Doctor of Philosophy (PhD)
Challenges in drug efficacy occur during the treatment of most types of cancer due to the heterogeneity of the tumor microenvironment. This has led to the development of personalized medicine. Due to the clinical success of the proteasome inhibitors Bortezomib and Carfilzomib in treatment of multiple myeloma, interest has shifted towards molecularly-targeted chemotherapeutics for ubiquitin-proteasome system (UPS). Deubiquitinating enzymes (DUBs) are an essential part of this pathway which have been found to promote Bortezomib resistance in multiple myeloma patients. Unfortunately, there is a lack of specific, high throughput biochemical assays to characterize DUB activity in patient samples before and after treatment with DUB inhibitors. Therefore, there is a need for novel biochemical assays for quantifying DUB activity in a single cell level. In this research, a long-lived, cell permeable, peptide-based reporter was developed to directly quantify DUB activity in intact single cells. A hallmark of this reporter is the use of a β-hairpin ‘protectide’ which can confer its stability to enzyme substrates. First, a study was performed to determine if the arginine-rich β-hairpin sequence motif could behave as a cell penetrating peptide (CPP). Chapter 2 highlights these findings and confirms that the RWRWR β-hairpin sequence could be rapidly internalized into intact cells. Chapter 3 summarizes findings from another study that investigated the use of incorporating D-chirality amino acids into CPPs. The D-chirality peptides were confirmed to be highly stabled under intracellular conditions and were found to be rapidly internalized. Next, a microfluidic droplet trapping array was developed to encapsulate and analyze the intracellular fluorescence of intact single cells. Chapter 4 summarizes the development of this device and its use to identify distinct subpopulations that emerge with respect to the CPP uptake. Finally, Chapter 5 utilizes the findings from Chapter 2 to develop a long-lived, cell permeable, fluorescent, peptide-based reporter for DUB activity. This chapter confirms the utility of this reporter in both cell lysates and intact cells. In summary, a set of toolkits including CPPs, a microfluidics droplet trapping array, and peptide-based DUB reporter were developed to provide a new platform for drug screening and personalized diagnostics.
Safabakhsh, Nora, "Direct Quantification of Deubiquitinating Enzyme Activity in Single Intact Cells" (2018). LSU Doctoral Dissertations. 4698.
Available for download on Wednesday, August 21, 2019
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