Exploring the Structure and Dynamics of Ceramide Channels

智力价值：本项目涉及三个主要领域：脂质在细胞功能中的作用，线粒体启动细胞凋亡的调节，以及纳米结构的自组装。在膜功能的背景下，脂质的作用通常归结为两个方面：在隔室之间产生物理屏障和调节蛋白质的作用。神经酰胺（一种鞘脂）的另一个作用是形成高度组织化的组装体，通过磷脂膜产生大的水孔。这些通道允许高达60kda的蛋白质穿过细胞膜。这种通道形成能力仅限于某些类型的膜：线粒体外膜，但不包括质膜。本研究旨在了解神经酰胺形成这些大型组织通道的结构基础。实验和计算（分子动力学模拟）方法的结合将用于测试神经酰胺分子各种特征的重要性。假设是各种特征的精确组合提供了形成大通道的正确形状和氢键能力。神经酰胺代谢途径中作为前体或产物的其他鞘脂的存在预计会促进或干扰这些通道的组装。了解这些相互作用对于理解这一途径中代谢酶活性变化的后果以及随之而来的生理变化非常重要。这些生理变化之一是线粒体外膜对蛋白质的渗透性。这种通透性被认为可以释放促凋亡蛋白，从而启动细胞凋亡的执行阶段。因此，了解神经酰胺通道如何形成可能使人们找到控制细胞凋亡起始的方法。计划中的实验将在磷脂膜上进行，在磷脂膜上，系统是明确的，没有蛋白质，以及在分离的线粒体上。前者提供了清晰详细的机械信息，没有在天然膜中发现的复杂因素。它还利用了电生理方法的高灵敏度和高精度。在天然膜上的实验提供了生物学背景，潜在地揭示了更复杂的相互作用的存在，然后将在确定的系统中进行测试。分子动力学模拟将用于测试假设的结构和它们的动力学，以便产生实验测试的预测。模拟退火的使用将允许我们在有限的计算机时间内探测各种结构。电子显微镜将用于将真实结构可视化，以便与从功能推断的结构进行比较。不同的方法有望协同作用，使我们能够对结构及其动力学有一个扎实的了解。更广泛的影响：这项工作将部分由寻求获得研究经验的本科生和优秀的高中生完成。这些学生将在至少一名受资助人员的直接监督下学习。从PI之前的经验来看，这些被选中的学生被证明是非常积极的，他们学得很快。离开时，学生的研究技能大大提高，而且通常对继续从事研究产生了新的兴趣。通常，这些学生将他们的研究成果作为海报展示在校园范围内的比赛中，或者作为高中班级的报告。因此，知识和经验与他人分享。通常情况下，PI的实验室有各种各样的学生，包括许多少数民族学生。PI经常与他的高级班级的学生分享在研究实验室中获得的见解。分子动力学模拟将由PI的实验室和Sergei Sukharev的实验室合作，他的博士后研究员将获得该奖项的支持。该建模工具主要用于研究拉伸敏感通道的结构和动力学。由于这种合作，新的分子动力学方法正在开发中。这是校园内唯一的分子动力学模拟设施，计划中的研究将扩大其实用性和知名度。就社会的潜在利益而言，了解神经酰胺形成这些大通道的结构特征可能对纳米结构的自组装研究产生影响，并导致实际应用。例如，这些知识可以用来帮助设计脂质体系统（例如，用于将内容物输送到特定部位），这些脂质体系统可以根据其货物的装载和卸载进行调节。

Intellectual Merit:This project impacts three major areas: the role of lipids in cell function, the regulation of the initiation of apoptosis by mitochondria, and the self-assembly of nanostructures. The roles of lipids, in the context of membrane function, are generally relegated to two areas: the generation of a physical barrier between compartments and the regulation of the action of proteins. An additional role is the ability of ceramide, a sphingolipid, to form highly organized assemblies that generate large aqueous pores through phospholipid membranes. These channels allow proteins up to 60 kDa to cross membranes. This channel forming ability is limited to certain types of membranes: mitochondrial outer membranes, but not plasma membranes. This research aims to understand the structural basis underlying the ability of ceramide to form these large organized channels. A combination of experimental and computational (molecular dynamic simulations) approaches will be used to test the importance of various features of the ceramide molecule. The hypothesis is that the precise combination of a variety of features provides exactly the right shape and hydrogen-bonding ability to form the large channels. The presence of other sphingolipids that are either precursors or products in the ceramide metabolic pathway is expected to either promote or interfere with the assembly of these channels. Understanding these interactions is important in understanding the consequences of changes in the activity of the metabolic enzymes in this pathway and the consequent physiological changes. Among such physiological changes is the permeabilization of the mitochondrial outer membrane to proteins. This permeabilization is believed to release pro-apoptotic proteins that initiate the execution phase of apoptosis. Thus, understanding how ceramide channels form may allow one to find ways to control the initiation of apoptosis. The planned experiments will be performed both on phospholipid membranes, where the system is defined and free of proteins, and on isolated mitochondria. The former provides clear detailed mechanistic information, free of the complicating factors found in natural membranes. It also takes advantage of the high sensitivity and precision of the electrophysiological approach. The experiments on natural membranes provide the biological context, potentially revealing the presence of more complex interactions that would then be tested in the defined system. Molecular dynamic simulations will be used to test hypothetical structures and their dynamics in order to generate predictions to test experimentally. The use of simulated annealing will allow us to probe a variety of structures with limited computer time. Electron microscopy will be used to visualize the real structures for comparison with those inferred from function. The different approaches are expected to be synergistic and allow us to gain a solid understanding of the structures and their dynamics. Broader impact: This work will, in part, be performed by undergraduate students seeking to gain research experience and by exceptional high school students. These students will be under the direct supervision of at least one of the personnel supported by this grant. From the PI's prior experience, such selected students have proven to be highly motivated and they learn quickly. The students leave with greatly improved research skills and usually a new-found interest in continuing to be involved in research. Typically these students present the results of their research as posters in campus-wide competitions or as reports to high school classes. Thus the knowledge and experience is shared with others. Typically the PI's lab has a great diversity of students, including many minority students. The PI often shares insight gained in the research lab with students in his upper-level classes. The molecular dynamics simulation will be a collaboration between the laboratory of the PI and that of Sergei Sukharev whose post-doctoral fellow is will be supported by this award. The modeling facility is primarily used to study the structure and dynamics of stretch-sensitive channels. As a result of this collaboration, new molecular dynamics methods are being developed. This is the only molecular dynamics simulation facility on campus and the planned research will expand its utility and visibility. In terms of potential benefits to society, understanding the structural features that allow ceramide to form these large channels may have implications on the study of the self-assembly of nanostructures and lead to practical applications. For example, this knowledge could be used to help design liposome systems (e.g., for delivery of contents to specific sites) that could be regulated in terms of the loading and unloading of their cargoes.