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Title page for ETD etd-07022014-114122


Type of Document Dissertation
Author Manna, Joseph Dominick
URN etd-07022014-114122
Title Identification and Characterization of a Major Prostaglandin Glycerol Ester Hydrolase in Human Cancer Cells
Degree PhD
Department Chemical and Physical Biology
Advisory Committee
Advisor Name Title
H. Alex Brown Committee Chair
Alan Brash Committee Member
Lawrence Marnett Committee Member
Ned Porter Committee Member
Richard Caprioli Committee Member
Keywords
  • Prostaglandin Glycerol Esters
  • Serine hydrolases
  • Lysophospholipase
  • Endocannabinoids
  • Activity Based Protein Profiling
Date of Defense 2014-05-19
Availability unrestricted
Abstract
Prostaglandin glycerol esters (PG-Gs) are produced by the oxygenation of the endocannabinoid, 2-arachidonoylglycerol, by cyclooxygenase 2. Understanding the role that PG-Gs play in a biological setting has been difficult because of the hydrolytic instability of the compounds. The main focus of this research was to identify the serine hydrolase responsible for PG-Gs hydrolysis and develop tools to assess the role this enzyme has in living systems.

Initially we sought to develop a new analytical method for the analysis and quantification of lipid metabolites. The most widely used methods employ chromatographic separation followed by mass spectrometric analysis, which requires significant time for sample preparation and sequential chromatography. We developed a novel high-throughput, separation-free methodology based on MALDI mass spectrometry that allows for the parallel analysis of targeted analytes. Proof-of-concept was demonstrated by analysis of prostaglandins and PG-Gs. Derivatization to incorporate a charged moiety into ketone-containing prostaglandins dramatically increased the signal-to-noise ratio relative to underivatized samples. This resulted in an increased dynamic range (15 fmol – 2000 fmol on plate) and improved linearity (r2= 0.99). The method was further adapted for high-throughput screening methods for enzymology and drug discovery.

Preliminary efforts to identify the hydrolase responsible for PG-G hydrolysis utilized protein purification techniques to try and isolate the enzyme. Upon purification, three enzymes were identified, 6-phosphoglucolactonase (PGLS), platelet activating factor acetyl hydrolase 1β3 (PAFAH1β3), and dimethylarginine dimethylaminohydrolase 2 (DDAH2). Protein expression in HEK293 and siRNA knockdown in MDA-MB-231 for each identified serine hydrolase made no significant contribution to PG-G hydrolysis. The inability to isolate a specific PG-G hydrolase by protein purification caused us to take a different approach to identify the hydrolase.

By comparing PG-G hydrolysis across human cancer cell types to serine hydrolase activities determined by activity-based protein profiling, we identified lysophospholipase A2 (LYPLA2) as a major enzyme responsible for PG-G hydrolysis. The principal role played by LYPLA2 in PGE2-G hydrolysis was confirmed by siRNA knockdown across multiple cancer cell types and expression in both HEK293 and E.coli. Additionally, LYPLA1, a homologue of LYPLA2, was shown to have no PGE2-G hydrolytic activity as determined by siRNA and overexpression.

Purified, recombinant LYPLA2 hydrolyzed PG-Gs with the following order of activity - PGE2-G > PGF2α-G > PGD2-G but, interestingly, did not hydrolyze the endocannabinoids, 2-AG or AEA. Chemical inhibition of LYPLA2 in RAW264.7 murine macrophages-like cell lines and 1483 human head and neck squamous cell carcinoma cells elicited an increase in PG-G production. Our data indicate that LYPLA2 serves as a major PG-G hydrolase in mammalian cells. Perturbation of this enzyme should enable selective modulation of PG-Gs without alterations in endocannabinoids, thereby providing a means to decipher the unique functions of PG-Gs in biology and disease.

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