ROLE OF THE NUCLEAR MATRIX PROTEIN NRB/B IN BRAIN

核基质蛋白 NRB/B 在大脑中的作用

• 批准号: 6198568
• 负责人: SHALOM AVRAHAM
• 金额: $ 30.45万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6198568
• 关键词: DNA binding protein; brain; cell cycle; gene expression; laboratory rat; microinjections; nerve /myelin protein; neurochemistry; neurogenesis; nuclear matrix; phosphorylation; protein localization; protein structure function; tissue /cell culture

DESCRIPTION(Verbatim from Applicant's Abstract): The nuclear matrix is defined as the insoluble framework of the nucleus and has been implicated in modulating gene expression, the cell cycle and nuclear structural integrity via linkage to intermediate filaments of the cytoskeleton. We have discovered and characterized a novel neuronal nuclear matrix protein, NRP/B (nuclear restricted protein/brain), which contains two major structural elements: a BTB domain-like structure in the predicted N-terminus, and a "kelch motif" in the predicted C-terminal domain. NRP/B mRNA (5.5 kb) is predominantly expressed in human fetal and adult brain. During mouse embryogenesis, NRP/B mRNA expression is upregulated in the nervous system. The NRP/B protein is also expressed in rat primary hippocampal neurons, but not in primary astrocytes. During the differentiation of murine Neuro 2A and human SH-SY5Y neuroblastoma cells, NRP/B protein expression is upregulated. Overexpression of NRP/B in these cells significantly induces growth arrest and augments neuronal process formation, while treatment with antisense NRP/B oligodeoxynucleotides inhibits neurite development of rat primary hippocampal neurons as well as neuronal process formation during differentiation of SH-SY5Y and Neuro 2A neuroblastoma cells. In addition, we have observed that microinjection of anti-NRP/B antibodies inhibited neurite outgrowth of PC12 cells in response to NGF stimulation, while microinjection of NRP/B cDNA promoted the neurite outgrowth of these cells. Both in vivo and in vitro experiments demonstrate that NRP/B can be phosphorylated and bind to the functionally active hypophosphorylated form of p110RB during neuronal differentiation of SH-SY5Y neuroblastoma cells induced by retinoic acid. The interaction of NRP/B with p110RB is mediated via the BTB/POZ domain of NRP/B. Furthermore, direct association of NRP/B with nuclear actin was observed, and this association is mediated via the "kelch" repeats. These data demonstrate that NRP/B is a novel nuclear matrix protein, specifically expressed in primary neurons, that interacts with p110RB and participates in the regulation of neuronal process formation. We aim to further characterize this novel neuronal nuclear matrix protein with regard to its role in regulatory pathways that control neuronal differentiation. We propose to specifically address: (1) the biological function(s) of the NRP/B protein; (2) the DNA binding properties of NRP/B and its structure/function relationships; and (3) the characterization of NRP/B protein during neuronal growth and differentiation. These studies should provide novel insights into the role of this nuclear matrix protein in neuronal differentiation.

描述（来自申请人摘要的逐字描述）：定义了核基质 作为细胞核的不溶性框架，并参与调节 基因表达，细胞周期和核结构的完整性，通过连接到 细胞骨架的中间丝。我们发现， NRP/B是一种新的神经元核基质蛋白， 限制性蛋白质/脑），其包含两个主要结构元件：BTB 在预测的N-末端的结构域样结构，和“kelch基序”， 预测的C-末端结构域。NRP/B mRNA（5.5 kb）主要在 人类胎儿和成人大脑。在小鼠胚胎发育过程中，NRP/B mRNA表达 在神经系统中上调。NRP/B蛋白也表达于 大鼠原代海马神经元，但不在原代星形胶质细胞中。期间 小鼠Neuro 2A和人SH-SY 5 Y神经母细胞瘤细胞NRP/B的分化 蛋白质表达上调。这些细胞中NRP/B的过表达 显著诱导生长停滞并增强神经元突起形成， 而反义NRP/B寡脱氧核苷酸处理抑制神经突 大鼠原代海马神经元及神经突起的发育 SH-SY 5 Y和Neuro 2A神经母细胞瘤细胞分化期间形成。 此外，我们还观察到，显微注射抗NRP/B抗体 抑制PC 12细胞对NGF刺激的神经突起生长， 显微注射NRP/B cDNA可促进这些细胞突起的生长。 体内和体外实验均表明，NRP/B可被 磷酸化并结合功能活性的低磷酸化形式的 p110 RB在SH-SY 5 Y神经母细胞瘤细胞神经元分化过程中的作用 视黄酸NRP/B与p110 RB的相互作用是通过 NRP/B的BTB/POZ结构域。此外，NRP/B与细胞核的直接结合， 观察到肌动蛋白，这种结合是通过“Kelch”重复介导的。 这些数据表明NRP/B是一种新的核基质蛋白， 在原代神经元中特异性表达，与p110 RB相互作用， 参与神经元突起形成的调节。 我们的目标是进一步表征这种新的神经元核基质蛋白， 关于它在控制神经元的调节途径中的作用， 分化我们建议具体解决：（1）生物 NRP/B蛋白的功能;（2）NRP/B的DNA结合特性， NRP/B的结构与功能关系 神经元生长和分化过程中的蛋白质。这些研究应 提供了新的见解，这种核基质蛋白的作用，在神经元 分化