Date of Award

Spring 5-18-2019

Degree Type

Thesis

Degree Name

Master of Science - Natural Sciences

Department

College of Science and Mathematics

First Advisor

Odutayo Odunuga

Second Advisor

Michele Harris

Third Advisor

Matibur Zamadar

Fourth Advisor

Rebecca Parr

Abstract

Ellis–van Creveld (EvC) syndrome is a rare autosomal recessive disorder that is characterized by short-limb dwarfism, extra fingers or toes, malformed teeth and nails, and congenital heart defects. Mutations in two genes, EVC and EVC2, which are located on chromosome 4, have been shown to be responsible for EvC syndrome. The protein products of these genes, EVC and EVC2 physically interact and together, they are tethered to the base of the primary cilium of cells. This multi-protein assembly positively regulates the Sonic and Indian hedgehog signaling pathways by promoting downstream processes after activation of the smoothened receptor (Smo), by the hedgehog proteins. Bioinformatic analysis revealed the presence of several structural motifs in EVC sequences including P-loop, leucine zipper, transmembrane domain, and nuclear localization signal. The putative nucleotide-binding and hydrolyzing activities of EVC were tested using fragments of the protein from mice and human that contain the P-loop and leucine zipper regions. The presence of GTP analog caused the quenching of the intrinsic fluorescence of the protein, suggesting interaction between the two molecules. The fluorescence gradually recovered as the nucleotide or its hydrolysis product dissociated from the protein.


GTP hydrolysis assays showed that EVC possessed a measurable intrinsic GTPase activity that obeyed Michaelis-Menten kinetics. The KM, kcat and catalytic efficiency of the enzyme were calculated to be 0.820 mM, 0.175 sec-1 and 213 M-1sec-1 respectively. Compared to other GTPases, the KM was higher, indicating a low affinity of the protein for GTP. The leucine zipper caused the protein to form dimers could be seen on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, the dimerization caused the protein to hydrolyze GTP to a mixture of GDP and GMP at unequal ratio. These kinetic parameters were similar to those displayed by large GTPases such as dynamin. It is proposed that the GTP-hydrolyzing activity of EVC protein is an important mechanistic step in the regulation of the hedgehog signaling pathways.

Included in

Biochemistry Commons

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