1.1 Biological functions of PE

PE is a key component of cell membranes and serves multiple fundamental biological functions. In the liver, PE acts as a substrate for the liver enzyme PE N-methyltransferase, which synthesises approximately one-third of the total PC.^{12, 13} Additionally, PE acts as a precursor in the production of anandamide, a ligand that activates cannabinoid receptors in the brain and is implicated in numerous neurophysiological processes.^{14, 15} Furthermore, PE serves as the donor of the ethanolamine group, a critical component in the glycosylphosphatidylinositol anchoring of proteins. This process is essential for the proper functioning and localisation of certain proteins within the cell membrane.¹⁶ Remarkably, PE has also been recognised as the only intrinsic component in the brain necessary for prion infectivity and facilitating its transmission in mammals.¹⁷

The contribution of each PE biosynthetic pathway varies depending on the cell type. In mammalian cells, PE is generated through four distinct biosynthetic pathways, although two of them contribute minimally under most physiological conditions (Figure 1). The primary pathways include: (i) the Kennedy pathway, also known as the CDP-ethanolamine pathway, which culminates on endoplasmic reticulum membranes, and (ii) the pathway involving phosphatidylserine (PS) decarboxylation, which occurs specifically at the inner mitochondrial membranes. A third pathway, prevalent in yeast and potentially significant in mammals, involves the acylation of lyso-PE. An alternative pathway for PE biosynthesis exists in the ER, involving calcium-mediated base exchange of PS with ethanolamine; however, this mechanism contributes minimally to overall PE production in mammalian systems.¹⁸ The relative activity of these pathways in different cell types may vary depending on cellular demands and metabolic state. Phosphate cytidylyltransferase 2 (Pcyt2) functions as the key regulatory enzyme in PE biosynthesis via the Kennedy pathway. Deficiency in Pcyt2 impairs PE-dependent lipidation of microtubule-associated protein 1 light chain 3 (LC3)-II during autophagy, thereby blocking autophagosome maturation.¹⁹ Studies have demonstrated that Pcyt2 operates through multilevel regulatory mechanisms and is indispensable for embryonic development, as systemic Pcyt2-knockout mice exhibit embryonic lethality before day 8.5 post-coitum.²⁰ Maternal asthma elevates PE levels in amniotic fluid, predominantly in oxidised species, which potentially enhances inflammatory mediator production. This establishes a pro-inflammatory fetal microenvironment that may predispose offspring to heightened allergic responses and exacerbate postnatal inflammation. Such conditions likely impair fetal lung development.²¹ PE derivatives demonstrate biomarker potential for assessing fetal growth and lung health in amniotic fluid, though causal pathological impacts require further elucidation. Recent researches underscore the therapeutic potential of inhibiting enzymes involved in PE biosynthesis in oncology. Specifically, targeting the Kennedy pathway, a primary route for PE production, could be particularly effective. This suggests that disruption of this pathway could suppress cancer cell growth and survival, offering a promising strategy in cancer chemotherapy.²²

1.2 Association of PE and pulmonary diseases

Studies have shown that changes in PE levels are associated with inflammatory responses, alveolar epithelial cell damage, and the progression of pulmonary fibrosis. PE plays a multifaceted role in maintaining pulmonary homeostasis, not only as a structural membrane component but also as a regulator of critical cellular processes such as autophagy (Figure 2) and surfactant function.

A notable example is Legionella pneumophila, an intracellular bacterium that circumvents the host's autophagy defence by introducing the effector protein RavZ (Figure 3). RavZ specifically detaches LC3 proteins from PE on autophagosomal membranes,²³ thereby disrupting autophagy initiation. The conversion of soluble LC3 to membrane-anchored LC3-PE (LC3-II) represents a critical step in the LC3 conjugation pathway, playing an indispensable role in autophagy. This post-translational modification regulates fundamental autophagic processes, from autophagosome biogenesis and cargo recognition to autophagosome-lysosome fusion.²⁴ Pulmonary surfactant consists of a blend of 10% specialised surfactant proteins, namely SP-A, SP-B, SP-C, and SP-D, along with 90% lipids, predominantly phospholipids.²⁵ Although PE comprises only about 1.5% of surfactant phospholipids, its conical molecular structure, resulting from a small polar head group, imparts biophysical properties that support negative membrane curvature and lateral pressure modulation. Beyond its structural roles, PE exerts anti-fibrotic effects by binding to putative cell surface receptors on lung fibroblasts, thereby activating a phospholipase C-dependent signalling cascade.²⁶ This activation triggers inositol trisphosphate (IP₃)-mediated calcium (Ca²⁺) release from the ER, followed by sustained Ca²⁺ influx via store-operated calcium entry. The resulting elevation in intracellular Ca²⁺ levels drives a dual anti-fibrotic response: (1) suppression of collagen type I gene transcription through Ca²⁺-dependent transcriptional regulators,²⁷ and (2) induction of caspase-3/7-dependent apoptosis. It has been shown to have a potential therapeutic role for PE in mitigating pulmonary fibrosis.

The PS receptor contributes to two key biological processes: the phagocytic removal of apoptotic cells and the cellular internalisation of numerous enveloped viral particles. Among these receptors, T-cell immunoglobulin and mucin-domain protein 1 (TIM1) has been shown to bind not only PS but also PE (Figure 3). TIM1 exhibits high-affinity binding to PE, a molecular interaction that mediates two distinct biological functions: (i) the efficient phagocytic clearance of apoptotic cells, and (ii) the enhancement of cellular entry for multiple pathogenic viruses spanning the Flaviviridae (e.g. West Nile virus and dengue virus) and Filoviridae (e.g. Ebola virus) families.²⁸ Further mechanistic investigations have revealed that duramycin, a PE-specific cyclic peptide antibiotic, can effectively inhibit host cell invasion by these viruses through selectively blocking the TIM1-mediated viral endocytic pathway,²⁸ thereby effectively inhibiting the invasion of host cells by these viruses. These findings imply that interfering with the interaction between PE and PS receptors could be a potential broad-spectrum antiviral approach. During the mid-to-late stages of infection, the levels of some PEs increase.²⁹ In sputum metabolomics studies of chronic obstructive pulmonary disease (COPD) patients, significant changes in some metabolites were observed in COPD patients compared to healthy individuals, including PE.³⁰ Specifically, among 24 COPD-depleted modules depleted in COPD, 275 metabolites were identified, of which 89 were PCs or PEs. This finding aligns with the catabolism of these phospholipids observed in the COPD metagenomes. A lipidomic study revealed that the levels of PE 42:7, PE 37:3, PE 35:1 and PE 39:6 were specifically elevated in severe acute pneumonia, but not in acute pulmonary embolism or acute exacerbations of COPD.³¹

Studies have demonstrated that PE can participate in biological processes such as ferroptosis, immune response²⁸ and neurotransmitter synthesis. During ferroptosis, PEs esterified with polyunsaturated fatty acids—specifically arachidonic acid (AA) or adrenic acid (AdA)—are selectively enriched through acyl-CoA synthetase long-chain family member 4 (ACSL4)-mediated esterification and lysophosphatidylcholine acyltransferase 3 (LPCAT3)-dependent membrane remodelling.³² These PE species are then oxidised by 15-lipoxygenase (15-LOX) within ER-associated compartments, where their non-bilayer hexagonal phase conformation facilitates enzymatic access. This process generates doubly or triply oxygenated 15-hydroperoxy-PE (PE-OOH) species, which act as direct execution signals of ferroptosis. When glutathione peroxidase 4 (GPX4) is inactivated or depleted, PE-OOH accumulation escalates due to failed reduction to non-toxic PE-OH derivatives. The resulting unresolved lipid peroxidation propagates membrane damage through reactive electrophilic mediators, ultimately executing iron-dependent cell death.³³ In this study, four PE oxygenates (PE-(C18:0/C20:4)+2[O], PE-(C18:0/C22:4)+2[O], PE-(C18:0/C20:4)+3[O], PE-(C18:0/C22:4)+3[O]) were screened as markers of ferroptosis. However, phospholipids (PLs) esterified with fatty acids with varying degrees of saturation exhibit distinct effects on ferroptosis. For instance, ACSL3 confers ferroptosis resistance by activating exogenous monounsaturated fatty acids (MUFAs) to MUFA-CoAs, which are preferentially incorporated into membrane phospholipids, displacing oxidatively vulnerable PUFA-PLs. This lipid remodelling diminishes the pool of peroxidation-susceptible PUFAs at the plasma membrane, thereby attenuating iron-dependent lethal lipid reactive oxygen species accumulation at this subcellular compartment. Consequently, ACSL3-dependent MUFA incorporation establishes a ferroptosis-resistant cellular state characterised by diminished plasma membrane oxidizability.³⁴ Although this study examined PLs collectively without specific isolation of PEs, it is plausible to speculate that PEs—as critical oxidizable components of PLs—contribute centrally to maintaining the observed effects. This hypothesis warrants further experimental confirmation in future studies. Notably, dysregulated PE metabolism has been shown to affect B-cell follicular recruitment and follicular helper T-cell differentiation, thereby interfering with humoral immune responses to organismal infection.³⁵

By regulating the PE metabolic pathway, it can affect cellular energy metabolism and lipid metabolism, and thus have an impact on the development of diverse disorders spanning cardiovascular (atherosclerosis), metabolic (insulin resistance and obesity)³⁶ and respiratory systems. Phospholipid-monounsaturated fatty acyl species, including PE-monounsaturated fatty acyl, serve as potent inhibitors of ferroptosis, primarily by reducing the accumulation of lipid peroxides in cells.³⁴ In contrast, recent studies have shown that ACSL4-mediated synthesis of PE-polyunsaturated fatty acyl is closely related to cellular metabolic adaptability and contributes to cancer cell metastasis.³⁷ This finding lays the foundation for the development of novel drugs that target tumour metastasis. This also provides new research directions for diseases related to PE metabolic disorders, including those affecting the lungs. However, the precise molecular mechanisms by which different acyl side chains influence PE function remain incompletely understood and require further exploration. In COPD patients, cigarette smoke exposure leads to the recruitment of macrophages, neutrophils, and lymphocytes to small airways and lung parenchyma. These infiltrating immune cells secrete inflammatory factors that induce oxidative stress and the death of pulmonary epithelial cells.³⁸ Specifically, the accumulation of monocyte-derived macrophages increases leukotriene B4 secretion and triggers epithelial cells to express ACSL4, thereby inducing ferroptosis in alveolar type 2 epithelial cells.³⁹