Emergent cellular functions of GPCRs and myosins

GPCR 和肌球蛋白的新兴细胞功能

• 批准号: 9919584
• 负责人: Sivaraj Sivaramakrishnan
• 金额: $ 33.14万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9919584
• 关键词: Actins; Address; Behavior; Biological; Biophysics; Biosensor; Cell division; Cell physiology; Cells; Complex; Computer Models; Crowding; DNA; Diabetes Mellitus; Disease; Elements; Engineering; Environment; Estrogen receptor positive; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Geometry; Goals; Heart failure; Hot Spot; In Vitro; Individual; Ligands; Link; Malignant Neoplasms; Mechanics; Mediating; Membrane Protein Traffic; Molecular; Molecular Conformation; Motor; Myosin ATPase; Nanotechnology; Neurodegenerative Disorders; Pathway interactions; Pattern; Pharmacology; Proteins; Regulation; Research; Role; Signal Transduction; Specificity; Structure; Techniques; Technology; base; cell motility; drug discovery; insight; macromolecule; mimetics; molecular dynamics; molecular scale; novel; novel therapeutics; protein complex; protein protein interaction; rab GTP-Binding Proteins; receptor; scaffold; stem

PROJECT SUMMARY Cellular processes such as signaling and membrane traffic emerge from an ensemble of dynamic, transient protein-protein interactions in crowded cellular environments. Aberrant protein-protein interactions are frequently implicated in debilitating or fatal diseases such as diabetes, neurodegenerative diseases, and cancer. Established structural and cell biological techniques are mostly limited to dissecting the function of stable protein complexes, and do not investigate emergent behavior stemming from multiple transient interactions. To address this challenge, we use DNA nanotechnology scaffolds to pattern macromolecules in vitro and a novel genetically encoded ER/K linker to probe and modulate protein interactions in live cells. Together, we leverage these technologies to dissect the molecular mechanisms of multiplicity in G protein- coupled receptor (GPCR) signaling and biophysical regulation of unconventional myosin function in cells. We have successfully investigated two distinct aspects of GPCR signaling specificity in cells using biosensors engineered by linking GPCR and G protein elements with an ER/K linker. First, we hypothesize that ligands stabilize GPCR conformational sub-states that selectively interface with one or more Gα C-termini, to tune ligand efficacy and potency for downstream pathways. Our goal is to use a combination of GPCR biosensors and multi-scale molecular dynamics simulations to define hot-spot residues, structural motifs, and allosteric pathways in both the GPCR and G protein that drive signaling specificity. Second, our research has advanced a role for concurrent and sequential interactions between GPCR and effectors on signaling specificity. Our goal is to combine GPCR biosensors and traditional pharmacology approaches to dissect the synergistic effects of G proteins on GPCR signaling. Together, our research provides insights into GPCR signaling specificity that can be leveraged in structure-based drug discovery efforts to identify functionally selective GPCR ligands. Unconventional myosins are essential in numerous cellular processes including membrane traffic, contractility, and cell division. Defining the motile function of myosins in cells is challenged by the myriad geometries of both actin networks and cargo, paired with a diversity of motor-cargo interfaces. We use cargo-mimetic DNA nanotechnology scaffolds combined with computational modeling to successfully dissect the mechanical coordination in myosin ensembles. We will use these approaches to gain insight into the biophysical regulation of myosin function through the motor-cargo interface. We hypothesize that cargo interfaces act as molecular modules that tune myosin function to match the functional requirements of individual cellular processes. Our goal is to dissect myosin regulation through interactions with distinct cargo adaptors, Rab GTPases, and cell- derived cargo complexes. Our research will advance our understanding of emergent myosin function in cells, while providing a broad theoretical framework for the cargo-mediated regulation of cytoskeletal motors.

项目摘要 蜂窝过程（例如信号传导和膜流量）从动态，瞬态的合奏中出现 拥挤的细胞环境中的蛋白质 - 蛋白质相互作用。异常的蛋白质 - 蛋白质相互作用是 经常在衰弱或致命疾病中实施，例如糖尿病，神经退行性疾病和 癌症。建立的结构和细胞生物学技术大多受到限制，以剖析 稳定的蛋白质复合物，并且不研究因多个瞬态而产生的新兴行为 互动。为了应对这一挑战，我们使用DNA纳米技术支架来模式大分子 体外和一种新型的遗传编码的ER/K连接器以探测并调节活细胞中的蛋白质相互作用。 我们共同利用这些技术在G蛋白中剖析了多重性的分子机制。 细胞中非常规肌球蛋白功能的耦合受体（GPCR）信号传导和生物物理调节。 我们已经成功地研究了使用生物传感器的细胞中GPCR信号特异性的两个不同方面 通过将GPCR和G蛋白质元素与ER/K连接器联系起来。首先，我们假设配体 稳定与一个或多个Gαc-termini接口的GPCR构象子态，以调整 下游途径的配体效率和效力。我们的目标是结合GPCR生物传感器 以及多尺度的分子动力学模拟，以定义热点保留，结构基序和变构。 驱动信号特异性的GPCR和G蛋白的途径。其次，我们的研究进步了 GPCR和对信号特异性的影响之间并发和顺序相互作用的作用。我们的目标 是结合GPCR生物传感器和传统药理学方法，以剖析 GPCR信号传导上的G蛋白。我们的研究共同提供了对GPCR信号特异性的见解 可以利用基于结构的药物发现工作，以识别功能性选择性GPCR配体。 非常规的肌醇在包括膜交通，收缩性， 和细胞分裂。在细胞中定义肌球蛋白的母体功能是由两者的无数几何形状挑战 肌动蛋白网络和货物，搭配多种电动货车接口。我们使用货物模拟的DNA 纳米技术支架与计算建模相结合，成功剖析机械 肌球蛋白合奏中的协调。我们将使用这些方法来深入了解生物物理调节 通过电动机量接口的肌球蛋白功能。我们假设货物界面充当分子 调整肌球蛋白功能以匹配单个细胞过程的功能要求的模块。我们的 目标是通过与不同的货物适配器，RAB GTPases和细胞的相互作用来剖析肌球蛋白调节。 派生的货物络合物。我们的研究将促进我们对细胞中新出现肌球蛋白功能的理解， 同时为货物介导的细胞骨架电机调节提供了广泛的理论框架。