scholarly journals SARS-CoV-2 treatment effects induced by ACE2-expressing microparticles are explained by the oxidized cholesterol-increased endosomal pH of alveolar macrophages

2022 ◽  
Author(s):  
Zhenfeng Wang ◽  
Jiadi Lv ◽  
Pin Yu ◽  
Yajin Qu ◽  
Yabo Zhou ◽  
...  
Keyword(s):  
Alveolar Macrophages ◽  
Ph Regulation ◽  
Proton Pumps ◽  
Lysosomal Ph ◽  
Oxidized Cholesterol ◽  
Endosomal Acidification ◽  
Endosomal Membranes ◽  
Endosomal Ph ◽  
Advanced Biomaterials ◽  
Potential Use

AbstractExploring the cross-talk between the immune system and advanced biomaterials to treat SARS-CoV-2 infection is a promising strategy. Here, we show that ACE2-overexpressing A549 cell-derived microparticles (AO-MPs) are a potential therapeutic agent against SARS-CoV-2 infection. Intranasally administered AO-MPs dexterously navigate the anatomical and biological features of the lungs to enter the alveoli and are taken up by alveolar macrophages (AMs). Then, AO-MPs increase the endosomal pH but decrease the lysosomal pH in AMs, thus escorting bound SARS-CoV-2 from phago-endosomes to lysosomes for degradation. This pH regulation is attributable to oxidized cholesterol, which is enriched in AO-MPs and translocated to endosomal membranes, thus interfering with proton pumps and impairing endosomal acidification. In addition to promoting viral degradation, AO-MPs also inhibit the proinflammatory phenotype of AMs, leading to increased treatment efficacy in a SARS-CoV-2-infected mouse model without side effects. These findings highlight the potential use of AO-MPs to treat SARS-CoV-2-infected patients and showcase the feasibility of MP therapies for combatting emerging respiratory viruses in the future.

scholarly journals Distinct uptake, amplification, and release of SARS-CoV-2 by M1 and M2 alveolar macrophages

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Jiadi Lv ◽  
Zhenfeng Wang ◽  
Yajin Qu ◽  
Hua Zhu ◽  
Qiangqiang Zhu ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Mouse Model ◽  
Alveolar Macrophages ◽  
Transgenic Mouse Model ◽  
Lysosomal Ph ◽  
Viral Spread ◽  
Lysosomal System ◽  
Endosomal Ph ◽  
Alternatively Activated ◽  
Classically Activated

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) invades the alveoli, where abundant alveolar macrophages (AMs) reside. How AMs respond to SARS-CoV-2 invasion remains elusive. Here, we show that classically activated M1 AMs facilitate viral spread; however, alternatively activated M2 AMs limit the spread. M1 AMs utilize cellular softness to efficiently take up SARS-CoV-2. Subsequently, the invaded viruses take over the endo-lysosomal system to escape. M1 AMs have a lower endosomal pH, favoring membrane fusion and allowing the entry of viral RNA from the endosomes into the cytoplasm, where the virus achieves replication and is packaged to be released. In contrast, M2 AMs have a higher endosomal pH but a lower lysosomal pH, thus delivering the virus to lysosomes for degradation. In hACE2 transgenic mouse model, M1 AMs are found to facilitate SARS-CoV-2 infection of the lungs. These findings provide insights into the complex roles of AMs during SARS-CoV-2 infection, along with potential therapeutic targets.

scholarly journals RAVE and Rabconnectin-3 Complexes as Signal Dependent Regulators of Organelle Acidification

2021 ◽  
Vol 9 ◽  
Author(s):  
Michael C. Jaskolka ◽  
Samuel R. Winkley ◽  
Patricia M. Kane
Keyword(s):  
Atpase Activity ◽  
Proton Transport ◽  
Atp Hydrolysis ◽  
Glucose Deprivation ◽  
Vacuolar Membrane ◽  
Proton Pumps ◽  
Endosomal Acidification ◽  
Endosomal Membranes ◽  
Higher Eukaryotes ◽  
And Function

The yeast RAVE (Regulator of H+-ATPase of Vacuolar and Endosomal membranes) complex and Rabconnectin-3 complexes of higher eukaryotes regulate acidification of organelles such as lysosomes and endosomes by catalyzing V-ATPase assembly. V-ATPases are highly conserved proton pumps consisting of a peripheral V1 subcomplex that contains the sites of ATP hydrolysis, attached to an integral membrane Vo subcomplex that forms the transmembrane proton pore. Reversible disassembly of the V-ATPase is a conserved regulatory mechanism that occurs in response to multiple signals, serving to tune ATPase activity and compartment acidification to changing extracellular conditions. Signals such as glucose deprivation can induce release of V1 from Vo, which inhibits both ATPase activity and proton transport. Reassembly of V1 with Vo restores ATP-driven proton transport, but requires assistance of the RAVE or Rabconnectin-3 complexes. Glucose deprivation triggers V-ATPase disassembly in yeast and is accompanied by binding of RAVE to V1 subcomplexes. Upon glucose readdition, RAVE catalyzes both recruitment of V1 to the vacuolar membrane and its reassembly with Vo. The RAVE complex can be recruited to the vacuolar membrane by glucose in the absence of V1 subunits, indicating that the interaction between RAVE and the Vo membrane domain is glucose-sensitive. Yeast RAVE complexes also distinguish between organelle-specific isoforms of the Vo a-subunit and thus regulate distinct V-ATPase subpopulations. Rabconnectin-3 complexes in higher eukaryotes appear to be functionally equivalent to yeast RAVE. Originally isolated as a two-subunit complex from rat brain, the Rabconnectin-3 complex has regions of homology with yeast RAVE and was shown to interact with V-ATPase subunits and promote endosomal acidification. Current understanding of the structure and function of RAVE and Rabconnectin-3 complexes, their interactions with the V-ATPase, their role in signal-dependent modulation of organelle acidification, and their impact on downstream pathways will be discussed.

scholarly journals Activation of vacuolar-type proton pumps by protein kinase C. Role in neutrophil pH regulation

1992 ◽  
Vol 267 (32) ◽  
pp. 22740-22746
Author(s):  
A Nanda ◽  
A Gukovskaya ◽  
J Tseng ◽  
S Grinstein
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ph Regulation ◽  
Proton Pumps ◽  
Kinase C

scholarly journals Niclosamide inhibits SARS-CoV2 entry by blocking internalization through pH-dependent CLIC/GEEC endocytic pathway

2020 ◽  
Author(s):  
Chaitra Prabhakara ◽  
Rashmi Godbole ◽  
Parijat Sil ◽  
Sowmya Jahnavi ◽  
Thomas S van Zanten ◽  
...  
Keyword(s):  
Viral Infection ◽  
Receptor Binding ◽  
Endocytic Pathway ◽  
Host Cells ◽  
Entry Inhibitor ◽  
Receptor Binding Domain ◽  
Endosomal Acidification ◽  
Independent Mechanism ◽  
Ph Dependent ◽  
Endosomal Ph

AbstractMany viruses utilize the host endo-lysosomal network to infect cells. Tracing the endocytic itinerary of SARS-CoV2 can provide insights into viral trafficking and aid in designing new therapeutic targets. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV2 is internalized via the clathrin and dynamin-independent, pH-dependent CLIC/GEEC (CG) endocytic pathway. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, strongly block the uptake of RBD. Using transduction assays with SARS-CoV2 Spike-pseudovirus, we confirmed that these acidification inhibitors also impede viral infection. By contrast, Chloroquine neither affects RBD uptake nor extensively alters the endosomal pH, yet attenuates Spike-pseudovirus entry, indicating a pH-independent mechanism of intervention. We screened a subset of FDA-approved acidification inhibitors and found Niclosamide to be a potential SARS-CoV2 entry inhibitor. Niclosamide, thus, could provide broader applicability in subverting infection of similar category viruses entering host cells via this pH-dependent endocytic pathway.

scholarly journals Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

2021 ◽  
Author(s):  
Joseph A Mindell ◽  
Xavier Leray ◽  
Jacob K Hilton ◽  
Kamsi Nwangwu ◽  
Alissa Becerril ◽  
...  
Keyword(s):  
Ion Channels ◽  
Narrow Range ◽  
Ph Regulation ◽  
Tonic Inhibition ◽  
Gain Of Function ◽  
Lysosomal Ph ◽  
Ion Movement ◽  
Luminal Ph

The acidic luminal pH of lysosomes, maintained within a narrow range, is essential for proper degrative function of the organelle and is generated by the action of a V-type H+ ATPase, but other pathways for ion movement are required to dissipate the voltage generated by this process. ClC-7, a Cl-/H+ antiporter responsible for lysosomal Cl- permeability, is a candidate to contribute to the acidification process as part of this “counterion pathway”. The signaling lipid PI(3,5)P2 modulates lysosomal dynamics, including by regulating lysosomal ion channels, raising the possibility that it could contribute to lysosomal pH regulation. Here we demonstrate that depleting PI(3,5)P2 by inhibiting the PIKfyve kinase causes lysosomal hyperacidification, primarily via an effect on ClC-7. We further show that PI(3,5)P2 directly inhibits ClC-7 transport and that this inhibition is eliminated in a disease-causing gain-of-function ClC-7 mutation. Together these observations suggest an intimate role for ClC-7 in lysosomal pH regulation.

The vacuolar proton pump of Dictyostelium discoideum: molecular cloning and analysis of the 100 kDa subunit

1996 ◽  
Vol 109 (5) ◽  
pp. 1041-1051 ◽  
Author(s):  
T. Liu ◽  
M. Clarke
Keyword(s):  
Dictyostelium Discoideum ◽  
Proton Pump ◽  
Single Gene ◽  
Consensus Sequence ◽  
Amino Acid Sequences ◽  
Contractile Vacuole ◽  
Proton Pumps ◽  
Variable Regions ◽  
Endosomal Membranes ◽  
Vacuolar Proton Pump

The vacuolar proton pump is a highly-conserved multimeric enzyme that catalyzes the translocation of protons across the membranes of eukaryotic cells. Its largest subunit (95-116 kDa) occurs in tissue and organelle-specific isoforms and thus may be involved in targeting the enzyme or modulating its function. In amoebae of Dictyostelium discoideum, proton pumps with a 100 kDa subunit are found in membranes of the contractile vacuole complex, an osmoregulatory organelle. We cloned the cDNA that encodes this 100 kDa protein and found that its sequence predicts a protein 45% identical (68% similar) to the corresponding mammalian proton pump subunit. Like the mammalian protein, the predicted Dictyostelium sequence contains six possible transmembrane domains and a single consensus sequence for N-linked glycosylation. Southern blot analysis detected only a single gene, which was designated vatM. Using genomic DNA and degenerate oligonucleotides based on conserved regions of the protein as primers, we generated products by polymerase chain reaction that included highly variable regions of this protein family. The cloned products were identical in nucleotide sequence to vatM, arguing that Dictyostelium cells contain only a single isoform of this proton pump subunit. Consistent with this interpretation, the amino acid sequences of peptides derived from a protein associated with endosomal membranes (Adessu et al. (1995) J. Cell Sci. 108, 3331–3337) match the predicted sequence of the protein encoded by vatM. Thus, a single isoform of the 100 kDa proton pump subunit appears to serve in both the contractile vacuole system and the endosomal/lysosomal system of Dictyostelium, arguing that this subunit is not responsible for regulating the differing abundance and function of proton pumps in these two compartments. Gene targeting experiments suggest that this subunit plays important (possibly essential) roles in Dictyostelium cells.

Kinetics of endosomal pH evolution in Dictyostelium discoideum amoebae. Study by fluorescence spectroscopy

1993 ◽  
Vol 105 (3) ◽  
pp. 861-866 ◽  
Author(s):  
L. Aubry ◽  
G. Klein ◽  
J.L. Martiel ◽  
M. Satre
Keyword(s):  
Dictyostelium Discoideum ◽  
Fluid Phase ◽  
Lag Phase ◽  
Nutritive Medium ◽  
Chase Period ◽  
Endosomal Acidification ◽  
Fluid Phase Endocytosis ◽  
Endosomal Ph ◽  
Efflux Kinetics ◽  
Secondary Lysosomes

The evolution of endo-lysosomal pH in Dictyostelium discoideum amoebae was examined during fluid-phase endocytosis. Pulse-chase experiments were conducted in nutritive medium or in non-nutritive medium using fluorescein labelled dextran (FITC-dextran) as fluid-phase marker and pH probe. In both conditions, efflux kinetics were characterized by an extended lag phase lasting for 45–60 min and corresponding to intracellular transit of FITC-dextran cohort. During the chase period, endosomal pH decreased during approximately 20 min from extracellular pH down to pH 4.6-5.0, then, it increased within the next 20–40 min to reach pH 6.0-6.2. It was only at this stage that FITC-dextran was released back into the medium with pseudo first-order kinetics. A vacuolar H(+)-ATPase is involved in endosomal acidification as the acidification process was markedly reduced in mutant strain HGR8, partially defective in vacuolar H(+)-ATPase and in parent type strain AX2 by bafilomycin A1, a selective inhibitor of this enzyme. Our data suggest that endocytic cargo is channeled from endosomes to secondary lysosomes that are actively linked to the plasma membrane via recycling vesicles.

Altered expression of the 100 kDa subunit of the Dictyosteliumvacuolar proton pump impairs enzyme assembly, endocytic function and cytosolic pH regulation

2002 ◽  
Vol 115 (9) ◽  
pp. 1907-1918 ◽  
Author(s):  
Tongyao Liu ◽  
Christian Mirschberger ◽  
Lilian Chooback ◽  
Quyen Arana ◽  
Zeno Dal Sacco ◽  
...  
Keyword(s):  
Proton Pump ◽  
Catalytic Domain ◽  
Ph Regulation ◽  
Proton Pumps ◽  
Cytosolic Ph ◽  
Acid Media ◽  
Dynamic Regulation ◽  
Atpase Subunit ◽  
Altered Expression ◽  
Mutant Cells

The vacuolar proton pump (V-ATPase) appears to be essential for viability of Dictyostelium cells. To investigate the function of VatM, the 100 kDa transmembrane V-ATPase subunit, we altered its level. By means of homologous recombination, the promoter for the chromosomal vatM gene was replaced with the promoter for the act6 gene, yielding the mutant strain VatMpr. The act6 promoter is much more active in cells growing axenically than on bacteria. Thus, transformants were selected under axenic growth conditions, then shifted to bacteria to determine the consequences of reduced vatM expression. When VatMpr cells were grown on bacteria,the level of the 100 kDa V-ATPase subunit dropped, cell growth slowed, and the A subunit, a component of the peripheral catalytic domain of the V-ATPase,became mislocalized. These defects were complemented by transformation of the mutant cells with a plasmid expressing vatM under the control of its own promoter. Although the principal locus of vacuolar proton pumps in Dictyostelium is membranes of the contractile vacuole system, mutant cells did not manifest osmoregulatory defects. However, bacterially grown VatMpr cells did exhibit substantially reduced rates of phagocytosis and a prolonged endosomal transit time. In addition, mutant cells manifested alterations in the dynamic regulation of cytosolic pH that are characteristic of normal cells grown in acid media, which suggested that the V-ATPase also plays a role in cytosolic pH regulation.

Different pathways of degradation of SP-A and saturated phosphatidylcholine by alveolar macrophages

2000 ◽  
Vol 279 (1) ◽  
pp. L91-L99 ◽  
Author(s):  
Aldo Baritussio ◽  
Antonella Alberti ◽  
Decio Armanini ◽  
Federica Meloni ◽  
Daniela Bruttomesso
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Alveolar Macrophages ◽  
Surfactant Protein ◽  
Phosphatidylinositol 3 Kinase ◽  
Lysosomal Compartment ◽  
Lysosomal Ph ◽  
Kinase C ◽  
Stimulation Of

Alveolar macrophages degrade surfactant protein (SP) A and saturated phosphatidycholine [dipalmitoylphosphatidylcholine (DPPC)]. To clarify this process, using rabbit alveolar macrophages, we analyzed the effect of drugs known to affect phagocytosis, pinocytosis, clathrin-mediated uptake, caveolae, the cytoskeleton, lysosomal pH, protein kinase C, and phosphatidylinositol 3-kinase (PI3K) on the degradation of SP-A and DPPC. We found the following: 1) SP-A binds to the plasma membrane, is rapidly internalized, and then moves toward degradative compartments. Uptake could be clathrin mediated, whereas phagocytosis, pinocytosis, or the use of caveolae are less likely. An intact cytoskeleton and an acidic milieu are necessary for the degradation of SP-A. 2) Stimulation of protein kinase C increases the degradation of SP-A. 3) PI3K influences the degradation of SP-A by regulating both the speed of internalization and subsequent intracellular steps, but its inhibition does not prevent SP-A from reaching the lysosomal compartment. 4) The degradation of DPPC is unaffected by most of the treatments able to influence the degradation of SP-A. Thus it appears that DPPC is degraded by alveolar macrophages through mechanisms very different from those utilized for the degradation of SP-A.

scholarly journals Regulated Assembly of Vacuolar ATPase Is Increased during Cluster Disruption-induced Maturation of Dendritic Cells through a Phosphatidylinositol 3-Kinase/mTOR-dependent Pathway

2013 ◽  
Vol 289 (3) ◽  
pp. 1355-1363 ◽  
Author(s):  
Rachel Liberman ◽  
Sarah Bond ◽  
Mara G. Shainheit ◽  
Miguel J. Stadecker ◽  
Michael Forgac
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Antigen Processing ◽  
Dendritic Cell Maturation ◽  
Proton Pumps ◽  
Phosphatidylinositol 3 Kinase ◽  
Lysosomal Ph ◽  
Concanamycin A ◽  
Phosphatidylinositol 3

The vacuolar (H+)-ATPases (V-ATPases) are ATP-driven proton pumps composed of a peripheral V1 domain and a membrane-embedded V0 domain. Regulated assembly of V1 and V0 represents an important regulatory mechanism for controlling V-ATPase activity in vivo. Previous work has shown that V-ATPase assembly increases during maturation of bone marrow-derived dendritic cells induced by activation of Toll-like receptors. This increased assembly is essential for antigen processing, which is dependent upon an acidic lysosomal pH. Cluster disruption of dendritic cells induces a semi-mature phenotype associated with immune tolerance. Thus, semi-mature dendritic cells are able to process and present self-peptides to suppress autoimmune responses. We have investigated V-ATPase assembly in bone marrow-derived, murine dendritic cells and observed an increase in assembly following cluster disruption. This increased assembly is not dependent upon new protein synthesis and is associated with an increase in concanamycin A-sensitive proton transport in FITC-loaded lysosomes. Inhibition of phosphatidylinositol 3-kinase with wortmannin or mTORC1 with rapamycin effectively inhibits the increased assembly observed upon cluster disruption. These results suggest that the phosphatidylinositol 3-kinase/mTOR pathway is involved in controlling V-ATPase assembly during dendritic cell maturation.

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