Structural determinants and functional consequences of protein partitioning to ordered membrane microdomains

项目摘要 质膜（PM）形成细胞与其细胞之间的物理屏障和功能界面。 环境为了适应这种复杂性，PM的功能被放大， 区室化成组成和功能不同的横向结构域，其中脂筏是 典型的例子筏介导的信号转导已广泛涉及多种细胞 功能”，调节异常导致癌症中的异常信号传导，过度炎症， 自身免疫和心血管疾病。尽管存在这种潜在影响，但缺乏一致的、定量的 方法学阻碍了筏组成的清晰定义或筏的明确机械描述 功能最近的方法学突破是直接观察大规模有序域， 从哺乳动物细胞分离的质膜。这个系统证实了哺乳动物的内在能力 PMs形成筏域，也提供了一个强大的实验平台，直接，定量 研究其组成和物理性质。我们提出一个全面的办法 结合生物物理学、生物信息学、计算机分子建模和细胞生物学来表征 结构决定因素和功能的蛋白质分区PM微域的后果。我们 广泛的初步数据表明，蛋白质跨膜结构域（TMD）包括必要的 筏亲和力的决定因素。在目标1中，我们将定义赋予筏亲和力的一般TMD物理特征， 特别关注TMD的长度和表面积，以检验相对长而薄的TMD 与有序的膜微环境有更有利的相互作用。的实验测量 筏划分将得到计算建模和生物信息学的支持，最终目标是 生成可以从氨基酸序列中识别筏偏好蛋白质的物理模型。在目标2中， 将从单一TMD扩展研究，以评估TMD寡聚化在驱动筏亲和力中的作用。我们 初步数据已经确定了一个特定的TMD序列基序，它显著增强了筏阶段 协会我们将评估这样的假设，即这种增强是由TMD寡聚化驱动的， 定量评价TMD寡聚化及其对活细胞、分离的PM中筏分配的影响， 合成模型系统这些观察结果背后的结构细节将由原子论研究。 分子模拟最后，我们的目标是明确证明筏亲和力作为亚细胞的主要调节因子， 膜交通目标3中提出的实验。为此，我们制作了一组蛋白质 除了它们的TMD编码的筏亲和力之外，缺乏任何分选决定簇的变体。对于这些蛋白质，PM 内吞作用后的再循环依赖于它们分配到有序的膜结构域，这意味着一个筏- 介导的蛋白质分选机制。这背后的贩运途径和分子机制 将TMD面板作为筏的有效探头， 非筏域。这些研究将鉴定依赖于微结构域结合的蛋白质， 定义这种关联的物理化学性质，并阐明PM组织 调节细胞生理学。长期目标是促进小分子的合理设计， 与异常PM信号转导所定义的疾病状态中的微结构域的蛋白质关联。 " " "

Project Summary The plasma membrane (PM) forms the physical barrier and functional interface between a cell and its environment. To accommodate this complexity, the functionality of the PM is amplified by compartmentalization into compositionally and functionally distinct lateral domains, of which lipid rafts are the archetypal example. Raft-mediated signal transduction has been extensively implicated in diverse cell functions," with dysregulation contributing to the aberrant signaling in cancer, hyperinflammation, autoimmunity, and cardiovascular disease. Despite this potential impact, a dearth of consistent, quantitative methodologies has prevented clear definition of raft composition or unequivocal mechanistic description of raft function. A recent methodological breakthrough is the direct observation of large-scale ordered domains in plasma membranes isolated from mammalian cells. This system confirms the inherent capacity of mammalian PMs to form raft domains and also provides a robust experimental platform for direct, quantitative investigations into their composition and physical properties. We propose a comprehensive approach combining biophysics, bioinformatics, in silico molecular modeling, and cell biology to characterize the structural determinants and functional consequences of protein partitioning to PM microdomains. Our extensive preliminary data reveal that protein transmembrane domains (TMDs) encompass the necessary determinants for raft affinity. In Aim 1, we will define the general TMD physical features that impart raft affinity, focusing specifically on TMD length and surface area to test the hypothesis that relatively long and thin TMDs have more favorable interactions with ordered membrane microenvironments. Experimental measurements of raft partitioning will be supported by computational modeling and bioinformatics with the ultimate goal of generating a physical model that can identify raft preferring proteins from amino acid sequence. In Aim 2, we will extend the study from single TMDs to evaluate the role of TMD oligomerization in driving raft affinity. Our preliminary data has identified a specific TMD sequence motif that significantly enhances raft phase association. We will evaluate the hypothesis that such enhancement is driven by TMD oligomerization via quantitative evaluation of TMD oligomerization and its effect on raft partitioning in live cells, isolated PMs, and synthetic model systems. The structural details behind these observations will be investigated by atomistic molecular modeling. Finally, we aim to definitively demonstrate raft affinity as a major regulator of subcellular membrane traffic by the experiments proposed in Aim 3. To this end, we have generated a panel of protein variants lacking any sorting determinants except their TMD-encoded raft affinity. For these proteins, PM recycling after endocytosis relies on their partitioning into ordered membrane domains, implying a raft- mediated protein sorting mechanism. The trafficking pathways and molecular machinery underlying this mechanism will be investigated by imaging experiments using the TMD panel as validated probes of raft and non-raft domains. These studies will identify proteins that rely on microdomain association for their function, define the physicochemical nature of this association, and clarify the mechanisms by which PM organization regulates cell physiology. The long-term goal is to facilitate rational design of small molecules that interfere with protein association with microdomains in disease states defined by aberrant PM signal transduction. " " "