Graduation Year

2020

Document Type

Thesis

Degree

M.S.

Degree Name

Master of Science (M.S.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Charles Chalfant, Ph.D.

Committee Member

Margaret Park, Ph.D.

Committee Member

Sandy Westerheide, Ph.D.

Keywords

Acid Sphingomyelinase, Cell Metabolism, Inflammation, Lipidomics

Abstract

Ceramide 1-Phosphate (C1P) is a sphingolipid metabolite which plays a large role in inflammation, cell survival and proliferation1. C1P is known to have both pro- and anti-apoptotic roles in lung cancer cells, governed by ceramide kinase (CERK), upstream of precursor ceramide (Cer)2. Previous work reveals C1P serves as the liaison between sphingolipid and eicosanoid synthesis, by decreasing the dissociation rate of group IVA cytosolic PLA2 (cPLA2α) from the Golgi membrane, C1P directly activates this phospholipase for downstream eicosanoid synthesis and subsequent inflammatory response3. CERK has been discovered to modulate eicosanoid synthesis, elucidating upstream enzymes may have similar regulation4. It is unknown the specifics behind C1P anabolism and catabolism, such as which isoform of ceramide synthase (CerS) yields the primary chain length of Cer for C1P synthesis, and exploration of the elusive phosphatases responsible for converting C1P back to Cer. The total amount of C1P which derives from the de novo pathway is unknown.

In this study, we hypothesize that ceramide synthase-1 and lipid phosphate phosphatase 3 are the primary enzymes for C1P de novo anabolism and catabolism, respectively, and exploiting their regulation can modulate wound healing. Observing loss of acid sphingomyelinase (ASM) may uncover how much Cer derives from the de novo pathway, phosphorylated downstream to yield C1P, as well as exploiting this enzymes effects on eicosanoid synthesis. Although it is proposed CerS5 is the prevalent isoform in lung epithelia5, preliminary data suggests loss of CerS1 reveals widespread losses in eicosanoids, unveiling C18 Cer as the possible chain length preferred by CERK. There are three isoforms of lipid phosphate phosphatases (LPPs), however, their regulation is not well understood. It has been revealed that LPP3 is structurally different from the other isoforms, found in a uniform distribution across cell types, and is involved within post-Golgi trafficking6. This suggests its larger role in sphingolipid metabolism. This study addresses our hypothesis by i) in vitro identification of anabolism and catabolism of C1P in lung epithelial cells to classify this novel pathway, and ii) in vitro analysis aims to explore amount of C1P generated from de novo sphingolipid synthesis.

By selective knockdown of each isoform of CerS, we expect to find the primary isoform responsible for C1P metabolism. Anabolism of C1P will be evaluated by eicosanoid analysis in absence LPP isoforms. C1P generation can occur by an S1P acylase7, it is suggested isoforms of CerS may harbor this activity. By comparing loss of CerS1 to ASM in lung epithelial cells, it can be identified which pathway C18 Cer commonly derives from, thereby revealing amount of C1P present from limiting ceramide synthesis to sphingomyelin or de novo specific utilizing eicosanoid analysis. ASM and NPC1 are similar in function8, we aim to determine whether NPC1 plays a role in C1P metabolism. Lastly, exploring the function of SM derived C1P and de novo derived C1P to compare how wound healing differs between these distinct pathways. This is significant because exploiting the metabolism of C1P can help identify new targets for therapeutics, as well as validating this enigmatic yet novel pathway.

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