Neurochemistry and Genetics of Drosophila CaMKII

果蝇 CaMKII 的神经化学和遗传学

基本信息

批准号：
8465358
负责人：
Leslie C Griffith
金额：
$ 2.29万
依托单位：
BRANDEIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-05-01 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8465358
关键词：
5&apos Splice Site Address Adult Affect Affinity Alternative Splicing Animals Area Axon Behavior Behavioral Binding Biochemical Biochemical Pathway Biochemical Reaction Biological Assay C-terminal Calcium Cells Complementary DNA Complex Dendrites Drosophila genus Drug Delivery Systems Drug Design Ethers Gene Expression Genes Genetic Goals Homeostasis In Vitro Individual Ion Channel Ions Kinetics Knock-in Mouse Knock-out Location Mass Spectrum Analysis Measures Membrane Membrane Potentials Messenger RNA Modification Molecular Molecular Conformation Motor Motor output Multiprotein Complexes Mutate Mutation Nervous system structure Neurons Output Phosphorylation Phosphotransferases Potassium Channel Process Protein Binding Proteins RNA Editing RNA Interference Regulation Role Scaffolding Protein Sequence Analysis Signal Transduction Signaling Protein Site Solutions Structure Subcellular structure Synapses Synaptic plasticity System Testing Therapeutic Intervention Transgenes Transgenic Organisms Voltage-Gated Potassium Channel Work calmodulin-dependent protein kinase II density fly homologous recombination in vivo information processing insight mutant neurochemistry novel postsynaptic prevent public health relevance research study response scaffold

项目摘要

DESCRIPTION (provided by applicant): The computational power of the nervous system relies on the ability of individual cells to integrate multiple inputs and generate an output targeted to the appropriate partner(s). These functions would be impossible without the asymmetric localization of proteins within the cell. For example, many of the proteins that are in the axon are specialized for functions like transport and electrical conduction. In contrast, many of the proteins in the dendrites are specialized for receiving information from other cells. Restriction of localization is important for limiting the activity of a particular protein to a specific cellular compartment, but it can also be an important regulator of biochemical reactions. Many proteins behave differently when localized to a multiprotein complex due to the ability of direct binding partners to alter their conformation. This function of localization has important implications for how neurons process information that is relevant to many therapeutic interventions since it means that the behavior or activity of a protein cannot always be predicted from in vitro studies. This proposal has the goal of understanding how CaMKII and two proteins that directly bind to it, dCASK, a scaffold protein, and Eag, a potassium channel, are regulated by localization and cellular context. To achieve an understanding of how cellular context can influence the activity of these proteins we propose two sets of experiments. In Aim #1 we will use a transfected cell system to understand how dCASK and CaMKII interact to change the location, activity and potential substrates of CaMKII in response to calcium influx. We will do a parallel set of experiments in dCASK mutant transgenic flies that express a YFP tagged dCASK transgene that restores normal behavior. We will determine where in the cell dCASK has to reside and what proteins it has to complex with to carry out its behavioral function. Aim #2 addresses how Eag, a voltage-gated potassium channel, is localized and regulated by other proteins that bind to it. We have discovered a novel role for RNA editing in localization that has implications for many other genes that undergo this type of modification. We will also investigate the function of an alternative splice product of the eag gene that does not conduct ions, but instead functions as a scaffold for CaMKII and other signaling proteins. We address these issues by using homologous recombination to create mutations in the eag gene that selectively alter each of these processes and assess the impact on neuronal gene expression, excitability and behavior. PUBLIC HEALTH RELEVANCE: The vast majority of drug targets are proteins, many of which have enzymatic activity. While in many cases we have extensive information about structure at the atomic level or kinetic parameters in solution, a critical gap exists in our understanding of how proteins actually work within the cell. Understanding how the context of a protein's location can affect or regulate activity is critical to rational drug design and may even provide new drug targets.

描述（由申请人提供）：神经系统的计算能力取决于单个细胞整合多个输入并生成针对适当合作伙伴的输出的能力。如果没有细胞中蛋白质的不对称定位，这些功能将是不可能的。例如，轴突中的许多蛋白质专门用于运输和电导等功能。相反，树突中的许多蛋白质专门用于从其他细胞中接收信息。限制定位对于将特定蛋白质的活性限制为特定的细胞区室很重要，但它也可能是生化反应的重要调节剂。由于直接结合伴侣改变其构象的能力，许多蛋白质在本地化到多蛋白复合物中的行为不同。这种定位的功能对神经元处理与许多治疗干预措施相关的信息具有重要意义，因为这意味着不能总是从体外研究中预测蛋白质的行为或活性。该建议的目的是了解CaMKII和两个直接与之结合的蛋白如何通过定位和细胞环境调节钾通道的DCASK，脚手架蛋白和EAG。为了了解细胞环境如何影响这些蛋白质的活性，我们提出了两组实验。在AIM＃1中，我们将使用转染的细胞系统来了解DCASK和CAMKII如何相互作用以改变CaMKII的位置，活动和潜在底物，以响应钙的流入。我们将在表达YFP标记的DCASK转基因的DCASK突变体转基因蝇中进行一组平行的实验，以恢复正常行为。我们将确定细胞DCASK中必须驻留的位置，以及它必须与执行其行为功能的复杂蛋白质。 AIM＃2解决了电压门控钾通道EAG如何被其他与之结合的蛋白质所定位和调节。我们发现了RNA编辑在本地化中的新作用，该作用对许多其他经历这种修饰的基因具有影响。我们还将研究不导致离子的EAG基因的替代剪接产物的功能，而是作为CAMKII和其他信号蛋白的支架。我们通过使用同源重组来在EAG基因中创建突变来解决这些问题，从而选择性地改变了这些过程，并评估对神经元基因表达，兴奋性和行为的影响。公共卫生相关性：绝大多数药物靶标是蛋白质，其中许多具有酶活性。尽管在许多情况下，我们在解决方案中有有关原子水平或动力学参数的结构的广泛信息，但我们对蛋白质如何在细胞中实际工作的理解中存在关键差距。了解蛋白质位置的背景如何影响或调节活动对于合理的药物设计至关重要，甚至可能提供新的药物靶标。