Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides

Lai, Ying; Fois, Giorgio; Flores, José Ramón Corcuera; Tuvim, Michael J.; Zhou, Qiuhong; Yang, Kailu; Leitz, Jeremy; Peters, John Jacob; Zhang, Yunxiang; Pfuetzner, Richard A.; Esquivies, Luis; Jones, Philip; Frick, Manfred; Dickey, Burton F.; Brunger, Axel T.

doi:10.1038/s41586-022-04543-1

Cited by 57 publications

(80 citation statements)

References 77 publications

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“…1,2). We therefore focused on these four stapled peptides in subsequent studies using a more advanced single-vesicle content-mixing assay that is capable of distinguishing between vesicle association, lipid-mixing, and content-mixing (Diao et al, 2012; Kyoung et al, 2011; Lai et al, 2022). The four stapled peptides reduced both Ca 2+ -triggered and Ca 2+ -independent fusion with reconstituted neuronal SNAREs and Syt1 (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…4, 5). For a more complete reconstitution involving Munc18 and Munc13 and using one of the stapled peptides (SP9), the effect on Ca 2+ -triggered fusion was also more pronounced than the effect on Ca 2+ -independent fusion (Lai et al, 2022). A possible explanation is that the airway system might lack complexin, a factor that both inhibits Ca 2+ -independent fusion, and activates Ca 2+ -triggered fusion (Lai et al, 2014; Maximov et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

“…Details for the single-vesicle content-mixing assays are described in refs. (Lai et al, 2022, 2017).…”

Section: Methodsmentioning

confidence: 99%

“…Single vesicle-vesicle content mixing assay, instrument setup, and data analysis Details for the single-vesicle content-mixing assays are described in refs. (Lai et al, 2022(Lai et al, , 2017.…”

Section: Ensemble Lipid Mixingmentioning

confidence: 99%

“…The detailed reconstitution protocols for the vesicles are described in refs. (Lai et al, 2022(Lai et al, , 2017.…”

Section: Vesicle Reconstitutionmentioning

confidence: 99%

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Screening of hydrocarbon-stapled peptides for inhibition of calcium-triggered exocytosis

Lai

Tuvim

Pfuetzner

et al. 2022

Preprint

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The so-called primary interface between the SNARE complex and synaptotagmin-1 (Syt1) is essential for Ca2+-triggered neurotransmitter release in neuronal synapses. The interacting residues of the primary interface are conserved across different species for synaptotagmins (Syt1, Syt2, Syt9), SNAP-25, and syntaxin-1A homologs involved in fast synchronous release. This Ca2+-independent interface forms prior to Ca2+-triggering and plays a role in synaptic vesicle priming. This primary interface is also conserved in the fusion machinery that is responsible for mucin granule membrane fusion. Ca2+ stimulated mucin secretion is mediated by the SNAREs syntaxin-3, SNAP-23, VAMP8, synaptotagmin-2, and other proteins. Here, we designed and screened a series of hydrocarbon-stapled peptides consisting of SNAP-25 fragments that included some of the key residues involved in the primary interface as observed in high-resolution crystal structures. We selected a subset of four stapled peptides that were highly α-helical as assessed by circular dichroism and that inhibited both Ca2+-independent and Ca2+-triggered ensemble lipid-mixing with neuronal SNAREs and Syt1. In a single-vesicle content-mixing assay with reconstituted neuronal SNAREs and synaptotagmin-1 or with reconstituted airway SNAREs and synaptotagmin-2, the selected peptides also suppressed Ca2+-triggered fusion. Taken together, hydrocarbon-stapled peptide that interfere with the primary interface consequently inhibit Ca2+-triggered exocytosis. Our inhibitor screen suggests that these compounds may be useful to combat mucus hypersecretion that is a major cause of airway obstruction in the pathophysiology of asthma and cystic fibrosis.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…Details for the single-vesicle content-mixing assays are described in refs. (Lai et al, 2022, 2017).…”

Section: Methodsmentioning

confidence: 99%

Section: Ensemble Lipid Mixingmentioning

confidence: 99%

“…The detailed reconstitution protocols for the vesicles are described in refs. (Lai et al, 2022(Lai et al, , 2017.…”

Section: Vesicle Reconstitutionmentioning

confidence: 99%

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Screening of hydrocarbon-stapled peptides for inhibition of calcium-triggered exocytosis

Lai

Tuvim

Pfuetzner

et al. 2022

Preprint

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On the difficulties of characterizing weak protein interactions that are critical for neurotransmitter release

et al. 2022

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The mechanism of neurotransmitter release has been extensively characterized, showing that vesicle fusion is mediated by the SNARE complex formed by syntaxin‐1, SNAP‐25 and synaptobrevin. This complex is disassembled by N‐ethylmaleimide sensitive factor (NSF) and SNAPs to recycle the SNAREs, whereas Munc18‐1 and Munc13s organize SNARE complex assembly in an NSF‐SNAP‐resistant manner. Synaptotagmin‐1 acts as the Ca2+ sensor that triggers exocytosis in a tight interplay with the SNAREs and complexins. Here, we review technical aspects associated with investigation of protein interactions underlying these steps, which is hindered because the release machinery is assembled between two membranes and is highly dynamic. Moreover, weak interactions, which are difficult to characterize, play key roles in neurotransmitter release, for instance by lowering energy barriers that need to be overcome in this highly regulated process. We illustrate the crucial role that structural biology has played in uncovering mechanisms underlying neurotransmitter release, but also discuss the importance of considering the limitations of the techniques used, including lessons learned from research in our lab and others. In particular, we emphasize: (a) the promiscuity of some protein sequences, including membrane‐binding regions that can mediate irrelevant interactions with proteins in the absence of their native targets; (b) the need to ensure that weak interactions observed in crystal structures are biologically relevant; and (c) the limitations of isothermal titration calorimetry to analyze weak interactions. Finally, we stress that even studies that required re‐interpretation often helped to move the field forward by improving our understanding of the system and providing testable hypotheses.

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Analysis of tripartite Synaptotagmin‐1‐SNARE‐complexin‐1 complexes in solution

et al. 2022

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Characterizing interactions of Synaptotagmin‐1 with the SNARE complex is crucial to understand the mechanism of neurotransmitter release. X‐ray crystallography revealed how the Synaptotagmin‐1 C2B domain binds to the SNARE complex through a so‐called primary interface and to a complexin‐1‐SNARE complex through a so‐called tripartite interface. Mutagenesis and electrophysiology supported the functional relevance of both interfaces, and extensive additional data validated the primary interface. However, ITC evidence suggesting that binding via the tripartite interface occurs in solution was called into question by subsequent NMR data. Here, we describe joint efforts to address this apparent contradiction. Using the same ITC approach with the same C2B domain mutant used previously (C2BKA‐Q) but including ion exchange chromatography to purify it, which is crucial to remove polyacidic contaminants, we were unable to observe the substantial endothermic ITC signal that was previously attributed to binding of this mutant to the complexin‐1‐SNARE complex through the tripartite interface. We were also unable to detect substantial populations of the tripartite interface in NMR analyses of the ITC samples or in measurements of paramagnetic relaxation effects, despite the high sensitivity of this method to detect weak protein complexes. However, these experiments do not rule out the possibility of very low affinity (KD > 1 mm) binding through this interface. These results emphasize the need to develop methods to characterize the structure of synaptotagmin‐1‐SNARE complexes between two membranes and to perform further structure–function analyses to establish the physiological relevance of the tripartite interface.

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Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides

Cited by 57 publications

References 77 publications

Screening of hydrocarbon-stapled peptides for inhibition of calcium-triggered exocytosis

Screening of hydrocarbon-stapled peptides for inhibition of calcium-triggered exocytosis

On the difficulties of characterizing weak protein interactions that are critical for neurotransmitter release

Analysis of tripartite Synaptotagmin‐1‐SNARE‐complexin‐1 complexes in solution

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