RUI: Interactions of Neurofilament Proteins During Filament Assembly

9723288 Cohlberg Research将就哺乳动物神经细丝从其组成蛋白组装的途径和机制，以及磷酸化在调控组装过程和细丝特性方面的作用进行研究。本研究的目的是确定和定量描述神经细丝蛋白相互作用的特异性和对细丝稳定起作用的相互作用，并获得有关表征神经丝动态特性的参数的信息。在中枢神经系统的大口径神经元中，神经营养因子由三种“核因子三联体”蛋白组成，根据其相对分子质量，分别称为核因子-H(高)、核因子-M(中)和核因子-L(低)。在某些神经元中，包括α-内联蛋白在内的其他蛋白质也存在于神经纤维中。神经丝蛋白含有α-螺旋杆状结构域，两侧是非螺旋的头部和尾部区域。它们聚集形成卷曲的线圈二聚体，然后进一步聚集形成NF-L/NF-M和NF-L/NF-H异构体，这些异构体反过来作为细丝组装的中间体。目前还不能确定盘绕的线圈二聚体是异二聚体还是同二聚体。将以二硫键交联为探针，检测均二聚体形成杂二聚体的情况，并表征单体交换动力学及其对溶剂条件的依赖关系。各种含有全长蛋白质及其杆状结构域的杂二聚体的稳定性将通过沉淀法和交联法研究，并将表征各种蛋白质结构域在二聚体与四聚体结合中的作用。使用非变性的聚丙烯酰胺凝胶电泳法将研究不同结构域在形成异四聚体组装中间体中的作用。需要回答的主要问题是，杆状结构域之间的特定相互作用是否完全导致了蛋白质在这一水平上的结合特异性(形成了LM和MH复合体，但没有形成MH复合体)，或者头部和尾部结构域是否也起到了作用。此外，各种蛋白质结构域在决定蛋白质结合形成细丝的特异性方面的作用将通过细丝重组实验来检验，并通过细丝成粒实验和电子显微镜检查重组细丝来分析，包括免疫金标记法研究。最后，将通过荧光团的共价吸附来修饰核因子蛋白，随后将进行荧光能量转移测量，从而实现细丝的组装和解体以及细丝之间的蛋白质交换。所有这些研究都将包括对α-网织蛋白和核因子三联体蛋白的实验。真核细胞的一个关键特征是细胞内存在丝状结构，统称为细胞骨架，它为细胞质及其功能提供了结构框架。细胞中的细胞骨架“元件”主要有三种，它们的分子组成和结构特征不同，它们是含有肌动蛋白的微丝、直径10 nm的中间丝和微管。神经丝(NF)是中间丝(IF)家族的成员，构成神经元(神经系统细胞)细胞骨架的一部分。它们主要沿称为轴突的长细胞突起与微管一起纵向运行，它们被认为负责促进轴突的径向生长(增厚)，建立轴突直径，维持轴突的结构完整性及其对压力的抵抗力。在中枢神经系统的大口径神经元中，神经纤维由三种“核因子三联体”蛋白组成，它们的分子量不同(重、中、低三重)。在某些神经元中，额外的离子蛋白，如α-内联蛋白，存在于神经纤维中。这些蛋白质在细胞体中合成，然后沿着轴突向下移动，这一过程被称为缓慢的轴突运输，最终离开运动阶段，并入轴突的固定细丝网络。许多运动神经元疾病都与异常的神经营养因子有关，在转基因小鼠中过表达轻或重的神经营养因子都会导致非常类似于肌萎缩侧索硬化症(ALS)或Lou Gherig病的病理。人们对这些核因子蛋白如何组装形成功能性细丝知之甚少。这项研究将提供有关核因子蛋白质组分的化学和物理性质以及它们之间相互作用的重要信息，这将有助于我们更好地了解活的神经细胞中微丝的组装和运输。***

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. ***