RUI: Interactions of Neurofilament Proteins During Filament Assembly

RUI：神经丝蛋白在丝组装过程中的相互作用

• 批准号: 9723288
• 负责人: Jeffrey Cohlberg
• 金额: $ 27.33万
• 依托单位: California State University-Long Beach Foundation
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-15 至 2002-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9723288&HistoricalAwards=false
• 关键词: RUI; Interactions; Neurofilament; Proteins; During

9723288 Cohlberg Research will be conducted on the pathway and mechanism of assembly of mammalian neurofilaments (NFs) from their constituent proteins and the role of phosphorylation in regulating the assembly process and the properties of the filaments. The goals of the research are to identify and quantitatively characterize the interactions which determine the specificity of interaction of neurofilament proteins and are responsible for filament stabilization, and to gain information about the parameters which characterize the dynamic properties of NFs. In large caliber neurons of the central nervous system, NFs are composed of three "NF triplet" proteins, termed NF-H (high), NF-M (middle), and NF-L (low) according to their molecular weights. In certain neurons, other proteins, including alpha-internexin, are present in NF. Neurofilament (NF) proteins contain alpha-helical rod domains flanked by nonhelical head and tail domains. They aggregate to form coiled coil dimers, which then aggregate further to form NF-L/NF-M and NF-L/NF-H heterotetramers, which in turn serve as intermediates in the assembly of filaments. It is not certain whether the coiled coil dimers are heterodimers or homodimers. Disulfide cross-linking will be used as a probe to detect the formation of heterodimers from homodimers, and the kinetics of monomer exchange and its dependence on solvent conditions will be characterized. The stabilities of various heterodimers containing both full-length proteins and their rod domains will be investigated by sedimentation and cross-linking studies, and the roles of various proteins domains in the association of dimers to tetramers will be characterized. The roles of the various domains in the formation of heterotetramer assembly intermediates will be investigated by the use of nondenaturing polyacrylamide gel electrophoresis. The principal question to be answered is whether specific interactions among rod domains are fully responsible for th e specificity of protein association at this level (LM and MH complexes are formed, but not MH) or whether the head and tail domains also play a role. Furthermore, the roles of the various protein domains in determining the specificity with which proteins associate to form filaments will be examined by filament reconstitution experiments and analyzed by filament pelleting experiments and by examination of reconstituted filaments by electron microscopy, including immunogold labeling studies. Finally, NF proteins will be modified by the covalent attachment of fluorophores, and the assembly and disassembly of filaments and the exchange of proteins between filaments will be followed by fluorescence energy transfer measurements. All of these studies will include experiments on alpha- internexin as well as the NF triplet proteins. A key attribute of eukaryotic cells is the presence of filamentous structures inside the cells, collectively termed the cytoskeleton, which provide a structural framework for the cytoplasm and its functions. The three major types of cytoskeletal "elements" in cells, distinguished by their molecular composition and structural features, are the actin- containing microfilaments, the 10-nm diameter intermediate filaments, and microtubules. Neurofilaments (NF) are those members of the family of intermediate filaments (IFs) that form part of the cytoskeleton in neurons (cells of the nervous system). They are found primarily running longitudinally down the long cellular projections known as axons, along with microtubules, and they are thought to be responsible for promoting the radial growth (thickening) of axons, establishing axonal diameter, and maintaining the structural integrity of the axon and its resistance to compressive forces. In large caliber neurons of the central nervous system, NFs are composed of three "NF triplet" proteins, distinguished by their molecular weights (heavy-, middle-, and low-weight). In certain neurons, add itional proteins, e.g., alpha-internexin, are present in NFs. The proteins are synthesized in the cell body and move down the axon in a process known as slow axonal transport, eventually leaving the moving phase and becoming incorporated into the stationary filament network of the axon. A number of diseases of motor neurons involve abnormal NFs, and overexpression of either the light or heavy weight NF in transgenic mice leads to pathology very similar to amyotrophic lateral sclerosis (ALS, or "Lou Gherig's Disease"). Very little is known about how these NF proteins assemble to form functional filaments. This research will provide vital information on the chemical and physical properties of NF protein components and their interactions with each other, which will help us better understand filament assembly and transport in living neuronal cells. ***

小行星9723288 研究哺乳动物神经丝（NF）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细丝的性质。 本研究的目的是确定和定量表征的相互作用，决定了特异性的相互作用的神经丝蛋白，并负责丝稳定，并获得有关的参数，表征的动态特性的NF。 在中枢神经系统的大口径神经元中，NF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）。 在某些神经元中，其他蛋白质，包括α-interexin，存在于NF中。 神经丝（NF）蛋白含有α-螺旋杆结构域两侧的非螺旋头部和尾部结构域。它们聚集以形成卷曲螺旋二聚体，然后进一步聚集以形成NF-L/NF-M和NF-L/NF-H异源四聚体，这些异源四聚体又充当细丝组装中的中间体。 卷曲螺旋二聚体是异源二聚体还是同源二聚体尚不确定。 二硫键交联将被用作探针，以检测异源二聚体从同源二聚体的形成，和单体交换的动力学及其对溶剂条件的依赖性将被表征。 各种异源二聚体含有全长蛋白质和他们的杆域的稳定性将通过沉降和交联研究进行调查，和各种蛋白质结构域的作用，在协会的二聚体的四聚体的特点。 异源四聚体组装中间体的形成中的各种结构域的作用将通过使用非变性聚丙烯酰胺凝胶电泳进行研究。 要回答的主要问题是，杆结构域之间的特异性相互作用是否完全负责蛋白质缔合的特异性（形成LM和MH复合物，但不形成MH），或者头部和尾部结构域是否也起作用。 此外，各种蛋白质结构域的作用，在确定与蛋白质相关联，形成细丝的特异性将检查细丝重建实验和分析细丝造粒实验和检查重建的细丝，通过电子显微镜，包括免疫金标记研究。 最后，NF蛋白将被修饰的共价连接的荧光团，和装配和拆卸的细丝和细丝之间的蛋白质交换将遵循荧光能量转移测量。 所有这些研究将包括α-内连接蛋白以及NF三联体蛋白的实验。 真核细胞的一个关键属性是细胞内存在丝状结构，统称为细胞骨架，其为细胞质及其功能提供结构框架。 细胞中的三种主要类型的细胞骨架“元件”，通过它们的分子组成和结构特征来区分，是含肌动蛋白的微丝、10 nm直径的中间丝和微管。 神经丝（NF）是中间丝（IF）家族的成员，其形成神经元（神经系统的细胞）中的细胞骨架的一部分。 发现它们主要沿着称为轴突的长细胞突起纵向向下延伸，沿着微管，并且它们被认为负责促进轴突的径向生长（增厚），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。 在中枢神经系统的大口径神经元中，NF由三种“NF三联体”蛋白组成，其通过其分子量（重、中和低重量）区分。 在某些神经元中，额外的蛋白质，例如， α-内连接蛋白存在于NF中。 蛋白质在细胞体内合成，并在称为缓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轴突的固定细丝网络。 许多运动神经元疾病涉及异常NF，并且转基因小鼠中轻或重NF的过表达导致与肌萎缩性侧索硬化症（ALS，或“Lou Gherig病”）非常相似的病理学。 关于这些NF蛋白如何组装形成功能性丝的知之甚少。 这项研究将提供有关NF蛋白组分的化学和物理性质及其相互作用的重要信息，这将有助于我们更好地了解活神经元细胞中的细丝组装和运输。 ***