PLC (phospholipase C) enzymes hydrolyze phosphatidylinositol lipids to generate inositol phosphates (IPx) and diacylglycerol (DAG). These second messengers stimulate intracellular calcium release and protein kinase C activation. PLCε is the most recently identified member of the PLC family, and is activated downstream of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) through direct interactions with G proteins. In the cardiovascular system, PLCε is necessary for normal cardiac contractility, and its dysregulation contributes to cardiac hypertrophy and heart failure. PLCε shares a highly conserved core that is observed in other PLC subfamilies, and contains a unique N-terminal CDC25 domain and two C-terminal Ras association (RA) domains. However, structural insights into the full-length protein, or catalytically active fragments that contain two or more regulatory domains, have been lacking. Using a combination of structural and functional studies, we previously showed that the PH domain and first two EF hands are dynamic in solution, providing an explanation as to why high-resolution structures have been elusive. We are now pursuing high-resolution structural studies of larger PLCε variants and the full-length enzyme using single particle cryo-electron microscopy (cryo-EM). To facilitate this process, we are utilizing domain-specific fab antibodies as fiduciary markers and to stabilize this conformationally heterogeneous protein. Understanding the structure of PLCε will be critical in identifying sites that can be targeted by small molecule chemical probes, ultimately validating this enzyme as a therapeutic target in cardiovascular disease.
Support or Funding Information
NIH NHLBI: 1RO1HL14076-01
Purdue University Center for Cancer Research: P30CA023168
lt;pgt;NIH NHLBI: 1RO1HL14076-01lt;/pgt;lt;pgt;Purdue University Center for Cancer Research: P30CA023168lt;/pgt;
