CHARACTERIZATION OF THE NEURITE PHOSPHOPROTEOME

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我的实验室的一个主要目标是了解控制生长锥和轴突形成的分子信号机制。神经元细胞伸展和收缩轴突的能力对于大脑的正常发育和脊髓损伤后的再生都是重要的。然而，对这一过程的研究一直很困难，因为还不可能通过生物化学方法分离轴突来进行蛋白质分析。最近，我们开发了一种新的生化方法，利用微孔技术从神经元细胞中大规模纯化轴突(BioTechniques)。2003年；35：254-256)。这一新的系统将使我们能够进行大规模的蛋白质组学，以确定分别促进生长锥形成和轴突延伸和收缩的关键调控蛋白，以响应化学诱导剂或化学排斥剂。我们将使用小鼠和人类神经母细胞瘤细胞进行这些研究，因为它们很容易伸展/收缩轴突。使用这些细胞的初步分析表明，与胞体相比，磷酸酪氨酸(PY)蛋白在突起部分高度激活和丰富。药物抑制酪氨酸磷酸化可以抑制生长锥的形成和轴突的延伸，这表明复杂的信号级联通过调节PY网络来控制这一过程。因此，我们的目标是确定与生长锥形成和轴突伸展/收缩有关的PY蛋白(轴突磷酸蛋白质组)。从分离的SoMAS和延伸或收回的轴突中提取的PY蛋白将用抗PY抗体进行免疫亲和纯化和/或使用IMac柱富集磷酸肽，然后使用NCRR高灵敏、高分辨率的LC-MS/MS分析关键蛋白和确定磷酸化残基的特定位置。 然后将使用siRNA蛋白敲除和定点突变进行功能测试，然后进行基于细胞的分析和我们实验室建立的轴突形成的动物模型。从这些实验中获得的信息将使用生物信息学和计算机建模进行分析，以揭示在脊髓损伤和神经元退化过程中促成轴突形成的潜在磷酸酪氨酸网络。我们的研究结果将为控制轴突形成的磷信号提供有价值的信息，并为神经退行性疾病的治疗和脊髓再生提供靶点。 具体目标： 1.鉴定PY蛋白及其在突起伸缩过程中酪氨酸磷酸化的特异性部位。 2.利用siRNA蛋白敲除和关键磷酸酪氨酸位点定点突变(经MS鉴定)对已鉴定的PY蛋白进行功能测试，然后建立基于细胞的分析和本实验室建立的神经突起形成的动物模型。 3.利用生物信息学和计算机模拟系统绘制PY蛋白之间可能的信号级联，并建立其功能关系。 方法1：利用PNNL的高灵敏度和高分辨率LC-MS/MS对免疫纯化的PY蛋白进行初步的“自下而上”分析，免疫纯化的PY蛋白将经过胰酶消化、多肽纯化和离线强阳离子交换分离与LC-MS/MS分析相结合，以鉴定PY蛋白。为了确定磷酸蛋白上的特定酪氨酸磷酸化位点，部分胰酶消化将通过IMAC柱，以在多肽甲醚化后富集消化中存在的磷酸肽。然后用LC-MS/MS分析这个富含磷酸肽的样品，以确定磷酸化残基的特性和特定位置，然后可以针对这些位置实现特定目标#2：使用siRNA导向的敲除方法和/或定点突变策略对关键的PY蛋白进行功能测试。然后，将使用生物信息学和计算机建模对从AIMS 1和2获得的信息进行分析，以揭示有助于轴突形成的潜在磷酸酪氨酸网络，这对损伤后正常的大脑发育和脊髓再生非常重要。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A major goal of my laboratory is to understand the molecular signaling mechanisms that control growth cone and neurite formation. The ability of neuronal cells to extend and retract neurites is important for proper brain development and is important for spinal cord regeneration after injury. However, it has been difficult to study this process because it has not been possible to biochemically isolate the neurite for protein analysis. Recently, we developed a new biochemical method using microporous technology to purify the neurite in large scale from neuronal cells (BioTechniques. 2003; 35:254-256). This novel system will allow us to perform large-scale proteomics to identify the key regulatory proteins that facilitate growth cone formation and neurite extension and retraction in response to chemoattractants or chemorepulsion agents, respectively. We will use mouse and human neuroblastoma cells for these studies as they readily extend/retract neurites. Initial analysis using these cells revealed that phosphotyrosine (PY) proteins are highly activated and enriched in the neurite fraction compared to the soma. Pharmacological inhibition of tyrosine phosphorylation inhibits growth cone formation and neurite extension indicating that complex signaling cascades control this process through modulation of PY networks. Therefore, our objective is to characterize the PY proteins (neurite phosphoproteome) responsible for growth cone formation and neurite extension/retraction. PY proteins from isolated somas and extending or retracting neurites will be immunoaffinity purified with anti-PY antibodies and/or enriched for phosphopeptides using an IMAC column and then analyzed using the NCRR high sensitivity, high resolution LC-MS/MS to identify key proteins and determine the specific locations of the phosphorylated residues. Functional testing will then be performed using siRNA protein knockdown and site directed mutagenesis followed by cell-based assays and animal models of neurite formation established in our laboratory. Information gained from these experiments will be analyzed using bioinformatics and computer modeling to reveal potential phosphotyrosine networks that contribute to neurite formation during spinal cord injury and neuronal degeneration. Results from our study will provide valuable information on the phosphosignals that control neurite formation and provide targets for therapeutic treatment of neurodegenerative diseases as well as spinal cord regeneration. Specific Aims: 1. To identify PY proteins and their specific sites of tyrosine phosphorylation in extending or retracting neurites. 2. To functionally test identified PY proteins using siRNA protein knockdown and site directed mutagenesis of key phosphotyrosine sites (as identified by MS) followed by cell-based assays and animal models of neurite formation established in our laboratory. 3. To map the putative signaling cascades and develop functional relationships among the PY proteins using bioinformatics and computer modeling systems. Methods for Specific Aim 1: An initial "bottom-up" analysis of immunopurified neurite PY proteins will be performed using PNNL's high sensitivity and high resolution LC-MS/MS. Immunopurified PY proteins will be subjected to tryptic digestion, peptide purification, and an off-line strong cation exchange separation coupled to LC-MS/MS analysis for the identification of PY proteins. To identify the specific tyrosine phosphorylation sites on the phosphoproteins, a portion of the tryptic digest will be passed through an IMAC column to enrich for phosphopeptides present in the digest following methyl etherification of the peptides. This phosphopeptide enriched sample will then be analyzed by LC-MS/MS to determine the identities of the phosphopeptides and the specific locations of the phosphorylated residues which can then be targeted for achieving specific aim #2: functional testing of key PY proteins using siRNA-directed knockdown approaches and/or site directed mutagenesis strategies. Information gained from aims 1 and 2 will then be analyzed using bioinformatics and computer modeling to reveal potential phosphotyrosine networks that contribute to neurite formation, which is important for proper brain development and spinal cord regeneration after injury.