Characterization of MICAL1-Mediated Oxidation of Cdc42

Rho GTPases are critically important and centrally positioned regulators of the actomyosin cytoskeleton. By influencing the organization and architecture of the cytoskeleton, Rho proteins play prominent roles in many cellular processes including adhesion, migration, intra-cellular transportation, and proliferation. Relative to other post-translational modifications (PTMs) such as lipidation or phosphorylation that have been extensively characterized, protein oxidation is a regulatory PTM that has been poorly studied. Protein oxidation primarily occurs from the reaction of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), with amino acid side chain thiols on cysteine (Cys) and methionine (Met) residues. The molecule interacting with CasL protein 1 (MICAL1) is a monooxygenase enzyme that produces diffusible H2O2 and influences F-actin disassembly. Cdc42 has been found to be a target of MICAL1 oxidation, and this thesis will examine how oxidation on conserved cysteine residues is affected by this monooxygenase enzyme. Furthermore, this thesis will characterize the use of a bifunctional sulfenic acid probe consisting of two bicyclo[6.1.0]nonyne (BCN) moieties linked with a short ethylenediamine linker (BCN-E-BCN) that will detect sulfenylated proteins in cultured cells and in vitro.