Human Eb peptide: not just a by-product of pre-pro-IGF1b processing?
Human Eb peptide: not just a by-product of pre-pro-IGF1b processing?
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DOI：
10.1055/s-0032-1331699
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发表时间：
2013-06
期刊：
影响因子：
--
通讯作者：
Barton ER
中科院分区：
文献类型：
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作者：
Durzyńska J;Wardziński A;Koczorowska M;Goździcka-Józefiak A;Barton ER
几种生理活性已被分配给前胰岛素样生长因子（IGF 1）加工的E-肽;然而，E-肽的功能的整个范围仍然是未知的。本研究的目的是研究人Eb肽（hEb）的生物活性，细胞定位，并利用人癌细胞的细胞内运输。与红色荧光蛋白（RFP）或绿色荧光蛋白（GFP）融合的人Eb强烈定位于HeLa和U2-OS细胞的核仁，并且在较小程度上定位于细胞核。hEb核仁定位序列（NoLS）的突变导致其从转染细胞的核和核仁到细胞质的部分离域。因此，NoLS不足以使hEb定位在细胞的核仁中，并且不同的机制可能涉及hEb靶向。BrdU ELISA显示表达hEb杂合蛋白的细胞的增殖指数增加高达28%。为了比较，使用用合成hEb细胞外处理的HeLa细胞进行相同的测定。观察到增殖指数显著增加（10-100 nM浓度范围分别为41- 58%）。此外，使用表达与RFP融合的hEb或单独的RFP作为阴性对照的稳定U2-OS细胞系进行细胞迁移测定。表达hEb的细胞的迁移指数高38.3%。表达hEb的细胞的细胞增殖指数和运动特性的增加表明hEb不仅仅是前-pro-IGF 1b加工产物，并且具有生物学意义的内在活性。
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.