Electron microscopy analysis of novel knob-pocket mechanism critical for intermediate filament assembly

项目总结 中间纤维是细胞内细胞骨架的基本纤维成分。 IF蛋白的突变会导致或使人类易患80多种疾病，这意味着IF具有 对人类健康和疾病起着至关重要的作用。此外，一些国际金融机构被认为与扩散和 癌症的转移。IF的例子包括角蛋白、波形蛋白、结蛋白、神经细丝和板层。至 在基于IF的疾病中，基因型与临床表型完全相关，重要的是了解 突变改变了IF蛋白的三维蛋白质结构和它们组装成的细丝。 目前，蛋白质如何组装成成熟的10纳米IF的原子分辨率基础尚不清楚 这代表着IF生物学中最关键的未满足的需求之一。从多个方面了解到的 生物物理研究表明，IF共享一个共同的螺旋线圈/螺旋杆域，该结构域分为四个螺旋 区域：表示螺旋1A、1B、2A或2B。该中心杆状结构域的两侧是可变的N端头 和C-末端尾部结构域。 这项建议旨在解决我们对IF蛋白质的原子分辨基础的理解上的不足。 组装成细丝。特别是，我们将重点放在锚定旋钮-疏水口袋IF组件上 这是我们实验室新发现的机制。这一发现是由两种x射线晶体结构得出的 角蛋白1/10螺旋1B四聚体-IF四聚体被认为是构建更高... 订购灯丝包装。这些结构提出了几个尚不清楚的问题：(1)旋钮- 口袋机构调节中频装配的速度和/或长度；(2)是旋钮口袋机构 在六种类型的IF中保守；(3)旋钮和口袋中的哪些残留物对 相互作用；(4)旋钮或口袋的突变体如何改变装配；以及(5)旋钮口袋是否可以 机制是以多肽或小分子为靶点来破坏IF组装。我们相信，专注于我们的 对这些重要问题的研究将促进我们对IF组装的机械化理解。 在这个项目中，我们将深入研究锚定旋钮的生物化学和结构特性- 我们实验室确定了疏水口袋IF的组装机制。在目标1中，我们将使用负片染色 用电子显微镜分析野生型和突变型的IF，以了解旋钮口袋如何丢失 相互作用影响成丝的速度和长度。多个IF系统将评估为 确定整个综合框架内这一机制的保守程度。在目标2中，我们将有选择地变异 疏水性口袋残留物，以确定哪些口袋残留物对旋钮结合最关键。然后, 我们将研究节肽是否能结合到口袋上，防止IF组装。实现这些目标 AIMS将为旋钮口袋机制如何管理IF组件提供新的见解，并建立 为通过其组装机制开发针对IFS的靶向疗法奠定基础。

PROJECT SUMMARY Intermediate filaments (IFs) are a fundamental fibrous component of the cytoskeleton within cells. Mutations in IF proteins cause or predispose humans to more than 80 diseases, meaning IFs have an essential role in human health and disease. Moreover, some IFs have been linked to proliferation and metastasis of cancers. Examples of IFs include keratins, vimentin, desmin, neurofilaments, and lamins. To fully correlate genotype with clinical phenotype for IF-based diseases, it is important to understand how mutations alter the three-dimensional protein structure of IF proteins and the filaments they assemble into. Currently, the atomic resolution basis for how IF proteins assemble into mature 10-nm IFs is not known and this represents one of the most critical unmet needs in IF biology. What is known from multiple biophysical studies is that IFs share a common coiled-coil/helical rod domain that is divided into four helical regions: denoted helix 1A, 1B, 2A, or 2B. This central rod domain is flanked by variable N-terminal head and C-terminal tail domains. This proposal aims to address a deficiency in our understanding of the atomic resolution basis for IF protein assembly into filaments. In particular, we focus on an anchoring knob-hydrophobic pocket IF assembly mechanism newly discovered in our lab. This discovery was made from two x-ray crystal structures of keratin 1/10 helix 1B tetrameric complexes – the IF tetramer is considered the building block for higher- order filament packing. These structures raised several questions that remain unclear: (1) does the knob- pocket mechanism regulate the rate and/or the length of IF assembly; (2) is the knob-pocket mechanism conserved across the six types of IFs; (3) which residues in the knob and pocket are most critical for the interaction; (4) how do mutants of the knob or pocket alter IF assembly; and (5) can the knob-pocket mechanism be targeted with peptides or small-molecules to disrupt IF assembly. We believe focusing our studies on these important questions will advance our mechanistic understanding of IF assembly. In this project, we examine in depth the biochemical and structural properties of the anchoring knob- hydrophobic pocket IF assembly mechanism identified our laboratory. In Aim 1 we will use negative-stain electron microscopy to analyze wild-type and mutant IFs to understand how the loss of the knob-pocket interaction affects the rate and length of filament formation. Multiple IF systems will be evaluated to establish the degree of conservation of this mechanism across IFs. In Aim 2 we will selectively mutate hydrophobic pocket residues to determine which pocket residues are most critical to knob binding. Then, we will study whether knob peptides can bind to the pocket and prevent IF assembly. Accomplishing these aims will provide novel insight into how the knob-pocket mechanism governs IF assembly and establish a foundation for developing targeted therapies of IFs through their assembly mechanisms.