Mechanisms guiding the fibrillar assembly of SNED1 in the extracellular matrix

项目摘要 细胞外基质(Ecm)是一个复杂的蛋白质网络，构成了所有细胞的结构支架。 纸巾。除了结构作用外，ECM还将生化信号传递给由受体解释的细胞， 像整合素一样，控制多种细胞功能，包括黏附和迁移。因此，ECM是一把钥匙 发育过程和组织动态平衡的调节器。因此，构图中的更改和 ECM网络的组装与包括纤维化和癌症在内的多种疾病有关。 在理解细胞外基质网络是如何构建以及细胞外基质是如何管理细胞方面的重要进展 通过研究主要的细胞外基质蛋白，如纤维连接蛋白和胶原蛋白，已经得出了表型。但是，使用 通过序列分析，我们预测了近300个蛋白质可以参与ECM网络。知识 关于这些其他ECM蛋白的作用仍是初步的。这代表着我们的 了解ECM以及我们纠正致病ECM缺陷的能力。我们最近变成了 感兴趣的是这些未被研究的ECM蛋白之一，SNED1，在发现它与 更具侵袭性的乳腺癌。临床相关性方面，我们发现SNED1的高表达与 乳腺癌患者的预后更差。为了深入了解SNED1的S函数，我们生成了 第一个Sned1基因敲除(KO)小鼠模型，表明Sned1是一个必不可少的基因，因为它导致了Sned1 在早期新生儿死亡中，部分原因是头面部畸形。重要的是，我们最近确定了第一个 携带SNED1变异的患者，他们表现为头面部畸形。尽管有这些观察，但 SNED1促进胚胎发育和癌症转移的机制尚不清楚。 使用我们开发的新工具(抗体、小鼠模型、细胞系、纯化蛋白)，我们已经证明 SNED1在ECM支架内形成纤维，并对其整体组织做出贡献。在这项提案中，我们将 验证SNED1在ECM中的组装及其在调节ECM结构中的作用依赖于 SNED1‘S与其他细胞外基质蛋白和整合素相互作用。利用我们独特的工具包，将我们的 在ECM蛋白质生物化学和ECM蛋白质组学方面的独特专业知识，以及最先进的显微镜，我们将 进行时间分辨的结构/功能分析，以绘制SNED1的哪些结构域参与其整合 在ECM中(AIM 1)；确定ECM蛋白/SNED1相互作用在SNED1 ECM组装中的作用(AIM 2)； 并鉴定细胞表面支配SNED1依赖的ECM组织的SNED1受体和 负责SNED1的粘接性能(目标3)？ 我们的目标是填补我们在理解导致ECM组装的基本机制方面的关键空白， 关于SNED1。这是破译这些机制的扰动是必要的一步 会导致发育缺陷和癌症进展。这项工作也将为未来的发展铺平道路。 未来的治疗策略，以纠正细胞外基质结构的致病变化。

Project Summary The extracellular matrix (ECM) is a complex protein meshwork that constitutes the architectural scaffold of all tissues. In addition to its structural role, the ECM conveys biochemical signals to cells interpreted by receptors, like integrins, and controlling diverse cellular functions including adhesion and migration. The ECM is thus a key regulator of developmental processes and tissue homeostasis. Consequently, alterations in the composition and assembly of the ECM meshwork have been linked to a plethora of diseases including fibrosis, and cancer. Important progress toward understanding how the ECM meshwork is built and how the ECM govern cellular phenotypes have been made by studying major ECM proteins such as fibronectin and collagens. However, using sequence analysis, we have predicted that nearly 300 proteins can contribute to the ECM meshwork. Knowledge regarding the roles of these other ECM proteins remains preliminary. This represents a significant gap in our understanding of the ECM and in our ability to correct disease-causing ECM defects. We have recently become interested in one of these understudied ECM proteins, SNED1, after having found that it was associated with more highly aggressive breast cancers. Of clinical relevance, we found that higher SNED1 expression correlated with a worse prognosis for breast cancer patients. To gain insights into SNED1’s functions, we generated the first knockout (KO) mouse model of Sned1 and showed that Sned1 is an essential gene, since its KO resulted in early neonatal lethality due, in part, to craniofacial malformations. Importantly, we recently identified the first patients with SNED1 variants and they present with craniofacial malformations. Despite these observations, the mechanisms by which SNED1 contributes to embryonic development and cancer metastasis are unknown. Using novel tools we developed (antibodies, mouse models, cell lines, purified proteins), we have shown that SNED1 forms fibers within the ECM scaffold and contributes to its overall organization. In this proposal, we will test the hypothesis that SNED1 assembly in the ECM and its role in regulating ECM architecture depend on SNED1’s interactions with other ECM proteins and integrins. Leveraging our unique toolkit and combining our unique expertise in ECM protein biochemistry and ECM proteomics with state-of-the-art microscopy, we will conduct a time-resolved structure/function analysis to map which domains of SNED1 mediate its incorporation in the ECM (Aim 1); determine the role of ECM proteins/SNED1 interactions in SNED1 ECM assembly (Aim 2); and identify the SNED1 receptors at the cell surface governing SNED1-dependent ECM organization and responsible for the adhesive property of SNED1 (Aim 3)? Our goal is to fill critical gaps in our understanding of the fundamental mechanisms leading to ECM assembly, with respect to SNED1. This is a necessary step toward deciphering how perturbations of these mechanisms can lead to developmental defects and cancer progression. This work will also pave the way to the development of future therapeutic strategies to correct disease-causing alterations in ECM structure.