Publication Date

12-2013

Advisor(s) - Committee Chair

Rajalingam Dakshinamurthy (Director), Kevin Williams, Bangbo Yan

Degree Program

Department of Chemistry

Degree Type

Master of Science

Abstract

Fibroblast growth factors (FGFs) are a family of growth factors which includes twenty three proteins. FGFs work as modulators for various cellular activities like mitosis, differentiation and survival. Among the FGF family, human fibroblast growth factor-1 (hFGF-1), which is also known as acidic fibroblast growth factor, is a potent angiogenic agent, involved in the formation of new blood vessels in various tissues. hFGF-1 is regarded as a prototype of the FGF family. It serves as one of the potential targets in tumor inhibition and obesity due to its involvement in new blood vessel formation in cancerous regions and adipose tissues. In general, FGFs exert their action by binding to heparin, forming FGF-heparin complex, which can then bind to fibroblast growth factor receptors (FGFRs). Inhibition of FGF dependent signal transduction by heparin mimicking compounds has shown promising results in control and treatment of tumor growth. Naturally occurring glycoside called phloridzin found to have anticancer property. Phloridzin (2-glucoside of phloretin) has structural resemblance to heparin; it is a natural antioxidant, widely known for its antidiabetic activity, besides controlling tumor growth. Phloridzin can mimic heparin and compete with it for FGF binding. This binding can be agonistic or antagonistic in nature on FGF signal transduction. In the present study, we investigated the molecular level interaction between phloridzin and hFGF-1 using various biophysical techniques like steady state fluorescence, limited trypsin digestion and protein-NMR spectroscopy. hFGF-1 needed for the study was expressed in recombinant Escherichia coli cells. The expressed protein was then purified using heparin sepharose affinity chromatography. Both expression and purification were monitored using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Conformational stability of purified hFGF-1 was assessed through steady state fluorescence. Purified hFGF-1 is in, its native, properly folded conformation. Interaction studies, such as thermal unfolding and limited trypsin digestion were performed to assess the thermal stability and solvent accessibility of hFGF-1 in the presence of phloridzin respectively. It was found from interaction studies that hFGF-1 in the presence of phloridzin shown increased thermal stability and increased resistance against trypsin digestion. In order to locate the sites of interaction on hFGF-1 surface, a protein-NMR study was performed. Exact sites of interaction of phloridzin on hFGF-1 surface were found. In future, isothermal titration calorimetry will be performed to determine kinetics of the enthalpy change and dissociation constant of phloridzin-hFGF-1 interaction. In vivo studies will also be performed after completion of in vitro studies, which will give an insight about possibility of phloridzin and hFGF-1 interaction under physiological condition

Disciplines

Chemistry | Medical Biochemistry | Medicinal-Pharmaceutical Chemistry | Organic Chemistry

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