CHARACTERIZATION OF THE NEURITE PHOSPHOPROTEOME

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 Klemke实验室的一个主要目标是了解控制生长锥和神经突形成的分子信号机制。 神经元细胞延伸和缩回神经突的能力对于适当的脑发育是重要的，并且对于损伤后的脊髓再生是重要的。 然而，研究这一过程一直很困难，因为还不可能通过生物化学方法分离神经突进行蛋白质分析。 最近，我们开发了一种新的生物化学方法，使用微孔技术从神经元细胞中大规模纯化神经突（BioTechniques. 2003; 35：254-256）。 这种新的系统将使我们能够进行大规模的蛋白质组学，以确定关键的调节蛋白，促进生长锥的形成和神经突的延伸和收缩，分别响应于化学引诱剂或化学排斥剂。 我们将使用小鼠和人类神经母细胞瘤细胞进行这些研究，因为它们容易伸展/收缩神经突。使用这些细胞的初步分析表明，磷酸酪氨酸（PY）蛋白是高度活化的，并在神经突馏分相比，索马丰富。 酪氨酸磷酸化的药理学抑制抑制生长锥形成和神经突延伸，表明复杂的信号级联通过PY网络的调节来控制该过程。 因此，我们的目标是表征PY蛋白（神经突磷酸化蛋白质组）负责生长锥的形成和神经突的延伸/收缩。 来自分离的胞体和延伸或收缩的神经突的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高灵敏度、高分辨率LC-MS/MS进行分析，以鉴定关键蛋白并确定磷酸化残基的具体位置。 然后使用siRNA蛋白敲低和定点诱变进行功能测试，然后进行基于细胞的测定和我们实验室建立的神经突形成动物模型。 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，以揭示潜在的磷酸酪氨酸网络，有助于脊髓损伤和神经元变性过程中的神经突形成。 我们的研究结果将为控制神经突形成的磷酸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蛋白及其酪氨酸磷酸化的特异位点。 2. 通过siRNA蛋白敲低和关键磷酸酪氨酸位点的定点诱变（通过MS鉴定），然后通过我们实验室建立的基于细胞的测定和神经突形成动物模型，对鉴定的PY蛋白进行功能性检测。 3. 利用生物信息学和计算机模拟系统对PY蛋白的信号通路进行定位，并建立PY蛋白之间的功能关系。 具体目标1的方法：将使用PNNL的高灵敏度和高分辨率LC-MS/MS对免疫纯化的神经突PY蛋白进行初始“自下而上”分析。免疫纯化的PY蛋白将进行胰蛋白酶消化、肽纯化和离线强阳离子交换分离，结合LC-MS/MS分析，以鉴定PY蛋白。 为了鉴定磷蛋白上的特异性酪氨酸磷酸化位点，将一部分胰蛋白酶消化物通过IMAC柱，以富集肽甲醚化后消化物中存在的磷酸肽。 然后通过LC-MS/MS分析该富含磷酸肽的样品，以确定磷酸肽的身份和磷酸化残基的具体位置，然后可以靶向磷酸化残基以实现具体目标#2：使用siRNA定向敲低方法和/或定点诱变策略对关键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分析从目标1和2获得的信息，以揭示有助于神经突形成的潜在磷酸酪氨酸网络，这对损伤后的适当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 the Klemke 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.