-
Scientific Reports May 2024The closely related endolysosomal tethering complexes HOPS and CORVET play pivotal roles in the homo- and heterotypic fusion of early and late endosomes, respectively,...
The closely related endolysosomal tethering complexes HOPS and CORVET play pivotal roles in the homo- and heterotypic fusion of early and late endosomes, respectively, and HOPS also mediates the fusion of lysosomes with incoming vesicles including late endosomes and autophagosomes. These heterohexameric complexes share their four core subunits that assemble with additional two, complex-specific subunits. These features and the similar structure of the complexes could allow the formation of hybrid complexes, and the complex specific subunits may compete for binding to the core. Indeed, our biochemical analyses revealed the overlap of binding sites for HOPS-specific VPS41 and CORVET-specific VPS8 on the shared core subunit VPS18. We found that the overexpression of CORVET-specific VPS8 or Tgfbrap1 decreased the amount of core proteins VPS11 and VPS18 that are assembled with HOPS-specific subunits VPS41 or VPS39, indicating reduced amount of assembled HOPS. In line with this, we observed the elevation of both lipidated, autophagosome-associated LC3 protein and the autophagic cargo p62 in these cells, suggesting impaired autophagosome-lysosome fusion. In contrast, overexpression of HOPS-specific VPS39 or VPS41 did not affect the level of assembled CORVET or autophagy. VPS8 or Tgfbrap1 overexpression also induced Cathepsin D accumulation, suggesting that HOPS-dependent biosynthetic delivery of lysosomal hydrolases is perturbed, too. These indicate that CORVET-specific subunit levels fine-tune HOPS assembly and activity in vivo.
Topics: Endosomes; Humans; Vesicular Transport Proteins; Lysosomes; Protein Subunits; Autophagy; Autophagosomes; HeLa Cells; Protein Binding
PubMed: 38698024
DOI: 10.1038/s41598-024-59775-0 -
Free Radical Biology & Medicine Aug 2024Hepatocellular carcinoma (HCC) is a global public health problem with increased morbidity and mortality. Agrimol B, a natural polyphenol, has been proved to be a...
Hepatocellular carcinoma (HCC) is a global public health problem with increased morbidity and mortality. Agrimol B, a natural polyphenol, has been proved to be a potential anticancer drug. Our recent report showed a favorable anticancer effect of agrimol B in HCC, however, the mechanism of action remains unclear. Here, we found agrimol B inhibits the growth and proliferation of HCC cells in vitro as well as in an HCC patient-derived xenograft (PDX) model. Notably, agrimol B drives autophagy initiation and blocks autophagosome-lysosome fusion, resulting in autophagosome accumulation and autophagy arrest in HCC cells. Mechanistically, agrimol B downregulates the protein level of NADH:ubiquinone oxidoreductase core subunit S1 (NDUFS1) through caspase 3-mediated degradation, leading to mitochondrial reactive oxygen species (mROS) accumulation and autophagy arrest. NDUFS1 overexpression partially restores mROS overproduction, autophagosome accumulation, and growth inhibition induced by agrimol B, suggesting a cytotoxic role of agrimol B-induced autophagy arrest in HCC cells. Notably, agrimol B significantly enhances the sensitivity of HCC cells to sorafenib in vitro and in vivo. In conclusion, our study uncovers the anticancer mechanism of agrimol B in HCC involving the regulation of oxidative stress and autophagy, and suggests agrimol B as a potential therapeutic drug for HCC treatment.
Topics: Animals; Humans; Mice; Apoptosis; Autophagosomes; Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Electron Transport Complex I; Indoles; Liver Neoplasms; Mice, Nude; Mitochondria; Reactive Oxygen Species; Sorafenib; Spiro Compounds; Xenograft Model Antitumor Assays
PubMed: 38697493
DOI: 10.1016/j.freeradbiomed.2024.04.242 -
Molecular Biology of the Cell Jul 2024Autophagy is a conserved catabolic process where double membrane-bound structures form around macromolecules or organelles targeted for degradation. Autophagosomes fuse...
Autophagy is a conserved catabolic process where double membrane-bound structures form around macromolecules or organelles targeted for degradation. Autophagosomes fuse with lysosomes to facilitate degradation and macromolecule recycling for homeostasis or growth in a cell autonomous manner. In cancer cells, autophagy is often up-regulated and helps cancer cells survive nutrient deprivation and stressful growth conditions. Here, we propose that the increased intracellular pH (pHi) common to cancer cells is sufficient to induce autophagic cell death. We previously developed tools to increase pHi in the eye via overexpression of resulting in aberrant patterning and reduced tissue size. We examined fly eyes at earlier stages of development and found fewer interommatidial cells. We next tested whether this decrease in cell number was due to increased cell death. We found that the -induced cell death was caspase independent, which is inconsistent with apoptosis. However, this cell death required autophagy genes, which supports autophagy as the mode of cell death. We also found that expression of molecular markers supports increased autophagy. Together, our findings suggest new roles for ion transport proteins in regulating conserved, critical developmental processes and provide evidence for new paradigms in growth control.
Topics: Animals; Drosophila Proteins; Autophagy; Autophagic Cell Death; Drosophila melanogaster; Sodium-Hydrogen Exchangers; Hydrogen-Ion Concentration; Eye; Apoptosis; Lysosomes; Drosophila; Autophagosomes
PubMed: 38696256
DOI: 10.1091/mbc.E24-02-0058 -
Biomacromolecules May 2024Intracellular bacteria in dormant states can escape the immune response and tolerate high-dose antibiotic treatment, leading to severe infections. To overcome this...
Intracellular bacteria in dormant states can escape the immune response and tolerate high-dose antibiotic treatment, leading to severe infections. To overcome this challenge, cascade-targeted nanoplatforms that can target macrophages and intracellular bacteria, exhibiting synergetic antibiotic/reactive oxygen species (ROS)/nitric oxide (NO)/immunotherapy, were developed. These nanoplatforms were fabricated by encapsulating trehalose (Tr) and vancomycin (Van) into phosphatidylserine (PS)-coated poly[(4-allylcarbamoylphenylboric acid)--(arginine-methacrylamide)--(,'-bisacryloylcystamine)] nanoparticles (PABS), denoted as PTVP. PS on PTVP simulates a signal of "eat me" to macrophages to promote cell uptake (the first-step targeting). After the uptake, the nanoplatform in the acidic phagolysosomes could release Tr, and the exposed phenylboronic acid on the nanoplatform could target bacteria (the second-step targeting). Nanoplatforms can release Van in response to infected intracellular overexpressed glutathione (GSH) and weak acid microenvironment. l-arginine (Arg) on the nanoplatforms could be catalyzed by upregulated inducible nitric oxide synthase (iNOS) in the infected macrophages to generate nitric oxide (NO). ,'-Bisacryloylcystamine (BAC) on nanoplatforms could deplete GSH, allow the generation of ROS in macrophages, and then upregulate proinflammatory activity, leading to the reinforced antibacterial capacity. This nanoplatform possesses macrophage and bacteria-targeting antibiotic delivery, intracellular ROS, and NO generation, and pro-inflammatory activities (immunotherapy) provides a new strategy for eradicating intracellular bacterial infections.
Topics: Reactive Oxygen Species; Nitric Oxide; Anti-Bacterial Agents; Mice; Animals; RAW 264.7 Cells; Nanoparticles; Macrophages; Immunotherapy; Vancomycin; Bacterial Infections; Trehalose
PubMed: 38693753
DOI: 10.1021/acs.biomac.4c00339 -
Nature May 2024Ischaemic diseases such as critical limb ischaemia and myocardial infarction affect millions of people worldwide. Transplanting endothelial cells (ECs) is a promising...
Ischaemic diseases such as critical limb ischaemia and myocardial infarction affect millions of people worldwide. Transplanting endothelial cells (ECs) is a promising therapy in vascular medicine, but engrafting ECs typically necessitates co-transplanting perivascular supporting cells such as mesenchymal stromal cells (MSCs), which makes clinical implementation complicated. The mechanisms that enable MSCs to facilitate EC engraftment remain elusive. Here we show that, under cellular stress, MSCs transfer mitochondria to ECs through tunnelling nanotubes, and that blocking this transfer impairs EC engraftment. We devised a strategy to artificially transplant mitochondria, transiently enhancing EC bioenergetics and enabling them to form functional vessels in ischaemic tissues without the support of MSCs. Notably, exogenous mitochondria did not integrate into the endogenous EC mitochondrial pool, but triggered mitophagy after internalization. Transplanted mitochondria co-localized with autophagosomes, and ablation of the PINK1-Parkin pathway negated the enhanced engraftment ability of ECs. Our findings reveal a mechanism that underlies the effects of mitochondrial transfer between mesenchymal and endothelial cells, and offer potential for a new approach for vascular cell therapy.
Topics: Animals; Humans; Male; Mice; Autophagosomes; Endothelial Cells; Energy Metabolism; Human Umbilical Vein Endothelial Cells; Ischemia; Mesenchymal Stem Cells; Mice, Nude; Mitochondria; Mitophagy; Protein Kinases; Ubiquitin-Protein Ligases; Cell- and Tissue-Based Therapy
PubMed: 38693258
DOI: 10.1038/s41586-024-07340-0 -
Frontiers in Neuroscience 2024In the eye, cells from the retinal pigment epithelium (RPE) facing the neurosensory retina exert several functions that are all crucial for long-term survival of...
In the eye, cells from the retinal pigment epithelium (RPE) facing the neurosensory retina exert several functions that are all crucial for long-term survival of photoreceptors (PRs) and vision. Among those, RPE cells phagocytose under a circadian rhythm photoreceptor outer segment (POS) tips that are constantly subjected to light rays and oxidative attacks. The MerTK tyrosine kinase receptor is a key element of this phagocytic machinery required for POS internalization. Recently, we showed that MerTK is subjected to the cleavage of its extracellular domain to finely control its function. In addition, monocytes in retinal blood vessels can migrate inside the inner retina and differentiate into macrophages expressing MerTK, but their role in this context has not been studied yet. We thus investigated the ocular phenotype of MerTK cleavage-resistant (MerTK) mice to understand the relevance of this characteristic on retinal homeostasis at the RPE and macrophage levels. MerTK retinae appear to develop and function normally, as observed in retinal sections, by electroretinogram recordings and optokinetic behavioral tests. Monitoring of MerTK and control mice between the ages of 3 and 18 months showed the development of large degenerative areas in the central retina as early as 4 months when followed monthly by optical coherence tomography (OCT) plus fundus photography (FP)/autofluorescence (AF) detection but not by OCT alone. The degenerative areas were associated with AF, which seems to be due to infiltrated macrophages, as observed by OCT and histology. MerTK RPE primary cultures phagocytosed less POS , while , the circadian rhythm of POS phagocytosis was deregulated. Mitochondrial function and energy production were reduced in freshly dissected RPE/choroid tissues at all ages, thus showing a metabolic impairment not present in macrophages. RPE anomalies were detected by electron microscopy, including phagosomes retained in the apical area and vacuoles. Altogether, this new mouse model displays a novel phenotype that could prove useful to understanding the interplay between RPE and PRs in inflammatory retinal degenerations and highlights new roles for MerTK in the regulation of the energetic metabolism and the maintenance of the immune privilege in the retina.
PubMed: 38680449
DOI: 10.3389/fnins.2024.1256522 -
Journal of Ethnopharmacology Sep 2024Tanreqing injection (TRQ) is widely used, traditional Chinese medicine (TCM) injection used in China to treat respiratory infections. Modern pharmacological studies have...
ETHNOPHARMACOLOGICAL RELEVANCE
Tanreqing injection (TRQ) is widely used, traditional Chinese medicine (TCM) injection used in China to treat respiratory infections. Modern pharmacological studies have confirmed that TRQ can protect against influenza viruses. However, the mechanism by which TRQ inhibits influenza viruses remains unclear.
AIM OF THE STUDY
To explore the therapeutic effects and possible mechanisms of TRQ inhibition by the influenza virus.
MATERIALS AND METHODS
Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) was used to determine the chemical composition of TRQ. Isobaric tags for relative and absolute quantification (iTRAQ) were used to define differential proteins related to TRQ inhibition of viruses. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed for functional annotation. For experimental validation, we established an in vitro model of the influenza virus infection by infecting A549 cells with the virus. The detection of the signaling pathway was carried out through qPCR, western blotting,and immunofluorescence.
RESULTS
Fifty one components were identified using UPLC/Q-TOF MS. We confirmed the inhibitory effect of TRQ on influenza virus replication in vitro. Ninety nine differentially expressed proteins related to the inhibitory effect of TRQ were identified using iTRAQ. KEGG functional enrichment analysis showed that the TRQ may inhibit influenza virus replication by affecting autophagy. Through network analysis, 29 targets were selected as major targets, and three key targets, HSPA5, PARP1, and GAPDH, may be the TRQ targets affecting autophagy. In vitro experiments showed that TRQ inhibits influenza virus replication by interfering with the expression and localization of STX17 and VAMP8 proteins, thereby promoting the fusion of autophagosomes with lysosomes.
CONCLUSION
TRQ inhibits influenza virus replication by promoting the fusion of autophagosomes with lysosomes. We additionally established potential gene and protein targets which are affected by TRQ. Therefore, our findings provide new therapeutic targets and a foundation further studies on influenza treatment with TRQ.
Topics: Drugs, Chinese Herbal; Virus Replication; Humans; A549 Cells; Antiviral Agents; Lysosomes; Autophagosomes; Endoplasmic Reticulum Chaperone BiP; Animals; Autophagy
PubMed: 38677572
DOI: 10.1016/j.jep.2024.118159 -
The Journal of Biological Chemistry Jun 2024Organelles and vesicular cargoes are transported by teams of kinesin and dynein motors along microtubules. We isolated endocytic organelles from cells at different...
Organelles and vesicular cargoes are transported by teams of kinesin and dynein motors along microtubules. We isolated endocytic organelles from cells at different stages of maturation and reconstituted their motility along microtubules in vitro. We asked how the sets of motors transporting a cargo determine its motility and response to the microtubule-associated protein tau. Here, we find that phagosomes move in both directions along microtubules, but the directional bias changes during maturation. Early phagosomes exhibit retrograde-biased transport while late phagosomes are directionally unbiased. Correspondingly, early and late phagosomes are bound by different numbers and combinations of kinesins-1, -2, -3, and dynein. Tau stabilizes microtubules and directs transport within neurons. While single-molecule studies show that tau differentially regulates the motility of kinesins and dynein in vitro, less is known about its role in modulating the trafficking of endogenous cargoes transported by their native teams of motors. Previous studies showed that tau preferentially inhibits kinesin motors, which biases late phagosome transport towards the microtubule minus-end. Here, we show that tau strongly inhibits long-range, dynein-mediated motility of early phagosomes. Tau reduces forces generated by teams of dynein motors on early phagosomes and accelerates dynein unbinding under load. Thus, cargoes differentially respond to tau, where dynein complexes on early phagosomes are more sensitive to tau inhibition than those on late phagosomes. Mathematical modeling further explains how small changes in the number of kinesins and dynein on cargoes impact the net directionality but also that cargoes with different sets of motors respond differently to tau.
Topics: Kinesins; tau Proteins; Dyneins; Animals; Microtubules; Phagosomes; Biological Transport; Mice; Humans; Endocytosis
PubMed: 38677516
DOI: 10.1016/j.jbc.2024.107323 -
International Journal of Molecular... Apr 2024Phagocytosis (and endocytosis) is an unusual cellular process that results in the formation of a novel subcellular organelle, the phagosome. This phagosome contains not...
Phagocytosis (and endocytosis) is an unusual cellular process that results in the formation of a novel subcellular organelle, the phagosome. This phagosome contains not only the internalised target of phagocytosis but also the external medium, creating a new border between extracellular and intracellular environments. The boundary at the plasma membrane is, of course, tightly controlled and exploited in ionic cell signalling events. Although there has been much work on the control of phagocytosis by ions, notably, Ca ions influxing across the plasma membrane, increasing our understanding of the mechanism enormously, very little work has been done exploring the phagosome/cytosol boundary. In this paper, we explored the changes in the intra-phagosomal Ca ion content that occur during phagocytosis and phagosome formation in human neutrophils. Measuring Ca ion concentration in the phagosome is potentially prone to artefacts as the intra-phagosomal environment experiences changes in pH and oxidation. However, by excluding such artefacts, we conclude that there are open Ca channels on the phagosome that allow Ca ions to "drain" into the surrounding cytosol. This conclusion was confirmed by monitoring the translocation of the intracellularly expressed YFP-tagged C2 domain of PKC-γ. This approach marked regions of membrane at which Ca influx occurred, the earliest being the phagocytic cup, and then the whole cell. This paper therefore presents data that have novel implications for understanding phagocytic Ca signalling events, such as peri-phagosomal Ca hotspots, and other phenomena.
Topics: Phagocytosis; Humans; Calcium; Phagosomes; Neutrophils; Calcium Signaling; Cytosol; Cell Membrane
PubMed: 38673839
DOI: 10.3390/ijms25084254 -
Journal of Fungi (Basel, Switzerland) Apr 2024is a facultative intracellular fungal pathogen. Ten-generation-old (10GEN) cells are more resistant to phagocytosis and killing by macrophages than younger daughter...
is a facultative intracellular fungal pathogen. Ten-generation-old (10GEN) cells are more resistant to phagocytosis and killing by macrophages than younger daughter cells. However, mechanisms that mediate this resistance and intracellular parasitism are poorly understood. Here, we identified important factors for the intracellular survival of 10GEN , such as urease activity, capsule synthesis, and DNA content using flow cytometry and fluorescent microscopy techniques. The real-time visualization of time-lapse imaging was applied to determine the phagosomal acidity, membrane permeability, and vomocytosis (non-lytic exocytosis) rate in J774 macrophages that phagocytosed of different generational ages. Our results showed that old exhibited higher urease activity and enhanced Golgi activity. In addition, old were more likely to be arrested in the G2 phase, resulting in the occasional formation of aberrant trimera-like cells. To finish, the advanced generational age of the yeast cells slightly reduced vomocytosis events within host cells, which might be associated with increased phagolysosome pH and membrane permeability. Altogether, our results suggest that old prevail within acidic phagolysosomes and can manipulate the phagosome pH. These strategies may be used by old to resist phagosomal killing and drive cryptococcosis pathogenesis. The comprehension of these essential host-pathogen interactions could further shed light on mechanisms that bring new insights for novel antifungal therapeutic design.
PubMed: 38667950
DOI: 10.3390/jof10040279