Nesiel-Nuttman Liron, Doron Shani, Schwartz Betty, Shoseyov Oded
The Robert H. Smith Institute of Plant Science and Genetics in Agriculture, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, ISRAEL.
School of Nutritional Sciences Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, ISRAEL.
Oncoscience. 2014 Nov 26;2(1):31-43. doi: 10.18632/oncoscience.100. eCollection 2015.
Human RNASET2 (hRNASET2) has been demonstrated to exert antiangiogenic and antitumorigenic effects independent of its ribonuclease capacity. We suggested that RNASET2 exerts its antiangiogenic and antitumorigenic activities via binding to actin and consequently inhibits cell motility. We focused herein on the identification of the actin binding site of hRNASET2 using defined sequences encountered within the whole hRNASET2 protein. For that purpose we designed 29 different hRNASET2-derived peptides. The 29 peptides were examined for their ability to bind immobilized actin. Two selected peptides-A103-Q159 consisting of 57 amino acids and peptide K108-K133 consisting of 26 amino acids were demonstrated to have the highest actin binding ability and concomitantly the most potent anti-angiogenic activity. Further analyses on the putative mechanisms associated with angiogenesis inhibition exerted by peptide K108-K133 involved its location during treatment within the HUVE cells. Peptide K108-K133 readily penetrates the cell membrane within 10 min of incubation. In addition, supplementation with angiogenin delays the entrance of peptide K108-K133 to the cell suggesting competition on the same cell internalization route. The peptide was demonstrated to co-localize with angiogenin, suggesting that both molecules bind analogous cellular epitopes, similar to our previously reported data for ACTIBIND and trT2-50.
人RNASET2（hRNASET2）已被证明具有抗血管生成和抗肿瘤作用，且与其核糖核酸酶能力无关。我们推测RNASET2通过与肌动蛋白结合发挥其抗血管生成和抗肿瘤活性，从而抑制细胞运动。我们在此专注于利用整个hRNASET2蛋白中遇到的特定序列来鉴定hRNASET2的肌动蛋白结合位点。为此，我们设计了29种不同的源自hRNASET2的肽段。检测了这29种肽段与固定化肌动蛋白结合的能力。两种选定的肽段——由57个氨基酸组成的A103 - Q159和由26个氨基酸组成的肽段K108 - K133被证明具有最高的肌动蛋白结合能力，同时具有最有效的抗血管生成活性。对肽段K108 - K133抑制血管生成相关潜在机制的进一步分析涉及到其在处理过程中在人脐静脉内皮细胞（HUVE细胞）内的定位。肽段K108 - K133在孵育10分钟内即可轻易穿透细胞膜。此外，添加血管生成素会延迟肽段K108 - K133进入细胞，这表明在相同的细胞内化途径上存在竞争。该肽段被证明与血管生成素共定位，这表明这两种分子结合类似的细胞表位，类似于我们之前报道的ACTIBIND和trT2 - 50的数据。
