Nuclear translocation of IGFBP3 by importin-β1 is a prerequisite for
IGFBP3-induced apoptosis. The neuroprotective peptide humanin (HN) counteracts
IGFBP3-induced cell death. However, the mechanism by which humanin protects cells
is currently unknown. The natural synthesis of this peptide decreases with age, coincident
with the likelihood for the development of Alzheimer’s Disease, making it a promising
target for therapeutics. We have examined the effect of full-length humanin and a
synthetic analogue (HN 3-19), known to be sufficient for its neuroprotective function, on
the interaction between IGFBP3 and importin-β1. Using competitive ligand dot blotting, co-immunoprecipitation, and an
ELISA-based binding assay, we determined that 1) humanin binds to IGFBP3 with a Kd of 5.05 µM and 2) both humanin
(IC50 of 18.1 µM) and HN 3-19 (IC50 of 10.3 μM) interfere with the binding of importin-β1 to IGFBP3 in vitro. We also
demonstrated that HN 3-19 is able to reduce the rate of apoptosis in a human lung adenocarcinoma cell line, suggesting a possible
mechanism of action for humanin as an inhibitor of IGFBP3 nuclear translocation. Understanding the exact mechanism
by which humanin and its analogue, HN 3-19, bind to IGFPB3 and regulate its interaction with importin-β1 will open the
door to modulating the protein-protein interactions involved in neuronal cell death.
Keywords: Alzheimer’s disease, apoptosis, humanin, IGFBP3, importin-β1, neuroprotective, peptide.
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