Structural and Functional Analysis of the CAPS SNARE-Binding Domain Required for SNARE Complex Formation and Exocytosis

Zhou, Hao; Wei, Ziqing; Wang, Shen; Yao, Deqiang; Zhang, Rongguang; Ma, Cong

doi:10.1016/j.celrep.2019.02.064

Cited by 13 publications

(13 citation statements)

References 58 publications

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“…Complete biochemical reconstitution approaches starting from all purified components have been powerful to dissect many elements of the mechanisms these proteins utilize to drive fusion 4–8 . SNARE-mediated fusion of secretory vesicles purified from endogenous sources has also been reconstituted into fusion assays with artificial membranes, but most of these fusion assays do not distinguish between docking, priming, and fusion 9–14 .…”

Section: Introductionmentioning

confidence: 99%

In vitro fusion of single synaptic and dense core vesicles reproduces key physiological properties

Kreutzberger¹,

Kiessling²,

Stroupe³

et al. 2019

Nat Commun

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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.

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Section: Introductionmentioning

confidence: 99%

In vitro fusion of single synaptic and dense core vesicles reproduces key physiological properties

Kreutzberger¹,

Kiessling²,

Stroupe³

et al. 2019

Nat Commun

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“…This activity increases the possibility that the initiation of DCVs in the early development stage of chromatin cells can be realized through the C 2 -PH domain, whereas the initiation function of CAPS needs the participation of other domains in more mature cells [ 185 ]. A subsequent study about the successful crystal structure analysis of the DAMH domain offers the possibility of further understanding the function of CAPS-1, thereby revealing the dual role of CAPS-1 in SNARE complex formation [ 186 ], as follows: (1) CAPS-1 DAMH domain interacts with Munc13-1 MUN, and the interaction hinders Munc13-1 activity to open Munc18-1/SNARE, which further leads to the assembly of the SNARE complex failure. (2) After syntaxin-1 is activated, CAPS-1 stabilizes the active state of syntaxin-1 through the interaction between the DAMH domain with the syntaxin-1/SNAP25 complex, thereby accelerating the assembly of the SNARE complex and finally promoting synaptic exocytosis [ 178 ].…”

Section: Regulatory Proteins and Mechanisms In Synaptic Secretionmentioning

confidence: 99%

“…Therefore, based on these studies, a model in which CAPS and Munc13 jointly regulate vesicle secretion was proposed ( Figure 2 ) [ 186 ]: in the resting state, CAPS-1 is first located on the cytoplasmic membrane through the calcium-independent interaction between PH and PIP2. Munc13-1 cannot bind to Munc18-1/syntaxin-1 complex due to the interaction of PH–PIP2 and DAMH–MUN [ 173 , 186 ]. Thus, the anchored DCVs and SVs cannot enter the vesicle priming stage.…”

Section: Regulatory Proteins and Mechanisms In Synaptic Secretionmentioning

confidence: 99%

Synaptic Secretion and Beyond: Targeting Synapse and Neurotransmitters to Treat Neurodegenerative Diseases

Wei

Yang

et al. 2022

Oxidative Medicine and Cellular Longevity

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The nervous system is important, because it regulates the physiological function of the body. Neurons are the most basic structural and functional unit of the nervous system. The synapse is an asymmetric structure that is important for neuronal function. The chemical transmission mode of the synapse is realized through neurotransmitters and electrical processes. Based on vesicle transport, the abnormal information transmission process in the synapse can lead to a series of neurorelated diseases. Numerous proteins and complexes that regulate the process of vesicle transport, such as SNARE proteins, Munc18-1, and Synaptotagmin-1, have been identified. Their regulation of synaptic vesicle secretion is complicated and delicate, and their defects can lead to a series of neurodegenerative diseases. This review will discuss the structure and functions of vesicle-based synapses and their roles in neurons. Furthermore, we will analyze neurotransmitter and synaptic functions in neurodegenerative diseases and discuss the potential of using related drugs in their treatment.

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“…In order to monitor the number of fusion events in per cell, a common highly K + stimulation buffer (15 mM HEPES, pH 7.4, 95 mM NaCl, 60 mM KCl, 2.2 mM CaCl2, 0.5 mM MgCl2, 5.6 mM glucose) was used to displace the basal buffer to depolarize PC12 cells and trigger vesicles fusion. We monitored exocytosis of NPY-td-mOrange2 at the single-vesicle level as described before (Zhou et al, 2019). Exocytosis events were scored manually.…”

Section: Dcvs Docking and Secretion Assay In Pc12 Cellsmentioning

confidence: 99%

Rabphilin 3A binds the N-peptide of SNAP-25 to promote SNARE complex assembly in exocytosis

Cheng

Wang

et al. 2022

Preprint

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Exocytosis of secretory vesicles requires the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and small GTPase Rabs. As a Rab3/Rab27 effector protein on secretory vesicles, Rabphilin 3A was implicated to interact with SNAP-25 to regulate vesicle exocytosis in neurons and neuroendocrine cells, yet the underlying mechanism remains unclear. In this study, we have characterized the physiologically relevant binding sites between Rabphilin 3A and SNAP-25. We found that an intramolecular interplay between the N-terminal Rab-binding domain and C-terminal C2AB domain enables Rabphilin 3A to strongly bind the SNAP-25 N-peptide region via its C2B bottom α-helix. Disruption of this interaction significantly impaired docking and fusion of vesicles with the plasma membrane in PC12 cells. In addition, we found that this interaction allows Rabphilin 3A to accelerate SNARE complex assembly. Furthermore, we revealed that this interaction accelerates SNARE complex assembly via inducing a conformational switch from random coils to α-helical structure in the SNAP-25 SNARE motif. Altogether, our data suggest that promotion of SNARE complex assembly by binding of the C2B bottom α-helix of Rabphilin 3A to the N-peptide of SNAP-25 underlies a pre-fusion function of Rabphilin 3A in vesicle exocytosis.

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Structural and Functional Analysis of the CAPS SNARE-Binding Domain Required for SNARE Complex Formation and Exocytosis

Abstract: Highlights d A structure of the DAMH domain of CAPS is presented d CAPS hinders the ability of Munc13to catalyze opening of syntaxin-1 d CAPS stabilizes the open state of syntaxin-1 to facilitate SNARE complex formation

Cited by 13 publications

References 58 publications

In vitro fusion of single synaptic and dense core vesicles reproduces key physiological properties

In vitro fusion of single synaptic and dense core vesicles reproduces key physiological properties

Synaptic Secretion and Beyond: Targeting Synapse and Neurotransmitters to Treat Neurodegenerative Diseases

Rabphilin 3A binds the N-peptide of SNAP-25 to promote SNARE complex assembly in exocytosis

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