Graduation Semester and Year

2018

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry

Department

Chemistry and Biochemistry

First Advisor

Jongyun Heo

Abstract

The Ras superfamily of small GTPases are monomeric proteins that bind guanine nucleotides, and regulate many cellular signaling pathways. They function as molecular switches by shuttling between active GTP-bound and inactive GDP-bound states. The Ras superfamily consists of its subfamilies including Ras and Rho GTPases. Ras and Rho GTPases function to regulate cellular growth, transformation, transport, motility, traffic, and adhesion. Small GTPases reactions often require regulators, such as guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs). GEFs mediate fast release of GDP and allow loading with GTP while GAPs accelerate the GTP hydrolysis to GDP. Redox agents also are capable to regulate Ras and Rho GTPases by targeting unique redox sensitive motifs that include NKCD and GXXXXGK(S/T)C. This thesis focuses on the cysteine (Cys) modification of GXXXXGK(S/T)C motif and how it affects cellular functions of Rho proteins. The inactivation mechanism of two redox-sensitive Rho GTPases, Ras-related C3 botulinum toxin substrate 1 (Rac1) and Ras homolog gene family member A (RhoA), is investigated. Many studies have shown that Rac1 is essential for cell transformation, tumor cell invasion, and metastasis. This study shows that thiopurine prodrugs (TPs) in combination with a redox agent effectively inhibit the metastasis of Rac1-overexpressed prostate cancer cells. The molecular mechanism for Rac1 inactivation via its redoxsensitive residue Cys18 and TPs-mediated treatment is also proposed. RhoA possesses Cys16 in addition to the redox-sensitive Cys20 in the GXXXXGK(S/T)C motif, where both Cys16 and Cys20 are confirmed to be redox-sensitive in GXXXC16GK(S/T)C20. Chapter 3 examines the possibility of inactivating RhoA by targeting Cys16 and Cys20. Phenylarsine Oxide (PAO) and 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) are thiol cross-linking reagents that oxidize vicinal thiols. During this study, PAO and 15d-PGJ2 significantly diminished the migration of breast cancer cells that overexpress RhoA GTPase. It was suggested that PAO and 15d-PGJ2 specifically target Cys16 and Cys20 side chains of RhoA, resulting in inactivation of RhoA protein and blockage in metastasis. The findings of this research reveal the therapeutic potentials of TPs, PAO, and 15d-PGJ2 that target the redox sensitive cysteines of Rac1 and RhoA.

Keywords

Metastasis, Rho GTPases, Redox regulation, Redox-sensitive GXXXXGK(S/T)C motif, Thiopurine prodrugs, Disulfide, Vicinal cysteines

Disciplines

Chemistry | Physical Sciences and Mathematics

Comments

Degree granted by The University of Texas at Arlington

Included in

Chemistry Commons

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