Molecular and cellular mechanisms in elastic nanofiber formation

弹性纤维的形成，称为弹性发生，是生物学中的一个基本过程，赋予几个重要器官如血管、肺和皮肤以弹性。这是一个依赖于许多单独组件的分层组装机制的过程。发现号计划的主要长期目标是破译这一过程中基本组件的属性，以及它们彼此之间以及与细胞之间的功能关系。它的目的是确定这些机制如何在纳米级上导致全功能弹性纤维的形成。尽管在理解弹性蛋白之间的功能关系方面取得了一些重要进展，但驱动弹性纳米纤维组装的关键潜在蛋白质-蛋白质和蛋白质-细胞相互作用仍然难以捉摸。下一个五年资助周期的短期目标集中在确定三种辅助蛋白在弹性纳米纤维形成中的作用，它们是微纤维相关糖蛋白4(MFAP4)、纤维蛋白-4和潜伏的转化生长因子结合蛋白4(LTBP4)。MFAP4定位于弹性纤维并与其相关，但其在弹性纤维生物发生中的作用尚不清楚。纤维蛋白-4和LTBP4是弹性发生中的关键辅助蛋白，我们最近确定了这些蛋白之间的关键功能关系，这是正确的弹性发生所必需的。我们发现纤维蛋白-4的多聚化对于细胞间的相互作用是必不可少的，并确定细胞表面的蛋白多糖是这两种蛋白的受体。利用一系列生物物理和细胞培养方法，我们旨在揭示这三种蛋白质形成复合体的重要机制信息，这些复合体是如何与其他弹性蛋白相互作用的，细胞后果，以及在弹性纳米纤维形成中的作用。在目标1中，我们建议定义MFAP4的结构、自结合和寡聚，并确定其在弹性形成中的意义。在目标2中，我们旨在研究MFAP4、纤毛蛋白-4和LTBP4蛋白复合体形成过程中的功能关系。我们还将分析复合体形成对细胞相互作用、弹性纳米纤维形成、细胞功能和收缩以及对细胞外基质重塑的功能影响。我们期望这个发现号研究项目将提供有关弹性发生中的分级过程的新的基础知识和分子机制。该计划将为研究生和博士后研究员提供在学术界和加拿大生物技术部门担任职位所需的一整套技能。我们预测，这将为合成弹性纳米纤维和先进纳米材料的生产开辟新的途径。

The formation of elastic fibers, termed elastogenesis, is a fundamental process in biology, conferring elasticity to several vital organs, such as blood vessels, lung, and skin. It is a process that depends on hierarchical assembly mechanisms of many individual components. The overarching long-term goal of the Discovery program is to decipher the properties of essential components in this process and their functional relationships with each other and with cells. It aims to determine how these mechanisms lead to formation of fully functional elastic fibers on the nanoscale level. Despite some important advances in understanding the functional relationships between elastogenic proteins, the critical underlying protein-protein and protein-cell interactions that drive elastic nanofiber assembly are still elusive. The short-term goals for the next five-year grant cycle focus on determining the role of three accessory proteins in elastic nanofiber formation, microfibrillar associated glycoprotein 4 (MFAP4), fibulin-4, and latent transforming growth factor binding protein 4 (LTBP4). MFAP4 localizes to and is associated with elastic fibers, but its role in elastic fiber biogenesis remains unclear. Fibulin-4 and LTBP4 are critical accessory proteins in elastogenesis, and we have recently identified key functional relationships between these proteins necessary for proper elastogenesis. We identified that multimerization of fibulin-4 is essential for cell interaction and determined cell surface proteoglycans as receptors for both proteins. Using a series of biophysical and cell culture methods, we aim to reveal important mechanistic information on complex formation of these three proteins, how these complexes interact with other elastogenic proteins, the cellular consequences, and the role in elastic nanofiber formation. In aim 1, we propose to define the structure, self-association, and oligomerization of MFAP4, and determine its significance in elastogenesis. In aim 2, we aim to study the functional relationship of protein complex formation of MFAP4, fibulin-4 and LTBP4. We also will analyze the functional consequences of complex formation on cell interactions, elastic nanofiber formation, cell function and contractility, and on extracellular matrix remodeling. We expect that this Discovery research program will provide novel fundamental knowledge and molecular mechanisms on the hierarchical processes in elastogenesis. The program will provide graduate students and postdoctoral fellows with skill sets needed for positions in academia and in the Canadian biotechnology sector. We predict that it will reveal new avenues to produce synthetic elastic nanofibers and advanced nanomaterials.