Regulated exocytosis of synaptic vesicles is substantially faster than of endocrine dense core vesicles despite similar molecular machineries. The reasons for this difference are unknown and could be ...due to different regulatory proteins, different spatial arrangements, different vesicle sizes, or other factors. To address these questions, we take a reconstitution approach and compare regulated SNARE-mediated fusion of purified synaptic and dense core chromaffin and insulin vesicles using a single vesicle-supported membrane fusion assay. In all cases, Munc18 and complexin are required to restrict fusion in the absence of calcium. Calcium triggers fusion of all docked vesicles. Munc13 (C1C2MUN domain) is required for synaptic and enhanced insulin vesicle fusion, but not for chromaffin vesicles, correlating inversely with the presence of CAPS protein on purified vesicles. Striking disparities in calcium-triggered fusion rates are observed, increasing with curvature with time constants 0.23 s (synaptic vesicles), 3.3 s (chromaffin vesicles), and 9.1 s (insulin vesicles) and correlating with rate differences in cells.
Cholesterol serves critical roles in enveloped virus fusion by modulating membrane properties. The glycoprotein (GP) of Ebola virus (EBOV) promotes fusion in the endosome, a process that requires the ...endosomal cholesterol transporter NPC1. However, the role of cholesterol in EBOV fusion is unclear. Here we show that cholesterol in GP-containing membranes enhances fusion and the membrane-proximal external region and transmembrane (MPER/TM) domain of GP interacts with cholesterol via several glycine residues in the GP2 TM domain, notably G660. Compared to wild-type (WT) counterparts, a G660L mutation caused a more open angle between MPER and TM domains in an MPER/TM construct, higher probability of stalling at hemifusion for GP2 proteoliposomes and lower cell entry of virus-like particles (VLPs). VLPs with depleted cholesterol show reduced cell entry, and VLPs produced under cholesterol-lowering statin conditions show less frequent entry than respective controls. We propose that cholesterol-TM interactions affect structural features of GP2, thereby facilitating fusion and cell entry.
Enzymes that cut proteins inside membranes regulate diverse cellular events, including cell signaling, homeostasis, and host-pathogen interactions. Adaptations that enable catalysis in this ...exceptional environment are poorly understood. We visualized single molecules of multiple rhomboid intramembrane proteases and unrelated proteins in living cells (human and
) and planar lipid bilayers. Notably, only rhomboid proteins were able to diffuse above the Saffman-Delbrück viscosity limit of the membrane. Hydrophobic mismatch with the irregularly shaped rhomboid fold distorted surrounding lipids and propelled rhomboid diffusion. The rate of substrate processing in living cells scaled with rhomboid diffusivity. Thus, intramembrane proteolysis is naturally diffusion-limited, but cells mitigate this constraint by using the rhomboid fold to overcome the "speed limit" of membrane diffusion.
Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an IFN-stimulated gene screen ...against vesicular stomatitis virus (VSV)-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of SARS-CoV-2 replication. Internalized 25HC accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion via blockade of cholesterol export. Our results highlight one of the possible antiviral mechanisms of 25HC and provide the molecular basis for its therapeutic development.
The role of cholesterol in membrane fusion Yang, Sung-Tae; Kreutzberger, Alex J.B.; Lee, Jinwoo ...
Chemistry and physics of lipids,
09/2016, Letnik:
199
Journal Article
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•Cholesterol is essential for fusion of secretory vesicles and some enveloped viruses with cell membranes.•Cholesterol alters the distribution of SNARE and viral fusion proteins in ...membranes.•Cholesterol changes the penetration of fusion peptides in membranes.•Cholesterol alters the intrinsic membrane curvature and bending in membrane fusion.•Cholesterol alters the lifetime of hemifusion intermediates in membrane fusion.
Cholesterol modulates the bilayer structure of biological membranes in multiple ways. It changes the fluidity, thickness, compressibility, water penetration and intrinsic curvature of lipid bilayers. In multi-component lipid mixtures, cholesterol induces phase separations, partitions selectively between different coexisting lipid phases, and causes integral membrane proteins to respond by changing conformation or redistribution in the membrane. But, which of these often overlapping properties are important for membrane fusion?—Here we review a range of recent experiments that elucidate the multiple roles that cholesterol plays in SNARE-mediated and viral envelope glycoprotein-mediated membrane fusion.
Repurposing FDA-approved inhibitors able to prevent infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could provide a rapid path to establish new therapeutic options to ...mitigate the effects of coronavirus disease 2019 (COVID-19). Proteolytic cleavages of the spike (S) protein of SARS-CoV-2, mediated by the host cell proteases cathepsin and TMPRSS2, alone or in combination, are key early activation steps required for efficient infection. The PIKfyve kinase inhibitor apilimod interferes with late endosomal viral traffic and through an ill-defined mechanism prevents
infection through late endosomes mediated by cathepsin. Similarly, inhibition of TMPRSS2 protease activity by camostat mesylate or nafamostat mesylate prevents infection mediated by the TMPRSS2-dependent and cathepsin-independent pathway. Here, we combined the use of apilimod with camostat mesylate or nafamostat mesylate and found an unexpected ∼5- to 10-fold increase in their effectiveness to prevent SARS-CoV-2 infection in different cell types. Comparable synergism was observed using both a chimeric vesicular stomatitis virus (VSV) containing S of SARS-CoV-2 (VSV-SARS-CoV-2) and SARS-CoV-2. The substantial ∼5-fold or higher decrease of the half-maximal effective concentrations (EC
s) suggests a plausible treatment strategy based on the combined use of these inhibitors.
Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the coronavirus disease 2019 (COVID-2019) global pandemic. There are ongoing efforts to uncover effective antiviral agents that could mitigate the severity of the disease by controlling the ensuing viral replication. Promising candidates include small molecules that inhibit the enzymatic activities of host proteins, thus preventing SARS-CoV-2 entry and infection. They include apilimod, an inhibitor of PIKfyve kinase, and camostat mesylate and nafamostat mesylate, inhibitors of TMPRSS2 protease. Our research is significant for having uncovered an unexpected synergism in the effective inhibitory activity of apilimod used together with camostat mesylate or nafamostat mesylate.
Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca
sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of ...synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca
binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place. Other regions of C2B influence the fusion probability and kinetics but do not control the expansion of the fusion pore. These data indicate that the C2B domain has at least two distinct molecular roles in the fusion event, and the data are consistent with a model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore.
Variable cooperativity in SNARE-mediated membrane fusion Hernandez, Javier M.; Kreutzberger, Alex J. B.; Kiessling, Volker ...
Proceedings of the National Academy of Sciences - PNAS,
08/2014, Letnik:
111, Številka:
33
Journal Article
Recenzirano
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The soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex drives the majority of intracellular and exocytic membrane fusion events. Whether and how SNAREs cooperate to ...mediate fusion has been a subject of intense study, with estimates ranging from a single SNARE complex to 15. Here we show that there is no universally conserved number of SNARE complexes involved as revealed by our observation that this varies greatly depending on membrane curvature. When docking rates of small (∼40 nm) and large (∼100 nm) liposomes reconstituted with different synaptobrevin (the SNARE present in synaptic vesicles) densities are taken into account, the lipid mixing efficiency was maximal with small liposomes with only one synaptobrevin, whereas 23–30 synaptobrevins were necessary for efficient lipid mixing in large liposomes. Our results can be rationalized in terms of strong and weak cooperative coupling of SNARE complex assembly where each mode implicates different intermediate states of fusion that have been recently identified by electron microscopy. We predict that even higher variability in cooperativity is present in different physiological scenarios of fusion, and we further hypothesize that plasticity of SNAREs to engage in different coupling modes is an important feature of the biologically ubiquitous SNARE-mediated fusion reactions.
Ca
-dependent secretion is a process by which important signaling molecules that are produced within a cell-including proteins and neurotransmitters-are expelled to the extracellular environment. The ...cellular mechanism that underlies secretion is referred to as exocytosis. Many years of work have revealed that exocytosis in neurons and neuroendocrine cells is tightly coupled to Ca
and orchestrated by a series of protein-protein/protein-lipid interactions. Here, we highlight landmark discoveries that have informed our current understanding of the process. We focus principally on reductionist studies performed using powerful model secretory systems and cell-free reconstitution assays. In recent years, molecular cloning and genetics have implicated the involvement of a sizeable number of proteins in exocytosis. We expect reductionist approaches will be central to attempts to resolve their roles. The
will continue to be an outlet for much of this work, befitting its tradition of publishing strongly mechanistic, basic research.
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