Proteomic Assays of Neuronal Protein Palmitoylation

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起着重要作用。测定蛋白棕榈酰化的标准方法相对不敏感，不定量，而且费力。鉴于这些困难，很明显，许多棕榈酰化蛋白仍未被识别。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切割，以及新生成的游离巯基与巯基特异性试剂（如3h - n -乙基马来酰亚胺（NEM）和生物素化试剂）反应的蛋白质棕榈酰化的替代分析方法。这些技术明显比3h -棕榈酸酯代谢标记更敏感，可用于定量测量蛋白棕榈酰化水平。它们还有一个额外的优点，即允许在大脑和其他神经组织的制剂中检测蛋白质棕榈酰化，而使用[3H]-棕榈酸盐标记是不可能的。本应用程序的目的是适应我们的新测定棕榈酰化到蛋白质组学规模，以实现以下两个具体目标。首先，我们建议修改我们的技术，以确定我们目前在实验室研究的不同嗜电性谷氨酸受体上棕榈酰化的特定位点，并且我们发现所有这些受体都被棕榈酰化了。其次，我们正在开发一种方案，允许从高度复杂的蛋白质提取物中特异性纯化一组棕榈酰化蛋白，以量化神经元制备中蛋白质棕榈酰化的差异。为了测试这些技术，我们将分析两种不同的制剂。首先，我们将从培养的大鼠皮质神经元中分离棕榈酰化蛋白，并测定化学诱导长期增强（LTP）时棕榈酰化的变化。在第二种制备中，我们将从大鼠伏隔核中分离棕榈酰化蛋白，并测试其棕榈酰化随安非他明暴露的变化，以确定行为致敏如何改变棕榈酰化。这项提议的研究具有创新性，因为它将开发新的基于蛋白质组学的技术来检测蛋白质翻译后修饰，这将为基本突触生物学和神经递质受体（如与药物滥用有关的谷氨酸受体）提供新的见解。

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.