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1.
Int J Antimicrob Agents ; 54(5): 601-609, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31356859

ABSTRACT

Rotavirus is one of the leading causes of severe acute gastroenteritis in children under 5 years of age, mainly affecting developing countries. Once the disease is acquired, no specific treatment is available; as such, the development of new drugs for effective antirotaviral treatment is critical. Ursolic acid is a pentacyclic triterpenoid with antiviral activity, which has been studied extensively in vitro and in vivo. To study the potential antirotaviral activity of ursolic acid, its toxic potential for viral particles (virucidal effect) and cultured cells (cytotoxicity) was analysed. No effect on virion infectivity was observed with treatments of up to 40 µM ursolic acid, while incipient cytotoxicity started to be evident with 20 µM ursolic acid. The antiviral potential of ursolic acid was evaluated in in-vitro rotavirus infections, demonstrating that 10 µM ursolic acid inhibits rotavirus replication (observed by a decrease in viral titre and the level of the main viral proteins) and affects viral particle maturation (a process associated with the endoplasmic reticulum) 15 h post infection. Interestingly, ursolic acid was also found to hamper the early stages of the viral replication cycle, as a significant reduction in the number and size of viroplasms, consistent with a decrease in VP6 and NSP2 viral proteins, was observed 4 h post infection. As such, these observations demonstrate that ursolic acid exhibits antiviral activity, suggesting that this chemical could be used as a new treatment for rotavirus.


Subject(s)
Antiviral Agents/therapeutic use , Gastroenteritis/drug therapy , Rotavirus Infections/drug therapy , Rotavirus/drug effects , Triterpenes/therapeutic use , Animals , Antigens, Viral/metabolism , Antiviral Agents/adverse effects , Capsid Proteins/metabolism , Cell Line , Child, Preschool , Chlorocebus aethiops , Gastroenteritis/virology , Humans , Microbial Sensitivity Tests , RNA-Binding Proteins/metabolism , Triterpenes/adverse effects , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects , Ursolic Acid
2.
Gene Ther ; 22(1): 9-19, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25410742

ABSTRACT

Secreted protein, acidic and rich in cysteine (SPARC) is involved in many biological process including liver fibrogenesis, but its role in acute liver damage is unknown. To examine the role of SPARC in acute liver injury, we used SPARC knock-out (SPARC(-/-)) mice. Two models of acute liver damage were used: concanavalin A (Con A) and the agonistic anti-CD95 antibody Jo2. SPARC expression levels were analyzed in liver samples from patients with acute-on-chronic alcoholic hepatitis (AH). SPARC expression is increased on acute-on-chronic AH patients. Knockdown of SPARC decreased hepatic damage in the two models of liver injury. SPARC(-/-) mice showed a marked reduction in Con A-induced necroinflammation. Infiltration by CD4+ T cells, expression of tumor necrosis factor-α and interleukin-6 and apoptosis were attenuated in SPARC(-/-) mice. Sinusoidal endothelial cell monolayer was preserved and was less activated in Con A-treated SPARC(-/-) mice. SPARC knockdown reduced Con A-induced autophagy of cultured human microvascular endothelial cells (HMEC-1). Hepatic transcriptome analysis revealed several gene networks that may have a role in the attenuated liver damaged found in Con A-treated SPARC(-/-) mice. SPARC has a significant role in the development of Con A-induced severe liver injury. These results suggest that SPARC could represent a therapeutic target in acute liver injury.


Subject(s)
Chemical and Drug Induced Liver Injury/metabolism , Endothelial Cells/physiology , Osteonectin/genetics , Animals , Chemical and Drug Induced Liver Injury/immunology , Concanavalin A , Endothelium, Vascular/pathology , Gene Knockdown Techniques , Lipopolysaccharides/pharmacology , Liver , Male , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Osteonectin/metabolism , Transcriptome
3.
Curr Mol Med ; 13(2): 241-51, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23228127

ABSTRACT

Bacterial protein toxins are important virulence factors. A particular class of toxins, the pore-form toxins (PFTs), shares the toxigenic mechanism of forming pores in the membrane of target cells. The relationship between autophagy and bacterial PFTs has been described for several toxin-secreting pathogens and in this review we have recapitulated the more recent findings on this issue. A common outcome is that the target cell, by a yet non-completely defined mechanism, senses the toxin attack and builds up complex responses as a protective mechanism for host survival. However, in some cases, this cellular response is beneficial to the microorganism by supplying an intracellular niche or by promoting host-cell death, which facilitates pathogen spreading.


Subject(s)
Autophagy/genetics , Bacterial Toxins/toxicity , Gram-Negative Bacteria/metabolism , Gram-Positive Bacteria/metabolism , Pore Forming Cytotoxic Proteins/toxicity , Virulence Factors/toxicity , Bacterial Toxins/metabolism , Cell Membrane/chemistry , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Survival/genetics , Gram-Negative Bacteria/pathogenicity , Gram-Positive Bacteria/pathogenicity , HeLa Cells , Host-Pathogen Interactions , Humans , Phagosomes/drug effects , Phagosomes/metabolism , Pore Forming Cytotoxic Proteins/metabolism , Signal Transduction/drug effects , Virulence Factors/metabolism
4.
Curr Mol Med ; 11(3): 197-203, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21375493

ABSTRACT

Autophagy is one of the major catabolic processes present in eukaryotic cells, conserved through evolution, by which damaged or superfluous organelles are degraded in response to different stimuli. A hallmark of the autophagic pathway is the formation of double or multiple layered membranes that engulf the material to be finally degraded in the lysosomes. Despite enormous advances in the last few years to understand the autophagic process at the molecular level, the origin of the sequestering membrane has remained elusive for more than forty years and it is still a matter of debate. In this review we have summarized recent experimental evidence indicating that more than one membrane source may exist. Even though de novo formation or assembly of the isolation membrane has been proposed, recent data points to the participation of specific organelles in the biogenesis of the sequestering membrane.


Subject(s)
Autophagy , Phagosomes/ultrastructure , Cell Membrane/physiology , Cell Membrane/ultrastructure , Endoplasmic Reticulum/ultrastructure , Golgi Apparatus , Humans , Intracellular Membranes/physiology , Intracellular Membranes/ultrastructure , Mitochondrial Membranes/physiology , Mitochondrial Membranes/ultrastructure , Phagosomes/metabolism
5.
Cell Death Differ ; 17(3): 421-38, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19798108

ABSTRACT

Coxiella burnetii is the etiological agent of the human disease, Q fever, and is an obligate intracellular bacterium that invades and multiplies in a vacuole with lysosomal characteristics. We have previously shown that Coxiella interacts with the autophagic pathway as a strategy for its survival and replication. In addition, recent studies have shown that Coxiella exerts anti-apoptotic activity to maintain the host cell viability, thus generating a persistent infection. In the present report, we have explored the role of Beclin 1 and Bcl-2 in C. burnetii infection to elucidate how this bacterium modulates autophagy and apoptosis to its own benefit. Beclin 1, a Bcl-2 interacting protein, is required for autophagy. In this study, we show that Beclin 1 is recruited to the Coxiella-membrane vacuole, favoring its development and bacterial replication. In contrast, the anti-apoptotic protein Bcl-2 alters the normal development of the Coxiella-replicative compartment, in spite of also being recruited to the vacuole membrane. Furthermore, both vacuole development and the anti-apoptotic effect of C. burnetii are affected by Beclin 1 depletion and by the expression of a Beclin 1 mutant defective in Bcl-2 binding. Overall, these findings indicate that C. burnetii infection modulates autophagy and apoptotic pathways through Beclin 1/Bcl-2 interplay to establish a successful infection in the host cell.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Apoptosis/physiology , Coxiella burnetii , Membrane Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Q Fever/metabolism , Apoptosis Regulatory Proteins/genetics , Autophagy/physiology , Beclin-1 , Coxiella burnetii/metabolism , Coxiella burnetii/pathogenicity , HeLa Cells , Humans , Membrane Proteins/genetics , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Vacuoles/metabolism
6.
Cell Death Differ ; 16(1): 70-8, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19008921

ABSTRACT

In the majority of cell types, multivesicular bodies (MVBs) are a special kind of late endosomes, crucial intermediates in the internalization of nutrients, ligands and receptors through the endolysosomal system. ESCRT-0, I, II and III (endosomal sorting complex required for transport) are involved in the sorting of proteins into MVBs, generating the intraluminal vesicles. Autophagy is a lysosomal degradation pathway for cytoplasmic components such as proteins and organelles. The autophagosome, a well-characterized structure of the autophagy pathway, can fuse with endocytic structures such as MVBs to generate the amphisome. Finally, the amphisome fuses with the lysosome to degrade the material wrapped inside. Currently, clear evidence suggests that efficient autophagic degradation requires functional MVBs. This review highlights the most recent advances in our understanding of the molecular machinery that participates in MVB biogenesis and regulates the interplay between autophagy and this organelle.


Subject(s)
Autophagy/physiology , Endosomes/metabolism , Lysosomes/metabolism , Phagosomes/metabolism , Animals , Biological Transport/physiology , Humans
7.
Infect Immun ; 74(12): 6599-614, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17000720

ABSTRACT

Helicobacter pylori colonizes the gastric epithelium of at least 50% of the world's human population, playing a causative role in the development of chronic gastritis, peptic ulcers, and gastric adenocarcinoma. Current evidence indicates that H. pylori can invade epithelial cells in the gastric mucosa. However, relatively little is known about the biology of H. pylori invasion and survival in host cells. Here, we analyze both the nature of and the mechanisms responsible for the formation of H. pylori's intracellular niche. We show that in AGS cells infected with H. pylori, bacterium-containing vacuoles originate through the fusion of late endocytic organelles. This process is mediated by the VacA-dependent retention of the small GTPase Rab7. In addition, functional interactions between Rab7 and its downstream effector, Rab-interacting lysosomal protein (RILP), are necessary for the formation of the bacterial compartment since expression of mutant forms of RILP or Rab7 that fail to bind each other impaired the formation of this unique bacterial niche. Moreover, the VacA-mediated sequestration of active Rab7 disrupts the full maturation of vacuoles as assessed by the lack of both colocalization with cathepsin D and degradation of internalized cargo in the H. pylori-containing vacuole. Based on these findings, we propose that the VacA-dependent isolation of the H. pylori-containing vacuole from bactericidal components of the lysosomal pathway promotes bacterial survival and contributes to the persistence of infection.


Subject(s)
Bacterial Proteins/physiology , Gastric Mucosa/microbiology , Helicobacter pylori/physiology , Membrane Fusion , Vacuoles/microbiology , Adaptor Proteins, Signal Transducing/metabolism , Animals , Bacterial Proteins/genetics , Cathepsin D/analysis , Cathepsin D/metabolism , Cells, Cultured , Cricetinae , Endocytosis , Endosomes/microbiology , Endosomes/physiology , Endosomes/ultrastructure , Gastric Mucosa/ultrastructure , Humans , Lysosomes/microbiology , Lysosomes/physiology , Lysosomes/ultrastructure , Mutation , Vacuoles/ultrastructure , rab GTP-Binding Proteins/metabolism , rab7 GTP-Binding Proteins
9.
J Cell Sci ; 114(Pt 20): 3619-29, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11707514

ABSTRACT

Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic portions and intracellular organelles in a membrane vacuole called the autophagosome. These vesicles fuse with lysosomes and the sequestered material is degraded. Owing to the complexity of the autophagic pathway and to its inaccessibility to external probes, little is known about the molecular mechanisms that regulate autophagy in higher eukaryotic cells. We used the autofluorescent drug monodansylcadaverine (MDC), a specific autophagolysosome marker to analyze at the molecular level the machinery involved in the autophagic process. We have developed a morphological and biochemical assay to study authophagy in living cells based on the incorporation of MDC. With this assay we observed that the accumulation of MDC was specifically induced by amino acid deprivation and was inhibited by 3-methlyadenine, a classical inhibitor of the autophagic pathway. Additionally, wortmannin, an inhibitor of PI3-kinases that blocks autophagy at an early stage, inhibited the accumulation of MDC in autophagic vacuoles. We also found that treatment of the cells with N-ethylmaleimide (NEM), an agent known to inhibit several vesicular transport events, completely blocked the incorporation of MDC, suggesting that an NEM-sensitive protein is required for the formation of autophagic vacuoles. Conversely, vinblastine, a microtubule depolymerizing agent that induces the accumulation of autophagic vacuoles by preventing their degradation, increased the accumulation of MDC and altered the distribution and size of the autophagic vacuoles. Our results indicate that in the presence of vinblastine very large MDC-vacuoles accumulated mainly under starvation conditions, indicating that the expansion of autophagosomes is upregulated by amino acid deprivation. Furthermore, these MDC-vacuoles were labeled with LC3, one of the mammalian homologues of the yeast protein Apg8/Aut7 that plays an important role in autophagosome formation.


Subject(s)
Adenine/analogs & derivatives , Autophagy/physiology , Cadaverine/analogs & derivatives , Phagosomes/metabolism , Vacuoles/metabolism , Adenine/pharmacology , Amino Acids/metabolism , Androstadienes/pharmacology , Animals , CHO Cells , Cadaverine/metabolism , Cricetinae , Cycloheximide/pharmacology , Enzyme Inhibitors/pharmacology , Ethylmaleimide/pharmacology , Fluorescent Dyes , Phagosomes/ultrastructure , Protein Synthesis Inhibitors/pharmacology , Recombinant Fusion Proteins/metabolism , Vacuoles/ultrastructure , Vinblastine/pharmacology , Wortmannin
10.
Exp Cell Res ; 271(1): 189-99, 2001 Nov 15.
Article in English | MEDLINE | ID: mdl-11697895

ABSTRACT

Phagocytosis is a receptor-mediated process by which specialized cell types engulf large extracellular particles. Phagosome maturation involves a series of intracellular membrane fusion and budding events resulting in the delivery of particles to compartments enriched in lysosomal hydrolases where they are digested. Substantial amounts of plasma membrane and many phagosomal proteins, such as receptors, rapidly recycle to the plasma membrane following phagosome formation. Despite the importance of this recycling pathway in phagosome maturation and in the retrieval of immunogenic peptides from phagosomes, the molecular machinery involved is largely unknown. To assess the participation of GTPases in phagocytosis and recycling from phagosomes we used aluminum fluoride (AIF(-)(4)), which activates the GDP-bound form of stimulatory and inhibitory trimeric G proteins. AlF(-)(4) inhibited both the uptake to and the recycling from the phagosomal compartment. Cholera toxin, which activates Galphas, and pertussis toxin, which uncouples Gi and Go from receptors, were effective inhibitors of phagocytosis. However, both toxins stimulated recycling from phagosomes. These results suggest that more than one GTP-binding protein participates either directly or indirectly not only in phagocytosis, but also in maturation and recycling from phagosomes, and thereby assign a role for heterotrimeric G proteins in controlling traffic through the phagocytic pathway.


Subject(s)
Aluminum Compounds/pharmacology , Fluorides/pharmacology , Heterotrimeric GTP-Binding Proteins/metabolism , Macrophages/physiology , Phagocytosis/physiology , Phagosomes/metabolism , Animals , Cell Line , Cholera Toxin/pharmacology , Macrophages/cytology , Macrophages/drug effects , Pertussis Toxin , Staphylococcus aureus/cytology , Staphylococcus aureus/drug effects , Staphylococcus aureus/metabolism , Virulence Factors, Bordetella/pharmacology
11.
Biochem J ; 355(Pt 2): 409-15, 2001 Apr 15.
Article in English | MEDLINE | ID: mdl-11284728

ABSTRACT

Particle internalization in macrophages is followed by a complex maturation process. We have previously observed that proteins bound to phagocytosed particles are sorted from phagosomes into a heterogeneous population of vesicles that fuse with endosomes. However, the mechanism and the protein machinery involved in the formation of these phagosome-derived vesicles are largely unknown. It has been shown that vesicles coated with coat protein complex type I (COPI) have a role in both secretion and endocytosis. To address the possibility that COPI proteins might participate in the formation of phagosome-derived vesicles we studied the recruitment of beta-COP to highly purified phagosomes. The binding of beta-COP to phagosomal membranes was regulated by nucleotides and inhibited by brefeldin A (BFA). An ADP-ribosylation factor 1 (ARF1) mutant defective in GTP hydrolysis supported the binding of beta-COP to phagosomes independently of added nucleotide. AlF(4) and Gbetagamma subunits, agents known to modulate heterotrimeric G-protein activity, were tested in the beta-COP binding assay. AlF(4) increased beta-COP association, whereas binding was inhibited by the addition of Gbetagamma subunits. Our results suggest that COP proteins are recruited to phagosomal membranes by a mechanism that involves heterotrimeric GTP-binding proteins and a BFA-sensitive ARF. In addition, our findings indicate that COPI proteins are involved in the recycling of components from phagosomes to the cell surface.


Subject(s)
ADP-Ribosylation Factors/metabolism , Brefeldin A/pharmacology , Phagosomes/metabolism , Animals , Cell Line , Coatomer Protein/metabolism , Intracellular Membranes/drug effects , Intracellular Membranes/metabolism , Microscopy, Electron , Phagosomes/ultrastructure
12.
Biochem Biophys Res Commun ; 249(2): 481-5, 1998 Aug 19.
Article in English | MEDLINE | ID: mdl-9712722

ABSTRACT

Solution phase combinatorial synthesis of flavone derivatives and evaluation of their affinity for the central benzodiazepine receptors is described. The libraries preparation is simple and provides a convenient method for rapid compound generation and screening. Thirty one new compounds were obtained of which the most promising, as high affinity benzodiazepine receptor ligands, were 6-bromo-3'-fluoroflavone; 6,3'-dichloroflavone; 6-bromo-3'-chloroflavone and 6-chloro-3'-bromoflavone.


Subject(s)
Flavonoids/chemical synthesis , Flavonoids/metabolism , Receptors, GABA-A/metabolism , Animals , Binding, Competitive , Cell Membrane/metabolism , Cerebellum/metabolism , Cerebral Cortex/metabolism , Chromatography, High Pressure Liquid , Flavonoids/chemistry , Flunitrazepam/metabolism , Mice , Molecular Structure , Rats , Solutions , gamma-Aminobutyric Acid/metabolism
13.
J Biol Chem ; 273(3): 1334-8, 1998 Jan 16.
Article in English | MEDLINE | ID: mdl-9430666

ABSTRACT

The N-ethylmaleimide-sensitive factor (NSF) is required for multiple intracellular vesicle transport events. In vitro biochemical studies have demonstrated that NSF, soluble NSF attachment proteins (SNAPs), and SNAP receptors from a 20 S particle. This complex is disassembled by the ATPase activity of NSF. We have studied particle disassembly in a membrane environment by examining the binding of recombinant SNAPs and NSF to endosomal membranes. We present evidence that alpha-SNAP is released from the membranes in a temperature- and time-dependent manner and that this release is mediated by the ATPase activity of NSF. Our results indicate that NSF mutants in the first ATP binding domain completely abrogate alpha-SNAP release, whereas no inhibitory effect is observed with a mutant in the second ATP binding domain. Interestingly, neither beta-SNAP nor gamma-SNAP are released by the ATPase activity of NSF, indicating that these proteins are retained on the membranes by interactions that differ from those that retain alpha-SNAP. Although the small Rab GTPases are known to play a role in SNARE complex assembly, our results indicate that these GTPases do not regulate the NSF-dependent release of alpha-SNAP.


Subject(s)
Carrier Proteins/metabolism , GTP Phosphohydrolases/metabolism , Guanine Nucleotide Dissociation Inhibitors , Membrane Proteins/metabolism , Vesicular Transport Proteins , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Animals , Binding Sites , Cell Membrane/metabolism , GTP-Binding Proteins/metabolism , Hydrolysis , N-Ethylmaleimide-Sensitive Proteins , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins , Temperature
14.
FEBS Lett ; 441(3): 373-8, 1998 Dec 28.
Article in English | MEDLINE | ID: mdl-9891974

ABSTRACT

Previous observations indicate that a zinc and phorbol ester binding factor is necessary for endosome fusion. To further characterize the role of this factor in the process, we used an in vitro endosome fusion assay supplemented with recombinant Rab5 proteins. Both zinc depletion and addition of calphostin C, an inhibitor of protein kinase C, inhibited endosome fusion in the presence of active Rab5. Addition of the phorbol ester PMA (phorbol 12-myristate 13-acetate) reversed the inhibition of endosome fusion caused by a Rab5 negative mutant. Moreover, PMA stimulated fusion in the presence of Rab5 immunodepleted cytosol. These results suggest that the phorbol ester binding protein is acting downstream of Rab5 in endosome fusion.


Subject(s)
Caenorhabditis elegans Proteins , Endosomes/metabolism , GTP-Binding Proteins/metabolism , Protein Kinase C/metabolism , Receptors, Drug/metabolism , Tetradecanoylphorbol Acetate/metabolism , Carrier Proteins , Membrane Fusion , rab5 GTP-Binding Proteins
15.
J Biol Chem ; 272(12): 7707-12, 1997 Mar 21.
Article in English | MEDLINE | ID: mdl-9065429

ABSTRACT

Calmodulin (CaM) has previously been implicated in regulated exocytosis, transcytosis, and receptor recycling. We have investigated the role of CaM in endocytic transport by examining the effects of several CaM antagonists in intact cells. We present evidence indicating that the mixing of sequentially internalized ligands is inhibited by CaM antagonists, indicating that CaM may play a general role in regulating endosomal membrane trafficking. To address the specific events that are affected by CaM we studied its role in an in vitro assay that reconstitutes fusion among endosomes. CaM antagonists inhibited endosome fusion, and the inhibition was reversed by the addition of CaM. Moreover, we found that Ca2+ stimulates fusion among endosomes and that addition of CaM stimulates fusion beyond that produced by Ca2+ alone. Our data indicate that one of the possible targets for CaM in endosome fusion is the CaM-dependent kinase II. We propose that CaM regulates endocytic transport by modulating an essential component(s) of the membrane traffic machinery.


Subject(s)
Calmodulin/pharmacology , Endosomes/drug effects , Membrane Fusion/drug effects , Benzylamines/pharmacology , Biological Transport , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Calmodulin/antagonists & inhibitors , Cell Line , Endocytosis , Endosomes/metabolism , Enzyme Inhibitors/pharmacology , Sulfonamides/pharmacology
17.
J Biol Chem ; 271(31): 18810-6, 1996 Aug 02.
Article in English | MEDLINE | ID: mdl-8702539

ABSTRACT

N-Ethylmaleimide-sensitive fusion protein (NSF) is an ubiquitous protein required for multiple vesicular transport events. We have investigated the role of the two nucleotide-binding regions of NSF in endosomal fusion by analyzing NSF mutants in a cell-free system. Our results indicate that mutations on the first ATP-binding domain, that render a protein defective in either ATP binding or ATP hydrolysis, results in almost complete inhibition of endosomal fusion. A mutation in the second ATP-binding site of NSF was only slightly inhibitory. The inhibitory effect was observed only when the mutant proteins were added at early times during the fusion reaction indicating that NSF may be required for an early step during the docking/fusion process. Binding studies using Western blotting reveal that the binding of NSF mutants to endosomal membranes is differentially affected by Ca2+. Our results indicate that NSF, depending on its nucleotide state, may interact with membranes via an alternate mechanism. Our findings suggest the existence of a predocking binding site either independent of the docking complex or a site that leads to the formation of the SNAP-SNARE complex (e.g. 20 S particle).


Subject(s)
Carrier Proteins/metabolism , Endosomes/metabolism , Vesicular Transport Proteins , Adenosine Triphosphate/metabolism , Animals , Binding Sites , Carrier Proteins/genetics , Cell Line , Ethylmaleimide/pharmacology , Immunohistochemistry , In Vitro Techniques , Membrane Fusion , Mice , N-Ethylmaleimide-Sensitive Proteins , Point Mutation
18.
Biochem J ; 312 ( Pt 3): 919-23, 1995 Dec 15.
Article in English | MEDLINE | ID: mdl-8554539

ABSTRACT

Fusion among endosomes is an important step for transport and sorting of internalized macromolecules. Working in a cell-free system, we previously reported that endosome fusion requires cytosol and ATP, and is sensitive to N-ethylmaleimide. Fusion is regulated by monomeric and heterotrimeric GTP-binding proteins. We now report that fusion can proceed at very low Ca2+ concentrations, i.e. < 30 nM. Moreover, fusion is not affected when intravesicular Ca2+ is depleted by preincubation of vesicles with calcium ionophores (5 microM ionomycin or A23187) in the presence of calcium chelators (5 mM EGTA or 60 mM EDTA). The results indicate that fusion can proceed at extremely low concentrations of intravesicular and extravesicular Ca2+. However, BAPTA [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid], a relatively specific Ca2+ chelator, inhibits fusion. BAPTA binds other metals besides Ca2+. We present evidence that BAPTA inhibition is due not to Ca2+ chelation but to Zn2+ depletion. TPEN [N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylenediamine], another metal-ion chelator with low affinity for Ca2+, also inhibited fusion. TPEN- and BAPTA-inhibited fusions were restored by addition of Zn2+. Zn(2+)-dependent fusion presents the same characteristics as control fusion. In intact cells, TPEN inhibited transport along the endocytic pathway. The results indicate that Zn2+ depletion blocks endosome fusion, suggesting that this ion is necessary for the function of one or more factors involved in the fusion process.


Subject(s)
Endosomes/physiology , Membrane Fusion/physiology , Zinc/physiology , Animals , Calcimycin/pharmacology , Calcium/administration & dosage , Chelating Agents/pharmacology , Edetic Acid/pharmacology , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Endosomes/ultrastructure , Ethylenediamines/pharmacology , Intracellular Membranes/physiology , Ionomycin/pharmacology , Membrane Fusion/drug effects , Mice , Zinc/pharmacology
19.
J Biol Chem ; 270(41): 24564-71, 1995 Oct 13.
Article in English | MEDLINE | ID: mdl-7592675

ABSTRACT

Trimeric G proteins have emerged as important regulators of membrane trafficking. To explore a role for G beta gamma in endosome fusion, we have taken advantage of beta-adrenergic receptor kinase (beta ARK), an enzyme translocated to membranes by interaction with G beta gamma. The COOH terminus of beta ARK (beta ARKct) has a G beta gamma-binding domain which blocks some G beta gamma-mediated processes. We found that beta ARKct and peptide G, a peptide derived from beta ARKct, inhibit in vitro endosome fusion. Interestingly, peptide G and ARF share sequence similarity. Peptide G and beta ARKct reversed ARF-mediated inhibition of endosome fusion and blocked ARF binding to membranes. Using an ARF fusion protein, we show that both G beta gamma and G alpha s interact with the small GTPase ARF, an interaction that is regulated by nucleotide binding. We conclude that G proteins may participate in the regulation of vesicular trafficking by directly interacting with ARF, a cytosolic factor required for transport.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , GTP-Binding Proteins/metabolism , ADP-Ribosylation Factors , Amino Acid Sequence , Animals , Cell Line , Cell Membrane , Endocytosis , GTP-Binding Proteins/chemistry , Glutathione Transferase/metabolism , Homeostasis , Macromolecular Substances , Macrophages , Membrane Fusion , Molecular Sequence Data , Organelles/physiology , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Sequence Homology, Amino Acid , beta-Adrenergic Receptor Kinases
20.
Arch Biochem Biophys ; 317(2): 337-42, 1995 Mar 10.
Article in English | MEDLINE | ID: mdl-7893147

ABSTRACT

We have assessed the role of heterotrimeric GTPases on in vitro fusion of phagosomes and endosomes. Highly purified phagosomes were found to contain G alpha s, G alpha i1, G alpha i2, G alpha i3, and G beta subunits of heterotrimeric GTP-binding proteins. A functional role for G proteins was established using an in vitro phagosome-endosome fusion assay. First, addition of AlF4- and purified G beta gamma subunits to the in vitro assay blocked fusion, indicating that heterotrimeric G proteins may play a role, either direct or indirect, in phagosome maturation. Second, a striking inhibitory effect was observed when the vesicles were incubated with peptides that preferentially activate G alpha s. A similar effect on phagosome-endosome fusion was observed with cholera toxin, a reagent known to activate G alpha s. Our results suggest that one or more heterotrimeric G proteins, including Gs, mediate and/or regulate phagosome-endosome fusion.


Subject(s)
Endosomes/physiology , GTP Phosphohydrolases/metabolism , GTP-Binding Proteins/physiology , Membrane Fusion , Phagosomes/physiology , Aluminum Compounds/pharmacology , Cell Line , Cholera Toxin/pharmacology , Endosomes/ultrastructure , Fluorides/pharmacology , GTP-Binding Proteins/analysis , GTP-Binding Proteins/chemistry , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Intracellular Membranes/chemistry , Intracellular Membranes/physiology , Macromolecular Substances , Membrane Fusion/drug effects , NAD/pharmacology , Phagosomes/ultrastructure , Potassium Chloride/pharmacology
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