Date of Graduation
1-2013
Document Type
Dissertation
Degree Name
Doctor of Philosophy in Chemistry (PhD)
Degree Level
Graduate
Department
Chemistry & Biochemistry
Advisor/Mentor
Suresh Kumar
Committee Member
Dan Davis
Second Committee Member
Roger Koeppe
Third Committee Member
Paul Adams
Fourth Committee Member
Colin Heyes
Abstract
Fibroblast growth factors (FGFs) are a superfamily of 23 heparin-binding proteins. Acidic fibroblast growth factor (FGF1) is a 16 kDa protein that is involved in many important biological processes such as cell differentiation, angiogenesis, and wound healing. FGF1 exhibits its activity by binding to cell-surface tyrosine kinase receptors (FGFRs). In addition to binding to FGFRs, FGF1 binds to heparin/HLGAGs. Heparin is proposed to have two functions: it protects FGFs from degradation by proteases and from thermal denaturation; and upon binding it induces FGF1 oligomerization, which is mandatory for binding to FGFR. However, it remains controversial whether or not heparin is required for both purposes.
The aim of this research is to fully characterize the heparin-binding region of FGF1 in terms of its contribution to the stability and function of the protein. In this context, mutational studies as well as various biophysical techniques were employed. To determine the role of heparin, several mutations within the heparin-binding region of FGF1 were designed. Structural studies were used to monitor changes in secondary and tertiary structure, while binding studies quantified the affinity of each mutant for heparin. Cell proliferation assays were performed to correlate heparin binding to cell proliferation activity promoted by FGF1. Within the heparin-binding region of FGF1 there is a thrombin-cleavage site, Arg136. Although this is not a classical thrombin-cleavage site, thrombin nevertheless cleaves at this site and decreases the activity of FGF1. This could serve as a possible regulatory mechanism of FGF1 in situations such as tumor growth when elevated FGF signaling and thrombin levels have been reported. Several mutations were made at this cleavage site and the structural and functional consequences were determined. To better understand the FGF-heparin interaction, binding studies were conducted to characterize the precise forces governing the heparin-FGF1 interaction. With the additional information obtained from these studies, a synthetic heparin with higher FGF1 binding-affinity could be designed and used in therapeutic settings. Finally, a heparin-binding peptide was synthesized and the binding to heparin was characterized. A method to overexpress this recombinant peptide was developed to isotope enrich the peptide for use in solution NMR studies.
Citation
Kerr, R. (2013). The Role of Heparin in Acidic Fibroblast Growth Factor Signaling. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/4814