SLLISWD Sequence in the 10FNIII Domain Initiates Fibronectin Fibrillogenesis
SLLISWD Sequence in the 10FNIII Domain Initiates Fibronectin Fibrillogenesis
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DOI：
10.1074/jbc.m113.462077
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发表时间：
2013-07-19
影响因子：
4.8
通讯作者：
Ingber, Donald E.
中科院分区：
文献类型：
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作者：
Gee, Elaine P. S.;Yueksel, Deniz;Ingber, Donald E.
纤维连接蛋白(FN)组装成细胞外基质受到严格调控，对胚胎发育和伤口愈合至关重要。FN纤维形成是通过跨膜整合素传递到第十个FN类型III(10FNIII)结构域内的RGD结合序列上的细胞骨架张力所启动的。这些力量展开10FNIII以暴露神秘的FN组装位点；然而，在10FNIII中还没有确定特定的序列。我们过去对10FNIII在其RGD环上用力展开的分子动力学模拟预测了一种机械中间体，其N末端暴露在溶剂中，横跨A和Bβ链。在这里，我们实验证实，预测的23个残基的隐肽1(CP1)启动了Fn多聚体，这是通过与10FNIII相互作用而介导的，10FNIII暴露了支持8-苯胺基-1-萘磺酸结合的疏水表面。C末端多聚化活性的定位导致发现了一个最小的7-氨基酸“多聚化序列”(SLLISWD)，它除了促进成纤维细胞中Fn的纤维形成外，还能诱导Fn和凝血蛋白原的聚合。Trp-6上的一个点突变，减少了8-苯胺基-1-萘磺酸结合疏水部位的暴露和β结构的形成，抑制了FN的多聚化，并阻止了培养中基于生理细胞的FN组装。我们提出了一个细胞介导的纤维形成模型，细胞牵引力启动了一系列分子间交换，从10FNIII的展开开始暴露多聚化序列，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，以稳定部分未折叠的中间体，传播FN自组装。
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 beta-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 beta-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 strandBof another 10FNIII domain via a Trp-mediated beta-strand exchange to stabilize a partially unfolded intermediate that propagates FN self-assembly.