Single-molecule manipulation of SNAREs

描述（由申请人提供）：思维、记忆和运动依赖于神经元之间快速准确的化学传递，这是由微妙的钙触发膜融合过程介导的。这一过程中的故障会导致各种精神障碍和神经退行性疾病。经过数十年的努力，人们发现了通常驱动膜融合的高度保守的核心机制（SNARE，或可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体），以及特异性控制突触融合的关键调控蛋白。最近，已经取得了重大进展，使重建的钙依赖性膜融合在体外。尽管如此，与体内观察到的融合相比，这种融合是缓慢的，并且潜在的调节机制仍存在争议。 作为膜融合的专门引擎，SNARE被认为产生显著的力，将膜吸引到非常接近的位置以进行融合。引人注目的是，这种力是由一对同源的SNARE像拉链一样逐渐折叠和组装而成的。这种拉链机制也有助于膜融合的特异性。然而，在SNARE和膜之间的拔河比赛中，力也可能对SNARE组装及其调节产生深远的影响，这在很大程度上被忽视了。通过使用从其膜环境中分离的蛋白质，促进了SNARE的结构和功能研究。但是由于缺乏它们的力负载，SNARE经常以不符合融合的不规则方式组装自己。 我们假设力是SNARE功能性组装和调节不可或缺的组成部分。它可以促进SNARE组装在正确的融合途径中，并促进其调节。为了验证这一假设，我们将提供一个可控的力负载的单一SNARE复合物，并在真实的时间检测其功能折叠/组装过程。使用高分辨率的光学镊子力显微镜，我们将以前所未有的时空分辨率，逐分子，一步一步地确定这些SNARE的折叠/组装反应。我们将测量伴随的力和能量的产生，并研究相反的力和调节蛋白质对组装过程的影响。新的方法将允许我们直接测试SNARE功能的主导力模型。我们的研究将为理解膜融合及其调节的分子基础提供基础，并有助于指导开发更好的药物用于各种膜运输相关疾病。 公共卫生相关性：SNARE或可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体是膜融合的引擎。目前的SNARE功能模型表明，这些蛋白质通过一种不寻常的蛋白质拉链机制产生驱动融合的力量。我们计划使用高分辨率光镊直接测量单个SNARE复合体产生的力，并以前所未有的分辨率精确定位其详细的拉链动力学。我们的研究将为SNARE功能模型提供最终测试，并为了解广泛参与人类疾病的各种膜运输过程奠定基础。

DESCRIPTION (provided by applicant): Thinking, memory, and motions rely on fast and accurate chemical transmission between neurons, which is mediated by a delicate calcium-triggered membrane fusion process. Malfunctions in this process cause various mental disorders and neurodegenerative diseases. Decades of effort has led to the discovery of highly conserved core machinery that generally drive membrane fusion (SNAREs, or soluble N-ethylmaleimide-sensitive factor attachment protein receptors), and key regulatory proteins that specifically control the synaptic fusion. Recently, major advances have been made that enables the reconstitution of the calcium-dependent membrane fusion in vitro. Nevertheless, this fusion is slow compared with that observed in vivo and the underlying regulatory mechanisms are in debate. As specialized engines for membrane fusion, SNAREs are believed to generate significant force that draws the membranes to close proximity for fusion. Strikingly, the force is produced by progressive folding and assembly of a cognate pair of SNAREs like a zipper. This zippering mechanism also contributes to the specificity of membrane fusion. However, in the tug-of-war between SNAREs and membranes, force may also have profound effects on SNARE assembly and its regulation, which has largely been neglected. Structural and functional studies of SNAREs are facilitated by using the proteins isolated from their membrane environments. But due to lack of their force load, the SNAREs often assemble themselves in an irregular manner that does not correspond to fusion. We hypothesize that force is an indispensible component for functional SNARE assembly and regulation. It can promote SNARE assembly in a correct pathway for fusion and facilitate its regulation. To test this hypothesis, we will provide single SNARE complexes with a controllable force load and detect their functional folding/assembly processes in real time. Using high-resolution optical tweezer force microscopy, we will pinpoint the folding/assembly reaction of these SNAREs at an unprecedented spatiotemporal resolution, molecule-by-molecule and step-by-step. We will measure the accompanying force and energy generation and examine the effects of the opposing force and regulatory proteins upon the assembly process. The novel approach will allow us to directly test the predominant force model for SNARE function. Our research will provide a foundation for understanding the molecular basis of membrane fusion and its regulation and help guide the development of better medicines for a variety of membrane-trafficking-related diseases. PUBLIC HEALTH RELEVANCE: SNAREs, or soluble N-ethylmaleimide-sensitive factor attachment protein receptors, are the engines for membrane fusion. The current model for SNARE function suggests that these proteins generate forces to drive the fusion through an unusual protein zippering mechanism. Using high-resolution optical tweezers, we plan to directly measure the force produced by a single SNARE complex and pinpoint its detailed zippering kinetics at unprecedented resolution. Our research will provide an ultimate test of the model for SNARE function and a foundation to understand various membrane trafficking processes widely involved in human diseases.