TY - JOUR
T1 - Screening of Hydrocarbon-Stapled Peptides for Inhibition of Calcium-Triggered Exocytosis
AU - Lai, Ying
AU - Tuvim, Michael J.
AU - Leitz, Jeremy
AU - Peters, John
AU - Pfuetzner, Richard A.
AU - Esquivies, Luis
AU - Zhou, Qiangjun
AU - Czako, Barbara
AU - Cross, Jason B.
AU - Jones, Philip
AU - Dickey, Burton F.
AU - Brunger, Axel T.
N1 - Funding Information:
This work was supported by the National Institutes of Health (NIH) (R37MH63105 to AB; R01 HL129795 and R21 AI137319 to BD), and the Cystic Fibrosis Foundation (DICKEY18G0 and DICKEY19P0). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH. This article is subject to HHMI’s Open Access to Publications policy. HHMI lab heads have previously granted a nonexclusive CC BY 4.0 license to the public and a sublicensable license to HHMI in their research articles. Pursuant to those licenses, the author-accepted manuscript of this article can be made freely available under a CC BY 4.0 license immediately upon publication.
Funding Information:
This work was supported by the National Institutes of Health (NIH) (R37MH63105 to AB; R01 HL129795 and R21 AI137319 to BD), and the Cystic Fibrosis Foundation (DICKEY18G0 and DICKEY19P0). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH. This article is subject to HHMI’s Open Access to Publications policy. HHMI lab heads have previously granted a nonexclusive CC BY 4.0 license to the public and a sublicensable license to HHMI in their research articles. Pursuant to those licenses, the author-accepted manuscript of this article can be made freely available under a CC BY 4.0 license immediately upon publication.
Publisher Copyright:
Copyright © 2022 Lai, Tuvim, Leitz, Peters, Pfuetzner, Esquivies, Zhou, Czako, Cross, Jones, Dickey and Brunger.
PY - 2022/6/17
Y1 - 2022/6/17
N2 - 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, Syt2, 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 Syt1 or with reconstituted airway SNAREs and Syt2, the selected peptides also suppressed Ca2+-triggered fusion. Taken together, hydrocarbon-stapled peptides 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, which is a major cause of airway obstruction in the pathophysiology of COPD, asthma, and cystic fibrosis.
AB - 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, Syt2, 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 Syt1 or with reconstituted airway SNAREs and Syt2, the selected peptides also suppressed Ca2+-triggered fusion. Taken together, hydrocarbon-stapled peptides 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, which is a major cause of airway obstruction in the pathophysiology of COPD, asthma, and cystic fibrosis.
KW - airway obstruction
KW - asthma
KW - cystic fibrosis
KW - mucin secretion
KW - neurotransmitter release
KW - stapled peptide
KW - stimulated membrane fusion
UR - http://www.scopus.com/inward/record.url?scp=85133795212&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85133795212&partnerID=8YFLogxK
U2 - 10.3389/fphar.2022.891041
DO - 10.3389/fphar.2022.891041
M3 - Article
C2 - 35814209
AN - SCOPUS:85133795212
SN - 1663-9812
VL - 13
JO - Frontiers in Pharmacology
JF - Frontiers in Pharmacology
M1 - 891041
ER -