Emergent cellular functions of GPCRs and myosins

GPCR 和肌球蛋白的新兴细胞功能

基本信息

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
项目类别：
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

项目摘要

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.

项目概要信号传导和膜交通等细胞过程是从动态、瞬态的集合中产生的拥挤的细胞环境中的蛋白质间相互作用是异常的蛋白质间相互作用。经常与糖尿病、神经退行性疾病等使人衰弱或致命的疾病有关已建立的结构和细胞生物学技术主要局限于剖析癌症的功能。稳定的蛋白质复合物，并且不研究由多个瞬态引起的紧急行为为了应对这一挑战，我们使用 DNA 纳米技术支架来构建大分子的图案。体外和一种新型基因编码的 ER/K 连接子来探测和调节活细胞中的蛋白质相互作用。我们共同利用这些技术来剖析 G 蛋白多样性的分子机制耦合受体（GPCR）信号传导和细胞中非常规肌球蛋白功能的生物物理调节。我们使用生物传感器成功研究了细胞中 GPCR 信号传导特异性的两个不同方面通过使用 ER/K 连接子连接 GPCR 和 G 蛋白元件进行设计。首先，我们利用了配体。 GPCR 构象亚状态选择性地与一个或多个 Gα C 末端结合，以调节我们的目标是结合使用 GPCR 生物传感器。和多尺度分子动力学模拟来定义热点残基、结构基序和变构其次，我们的研究取得了进展。 GPCR 和效应器之间的并发和顺序相互作用对信号特异性的作用。是结合GPCR生物传感器和传统药理学方法来剖析协同效应我们的研究共同提供了对 GPCR 信号传导特异性的见解。可用于基于结构的药物发现工作，以识别功能选择性 GPCR 配体。非常规肌球蛋白在许多细胞过程中至关重要，包括膜运输、收缩性、细胞中肌球蛋白的运动功能的定义受到两者无数几何形状的挑战。肌动蛋白网络和货物，与多种汽车-货物接口配对我们使用货物模拟DNA。纳米技术支架与计算模型相结合，成功剖析了机械结构我们将使用这些方法来深入了解生物物理调节。我们发现货物界面充当分子的作用。调节肌球蛋白功能以满足单个细胞过程的功能要求的模块。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的调节。我们的研究将增进我们对细胞中新兴肌球蛋白功能的理解，同时为细胞骨架马达的货物介导的调节提供了广泛的理论框架。