Aminophospholipids, including phosphatidylserines (PS) and phosphatidylethanolamines (PE), are major components of the inner leaflet of cellular membranes with emerging immunomodulatory functions, yet the contribution of their polar headgroups remains poorly defined. To address this, we compared PS16:0/20:4 and PE16:0/20:4, two phospholipids with identical fatty-acyl composition, in resting and lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages using LC-MS/MS proteomics, gene set enrichment analysis, and targeted qPCR. Among the 1611 proteins retained for statistical analysis, both phospholipids produced distinct basal proteomic profiles and substantially modified the subsequent response to LPS. A core of 667 LPS-responsive proteins was preserved across control, PS-, and PE-supplemented macrophages, while interaction analyses revealed marked headgroup-dependent differences in response magnitude. These differences mapped onto distinct pathways. PS supplementation was associated with vesicle trafficking, lipid recognition, redox regulation, and ER proteostasis, whereas PE preferentially affected glutathione and redox-related proteins. Both phospholipids reduced selected inflammatory markers and LPS-induced nitric oxide production. Together, these findings demonstrate headgroup-dependent modulation of macrophage proteomic responses by matched arachidonoyl phospholipids, underscoring the functional relevance of phospholipid headgroup.Significance: Membrane phospholipids are increasingly recognized as active regulators of immune cell function, yet the contribution of the polar headgroup remains poorly defined. By directly comparing PS16:0/20:4 and PE16:0/20:4, this study shows that two native phospholipids carrying identical acyl chains generate distinct macrophage proteomic responses. Both lipids remodel the resting proteome and modify subsequent LPS responsiveness, but with different associations with redox regulation, membrane trafficking, ER proteostasis, and inflammatory signalling. These findings advance our understanding of the role of phospholipid headgroup as an important modulator of macrophage responses and support the development of phospholipid-based approaches to modulate inflammation and immunometabolism.
Anticoccidial drugs remain the primary strategy for controlling avian coccidiosis caused by Eimeria parasites. However, widespread drug resistance and limited understanding of the molecular mechanisms underlying the action of existing anticoccidials severely impede the development of novel agents. In this study, simplified thermal proteome profiling coupled with precipitate-supported thermal proteome profiling was employed to globally identify halofuginone-binding proteins in Eimeria tenella. Sixteen candidate interacting proteins were prioritized through integrated bioinformatics analyses and molecular docking simulations. Three targets—U6L2Z8, U6L2C0, and U6L6M2—were recombinantly produced and purified, and their direct, dose-dependent binding to halofuginone was rigorously confirmed using thermal shift assays, differential scanning fluorimetry, and surface plasmon resonance, yielding dissociation constants in the micromolar range. RT-qPCR analysis showed that halofuginone induces significant changes in the mRNA expression profiles of target genes in Eimeria tenella. These transcriptional alterations provide a plausible explanation that HAL interferes with mitochondrial function and activates phosphorylation-related compensatory signaling pathways via transcriptional perturbation of these target genes, thereby inhibiting the growth and development of parasites. Collectively, this work identifies three novel, experimentally validated direct targets of halofuginone, significantly advancing the mechanistic understanding of its anticoccidial activity and establishing a robust foundation for target-informed rational design of next-generation anticoccidial drugs. Significance Anticoccidial agents serve as the primary measure to control Eimeria coccidiosis in modern poultry farming. The widespread drug resistance and poorly understood action mechanisms of currently commercialized anticoccidials have severely hindered the discovery of novel drug candidates. This study, using the anticoccidial drug halofuginone as an example, based on thermal proteomics analysis, for the first time constructs an in vitro screening and identification model for anticoccidial drug target proteins. By integrating structural thermal proteome profiling with molecular docking, in vitro recombinant protein synthesis, thermal proteome profiling - western blotting, differential scanning fluorimetry and surface plasmon resonance, this study successfully screened and validated U6L2Z8, U6L2C0 and U6L6M2 as novel direct binding targets of halofuginone in Eimeria tenella. These direct target-binding identifications fill a critical knowledge gap regarding the anticoccidial mechanism of halofuginone. Meanwhile, our research findings provide innovative strategies for the rational pharmacophore design targeting parasite-specific proteins and the development of novel anticoccidial drugs.
Vestibular schwannomas (VS) are benign neoplasms of the VIII cranial nerve; they account for 6–8% of all intracranial tumors and about 80% of tumors of the cerebellopontine angle. Cystic forms of VS may be particularly dangerous, as they demonstrate faster growth and worse postoperative outcomes. In this study, we performed proteomic analysis of cerebrospinal fluid (CSF) samples obtained from patients with solid and cystic forms of VS, as well as cyst fluid from patients with cystic VS. CSF samples from patients with hemifacial spasm were used as controls. Mass spectrometric (MS) analysis resulted in identification of 816 proteins in all samples and 111 proteins were upregulated in samples obtained from VS patients compared to the control group. The upregulated proteins belonged to the groups of components and regulators of the complement system, proteins associated with glucose and lipid metabolism. MS analysis of CSF obtained from patients with cystic and solid forms of VS resulted in identification of two proteomic clusters enriched in proteins associated with the development of the nervous system, cell adhesion, and myelination processes. The data obtained suggest the involvement of these biological processes in the pathogenesis of cystic VS. Significance This study provides a comprehensive proteomic characterization of cerebrospinal fluid (CSF) and cyst fluid from patients with solid and cystic vestibular schwannomas (VS), offering valuable insights into the molecular mechanisms underlying these tumors. Our discovery that complement system components and regulators are consistently upregulated in CSF and cyst fluid from VS patients, along with proteins involved in lipid metabolism, points to a potential mechanism of blood-brain barrier disruption and cyst formation, which is particularly relevant given the more aggressive clinical course of cystic VS. These findings not only contribute to understanding of VS pathogenesis but also suggest candidate protein targets for future diagnostic, prognostic, and therapeutic strategies, particularly for the more challenging cystic form of the disease.
