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

项目摘要 质膜(PM)形成了细胞和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的功能通过 划分为成分和功能上不同的侧区，其中脂筏是 典型的例子。RAFT介导的信号转导广泛涉及多种细胞 功能“，调节失调导致癌症、过度炎症、 自身免疫和心血管疾病。尽管有这种潜在的影响，但缺乏一致的、量化的 方法论阻碍了对筏子组成的明确定义或对筏子的明确机械描述 功能。最近的一项方法学突破是对大规模有序区域的直接观察 从哺乳动物细胞中分离出的质膜。这一系统证实了哺乳动物的先天能力 PMS形成RAFT结构域，并为直接、定量 对它们的组成和物理性质进行研究。我们提出了一个全面的方法 结合生物物理学、生物信息学、电子分子模拟和细胞生物学来表征 蛋白质分配到PM微区的结构决定因素和功能后果。我们的 大量的初步数据显示，蛋白质跨膜结构域(TMD)包含了必要的 浮筏亲和力的决定因素。在目标1中，我们将定义赋予RAFT亲和力的一般TMD物理特征， 特别关注TMD的长度和表面积，以检验相对较长和较薄的TMD的假设 与有序的膜微环境有更有利的相互作用。实验测量结果表明 浮筏分割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RAFT偏好蛋白质的物理模型。在目标2中，我们 将把这项研究从单一的TMD扩展到评估TMD齐聚在推动RAFT亲和力方面的作用。我们的 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RAFT阶段 协会。我们将评估这样一个假设，即这种增强是由TMD齐聚通过 定量评估TMD寡聚及其对活细胞、分离的PM和 合成模型系统。这些观察背后的结构细节将由原子论进行调查 分子建模。最后，我们的目标是明确地证明RAFT亲和力是亚细胞的主要调节因子。 通过目标3中提出的实验进行膜交通。为此，我们产生了一组蛋白质 除TMD编码的RAFT亲和力外，变异体缺乏任何排序决定因素。对于这些蛋白质，PM 内吞作用后的循环依赖于它们划分成有序的膜域，这意味着- 介导的蛋白质分选机制。其背后的贩运途径和分子机制 机理将通过使用TMD面板作为浮筏和 非RAFT域。这些研究将确定依赖微域关联实现其功能的蛋白质， 定义这种关联的物理化学性质，并阐明PM组织的机制 调节细胞生理学。长期目标是促进干扰小分子的合理设计 在由异常PM信号转导定义的疾病状态中，蛋白质与微域相关。 “ “ “

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. " " "