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Probing SNARE assembly and disassembly in vitro and in live cells

Award Number: F31GM147951
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS
PERIOD OF PERFORMANCE START DATE: 04/14/2023
PERIOD OF PERFORMANCE END DATE: 04/13/2025
AWARD TERMINATION DATE: 06/05/2025

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Probing SNARE assembly and disassembly in vitro and in live cells - PROJECT ABSTRACT/SUMMARY Intracellular vesicle fusion is primarily mediated by SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors), which consist of vesicle membrane protein synaptobrevin II (syb2, VAMP2 or v- SNARE), and target membrane proteins syntaxin (syx) and SNAP-25 (collectively called t-SNAREs). As the vesicle approaches the target membrane, the v-SNARE and t-SNAREs assemble in a zipper-like manner to form a vesicle fusion machine, enabling release of the molecular cargo. After fusion, the assembled SNARE complex is disassembled by the recycling machinery, composed of NSF and α-SNAP, into individual proteins for the next round of fusion. Since vesicle fusion is a ubiquitous process and is critical for cell-cell communication, mutations in SNAREs lead to numerous neuronal and metabolic diseases. Over the past few decades, extensive studies have revealed great details of individual steps in the SNARE cycle. However, the molecular mechanism that orders the sequence of each step remains unclear. A pressing need exists to probe this relationship. Here, we seek to understand the assembly and disassembly of SNAREs in vitro and in live cells. In Aim 1, we will develop a hydrogen deuterium exchange-mass spectrometry (HDX-MS) approach to analyze SNARE complex formation and deformation in vitro under physiologically relevant conditions. We have shown that the sequence coverage collected after MS of the individual SNARE proteins includes their SNARE motifs, supporting that subsequent HDX-MS will reveal details into their mechanism. We will use this approach to study SNARE binary and ternary complex to examine assembly, then introduce the recycling proteins to assess complex disassembly. In Aim 2, our objective is to understand the spatiotemporal regulation of the SNARE cycle during vesicle exocytosis by developing a genetically encoded intensity-based conformational sensor for SNAREs assembly and disassembly (icsenSNARE). Using reconstituted assays, we have identified the first design, icsenSNARE000 that showed a 1.5-fold fluorescence increase upon assembly of the SNARE complex and could be reversed by the disassembly machinery. Finally, we plan to employ icsenSNARE in live cells to reveal how the spatiotemporal regulation of the SNARE cycle is coupled to vesicle fusion. Together, the proposed study will advance mechanistic understandings of vesicle exocytosis, develop a new approach for studying SNARE assembly and disassembly, and generate a new probe for the SNARE proteins. Achievement of this work has the potential to help study SNARE-related pathologies to improve human health.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $0 )
2026	2024	UNIVERSITY OF FLORIDA	1523 UNION RD RM 207	GAINESVILLE	FL	32611	ALACHUA	USA	Biomedical Research and Research Training	000	2	2/24/2026	TERMINATION	$0
														Subtotal = $0

Issue Date FY: 2025 ( Subtotal = $0 )
2025	2024	UNIVERSITY OF FLORIDA	1523 UNION RD RM 207	GAINESVILLE	FL	32611	ALACHUA	USA	Biomedical Research and Research Training	000	2	6/3/2025	TERMINATION	$0
														Subtotal = $0

Issue Date FY: 2024 ( Subtotal = $35,974 )
2024	2024	UNIVERSITY OF FLORIDA	1523 UNION RD RM 207	GAINESVILLE	FL	32611	ALACHUA	USA	Biomedical Research and Research Training	001	2	3/20/2024	NON-COMPETING CONTINUATION	$34,694
2024	2024	UNIVERSITY OF FLORIDA	1523 UNION RD RM 207	GAINESVILLE	FL	32611	ALACHUA	USA	Biomedical Research and Research Training	002	2	6/11/2024	NON-COMPETING CONTINUATION	$1,280
2024	2023	UNIVERSITY OF FLORIDA	1523 UNION RD RM 207	GAINESVILLE	FL	32611	ALACHUA	USA	Biomedical Research and Research Training	000	1	10/31/2023	NEW	$0
														Subtotal = $35,974

Issue Date FY: 2023 ( Subtotal = $34,694 )
2023	2023	UNIVERSITY OF FLORIDA	1523 UNION RD RM 207	GAINESVILLE	FL	32611	ALACHUA	USA	Biomedical Research and Research Training	001	1	4/1/2023	NEW	$0
2023	2023	UNIVERSITY OF FLORIDA	1523 UNION RD RM 207	GAINESVILLE	FL	32611	ALACHUA	USA	Biomedical Research and Research Training	000	1	4/1/2023	NEW	$34,694
														Subtotal = $34,694

Grand Total All Awards = $70,668

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Probing SNARE assembly and disassembly in vitro and in live cells

Award Number: F31GM147951

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

PERIOD OF PERFORMANCE START DATE: 04/14/2023

PERIOD OF PERFORMANCE END DATE: 04/13/2025

AWARD TERMINATION DATE: 06/05/2025

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