ROLES OF THE MYELIN BASIC PROTEINS IN MYELINOGENESIS

我们的长期目标是了解其背后的分子机制 成熟髓鞘的形成，意识到这可能 建议在髓鞘后促进修复过程的方法 毁灭。实现这一目标的关键一步是 准确定义髓鞘碱性蛋白所起的功能作用 (Mbps)在由实施的髓鞘生成计划的过程中 少突胶质细胞。对于下面概述的许多研究，我们将依赖于 关于我们在过去的几年中开发和完善的cdna导入系统 在过去的两年里，使用了特定的MBP抗血清和共聚焦 免疫显微镜定位表达的细胞内分布 蛋白质。我们的具体目标是： I.确定14K和18.5K Mbps内的功能域 介导这些异构体与质膜的结合。 使用定点突变，我们将(1)删除内部片段或 (2)截断14K和18.5K MBP cDNA的N或C末端，以及 在非胶质细胞和寒战细胞中表达突变的结构 少突胶质细胞。每种突变的细胞内分布 多肽将被绘制成免疫细胞化学图谱，以确定最低 膜结合所需的多肽长度和序列。在……里面 平行，(3)突变的多肽将在 细胞结合能力的生化和超微结构实验 来自颤抖的脑浆膜的聚集定向囊泡。 2.开始探索含有21.5K的外显子II的作用机制 和17K Mbps从它们在细胞内的合成部位移位 细胞质穿过核孔复合体进入核基质。 在非胶质细胞中，以及在颤抖的小鼠少突胶质细胞中， 在21.5K或17K MBP的cDNA中，表达的蛋白质位于 细胞质和核质。值得注意的是，在正常发育的小鼠中 脑内，少突胶质细胞很容易被检测到，其细胞核含有高含量 Mbps的浓度。在许多细胞类型中，转位的蛋白质 核内含有初级氨基酸序列中的元素 它们充当核定位信号(NLS)，从而能够选择性地 通过核孔复合体进入核室，在那里 转位的蛋白质可以发挥调节功能。我们有 在含有外显子II的MBP亚型中发现了一个区域，该区域 与已知的NLSS有很强的同源性。我们将测试它的能力，并 这些Mbps中的其他序列在瞬变中用作可信的NLSS 转染者。中用于严格定义NLS的三个标准 其他系统将被应用：(1)NLS-调解的MBP进入 原子核很可能是一个需要能量的过程，因此应该 对(A)冷藏或(B)ATP耗尽造成的干扰敏感；(2) MBP NLS序列的缺失或突变(S)应引起细胞质 突变蛋白的积累；以及(3)当假定的MBP NLS 序列被改造成一种无关的非核蛋白，例如 牛血清白蛋白，嵌合蛋白应转位到 原子核。

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.