PROJECT SUMMARY
Proteins are known to play a crucial role in the undertaking of numerous cellular processes and a variety of
human diseases result from disruption of their function. Many gaps in knowledge need to be filled, however, to
gain a better understanding of how proteins regulate and fine-tune signaling of glycosaminoglycans (GAGs),
components of the highly dynamic extracellular matrix. Insulin-like growth factor-binding protein-3 (IGFBP-3) is
known to bind certain GAGs and operate in either an insulin-like growth factor (IGF)-dependent or independent
manner to exert its cytotoxic functions. How dysregulation of the IGFBP-3-GAGs interactions underlies cytotoxic
mechanisms, is largely unknown, needs to be elucidated, and currently represents a significant gap in our
knowledge. Our long-term goal is to develop tools to switch protein-GAGs interactions ‘on and off’ to regulate
cell survival. The overall objective of this proposal is to shed light on the mechanisms employed by IGFBP-3 in
modulating hyaluronan (HA) and heparan sulfate (HS) signaling, and how modulation of this signaling ultimately
leads to cytotoxicity. Our central hypothesis is that IGFBP-3 is a key and vital player in regulating diverse aspects
of GAG signaling. Accomplishing our aims in this proposal will lead to novel insights into the molecular
mechanisms underlying fundamental protein-GAGs interactions. Our specific aims are to: 1. Test the hypothesis
that IGFBP-3 blocks HA-CD44 signaling resulting in a p53-dependent increase in its own levels and signaling,
and a p53-dependent decrease in the levels of heparanase, promoting apoptosis and decreasing cell survival,
2. Test the hypothesis that bacterial attachment and docking to the mammalian cell surface is hindered by both
binding of extracellular IGFBP-3 to HS and by the enzymatic activity of heparanase, 3. Examine the hypothesis
that blocking HA-CD44 signaling with either the HA synthesis inhibitor 4-Methylumbelliferone (4-MU) or IGFBP-
3, leads to decreased matrix metalloproteinases-2 and -9 (MMP2/9) levels and increased extracellular
accumulation of Pro-BDNF which is now able to bind its receptor, decreasing cell survival, 4. Test the hypothesis
that phosphorylation of IGFBP-3 by casein kinase 2 (CK2) results in increased cell survival by two mechanisms:
phosphorylation a) inhibits IGFBP-3’s ability to bind HA allowing the sugar to re-bind its receptor, CD44; b)
inhibits binding of the cytoprotective peptide, humanin (HN) to IGFBP-3, allowing HN to bind the amyloid-ß
peptide (Aß), recruiting acetylcholinesterase (AChE) into a ternary complex, decreasing AChE activity,
increasing ACh levels, and activating the a7nAChR. Better understanding of these basic mechanisms will
advance our knowledge of diseases resulting from dysregulation of protein-peptide-carbohydrate signaling and
is likely to provide valuable clues into novel therapeutic strategies targeting protein-GAGs interactions in
fundamental cellular processes. This R15 application provides an effective vehicle for introducing undergraduate
and graduate students to an authentic and extensive hands-on research training at an early stage of their
education and cultivates confidence, resilience, appreciation for and interest in biomedical research careers.
