Regulation of The Expression and Lysine Acetylation of Pro-Inflammatory Molecules By Lipid-Modifying Enzyme (LPCAT2) in RAW264.7 Cells
Lysophosphatidylcholine Acyltransferases (LPCATs) are Lipid modifying enzymes that significantly regulate changes in the glycerophospholipid components of the macrophage membrane phospholipid bilayer. LPCAT2 is a subtype of LPCAT that regulates inflammatory responses to infection by lipopolysaccharides and lipopeptides. LPCAT2 co-localises with TLR4 and prevents its translocation to the signalling site (lipid rafts) of macrophage membranes. The first response to inflammation involves infiltration of macrophages to the infected site, where they fight infection by phagocytosing the microbial pathogen. Changes in the macrophage membrane phospholipid bilayer are essential for phagocytosis and receptor signalling. Regulation of protein acylation is also an integral mechanism for macrophage function. LPCAT2 influences acylation of proteins such as RNF19B – An E3 Ubiquitin Ligase. This research aims to understand further the molecular mechanisms of the enzyme – LPCAT2, that leads to its involvement in regulating macrophage inflammatory responses. Building on pre-existing work on the role of LPCAT2 in inflammation, the primary research question is – “How Does LPCAT2 regulate the expression and function of receptors that mediate macrophage inflammatory responses?” Comparison of the expression of proteins and genes in RAW264.7 cells with or without LPCAT2 silenced determined the possible mechanisms by which LPCAT2 regulates inflammation in macrophages. This study analysed the expression of TLR4 and its co-receptors (CD14 and MD2) – which play a significant role in macrophage inflammatory responses to infection by gram-negative bacteria. The expression of inflammatory cytokines such as IL6, TNFα, IP10, and IFNβ, which are synthesised following activation of TLR4, was also studied. Furthermore, the influence of LPCAT2 in the expression and function of acylated proteins such as RNF19B was studied. Analysis of the regulation of lysine acetylation by LPCAT2 helps to understand the role of LPCAT2 in protein acylation. The results show that LPCAT2 regulates the expression of CD14 and RNF19B. It also influences the expression of lysine-acetylated proteins. Finally, LPCAT2 and RNF19B could modulate macrophage inflammatory responses to both bacterial and viral infection. This study shows that LPCAT2 modulates macrophage inflammatory responses by influencing CD14 and RNF19B expression. It also suggests a novel basis for further studies on the role of LPCAT2 in lysine acetylation of proteins during macrophage inflammatory responses. This thesis contributes novel knowledge to the fields of molecular immunology and biochemistry. The future establishment of the role of LPCAT2 in regulating inflammation can lead to its discovery as a biomarker or a drug-target for various immunological diseases.
LPCAT2, Macrophage, Signalling, Phospholipid, CD14, RNF19B, TLR, Lipopeptide, Lipopolysaccharide, PolyIC
Networked Digital Library of Theses and Dissertations