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

蛋白质分配到有序膜微域的结构决定因素和功能后果

基本信息

批准号：
9363982
负责人：
Ilya Levental
金额：
$ 30.26万
依托单位：
UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-30 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9363982
关键词：
Affect Affinity Alpha Cell Amino Acid Sequence Area Autoimmune Diseases Autoimmunity Automobile Driving Bioinformatics Biological Models Biophysics Cardiovascular Diseases Cell membrane Cell physiology Cells Cellular Membrane Cellular biology Characteristics Charge Computer Simulation Data Disease Drug Design Endocytosis Endosomes Ensure Environment Face Functional disorder Goals Image Immune Integral Membrane Protein Investigation Lateral Length Lipids Malignant Neoplasms Mammalian Cell Measurement Mediating Membrane Membrane Lipids Membrane Microdomains Membrane Protein Traffic Membrane Proteins Methodology Modeling Molecular Molecular Models Mutation Nature Pathway interactions Phase Plasma Protein Sortings Proteins Proteome Quantitative Evaluations Recruitment Activity Recycling Role Signal Transduction Sorting - Cell Movement Structure Surface System Testing Thinness Transmembrane Domain Variant base design experimental study heuristics insight molecular modeling physical model physical property preference prevent protein transport public health relevance small molecule synergism trafficking transmission process

项目摘要

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. " " "

项目概要质膜 (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 的功能被放大了：划分为组成和功能不同的侧域，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”，失调导致癌症、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、定量的方法论阻碍了筏组成的明确定义或筏的明确机械描述功能。最近的方法论突破是直接观察大规模有序域从哺乳动物细胞中分离出质膜。该系统证实了哺乳动物的固有能力 PM 形成筏域，还为直接定量提供了强大的实验平台对其成分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、计算机分子建模和细胞生物学来表征蛋白质分配到 PM 微结构域的结构决定因素和功能后果。我们的大量初步数据表明，蛋白质跨膜结构域 (TMD) 包含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 长度和表面积，以检验相对较长且较薄的 TMD 的假设与有序膜微环境有更有利的相互作用。实验测量筏分区将得到计算建模和生物信息学的支持，最终目标是生成一个物理模型，可以从氨基酸序列中识别筏偏好蛋白。在目标 2 中，我们将把研究从单一 TMD 扩展到评估 TMD 寡聚在驱动筏亲和力中的作用。我们的初步数据已确定了显着增强筏相的特定TMD序列基序协会。我们将通过以下方式评估这种增强是由 TMD 寡聚驱动的假设定量评估 TMD 寡聚及其对活细胞、分离的 PM 和筏分配的影响综合模型系统。这些观察结果背后的结构细节将通过原子论来研究分子建模。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剂通过目标 3 中提出的实验进行膜运输。为此，我们生成了一组蛋白质除了TMD编码的筏亲和力之外，缺乏任何分选决定因素的变体。对于这些蛋白质，PM 内吞作用后的回收依赖于它们划分成有序的膜域，这意味着筏- 介导的蛋白质分选机制。其背后的贩运途径和分子机制将使用TMD面板作为筏和的有效探针通过成像实验来研究机制非 raft 域。这些研究将鉴定依赖于微域关联来发挥其功能的蛋白质，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，蛋白质与微结构域相关。 ” ” ”