Lipidomics Issues on Human Positive ssRNA Virus Infection: An Update

The pathogenic mechanisms underlying the Biology and Biochemistry of viral infections are known to depend on the lipid metabolism of infected cells. From a lipidomics viewpoint, there are a variety of mechanisms involving virus infection that encompass virus entry, the disturbance of host cell lipid metabolism, and the role played by diverse lipids in regard to the infection effectiveness. All these aspects have currently been tackled separately as independent issues and focused on the function of proteins. Here, we review the role of cholesterol and other lipids in ssRNA+ infection.

A sensitive S-Trap-based approach to the analysis of T cell lipid raft proteome

The analysis of T cell lipid raft proteome is challenging due to the highly dynamic nature of rafts and the hydrophobic character of raft-resident proteins. We explored an innovative strategy for bottom-up lipid raftomics based on suspension-trapping (S-Trap) sample preparation. Mouse T cells were prepared from splenocytes by negative immunoselection, and rafts were isolated by a detergent-free method and OptiPrep gradient ultracentrifugation. Microdomains enriched in flotillin-1, LAT, and cholesterol were subjected to proteomic analysis through an optimized protocol based on S-Trap and high pH fractionation, followed by nano-LC-MS/MS. Using this method, we identified 2,680 proteins in the raft-rich fraction and established a database of 894 T cell raft proteins. We then performed a differential analysis on the raft-rich fraction from nonstimulated versus anti-CD3/CD28 T cell receptor (TCR)-stimulated T cells. Our results revealed 42 proteins present in one condition and absent in the other. For the first time, we performed a proteomic analysis on rafts from ex vivo T cells obtained from individual mice, before and after TCR activation. This work demonstrates that the proposed method utilizing an S-Trap-based approach for sample preparation increases the specificity and sensitivity of lipid raftomics.

CHO/LY-B cell growth under limiting sphingolipid supply: correlation between lipid composition and biophysical properties of sphingolipid-restricted cell membranes+

ABSTRACTSphingolipids (SL) are ubiquitous in mammalian cell membranes, yet there is little data on the behavior of cells under SL-restriction conditions. LY-B cells derive from a CHO line in which serine palmitoyl transferase (SPT), thus de novo SL synthesis, is suppressed, while maintaining the capacity of taking up and metabolizing exogenous sphingoid bases from the culture medium. In the present study LY-B cells were adapted to grow in a fetal bovine serum (FBS)-deficient medium to avoid external uptake of lipids. The lowest FBS concentration that allowed LY-B cell growth, though at a slow rate, under our conditions was 0.04%, i.e. 250-fold less than the standard (10%) concentration. Cells grown under limiting SL concentrations remained viable for at least 72 h. Enriching with sphingomyelin the SL-deficient medium allowed the recovery of control LY-B cell growth rates. Studies including whole cells, plasma membrane preparations, and derived lipid vesicles were carried out. Laurdan fluorescence was recorded to measure membrane molecular order, showing a significant decrease in the rigidity of LY-B cells, not only in plasma membrane but also in whole cell lipid extract, as a result of SL limitation in the growth medium. Plasma membrane preparations and whole cell lipid extracts were also studied using atomic force microscopy in the force spectroscopy mode. Force measurements demonstrated that lower breakthrough forces were required to penetrate samples obtained from SL-poor LY-B cells than those obtained from control cells. Mass-spectroscopic analysis was also a helpful tool to understand the rearrangement undergone by the LY-B cell lipid metabolism. The most abundant SL in LY-B cells, sphingomyelin, decreased by about 85% as a result of SL limitation in the medium, the bioactive lipid ceramide and the ganglioside precursor hexosylceramide decreased similarly, together with cholesterol. Quantitative SL analysis showed that a 250-fold reduction in sphingolipid supply to LY-B cells led to a 6-fold decrease in membrane sphingolipids, underlining the resistance to changes in composition of these cells. Plasma membrane compositions exhibited similar changes, at least qualitatively, as the whole cells with SL restriction. A linear correlation was observed between the sphingomyelin concentration in the membranes, the degree of lipid order as measured by laurdan fluorescence, and membrane breakthrough forces assessed by atomic force microscopy. Concomitant changes were detected in glycerophospholipids under SL-restriction conditions.

Identification of a novel regulatory pathway for PPARα by RNA-seq characterization of the endothelial cell lipid peroxidative injury transcriptome

Endothelial dysfunction caused by endothelial cell injuries is the initiating factor for atherosclerosis (AS), and lipid peroxidative injury is one of a dominant factor for AS pathogenesis. Using RNA-seq, we compared changes in transcriptome expression before and after endothelial cell injury, and found 311 differentially expressed genes (DEGs), of which 258 genes were upregulated and 53 genes were downregulated. The protein–protein interactions (PPIs) between the genes were analysed using the STRING database, and a PPI network of DEGs was constructed. The relationship distributions among these PPIs were analysed by performing network node statistics. We found that in the top 20 DEGs with high connected protein nodes in the PPI network, 16 were upregulated and 4 were downregulated. Gene ontology (GO) functional enrichment analysis and KEGG pathway enrichment analysis on the DEGs were also performed. By comparing the upregulated expressed genes with high connected protein nodes in the PPI network to those related to endothelial cell lipid damage and repair in the GO analysis, we identified seven genes (NOX4, PPARA, CCL2, PDGFB, IL8, VWF, CD36) and verified their expression levels by real-time polymerase chain reaction. The protein interactions between the seven genes were then analysed using the STRING database. The results predicted that CCL2 interacts with NOX4, PPARα, PDGFβ and VWF individually. Thus, we examined the protein expression levels of CCL2, NOX4, PPARα, PDGFβ and VWF, and found that the expression levels of all proteins were significantly upregulated after the lipid peroxidative injury, with CCL2 and PPARα exhibiting the highest expression levels. Therefore, we investigated the interregulatory relationship between CCL2 and PPARα and their roles in the repair of endothelial cell injury. With the help of gene overexpression and knockdown techniques, we discovered that PPARα promotes the repair of endothelial cell injury by upregulating CCL2 expression in human umbilical vein endothelial cells but that CCL2 cannot regulate PPARα expression. Therefore, we believe that PPARα participates in the repair of endothelial cell lipid peroxidative injury through regulating the expression of CCL2.