Abstract
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.
Key words
NoLS - nucleolus - protein trafficking - stable cell lines - proliferation and motility
index
