AS_7.12_final.pdf

Reason: The data in this thesis may be including or used in future publications from the Lyon lab.

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STRUCTURAL AND MECHANISTIC INSIGHTS INTO THE ACTIVATION OF PHOSPHOLIPASE C EPSILON BY SMALL GTPASE RAP1A

thesis
posted on 13.07.2021, 00:23 by Arielle F SelviaArielle F Selvia

Phospholipase Cε (PLCε) is an enzyme that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PI4,5P2 or PIP2) and phosphatidylinositol 4-phosphate (PI4P) lipids at the plasma membrane and perinuclear membrane, respectively, producing the second messengers diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3). PLCε has low basal activity, and is activated by direct interactions with the small GTPase Rap1A. Rap1A is activated downstream of β-adrenergic receptors and binds to the Ras association (RA) domain 2 (RA2) of PLCε. This interaction translocates the Rap1A-PLCε complex to the perinuclear membrane for lipid hydrolysis. PLCε is expressed in the heart, where it is vital for cardiomyocyte function, including processes such as proliferation, differentiation, and survival. The DAG produced from PI4P hydrolysis is required for Ca2+-induced Ca2+ release in the cardiovascular system. In addition, mutations and/or increased expression of PLCε is implicated in cardiac hypertrophy and heart disease. However, the exact role that Rap1A-dependent activation of PLCε plays in the disease process is not fully understood. The RA2 domain is essential for Rap1A binding and activation, and a recent study demonstrated that the PLCε pleckstrin homology (PH) domain and first two EF hands (EF 1/2) are also required. Small-angle X-ray scattering was used to investigate the solution structures of Rap1A-PLCε complexes that varied in their sensitivity to activation and showed that Rap1A can stabilizes unique conformational states. SAXS and activity assays were also used to investigate the roles of six conserved hydrophobic residues on the RA2 surface that were implicated in Rap1A-dependent activation. Taken together, these studies provide novel insights into the allosteric activation of PLCε by Rap1A.

History

Degree Type

Master of Science

Department

Chemistry

Campus location

West Lafayette

Advisor/Supervisor/Committee Chair

Angeline Lyon

Additional Committee Member 2

Nicholas Noinaj

Additional Committee Member 3

Elizabeth Parkinson

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