An Investigation into the Function of CDS2 in Phosphoinositide Synthesis in Murine Macrophages

Phosphoinositides (PIPn) are a branch of membrane phospholipids involved in regulating critical cellular functions, such as Phosphoinositide 3-kinase (PI3K) and Phospholipase C (PLC) signalling pathways, the definition of sub-cellular membrane identity and the sorting of proteins and lipids within intracellular compartments. GPCR-stimulated PLC generates the second messengers inositol (1,4,5)-trisphosphate and diacylglycerol (DAG) from phosphatidylinositol (4,5)-bisphosphate, resulting in a net loss in the cellular PIPn pool which is replenished by the activation of phosphatidylinositol (PI) synthesis. A critical intermediate for PI synthesis is cytidine diphosphate diacylglycerol (CDP-DAG), synthesized from phosphatidic acid (PA) in the endoplasmic reticulum by CDP-DAG synthase 1 & 2 (CDS1 & 2). The aim of this work is to investigate whether CDS2-dependent PI synthesis plays an active role in PIPn signalling and homeostasis under basal and PLC-activated conditions. In CDS2-KO bone marrow derived macrophages (BMDMs), basal PIPn levels are maintained, albeit with a clear steady-state accumulation of certain molecular species of PA, DAG and triacylglycerol (TAG). To interrogate the changes in pathway activity that underlie these effects, mass spectrometry coupled with metabolic tracing techniques (¹³C-glucose and ¹⁸O-H2O) were used. The results point to significant increases in both *de novo* PA synthesis and the phosphorylation of DAG by diacylglycerol kinases in CDS2-KO cells. This suggests that increased levels of PA synthesis support normal rates of CDP-DAG production and ultimately PI synthesis by CDS1, in the absence of CDS2. The effects of CDS2 deletion during stimulation of PLC by purinergic receptors was also investigated. Under conditions of chronic stimulation, CDS2-KO cells were unable to maintain their PI and PIP pools. This was also accompanied by an increase in PA accumulation and a substantial reduction in new PI synthesis, suggesting that loss of CDS2 compromised the ability of macrophages to sustain their PIPn pool under these conditions. In contrast, PIP2 pools were maintained in CDS2-KO cells and stimulated inositol trisphosphate production was normal. Interestingly, Ca²⁺ re-uptake into ER pools after PLC stimulation in CDS2-KO cells was found to be reduced, but the origin of this effect is unknown. Overall, the work in this dissertation defines an important role for the CDS2 isoform in supporting increased PI synthesis under conditions of chronic PLC stimulation and also highlights new points of regulation in lipid biosynthetic pathways that are used to maintain homeostasis in PIPn function.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Collins, Daniel

Advisor dc:contributor.advisor

• Hawkins, Phillip

• CDS2
• Macrophages
• Phosphoinositde

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/9b998d02-3178-4e9d-8541-355e8b5e70d7/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.105988

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/364240