Proteomic Assays of Neuronal Protein Palmitoylation

神经元蛋白棕榈酰化的蛋白质组学测定

• 批准号: 7839651
• 负责人: WILLIAM GREEN
• 金额: $ 22.19万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-04 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7839651
• 关键词: Address; Alzheimer' s Disease; Amphetamines; Biological Assay; Biology; Brain; Cysteine; Development; Drug Addiction; Drug abuse; Enzymes; Fatty Acids; Fluorescence; Gel; Glutamate Receptor; Goals; Huntington Disease; Hydroxylamine; Label; Laboratories; Lipids; Long-Term Potentiation; Maleimides; Mass Spectrum Analysis; Measures; Membrane; Membrane Microdomains; Metabolic; Methods; Modification; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; Nerve Tissue; Nervous system structure; Neurons; Neurotransmitter Receptor; Nicotinic Receptors; Nucleus Accumbens; Palmitates; Palmitic Acylation Site; Play; Post-Translational Protein Processing; Preparation; Process; Proteins; Proteome; Proteomics; Protocols documentation; Rat Protein; Rattus; Reaction; Reagent; Regulation; Research; Role; Schizophrenia; Serotonin; Side; Solutions; Synapses; Techniques; Technology; Testing; Time; base; behavioral sensitization; gel electrophoresis; in vivo; innovation; insight; nervous system disorder; neuron development; palmitoylation; protein complex; public health relevance; receptor; synaptic function; synaptogenesis; thioester

DESCRIPTION (provided by applicant): Evidence is mounting that the post-translational process, palmitoylation, has a major role in neuronal development and function, in particular, the formation and functioning of synapses. The standard methods for assaying protein palmitoylation are relatively insensitive, not quantitative, as well as laborious. Given these difficulties, it is apparent that many palmitoylated proteins remained unidentified. We have recently developed alternative assays of protein palmitoylation based on hydroxylamine cleavage of the thioester bond between the fatty acid and cysteine side chain followed by reaction of the newly generated free sulfhydryl with sulfhydryl-specific reagents, such as 3H-N-ethyl maleimide (NEM) and biotinylated reagents. These techniques are significantly more sensitive than metabolic labeling with 3H-palmitate and can be used quantitatively to measure levels of protein palmitoylation. They have the additional advantage of allowing protein palmitoylation to be assayed on preparations from brain and other nervous tissue, which is not possible using [3H]-palmitate labeling. The goal of this application is to adapt our new assays for palmitoylation to a proteomic scale in order to accomplish the following two specific aims. First, we propose to modify our techniques in order to identify the specific sites of palmitoylation on the different ionotropic glutamate receptors we are currently studying in our laboratory and all of which we have found to be palmitoylated. Second, we are developing protocols that allow a set of palmitoylated proteins to be specifically purified from highly complex protein extracts in order to quantify protein palmitoylation differences in neuronal preparations. To test these techniques, two different preparations will be analyzed. First, we will isolate palmitoylated proteins from rat cultured cortical neurons and assay for changes in their palmitoylation when long-term potentiation (LTP) has been chemically induced. In the second preparation, we will isolate palmitoylated proteins from rat nucleus accumbens punches and test for changes in their palmitoylation with amphetamine exposure in order to determine how behavioral sensitization alters palmitoylation. The proposed research is innovative because it will develop new proteomic-based technology to assay a protein post-translational modification that should provide new insights into basic synaptic biology and the neurotransmitter receptors, such as gluatamate receptors that are involved in drug abuse. PUBLIC HEALTH RELEVANCE: Protein palmitoylation is a reversible lipid modification on cysteine (Cys) residues and is involved in targeting proteins to membrane domains. The regulation of this modification has been shown to play a significant role in synapse formation and function. The large scale analysis of palmitoylated proteins will provide a comprehensive view of the scope of this modification by identifying many unknown protein targets, which will promote the development of subsequent hypothesis-driven research.

描述（由申请人提供）：越来越多的证据表明，翻译后过程棕榈酰化在神经元发育和功能，特别是突触的形成和功能中具有重要作用。用于测定蛋白质棕榈酰化的标准方法相对不敏感，不能定量，并且费力。考虑到这些困难，很明显，许多棕榈酰化蛋白质仍未被鉴定。我们最近开发了基于脂肪酸和半胱氨酸侧链之间的硫酯键的羟胺裂解的蛋白质棕榈酰化的替代测定，随后新产生的游离巯基与巯基特异性试剂（如3 H-N-乙基马来酰亚胺（NEM）和生物素化试剂）反应。这些技术比用3 H-棕榈酸酯的代谢标记显著更灵敏，并且可以定量地用于测量蛋白质棕榈酰化的水平。它们具有允许在来自脑和其他神经组织的制备物上测定蛋白质棕榈酰化的额外优点，这是使用[3 H]-棕榈酸酯标记不可能的。本申请的目标是使我们的棕榈酰化新测定适应蛋白质组学规模，以实现以下两个特定目标。首先，我们建议修改我们的技术，以确定我们目前正在我们的实验室研究的不同离子型谷氨酸受体上的棕榈酰化的特定位点，我们发现所有这些都是棕榈酰化的。第二，我们正在开发的协议，允许一组棕榈酰化的蛋白质被专门从高度复杂的蛋白质提取物中纯化，以量化神经元制剂中的蛋白质棕榈酰化差异。为了测试这些技术，将分析两种不同的制备物。首先，我们将从大鼠培养的皮层神经元中分离棕榈酰化蛋白，并测定长时程增强（LTP）被化学诱导时棕榈酰化的变化。在第二个准备中，我们将从大鼠核内分离棕榈酰化蛋白质，并测试其棕榈酰化与安非他明暴露的变化，以确定行为敏化如何改变棕榈酰化。拟议的研究是创新的，因为它将开发新的基于蛋白质组学的技术来分析蛋白质翻译后修饰，这应该为基本突触生物学和神经递质受体提供新的见解，例如参与药物滥用的谷氨酸受体。 公共卫生相关性：蛋白质棕榈酰化是半胱氨酸（Cys）残基上的可逆脂质修饰，并且参与将蛋白质靶向膜结构域。这种修饰的调节已被证明在突触形成和功能中起重要作用。对棕榈酰化蛋白的大规模分析将通过识别许多未知的蛋白质靶点来全面了解这种修饰的范围，这将促进后续假设驱动研究的发展。