Emergent cellular functions of GPCRs and myosins

项目摘要 细胞过程，如信号和膜交通出现从一个动态的，短暂的 拥挤的细胞环境中的蛋白质相互作用。异常蛋白质-蛋白质相互作用 经常涉及使人衰弱或致命的疾病，如糖尿病、神经退行性疾病， 癌已建立的结构和细胞生物学技术大多限于解剖细胞的功能， 稳定的蛋白质复合物，并没有调查紧急行为源于多个瞬态 交互.为了应对这一挑战，我们使用DNA纳米技术支架来图案化大分子， 体外和一种新的遗传编码的ER/K接头，以探测和调节活细胞中的蛋白质相互作用。 我们一起利用这些技术来剖析G蛋白多样性的分子机制- 偶联受体（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。 我们已经成功地研究了两个不同方面的GPCR信号特异性在细胞中使用生物传感器 通过用ER/K接头连接GPCR和G蛋白元件而工程化。首先，我们假设配体 稳定选择性地与一个或多个Gα C-末端相互作用GPCR构象亚状态， 配体功效和下游途径的效力。我们的目标是使用GPCR生物传感器 和多尺度分子动力学模拟，以确定热点残基，结构基序，和变构 GPCR和G蛋白中驱动信号特异性的途径。第二，我们的研究取得了进展， GPCR和效应子之间的并发和顺序相互作用对信号传导特异性的作用。我们的目标 是将联合收割机GPCR生物传感器和传统药理学方法结合起来， G蛋白对GPCR信号转导的影响。总之，我们的研究提供了对GPCR信号特异性的见解， 可以在基于结构的药物发现工作中利用，以鉴定功能选择性GPCR配体。 非常规肌球蛋白在许多细胞过程中是必不可少的，包括膜运输，收缩， 和细胞分裂。定义肌球蛋白在细胞中的运动功能受到无数几何形状的挑战， 肌动蛋白网络和货物，以及各种各样的马达-货物接口。我们使用货物模拟DNA 纳米技术支架与计算建模相结合，成功地解剖了机械 肌球蛋白集合体的协调。我们将使用这些方法来深入了解生物物理调节 肌球蛋白的功能。我们假设货物接口作为分子 调节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 目的是通过与不同的货物衔接子，Rab GTP酶和细胞- 衍生的货物复合物。我们的研究将促进我们对细胞中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。

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.