Arf Functional Landscapes

Arf功能景观

基本信息

批准号：
10408843
负责人：
CATHERINE A ROYER
金额：
$ 34.95万
依托单位：
RENSSELAER POLYTECHNIC INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10408843
关键词：
ADP-Ribosylation Factors Alzheimer&apos s Disease Autoimmune Diseases Bacteria Binding Biophysics Brefeldin A Cell Shape Cell membrane Cell physiology Cells Cerebral cortex Chemicals Communicable Diseases Cytokinesis Data Development Disease Endosomes Enzymes Eukaryotic Cell Exhibits Fluorescence Free Energy GTP Binding GTPase-Activating Proteins Genetic Goals Golgi Apparatus Grain Guanine Nucleotide Exchange Factors Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Hepatitis C virus Human Infection Invaded Investigation Link Lipids Malignant Neoplasms Maps Membrane Mental Retardation Mitochondria Modeling Modification Molecular Molecular Conformation Monomeric GTP-Binding Proteins Multidimensional NMR Techniques Mutation Mutation Analysis N-terminal Nucleotides Organelles Parkinson Disease Pathway interactions Pharmaceutical Preparations Population Positioning Attribute Protein Conformation Proteins Regulation of Cell Size Salmonella Signal Pathway Signal Transduction Site Solvents Specificity Structure System Therapeutic Virus Yeasts base biophysical tools lipid transport malformation molecular dynamics molecular recognition nervous system disorder novel pathogenic bacteria pathogenic virus pressure protein structure three dimensional structure tool trafficking vesicle transport

项目摘要

Summary Membrane organization in eukaryotic cells is controlled by ADP ribosylation factors (Arfs), small GTPases that function as molecular switches to activate signaling cascades. Arfs regulate vesicular transport of lipids and proteins between the ER and the Golgi (Class I-Arf1) and endosome-plasma membrane trafficking (Class III-Arf6), implicating Arf function in cytokinesis, cell shape, organelle transport, mitochondrial and lipid droplet function and pH-dependent regulation of cell size. Mutations in Arfs or their partners have been linked to genetic neurological diseases causing severe malformation of the cerebral cortex or mental retardation. Moreover, many pathogenic bacteria and viruses commandeer Arfs as they invade cells, thereby promoting infection. Our overall goal is to understand the nucleotide exchange transitions of Arf GTPases, the mechanisms of which cannot be deduced from their static structures. We hypothesize that the Arf conformations specifically recognized by their cognate exchange factors correspond to significantly disrupted excited states that are populated at very low levels under standard conditions. Specifically, we aim to map the GDP/GTP switches of Arf1 and Arf6 (Aims 1 and 2), and using mutational analysis, establish the underlying molecular mechanisms of their functional specificity (Aim 3). Our approach combines experimental biophysical tools (multi- dimensional NMR, SAXS and fluorescence) with pressure perturbation and coarse-grained molecular dynamics simulations constrained by our data, to provide structural ensembles and pseudo-free energy landscapes that will reveal functionally relevant excited states implicated in Arf function and specificity. These excited state structures will provide novel target sites for inhibiting Arf signaling pathways, offering new avenues for developing approaches to mitigate the invasive capacity of bacteria and viruses. More generally, the pressure-based mapping approach proposed here represents a powerful means to characterize elusive states of proteins implicated in their functions.

概括真核细胞中的膜组织由ADP核糖基化因子（ARF）控制，小充当分子开关的GTP酶可激活信号级联。 ARFS调节囊泡 ER和高尔基体之间的脂质和蛋白质的运输（I-ARF1类）和内体质量膜运输（III-ARF6级），与细胞因子，细胞形状，细胞器有关ARF功能传输，线粒体和脂质液滴功能以及细胞大小的pH依赖性调节。突变在ARF或其伴侣中，与遗传神经系统疾病有关，导致严重畸形大脑皮质或智力低下。此外，许多致病细菌和病毒指挥生在入侵细胞时，从而促进感染。我们的总体目标是了解 ARF GTPases的核苷酸交换过渡，其机制不能从中推导它们的静态结构。我们假设ARF构象由其专门认可同源交换因子对应于在非常破坏的激发态，这些状态在在标准条件下低水平。具体而言，我们旨在绘制ARF1和ARF1的GDP/GTP开关 ARF6（目标1和2），并使用突变分析建立了基本的分子机制它们的功能特异性（AIM 3）。我们的方法结合了实验生物物理工具（多尺寸NMR，SAXS和荧光），带压力扰动和粗粒分子受我们数据约束的动力模拟，提供结构合奏和无伪能将揭示与ARF功能和特异性有关的功能相关激发态的景观。这些激发的状态结构将为抑制ARF信号通路提供新的目标位点，提供新的途径来开发方法来降低细菌和病毒。更普遍地，此处提出的基于压力的映射方法代表了一个强大的表征与其功能有关的蛋白质难以捉摸状态的手段。