scholarly journals NSF-mediated disassembly of on- and off-pathway SNARE complexes and inhibition by complexin

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Ucheor B Choi ◽  
Minglei Zhao ◽  
K Ian White ◽  
Richard A Pfuetzner ◽  
Luis Esquivies ◽  
...  
Keyword(s):  
Quality Control ◽  
Ionic Strength ◽  
Single Molecule ◽  
Membrane Trafficking ◽  
Neurotransmitter Release ◽  
Single Step ◽  
High Ionic Strength ◽  
Snare Complex ◽  
Single Molecule Fret ◽  
Cis And Trans

SNARE complex disassembly by the ATPase NSF is essential for neurotransmitter release and other membrane trafficking processes. We developed a single-molecule FRET assay to monitor repeated rounds of NSF-mediated disassembly and reassembly of individual SNARE complexes. For ternary neuronal SNARE complexes, disassembly proceeds in a single step within 100 msec. We observed short- (<0.32 s) and long-lived (≥0.32 s) disassembled states. The long-lived states represent fully disassembled SNARE complex, while the short-lived states correspond to failed disassembly or immediate reassembly. Either high ionic strength or decreased αSNAP concentration reduces the disassembly rate while increasing the frequency of short-lived states. NSF is also capable of disassembling anti-parallel ternary SNARE complexes, implicating it in quality control. Finally, complexin-1 competes with αSNAP binding to the SNARE complex; addition of complexin-1 has an effect similar to that of decreasing the αSNAP concentration, possibly differentially regulating cis and trans SNARE complexes disassembly.

scholarly journals Molecular basis of ubiquitin-specific protease 8 autoinhibition by the WW-like domain

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Keijun Kakihara ◽  
Kengo Asamizu ◽  
Kei Moritsugu ◽  
Masahide Kubo ◽  
Tetsuya Kitaguchi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Membrane Trafficking ◽  
Cushing’S Disease ◽  
Molecular Basis ◽  
Binding Pocket ◽  
Cushing's Disease ◽  
Binding Motif ◽  
Single Molecule Fret ◽  
Specific Protease ◽  
Ubiquitin Specific Protease

AbstractUbiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme involved in multiple membrane trafficking pathways. The enzyme activity is inhibited by binding to 14-3-3 proteins. Mutations in the 14-3-3-binding motif in USP8 are related to Cushing’s disease. However, the molecular basis of USP8 activity regulation remains unclear. This study identified amino acids 645–684 of USP8 as an autoinhibitory region, which might interact with the catalytic USP domain, as per the results of pull-down and single-molecule FRET assays performed in this study. In silico modelling indicated that the region forms a WW-like domain structure, plugs the catalytic cleft, and narrows the entrance to the ubiquitin-binding pocket. Furthermore, 14-3-3 inhibited USP8 activity partly by enhancing the interaction between the WW-like and USP domains. These findings provide the molecular basis of USP8 autoinhibition via the WW-like domain. Moreover, they suggest that the release of autoinhibition may underlie Cushing’s disease due to USP8 mutations.

scholarly journals Polybasic patches in both C2 domains of Synaptotagmin-1 are required for evoked neurotransmitter release

2021 ◽  
Author(s):  
Zhenyong Wu ◽  
Lu Ma ◽  
Nicholas A Courtney ◽  
Jie Zhu ◽  
Yongli Zhang ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Calcium Binding ◽  
Neurotransmitter Release ◽  
Transmembrane Domain ◽  
Snare Complex ◽  
Critical Feature ◽  
C2 Domains ◽  
Electrophysiological Recordings ◽  
Synaptotagmin 1

Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release. It is composed of a single-pass transmembrane domain linked to two tandem C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is not well understood. Calcium binding to C2B, but not to C2A, is critical for synchronous release and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here we asked what critical feature of C2A may be responsible for its functional role, and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1K189-192A and Syt1K326-327A) side-by-side to determine their relative contributions to Syt1 function in cultured cortical mouse neurons and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain-membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The readily releasable vesicle pool or spontaneous release were not affected, so both patches are specifically required for synchronization of release. We suggest these patches contribute to cooperative binding to membranes, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release.

scholarly journals Complexin induces a conformational change at the membrane-proximal C-terminal end of the SNARE complex

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Ucheor B Choi ◽  
Minglei Zhao ◽  
Yunxiang Zhang ◽  
Ying Lai ◽  
Axel T Brunger
Keyword(s):  
Conformational Change ◽  
Single Molecule ◽  
Neurotransmitter Release ◽  
Plasma Membranes ◽  
Molecular Mechanisms ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Snare Complex ◽  
Spontaneous Release ◽  
Trans Conformation

Complexin regulates spontaneous and activates Ca2+-triggered neurotransmitter release, yet the molecular mechanisms are still unclear. Here we performed single molecule fluorescence resonance energy transfer experiments and uncovered two conformations of complexin-1 bound to the ternary SNARE complex. In the cis conformation, complexin-1 induces a conformational change at the membrane-proximal C-terminal end of the ternary SNARE complex that specifically depends on the N-terminal, accessory, and central domains of complexin-1. The complexin-1 induced conformation of the ternary SNARE complex may be related to a conformation that is juxtaposing the synaptic vesicle and plasma membranes. In the trans conformation, complexin-1 can simultaneously interact with a ternary SNARE complex via the central domain and a binary SNARE complex consisting of syntaxin-1A and SNAP-25A via the accessory domain. The cis conformation may be involved in activation of synchronous neurotransmitter release, whereas both conformations may be involved in regulating spontaneous release.

scholarly journals In vivo single-molecule imaging of syntaxin1A reveals polyphosphoinositide- and activity-dependent trapping in presynaptic nanoclusters

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Adekunle T. Bademosi ◽  
Elsa Lauwers ◽  
Pranesh Padmanabhan ◽  
Lorenzo Odierna ◽  
Ye Jin Chai ◽  
...  
Keyword(s):  
Single Molecule ◽  
Neurotransmitter Release ◽  
Motor Nerve ◽  
Living Organism ◽  
Neurosecretory Cells ◽  
Single Particle Tracking ◽  
Snare Complex ◽  
Motor Nerve Terminal ◽  
Activity Dependent

Abstract Syntaxin1A is organized in nanoclusters that are critical for the docking and priming of secretory vesicles from neurosecretory cells. Whether and how these nanoclusters are affected by neurotransmitter release in nerve terminals from a living organism is unknown. Here we imaged photoconvertible syntaxin1A-mEos2 in the motor nerve terminal of Drosophila larvae by single-particle tracking photoactivation localization microscopy. Opto- and thermo-genetic neuronal stimulation increased syntaxin1A-mEos2 mobility, and reduced the size and molecular density of nanoclusters, suggesting an activity-dependent release of syntaxin1A from the confinement of nanoclusters. Syntaxin1A mobility was increased by mutating its polyphosphoinositide-binding site or preventing SNARE complex assembly via co-expression of tetanus toxin light chain. In contrast, syntaxin1A mobility was reduced by preventing SNARE complex disassembly. Our data demonstrate that polyphosphoinositide favours syntaxin1A trapping, and show that SNARE complex disassembly leads to syntaxin1A dissociation from nanoclusters. Lateral diffusion and trapping of syntaxin1A in nanoclusters therefore dynamically regulate neurotransmitter release.

scholarly journals The molecular basis of ubiquitin-specific protease 8 autoinhibition by the WW-like domain

2021 ◽  
Author(s):  
Keijun Kakihara ◽  
Kengo Asamizu ◽  
Kei Moritsugu ◽  
Masahide Kubo ◽  
Tetsuya Kitaguchi ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Single Molecule ◽  
Membrane Trafficking ◽  
Molecular Basis ◽  
Binding Pocket ◽  
Binding Motif ◽  
Deubiquitinating Enzyme ◽  
Single Molecule Fret ◽  
Specific Protease ◽  
Ubiquitin Specific Protease

Ubiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme involved in multiple membrane trafficking pathways. The enzyme activity is inhibited by binding to 14-3-3 proteins, and mutations of the 14-3-3 binding motif in USP8 are related to Cushing′s disease. However, the molecular basis of USP8 enzyme activity regulation remains unclear. Here, we identified amino acids 645–684 of USP8 as an autoinhibitory region, which our pull-down and single-molecule FRET assay results suggested interacts with the catalytic USP domain. In silico modelling indicated that the region forms a WW-like domain structure, plugs the catalytic cleft, and narrows the entrance to the ubiquitin-binding pocket. Furthermore, 14-3-3 was found to inhibit USP8 enzyme activity partly by enhancing the interaction between the WW-like and USP domains. These findings provide the molecular basis of USP8 autoinhibition via the WW-like domain. Moreover, they suggest that the release of autoinhibition may underlie Cushing′s disease caused by USP8 mutations.

scholarly journals High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study

2014 ◽  
Vol 1343 ◽  
pp. 135-142 ◽  
Author(s):  
Lydia Kisley ◽  
Jixin Chen ◽  
Andrea P. Mansur ◽  
Sergio Dominguez-Medina ◽  
Eliona Kulla ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Ion Exchange ◽  
Single Molecule ◽  
High Ionic Strength ◽  
Exchange Adsorption

Importance of Protease Inhibition in Studies on Purified Factor VIII (Antihaemophilic Factor)

1976 ◽  
Vol 35 (01) ◽  
pp. 186-190 ◽  
Author(s):  
Eugen A. Beck ◽  
Peter Bachmann ◽  
Peter Barbier ◽  
Miha Furlan
Keyword(s):  
Ionic Strength ◽  
Factor Viii ◽  
Gel Filtration ◽  
High Ionic Strength ◽  
Procoagulant Activity ◽  
Carrier Protein ◽  
Protease Inhibition ◽  
Functional Sites ◽  
Molecular Weight Peak ◽  
Von Willebrand

SummaryAccording to some authors factor VIII procoagulant activity may be dissociable from carrier protein (MW~ 2 × 106) by agarose gel filtration, e.g. at high ionic strength. We were able to reproduce this phenomenon. However, addition of protease inhibitor (Trasylol) prevented the appearance of low molecular weight peak of factor VIII procoagulant activity both at high ionic strength and elevated temperature (37°C). We conclude from our results that procoagulant activity and carrier protein (von Willebrand factor, factor VIII antigen) are closely associated functional sites of native factor VIII macro molecule. Consequently, proteolytic degradation should be avoided in functional and structural studies on factor VIII and especially in preparing factor VIII concentrate for therapeutic use.

THE RELATIVE DISTRIBUTION OF THYROXINE, TRIIODOTHYRONINE AND 3,3′,5′-(REVERSE)-TRIIODOTHYRONINE IN VARIOUS FRACTIONS OF THYROGLOBULIN

1978 ◽  
Vol 88 (2) ◽  
pp. 298-305 ◽  
Author(s):  
Peter Laurberg
Keyword(s):  
Ionic Strength ◽  
Guinea Pig ◽  
Sucrose Gradient ◽  
Deae Cellulose ◽  
High Ionic Strength ◽  
Relative Distribution ◽  
Thyroid Extract ◽  
Reverse Triiodothyronine ◽  
Thyroid Secretion ◽  
Stable Iodine

ABSTRACT Thyroglobulin fractions rich and poor in new thyroglobulin were separated by means of DEAE-cellulose chromatography of dog thyroid extracts and by zonal ultracentrifugation in a sucrose gradient of guinea pig thyroid extract incubated at low temperature. The distribution of thyroxine, triiodothyronine and 3,3′,5′-(reverse)-triiodothyronine in hydrolysates of the different fractions was estimated by radioimmunoassays. Following DEAE-cellulose chromatography there was a small but statistically significant increase in the T4/T3 ratio in thyroglobulin fractions eluted at high ionic strength - that is fractions relatively rich in stable iodine but poor in fresh thyroglobulin. There were no differences in the T4/rT3 ratios between the different fractions. The ratios between iodothyronines were almost identical in the various thyroglobulin fractions following zonal ultracentrifugation in a sucrose gradient of cold treated guinea pig thyroid extract. These findings lend no support to the possibility that a relatively high content of triiodothyronines in freshly synthesized thyroglobulin modulates the thyroid secretion towards a preferential secretion of triiodothyronine and 3,3′,5′-(reverse)-triiodothyronine at the expense of the secretion of thyroxine.

Sign in / Sign up

Export Citation Format

Share Document