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Gβγ directly modulates vesicle fusion by competing with synaptotagmin for binding to neuronal SNARE proteins embedded in membranes.
Zurawski Z, Page B, Chicka MC, Brindley RL, Wells CA, Preininger AM, Hyde K, Gilbert JA, Cruz-Rodriguez O, Currie KPM, Chapman ER, Alford S, Hamm HE
(2017) J Biol Chem 292: 12165-12177
MeSH Terms: Animals, Binding, Competitive, Calcium Signaling, Cattle, Cell Line, GTP-Binding Protein beta Subunits, GTP-Binding Protein gamma Subunits, Humans, Lipid Bilayers, Liposomes, Membrane Fusion, Models, Molecular, Mutation, Nerve Tissue Proteins, Peptide Fragments, Protein Conformation, Protein Interaction Domains and Motifs, Protein Multimerization, Rats, Recombinant Fusion Proteins, Recombinant Proteins, Synaptosomal-Associated Protein 25, Synaptotagmin I, Syntaxin 1
Show Abstract · Added July 12, 2017
G-coupled G protein-coupled receptors can inhibit neurotransmitter release at synapses via multiple mechanisms. In addition to Gβγ-mediated modulation of voltage-gated calcium channels (VGCC), inhibition can also be mediated through the direct interaction of Gβγ subunits with the soluble -ethylmaleimide attachment protein receptor (SNARE) complex of the vesicle fusion apparatus. Binding studies with soluble SNARE complexes have shown that Gβγ binds to both ternary SNARE complexes, t-SNARE heterodimers, and monomeric SNAREs, competing with synaptotagmin 1(syt1) for binding sites on t-SNARE. However, in secretory cells, Gβγ, SNAREs, and synaptotagmin interact in the lipid environment of a vesicle at the plasma membrane. To approximate this environment, we show that fluorescently labeled Gβγ interacts specifically with lipid-embedded t-SNAREs consisting of full-length syntaxin 1 and SNAP-25B at the membrane, as measured by fluorescence polarization. Fluorescently labeled syt1 undergoes competition with Gβγ for SNARE-binding sites in lipid environments. Mutant Gβγ subunits that were previously shown to be more efficacious at inhibiting Ca-triggered exocytotic release than wild-type Gβγ were also shown to bind SNAREs at a higher affinity than wild type in a lipid environment. These mutant Gβγ subunits were unable to inhibit VGCC currents. Specific peptides corresponding to regions on Gβ and Gγ shown to be important for the interaction disrupt the interaction in a concentration-dependent manner. In fusion assays using full-length t- and v-SNAREs embedded in liposomes, Gβγ inhibited Ca/synaptotagmin-dependent fusion. Together, these studies demonstrate the importance of these regions for the Gβγ-SNARE interaction and show that the target of Gβγ, downstream of VGCC, is the membrane-embedded SNARE complex.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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24 MeSH Terms
Extracellular Vesicles: Unique Intercellular Delivery Vehicles.
Maas SLN, Breakefield XO, Weaver AM
(2017) Trends Cell Biol 27: 172-188
MeSH Terms: Biological Transport, Disease, Extracellular Space, Extracellular Vesicles, Humans, Membrane Fusion, Models, Biological
Show Abstract · Added April 26, 2017
Extracellular vesicles (EVs) are a heterogeneous collection of membrane-bound carriers with complex cargoes including proteins, lipids, and nucleic acids. While the release of EVs was previously thought to be only a mechanism to discard nonfunctional cellular components, increasing evidence implicates EVs as key players in intercellular and even interorganismal communication. EVs confer stability and can direct their cargoes to specific cell types. EV cargoes also appear to act in a combinatorial manner to communicate directives to other cells. This review focuses on recent findings and knowledge gaps in the area of EV biogenesis, release, and uptake. In addition, we highlight examples whereby EV cargoes control basic cellular functions, including motility and polarization, immune responses, and development, and contribute to diseases such as cancer and neurodegeneration.
Copyright © 2016 Elsevier Ltd. All rights reserved.
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7 MeSH Terms
The human metapneumovirus fusion protein mediates entry via an interaction with RGD-binding integrins.
Cox RG, Livesay SB, Johnson M, Ohi MD, Williams JV
(2012) J Virol 86: 12148-60
MeSH Terms: Amino Acid Motifs, Cell Line, Flow Cytometry, Humans, Integrins, Membrane Fusion, Metapneumovirus, Microscopy, Electron, Oligopeptides, Protein Binding, Receptors, Vitronectin, Recombinant Proteins, Viral Fusion Proteins, Virion, Virus Internalization
Show Abstract · Added March 7, 2014
Paramyxoviruses use a specialized fusion protein to merge the viral envelope with cell membranes and initiate infection. Most paramyxoviruses require the interaction of two viral proteins to enter cells; an attachment protein binds cell surface receptors, leading to the activation of a fusion (F) protein that fuses the viral envelope and host cell plasma membrane. In contrast, human metapneumovirus (HMPV) expressing only the F protein is replication competent, suggesting a primary role for HMPV F in attachment and fusion. We previously identified an invariant arginine-glycine-aspartate (RGD) motif in the HMPV F protein and showed that the RGD-binding integrin αVβ1-promoted HMPV infection. Here we show that both HMPV F-mediated binding and virus entry depend upon multiple RGD-binding integrins and that HMPV F can mediate binding and fusion in the absence of the viral attachment (G) protein. The invariant F-RGD motif is critical for infection, as an F-RAE virus was profoundly impaired. Further, F-integrin binding is required for productive viral RNA transcription, indicating that RGD-binding integrins serve as receptors for the HMPV fusion protein. Thus, HMPV F is triggered to induce virus-cell fusion by interactions with cellular receptors in a manner that is independent of the viral G protein. These results suggest a stepwise mechanism of HMPV entry mediated by the F protein through its interactions with cellular receptors, including RGD-binding integrins.
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15 MeSH Terms
Cell-cell fusion: a new function for invadosomes.
Sung BH, Weaver A
(2011) Curr Biol 21: R121-3
MeSH Terms: Actins, Animals, Cell Membrane, Cell Surface Extensions, Drosophila, Drosophila Proteins, Membrane Fusion, Microfilament Proteins, Models, Biological, Wiskott-Aldrich Syndrome Protein
Show Abstract · Added May 19, 2014
Podosomes are cytoskeletal-based structures involved in extracellular matrix remodeling and cellular motility. A new study now implicates podosomes in pore formation during myoblast fusion.
Copyright © 2011 Elsevier Ltd. All rights reserved.
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10 MeSH Terms
Fragmented mitochondria are sensitized to Bax insertion and activation during apoptosis.
Brooks C, Cho SG, Wang CY, Yang T, Dong Z
(2011) Am J Physiol Cell Physiol 300: C447-55
MeSH Terms: Animals, Apoptosis, Apoptosis Regulatory Proteins, Cell Line, Cells, Cultured, Enzyme Activation, GTP Phosphohydrolases, Gene Knockdown Techniques, HeLa Cells, Humans, Membrane Fusion, Membrane Transport Proteins, Mice, Mice, Knockout, Microtubule-Associated Proteins, Mitochondria, Mitochondrial Diseases, Mitochondrial Membrane Transport Proteins, Mitochondrial Proteins, RNA Interference, Rats, Stress, Physiological, bcl-2-Associated X Protein
Show Abstract · Added September 12, 2016
Recent studies have shown mitochondrial fragmentation during cell stress and have suggested a role for the morphological change in mitochondrial injury and ensuing apoptosis. However, the underlying mechanism remains elusive. Here we demonstrate that mitochondrial fragmentation facilitates Bax insertion and activation in mitochondria, resulting in the release of apoptogenic factors. In HeLa cells, overexpression of mitofusins attenuated mitochondrial fragmentation during cisplatin- and azide-induced cell injury, which was accompanied by less apoptosis and less cytochrome c release from mitochondria. Similar effects were shown by inhibiting the mitochondrial fission protein Drp1 with a dominant negative mutant (dn-Drp1). Mitofusins and dn-Drp1 did not seem to significantly affect Bax translocation/accumulation to mitochondria; however, they blocked Bax insertion and activation in mitochondrial membrane. Consistently, in rat kidney proximal tubular cells, small interfering RNA knockdown of Drp1 prevented mitochondrial fragmentation during azide-induced ATP depletion, which was accompanied by less Bax activation, insertion, and oligomerization in mitochondria. These cells released less cytochrome c and AIF from mitochondria and showed significantly lower apoptosis. Finally, mitofusin-null mouse embryonic fibroblasts (MEF) had fragmented mitochondria. These MEFs were more sensitive to cisplatin-induced Bax activation, release of cytochrome c, and apoptosis. Together, this study provides further support for a role of mitochondrial fragmentation in mitochondrial injury and apoptosis. Mechanistically, mitochondrial fragmentation may sensitize the cells to Bax insertion and activation in mitochondria, facilitating the release of apoptogenic factors and consequent apoptosis.
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23 MeSH Terms
Crystal structure of the Epstein-Barr virus (EBV) glycoprotein H/glycoprotein L (gH/gL) complex.
Matsuura H, Kirschner AN, Longnecker R, Jardetzky TS
(2010) Proc Natl Acad Sci U S A 107: 22641-6
MeSH Terms: Amino Acid Sequence, Animals, Binding Sites, Cell Line, Crystallization, Cysteine, Disulfides, Herpesvirus 4, Human, Humans, Membrane Fusion, Membrane Glycoproteins, Microscopy, Electron, Models, Molecular, Molecular Chaperones, Molecular Sequence Data, Multiprotein Complexes, Protein Binding, Protein Multimerization, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Spodoptera, Viral Envelope Proteins, Viral Proteins
Show Abstract · Added April 12, 2015
The Epstein-Barr virus (EBV) is a γ-herpesvirus that infects B cells and epithelial cells and that has been linked to malignancies in both cell types in vivo. EBV, like other herpesviruses, has three glycoproteins, glycoprotein B (gB), gH, and gL, that form the core membrane fusion machinery mediating viral penetration into the cell. The gH and gL proteins associate to form a heterodimeric complex, which is necessary for efficient membrane fusion and also implicated in direct binding to epithelial cell receptors required for viral entry. To gain insight into the mechanistic role of gH/gL, we determined the crystal structure of the EBV gH/gL complex. The structure is comprised of four domains organized along the longest axis of the molecule. Comparisons with homologous HSV-2 gH/gL and partial pseudorabies virus gH structures support the domain boundaries determined for the EBV gH/gL structure and illustrate significant differences in interdomain packing angles. The gL subunit and N-terminal residues of gH form a globular domain at one end of the structure, implicated in interactions with gB and activation of membrane fusion. The C-terminal domain of gH, proximal to the viral membrane, is also implicated in membrane fusion. The gH/gL structure locates an integrin binding motif, implicated in epithelial cell entry, on a prominent loop in the central region of the structure. Multiple regions of gH/gL, including its two extreme ends, are functionally important, consistent with the multiple roles of gH/gL in EBV entry.
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23 MeSH Terms
Mapping the N-terminal residues of Epstein-Barr virus gp42 that bind gH/gL by using fluorescence polarization and cell-based fusion assays.
Liu F, Marquardt G, Kirschner AN, Longnecker R, Jardetzky TS
(2010) J Virol 84: 10375-85
MeSH Terms: Amino Acid Sequence, Animals, Binding Sites, CHO Cells, Cell Line, Cricetinae, Cricetulus, Fluorescence Polarization, Herpesvirus 4, Human, Humans, Membrane Fusion, Molecular Sequence Data, Mutagenesis, Site-Directed, Peptide Mapping, Protein Binding, Recombinant Proteins, Viral Proteins, Virus Internalization
Show Abstract · Added April 12, 2015
Epstein-Barr virus (EBV) requires at a minimum membrane-associated glycoproteins gB, gH, and gL for entry into host cells. B-cell entry additionally requires gp42, which binds to gH/gL and triggers viral entry into B cells. The presence of soluble gp42 inhibits membrane fusion with epithelial cells by forming a stable heterotrimer of gH/gL/gp42. The interaction of gp42 with gH/gL has been previously mapped to residues 36 to 81 at the N-terminal region of gp42. In this study, we further mapped this region to identify essential features for binding to gH/gL by use of synthetic peptides. Data from fluorescence polarization, cell-cell fusion, and viral infection assays demonstrated that 33 residues corresponding to 44 to 61 and 67 to 81 of gp42 were indispensable for maintaining low-nanomolar-concentration gH/gL binding affinity and inhibiting B-cell fusion and epithelial cell fusion as well as viral infection. Overall, specific, large hydrophobic side chain residues of gp42 appeared to provide critical interactions, determining the binding strength. Mutations of these residues also diminished the inhibition of B-cell and epithelial cell fusions as well as EBV infection. A linker region (residues 62 to 66) between two gH/gL binding regions served as an important spacer, but individual amino acids were not critical for gH/gL binding. Probing the binding site of gH/gL and gp42 with gp42 peptides is critical for a better understanding of the interaction of gH/gL with gp42 as well as for the design of novel entry inhibitors of EBV and related human herpesviruses.
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18 MeSH Terms
Residues in the heptad repeat a region of the fusion protein modulate the virulence of Sendai virus in mice.
Luque LE, Bridges OA, Mason JN, Boyd KL, Portner A, Russell CJ
(2010) J Virol 84: 810-21
MeSH Terms: Animals, Cell Line, Cercopithecus aethiops, Female, Gene Expression Regulation, Viral, Lung, Membrane Fusion, Mice, Mice, Inbred DBA, Point Mutation, Respirovirus Infections, Sendai virus, Vero Cells, Viral Fusion Proteins, Virulence
Show Abstract · Added March 20, 2014
While the molecular basis of fusion (F) protein refolding during membrane fusion has been studied extensively in vitro, little is known about the biological significance of membrane fusion activity in parainfluenza virus replication and pathogenesis in vivo. Two recombinant Sendai viruses, F-L179V and F-K180Q, were generated that contain F protein mutations in the heptad repeat A region of the ectodomain, a region of the protein known to regulate F protein activation. In vitro, the F-L179V virus caused increased syncytium formation (cell-cell membrane fusion) yet had a rate of replication and levels of F protein expression and cleavage similar to wild-type virus. The F-K180Q virus had a reduced replication rate along with reduced levels of F protein expression, cleavage, and fusogenicity. In DBA/2 mice, the hyperfusogenic F-L179V virus induced greater morbidity and mortality than wild-type virus, while the attenuated F-K180Q virus was much less pathogenic. During the first week of infection, virus replication and inflammation in the lungs were similar for wild-type and F-L179V viruses. After approximately 1 week of infection, the clearance of F-L179V virus was delayed, and more extensive interstitial inflammation and necrosis were observed in the lungs, affecting entire lobes of the lungs and having significantly greater numbers of syncytial cell masses in alveolar spaces on day 10. On the other hand, the slower-growing F-K180Q virus caused much less extensive inflammation than wild-type virus, presumably due to its reduced replication rate, and did not cause observable syncytium formation in the lungs. Overall, the results show that residues in the heptad repeat A region of the F protein modulate the virulence of Sendai virus in mice by influencing both the spread and clearance of the virus and the extent and severity of inflammation. An understanding of how the F protein contributes to infection and inflammation in vivo may assist in the development of antiviral therapies against respiratory paramyxoviruses.
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15 MeSH Terms
Regulation of mitochondrial morphological dynamics during apoptosis by Bcl-2 family proteins: a key in Bak?
Brooks C, Dong Z
(2007) Cell Cycle 6: 3043-7
MeSH Terms: Animals, Apoptosis, Humans, Membrane Fusion, Mitochondria, Mitochondrial Membranes, bcl-2 Homologous Antagonist-Killer Protein, bcl-2-Associated X Protein
Show Abstract · Added September 12, 2016
Early during apoptosis, the mitochondrial network collapses into short punctuate fragments. The seemingly morphological change, called mitochondrial fragmentation, contributes to mitochondrial injury. Mitochondrial morphology is dictated by two opposing processes, fission and fusion. It is unclear how the fission-fusion balance is tilted during apoptosis, resulting in mitochondrial fragmentation. Emerging evidence has now suggested a regulation of mitochondrial morphological dynamics by Bcl-2 family proteins. In this regulation, Bak appears to be a key. Through interaction with mitofusins, Bak may block mitochondrial fusion to induce fragmentation. By this function, Bak may collaborate with Bax to permeabilize mitochondrial outer membrane, leading to the release of apoptogenic factors.
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8 MeSH Terms
Naked2 acts as a cargo recognition and targeting protein to ensure proper delivery and fusion of TGF-alpha containing exocytic vesicles at the lower lateral membrane of polarized MDCK cells.
Li C, Hao M, Cao Z, Ding W, Graves-Deal R, Hu J, Piston DW, Coffey RJ
(2007) Mol Biol Cell 18: 3081-93
MeSH Terms: Adaptor Protein Complex 1, Animals, Carrier Proteins, Cell Membrane, Cell Polarity, Cell Survival, Cytosol, Dogs, Exocytosis, Humans, Membrane Fusion, Microscopy, Fluorescence, Protein Structure, Tertiary, Protein Transport, RNA, Small Interfering, Swine, Tannins, Transforming Growth Factor alpha, Transport Vesicles
Show Abstract · Added August 12, 2010
Transforming growth factor-alpha (TGF-alpha) is the major autocrine EGF receptor ligand in vivo. In polarized epithelial cells, proTGF-alpha is synthesized and then delivered to the basolateral cell surface. We previously reported that Naked2 interacts with basolateral sorting determinants in the cytoplasmic tail of a Golgi-processed form of TGF-alpha and that TGF-alpha is not detected at the basolateral surface of Madin-Darby canine kidney (MDCK) cells expressing myristoylation-deficient (G2A) Naked2. By high-resolution microscopy, we now show that wild-type, but not G2A, Naked2-associated vesicles fuse at the plasma membrane. We further demonstrate that Naked2-associated vesicles are delivered to the lower lateral membrane of polarized MDCK cells independent of mu1B adaptin. We identify a basolateral targeting segment within Naked2; residues 1-173 redirect NHERF-1 from the apical cytoplasm to the basolateral membrane, and internal deletion of residues 37-104 results in apical mislocalization of Naked2 and TGF-alpha. Short hairpin RNA knockdown of Naked2 leads to a dramatic reduction in the 16-kDa cell surface isoform of TGF-alpha and increased cytosolic TGF-alpha immunoreactivity. We propose that Naked2 acts as a cargo recognition and targeting (CaRT) protein to ensure proper delivery, tethering, and fusion of TGF-alpha-containing vesicles to a distinct region at the basolateral surface of polarized epithelial cells.
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19 MeSH Terms