Lysophosphatidylcholine acyltransferase-2 (LPCAT-2) & protein S-acylation in macrophage innate immunity

Abstract

Lysophosphatidylcholine acyltransferase (LPCAT)2 is an enzyme involved in remodelling glycerophospholipids within cell membranes and the generation of platelet-activating factor. LPCAT2 also mediates lipopolysaccharide (LPS)-induced cytokine responses in both murine and human macrophages. Investigations into the mechanism behind LPCAT2 involvement into LPS-induced cytokine responses identified that LPCAT2 translocates to membrane lipid raft domains and associates with toll-like receptor (TLR)4 following activation by LPS. The mechanism behind this is not known, however LPCAT2 appears to have a role in regulating innate immune responses via lipid raft signalling complexes and understanding the molecular details of these mechanisms could help in the development of targeted anti-inflammatory therapies.

The aim of this thesis is to build on these findings to develop a detailed molecular analysis of the mechanisms by which LPCAT2 can regulate inflammatory responses in macrophages. It has been reported that LPCAT1 can catalyse the acylation of histone proteins. The addition of lipid groups to a protein can impact membrane-protein association, protein structure and protein stability ultimately affecting a proteins function. This gave rise to the main research question explored in this thesis: Does LPCAT2 play a role in the S-acylation of proteins involved in LPS signalling?

To identify if LPCAT2 plays a role in protein S-acylation in LPS stimulated macrophages, siRNAs were used to knockdown the protein expression of LPCAT2 in LPS stimulated RAW264.7 cells combined with acyl-biotin exchange chemistry, a method of purifying S-acylated proteins. The purified proteins could then be identified by tandem mass spectrometry and western blotting. Changes in the level of protein S-acylation could then be compared between cells expressing normal levels of LPCAT2 and cells expressing reduced levels of LPCAT2.

This thesis identified for the first time a link between LPCAT2 and protein S-acylation in LPS stimulated macrophages. Western blot analysis confirmed the acylation of the scavenger receptors CD36 and Lysosome membrane protein II. Further investigation established that CD36 positively regulates LPS induced cytokine responses in macrophages and that S-acylation of CD36 regulates both lipid raft association and surface expression. This highlights a mechanism in which LPCAT2 mediates the S-acylation of CD36 which is required for surface and lipid raft association where CD36 most likely influences LPS signalling. These findings are not only relevant to inflammatory conditions that are mediated by LPS and TLR4 signalling but could also have implications for various inflammatory diseases such as Atherosclerosis and Alzheimer’s disease in which CD36 plays a major role in their pathology.