Phosphoinositide-calcium Signaling In Cell Regulation

细胞调节中的磷酸肌醇-钙信号转导

• 批准号: 8553830
• 负责人: TAMAS BALLA
• 金额: $ 110.69万
• 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/8553830
• 关键词: 1-Phosphatidylinositol 4-Kinase; ARRB2; Acute; Adrenergic Agents; Affect; Angiotensins; Antiviral Agents; Apoptosis; Attenuated; Benefits and Risks; Binding; Biochemical Process; Calcium Signaling; Cell Differentiation process; Cell Proliferation; Cell Surface Receptors; Cell membrane; Cell physiology; Cells; Cellular Membrane; Clathrin; Cleaved cell; Collaborations; Commit; Complex; Data; Defect; Diabetes Mellitus; Disease; Early Endosome; Electrostatics; Elements; Endoplasmic Reticulum; Ensure; Environment; Enzymes; Eukaryotic Cell; Excision; Family; Focus Groups; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gaucher Disease; Golgi Apparatus; Growth Factor; Heating; Homeostasis; Hormones; Human; Human body; Hungary; Hydrolysis; Individual; Inositol; Intercept; Ion Channel; Laboratories; Life; Life Cycle Stages; Ligands; Lipids; Lysosomal Storage Diseases; Lysosomes; Malignant Neoplasms; Mammalian Cell; Mediating; Medicine; Membrane; Membrane Proteins; Membrane Transport Proteins; Menthol; Metabolic; Metabolism; Molecular; Multienzyme Complexes; Mutate; Names; Neurodegenerative Disorders; Neurotransmitters; Normal Cell; Oncogenic; PIK4CB gene; Pathway interactions; Peripheral; Pharmaceutical Preparations; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phospholipids; Phosphoric Monoester Hydrolases; Phosphotransferases; Physiological; Play; Process; Protein Kinase; Proteins; Proteomics; Receptor Protein-Tyrosine Kinases; Recruitment Activity; Regulation; Reporting; Research; Robin bird; Role; Signal Transduction; Signal Transduction Pathway; Signaling Protein; Specificity; Sphingolipids; Surface; System; Vesicle; Virus; Yeasts; adrenergic; antimicrobial drug; base; capsaicin receptor; cell growth regulation; cell motility; cellular targeting; design; fighting; glucosylceramidase; human disease; hybrid enzyme; information processing; inhibitor/antagonist; kinase inhibitor; novel; pathogen; phosphatidylinositol 4-phosphate; protein complex; ras Proteins; receptor; receptor internalization; receptor sensitivity; research study; response; tool; trafficking; transmission process

Every biochemical process that happens in an 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 with the structural framework on which molecular signaling complexes assemble to ensure proper information processing. These cellular processes 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. The first set of experiments focused on the question of how the beta-glucocerebrosidase enzyme (GBA) reaches the lysosomes. Defects in the function of this enzyme cause Gaucher disease, one of the most common human lysosomal storage diseases. GBA is synthesized in the endoplasmic reticulum (ER) and binds to a receptor protein, called LIMP2 that carries GBA all the way to the lysosome. In a proteomic analysis both GBA and LIMP2 was found in a complex with one of the phosphatidylinositol kinase enzymes, PI4KB. Inositol lipids are a small fraction of membrane phospholipids with important regulatory functions and we determined whether phosphatidylinositol 4-phosphate (PI4P), the lipid product of PI4KB, is important for LIMP2/GBA transport to the lysosome. We found that distinct phosphatidylinositol 4-kinases (PI4Ks) play important roles at multiple steps in the trafficking pathway of the LIMP-2/GBA complex. Acute depletion of phosphatidylinositol 4-phosphate in the Golgi caused accumulation of LIMP-2 in this compartment, and PI4KB was found to be responsible for controlling the exit of LIMP-2 from the Golgi. In contrast, depletion of PI4K2A, another PI4K, blocked trafficking at a post-Golgi compartment, leading to accumulation of LIMP-2 in enlarged endosomal vesicles. PI4K2A depletion also caused secretion of missorted GBA into the medium, which was attenuated by limiting LIMP-2/GBA exit from the Golgi by PI4KB inhibitors. These studies identified PI4KB and PI4K2A as important regulators of lysosomal delivery of GBA, revealing a new element of control to sphingolipid homeostasis by phosphoinositides. The importance of these studies is that PI4P and sphingolipids are not only critical for cellular functions, but also for replication of certain viruses within the cell. Several Pharma companies are developing PI4K inhibitors as potential antiviral agents. It is critically important that we understand what processes are controlled by these enzymes in the cell to properly evaluate the potential risks and benefits from the use of such inhibitors. In a second set of studies the role of plasma membrane phosphoinositides, PI4P and PI(4,5)P2 was studied in cellular regulation. PI4P is a metabolic precursor of PI(4,5)P2 in the plasma membrane and the two lipids often change in parallel during physiological stimulation of cell surface receptors. To be able to alter the levels of these two lipids separately, a novel molecular tool was developed, which consisted of a hybrid enzyme formed from a PI(4,5)P2 5-phosphatase and the yeast Sac1 phosphatase that hydrolyses PI4P. This hybrid enzyme, called pseudojanin (PJ), was acutely recruited to the plasma membrane by a drug-induced heterodimerizetion system developed in our laboratory. Versions of PJ with defective 5-phosphatase or 4-phosphatase activities (or lacking both activities) were generated and with these tools PI4P and PI(4,5)P2 levels could be selectively manipulated in a rapidly controlled manner. These studies showed that cells are able to maintain their PI(4,5)P2 pools at a wide range of PI4P levels provided that the PI4K enzyme(s) that generate PI4P at the plasma membrane are functional. However, PI4P in the plasma membrane turned out to play a very important role in supplying an electrostatic interaction that is required for proper functions of some ion channels and for the membrane targeting of a whole set of peripheral membrane proteins such as several small GTP binding proteins. These studies uncovered an important and hitherto unrecognized role of PI4P at the plasma membrane, namely its ability to fulfill functions that had been traditionally attributed to PI(4,5)P2. It is also notable that differences exist between proteins that selectively require PI(4,5)P2 (such as the cold-sensing menthol channels) and those that can use either lipids to support their functions (such as the heat-sensing vanilloid receptors). These studies were done in close collaboration with Dr. Robin Irvines group in Cambridge, UK. The importance of these studies is that they help us better understand how some of the most important regulators of cell proliferation, such as the Ras proteins that are mutated in a significant number of cancers bind to the plasma membrane, since membrane binding is absolutely critical for their normal and oncogenic activities. In related studies using similar approaches performed in collaboration with Dr. Peter Varnais group in Budapest, Hungary, the PI(4,5)P2 requirement of G protein-coupled receptor internalization was analyzed using AT1 angiotensin, beta-2 adrenergic and type-2C serotoninergic receptors. These studies showed that ligand-induced interaction of AT1, 5HT2C and beta2A receptors with beta-arrestin-2 was unaffected by PI(4,5)P2 depletion. However, trafficking of the receptors to Rab5-positive early endosomes was completely abolished in the absence of PI(4,5)P2. Remarkably, removal of the receptors from the plasma membrane was reduced but not eliminated after PI(4,5)P2 depletion and here the stimulated AT1 receptors clustered along the plasma membrane without entering the cells. These data suggest that in the absence of PI(4,5)P2 these receptors move into clathrin-coated membrane structures that are not cleaved efficiently and hence cannot reach the early endosomal compartment. The significance of these studies is that G protein-coupled receptors regulate almost every process in the human body and they are targeted by a large fraction of human medicines. Cells regulate the sensitivity of these receptors by the very processes that are the focus of these studies and better understanding of these pathways will help us decipher disease mechanisms and design better drugs.

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，这些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，以提供结构框架，该结构框架在哪种分子信号传导复合物组装以确保适当的信息处理的问题上。 这些细胞过程通常由细胞病原体（例如病毒）靶向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。 第一组实验的重点是β-葡萄糖核苷酶（GBA）如何达到溶酶体的问题。 该酶功能的缺陷引起的Gaucher病，Gaucher病是最常见的人类溶酶体储存疾病之一。 GBA在内质网（ER）中合成，并与受体蛋白结合，称为LIMP2，该蛋白将GBA一直携带到溶酶体。在蛋白质组学分析中，GBA和LIMP2都在与一种磷脂酰肌醇激酶酶PI4KB的复合物中发现。 肌醇脂质是具有重要调节功能的膜磷脂的一小部分，我们确定PI4KB的脂质产物（PI4P）是否对LIMP2/GBA转运至溶酶体很重要。我们发现，在LIMP-2/GBA复合物的运输途径中，不同的磷脂酰肌醇4-激酶（PI4K）在多个步骤中起着重要作用。高尔基体中磷脂酰肌醇4-磷酸的急性耗竭导致LIMP-2在该室中的积累，并且发现PI4KB被发现负责控制高尔基体的Limp-2出口。 相比之下，另一个PI4K PI4K2A的耗尽阻止了贩运后高尔基腔室的贩运，导致内体囊泡扩大的LIMP-2积累。 PI4K2A的耗竭也导致遗体GBA分泌到该培养基中，通过PI4KB抑制剂限制了从高尔基体退出LIMP-2/GBA，这会减弱。这些研究将PI4KB和PI4K2A确定为GBA溶酶体递送的重要调节剂，揭示了磷酸肌醇对鞘脂稳态的新元素。 这些研究的重要性在于，PI4P和鞘脂不仅对细胞功能至关重要，而且对于细胞内某些病毒的复制也至关重要。 几家制药公司正在开发PI4K抑制剂作为潜在的抗病毒剂。 至关重要的是，我们了解细胞中这些酶控制哪些过程，以正确评估使用这种抑制剂的潜在风险和益处。 在第二组研究中，在细胞调节中研究了质膜磷酸肌醇，PI4P和PI（4,5）P2的作用。 PI4P是质膜中PI（4,5）P2的代谢前体，在细胞表面受体的生理刺激期间，这两种脂质经常平行地变化。 为了能够分别改变这两种脂质的水平，开发了一种新型的分子工具，该工具由由PI（4,5）P2 5-磷酸酶和酵母SAC1磷酸酶形成的杂化酶组成，该酶的水解PI4P。 这种称为假果宁（PJ）的杂化酶被我们实验室中开发的药物诱导的异二酰胺促性系统急剧募集到质膜上。 生成具有缺陷的5磷酸酶或4-磷酸酶活性（或缺乏两种活动）的PJ版本，使用这些工具，可以以快速控制的方式选择性地操纵PI4P和PI（4,5）P2水平。 这些研究表明，细胞能够将其PI（4,5）P2池保持在广泛的PI4P水平上，前提是在质膜上产生PI4P的PI4K酶是功能的。 但是，质膜中的PI4P在提供某些离子通道的正确功能以及整个外围膜蛋白（例如几种小GTP结合蛋白）的膜靶向的膜靶向中所需的静电相互作用中起着非常重要的作用。 这些研究发现了PI4P在质膜上的重要且迄今未识别的作用，即其实现传统上归因于PI（4,5）P2的功能的能力。 还值得注意的是，有选择地需要PI（4,5）P2（例如冷感应薄荷醇通道）的蛋白质存在差异，以及那些可以使用任何一种脂质支持其功能的蛋白质（例如热感应香草片受体）。这些研究是与英国剑桥的Robin Irvines Group密切合作进行的。 这些研究的重要性是，它们有助于我们更好地理解一些最重要的细胞增殖调节剂，例如在大量癌症中突变的RAS蛋白与质膜结合，因为膜结合对其正常和致癌活性绝对至关重要。 在相关研究中，使用与匈牙利布达佩斯的彼得·瓦纳（Peter Varnais）组合作执行的类似方法，使用AT1血管紧张素，Beta-2肾上腺素能和2C羟色胺素受体分析了G蛋白偶联受体内在的PI（4,5）P2 P2要求。这些研究表明，配体诱导的AT1，5HT2C和BETA2A受体与β-arrestin-2的相互作用不受PI（4,5）P2耗竭影响。然而，在没有PI（4,5）P2的情况下，完全废除了将受体运输到Rab5阳性早期内体上。值得注意的是，从质膜中去除受体，但在Pi（4,5）P2耗竭后没有消除受体，在这里沿着质膜聚集的刺激的AT1受体而无需进入细胞。这些数据表明，在没有PI（4,5）P2的情况下，这些受体转移到网状蛋白包被的膜结构中，这些结构没有有效裂解，因此无法达到早期内体室。 这些研究的意义在于，G蛋白偶联受体几乎调节人体中的每个过程，并且它们是大量人类药物的目标。 细胞通过这些研究重点的过程来调节这些受体的敏感性，并且对这些途径的更好理解将有助于我们破译疾病机制并设计更好的药物。