Biophysical Characterization of Complexin C-terminal Domain Lipid Interactions

描述(申请人提供)：正确的突触沟通对神经系统功能至关重要，依赖于神经递质通过突触小泡融合到突触前质膜的释放。这一高度调控的过程是由SNARE蛋白组成的核心膜融合机制介导的，并受到许多辅助蛋白的进一步调节。一种这样的蛋白质，复合素(CPX)，以对正常突触功能至关重要的方式直接调节SNARE介导的突触小泡融合。这项建议侧重于蛋白质/脂类相互作用在络合蛋白功能中所起的机制作用。Cpx1或 Cpx2单基因敲除小鼠表现出明显的神经和/或行为缺陷，表明复合蛋白的活动是正常神经功能所必需的。事实上，完全缺乏Cpx功能是致命的，因为Cpx1/Cpx2双基因敲除小鼠在出生后不久就会死亡。在蠕虫和苍蝇中，CPX基因敲除会产生类似的严重神经损伤。CPX在多种精神疾病和神经退行性疾病中的表达水平发生改变，包括亨廷顿病、帕金森病和阿尔茨海默病、精神分裂症、抑郁症和双相情感障碍，已有研究表明，CPX表达水平的改变可能有助于这些疾病的症状。CPX与三元SNARE复合体结合，直接调节突触囊泡的胞吐作用。然而，关于CPX是抑制还是刺激突触囊泡融合一直存在很大的争议。Cpx的C-末端结构域(CTD)被证明介导了对蠕虫自发性突触囊泡融合的抑制，我们最近发现CTD与磷脂双层的结合似乎是这种抑制所必需的。我们的数据表明，突触小泡可能是CPX CTD的相关体内靶点，但目前尚不清楚是什么可能将CPX特异性地导向突触小泡而不是其他细胞膜。我们认为，膜的组成和曲率可能在将CPX靶向囊泡的过程中发挥关键作用。此外，CPX-Vesicl相互作用在CPX功能中的确切作用目前还不清楚。我们假设，囊泡结合使CPX定位于对接的突触小泡，从而促进组装SNARE复合体的快速拦截。为了探索这些假说，我将利用强大的生物物理和生化技术来研究关键的突触前蛋白复合体与磷脂双层之间的相互作用，并努力了解这种相互作用是如何促进突触功能的。具体地说，我将(1)通过表征结合的蛋白质结构决定因素，以及脂质成分和曲率如何影响结合，来确定CPX如何被导向囊泡，以及(2)评估CPX-脂质结合的能量学和动力学可能如何影响CPX的功能。这些结果将为后续关键的体内研究评估结果铺平道路，有助于加深对突触中CPX功能的了解，并有助于更好地理解复杂蛋白水平和/或功能的变化如何影响疾病状态下的突触功能和突触生物学。

DESCRIPTION (provided by applicant): Proper synaptic communication is critical for nervous system function and depends on neurotransmitter release through synaptic vesicle fusion to the presynaptic plasma membrane. This highly regulated process is mediated by a core membrane fusion machinery consisting of the SNARE proteins, and is further modulated by a number of accessory proteins. One such protein, complexin (Cpx), directly regulates SNARE-mediated synaptic vesicle fusion in a manner that is crucial for proper synaptic function. This proposal focuses on the mechanistic role that protein/lipid interactions play in complexin function. Cpx1 or Cpx2 single knockout mice display clear neurological and/or behavioral deficits, demonstrating the requirement of complexin's activities for proper neuronal function. Indeed, complete absence of Cpx function is lethal, as Cpx1/Cpx2 double knockout mice die shortly after birth. In worms and flies, Cpx knockout produces similarly severe neurological impairments. Cpx expression levels are altered in a variety of psychiatric and neurodegenerative diseases, including Huntington's, Parkinson's and Alzheimer's diseases, schizophrenia, depression, and bipolar disorder, and it has been suggested that altered Cpx expression levels may contribute to the symptomatology of these disorders. Cpx binds to the ternary SNARE complex and is known to directly regulate synaptic vesicle exocytosis. However, there has been great controversy regarding whether Cpx inhibits or stimulates synaptic vesicle fusion. The Cpx C-terminal domain (CTD) has been shown to mediate inhibition of spontaneous synaptic vesicle fusion in worms, and we have recently shown that binding of the CTD to phospholipid bilayers appears to be necessary for such inhibition. Our data suggest that synaptic vesicles are likely the relevant in vivo targets for the Cpx CTD, but it is unclear what might direct Cpx specifically to synaptic vesicles vs. other cellular membranes. We propose that membrane composition and curvature may play a critical role in targeting Cpx to vesicles. Additionally, the precise role of Cpx-vesicl interactions in Cpx function is unclear at present. We hypothesize that vesicle binding localizes Cpx to docking synaptic vesicles and thereby facilitates rapid interception of assembling SNARE complexes. To explore these hypotheses, I will study the interactions of the critical presynaptic protein complexin with phospholipid bilayers using powerful biophysical and biochemical techniques, and I will work to understand how this interaction contributes to synaptic function. Specifically, I will (1) determine how Cpx is directed to vesicles by characterizing the protein structural determinants of binding, and how lipid composition and curvature affect binding, and (2) assess how the energetics and kinetics of Cpx- lipid binding might influence Cpx function. These results will pave the way for critical subsequent in vivo studies to evaluate the resulting conclusions, facilitate increased understanding of Cpx function at the synapse, and contribute to an improved understanding of how alterations in complexin levels and/or function might affect synaptic function and synaptic biology in disease states.