Date of Graduation

12-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Fan, Chenguang

Committee Member

Koeppe, Roger E. II

Second Committee Member

Ivey, D. Mack

Third Committee Member

Thallapuranam, Suresh

Keywords

Acetylation; Citric acid cycle; Genetic code expansion; Krebs cycle; Post-translational modifications; TCA cycle

Abstract

The tricarboxylic acid (TCA) cycle is a central route for the energy production via oxidative phosphorylation and is ubiquitous throughout nature. Apart from playing a major role in energy provision, it is also essential for a wide range of physiological functions in any organism. Although an increasing emphasis is laid on the interlinking role of the cycle, its regulation and control are less very less understood. As the field progresses, the consequences of TCA cycle anomalies are shown to be tied to various cancers and other disorders. The matter becomes more complex when considering the varying functions of the isozymes present in the cycle.

The repertoire of twenty amino acids that is present in almost all organisms makes up for the hundreds of proteins responsible for the basic functioning of an organism. In order to synthesize a wide spectrum of viable proteins, one of the methods that the cells utilize is post-translational modifications. These modifications can be reversible depending on the conditions and they render conformational changes to the protein, thereby influencing its enzyme activity or labeling them for transportation or degradation. One such post-translational modification is acetylation, which is the main focus of this project. Acetylation is predominantly seen across all domains of life, and in spite of the prevalence of this modification, it is only recently that with the advances in technology the basic role of acetylation is being understood. However, there are still many questions that need to be answered to recognize the complete role of protein acetylation.

Recently, a newer technique has been adopted to study the impact of post-translational modifications. The genetic code expansion strategy helps in the incorporation of a noncanonical amino acid into a distinct position, thereby giving rise to a customized protein. It uses stop codon suppression to insert the unnatural amino acid. The pyrrolysyl-tRNA synthetase and its cognate tRNA were used in this project to create TCA cycle enzymes with acetyllysine.

In this dissertation, I examine the changes caused in the enzymes, malate dehydrogenase, isocitrate dehydrogenase, and citrate synthase, of the TCA cycle in response to acetylation of specific lysine residues and deduce the potential mechanisms regulating these changes. In doing so in E. coli, a greater understanding of protein acetylation is achieved and its implications on modulating functions are examined.

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