scholarly journals The role of the C terminus of the SNARE protein SNAP-25 in fusion pore opening and a model for fusion pore mechanics

2008 ◽  
Vol 105 (40) ◽  
pp. 15388-15392 ◽  
Author(s):  
Qinghua Fang ◽  
Khajak Berberian ◽  
Liang-Wei Gong ◽  
Ismail Hafez ◽  
Jakob B. Sørensen ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Mechanistic Model ◽  
Snare Complex ◽  
Fusion Pore ◽  
Snare Protein ◽  
C Terminus ◽  
Target Membrane ◽  
Pore Opening ◽  
Fusion Pores

Formation of a fusion pore between a vesicle and its target membrane is thought to involve the so-called SNARE protein complex. However, there is no mechanistic model explaining how the fusion pore is opened by conformational changes in the SNARE complex. It has been suggested that C-terminal zipping triggers fusion pore opening. A SNAP-25 mutant named SNAP-25Δ9 (lacking the last nine C-terminal residues) should lead to a less-tight C-terminal zipping. Single exocytotic events in chromaffin cells expressing this mutant were characterized by carbon fiber amperometry and cell-attached patch capacitance measurements. Cells expressing SNAP-25Δ9 displayed smaller amperometric “foot-current” currents, reduced fusion pore conductances, and lower fusion pore expansion rates. We propose that SNARE/lipid complexes form proteolipid fusion pores. Fusion pores involving the SNAP-25Δ9 mutant will be less tightly zipped and may lead to a longer fusion pore structure, consistent with the observed decrease of fusion pore conductance.

scholarly journals The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

2019 ◽  
Author(s):  
Zhenyong Wu ◽  
Nadiv Dharan ◽  
Sathish Thiyagarajan ◽  
Ben O’Shaughnessy ◽  
Erdem Karatekin
Keyword(s):  
Membrane Fusion ◽  
Snare Complex ◽  
Snare Proteins ◽  
Fusion Pore ◽  
Calcium Sensor ◽  
Fusion Reactions ◽  
Synaptotagmin 1 ◽  
Pore Opening ◽  
Pore Dilation ◽  
Fusion Pores

ABSTRACTAll membrane fusion reactions proceed through an initial fusion pore, including calcium-triggered vesicular release of neurotransmitters and hormones. Expansion of this small pore to release cargo molecules is energetically costly and regulated by cells, but the mechanisms are poorly understood. Here we show that the neuronal/exocytic calcium sensor Synaptotagmin-1 (Syt1) promotes expansion of fusion pores induced by SNARE proteins, beyond its established role in coupling calcium influx to fusion pore opening. Our results suggest that fusion pore dilation by Syt1 requires interactions with SNAREs, PI(4,5)P2, and calcium. Pore opening was abolished by a mutation of the tandem C2 domain (C2AB) hydrophobic loops of Syt1, suggesting that their calcium-induced insertion into the membrane is required for pore opening. We propose that loop insertion is also required for pore expansion, but through a distinct mechanism. Mathematical modelling suggests that membrane insertion re-orients the C2 domains bound to the SNARE complex, rotating the SNARE complex so as to exert force on the membranes in a mechanical lever action that increases the intermembrane distance. The increased membrane separation provokes pore dilation to offset a bending energy penalty. We conclude that Syt1 assumes a critical role in calcium-dependent fusion pore dilation during neurotransmitter and hormone release.SIGNIFICANCE STATEMENTMembrane fusion is a fundamental biological process, required for development, infection by enveloped viruses, fertilization, intracellular trafficking, and calcium-triggered release of neurotransmitters and hormones when cargo-laden vesicles fuse with the plasma membrane. All membrane fusion reactions proceed through an initial, nanometer-sized fusion pore which can flicker open-closed multiple times before expanding or resealing. Pore expansion is required for efficient cargo release, but underlying mechanisms are poorly understood. Using a combination of single-pore measurements and quantitative modeling, we suggest that a complex between the neuronal calcium sensor Synaptotagmin-1 and the SNARE proteins together act as a calcium-sensitive mechanical lever to force the membranes apart and enlarge the pore.

scholarly journals Structural transitions in the synaptic SNARE complex during Ca2+-triggered exocytosis

2006 ◽  
Vol 172 (2) ◽  
pp. 281-293 ◽  
Author(s):  
Xue Han ◽  
Meyer B. Jackson
Keyword(s):  
Plasma Membranes ◽  
Structural Transition ◽  
Snare Complex ◽  
Fusion Pore ◽  
Hydrophobic Core ◽  
Structural Transitions ◽  
Triggered Release ◽  
Rate Processes ◽  
Pore Opening ◽  
Fusion Pores

The synaptic SNARE complex is a highly stable four-helix bundle that links the vesicle and plasma membranes and plays an essential role in the Ca2+-triggered release of neurotransmitters and hormones. An understanding has yet to be achieved of how this complex assembles and undergoes structural transitions during exocytosis. To investigate this question, we have mutated residues within the hydrophobic core of the SNARE complex along the entire length of all four chains and examined the consequences using amperometry to measure fusion pore opening and dilation. Mutations throughout the SNARE complex reduced two distinct rate processes before fusion pore opening to different degrees. These results suggest that two distinct, fully assembled conformations of the SNARE complex drive transitions leading to open fusion pores. In contrast, a smaller number of mutations that were scattered through the SNARE complex but were somewhat concentrated in the membrane-distal half stabilized open fusion pores. These results suggest that a structural transition within a partially disassembled complex drives the dilation of open fusion pores. The dependence of these three rate processes on position within the SNARE complex does not support vectorial SNARE complex zipping during exocytosis.

scholarly journals Nascent fusion pore opening monitored at single-SNAREpin resolution

2021 ◽  
Vol 118 (5) ◽  
pp. e2024922118
Author(s):  
Paul Heo ◽  
Jeff Coleman ◽  
Jean-Baptiste Fleury ◽  
James E. Rothman ◽  
Frederic Pincet
Keyword(s):  
Energy Landscape ◽  
Three Dimensional ◽  
Fusion Pore ◽  
Fusion Event ◽  
Direct Estimate ◽  
Target Membrane ◽  
Pore Opening ◽  
Two Peaks ◽  
Fusion Pores ◽  
Second Peak

Vesicle fusion with a target membrane is a key event in cellular trafficking and ensures cargo transport within the cell and between cells. The formation of a protein complex, called SNAREpin, provides the energy necessary for the fusion process. In a three-dimensional microfluidic chip, we monitored the fusion of small vesicles with a suspended asymmetric lipid bilayer. Adding ion channels into the vesicles, our setup allows the observation of a single fusion event by electrophysiology with 10-μs precision. Intriguingly, we identified that small transient fusion pores of discrete sizes reversibly opened with a characteristic lifetime of ∼350 ms. The distribution of their apparent diameters displayed two peaks, at 0.4 ± 0.1 nm and 0.8 ± 0.2 nm. Varying the number of SNAREpins, we demonstrated that the first peak corresponds to fusion pores induced by a single SNAREpin and the second peak is associated with pores involving two SNAREpins acting simultaneously. The pore size fluctuations provide a direct estimate of the energy landscape of the pore. By extrapolation, the energy landscape for three SNAREpins does not exhibit any thermally significant energy barrier, showing that pores larger than 1.5 nm are spontaneously produced by three or more SNAREpins acting simultaneously, and expand indefinitely. Our results quantitatively explain why one SNAREpin is sufficient to open a fusion pore and more than three SNAREpins are required for cargo release. Finally, they also explain why a machinery that synchronizes three SNAREpins, or more, is mandatory to ensure fast neurotransmitter release during synaptic transmission.

scholarly journals Membrane Binding of α-Synuclein Stimulates Expansion of SNARE-Dependent Fusion Pore

2021 ◽  
Vol 9 ◽  
Author(s):  
Ryan Khounlo ◽  
Brenden J. D. Hawk ◽  
Tung-Mei Khu ◽  
Gyeongji Yoo ◽  
Nam Ki Lee ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Membrane Binding ◽  
Snare Complex ◽  
Fusion Pore ◽  
Fusion Inhibitors ◽  
Important Regulator ◽  
Fusion Pores ◽  
Single Vesicle ◽  
Pore Expansion

SNARE-dependent membrane fusion is essential for neurotransmitter release at the synapse. Recently, α-synuclein has emerged as an important regulator for membrane fusion. Misfolded α-synuclein oligomers are potent fusion inhibitors. However, the function of normal α-synuclein has been elusive. Here, we use the single vesicle-to-supported bilayer fusion assay to dissect the role of α-synuclein in membrane fusion. The assay employs 10 kD Rhodamine B-dextran as the content probe that can detect fusion pores larger than ∼6 nm. We find that the SNARE complex alone is inefficient at dilating fusion pores. However, α-synuclein dramatically increases the probability as well as the duration of large pores. When the SNARE-interacting C-terminal region of α-synuclein was truncated, the mutant behaves the same as the wild-type. However, the double proline mutants compromising membrane-binding show significantly reduced effects on fusion pore expansion. Thus, our results suggest that α-synuclein stimulates fusion pore expansion specifically through its membrane binding.

scholarly journals Conformational Landscape Reduction of a Dynamic 29-Residue Peptide by a Perfluoroaromatic Small Molecule

2020 ◽  
Author(s):  
ethan evans
Keyword(s):  
Small Molecule ◽  
Conformational Changes ◽  
Dynamic Systems ◽  
Biochemical Properties ◽  
State Dependent ◽  
C Terminus ◽  
Explicit Solvent ◽  
Conformational Landscape

Conformationally dynamic peptides and proteins display both important biochemical properties and present a challenge for computational modeling. Characterizing the accessible structural landscape represents one route to understand their function with molecular level detail. We characterize a self-labeling 29-residue peptide, MP01-Gen4, that undergoes structural alterations in the presence of a perfluoroaromatic reaction partner. Replica exchange molecular dynamics (REMD) shows MP01 to access a broad set of states, that microsecond-long explicit solvent simulations only minimally sample. REMD and structural network analysis find an altered and reduced conformational landscape when MP01 interacts non-covalently or is covalently attached to the perfluoroaromatic small molecule. Residues throughout the peptide, notably at the C-terminus, interact with the small molecule in conformational state-dependent manners. The results help explain and generate hypotheses for experimental observations including the importance of flexibility and the role of the N- and C-terminal regions, both of which are distant from the active cysteine. The simulations highlight the importance of substantial sampling in minimally stabilized, conformationally dynamic systems and supplies a case study for small molecule-mediated, peptide conformational changes.<br>

scholarly journals Epigenetic modification in the expression of p73 p73 - epigenetic target for anticancer therapy

2019 ◽  
Vol 13 (2) ◽  
Author(s):  
Faiza Naseer ◽  
Mohammad Saleem
Keyword(s):  
Conformational Changes ◽  
Posttranslational Modifications ◽  
Epigenetic Modification ◽  
Amino Acid Residues ◽  
P53 Family ◽  
After Effects ◽  
Cycle Arrest ◽  
C Terminus ◽  
Upstream Promoter

A p73 is a new member of p53 family of transcription factor, having two types. First is TAp73, transcriptionally active and expressed via upstream promoter as a tumor suppressor and vital apoptotic inductor, it also has a key role in cell cycle arrest/differentiation and Second is ΔNp73 that is transcriptionally inactive and expressed via downstream regulator as oncogenes. Both types are expressed in various isoforms, which originate from alternative splicing events at the C-terminus. Upon DNA damage, posttranslational modifications cause conformational changes in various amino acid residues via induction or inhibition of various proteins, which are present in the structural domains of p73. These modifications may cause up- or down-regulation of p73 expression levels, as well as alters the transcriptional activity and/or stability of the protein. In this review, we have made an effort to assemble all existing data regarding the role of p73, its modification and after effects in cancer.

scholarly journals Role of C-Terminal Cysteine Residues of Aspergillus fumigatus Allergen Asp f 4 in Immunoglobulin E Binding

2004 ◽  
Vol 11 (2) ◽  
pp. 261-265 ◽  
Author(s):  
Harikrishnan Ramachandran ◽  
Banani Banerjee ◽  
Paul A. Greenberger ◽  
Kevin J. Kelly ◽  
Jordan N. Fink ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Conformational Changes ◽  
Immunoglobulin E ◽  
Allergic Bronchopulmonary Aspergillosis ◽  
Allergic Diseases ◽  
Cysteine Residues ◽  
Conformational Structure ◽  
C Terminus ◽  
Function Relationship

ABSTRACT Among the several allergens cloned and expressed from Aspergillus fumigatus, Asp f 4 is a major one associated with allergic bronchopulmonary aspergillosis (ABPA). The structure-function relationship of allergens is important in understanding the immunopathogenesis, diagnosis, and treatment of allergic diseases. These include the epitopes, conformational or linear, deletion of the N or C terminus or both N and C termini, and glycosylation or nonglycosylation, all of which affect immune responses. Similarly, the role of cysteine residues present in allergens may yield useful information regarding the conformational structure of allergens and the immunoglobulin E (IgE) epitope interaction. Such information may help in developing new strategies towards immunotherapy. In order to define the role of cysteine in the interaction of the antibody with Asp f 4, we have constructed mutants by selectively deleting cysteine residues from the C-terminal region of the Asp f 4. Immunological evaluation of these engineered recombinant constructs was conducted by using sera from patients with ABPA, Aspergillus skin test-positive asthmatics, and healthy controls. The results demonstrate strong IgE binding with Asp f 4 and two truncated mutants, Asp f 41-234 (amino acids [aa] 1 to 234) and Asp f 41-241 (aa 1 to 241), while another mutant, Asp f 41-196 (aa 1 to 196), showed reactivity with fewer patients. The result suggests that deletion of cysteines and the alteration of IgE epitopes at the C-terminal end resulted in conformational changes, which may have a potential role in the immunomodulation of the disease.

scholarly journals Phosphorylation and O-GlcNAcylation of the PHF-1 Epitope of Tau Protein Induce Local Conformational Changes of the C-Terminus and Modulate Tau Self-Assembly Into Fibrillar Aggregates

2021 ◽  
Vol 14 ◽  
Author(s):  
François-Xavier Cantrelle ◽  
Anne Loyens ◽  
Xavier Trivelli ◽  
Oliver Reimann ◽  
Clément Despres ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Tau Protein ◽  
Posttranslational Modifications ◽  
Self Assembly ◽  
Critical Role ◽  
Small Peptides ◽  
C Terminus ◽  
The Impact

Phosphorylation of the neuronal microtubule-associated Tau protein plays a critical role in the aggregation process leading to the formation of insoluble intraneuronal fibrils within Alzheimer’s disease (AD) brains. In recent years, other posttranslational modifications (PTMs) have been highlighted in the regulation of Tau (dys)functions. Among these PTMs, the O-β-linked N-acetylglucosaminylation (O-GlcNAcylation) modulates Tau phosphorylation and aggregation. We here focus on the role of the PHF-1 phospho-epitope of Tau C-terminal domain that is hyperphosphorylated in AD (at pS396/pS404) and encompasses S400 as the major O-GlcNAc site of Tau while two additional O-GlcNAc sites were found in the extreme C-terminus at S412 and S413. Using high resolution NMR spectroscopy, we showed that the O-GlcNAc glycosylation reduces phosphorylation of PHF-1 epitope by GSK3β alone or after priming by CDK2/cyclin A. Furthermore, investigations of the impact of PTMs on local conformation performed in small peptides highlight the role of S404 phosphorylation in inducing helical propensity in the region downstream pS404 that is exacerbated by other phosphorylations of PHF-1 epitope at S396 and S400, or O-GlcNAcylation of S400. Finally, the role of phosphorylation and O-GlcNAcylation of PHF-1 epitope was probed in in-vitro fibrillization assays in which O-GlcNAcylation slows down the rate of fibrillar assembly while GSK3β phosphorylation stimulates aggregation counteracting the effect of glycosylation.

scholarly journals The initial fusion pore induced by baculovirus GP64 is large and forms quickly.

1996 ◽  
Vol 135 (6) ◽  
pp. 1831-1839 ◽  
Author(s):  
I Plonsky ◽  
J Zimmerberg
Keyword(s):  
Wide Distribution ◽  
Fusion Pore ◽  
High Time Resolution ◽  
Viral Fusion ◽  
Time Resolved ◽  
Cell Recording ◽  
Resolution Model ◽  
Pore Opening ◽  
Lag Times ◽  
Fusion Pores

The formation of the fusion pore is the first detectable event in membrane fusion (Zimmerberg, J., R. Blumenthal, D.P. Sarkar, M. Curran, and S.J. Morris. 1994. J. Cell Biol. 127:1885-1894). To date, fusion pores measured in exocytosis and viral fusion have shared features that include reversible closure (flickering), highly fluctuating semistable stages, and a lag time of at least several seconds between the triggering and the pore opening. We investigated baculovirus GP64-induced Sf9 cell-cell fusion, triggered by external acid solution, using two different electrophysiological techniques: double whole-cell recording (for high time resolution, model-independent measurements), and the more conventional time-resolved admittance recordings. Both methods gave essentially the same results, thus validating the use of the admittance measurements for fusion pore conductance calculations. Fusion was first detected by abrupt pore formation with a wide distribution of initial conductance, centered around 1 nS. Often the initial fusion pore conductance was stable for many seconds. Fluctuations in semistable conductances were much less than those of other fusion pores. The waiting time distribution, measured between pH onset and initial pore appearance, fits best to a model with many (approximately 19) independent elements. Thus, unlike previously measured fusion pores, GP64-mediated pores do not flicker, can have large, stable initial pore conductances lasting up to a minute, and have typical lag times of &lt; 1 s. These findings are consistent with a barrel-shaped model of an initial fusion pore consisting of five to eight GP64 trimers that is lined with lipid.

scholarly journals Molecular mechanism of fusion pore formation driven by the neuronal SNARE complex

2018 ◽  
Vol 115 (50) ◽  
pp. 12751-12756 ◽  
Author(s):  
Satyan Sharma ◽  
Manfred Lindau
Keyword(s):  
Pore Formation ◽  
Structural Model ◽  
Snare Complex ◽  
Coarse Grained ◽  
Fusion Pore ◽  
Protein Mutants ◽  
Experimental Values ◽  
Dynamics Simulations ◽  
External Forces ◽  
Fusion Pores

Release of neurotransmitters from synaptic vesicles begins with a narrow fusion pore, the structure of which remains unresolved. To obtain a structural model of the fusion pore, we performed coarse-grained molecular dynamics simulations of fusion between a nanodisc and a planar bilayer bridged by four partially unzipped SNARE complexes. The simulations revealed that zipping of SNARE complexes pulls the polar C-terminal residues of the synaptobrevin 2 and syntaxin 1A transmembrane domains to form a hydrophilic core between the two distal leaflets, inducing fusion pore formation. The estimated conductances of these fusion pores are in good agreement with experimental values. Two SNARE protein mutants inhibiting fusion experimentally produced no fusion pore formation. In simulations in which the nanodisc was replaced by a 40-nm vesicle, an extended hemifusion diaphragm formed but a fusion pore did not, indicating that restricted SNARE mobility is required for rapid fusion pore formation. Accordingly, rapid fusion pore formation also occurred in the 40-nm vesicle system when SNARE mobility was restricted by external forces. Removal of the restriction is required for fusion pore expansion.

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