Publication Date

8-2021

Date of Final Oral Examination (Defense)

6-18-2021

Type of Culminating Activity

Dissertation

Degree Title

Doctor of Philosophy in Biomolecular Sciences

Department

Biology

Supervisory Committee Chair

Owen M. McDougal, Ph.D.

Supervisory Committee Co-Chair

Lisa R. Warner, Ph.D.

Supervisory Committee Member

Cheryl L. Jorcyk, Ph.D.

Supervisory Committee Member

Matthew King, Ph.D.

Supervisory Committee Member

Xinzhu Pu, Ph.D.

Abstract

Cardiovascular disease (CVD) is the leading cause of death worldwide affecting approximately 40% of all adults over the age of 20 and is responsible for an economic burden upwards of $3 billion annually. Treatments for CVD are limited to either hypertension medication to treat symptoms, and/or statin-based drugs to reduce low-density lipoprotein (LDL) cholesterol formation. However, recent studies suggest that approximately 50% of patients diagnosed with CVD have normal to low LDL cholesterol levels. Therefore, a critical need exists to develop new treatments for CVD that are independent of cholesterol lowering statins. Fortilin, also known as translationally controlled tumor protein (TCTP), is a 19kDa, 172 amino acid cytosolic protein ubiquitously expressed in all cell types, at all stages of life. Fortilin overexpression in arterial walls has been shown to propagate atherosclerotic plaque formation, a major component of CVD. Fortilin is therefore a promising target for the rational design of drugs to prevent formation of new plaques.

For a structure-based drug development process, recombinant protein is required to characterize potential protein-drug interactions. Recombinant expression and purification of fortilin has proven to be effectual, provided any affinity tag used for purification is not cleaved. Here, we designed several constructs of recombinant fortilin fusion protein and were the first to successfully cleave the affinity tag, making structural activity studies more meaningful due to greater semblance to the native protein. Our new construct, GGS-fortilin, can be produced in high yield with greater than 85% tag cleavage, but still contains the three amino acid linkers at the N-terminus. To determine whether these three amino acids, Gly-Gly-Ser interfered with small molecule inhibitor (SMI) binding, loop constructs were designed, wherein the affinity tag, a Strep-Tactin peptide with the sequence WSHPQFEK, was placed at either the C terminal side of Arg37, Ser46 or Gly56 within the flexible loop of fortilin’s highly conserved structure and activity validated via calcium titration. Structural analysis and SMI binding studies were performed for the loop constructs using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy.

NMR spectroscopy is a dynamic tool used in structure-based drug development to characterize the structure of recombinantly expressed protein and to determine binding sites for SMIs. The results presented here portend to the development of novel fortilin constructs and structural studies with SMIs. Structural integrity was validated for the loop constructs using CD and NMR, and activity validated with an NMR calcium binding assay. Binding studies were also attempted using differential scanning fluorimetry (DSF) and NMR.

DOI

https://doi.org/10.18122/td.1861.boisestate

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