Phosphoinositide-calcium Signaling In Cell Regulation

细胞调节中的磷酸肌醇-钙信号转导

• 批准号: 10266455
• 负责人: TAMAS BALLA
• 金额: $ 185万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10266455
• 关键词: 1-Phosphatidylinositol 4-Kinase; ADP-Ribosylation Factor 1; Active Sites; Acute; Affect; Apoptosis; Bacillus cereus; Binding; Binding Proteins; Biochemical Process; Bioluminescence; Biosensor; British Columbia; Calcium Signaling; Canada; Catalytic Domain; Cell Differentiation process; Cell membrane; Cell physiology; Cells; Cellular Membrane; Cellular Metabolic Process; Complex; Confocal Microscopy; Cyclic AMP-Dependent Protein Kinases; Cytosol; Detection; Deuterium; Diabetes Mellitus; Diglycerides; Disease; Endoplasmic Reticulum; Energy Transfer; Ensure; Enterovirus; Environment; Enzymes; Eukaryotic Cell; Family; Family Picornaviridae; Focus Groups; Future; GTP-Binding Proteins; Generations; Golgi Apparatus; Green Fluorescent Proteins; Growth Factor; Health; Heterodimerization; Hormones; Human; Hydrogen; Hydrolysis; Individual; Inositol; Integration Host Factors; Intercept; Interruption; Ion Channel; Knowledge; Life Cycle Stages; Lipids; Maintenance; Malignant Neoplasms; Mammalian Cell; Maps; Mass Spectrum Analysis; Mediating; Membrane; Membrane Lipids; Membrane Transport Proteins; Methods; Mitochondria; Molecular; Monitor; Names; Netherlands; Neurodegenerative Disorders; Neurotransmitters; Organelles; PLCG2 gene; Pathway interactions; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phospholipase C; Phospholipid Metabolism; Phospholipids; Phosphorylation; Phosphotransferases; Population; Positioning Attribute; Process; Protein Engineering; Protein Kinase; Proteins; Proxy; Regulation; Reporting; Research; Research Personnel; Risk; Role; Signal Transduction; Signal Transduction Pathway; Signaling Protein; Sirolimus; Site; Specificity; Structure; Surface; System; Time; Universities; Virus; Virus Diseases; Virus Replication; Visualization; antimicrobial drug; base; biophysical properties; cell motility; cell type; cellular targeting; combat; design; fighting; human disease; information processing; inhibitor/antagonist; interfacial; mutant; pathogen; peroxisome; phosphatidylinositol 4-phosphate; protein complex; protein distribution; protein expression; receptor; recruit; response; tool; trafficking; transmission process

Every biochemical process that happens in a eukaryotic cell relies upon a molecular information flow that leads from receptors that inform the cell about its environment all the way to the molecular effectors that determine the appropriate cellular response. A proper information transmission requires a high degree of organization where the molecular players are organized into different cellular compartments so that the specificity of the cellular response can be properly maintained. Breakdown of this organization is the ultimate cause of all human diseases even if the affected molecular pathways differ according to the kind of disease, such as cancer, diabetes or neurodegenerative diseases just to name a few. Research described in this report has focused on the question of how cells organize their internal membranes to provide a structural framework on which molecular signaling complexes assemble to ensure proper information processing.The lipid composition of the cellular membranes is a major determinant of their biophysical properties and is unique to the different cellular organelles. How cells achieve and maintain the proper lipid composition of their membranes is poorly understood. Cellular processes that affect membrane lipid composition of organelles are often targeted by cellular pathogens such as viruses to force the cells to produce the pathogen instead of performing the cells normal functions. Better understanding of these processes not only can provide new strategies to fight various human diseases but also to intercept the life cycle of cellular pathogens offering an alternative to antimicrobial drugs. Phosphorylated inositol phospholipids (PPIn) are a class of phospholipids that are present in tiny amounts but have very important regulatory functions as they organize protein signaling complexes on specific membrane compartments. They are produced by phosphoinositide kinases that can phosphorylate specific positions of the inositol ring in one of the major classes of phospholipids, phosphatidylinositol (PI). Although the cellular localization of most PPIn has been thoroughly studied and mapped, the cellular distribution of their more abundant precursor, PI has not been determined. Here we developed new molecular tools to gain information on PI distribution in intact live mammalian cells without disrupting their membrane integrity. Using structural information available for bacterial PI-specific phospholipase C enzymes (PI-PLCs), we used the enzyme from Bacillus cereus as a platform for protein engineering. Bacillus cereus (Bc)PI-PLC shows remarkable specificity for PI and does not display catalytic activity towards phosphorylated PPIn species or other phospholipids. We generated mutant forms of this enzyme targeting residues within the conserved catalytic domain that would abolish enzymatic activity but maintain substrate coordination within the active site in order to mark the intracellular distribution of PI. Fusion of this mutant Bc-PI-PLC to the green fluorescent protein (GFP) and expression in mammalian cells allowed visualization of the distribution of the protein by confocal microscopy. We also designed BcPI-PLC constructs with minimal interfacial binding, and therefore low basal catalytic activity from the cytosol. Such modified enzymes were capable of rapidly hydrolyzing PI when recruited in the proximity of membrane-embedded substrate using the rapamycin-inducible heterodimerization system. Acute targeting of this recruitable BcPI-PLC to various organelles, together with detection of diacylglycerol (DAG), the product of PI hydrolysis could serve as a proxy to assess the PI content of that membrane. Using these tools, we showed that PI is localized to the endoplasmic reticulum (ER), the site of its synthesis, but is also enriched in the cytosolic leaflets of the Golgi complex, peroxisomes, and mitochondria. Strikingly, we did not find significant amounts of PI within the plasma membrane (PM) or in endosomal compartments in any of the mammalian cell types examined. As part of these studies, we also developed bioluminescence energy-transfer (BRET)-based biosensors to monitor organelle-specific PI, DAG, and PPIn dynamics at the level of cell populations and combined this method with the use of the recruitable BcPI-PLC construct to characterize the role of PI availability and supply for the generation of PPIn species within distinct membrane compartments. These studies reveal the explicit need for the sustained delivery of PI from the ER, rather than its absolute steady-state content for the maintenance of monophosphorylated PPIn species within the PM, Golgi complex, and endosomal compartments. Overall, our findings for the first time have mapped the PI distribution in mammalian cells and revealed a suspected yet never formally proven role for PI transfer and substrate channeling in the spatial control of PPIn metabolism. The importance of these studies is that using these new molecular tools researchers will be able to characterize the biochemical processes and their molecular machinery that are responsible for the transport of PI from the ER and how these molecules contribute to establish the proper lipid composition of organelle membranes. As mentioned above, viral replication requires host factors that many viruses hijack to establish their optimal membrane niche for their replication. Targeting these processes would be an effective means of combating virus infections and propagation. In a separate set of studies, we collaborated with the group of Dr. John Burke (University of Victoria, British Columbia, Canada) and Frank van Kuppeveld (University of Utrecht, The Netherlands) to understand the role of the enigmatic c10orf76 protein in viral replication with respect to its interaction with the lipid kinase, phosphatidylinositol 4-kinase B (PI4KB). PI4KB is a lipid kinase that generates phosphatidylinositol 4-phosphate (PI4P), a critical regulatory lipid in the Golgi complex. PI4KB has been identified as one of the essential host factors necessary for replication of a number of small picornaviruses in mammalian cells. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB-associated protein that increases PI4P levels at the Golgi and is essential for the viral replication of specific enteroviruses. In this collaborative effort, the Burke lab has used hydrogen-deuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex. These studies revealed that c10orf76 and PI4KB directly interact and binding is mediated by the kinase linker region of PI4KB, and that formation of the heterodimeric complex was modulated by protein kinase A (PKA)-dependent phosphorylation. Using mutant proteins that interrupt the association between PI4KB and c10orf76, we found that PI4KB is required for the recruitment of c10orf76 to the Golgi, but PI4KB will still bind to the Golgi, without c10orf76 protein. Dr. Kupeveld's group showed that while all enteroviruses require PI4KB for replication, replication of c10orf76dependent enteroviruses requires intact c10orf76PI4KB interaction, whereas viruses whose replication is independent of c10orf76 still require PI4KB but can replicate with PI4KB mutants that are unable to recruit c10-rf76. These studies also revealed that c10orf76 controls the small GTP binding protein Arf1 and this regulation is important to maintain the level of PI4P in the Golgi complex. The importance of these studies is that they characterized important protein components of human cells that are used by some viruses for their replication. This knowledge will be used to assess the host protein requirement for the replication of any future viruses that pose a health risk for the public.

在真核细胞中发生的每一个生化过程都依赖于分子信息流，该分子信息流来自受体，该信息一直通知细胞的环境，一直到确定适当细胞反应的分子效应子。适当的信息传输需要高度的组织，其中分子玩家被组织到不同的细胞室中，以便可以正确维护细胞反应的特异性。即使受影响的分子途径因癌症，糖尿病或神经退行性疾病等疾病的类型而异，该组织的崩溃是所有人类疾病的最终原因。 本报告中描述的研究集中在细胞如何组织其内部膜的问题上，以提供一个结构框架，分子信号传导复合物组装以确保信息处理。细胞膜的脂质组成是其生物物理特性的主要决定因素，并且是不同细胞细胞器的独特性。细胞如何实现和维持其膜的适当脂质组成的理解很少。影响细胞器的膜脂质组成的细胞过程通常是由病毒等细胞病原体靶向的，迫使细胞产生病原体，而不是执行细胞正常功能。更好地了解这些过程不仅可以提供与各种人类疾病作斗争的新策略，而且还可以拦截提供抗菌药物替代品的细胞病原体生命周期。 磷酸化的肌醇磷脂（PPIN）是一类磷脂的类别，其量很小，但在特定膜室上组织蛋白质信号复合物时具有非常重要的调节功能。它们是由磷酸肌醇激酶产生的，可以在主要类别的磷脂磷脂磷脂酰肌醇（PI）中磷酸化肌醇环的特定位置。尽管大多数PPIN的细胞定位已经进行了彻底研究和映射，但其更丰富的前体的细胞分布尚未确定。在这里，我们开发了新的分子工具，以获取有关完整活哺乳动物细胞中PI分布的信息，而不会破坏其膜完整性。使用可用于细菌Pi特异性磷脂酶C酶（PI-PLC）的结构信息，我们将蜡状芽孢杆菌的酶用作蛋白质工程的平台。蜡状芽孢杆菌（BC）PI-PLC显示出对PI的显着特异性，并且对磷酸化的PPIN物种或其他磷脂没有表现出催化活性。我们在保守的催化结构域中产生了该酶的突变形式，该酶靶向残基，该残基将废除酶促活性，但要在活性位点内维持底物配位，以便标记PI的细胞内分布。该突变体BC-PI-PLC融合到绿色荧光蛋白（GFP）和哺乳动物细胞中的表达允许通过共聚焦显微镜可视化蛋白质的分布。我们还设计了具有最小界面结合的BCPI-PLC构建体，因此来自细胞质的基底催化活性低。当使用雷帕霉素诱导的异二聚化系统募集在膜上包裹的底物中，这种修饰的酶能够快速水解Pi。将这种可募集的BCPI-PLC急性靶向各种细胞器，再加上二酰基甘油（DAG），PI水解的乘积可以作为评估该膜的PI含量的代理。使用这些工具，我们表明PI位于内质网（ER）（其合成部位），但也富含Golgi复合物，过氧化物酶体和线粒体的胞质小叶。引人注目的是，我们在质膜（PM）中没有发现大量的PI或在检查的任何哺乳动物细胞类型中的内体室中。作为这些研究的一部分，我们还开发了基于生物发光的能量转移（BRET）生物传感器，以监测细胞群体的细胞器特异性PI，DAG和PPIN动力学，并将这种方法与可募集的BCPI-PLC结构相结合，以使PPIN在ppin inters of Membrane commant cons的作用表征bcpi-plc构建体的作用。这些研究揭示了PI从ER中持续传递的明确需求，而不是其绝对稳态含量以维持PM，高尔基体复合物和内体室内的单磷酸化PPIN物种。总体而言，我们的发现首次绘制了哺乳动物细胞中的PI分布，并揭示了可疑但从未正式证明的PI转移和底物通道的作用在PPIN代谢的空间控制中。这些研究的重要性是，使用这些新的分子工具研究人员将能够表征生化过程及其分子机械，这些过程及其分子机制负责从ER中运输PI，以及这些分子如何促进细胞器膜的适当脂质组成。 如上所述，病毒复制需要许多病毒劫持的宿主因素来为其复制建立最佳的膜生态位。针对这些过程将是打击病毒感染和传播的有效手段。 In a separate set of studies, we collaborated with the group of Dr. John Burke (University of Victoria, British Columbia, Canada) and Frank van Kuppeveld (University of Utrecht, The Netherlands) to understand the role of the enigmatic c10orf76 protein in viral replication with respect to its interaction with the lipid kinase, phosphatidylinositol 4-kinase B (PI4KB). PI4KB是一种脂质激酶，可产生4-磷酸磷脂酰肌醇（PI4P），这是高尔基体复合物中的关键调节脂质。 PI4KB已被确定为复制哺乳动物细胞中许多小picornavires所必需的宿主因素之一。 PI4KB可以与多种蛋白质结合伴侣相互作用，这些蛋白质结合伴侣通过picornavires差异地操纵以促进复制。蛋白C10ORF76是一种与PI4KB相关的蛋白质，可在高尔基体上增加PI4P水平，对于特定肠病毒的病毒复制至关重要。在这项合作的工作中，伯克实验室使用氢 - 偏见交换质谱法来表征C10ORF76-PI4KB复合体。这些研究表明，C10ORF76和PI4KB直接相互作用和结合是由PI4KB的激酶接头区域介导的，并且异二聚体复合物的形成是由蛋白激酶A（PKA）依赖性磷酸化调节的。使用中断PI4KB和C10ORF76之间关联的突变蛋白，我们发现PI4KB是募集C10orf76与高尔基体募集所必需的，但是PI4KB仍将与GOLGI结合，没有C10orf76蛋白。 Kupeveld博士的小组表明，尽管所有肠病毒都需要PI4KB进行复制，但复制C10ORF76依赖性肠病毒需要完整的C10ORF76PI4KB相互作用，而该病毒的复制病毒复制与C10orf76的复制仍然需要PI4KB，但可以与PI4KB相关，但可以与PI4KB的co and-tos-kns and-kbb to and-kbb to and-kbb to and-kbb to and-kbb cocbb and knykB这些研究还表明，C10ORF76控制着小的GTP结合蛋白ARF1，该调节对于维持高尔基体复合物中的PI4P水平很重要。这些研究的重要性是它们表征了某些病毒用于复制的人类细胞的重要蛋白质成分。这些知识将用于评估宿主蛋白质的需求，以复制任何对公众构成健康风险的未来病毒。