Department of Physiology and Cell Biology, The Integrated Biomedical Sciences Graduate Program, and the Ohio State Biochemistry Program, Dorothy M. Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University, Columbus, Ohio, 43210.
J Cell Biochem. 2013 Dec;114(12):2753-69. doi: 10.1002/jcb.24624.
Myofibroblast differentiation is required for wound healing and accompanied by activation of smooth muscle α-actin (SMαA) gene expression. The stress-response protein, Y-box binding protein-1 (YB-1) binds SMαA mRNA and regulates its translational activity. Activation of SMαA gene expression in human pulmonary myofibroblasts by TGFβ1 was associated with formation of denaturation-resistant YB-1 oligomers with selective affinity for a known translation-silencer sequence in SMαA mRNA. We have determined that YB-1 is a substrate for the protein-crosslinking enzyme transglutaminase 2 (TG2) that catalyzes calcium-dependent formation of covalent γ-glutamyl-isopeptide linkages in response to reactive oxygen signaling. TG2 transamidation reactions using intact cells, cell lysates, and recombinant YB-1 revealed covalent crosslinking of the 50 kDa YB-1 polypeptide into protein oligomers that were distributed during SDS-PAGE over a 75-250 kDa size range. In vitro YB-1 transamidation required nanomolar levels of calcium and was enhanced by the presence of SMαA mRNA. In human pulmonary fibroblasts, YB-1 crosslinking was inhibited by (a) anti-oxidant cystamine, (b) the reactive-oxygen antagonist, diphenyleneiodonium, (c) competitive inhibition of TG2 transamidation using the aminyl-surrogate substrate, monodansylcadaverine, and (d) transfection with small-interfering RNA specific for human TG2 mRNA. YB-1 crosslinking was partially reversible as a function of oligomer-substrate availability and TG2 enzyme concentration. Intracellular calcium accumulation and peroxidative stress in injury-activated myofibroblasts may govern SMαA mRNA translational activity during wound healing via TG2-mediated crosslinking of the YB-1 mRNA-binding protein.
肌成纤维细胞分化是伤口愈合所必需的，伴随着平滑肌α-肌动蛋白（SMαA）基因表达的激活。应激反应蛋白 Y 盒结合蛋白-1（YB-1）结合 SMαA mRNA 并调节其翻译活性。TGFβ1 激活人肺肌成纤维细胞中 SMαA 基因表达与形成抗变性 YB-1 寡聚体有关，该寡聚体对 SMαA mRNA 中已知的翻译沉默序列具有选择性亲和力。我们已经确定 YB-1 是蛋白交联酶转谷氨酰胺酶 2（TG2）的底物，该酶在活性氧信号的作用下催化钙依赖性形成共价γ-谷氨酰异肽键。使用完整细胞、细胞裂解物和重组 YB-1 进行的 TG2 转酰胺反应揭示了 50 kDa YB-1 多肽被共价交联成蛋白寡聚体，在 SDS-PAGE 中分布在 75-250 kDa 的大小范围内。体外 YB-1 转酰胺需要纳摩尔水平的钙，并且 SMαA mRNA 的存在增强了该反应。在人肺成纤维细胞中，YB-1 交联被（a）抗氧化剂胱胺、（b）活性氧拮抗剂二苯并碘onium、（c）使用氨酰-替代底物单丹磺酰尸胺进行的 TG2 转酰胺竞争抑制以及（d）针对人 TG2 mRNA 的小干扰 RNA 转染所抑制。YB-1 交联是作为寡聚体-底物可用性和 TG2 酶浓度的函数部分可逆的。损伤激活的肌成纤维细胞中的细胞内钙积累和过氧化应激可能通过 TG2 介导的 YB-1 mRNA 结合蛋白交联来控制伤口愈合过程中 SMαA mRNA 的翻译活性。
