The transmembrane domain peptide of vesicular stomatitis virus promotes both intermediate and pore formation during PEG-mediated vesicle fusion.
The transmembrane domain peptide of vesicular stomatitis virus promotes both intermediate and pore formation during PEG-mediated vesicle fusion.
复制标题
水疱性口炎病毒的跨膜结构域肽在 PEG 介导的囊泡融合过程中促进中间体和孔的形成。
DOI：
10.1016/j.bpj.2014.03.053
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发表时间：
2014
影响因子：
3.4
通讯作者：
Lentz,BarryR
中科院分区：
文献类型：
--
作者：
Sengupta,Tanusree;Chakraborty,Hirak;Lentz,BarryR
我们提出了水泡性口炎病毒（VSV-TMD）的跨膜结构域促进融合起始和融合孔形成的机制。记录了在pH 7.4、5种不同温度（17℃~ 37℃）下，由二油基磷脂酰胆碱、二油基磷脂酰乙醇胺（DOPE）、牛脑鞘磷脂和胆固醇（35:30:15:20摩尔比）组成的25 nm单层小囊泡融合的时间过程，并与含有VSV G蛋白融合活性TMD的相同囊泡的时间过程进行了比较。将多个时间过程全局拟合到一个单中间体系综动力学模型中，以估计聚合态向中间半融合态（k1、stalk或I1）转化的速率常数，这些中间半融合态迅速过渡到不稳定的中间态（i2态），然后以组合速率k3转化为最终融合孔态。通过分析得到了三种状态下脂质混合、内容物混合和内容物泄漏的概率。每个步骤的活化热力学与先前发表的中间和孔隙形成过程中的脂质重排模型一致。VSV-TMD、十六烷和VSV-TMD +十六烷对动力学、活化热力学和膜结构的影响支持了这两种试剂不通过共同机制催化融合的假设，除非在最低温度下。VSV-TMD主要催化初始中间产物的形成，尽管它大大增加了中间产物混合的可能性。我们的研究结果支持了这样的假设，即VSV-TMD对peg介导的融合的初始-中间和成孔步骤的催化作用源于其施加正固有曲率的能力，从而对小的单层囊泡外小叶以及中间微结构的外围施加应力。
We propose mechanisms by which the transmembrane domain of vesicular stomatitis virus (VSV-TMD) promotes both initiation of fusion and formation of a fusion pore. Time courses of polyethyleneglycol (PEG)-mediated fusion of 25 nm small unilamellar vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), bovine brain sphingomyelin, and cholesterol (35:30:15:20 molar ratio) were recorded at pH 7.4 at five different temperatures (from 17°C to 37°C) and compared with time courses obtained with the same vesicles containing the fusion-active TMD of the G protein of VSV. Multiple time courses were fitted globally to a one-intermediate ensemble kinetic model to estimate the rate constants for conversion of the aggregated state to an intermediate hemifused state (k1, stalk, or I1) that rapidly transits to an unstable intermediate (I2state) that converts to a final fusion pore state with a combined ratek3. The probabilities of lipid mixing, contents mixing, and contents leakage in the three states were also obtained from this analysis. The activation thermodynamics for each step were consistent with previously published models of lipid rearrangements during intermediate and pore formation. The influences of VSV-TMD, hexadecane, and VSV-TMD + hexadecane on the kinetics, activation thermodynamics, and membrane structure support the hypothesis that these two agents do not catalyze fusion by a common mechanism, except possibly at the lowest temperatures examined. VSV-TMD primarily catalyzed initial intermediate formation, although it substantially increased the probability of contents mixing in the intermediate state. Our results support the hypothesis that the catalytic influence of VSV-TMD on the initial-intermediate- and pore-forming steps of PEG-mediated fusion derives from its ability to impose a positive intrinsic curvature and thereby stress small unilamellar vesicle outer leaflets as well as the periphery of intermediate microstructures.