Graduate Program

Chemistry

Degree Name

Master of Science (MS)

Semester of Degree Completion

Spring 2021

Thesis Director

Michael W. Beck

Thesis Committee Member

Thomas Canam

Thesis Committee Member

Radu F. Semeniuc

Thesis Committee Member

Gopal R. Periyannan

Abstract

Understanding the subcellular localization of proteins and their activity is important in understanding their normal function in eukaryotic cells. Fluorescence cellular imaging techniques can selectively and sensitively visualize subcellular biochemistry. Using this approach, two different methods were employed in this thesis. The first focused on studying protein import into peroxisome and the other on monitoring the activity of an endoplasmic reticulum (ER)-localized enzyme, human carboxylesterase 1 (CES1).

Peroxisomes are mainly known as the center for long chain fatty acid b-oxidation as well as the production and detoxification of hydrogen peroxide. Proteins which are needed in the peroxisomes are encoded in the nucleus and synthesized in the cytosol which are then transported to the peroxisomes with the help of a sophisticated protein-transport machinery. Pex5 is one of the key transport proteins responsible for carrying proteins tagged with the peroxisome targeting sequence 1 (PTS-1). The peptide signals that target proteins to the peroxisomes, however, are poorly studied in human cell lines. While different PTS-1 targeting sequences are known to have different efficiencies in being delivered to the peroxisomes, no study has cataloged all known localization signals under the same conditions in human cells. Thus, the aim of the first part of thesis is to design a method to quantify the peroxisome targeting ability of a peptide sequence.

In second part of this thesis, ER-localized CES1 is an enzyme that plays a key role in ester-containing drug metabolism. CES1 is known to have a high degree of genetic variation through a variety of mechanisms including single nucleotide polymorphisms (SNPs) producing CES1 forms that have reduced metabolism of ester-containing drugs, such as methylphenidate (Ritalin; treats ADD), oseltamivir (Tamiflu; treats the flu), and trandolapril (Mavik; treats high blood pressure). Thus, depending on a patient’s genetics they may produce a form of CES1 that metabolizes a drug at different rate than somebody who has another genetic form of CES1. This has led to different clinical outcomes for patients. Current methods for studying CES1 typically require relatively large sample sizes and purification steps to isolate CES1 metabolites before analysis. To address this, fluorescent probes have been developed but to date there are only two well-characterized probes that can report on CES1 activity in live cells. Overall, having a variety of fluorescent CES1 probes with different properties would allow researchers to choose the one that is best suited for use in the system of their interest. To address this, Fluorescein-based CES1 Probe 1 (FCP-1) was developed and evaluated for its ability to monitor CES1 activity in living cells using epifluorescence microscopy. Studies with small molecule inhibitors and genetic manipulations indicate that FCP-1 can specifically report on CES1 activity in human cell lines. As a whole, this thesis demonstrates that fluorescence microscopy can be a powerful tool in studying subcellular biochemical processes in live cells.

Available for download on Saturday, May 27, 2023

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