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Membrane Fusion (membrane + fusion)
Selected AbstractsPhotoinactivation of Sindbis Virus Infectivity Without Inhibition of Membrane FusionPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2009Wor Thongthai Photoinactivation of enveloped viruses is commonly associated with damage to fusion proteins and inhibition of membrane fusion capacity. Here we show that photobleaching of Sindbis virus labeled with the membrane localized dye, R18 (octadecyl rhodamine B) causes a dramatic loss of infectivity without observable changes in low-pH triggered membrane fusion to liposomes. Sindbis labeled with DiI (1,1,-dioctadecyl-3,3,3,,3,-tetramethylindocarbocyanine perchlorate) also maintains low-pH triggered membrane fusion capacity, but in contrast to R18, extensive photobleaching of DiI-labeled virus has little effect on infectivity. Electrophoretic gel analysis suggests no cross-linking of viral fusion proteins following photobleaching of dye-labeled Sindbis. These observations have implications for live-cell, single particle tracking studies of dye-labeled Sindbis virus. Our observations suggest that R18 and DiI have different propensities for spontaneous flip-flop in lipid bilayers. [source] Timing of early amniocentesis as a function of membrane fusionJOURNAL OF CLINICAL ULTRASOUND, Issue 1 2004Michael G. Pinette MD Abstract Purpose The purpose of this prospective study was to evaluate sonographically the timing of membrane fusion and to determine its possible effect on the timing of amniocentesis. Methods Between May 18, 1998, and January 31, 2002, the status of amnion fusion in pregnant patients at 9,15 weeks' menstrual age was identified in women who were to undergo obstetric sonography. Amniocentesis was performed if even a small area of fused membranes that could be traversed was identified; if the membranes were completely unfused, amniocentesis was delayed. The effect of membrane fusion in terms of the need to reschedule amniocentesis was evaluated. Results We examined a total of 594 patients. Membrane fusion occurred progressively with increasing menstrual age. One hundred six early amniocenteses were scheduled, and 70 were performed; the others were delayed because the membranes were unfused. Our requirement that an area of membrane fusion be found before we would perform amniocentesis resulted in rescheduling the procedure 24,38% of the time. Conclusions Membrane fusion, as seen sonographically, is a function of menstrual age. Even by 15 weeks, a portion of the amnion may be unfused with the chorion. Amniocenteses scheduled for early in the pregnancy may need to be delayed until later, when the membranes are at least partially fused, allowing safe passage of a needle. Delaying the procedure may incur higher expense but may be important in terms of lessening the risk involved. © 2003 Wiley Periodicals, Inc. J Clin Ultrasound 32:8,11, 2004 [source] The application of perfluorooctanoate to investigate trimerization of the human immunodeficiency virus-1 gp41 ectodomain by electrophoresisELECTROPHORESIS, Issue 15 2008Chi-Hui Lin Abstract The transmembrane glycoprotein gp41 of human immunodeficiency virus has been proposed to form trimer-of-hairpin during virus-cell membrane fusion. To investigate its oligomerization propensity under soluble and membrane-mimic conditions, sodium salt of perfluorooctanoate (PFO) was applied. A recombinant gp41 ectodomain devoid of disulfide linkage was overexpressed in Escherichia coli and characterized by MS and circular dichroism spectropolarimetry in PFO solution in comparison to SDS. The helical content of this ectodomain in PFO is higher than that in SDS. Notably, PFO employed in PAGE clearly conduced to the formation of trimer under the optimized condition as visualized in the gel. In addition, the proteins expressed from the two mutants in the heptad repeat (HR) domains of gp41, I62P, and N126K, were also examined by the PFO-PAGE analysis for functional ramification of molecular organization. Remarkably, the I62P mutation completely abolished the gp41 trimer formation, whereas the N126K mutation resulted in a more stable trimer. The data suggested that PFO-PAGE analysis is appropriate for evaluating the effect of mutations on the trimerization of gp41 and other fusion proteins which may be implicated in the alteration of their fusogenicity. [source] Crystal structures of Nipah and Hendra virus fusion core proteinsFEBS JOURNAL, Issue 19 2006Zhiyong Lou The Nipah and Hendra viruses are highly pathogenic paramyxoviruses that recently emerged from flying foxes to cause serious disease outbreaks in humans and livestock in Australia, Malaysia, Singapore and Bangladesh. Their unique genetic constitution, high virulence and wide host range set them apart from other paramyxoviruses. These characteristics have led to their classification into the new genus Henpavirus within the family Paramyxoviridae and to their designation as Biosafety Level 4 pathogens. The fusion protein, an enveloped glycoprotein essential for viral entry, belongs to the family of class I fusion proteins and is characterized by the presence of two heptad repeat (HR) regions, HR1 and HR2. These two regions associate to form a fusion-active hairpin conformation that juxtaposes the viral and cellular membranes to facilitate membrane fusion and enable subsequent viral entry. The Hendra and Nipah virus fusion core proteins were crystallized and their structures determined to 2.2 Å resolution. The Nipah and Hendra fusion core structures are six-helix bundles with three HR2 helices packed against the hydrophobic grooves on the surface of a central coiled coil formed by three parallel HR1 helices in an oblique antiparallel manner. Because of the high level of conservation in core regions, it is proposed that the Nipah and Hendra virus fusion cores can provide a model for membrane fusion in all paramyxoviruses. The relatively deep grooves on the surface of the central coiled coil represent a good target site for drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation. [source] Altering the surface properties of baculovirus Autographa californica NPV by insertional mutagenesis of the envelope protein gp64FEBS JOURNAL, Issue 18 2002Alexandra Spenger The envelope protein gp64 of the baculovirus Autographa californica nuclear polyhedrosis virus is essential for viral entry into insect cells, as the glycoprotein both mediates pH-dependent membrane fusion and binds to host cell receptors. Surface modification of baculovirus particles by genetic engineering of gp64 has been demonstrated by various strategies and thus has become an important and powerful tool in molecular biology. To improve further the presentation of peptides on the surface of baculovirus particles, several insertion sites within the gp64 envelope protein were selected by their theoretical maximum surface probability and investigated for efficient peptide presentation. The ELDKWA peptide of the gp41 of HIV-1, specific for the human mAb 2F5, was inserted into 17 different positions of the glycoprotein gp64. Propagation of viruses was successful in 13 cases, mutagenesis at four positions did not result in production of intact virus particles. Western blotting, FACS analysis and ELISA were used for characterization of the different binding properties of the mutants. Insertion of this peptide into the native envelope protein resulted in high avidity display on the surface of baculovirus particles. This approach offers the possibility of effective modification of surface properties in regard to host range specificity and antigen display. [source] AAA+ superfamily ATPases: common structure,diverse functionGENES TO CELLS, Issue 7 2001Teru Ogura The AAA+ superfamily of ATPases, which contain a homologous ATPase module, are found in all kingdoms of living organisms where they participate in diverse cellular processes including membrane fusion, proteolysis and DNA replication. Recent structural studies have revealed that they usually form ring-shaped oligomers, which are crucial for their ATPase activities and mechanisms of action. These ring-shaped oligomeric complexes are versatile in their mode of action, which collectively seem to involve some form of disruption of molecular or macromolecular structure; unfolding of proteins, disassembly of protein complexes, unwinding of DNA, or alteration of the state of DNA,protein complexes. Thus, the AAA+ proteins represent a novel type of molecular chaperone. Comparative analyses have also revealed significant similarities and differences in structure and molecular mechanism between AAA+ ATPases and other ring-shaped ATPases. [source] Topology of the Mitochondrial Inner Membrane: Dynamics and Bioenergetic ImplicationsIUBMB LIFE, Issue 3-5 2001Carmen A. Mannella Abstract Electron tomography indicates that the mitochondrial inner membrane is not normally comprised of baffle-like folds as depicted in textbooks. In actuality, this membrane is pleomorphic, with narrow tubular regions connecting the internal compartments (cristae) to each other and to the membrane periphery. The membrane topologies observed in condensed (matrix contracted) and orthodox (matrix expanded) mitochondria cannot be interconverted by passive folding and unfolding. Instead, transitions between these morphological states likely involve membrane fusion and fission. Formation of tubular junctions in the inner membrane appears to be energetically favored, because they form spontaneously in yeast mitochondria following large-amplitude swelling and recontraction. However, aberrant, unattached, vesicular cristae are also observed in these mitochondria, suggesting that formation of cristae junctions depends on factors (such as the distribution of key proteins and/or lipids) that are disrupted during extreme swelling. Computer modeling studies using the "Virtual Cell" program suggest that the shape of the inner membrane can influence mitochondrial function. Simulations indicate that narrow cristae junctions restrict diffusion between intracristal and external compartments, causing depletion of ADP and decreased ATP output inside the cristae. [source] Discovery of the Porosome: revealing the molecular mechanism of secretion and membrane fusion in cellsJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 1 2004B. P. Jena Abstract Secretion and membrane fusion are fundamental cellular processes involved in the physiology of health and disease. Studies within the past decade reveal the molecular mechanism of secretion and membrane fusion in cells. Studies reveal that membrane-bound secretory vesicles dock and fuse at porosomes, which are specialized plasma membrane structures. Swelling of secretory vesicles result in a build-up of intravesicular pressure, which allows expulsion of vesicular contents. The discovery of the porosome, its isolation, its structure and dynamics at nm resolution and in real time, its biochemical composition and functional reconstitution, are discussed. The molecular mechanism of secretory vesicle fusion at the base of porosomes, and vesicle swelling, have been resolved. With these findings a new understanding of cell secretion has emerged and confirmed by a number of laboratories. [source] Syntaxin 16: Unraveling cellular physiology through a ubiquitous SNARE moleculeJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2010Yanan Chen Syntaxin 16 (Syx16) is member of the soluble N -ethylmaleimide sensitive factor attachment protein receptor (SNARE) family of molecules that functions in membrane fusion in eukaryotic cells. A rather ubiquitously expressed, tail-anchored membrane protein localized mainly at the trans-Golgi network (TGN), it mediates primarily retrograde endosomal-TGN transport. In spite of its ubiquitous expression, Syx16 has specific and interesting roles in the physiology of specialized cells, including Glut4 dynamics, dendritic outgrowth-related membrane traffic, and cytokinesis. We discussed these physiological functions of Syx16 in the light of what is known of its subcellular localization, vesicular trafficking pathways involved, cognate SNARE partners and other interacting proteins. Further, we speculate on some possible pathophysiological roles of Syx16. J. Cell. Physiol. 225: 326,332, 2010. © 2010 Wiley-Liss, Inc. [source] Acrosomal exocytosis of mouse sperm progresses in a consistent direction in response to zona pellucidaJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2009Mariano G. Buffone Sperm acrosomal exocytosis is essential for successful fertilization, and the zona pellucida (ZP) has been classically considered as the primary initiator in vivo. At present, following what is referred to as primary binding of the sperm to the ZP, the acrosome reaction paradigm posits that the outer acrosomal membrane and plasma membrane fuse at random points, releasing the contents of the acrosome. It is then assumed that the inner acrosomal membrane mediates secondary binding of the sperm to the ZP. In the present work we used a live fluorescence imaging system and mouse sperm containing enhanced green fluorescent protein (EGFP) in their acrosomes. We compared the processes of acrosomal exocytosis stimulated by the calcium ionophore ionomycin or by solubilized ZP. As monitored by the loss of EGFP from the sperm, acrosomal exocytosis driven by these two agents occurred differently. When ionomycin was used, exocytosis started randomly (no preference for the anterior, middle or posterior acrosomal regions). In contrast, following treatment with solubilized ZP, the loss of acrosomal components always started at the posterior zone of the acrosome and progressed in an anterograde direction. The exocytosis was slower when stimulated with ZP and on the order of 10 sec, which is in accordance with other reports. These results demonstrate that ZP stimulates acrosomal exocytosis in an orderly manner and suggest that a receptor-mediated event controls this process of membrane fusion and release of acrosomal components. These findings are incorporated into a model. J. Cell. Physiol. 220: 611,620, 2009. © 2009 Wiley-Liss, Inc. [source] Timing of early amniocentesis as a function of membrane fusionJOURNAL OF CLINICAL ULTRASOUND, Issue 1 2004Michael G. Pinette MD Abstract Purpose The purpose of this prospective study was to evaluate sonographically the timing of membrane fusion and to determine its possible effect on the timing of amniocentesis. Methods Between May 18, 1998, and January 31, 2002, the status of amnion fusion in pregnant patients at 9,15 weeks' menstrual age was identified in women who were to undergo obstetric sonography. Amniocentesis was performed if even a small area of fused membranes that could be traversed was identified; if the membranes were completely unfused, amniocentesis was delayed. The effect of membrane fusion in terms of the need to reschedule amniocentesis was evaluated. Results We examined a total of 594 patients. Membrane fusion occurred progressively with increasing menstrual age. One hundred six early amniocenteses were scheduled, and 70 were performed; the others were delayed because the membranes were unfused. Our requirement that an area of membrane fusion be found before we would perform amniocentesis resulted in rescheduling the procedure 24,38% of the time. Conclusions Membrane fusion, as seen sonographically, is a function of menstrual age. Even by 15 weeks, a portion of the amnion may be unfused with the chorion. Amniocenteses scheduled for early in the pregnancy may need to be delayed until later, when the membranes are at least partially fused, allowing safe passage of a needle. Delaying the procedure may incur higher expense but may be important in terms of lessening the risk involved. © 2003 Wiley Periodicals, Inc. J Clin Ultrasound 32:8,11, 2004 [source] Complex molecular assemblies at hand via interactive simulationsJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 15 2009Olivier Delalande Abstract Studying complex molecular assemblies interactively is becoming an increasingly appealing approach to molecular modeling. Here we focus on interactive molecular dynamics (IMD) as a textbook example for interactive simulation methods. Such simulations can be useful in exploring and generating hypotheses about the structural and mechanical aspects of biomolecular interactions. For the first time, we carry out low-resolution coarse-grain IMD simulations. Such simplified modeling methods currently appear to be more suitable for interactive experiments and represent a well-balanced compromise between an important gain in computational speed versus a moderate loss in modeling accuracy compared to higher resolution all-atom simulations. This is particularly useful for initial exploration and hypothesis development for rare molecular interaction events. We evaluate which applications are currently feasible using molecular assemblies from 1900 to over 300,000 particles. Three biochemical systems are discussed: the guanylate kinase (GK) enzyme, the outer membrane protease T and the soluble N -ethylmaleimide-sensitive factor attachment protein receptors complex involved in membrane fusion. We induce large conformational changes, carry out interactive docking experiments, probe lipid,protein interactions and are able to sense the mechanical properties of a molecular model. Furthermore, such interactive simulations facilitate exploration of modeling parameters for method improvement. For the purpose of these simulations, we have developed a freely available software library called MDDriver. It uses the IMD protocol from NAMD and facilitates the implementation and application of interactive simulations. With MDDriver it becomes very easy to render any particle-based molecular simulation engine interactive. Here we use its implementation in the Gromacs software as an example. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2009 [source] Comparing the antibody responses against recombinant hemagglutinin proteins of avian influenza A (H5N1) virus expressed in insect cells and bacteria,JOURNAL OF MEDICAL VIROLOGY, Issue 11 2008Shuo Shen Abstract The hemagglutinin (HA) of influenza A virus plays an essential role in mediating the entry of the virus into host cells. Here, recombinant full-length HA5 protein from a H5N1 isolate (A/chicken/hatay/2004(H5N1)) was expressed and purified from the baculovirus-insect cell system. As expected, full-length HA5 elicits strong neutralizing antibodies, as evaluated in micro-neutralization tests using HA5 pseudotyped lentiviral particles. In addition, two fragments of HA5 were expressed in bacteria and the N-terminal fragment, covering the ectodomain before the HA1/HA2 polybasic cleavage site, was found to elicit neutralizing antibodies. But the C-terminal fragment, which covers the remaining portion of the ectodomain, did not. Neutralizing titer of the anti-serum against the N-terminal fragment is only four times lower than the anti-serum against the full-length HA5 protein. Using a novel membrane fusion assay, the abilities of these antibodies to block membrane fusion were found to correlate well with the neutralization activities. J. Med. Virol. 80:1972,1983, 2008. © 2008 Wiley-Liss, Inc. [source] Munc18-1 as a key regulator of neurosecretionJOURNAL OF NEUROCHEMISTRY, Issue 1 2010Gayoung A. Han J. Neurochem. (2010) 115, 1,10. Abstract Munc18-1 plays essential roles in neurosecretion by interacting with syntaxin-1 and controlling the formation of the soluble N -ethylmaleimide-sensitive factor attachment protein receptors (SNARE) complex. At least three important functions of Munc18-1 have been proposed: (i) molecular chaperone of syntaxin-1 for appropriate localization and expression of syntaxin-1, (ii) priming/stimulation of the SNARE-mediated membrane fusion, and (iii) docking of large dense-core vesicles to the plasma membrane. Similarly, at least two different binding modes have been proposed for the interaction between Munc18-1 and syntaxin-1: (i) binary binding to a ,closed' conformation of syntaxin-1, and (ii) binding to the N-terminal peptide of syntaxin-1, which is thought to enable an interaction with the quaternary SNARE complex and/or further stabilize the binary interaction between Munc18-1 and closed syntaxin-1. Recent structural analyses have identified critical Munc18-1 residues implicated in these different modes of binding. These have recently been tested functionally in rescue experiments using Munc18-1 null neurons, chromaffin cells and Munc18-1/-2 knockdown PC12 cells, allowing remarkable progress to be made in the structural/functional understanding of Munc18-1. In this review, we summarize these recent advances and attempt to propose an updated model of the pleiotropic functions of Munc18-1 in neuroexocytosis. [source] Emerging functions of the calpain superfamily of cysteine proteases in neuroendocrine secretory pathwaysJOURNAL OF NEUROCHEMISTRY, Issue 3 2007Joanne S. Evans Abstract The first calpain protease was discovered over 40 years ago now, yet despite the vast amount of literature that has subsequently emerged detailing their involvement in the pathophysiology of a variety of human diseases, it is only in the last decade that calpain-mediated actions along the secretory pathway have begun to emerge. However, the number of secretory pathway substrates identified and their diversity of function continues to grow. This review summarizes our current knowledge of calpain-mediated mechanisms of action that are pertinent to synaptic vesicle assembly and budding, cytoskeletal organization, endosomal recycling, and exocytotic membrane fusion. [source] A minor ,-structured conformation is the active state of a fusion peptide of vesicular stomatitis virus glycoprotein,JOURNAL OF PEPTIDE SCIENCE, Issue 4 2008Carolina G. Sarzedas Abstract Entry of enveloped animal viruses into their host cells always depends on a step of membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in the VSV G protein, comprising the residues 145,164, directly involved in membrane interaction and fusion. In the present work we studied the interaction of pep[145,164] with membranes using NMR to solve the structure of the peptide in two membrane-mimetic systems: SDS micelles and liposomes composed of phosphatidylcholine and phosphatidylserine (PC:PS vesicles). The presence of medium-range NOEs showed that the peptide has a tendency to form N - and C -terminal helical segments in the presence of SDS micelles. Analysis of the chemical shift index indicated helix,coil equilibrium for the C -terminal helix under all conditions studied. At pH 7.0, the N -terminal helix also displayed a helix,coil equilibrium when pep[145-164] was free in solution or in the presence of PC:PS. Remarkably, at the fusogenic pH, the region of the N -terminal helix in the presence of SDS or PC:PS presented a third conformational species that was in equilibrium with the helix and random coil. The N -terminal helix content decreases pH and the minor ,-structured conformation becomes more prevalent at the fusogenic pH. These data point to a ,-conformation as the fusogenic active structure-which is in agreement with the X-ray structure, which shows a ,-hairpin for the region corresponding to pep[145-164]. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [source] Botulinum toxins in neurological diseaseMUSCLE AND NERVE, Issue 5 2004Cynthia L. Comella MD Abstract Botulinum toxins are among the most potent neurotoxins known to humans. In the past 25 years, botulinum toxin has emerged as both a potential weapon of bioterrorism and as a powerful therapeutic agent, with growing applications in neurological and non-neurological disease. Botulinum toxin is unique in its ability to target peripheral cholinergic neurons, preventing the release of acetylcholine through the enzymatic cleavage of proteins involved in membrane fusion, without prominent central nervous system effects. There are seven serotypes of the toxin, each with a specific activity at the molecular level. Currently, serotypes A (in two preparations) and B are available for clinical use, and have been shown to be safe and effective for the treatment of dystonia, spasticity, and other disorders in which muscle overactivity gives rise to symptoms. This review focuses on the pharmacology, electrophysiology, immunology, and application of botulinum toxin in selected neurological disorders. Muscle Nerve 29: 628,644, 2004 [source] Photoinactivation of Sindbis Virus Infectivity Without Inhibition of Membrane FusionPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2009Wor Thongthai Photoinactivation of enveloped viruses is commonly associated with damage to fusion proteins and inhibition of membrane fusion capacity. Here we show that photobleaching of Sindbis virus labeled with the membrane localized dye, R18 (octadecyl rhodamine B) causes a dramatic loss of infectivity without observable changes in low-pH triggered membrane fusion to liposomes. Sindbis labeled with DiI (1,1,-dioctadecyl-3,3,3,,3,-tetramethylindocarbocyanine perchlorate) also maintains low-pH triggered membrane fusion capacity, but in contrast to R18, extensive photobleaching of DiI-labeled virus has little effect on infectivity. Electrophoretic gel analysis suggests no cross-linking of viral fusion proteins following photobleaching of dye-labeled Sindbis. These observations have implications for live-cell, single particle tracking studies of dye-labeled Sindbis virus. Our observations suggest that R18 and DiI have different propensities for spontaneous flip-flop in lipid bilayers. [source] Zinc improves gene transfer mediated by DNA/cationic polymer complexesTHE JOURNAL OF GENE MEDICINE, Issue 5 2002Chantal Pichon Abstract Background The weak efficiency of plasmid transfer into the cytosol remains one of the major limiting factors to achieve an efficient transfection with DNA/cationic polymer complexes. We found that divalent metal Zn2+ can improve the polyfection efficiency, especially with DNA/histidylated polylysine (His-pLK) complexes. Methods and results The supplementation of the transfection medium with 250 µM ZnCl2 increased the polyfection of human hepatocarcinoma (HepG2) cells with a plasmid encoding EGFP complexed with pLK, polyethyleneimine and His-pLK. Zn2+ is more efficient on DNA/His-pLK complexes: the number of EGFP-positive cells increased from 1% to more than 40%. This phenomenon is selective to Zn2+ because no effect was obtained with other divalent cations. The effect of zinc varies from cell to cell. The binding of Zn2+ to histidyl residues might increase zinc endosomal concentration favoring membrane fusion. Flow cytometry and confocal microscopy studies clearly indicate that with His-pLK, the plasmid is better delivered in the cytosol as well as in the cell nucleus in zinc-treated cells. An investigation conducted with the histidine-rich peptide H5WYG showed that zinc inhibits membrane permeabilization but promotes membrane fusion as evidenced by resonance energy transfer. Conclusions Data reported here imply that the addition of zinc ions in the transfection medium can trigger an increase of the fusion of endosomes containing polyplexes which is more effective in the presence of histidine-rich molecules. Consequently, the amount of plasmid in the cytosol available to reach the nucleus is increased leading to an improvement of polyfection. Copyright © 2002 John Wiley & Sons, Ltd. [source] Identification of novel proteins in isolated polyphosphate vacuoles in the primitive red alga Cyanidioschyzon merolaeTHE PLANT JOURNAL, Issue 5 2009Fumi Yagisawa Summary Plant vacuoles are organelles bound by a single membrane, and involved in various functions such as intracellular digestion, metabolite storage, and secretion. To understand their evolution and fundamental mechanisms, characterization of vacuoles in primitive plants would be invaluable. Algal cells often contain polyphosphate-rich compartments, which are thought to be the counterparts of seed plant vacuoles. Here, we developed a method for isolating these vacuoles from Cyanidioschyzon merolae, and identified their proteins by MALDI TOF-MS. The vacuoles were of unexpectedly high density, and were highly enriched at the boundary between 62 and 80% w/v iodixanol by density-gradient ultracentrifugation. The vacuole-containing fraction was subjected to SDS,PAGE, and a total of 46 proteins were identified, including six lytic enzymes, 13 transporters, six proteins for membrane fusion or vesicle trafficking, five non-lytic enzymes, 13 proteins of unknown function, and three miscellaneous proteins. Fourteen proteins were homologous to known vacuolar or lysosomal proteins from seed plants, yeasts or mammals, suggesting functional and evolutionary relationships between C. merolae vacuoles and these compartments. The vacuolar localization of four novel proteins, namely CMP249C (metallopeptidase), CMJ260C (prenylated Rab receptor), CMS401C (ABC transporter) and CMT369C (o -methyltransferase), was confirmed by labeling with specific antibodies or transient expression of hemagglutinin-tagged proteins. The results presented here provide insights into the proteome of C. merolae vacuoles and shed light on their functions, as well as indicating new features. [source] Fab crystallization and preliminary X-ray analysis of NC-1, an anti-HIV-1 antibody that recognizes the six-helix bundle core of gp41ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Lei Jin NC-1 is a murine monoclonal antibody that specifically recognizes the six-helix bundle core of the human immunodeficiency virus type 1 (HIV-1) gp41. As such, it is a useful tool for probing gp41 conformations in HIV-1 membrane fusion. To establish the structural basis underlying the NC-1 specificity, X-ray crystallography was employed to solve its three-dimensional structure. To accomplish this, hybridoma-produced NC-1 antibody was first purified and digested with papain. Its Fab fragment was then purified using size-exclusion chromatography following Fc depletion using a Protein A affinity column. Finally, crystallization of NC-1 Fab was performed by the hanging-drop vapour-diffusion method and the protein was crystallized at pH 8.0 using PEG 6000 as precipitant. The results showed that the NC-1 Fab crystals belonged to the trigonal space group P3221, with unit-cell parameters a = b = 118.7, c = 106.0,Å. There is one Fab molecule in the asymmetric unit, with 67.5% solvent content. An X-ray diffraction data set was collected at 3.2,Å resolution and a clear molecular-replacement solution was obtained for solution of the structure. [source] Molecular engineering of exocytic vesicle traffic enhances the productivity of Chinese hamster ovary cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009Ren-Wang Peng Abstract A complex vesicle trafficking system manages the precise and regulated distribution of proteins, membranes and other molecular cargo between cellular compartments as well as the secretion of (heterologous) proteins in mammalian cells. Sec1/Munc18 (SM) proteins are key components of the system by regulating membrane fusion. However, it is not clear how SM proteins contribute to the overall exocytosis. Here, functional analysis of the SM protein Sly1 and Munc18c suggested a united, positive impact upon SNARE-based fusion of ER-to-Golgi- and Golgi-to-plasma membrane-addressed exocytic vesicles and increased the secretory capacity of different therapeutic proteins in Chinese hamster ovary cells up to 40 pg/cell/day. Sly1- and Munc18c-based vesicle traffic engineering cooperated with Xbp-1-mediated ER/Golgi organelle engineering. Our study supports a model for united function of SM proteins in stimulating vesicle trafficking machinery and provides a generic secretion engineering strategy to improve biopharmaceutical manufacturing of important protein therapeutics. Biotechnol. Bioeng. 2009;102: 1170-1181. © 2008 Wiley Periodicals, Inc. [source] Cloning, expression, purification, crystallization and preliminary crystallographic study of the protein module (BIV2-Helix) in the fusion core of bovine immunodeficiency-like virus (BIV) gp40ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2005Xiaodong Zhao The fusion core of bovine immunodeficiency virus (BIV) gp40 is proposed to be involved in membrane fusion. However, no crystal structures are yet available. A predicted protein module BIV2-Helix of BIVgp40 has been expressed in Escherichia coli and purified by chromatography. Recombinant BIV2-Helix was crystallized using the hanging-drop vapour-diffusion technique at 291,K. The crystals were grown in MPD and belonged to the primitive rhombohedral space group R3, with unit-cell parameters a = 39.17, b = 39.17, c = 295.05,Å and two molecules per asymmetric unit. X-ray diffraction data were collected to 1.76,Å in the home laboratory from a single crystal. [source] Cell entry by human pathogenic arenavirusesCELLULAR MICROBIOLOGY, Issue 4 2008Jillian M. Rojek Summary The arenaviruses Lassa virus (LASV) in Africa and Machupo (MACV), Guanarito (GTOV) and Junin viruses (JUNV) in South America cause severe haemorrhagic fevers in humans with fatality rates of 15,35%. The present review focuses on the first steps of infection with human pathogenic arenaviruses, the interaction with their cellular receptor molecules and subsequent entry into the host cell. While similarities exist in genomic organization, structure and clinical disease caused by pathogenic Old World and New World arenaviruses these pathogens use different primary receptors. The Old World arenaviruses employ ,-dystroglycan, a cellular receptor for proteins of the extracellular matrix, and the human pathogenic New World arenaviruses use the cellular cargo receptor transferrin receptor 1. While the New World arenavirus JUNV enters cells via clathrin-dependent endocytosis, evidence occurred for clathrin-independent entry of the prototypic Old World arenavirus lymphocytic choriomeningitis virus. Upon internalization, arenaviruses are delivered to the endosome, where pH-dependent membrane fusion is mediated by the envelope glycoprotein (GP). While arenavirus GPs share characteristics with class I fusion GPs of other enveloped viruses, unusual mechanistic features of GP-mediated membrane fusion have recently been discovered for arenaviruses with important implications for viral entry. [source] Cell vacuolization induced by Helicobacter pylori VacA cytotoxin does not depend on late endosomal SNAREs,CELLULAR MICROBIOLOGY, Issue 1 2002M. de Bernard Summary Cellular vacuoles induced by the Helicobacter pylori vacuolating cytotoxin VacA originate from late endosomal compartments. Their biogenesis requires the activity of both rab7 GTPase and the ATPase proton pump. The toxin has been suggested to cause an increased luminal osmotic pressure via its anion-specific channel activity localized on late endosomal compartments after endocytosis. Here, we show that the extensive membrane fusion that takes place in the transition from the small late endosomal compartments to the large vacuoles does not depend on soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) proteins. The process of vacuolization leads to disappearance of the large array of internal membranes of late endosomes. We suggest that most of the vacuole-limiting membrane derives from internal membranes. [source] | ||||||||||||||||