ROLES OF THE MYELIN BASIC PROTEINS IN MYELINOGENESIS

髓鞘碱性蛋白在髓鞘形成中的作用

• 批准号: 3418016
• 负责人: DAVID R COLMAN
• 金额: $ 17.62万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3418016
• 关键词: binding proteins; complementary DNA; developmental neurobiology; gene expression; genetic manipulation; genetic mapping; immunocytochemistry; immunoelectron microscopy; laboratory mouse; myelin; myelin basic proteins; oligodendroglia; protein sequence; protein structure function; radiotracer; site directed mutagenesis; tissue /cell culture; transfection /expression vector

Our long range goal is to understand the molecular mechanisms that underlie the formation of the mature myelin sheath, in the realization that this may suggest ways by which the repair process can be encouraged after myelin destruction. An essential step towards achievement of this goal is to precisely define the functional roles played by the myelin basic proteins (MBPs) over the course of the myelinogenesis program that is implemented by the oligodendrocyte. For many of the studies outlined below, we will rely on the cDNA transfection systems we have developed and refined over the past two years, that employ specific MBP antisera and confocal immunomicroscopy to map intracellular distributions of the expressed proteins. Our specific aims are: I.To identify the functional domains within the 14K and 18.5K MBPs that mediate association of these isoforms with the plasma membrane. Using site-directed mutagenesis, we will (1) delete internal segments or (2) truncate the N or C terminus of the 14K and 18.5K MBP cDNAs, and express the mutated constructs in non-glial cells, and in shiverer oligodendrocytes. the intracellular distribution of each mutated polypeptide will be mapped immunocytochemically to determine minimum polypeptide lengths and sequences required for membrane association. In parallel, (3) the mutated polypeptides will be expressed and tested in biochemical and ultrastructural experiments for the capacity to bind to and aggregate oriented vesicles derived from shiverer brain plasma membranes. II.To begin to explore the mechanisms by which the exon II-containing 21.5K and 17K MBPs are translocated from their site of synthesis in the cell cytoplasm across the nuclear pore complex into the nuclear matrix. In non-glial cells, and in shiverer mouse oligodendrocytes transfected with the 21.5K or 17K MBP cDNAs, the expressed proteins are found within the cytoplasm and nucleoplasm. Significantly, in the normal developing mouse brain, oligodendrocytes are readily detected whose nuclei contain high concentrations of MBPs. In many cell types, proteins that are translocated into the nucleus contain elements within their primary amino acid sequences that act as nuclear localization signals (NLSs) which enable selective entry through the nuclear pore complex into the nuclear compartment where the translocated proteins can exert regulatory functions. We have identified a region in the exon II-containing MBP isoforms that bears strong homology to known NLSs. We will test the capacity of this, and other sequences in these MBPs to function as authentic NLSs in transient transfectants. Three criteria that are used to rigorously define NLSs in other systems will be applied: (1) NLS-mediated entry of MBP into the nucleus is likely to be an energy-requiring process, and therefore should be sensitive to disruption by (a) chilling, or (b) ATP depletion; (2) deletion or mutation of MBP NLS sequence(s) should cause cytoplasmic accumulation of the mutated protein; and (3) when the putative MBP NLS sequence is engineered into an unrelated non-nuclear protein, such as bovine serum albumin, the chimeric protein should be translocated into the nucleus.

我们的远距离目标是了解基于的分子机制 成熟的髓鞘的形成，意识到这可能 建议在髓线后鼓励维修过程的方法 破坏。 实现这一目标的重要一步是 精确定义了髓磷脂碱性蛋白扮演的功能角色 （Mbps）在骨髓生成计划的过程中 少突胶质细胞。 对于以下概述的许多研究，我们将依靠 在cDNA转染系统上，我们已经开发并完善了 过去两年，使用特定的MBP抗血清和共焦 免疫显微镜以绘制表达的细胞内分布 蛋白质。 我们的具体目的是： I.确定14K和18.5K Mbps中的功能域 这些同工型与质膜的介导关联。 使用定向诱变，我们将（1）删除内部段或 （2）截断14K和18.5K MBP cDNA的N或C末端，以及 在非胶质细胞中表达突变的构建体和颤抖者 少突胶质细胞。 每个突变的细胞内分布 多肽将通过免疫细胞化学映射以确定最小 膜关联所需的多肽长度和序列。 在 平行，（3）突变的多肽将在 生化和超微结构实验的结合能力和 源自颤抖的脑质膜的聚集囊泡。 II.开始探索含外II的21.5K的机制 和17K Mbps从其在细胞中的合成部位转移 细胞质穿过核孔复合物进入核基质。 在非胶质细胞中，在shiverer小鼠中转染的少突胶质细胞 21.5K或17K MBP CDNA，在 细胞质和核质。 值得注意的是，在正常发育的鼠标中 大脑，少突胶质细胞很容易检测到其核包含高的核 Mbps的浓度。 在许多细胞类型中，易位的蛋白质 进入细胞核中包含其主要氨基酸序列中的元素 充当核定位信号（NLSS），可以选择性 通过核孔复合体进入核室 易位的蛋白质可以发挥调节功能。 我们有 鉴定出含有外显子II的MBP同工型中的区域 与已知NLSS的强大同源。 我们将测试其能力，并 这些Mbps中的其他序列可作为瞬态的真实NLS起作用 转染物。 三个用于严格定义NLSS的标准 将应用其他系统：（1）NLS介导的MBP进入 细胞核可能是一个征用能量的过程，因此应 对（a）冷却或（b）ATP耗竭敏感； （2） MBP NLS序列的缺失或突变应引起细胞质 突变蛋白的积累； （3）推定的MBP NLS 序列被设计成无关的非核蛋白，例如 牛血清白蛋白，嵌合蛋白应易位 核。