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Biology Open Nov 2023Autotaxin, encoded by the Enpp2 gene, is an exoenzyme that produces lysophosphatidic acid, thereby regulating many biologic functions. We previously reported that Enpp2...
Autotaxin, encoded by the Enpp2 gene, is an exoenzyme that produces lysophosphatidic acid, thereby regulating many biologic functions. We previously reported that Enpp2 mRNA was abundantly expressed in yolk sac visceral endoderm (VE) cells and that Enpp2-/- mice were lethal at embryonic day 9.5 owing to angiogenic defects in the yolk sac. Enpp2-/- mice showed lysosome fragmentation in VE cells and embryonic abnormalities including allantois malformation, neural tube defects, no axial turning, and head cavity formation. However, whether the defects in endocytic vesicle formation affect membrane trafficking in VE cells remained to be directly examined. In this study, we found that pinocytosis, transcytosis, and secretion of angiogenic factors such as vascular endothelial growth factor and transforming growth factor β1 were impaired in Enpp2-/- VE cells. Moreover, pharmacologic inhibition of membrane trafficking phenocopied the defects of Enpp2-/- mice. These findings demonstrate that Enpp2 promotes endocytosis and secretion of angiogenic factors in VE cells, thereby regulating angiogenesis/vasculogenesis and embryonic development.
Topics: Animals; Female; Mice; Pregnancy; Cell Differentiation; Embryonic Development; Endoderm; Vascular Endothelial Growth Factor A; Yolk Sac; Phosphoric Diester Hydrolases
PubMed: 37795611
DOI: 10.1242/bio.060081 -
The Science of the Total Environment Dec 2023Microplastics (MPs) and nanoplastics (NPs) are ubiquitous in the marine environments due to the wide use and mismanagement of plastics. However, the effect of MPs/NPs on...
Microplastics (MPs) and nanoplastics (NPs) are ubiquitous in the marine environments due to the wide use and mismanagement of plastics. However, the effect of MPs/NPs on the nutrition quality of economic species is poorly understood, and their underlying mechanisms remained unclear. We therefore investigated the impacts of polystyrene MPs/NPs on the nutrition composition of marine jacopever Sebastes schlegelii from the perspective of assimilation and metabolism. Results showed that NPs reduced more nutrition quality than MPs. Despite no notable impact on intestinal microbiota function, MPs/NPs influenced the assimilation of fish through intestinal damage. Furthermore, NPs induced greater damage to hepatocyte metabolism than MPs, caused by hepatocyte uptake through membrane protein pumps/channels and clathrin/caveolin-mediated endocytosis for NPs, while through phagocytosis/pinocytosis for MPs. NPs triggered more cell apoptosis signals in Ferroptosis and FoxO signaling pathways than MPs, destroying mitochondria structure. Compared with MP treatments, a significant upregulation of genes (PRODH and SLC25A25A) associated with the electron transfer chain of mitochondria was detected in the NP treatments, influencing the tricarboxylic acid cycle and interfering with liver metabolism of proteins, fatty acid, glycerol phospholipids, and carbohydrates. This work provides new insights into the potential impacts of MPs/NPs on the quality and safety of seafood.
PubMed: 37633373
DOI: 10.1016/j.scitotenv.2023.166560 -
Frontiers in Immunology 2023Invariant 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...
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.
Topics: Tretinoin; Histocompatibility Antigens Class II; Pinocytosis; Cytoskeletal Proteins; Myosin Type II
PubMed: 37545539
DOI: 10.3389/fimmu.2023.1182180 -
Insects Jun 2023In mosquitoes, the utilization of RNAi for functional genetics is widespread, usually mediated through introduced double-stranded RNAs (dsRNAs) with sequence identity to...
In mosquitoes, the utilization of RNAi for functional genetics is widespread, usually mediated through introduced double-stranded RNAs (dsRNAs) with sequence identity to a gene of interest. However, RNAi in mosquitoes is often hampered by inconsistencies in target gene knockdown between experimental setups. While the core RNAi pathway is known to function in most mosquito strains, the uptake and biodistribution of dsRNAs across different mosquito species and life stages have yet to be extensively explored as a source of variation in RNAi experiments. To better understand mosquito-RNAi dynamics, the biodistribution of a dsRNA to a heterologous gene, LacZ (iLacZ), was tracked following various routes of exposure in the larval and adult stages of , , and . iLacZ was largely limited to the gut lumen when exposed per os, or to the cuticle when topically applied, but spread through the hemocoel when injected. Uptake of dsRNA was noted in a subset of cells including: hemocytes, pericardial cells of the dorsal vessel, ovarian follicles, and ganglia of the ventral nerve cord. These cell types are all known to undergo phagocytosis, pinocytosis, or both, and as such may actively take up RNAi triggers. In , iLacZ was detected for up to one week post exposure by Northern blotting, but uptake and degradation drastically differed across tissues. The results presented here reveal that the uptake of RNAi triggers is distinct and specific to the cell type in vivo.
PubMed: 37367372
DOI: 10.3390/insects14060556 -
ACS Nano May 2023The RAS-transformed cells utilize macropinocytosis to acquire amino acids to support their uncontrolled growth. However, targeting RAS to inhibit macropinocytosis...
The RAS-transformed cells utilize macropinocytosis to acquire amino acids to support their uncontrolled growth. However, targeting RAS to inhibit macropinocytosis remains a challenge. Here, we report that gold nanoparticles (GNP) inhibit macropinocytosis by decreasing KRAS activation. Using surface-modified and unmodified GNP, we showed that unmodified GNP specifically sequestered both wild-type and mutant KRAS and inhibited its activation, irrespective of growth factor stimulation, while surface-passivated GNP had no effect. Alteration of KRAS activation is reflected on downstream signaling cascades, macropinocytosis and tumor cell growth , and two independent preclinical human xenograft models of pancreatic cancer . The current study demonstrates NP-mediated inhibition of macropinocytosis and KRAS activation and provides translational opportunities to inhibit tumor growth in a number of cancers where activation of KRAS plays a major role.
Topics: Humans; Gold; Proto-Oncogene Proteins p21(ras); Metal Nanoparticles; Pinocytosis; Pancreatic Neoplasms; Cell Proliferation; Cell Line, Tumor; Mutation
PubMed: 37129853
DOI: 10.1021/acsnano.3c00920 -
Journal of Virology Apr 2023Porcine enteric alphacoronavirus (PEAV) is a new bat HKU2-like porcine coronavirus, and its endemic outbreak has caused severe economic losses to the pig industry. Its...
Porcine enteric alphacoronavirus (PEAV) is a new bat HKU2-like porcine coronavirus, and its endemic outbreak has caused severe economic losses to the pig industry. Its broad cellular tropism suggests a potential risk of cross-species transmission. A limited understanding of PEAV entry mechanisms may hinder a rapid response to potential outbreaks. This study analyzed PEAV entry events using chemical inhibitors, RNA interference, and dominant-negative mutants. PEAV entry into Vero cells depended on three endocytic pathways: caveolae, clathrin, and macropinocytosis. Endocytosis requires dynamin, cholesterol, and a low pH. Rab5, Rab7, and Rab9 GTPases (but not Rab11) regulate PEAV endocytosis. PEAV particles colocalize with EEA1, Rab5, Rab7, Rab9, and Lamp-1, suggesting that PEAV translocates into early endosomes after internalization, and Rab5, Rab7, and Rab9 regulate trafficking to lysosomes before viral genome release. PEAV enters porcine intestinal cells (IPI-2I) through the same endocytic pathway, suggesting that PEAV may enter various cells through multiple endocytic pathways. This study provides new insights into the PEAV life cycle. Emerging and reemerging coronaviruses cause severe human and animal epidemics worldwide. PEAV is the first bat-like coronavirus to cause infection in domestic animals. However, the PEAV entry mechanism into host cells remains unknown. This study demonstrates that PEAV enters into Vero or IPI-2I cells through caveola/clathrin-mediated endocytosis and macropinocytosis, which does not require a specific receptor. Subsequently, Rab5, Rab7, and Rab9 regulate PEAV trafficking from early endosomes to lysosomes, which is pH dependent. The results advance our understanding of the disease and help to develop potential new drug targets against PEAV.
Topics: Virus Internalization; Alphacoronavirus; rab GTP-Binding Proteins; Endosomes; Coronavirus Infections; Hydrogen-Ion Concentration; Dynamins; Caveolae; Cholesterol; Clathrin; Pinocytosis; Vero Cells; Chlorocebus aethiops; Animals
PubMed: 36975780
DOI: 10.1128/jvi.00210-23 -
Drug Discoveries & Therapeutics May 2023Since nanoparticles are taken up into cells by endocytosis, phagocytosis, or pinocytosis, they have been studied as intracellular drug carriers. Janus particles have an...
Since nanoparticles are taken up into cells by endocytosis, phagocytosis, or pinocytosis, they have been studied as intracellular drug carriers. Janus particles have an anisotropic structure composed of two or more distinct domains and have been proposed for use in various applications, including use as imaging agents or nanosensors. This study aimed to clarify the influence of the type of nanoparticles on their distribution in a human Caucasian colon adenocarcinoma (Caco-2) cell monolayer. We fabricated Janus and conventional spherical nanoparticles composed of pharmaceutically applicable ingredients. Janus and spherical nanoparticles composed of a cationic polymer and surfactant lipids were prepared by controlling the solvent removal pattern from the oil phase in the solvent removal process using the solvent evaporation and solvent diffusion methods. The distribution of nanoparticles in the Caco-2 cell monolayer was then evaluated using confocal laser microscopy. The mean hydrodynamic size of the fabricated Janus nanoparticles was 119.2 ± 4.6 nm. Distribution analysis using Caco-2 cells suggested that Janus nanoparticles were localized around the adherens junctions located just below the tight junction. Clear localization was not observed in non-Janus nanoparticles with the same composition. The clear localization of the Janus nanoparticles around the adherens junction may be due to their positive charge and asymmetric structure. Our results suggest the considerable potential for the development of nanoparticulate drug carriers to target cellular gaps.
Topics: Humans; Surface-Active Agents; Multifunctional Nanoparticles; Caco-2 Cells; Polymers; Adenocarcinoma; Colonic Neoplasms; Nanoparticles; Drug Carriers; Solvents; Lipids; Particle Size
PubMed: 36948641
DOI: 10.5582/ddt.2022.01118 -
The Journal of Biological Chemistry Apr 2023Pseudorabies virus (PRV) has become a "new life-threatening zoonosis" since the human-originated PRV strain was first isolated in 2020. To identify novel anti-PRV...
Pseudorabies virus (PRV) has become a "new life-threatening zoonosis" since the human-originated PRV strain was first isolated in 2020. To identify novel anti-PRV agents, we screened a total of 107 β-carboline derivatives and found 20 compounds displaying antiviral activity against PRV. Among them, 14 compounds showed better antiviral activity than acyclovir. We found that compound 45 exhibited the strongest anti-PRV activity with an IC value of less than 40 nM. Our in vivo studies showed that treatment with 45 significantly reduced the viral loads and protected mice challenged with PRV. To clarify the mode of action of 45, we conducted a time of addition assay, an adsorption assay, and an entry assay. Our results indicated that 45 neither had a virucidal effect nor affected viral adsorption while significantly inhibiting PRV entry. Using the FITC-dextran uptake assay, we determined that 45 inhibits macropinocytosis. The actin-dependent plasma membrane protrusion, which is important for macropinocytosis, was also suppressed by 45. Furthermore, the kinase DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1A) was predicted to be a potential target for 45. The binding of 45 to DYRK1A was confirmed by drug affinity responsive target stability and cellular thermal shift assay. Further analysis revealed that knockdown of DYRK1A by siRNA suppressed PRV macropinocytosis and the tumor necrosis factor alpha-TNF-induced formation of protrusions. These results suggested that 45 could restrain PRV macropinocytosis by targeting DYRK1A. Together, these findings reveal a unique mechanism through which β-carboline derivatives restrain PRV infection, pointing to their potential value in the development of anti-PRV agents.
Topics: Animals; Humans; Mice; Acyclovir; Antiviral Agents; Carbolines; Gene Knockdown Techniques; Herpesvirus 1, Suid; Inhibitory Concentration 50; Pinocytosis; Protein-Tyrosine Kinases; Pseudorabies; Virus Internalization; HeLa Cells; Models, Chemical; Dyrk Kinases
PubMed: 36918100
DOI: 10.1016/j.jbc.2023.104605 -
Experimental & Molecular Medicine Mar 2023The osmosensitive transcription factor nuclear factor of activated T cells 5 (NFAT5; or tonicity-responsive enhancer binding protein; TonEBP) plays a key role in...
The osmosensitive transcription factor nuclear factor of activated T cells 5 (NFAT5; or tonicity-responsive enhancer binding protein; TonEBP) plays a key role in macrophage-driven regulation of cutaneous salt and water balance. In the immune-privileged and transparent cornea, disturbances in fluid balance and pathological edema result in corneal transparency loss, which is one of the main causes of blindness worldwide. The role of NFAT5 in the cornea has not yet been investigated. We analyzed the expression and function of NFAT5 in naive corneas and in an established mouse model of perforating corneal injury (PCI), which causes acute corneal edema and transparency loss. In uninjured corneas, NFAT5 was mainly expressed in corneal fibroblasts. In contrast, after PCI, NFAT5 expression was highly upregulated in recruited corneal macrophages. NFAT5 deficiency did not alter corneal thickness in steady state; however, loss of NFAT5 led to accelerated resorption of corneal edema after PCI. Mechanistically, we found that myeloid cell-derived NFAT5 is crucial for controlling corneal edema, as edema resorption after PCI was significantly enhanced in mice with conditional loss of NFAT5 in the myeloid cell lineage, presumably due to increased pinocytosis of corneal macrophages. Collectively, we uncovered a suppressive role for NFAT5 in corneal edema resorption, thereby identifying a novel therapeutic target to combat edema-induced corneal blindness.
Topics: Mice; Animals; Corneal Edema; Gene Expression Regulation; NFATC Transcription Factors; Transcription Factors
PubMed: 36869067
DOI: 10.1038/s12276-023-00954-w -
International Journal of Molecular... Feb 2023Endocytosis in mammalian cells is a fundamental cellular machinery that regulates vital physiological processes, such as the absorption of metabolites, release of... (Review)
Review
Endocytosis in mammalian cells is a fundamental cellular machinery that regulates vital physiological processes, such as the absorption of metabolites, release of neurotransmitters, uptake of hormone cellular defense, and delivery of biomolecules across the plasma membrane. A remarkable characteristic of the endocytic machinery is the sequential assembly of the complex proteins at the plasma membrane, followed by internalization and fusion of various biomolecules to different cellular compartments. In all eukaryotic cells, functional characterization of endocytic pathways is based on dynamics of the protein complex and signal transduction modules. To coordinate the assembly and functions of the numerous parts of the endocytic machinery, the endocytic proteins interact significantly within and between the modules. Clathrin-dependent and -independent endocytosis, caveolar pathway, and receptor mediated endocytosis have been attributed to a greater variety of physiological and pathophysiological roles such as, autophagy, metabolism, cell division, apoptosis, cellular defense, and intestinal permeabilization. Notably, any defect or alteration in the endocytic machinery results in the development of pathological consequences associated with human diseases such as cancer, cardiovascular diseases, neurological diseases, and inflammatory diseases. In this review, an in-depth endeavor has been made to illustrate the process of endocytosis, and associated mechanisms describing pathological manifestation associated with dysregulated endocytosis machinery.
Topics: Animals; Humans; Endocytosis; Caveolae; Cell Membrane; Signal Transduction; Biological Transport; Mammals
PubMed: 36769293
DOI: 10.3390/ijms24032971