C-terminal propeptide is required for fibrillin-1 secretion and blocks premature assembly through linkage to domains cbEGF41-43
C-terminal propeptide is required for fibrillin-1 secretion and blocks premature assembly through linkage to domains cbEGF41-43
复制标题
DOI：
10.1073/pnas.1401697111
复制
发表时间：
2014-07-15
影响因子：
11.1
通讯作者：
Handford, Penny A.
中科院分区：
文献类型：
--
作者：
Jensen, Sacha A.;Aspinall, Georgia;Handford, Penny A.
纤维蛋白微纤维直径为10-12 nm，是细胞外基质组件，为后生动物物种的动态组织提供了许多生物力学特性以及隔离生长因子和细胞因子。纤维蛋白单体组装成微纤维被认为发生在细胞表面，最初的步骤包括原蛋白加工，由C末端驱动的多聚化，以及相邻分子从头到尾的排列。目前，微纤丝组装的调控机制仍有待阐明。我们利用结构信息蛋白质工程创造了重组的GFP标记的纤维蛋白-1(GFP-FBN)来研究这一过程。使用瞬时转染GFP-FBN构建物的HEK293T细胞，我们证明：(I)C-末端前肽是细胞分泌全长纤维蛋白-1的必要条件；(Ii)未能切割C-末端前肽阻止纤维蛋白-1组装成真皮成纤维细胞产生的微纤维；(Iii)分泌前肽的需要与cbEGF41-43结构域的存在有关，因为该区域的结构域的缺失或交换都会导致细胞保留。总而言之，这些数据表明了一种机制，在这种机制中，前肽阻止了C末端的一个关键位点，以防止过早地组装微原纤维。
Fibrillin microfibrils are 10-12 nm diameter, extracellular matrix assemblies that provide dynamic tissues of metazoan species with many of their biomechanical properties as well as sequestering growth factors and cytokines. Assembly of fibrillin monomers into microfibrils is thought to occur at the cell surface, with initial steps including proprotein processing, multimerization driven by the C terminus, and the head-to-tail alignment of adjacent molecules. At present the mechanisms that regulate microfibril assembly are still to be elucidated. We have used structure-informed protein engineering to create a recombinant, GFP-tagged version of fibrillin-1 (GFP-Fbn) to study this process. Using HEK293T cells transiently transfected with GFP-Fbn constructs, we show that (i) the C-terminal propeptide is an essential requirement for the secretion of full-length fibrillin-1 from cells; (ii) failure to cleave off the C-terminal propeptide blocks the assembly of fibrillin-1 into microfibrils produced by dermal fibroblasts; and (iii) the requirement of the propeptide for secretion is linked to the presence of domains cbEGF41-43, because either deletion or exchange of domains in this region leads to cellular retention. Collectively, these data suggest a mechanism in which the propeptide blocks a key site at the C terminus to prevent premature microfibril assembly.