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<p>The phospholipase C (PLC) family of enzymes canonically hydrolyzes the inner plasma membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG are crucial secondary messengers that activate multiple signaling pathways and modulate gene expression to control cellular function and behavior. The PLCe subfamily is essential for normal cardiovascular function, where it is activated through direct interactions with the RhoA and Rap1A small GTPases, linking lipase activity to the stimulation of G protein-coupled receptors (GPCRs. RhoA activates PLCe at the plasma membrane, whereas Rap1A translocates and activates PLCe at the perinuclear membrane, where phosphatidylintol-4-phosphate (PI4P) is hydrolyzed. The domains of PLCe involved in G protein binding, activation, and translocation to different subcellular membranes are largely unknown. In this work, we use cell-based activity assays, epifluorescence, and confocal microscopy to identify the domains of PLCe involved in basal activity, subcellular localization, and regulation by RhoA and Rap1A GTPases. Our preliminary studies demonstrate that the unique N- and C-terminal regulatory domains of PLCe dictate its location within the cell and contribute differently to basal and G protein-dependent activity. These studies will provide needed insights into the regulation and localization of PLCe in cells, which is critical for its roles in cardiovascular function. </p>
<p>Lipids on membranes are known to regulate the function of lipases in cells, however their contribution towards this phenomenon is not well understood. It is difficult to explore this mechanism due to the dynamic behavior of lipid bilayers in cells and most importantly the unknown variable of the amount of lipids present locally when lipases are activated. This study utilizes an approach, to use in vitro reconstitution methods to understand the contribution of lipids in activation of PLCb3 which is known to hydrolyze PIP2 in cell to generate IP3 and DAG. We also use single enzyme kinetics using total internal reflection microscopy to understand recruitment of single enzyme to the membrane, its association and disassociation rates at the membrane. </p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/20402052 |
| Date | 29 July 2022 |
| Creators | Kaushik Muralidharan (13169904) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/thesis/MOLECULAR_MECHANISMS_OF_PHOSPHOLIPASE_C_AND_REGULATION/20402052 |
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