Flynn Charles R, Brophy Colleen M, Furnish Elizabeth J, Komalavilas Padmini, Tessier Deron, Thresher Jeffrey, Joshi Lokesh
The Biodesign Institute, Arizona State Univeristy, Tempe, AZ 85287-9709, USA.
J Appl Physiol (1985). 2005 May;98(5):1836-45. doi: 10.1152/japplphysiol.01043.2004.
Activation of cyclic nucleotide-dependent signaling pathways inhibits agonist-induced contraction of most vascular smooth muscles except human umbilical artery smooth muscle (HUASM). This impaired vasorelaxation may contribute to complications associated with preeclampsia, intrauterine growth restriction, and preterm delivery. Cyclic nucleotide-dependent signaling pathways converge at the phosphorylation of the small heat shock-related protein HSP20, causing relaxation of vascular smooth muscle. We produced recombinant proteins containing a protein transduction domain linked to HSP20 (rTAT-HSP20). Pretreatment of HUASM with in vitro phosphorylated rTAT-HSP20 (rTAT-pHSP20) significantly inhibited serotonin-induced contraction, without a decrease in myosin light chain phosphorylation. rTAT-pHSP20 remained phosphorylated upon transduction into isolated HUASM as demonstrated by two-dimensional gel electrophoresis. Transduction of peptide analogs of phospho-HSP20 containing the phosphorylation site on HSP20 and phosphatase-resistant mimics of the phosphorylation site (S16E) also inhibited HUASM contraction. These data suggest that impaired relaxation of HUASM may result from decreased levels of phosphorylated HSP20. Protein transduction can be used to restore intracellular expression levels and the associated physiological response. Transduction of posttranslationally modified substrate proteins represents a proteomic-based therapeutic approach that may be particularly useful when the expression of downstream substrate proteins is downregulated.
环核苷酸依赖性信号通路的激活可抑制除人脐动脉平滑肌（HUASM）外的大多数血管平滑肌的激动剂诱导收缩。这种血管舒张功能受损可能导致与子痫前期、宫内生长受限和早产相关的并发症。环核苷酸依赖性信号通路在小热休克相关蛋白HSP20的磷酸化处汇聚，从而引起血管平滑肌舒张。我们制备了含有与HSP20相连的蛋白转导结构域的重组蛋白（rTAT-HSP20）。用体外磷酸化的rTAT-HSP20（rTAT-pHSP20）预处理HUASM可显著抑制5-羟色胺诱导的收缩，而肌球蛋白轻链磷酸化没有降低。二维凝胶电泳显示，rTAT-pHSP20转导至分离的HUASM后仍保持磷酸化状态。转导含有HSP20磷酸化位点的磷酸化HSP20肽类似物以及该磷酸化位点的磷酸酶抗性模拟物（S16E）也可抑制HUASM收缩。这些数据表明，HUASM舒张功能受损可能是由于磷酸化HSP20水平降低所致。蛋白转导可用于恢复细胞内表达水平及相关生理反应。翻译后修饰底物蛋白的转导代表了一种基于蛋白质组学的治疗方法，当下游底物蛋白表达下调时可能特别有用。
