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

项目摘要 中间丝（IF）是细胞内细胞骨架的基本纤维成分。 IF蛋白质的突变导致或使人类易患80多种疾病，这意味着IF蛋白质具有 对人类健康和疾病的重要作用。此外，一些国际单项体育联合会与扩散有关， 癌症的转移。IF的实例包括角蛋白、波形蛋白、结蛋白、神经丝和核纤层蛋白。到 对于基于IF的疾病，基因型与临床表型完全相关，重要的是要了解如何 突变改变IF蛋白的三维蛋白质结构和它们组装成的细丝。 目前，IF蛋白如何组装成成熟的10-nm IF的原子分辨率基础尚不清楚 这代表了IF生物学中最关键的未满足的需求之一。从多个已知的 生物物理学研究表明，IF共享一个共同的卷曲螺旋/螺旋杆结构域，该结构域分为四个螺旋结构域， 区域：表示为螺旋1A、1B、2A或2B。这个中心杆域两侧是可变的N-末端头部 和C-末端尾部结构域。 该建议旨在解决我们对IF蛋白的原子分辨率基础理解的不足 组装成细丝。特别是，我们专注于锚定旋钮疏水口袋IF组装 新发现的机制。这一发现是从两个X射线晶体结构的 角蛋白1/10螺旋1B四聚体复合物-IF四聚体被认为是更高- 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- 调节IF组装的速率和/或长度的口袋机制;（2）是旋钮口袋机制 保守的六种类型的IF;（3）在旋钮和口袋中的残基是最关键的 相互作用;（4）旋钮或口袋的突变体如何改变IF组装;以及（5）旋钮口袋是否可以改变IF组装。 可以用肽或小分子靶向该机制以破坏IF组装。我们相信， 对这些重要问题的研究将有助于我们对IF组装机理的理解。 在这个项目中，我们深入研究了锚结的生化和结构特性- 疏水口袋IF组装机制确定了我们的实验室。在目标1中，我们将使用负染色 用电子显微镜分析野生型和突变型IFs，以了解knob-pocket的丢失是如何导致 相互作用影响细丝形成的速率和长度。将评估多个IF系统， 确定该机制在国际单项体育联合会中的保守程度。在目标2中，我们将选择性地 疏水口袋残基，以确定哪些口袋残基对结结合最关键。然后， 我们将研究结肽是否可以结合到口袋，并阻止IF组装。完成这些 目的将提供新的见解如何旋钮口袋机制支配IF组装，并建立一个 为通过其组装机制开发IF靶向治疗奠定了基础。

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.