RUI: Assembly and Phosphorylation of Neurofilaments

将从哺乳动物神经丝的组成蛋白出发，研究其组装的途径和机制，以及磷酸化在调节神经丝组装过程中的作用和神经丝的性质。目标是：1，获得关于三种蛋白质在神经纤维丝（NF）中的排列，它们的结构作用，以及它们组装成纤维丝的途径以及它们通过何种途径与先前存在的纤维丝结合的信息；2、研究磷酸化在调节蛋白质组装和分子构象中的作用；第三，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于某些混合四聚体组装中间体的作用，一种含有NF-L和NF-M，另一种含有NF-L和NF-H。这些物种的存在是通过聚丙烯酰胺“天然凝胶”电泳检测的。多肽链在复合物中的排列将通过旋转阴影蛋白制备的电子显微镜和通过化学交联蛋白形成四聚体复合物的能力的实验来研究。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，这种化学相互作用决定了三种蛋白质结合的规则。NF蛋白包含三个结构域——形成丝体的中心棒结构域，在棒的组装中重要的头部结构域，以及从丝中伸出的尾部结构域。通过将蛋白质基因中的DNA切割成编码单个结构域的片段，然后将这些片段以各种组合方式粘贴在一起，然后在细菌中表达所得到的DNA，一次就可以交换结构域，并产生具有来自NF三元组不同成员的结构域的模块化蛋白质。确定这些模块蛋白相互结合的能力应该有助于确定不同结构域在特定相互作用中的作用，这些相互作用决定了三种NF蛋白在细丝中的排列。NF-M和NF-H头部结构域磷酸化对组装的影响将通过添加磷酸盐和使用已知磷酸化这些结构域的酶来确定，然后进行离心实验以检查磷酸化蛋白质形成球团细丝的能力。利用激酶和磷酸酶制备不同磷酸化状态的NF，并测量其圆二色性和红外光谱，研究尾结构域磷酸化对蛋白质构象的影响。催化NF磷酸化的蛋白激酶将从神经元提取物中鉴定出来。糖原合成酶激酶3的作用，在先前的工作中显示，在体外磷酸化NF，将进一步表征。神经丝（NF）是“中间丝”家族的成员，构成中枢神经系统神经元细胞骨架的一部分。它们主要与微管一起沿轴突纵向分布，它们被认为负责促进轴突的径向生长，建立轴突直径，维持轴突的结构完整性及其对压缩力的抵抗力。它们由三种“NF三重态”蛋白质组成，根据分子量分别命名为NF- h（高）、NF- m（中）和NF- l（低）。这些蛋白质在细胞体中合成，并沿着轴突向下移动，这一过程被称为“慢轴突运输”，最终离开移动阶段，并入静止的细丝网络。当它们沿着轴突向下移动时，磷酸基团被添加，主要是在从细丝伸出的羧基末端“尾部”区域。许多运动神经元疾病涉及NF异常，小鼠中NF- l或NF- h的过度表达导致的病理与肌萎缩性侧索硬化症（“卢盖里格病”）非常相似。此外，在这种和其他神经病变中，异常磷酸化的NF积聚在神经元的不同部位。除了与中枢神经系统神经元的健康和正常功能明显相关外，这些对NF的生化研究还有望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是生物分子材料、材料科学、材料和纳米制造工程领域特别感兴趣的，因为其基本原理，甚至生物聚合物本身，都具有作为各种用途的“智能”材料的适应性和商业开发潜力。＊＊＊

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. ***