RUI: Assembly and Phosphorylation of Neurofilaments

RUI：神经丝的组装和磷酸化

• 批准号: 9316365
• 负责人: Jeffrey Cohlberg
• 金额: $ 25.35万
• 依托单位: California State University-Long Beach Foundation
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-15 至 1998-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9316365&HistoricalAwards=false
• 关键词: RUI; Assembly; Phosphorylation; Neurofilaments

9316365 Cohlberg Research will be conducted on the pathway and mechanisms of assembly of mammalian neurofilaments from their constituent proteins and the role of phosphorylation in regulating the assembly process and the properties of the filaments. The goals are to: 1, gain information about the arrangement of the three proteins in neurofilaments (NF), their structural roles, and the pathway by which they assemble into filaments and by which they become incorporated in preexisting filaments; 2, investigate the role of phosphorylation in regulating assembly and in molecular conformation of the proteins; and 3, gain information on the protein kinase enzymes which regulate neurofilament phosphorylation. The first phase of the project will center on the role of certain mixed tetramer assembly intermediates, one containing both NF-L and NF-M and another containing NF-L and NF-H. The presence of these species is detected by polyacrylamide "native gel" electrophoresis. The arrangement of polypeptide chains in the complex will be investigated by electron microscopy of rotary shadowed protein preparations and by experiments examining the ability of chemically cross-linked proteins to form the tetrameric complexes. Proteins produced by recombinant DNA techniques will be used to determine the nature of the specific chemical interactions which determine the rules by which the three proteins combine. NF proteins contain three domains -- a central rod domain which forms the body of the filament, a head domain important in the assembly of the rods, and tail domains which project from the filament. By cutting the DNA from the genes for the proteins into fragments encoding the individual domains and then pasting the fragments together in various combinations, and then expressing the resultant DNAs in bacteria, once can swap domains and produce a modular protein with domains derived from different members of the NF triplet. Determining the ability of these modular proteins to combine with each other should help to determine the roles of different domains in the specific interactions which determine the arrangement of the three NF proteins in filaments. The effect of phosphorylation of the head domains of NF-M and NF-H on assembly will be determined by adding phosphates with the use of enzymes known to phosphorylate these domains and then conducting centrifugation experiments to examine the ability of the phosphorylated proteins to form pelletable filaments. The effect of tail domain phosphorylation on protein conformation will be investigated by using kinase and phosphatase enzymes to prepare NF in different states of phosphorylation and then to measure their circular dichroism and infrared spectra. Protein kinases which catalyze NF phosphorylation will be identified from neuronal extracts. The action of the enzyme, glycogen synthase kinase 3, shown in previous work to phosphorylate NF in vitro, will be further characterized. %%% Neurofilaments (NF) are those members of the family of "intermediate filaments" which form part of the cytoskeleton in neurons of the central nervous system. They are found primarily running longitudinally down the axon along with microtubules, and they are thought to be responsible for promoting the radial growth of axons, establishing axonal diameter, and maintaining the structural integrity of the axon and its resistance to compressive forces. The are composed of three "NF triplet" proteins, termed NF-H (high), NF-M (middle), and NF-L (low) according to their molecular weights. These proteins are synthesized in the cell body and move down the axon in a process termed "slow axonal transport," eventually leaving the moving phase and becoming incorporated into the stationary filament network. As they move down the axon, phosphate groups are added, primarily to the carboxyl-terminal "tail" domains which project from the filaments. A number of diseases of motor neurons involve abnormal NF, and overexpression of either NF-L or NF-H in mice leads to pathology very similar to amyotrophic lateral sclerosis ("Lou Gherig's disease"). In addition, in this and other neuropathologies, abnormally phosphorylated NF accumulate in different parts of the neuron. In addition to the obvious relevance to health and the normal function of CNS neurons, these biochemical studies of NF can be expected to lead to insights into "general principles" of cytoskeletal assembly in other intermediate filament systems. The mechanism of self-assembly of biological polymers is of particular interest in the area of biomolecular materials, materials science, and materials and nanofabrication engineering, since the underlying principles, and even the biological polymers themselves, have potential for adaptation and commercial exploitation as "smart" materials for a variety of uses. ***

小行星9316365 将研究哺乳动物神经丝从其组成蛋白质组装的途径和机制，以及磷酸化在调节组装过程中的作用和丝的性质。 目标是：1、获得关于这三种蛋白在神经丝（NF）中的排列、它们的结构作用以及它们组装成纤维和整合到预先存在的纤维中的途径的信息：2、研究磷酸化在调节蛋白组装和分子构象中的作用;和3，获得关于调节神经丝磷酸化的蛋白激酶的信息。 该项目的第一阶段将集中在某些混合四聚体组装中间体的作用上，一种含有NF-L和NF-M，另一种含有NF-L和NF-H。 通过聚丙烯酰胺“天然凝胶”电泳检测这些物质的存在。 复合物中多肽链的排列将通过旋转阴影蛋白制剂的电子显微镜和通过检查化学交联蛋白形成四聚体复合物的能力的实验来研究。 通过重组DNA技术产生的蛋白质将用于确定特定化学相互作用的性质，所述化学相互作用确定三种蛋白质联合收割机结合的规则。 NF蛋白含有三个结构域--形成丝状体的中心杆状结构域、在杆状体组装中重要的头部结构域和从丝状体突出的尾部结构域。 通过将来自蛋白质基因的DNA切割成编码各个结构域的片段，然后将片段以各种组合粘贴在一起，然后在细菌中表达所得的DNA，一旦可以交换结构域并产生具有来自NF三联体的不同成员的结构域的模块蛋白。 确定这些模块化蛋白相互联合收割机的能力将有助于确定不同结构域在决定丝中三种NF蛋白排列的特定相互作用中的作用。 NF-M和NF-H的头部结构域的磷酸化对组装的影响将通过使用已知磷酸化这些结构域的酶加入磷酸盐，然后进行离心实验以检查磷酸化蛋白质形成可沉淀细丝的能力来确定。 通过使用激酶和磷酸酶制备不同磷酸化状态的NF，然后测量其圆二色谱和红外光谱，研究尾域磷酸化对蛋白质构象的影响。 将从神经元提取物中鉴定催化NF磷酸化的蛋白激酶。 在以前的工作中所示的酶，糖原合成酶激酶3，磷酸化NF在体外的行动，将进一步的特点。 神经丝（NF）是“中间丝”家族的成员，其形成中枢神经系统的神经元中的细胞骨架的一部分。 发现它们主要沿着轴突沿着微管纵向延伸，并且它们被认为负责促进轴突的径向生长，建立轴突直径，并保持轴突的结构完整性及其对压缩力的抵抗力。 由三种“NF三联体”蛋白组成，根据其分子量称为NF-H（高），NF-M（中）和NF-L（低）。 这些蛋白质在细胞体中合成，并在称为“慢轴突运输”的过程中沿着轴突向下移动，最终离开移动相并并入固定的细丝网络。 当它们沿着轴突向下移动时，磷酸基团被添加，主要是添加到从细丝突出的羧基末端“尾”结构域。 许多运动神经元疾病涉及异常NF，并且小鼠中NF-L或NF-H的过表达导致非常类似于肌萎缩性侧索硬化症（“Lou Gherig病”）的病理学。 此外，在这种和其他神经病理中，异常磷酸化的NF在神经元的不同部位积累。 除了与健康和CNS神经元的正常功能的明显相关性之外，NF的这些生化研究可以预期导致对其他中间丝系统中细胞骨架组装的“一般原理”的深入了解。 生物聚合物的自组装机制在生物分子材料、材料科学以及材料和纳米纤维工程领域中特别令人感兴趣，因为基本原理，甚至生物聚合物本身，具有作为用于各种用途的“智能”材料的适应和商业开发的潜力。 ***