Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Horm Metab Res. 2013 Jun;45(6):415-22. doi: 10.1055/s-0032-1331699. Epub 2013 Jan 18.
Several physiological activities have been assigned to E-peptides derived from pre-pro-insulin-like growth factor (IGF1) processing; however, the whole range of the E-peptides' functions is still unknown. The objective of this study was to investigate human Eb peptide (hEb) in terms of its bioactivity, cellular localization, and intracellular trafficking using human cancer cells. Human Eb fused with red fluorescence protein (RFP) or green fluorescence protein (GFP) localizes strongly to nucleoli and to a lesser extent to nuclei of HeLa and U2-OS cells. Mutagenesis of hEb nucleolus localization sequence (NoLS) leads to its partial delocalization from nuclei and nucleoli to cytoplasm of transfected cells. Thus, NoLS is not sufficient for the hEb to be localized in nucleoli of the cells and a different mechanism may be involved in hEb targeting. A BrdU ELISA showed that the proliferation index of cells expressing hEb hybrid proteins increased up to 28%. For comparison, the same assay was performed using HeLa cells treated extracellularly with synthetic hEb. A significant increase in the proliferation index was observed (41-58% for concentrations ranging from 10-100 nM, respectively). Additionally, a cell migration assay was performed using stable U2-OS cell lines expressing hEb fused with RFP or RFP alone as a negative control. The migration index of hEb expressing cells was 38.3% greater. The increase in cell proliferation index and in motile properties of hEb expressing cells demonstrate that hEb is more than a pre-pro-IGF1b processing product, and has intrinsic activity of biological significance.
几种生理活性已被分配给前胰岛素样生长因子（IGF1）加工衍生的 E 肽；然而，E 肽的全部功能仍不清楚。本研究旨在使用人类癌细胞研究人类 Eb 肽（hEb）的生物活性、细胞定位和细胞内运输。与人红色荧光蛋白（RFP）或绿色荧光蛋白（GFP）融合的 hEb 强烈定位于核仁，在一定程度上也定位于 HeLa 和 U2-OS 细胞的核。hEb 核仁定位序列（NoLS）的突变导致其部分从细胞核和核仁脱定位到转染细胞的细胞质中。因此，NoLS 不足以使 hEb 定位于细胞的核仁中，可能涉及不同的机制。BrdU ELISA 显示表达 hEb 杂合蛋白的细胞增殖指数增加了 28%。相比之下，使用用合成 hEb 处理的 HeLa 细胞进行了相同的测定。在浓度范围为 10-100 nM 时，观察到增殖指数显著增加（分别为 41-58%）。此外，使用表达 hEb 的稳定 U2-OS 细胞系进行了细胞迁移测定，将其与单独表达 RFP 的 RFP 融合作为阴性对照。表达 hEb 的细胞的迁移指数增加了 38.3%。表达 hEb 的细胞增殖指数增加和运动特性增加表明，hEb 不仅仅是前胰岛素样生长因子（IGF1b）加工产物，而且具有内在的生物学意义的活性。
