Arf Functional Landscapes

Arf功能景观

• 批准号: 10248460
• 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
• 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/10248460
• 关键词: ADP-Ribosylation Factors; Alzheimer' 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

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.

总结 真核细胞的膜组织由ADP核糖基化因子（Arfs）控制，小分子 作为分子开关激活信号级联的GTP酶。Arfs调节囊泡 脂质和蛋白质在内质网和高尔基体（I-Arf 1类）和内体-血浆之间的转运 膜运输（III类-Arf 6），涉及Arf在胞质分裂、细胞形状、细胞器中的功能 转运、线粒体和脂滴功能以及细胞大小的pH依赖性调节。突变 在Arfs或他们的伴侣中，与导致严重畸形的遗传神经疾病有关 大脑皮层或智力迟钝。此外，许多致病细菌和病毒 当Arfs侵入细胞时，它们会控制Arfs，从而促进感染。我们的总体目标是了解 Arf GTP酶的核苷酸交换转换，其机制不能从 静态结构。我们假设，Arf构象特异性识别的，他们的 同源交换因子对应于显著中断的激发态， 标准条件下的低水平。具体来说，我们的目标是映射Arf 1和Arf 2的GDP/GTP开关。 Arf 6（目的1和2），并使用突变分析，建立 其功能特异性（目标3）。我们的方法结合了实验生物物理工具（多个， 三维NMR，SAXS和荧光）与压力扰动和粗粒分子 我们的数据约束的动力学模拟，以提供结构合奏和伪自由能 景观，将揭示功能相关的激发态牵连Arf的功能和特异性。 这些激发态结构将为抑制Arf信号通路提供新的靶位点， 为开发减轻细菌入侵能力的方法提供了新的途径， 病毒更一般地，这里提出的基于压力的映射方法代表了一种强大的 手段来表征与其功能有关的蛋白质的难以捉摸的状态。