Dissecting the role of phospholipase A2 in the late stages of regulated exocytosis

Abstract

This thesis is divided into five chapters. The overarching working hypothesis is published as a 'perspective' article in International Journal of Biochemistry and Cell biology, 2017 https://doi.org/10.1016/j.biocel.2017.01.011 and is also discussed in the last section of Chapter - 1. Hence, there are similarities in the published article and sections of Chapter - 1. The working hypothesis states that endogenous PLA2 near docking/ fusion sites provide basal levels of LPC that acts as a 'molecular brake' against spontaneous fusion, and FFA that aids in trans-SNARE complex formation to ensure efficient docking of secretory vesicles to the plasma membrane (PM) prior to triggering. The data in Chapters - 2 and - 3 then test the working hypothesis and are also published as original articles in cells, 2019 https://doi.org/10.3390/cells8040303 and Biochemical and Biophysical Research Communications, 2019 https://doi.org/10.1016/j.bbrc.2019.05.106, respectively. The data presented in Chapter - 2, indicate that vesicle-associated endogenous PLA2 isozymes - sPLA2 in the CV lumen and iPLA2 on the CV surface - maintain docking/priming steps in regulated exocytosis. The specific CV localization of sPLA2 and iPLA2 is also shown, using highly sensitive two-dimensional gel electrophoresis (2DE) immuno-blotting with selective antibodies on separated luminal and membrane fractions. Biochemical assays confirmed that these isozymes are catalytically active; sPLA2 showed higher efficiency in catalyzing PE than PC, while iPLA2 showed no such preference. Notably, a significant increase in the de novo metabolite TAG upon iPLA2 inhibition confirmed the role of this isozyme in maintaining membrane lipid homeostasis. Removal of CV surface proteins by treatment with trypsin, coupled with the use of a selective PLA2 substrate (PED6) also confirmed the presence of an active PLA2 isozyme in the outer CV membrane. Overall, the data presented in Chapter - 2, provide evidence in support of the working hypothesis. To test the hypothesis further, in Chapter - 3, exogenous arachidonic acid (ARA) and lysophosphatidylcholine (LPC), canonical PLA2 metabolites, are shown to impair docking/priming and actual membrane merger steps, respectively. The observed decrease in docking/priming induced by exogenous ARA is contradictory to part of the working hypothesis that was put forth based on existing literature. That exogenous LPC and ARA were to a significant extent converted into endogenous PC and FFA, respectively, indicated that endogenous enzymes maintain lipid homeostasis in the native vesicle membrane. With the intension of activating CV luminal sPLA2, Phospholipase A2 activating peptide (PLAP) - a melittin homolog, was used in Chapter - 4. The data presented indicates that PLAP inhibited CSC, and CV fusion by impairing docking/priming and also altered endogenous PE and FFA levels in the CV membrane. This suggests that PLAP caused no change in the CV luminal sPLA2 activity; an observation contrary to the earlier in vitro studies. To test further, PLAP was delivered to mouse chromaffin cells and was seen to reduce membrane capacitance and readily releasable pool (RRP) size, when triggered with depolarizing pulses. This substantiate that the PLAP blocked a common underlying mechanism associated with the late stages of Ca2+ triggered exocytosis. The Chapter - 4 is another manuscript in preparation and will be submitted to an appropriate journal. Finally, in Chapter - 5, the Discussion and Future Directions, all key finding in this thesis are reviewed and integrated with a critical evaluation of the existing literature, and prospective future experiments are outlined.

Date of Award	2019
Original language	English