in the palmitoylation cycle of STAT3 upon internalization into cells. Following cellular uptake through CD36, LCFAs are converted to palmitoyl-CoA. In the presence of DHHC7, palmitoyl-CoA binds to STAT3 at the C108 site, forming palmitoylated STAT3. Palmitoylation further promotes phosphorylation at the Y705 site of STAT3. Subsequently, palmitoylated STAT3 undergoes depalmitoylation by APT2 and translocates to the nucleus to exert its biological functions.


Assuntos
Colite , Doenças Inflamatórias Intestinais , Animais , Camundongos , Colite/induzido quimicamente , Dieta Hiperlipídica/efeitos adversos , Endocitose , Ácidos Graxos/metabolismo , Lipoilação , Fator de Transcrição STAT3/metabolismo
11.
Cell Mol Life Sci ; 81(1): 43, 2024 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-38217571

RESUMO

Adherent cells ensure membrane homeostasis during de-adhesion by various mechanisms, including endocytosis. Although mechano-chemical feedbacks involved in this process have been studied, the step-by-step build-up and resolution of the mechanical changes by endocytosis are poorly understood. To investigate this, we studied the de-adhesion of HeLa cells using a combination of interference reflection microscopy, optical trapping and fluorescence experiments. We found that de-adhesion enhanced membrane height fluctuations of the basal membrane in the presence of an intact cortex. A reduction in the tether force was also noted at the apical side. However, membrane fluctuations reveal phases of an initial drop in effective tension followed by saturation. The area fractions of early (Rab5-labelled) and recycling (Rab4-labelled) endosomes, as well as transferrin-labelled pits close to the basal plasma membrane, also transiently increased. On blocking dynamin-dependent scission of endocytic pits, the regulation of fluctuations was not blocked, but knocking down AP2-dependent pit formation stopped the tension recovery. Interestingly, the regulation could not be suppressed by ATP or cholesterol depletion individually but was arrested by depleting both. The data strongly supports Clathrin and AP2-dependent pit-formation to be central to the reduction in fluctuations confirmed by super-resolution microscopy. Furthermore, we propose that cholesterol-dependent pits spontaneously regulate tension under ATP-depleted conditions.


Assuntos
Clatrina , Invaginações Revestidas da Membrana Celular , Humanos , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Células HeLa , Endocitose/fisiologia , Colesterol/metabolismo , Trifosfato de Adenosina/metabolismo , Membrana Celular/metabolismo
12.
ACS Appl Mater Interfaces ; 16(5): 5451-5461, 2024 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-38265005

RESUMO

Nonendocytic cell uptake of nanomaterials is challenging, which requires specific surface chemistry and smaller particle size. Earlier works have shown that an arginine-terminated nanoparticle of <10-20 nm size shows nonendocytic uptake via direct membrane penetration. However, the roles of surface arginine density and the arginine-arginine distance at the nanoparticle surface in controlling such nonendocytic uptake mechanism is not yet explored. Here we show that a higher arginine density at the nanoparticle surface with an arginine-arginine distance of <3 nm is the most critical aspect for such nonendocytic uptake. We have used quantum dot (QD)-based nanoparticles as a model for fluorescent tracking inside cells and for quantitative estimation of cellular uptake. We found that arginine-terminated nanoparticles of 10 nm size can opt for the energy-dependent endocytosis pathway if the arginine-arginine distance is >3 nm. In contrast, nanoparticles with <3 nm arginine-arginine distance rapidly enter into the cell via the nonendocytic approach, are freely available in the cytosol in large amounts to capture the cellular adenosine triphosphate (ATP), generate oxidative stress, and induce ATP-deficient cellular autophagy. This work shows that arginine-arginine distance at the nanoparticle surface is another fundamental parameter, along with the particle size, for the nonendocytic cell uptake of foreign materials and to control intracellular activity. This approach may be utilized in designing nanoprobes and nanocarriers with more efficient biomedical performances.


Assuntos
Nanopartículas , Pontos Quânticos , Arginina , Nanopartículas/metabolismo , Autofagia , Trifosfato de Adenosina , Tamanho da Partícula , Endocitose
13.
Cell Res ; 34(2): 140-150, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38182887

RESUMO

Crimean-Congo hemorrhagic fever virus (CCHFV) is the most widespread tick-born zoonotic bunyavirus that causes severe hemorrhagic fever and death in humans. CCHFV enters the cell via clathrin-mediated endocytosis which is dependent on its surface glycoproteins. However, the cellular receptors that are required for CCHFV entry are unknown. Here we show that the low density lipoprotein receptor (LDLR) is an entry receptor for CCHFV. Genetic knockout of LDLR impairs viral infection in various CCHFV-susceptible human, monkey and mouse cells, which is restored upon reconstitution with ectopically-expressed LDLR. Mutagenesis studies indicate that the ligand binding domain (LBD) of LDLR is necessary for CCHFV infection. LDLR binds directly to CCHFV glycoprotein Gc with high affinity, which supports virus attachment and internalization into host cells. Consistently, a soluble sLDLR-Fc fusion protein or anti-LDLR blocking antibodies impair CCHFV infection into various susceptible cells. Furthermore, genetic knockout of LDLR or administration of an LDLR blocking antibody significantly reduces viral loads, pathological effects and death following CCHFV infection in mice. Our findings suggest that LDLR is an entry receptor for CCHFV and pharmacological targeting of LDLR may provide a strategy to prevent and treat Crimean-Congo hemorrhagic fever.


Assuntos
Vírus da Febre Hemorrágica da Crimeia-Congo , Febre Hemorrágica da Crimeia , Receptores de LDL , Animais , Humanos , Camundongos , Endocitose , Glicoproteínas/metabolismo , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Vírus da Febre Hemorrágica da Crimeia-Congo/metabolismo , Febre Hemorrágica da Crimeia/prevenção & controle , Receptores de LDL/metabolismo , Internalização do Vírus
14.
BMC Biol ; 22(1): 12, 2024 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-38273307

RESUMO

BACKGROUND: Many viruses enter host cells by hijacking endosomal trafficking. CapZ, a canonical actin capping protein, participates in endosomal trafficking, yet its precise role in endocytosis and virus infection remains elusive. RESULTS: Here, we showed that CapZ was transiently associated with early endosomes (EEs) and was subsequently released from the matured EEs after the fusion of two EEs, which was facilitated by PI(3)P to PI(3,5)P2 conversion. Vacuolin-1 (a triazine compound) stabilized CapZ at EEs and thus blocked the transition of EEs to late endosomes (LEs). Likewise, artificially tethering CapZ to EEs via a rapamycin-induced protein-protein interaction system blocked the early-to-late endosome transition. Remarkably, CapZ knockout or artificially tethering CapZ to EEs via rapamycin significantly inhibited flaviviruses, e.g., Zika virus (ZIKV) and dengue virus (DENV), or beta-coronavirus, e.g., murine hepatitis virus (MHV), infection by preventing the escape of RNA genome from endocytic vesicles. CONCLUSIONS: These results indicate that the temporal association of CapZ with EEs facilitates early-to-late endosome transition (physiologically) and the release of the viral genome from endocytic vesicles (pathologically).


Assuntos
Fosfatos de Fosfatidilinositol , Infecção por Zika virus , Zika virus , Animais , Humanos , Camundongos , Endocitose/fisiologia , Endossomos/metabolismo , Sirolimo/farmacologia , Sirolimo/metabolismo , Vesículas Transportadoras , Internalização do Vírus , Infecção por Zika virus/metabolismo
15.
Mol Biol Cell ; 35(3): ar40, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38198575

RESUMO

The multisubunit HOPS tethering complex is a well-established regulator of lysosome fusion with late endosomes and autophagosomes. However, the role of the HOPS complex in other stages of endo-lysosomal trafficking is not well understood. To address this, we made HeLa cells knocked out for the HOPS-specific subunits Vps39 or Vps41, or the HOPS-CORVET-core subunits Vps18 or Vps11. In all four knockout cells, we found that endocytosed cargos were trapped in enlarged endosomes that clustered in the perinuclear area. By correlative light-electron microscopy, these endosomes showed a complex ultrastructure and hybrid molecular composition, displaying markers for early (Rab5, PtdIns3P, EEA1) as well as late (Rab7, CD63, LAMP1) endosomes. These "HOPS bodies" were not acidified, contained enzymatically inactive cathepsins and accumulated endocytosed cargo and cation-independent mannose-6-phosphate receptor (CI-MPR). Consequently, CI-MPR was depleted from the TGN, and secretion of lysosomal enzymes to the extracellular space was enhanced. Strikingly, HOPS bodies also contained the autophagy proteins p62 and LC3, defining them as amphisomes. Together, these findings show that depletion of the lysosomal HOPS complex has a profound impact on the functional organization of the entire endosomal system and suggest the existence of a HOPS-independent mechanism for amphisome formation.


Assuntos
Endocitose , Endossomos , Humanos , Células HeLa , Endossomos/metabolismo , Membranas Intracelulares , Lisossomos/metabolismo
16.
Exp Cell Res ; 435(1): 113906, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38176465

RESUMO

Quiescin/sulfhydryl oxidase (QSOX1) is a secreted flavoprotein that modulates cellular proliferation, migration and adhesion, roles attributed to its ability to organize the extracellular matrix. We previously showed that exogenously added QSOX1b induces smooth muscle cells migration in a process that depends on its enzymatic activity and that is mediated by hydrogen peroxide derived from Nox1, a catalytic subunit of NAD(P)H oxidases. Here, we report that exogenous QSOX1b also stimulates the migration of L929 fibroblasts and that this effect is regulated by its endocytosis. The use of endocytosis inhibitors and caveolin 1-knockdown demonstrated that this endocytic pathway is caveola-mediated. QSOX1b colocalized with Nox1 in intracellular vesicles, as detected by confocal fluorescence, suggesting that extracellular QSOX1b is endocytosed with the transmembrane Nox1. These results reveal that endosomal QSOX1b is a novel intracellular redox regulator of cell migration.


Assuntos
Cavéolas , NADPH Oxidases , Fibroblastos , Endocitose , Proliferação de Células
17.
Sci Total Environ ; 908: 168374, 2024 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-37956851

RESUMO

Cadmium (Cd) and lead (Pb) accumulate easily in leafy vegetables and can harm human health. Lanthanum (La) have been used to improve agricultural yield and quality, but the effect of La application on Cd/Pb enrichment in leafy vegetables remains incomplete currently. A previous study reported that the endocytosis in lettuce leaf cells can be activated by La, leading to an increase in Pb accumulation in lettuce leaves. However, it has not been investigated whether foliar application of La enhances root cellular endocytosis and promotes its uptake of Cd and Pb. In this study, the influence of La on the uptake of Cd and Pb, Cd bioaccessibility, and the safety risks of cultivating lettuce under Cd and Pb stress were explored. It was found that La increased Cd (16-30 % in shoot, 16-34 % in root) and Pb (25-29 % in shoot, 17-23 % in root) accumulation in lettuce. The increased accumulation of Cd and Pb could be attributed to La-enhanced endocytosis. Meanwhile, La enhanced the toxicity of both Cd and Pb, inhibited lettuce growth, and aggravated the damage to the photosynthetic and antioxidant systems. Finally, gastrointestinal simulation experiments showed that La increased the Cd bioaccessibility in both gastric and intestinal phase by 7-108 % and 9-87 %, respectively. These results offer valuable insights into the safety of REEs for agricultural applications.


Assuntos
Cádmio , Poluentes do Solo , Humanos , Cádmio/análise , Lantânio/toxicidade , Chumbo/toxicidade , Verduras , Endocitose , Poluentes do Solo/análise , Solo
18.
Mol Biol Cell ; 35(1): ar9, 2024 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-37938925

RESUMO

The HIV-1 accessory protein Nef hijacks clathrin adaptors to degrade or mislocalize host proteins involved in antiviral defenses. Here, using quantitative live-cell microscopy in genome-edited Jurkat cells, we investigate the impact of Nef on clathrin-mediated endocytosis (CME), a major pathway for membrane protein internalization in mammalian cells. Nef is recruited to CME sites on the plasma membrane, and this recruitment is associated with an increase in the recruitment and lifetime of the CME coat protein AP-2 and the late-arriving CME protein dynamin2. Furthermore, we find that CME sites that recruit Nef are more likely to recruit dynamin2 and transferrin, suggesting that Nef recruitment to CME sites promotes site maturation to ensure high efficiency in host protein downregulation. Implications of these observations for HIV-1 infection are discussed.


Assuntos
Clatrina , Endocitose , HIV-1 , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Animais , Humanos , Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitose/fisiologia , HIV-1/metabolismo , Células Jurkat , Proteínas de Membrana/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo
19.
J Neurochem ; 168(2): 100-114, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38102893

RESUMO

The aquaporin-4 (AQP4) water channel is abundantly expressed in the glial cells of the central nervous system and facilitates brain swelling following diverse insults, such as traumatic injury or stroke. Lack of specific and therapeutic AQP4 inhibitors highlights the need to explore alternative routes to control the water permeability of glial cell membranes. The cell surface abundance of AQP4 in mammalian cells fluctuates rapidly in response to changes in oxygen levels and tonicity, suggesting a role for vesicular trafficking in its translocation to and from the cell surface. However, the molecular mechanisms of AQP4 trafficking are not fully elucidated. In this work, early and recycling endosomes were investigated as likely candidates of rapid AQP4 translocation together with changes in cytoskeletal dynamics. In transiently transfected HEK293 cells a significant amount of AQP-eGFP colocalised with mCherry-Rab5-positive early endosomes and mCherry-Rab11-positive recycling endosomes. When exposed to hypotonic conditions, AQP4-eGFP rapidly translocated from intracellular vesicles to the cell surface. Co-expression of dominant negative forms of the mCherry-Rab5 and -Rab11 with AQP4-eGFP prevented hypotonicity-induced AQP4-eGFP trafficking and led to concentration at the cell surface or intracellular vesicles respectively. Use of endocytosis inhibiting drugs indicated that AQP4 internalisation was dynamin-dependent. Cytoskeleton dynamics-modifying drugs also affected AQP4 translocation to and from the cell surface. AQP4 trafficking mechanisms were validated in primary human astrocytes, which express high levels of endogenous AQP4. The results highlight the role of early and recycling endosomes and cytoskeletal dynamics in AQP4 translocation in response to hypotonic and hypoxic stress and suggest continuous cycling of AQP4 between intracellular vesicles and the cell surface under physiological conditions.


Assuntos
Endocitose , Endossomos , Animais , Humanos , Células HEK293 , Transporte Proteico , Endossomos/metabolismo , Astrócitos/metabolismo , Aquaporina 4/genética , Aquaporina 4/metabolismo , Mamíferos/metabolismo
20.
Biochem Pharmacol ; 220: 116013, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38151077

RESUMO

GPR101 is an orphan G protein-coupled receptor that promotes growth hormone secretion in the pituitary. The microduplication of the GPR101 gene has been linked with the X-linked acrogigantism, or X-LAG, syndrome. This disease is characterized by excessive growth hormone secretion and abnormal rapid growth beginning early in life. Mechanistically, GPR101 induces growth hormone secretion through constitutive activation of multiple heterotrimeric G proteins. However, the full scope of GPR101 signaling remains largely elusive. Herein, we investigated the association of GPR101 to multiple transducers and uncovered an important basal interaction with Arrestin 2 (ß-arrestin 1) and Arrestin 3 (ß-arrestin 2). By using a GPR101 mutant lacking the C-terminus and cell lines with an Arrestin 2/3 null background, we show that the arrestin association leads to constitutive clathrin- and dynamin-mediated GPR101 internalization. To further highlight GPR101 intracellular fate, we assessed the colocalization of GPR101 with Rab protein markers. Internalized GPR101 was mainly colocalized with the early endosome markers, Rab5 and EEA-1, and to a lesser degree with the late endosome marker Rab7. However, GPR101 was not colocalized with the recycling endosome marker Rab11. These findings show that the basal arrestin recruitment by GPR101 C-terminal tail drives the receptor constitutive clathrin-mediated internalization. Intracellularly, GPR101 concentrates in the endosomal compartment and is degraded through the lysosomal pathway. In conclusion, we uncovered a constitutive intracellular trafficking of GPR101 that potentially represents an important layer of regulation of its signaling and function.


Assuntos
Arrestinas , Receptores Acoplados a Proteínas G , beta-Arrestina 1/metabolismo , beta-Arrestinas/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Arrestinas/genética , Arrestinas/metabolismo , beta-Arrestina 2/metabolismo , Hormônio do Crescimento , Clatrina/metabolismo , Endocitose
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