INVESTIGATING RHOA-DEPENDENT REGULATION OF PHOSPHOLIPASE C EPSILON IN CARDIOVASCULAR DISEASE

Phospholipase Cε (PLCε) is required for normal cardiovascular function, and dysregulation of its expression or activity has been shown to cause cardiac hypertrophy and heart failure. However, regulation of PLCε by the RhoA small GTPase protects the heart against ischemia-reperfusion injury, particularly downstream of G_12/13-coupled receptors. Despite the role of RhoA and PLCε in driving the cardioprotective response, little is known about how these proteins interact to increase lipase activity. RhoA was initially thought to bind to PLCε through one of its C-terminal Ras association (RA) domains, which are essential for its regulation by other GTPases. However, the RA domains are dispensable for both RhoA binding and activation, and further truncations of PLCε narrowed its binding site to the highly conserved PLC catalytic core. Functional studies implicated an insertion within the catalytic TIM barrel domain, known as the Y-box, as a requirement for RhoA-dependent activation of PLCε. However, the Y-box does not bind the GTPase. The goal of this dissertation is to identify the molecular mechanism by which RhoA binds to PLCε and increases its activity using structural and functional studies. The successful completion of these studies will map the interaction between these two critical signaling proteins, as well as identify elements in PLCε required for activation at the membrane. Ultimately, this knowledge can be exploited to develop lead therapeutic compounds that modulate this interaction to improve cardiovascular health.