From the Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115.
From the Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115,; the Vascular Biology Program and Departments of Pathology and Surgery, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 2013 Jul 19;288(29):21329-21340. doi: 10.1074/jbc.M113.462077. Epub 2013 Jun 5.
Fibronectin (FN) assembly into extracellular matrix is tightly regulated and essential to embryogenesis and wound healing. FN fibrillogenesis is initiated by cytoskeleton-derived tensional forces transmitted across transmembrane integrins onto RGD binding sequences within the tenth FN type III (10FNIII) domains. These forces unfold 10FNIII to expose cryptic FN assembly sites; however, a specific sequence has not been identified in 10FNIII. Our past steered molecular dynamics simulations modeling 10FNIII unfolding by force at its RGD loop predicted a mechanical intermediate with a solvent-exposed N terminus spanning the A and B β-strands. Here, we experimentally confirm that the predicted 23-residue cryptic peptide 1 (CP1) initiates FN multimerization, which is mediated by interactions with 10FNIII that expose hydrophobic surfaces that support 8-anilino-1-napthalenesulfonic acid binding. Localization of multimerization activity to the C terminus led to the discovery of a minimal 7-amino acid "multimerization sequence" (SLLISWD), which induces polymerization of FN and the clotting protein fibrinogen in addition to enhancing FN fibrillogenesis in fibroblasts. A point mutation at Trp-6 that reduces exposure of hydrophobic sites for 8-anilino-1-napthalenesulfonic acid binding and β-structure formation inhibits FN multimerization and prevents physiological cell-based FN assembly in culture. We propose a model for cell-mediated fibrillogenesis whereby cell traction force initiates a cascade of intermolecular exchange starting with the unfolding of 10FNIII to expose the multimerization sequence, which interacts with strand B of another 10FNIII domain via a Trp-mediated β-strand exchange to stabilize a partially unfolded intermediate that propagates FN self-assembly.
纤连蛋白（FN）组装到细胞外基质中受到严格调控，这对于胚胎发生和伤口愈合至关重要。FN 原纤维的形成是由细胞骨架衍生的张力通过跨膜整合素传递到第十个 FN 三型（10FNIII）结构域中的 RGD 结合序列而启动的。这些力使 10FNIII 展开，暴露出隐藏的 FN 组装位点；然而，在 10FNIII 中尚未确定特定的序列。我们过去的导向分子动力学模拟通过力在其 RGD 环处对 10FNIII 进行展开，预测了一种机械中间物，其中溶剂暴露的 N 端跨越 A 和 B β-链。在这里，我们通过实验证实了预测的 23 个残基隐藏肽 1（CP1）启动 FN 多聚化，这是由与暴露出支持 8-苯胺-1-萘磺酸结合的疏水面的 10FNIII 相互作用介导的。多聚化活性的定位导致发现了一个最小的 7 个氨基酸“多聚化序列”（SLLISWD），它除了增强成纤维细胞中的 FN 原纤维形成外，还诱导 FN 和凝血蛋白纤维蛋白原的聚合。在降低 8-苯胺-1-萘磺酸结合和 β-结构形成的疏水面暴露的 Trp-6 处的点突变抑制 FN 多聚化并防止生理细胞中 FN 在培养物中的组装。我们提出了一种细胞介导的原纤维形成模型，其中细胞牵引力引发了一个分子间交换的级联反应，首先是 10FNIII 的展开以暴露出多聚化序列，该序列通过色氨酸介导的 β-链交换与另一个 10FNIII 结构域的链 B 相互作用，以稳定部分展开的中间体，该中间体可传播 FN 自组装。
