ATP8B2-mediated asymmetric distribution of plasmalogens regulates plasmalogen homeostasis and plays a role in intracellular signaling (Part 1)

Representative image of a P4-ATPase.

Honsho M, Mawatari S, and Fujiki Y. (2022) ATP8B2-mediated asymmetric distribution of plasmalogens regulates plasmalogen homeostasis and plays a role in intracellular signaling. Frontiers in Molecular Biosciences

Plasmalogens are a class of phospholipids with a vinyl-ether bond at sn-1, which causes them to have a more compact structure. Plasmalogens are involved in membrane structure and fluidity, protection against oxidative stress, and vesicular fusion. Plasmalogen biosynthesis occurs in the peroxisome and endoplasmic reticulum and was previously found to be regulated through a feedback mechanism that detects plasmalogen levels; however, this specific mechanism is not well understood. Honsho et al were interested in understanding the mechanism that senses plasmalogen levels. Plasmalogens have an asymmetric distribution across the bilayer with an enrichment on the inner leaflet and this may occur by phospholipid flippases called type IV P-type adenosine triphosphatases (P4-ATPase) in plasma membranes. Previously Honsho et al found that knocking down CDC50A, a β-subunit of P4-ATPases, results in an increase in fatty acyl-CoA reductase 1 (FAR1) protein levels and an interference in the asymmetric distribution of plasmalogens. In addition to this finding, P4-ATPases require an association with CDC50A to exit the endoplasmic reticulum, leading the authors to be interested in P4-ATPases.

Firstly, Honsho et al wanted to determine which, if any P4-ATPases were translocating plasmalogens. To accomplish this, HeLa cells were studied and of the eleven P4-ATPases in this cell line, four were chosen due to their association with CDC50A and their presence on plasma membranes: ATP8B2, ATP10D, ATP11A, and ATP11B. To determine which are needed for plasmalogens to be localized to the inner leaflet, the protein level of FAR1 in mutant lines was measured in cell lines that were treated to knock down each P4-ATPase. Although all were found to reduce mRNA levels of their respective P4-ATPase, only transfection of siRNA against ATP8B2 caused an increase in FAR1 protein level, suggesting that the mechanism used to sense plasmalogen levels that regulates FAR1 is influenced by downregulating ATP8B2. To confirm that plasmalogen distribution is also affected in ATP8B2-knocked down cells, cells were treated with a membrane impermeable amine-reactive reagent, TNBS, which converts phosphotidylethanolamine and plasmalogens on the outer leaflet, followed by acid hydrolysis to allow separation of phosphotidylethanolamines and plasmalogens through thin layer chromatography. They found that only TNBS-plasmalogens were increased in the ATP8B2-knocked down cells, but TNBS-phosphotidylethanolamine was not. In comparison, ATP11B-knocked down cells did not show an increase of either. This would support the theory that ATP8B2 flips plasmalogens from the outer leaflet to the inner leaflet and when there are less plasmalogens on the inner leaflet, FAR1 is upregulated so that more plasmalogens can be synthesized.

Honsho et al were interested in the mechanism that translocates plasmalogens to the inner leaflet of the membrane and the feedback mechanism used to sense plasmalogens levels. The authors determined which P4-ATPase has the ability to flip plasmalogens from the outer leaflet to the inner leaflet of the membrane bilayer, ATP8B2, since knocking this out in HeLa cells which showed an increase in FAR1 protein levels and an increase in plasmalogen levels on the inner leaflet. The loss of ATP8B2 caused an upregulation of FAR1 and plasmalogen levels, suggesting that the mechanism used to regulate FAR1 and plasmalogen production must also downregulate ATP8B2. This could also be an interesting target for ways to increase plasmalogen production to combat plasmalogen deficiency. The next blog post will continue to explore the work presented in this article where the importance of the asymmetric distribution of plasmalogens in the phosphorylation of protein kinase B (AKT), a regulator for cellular metabolism, is analyzed.