The neuron is remarkable in that upon the influx of Ca2+ it synchronizes vesicle fusion, and releases many quanta of neurotransmitters to the synaptic cleft in less than 1 msec. The fast synchronization is orchestrated by interactions between the core fusion machinery SNAREs and auxiliary proteins including a major Ca2+-sensor synaptotagmin 1 (Syt1) and a clamping factor complexin (Cpx). Because release underlies cognition and behavior, toxic agents that undermine the release of neurotransmitter might lead to the symptoms of neurodegenerative diseases such as Parkinson's and Alzheimer's. In this project we use innovative approaches to investigate the mechanism whereby the fusion machinery achieves the synchronization of vesicle fusion. To mimic the native membrane environment we prepare a nanodisc sandwich that harbors a single trans SNARE complex in the middle. Single molecule (sm)FRET and site-directed spin labeling (SDSL) EPR are used to characterize the interactions of SNAREs with auxiliary proteins in the chasm of two nanodiscs. In addition, the drastically improved single vesicle-vesicle fusion assay that can resolve docking, lipid mixing, fusion pore opening, and pore expansion steps, is used to delineate the intervention of regulatory factors onto individual fusion steps. Taken altogether, a comprehensive picture of how synchronization of vesicle fusion is choreographed by the interactions among individual components of the fusion machinery would emerge. For neurodegenerative diseases such as Parkinson's there is an emerging theme of pathophysiology that toxic misfolded oligomers are tampering with the vesicle fusion machinery, leading to disease symptoms. We use EPR to investigate the interaction between ?-synuclein and vesicle (v-)SNARE VAMP2 that takes place on the membrane surface. The outcomes of these investigations are expected to reveal new therapeutic targets for treating symptoms of the Parkinson's disease.

Public Health Relevance

Neurons release chemical messengers called neurotransmitters to synapses via the pathway called vesicle fusion when stimulated by the action potential. Communication among neurons constitutes the fundamental basis for cognitive activities. This project is aimed at elucidating the mechanism, at a molecular level, by which vesicle fusion is regulated. The study will ultimately provide important insights into mental illnesses. Furthermore, toxic misfolded proteins cause altercation of vesicle fusion, leading to symptoms of neurodegenerative diseases such as Parkinson's and Alzheimer's. The study is also directed toward the understanding of molecular interactions that cause neurodegenerative diseases, which will identify new drug targets for treating these diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM051290-22
Application #
9093054
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Ainsztein, Alexandra M
Project Start
1994-08-01
Project End
2020-04-30
Budget Start
2016-05-01
Budget End
2017-04-30
Support Year
22
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Iowa State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
005309844
City
Ames
State
IA
Country
United States
Zip Code
50011
Lou, Xiaochu; Kim, Jaewook; Hawk, Brenden J et al. (2017) ?-Synuclein may cross-bridge v-SNARE and acidic phospholipids to facilitate SNARE-dependent vesicle docking. Biochem J 474:2039-2049
Su, Chih-Chia; Yin, Linxiang; Kumar, Nitin et al. (2017) Structures and transport dynamics of a Campylobacter jejuni multidrug efflux pump. Nat Commun 8:171
Choi, Bong-Kyu; Kim, Jae-Yeol; Cha, Moon-Yong et al. (2017) Retraction of ""?-Amyloid and ?-Synuclein Cooperate To Block SNARE-Dependent Vesicle Fusion"". Biochemistry 56:1026
Khounlo, Ryan; Kim, Jaewook; Yin, Linxiang et al. (2017) Botulinum Toxins A and E Inflict Dynamic Destabilization on t-SNARE to Impair SNARE Assembly and Membrane Fusion. Structure 25:1679-1686.e5
Xue, Chaoyou; Zhu, Yicheng; Zhang, Xiangmei et al. (2017) Real-Time Observation of Target Search by the CRISPR Surveillance Complex Cascade. Cell Rep 21:3717-3727
Na, Jung-Hyun; Lee, Won-Kyu; Kim, Yuyoung et al. (2016) Biophysical characterization of the structural change of Nopp140, an intrinsically disordered protein, in the interaction with CK2?. Biochem Biophys Res Commun 477:181-7
Yin, Linxiang; Kim, Jaewook; Shin, Yeon-Kyun (2016) Complexin splits the membrane-proximal region of a single SNAREpin. Biochem J 473:2219-24
Lou, Xiaochu; Shin, Yeon-Kyun (2016) SNARE zippering. Biosci Rep 36:
Kim, Jaewook; Zhu, Yicheng; Shin, Yeon-Kyun (2016) Preincubation of t-SNAREs with Complexin I Increases Content-Mixing Efficiency. Biochemistry 55:3667-73
Heo, Paul; Yang, Yoosoo; Han, Kyu Young et al. (2016) A Chemical Controller of SNARE-Driven Membrane Fusion That Primes Vesicles for Ca(2+)-Triggered Millisecond Exocytosis. J Am Chem Soc 138:4512-21

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