Unique acyl chains mark phosphoinositides for recycling

Abstract

Two recent complementary studies show that, after phospholipase C cleavage, the characteristic acyl chain composition of phosphoinositide‐derived diacylglycerol funnels them back into the PI cycle.

Phosphoinositides (PIPs), membrane lipids generated by phosphatidylinositol (PI) head group phosphorylation, regulate multiple biological functions, including cell signaling, membrane dynamics, ion channel activity, and actin cytoskeleton integrity (Schink et al, 2016). These functions are mainly mediated by electrostatic interactions between their head groups and target proteins. When phospholipase C (PLC) is activated, PI(4,5)P₂ (a PIP abundant in the plasma membrane) is converted into the signaling molecules diacylglycerol (DAG) and inositol 1,4,5‐triphosphate, and PI(4,5)P₂ levels fall (Barneda et al, 2019). DAG can be reconverted into PI(4,5)P₂ through a series of metabolic steps that are collectively called the PI cycle (Fig 1), which replenishes PIP pools (Epand, 2016). Importantly, among PI cycle metabolites, DAG, phosphatidic acid (PA), and cytidine diphosphate DAG (CDP‐DAG) are precursors of other lipids too (Fig 1), raising the question of how PIPs are targeted back through the PI cycle and not to other lipids.

Figure 1. The acyl chain signature of PIPs helps the recycling via the PI cycle.

(Left) The structure of a glycerol backbone with 38:4 acyl chains that are enriched in PI and PIPs. The ‐R group defines the lipid class and can be ‐H (for DAG), phosphate (for PA), CDP (for CDP‐DAG), and phosphoinositol (for PI and PIPs). (Right) Lipid metabolism associated with the PI cycle. Red arrows are metabolic steps that have preference for 38:4 species. Steps in which metabolism diverges and PI cycle intermediates could potentially be converted into other lipids are also depicted. Note that the selectivity for 38:4 species occurs at steps in which metabolic pathways diverge, which helps the efficient recycling of PLC‐derived metabolites via the PI cycle. In blue are depicted the metabolites that were used for stable isotope labeling in the study of Barneda et al (2022). CL, cardiolipin; G3P, glycerol 3‐phosphate; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine. [Colour figure can be viewed at wileyonlinelibrary.com]

In contrast to most phospholipids, which have diverse acyl chain compositions (Harayama & Riezman, 2018), mammalian PIPs and PI acyl chains are biased towards species containing stearic and arachidonic acids (Fig 1, hereafter labeled 38:4 for the combined chain length: double‐bond numbers of acyl chains) (Barneda et al, 2019). This bias partly arises from the removal and subsequent reincorporation of PI acyl chains: a process named remodeling. Loss of MBOAT7 function, an enzyme that remodels PI, causes neurodevelopmental disorders (Lee et al, 2012), suggesting that the specific composition of PI acyl chains is biologically important. However, the reason why remains mysterious. Previous studies revealed that some enzymes of the PI cycle, DGKε (which converts DAG to PA) and CDS2 (which converts PA to CDP‐DAG), prefer 38:4 substrates, suggesting that DAG derived from PIPs enter the PI cycle efficiently thanks to their acyl chains (Epand, 2016). This concept is now supported by two recent studies (Barneda et al, 2022; Kim et al, 2022), which provide clear evidence that the 38:4 acyl chains of PIPs are crucial for their efficient replenishment via the PI cycle upon PLC activation.

Barneda et al combined mass spectrometry and heavy isotope labeling to monitor metabolic fluxes of PI synthesis via the PI cycle or de novo synthesis (Fig 1, labeling strategies shown in blue). Three isotopes were used to label PI at different stages of biosynthesis, and while no strategies monitored the PI cycle exclusively, the authors blocked the PI cycle to dissect different pathways and measure how acyl chains affect the channeling of lipids. Data were compared for bone marrow‐derived macrophages (BMDMs), HEK293, and MCF7 cells, which have high, medium, and low enrichment of 38:4 acyl chains in PI, respectively.

In HEK293 cells at a steady state, the PI cycle mainly contributed to PI synthesis and had a preference for 38:4 species. De novo‐synthesized PI had no such enrichment for 38:4 species in these cells. By contrast, 38:4 PI was efficiently produced after de novo synthesis in wild‐type BMDMs, but not in BMDMs lacking the remodeling enzyme MBOAT7 (also named LPIAT1), showing that de novo‐synthesized PI is rapidly remodeled into PI 38:4 in these cells. However, even in MBOAT7‐deficient BMDMs, PI 38:4 was efficiently produced in the PI cycle, showing again the selectivity of this pathway for 38:4 species. On the contrary, none of the labeling strategies led to dominant labeling of 38:4 species in MCF7 cells, suggesting low rates of remodeling and 38:4 specificity in the PI cycle in these cells.

PI cycle fluxes were then measured after PLC activation. PLC activation decreased PIP₂ irrespective of its acyl chains, but their recovery was fastest for 38:4 species in both HEK293 and MCF7 cells. The rapid recovery of PIP₂ 38:4 could be attributed to the high levels of DAG 38:4 formed by PLC in HEK293 cells (due to 38:4 enrichment in PIPs). In MCF7 cells, however, despite DAG produced by PLC being poor in 38:4 species, CDS2 and DGKs (the enzymes which convert PA to CDP‐DAG and DAG to PA, respectively) were found to contribute significantly to the selective resynthesis of PI 38:4. In summary, Barneda et al showed that the specificity of DGKs and CDS2 channels DAG 38:4 was generated by PLC towards PIP₂ resynthesis, helping their efficient recycling and preventing PI cycle metabolites to be converted into other lipids.

Kim et al (2022) reached similar conclusions using a partially overlapping strategy. By stimulating PLC, they induced acute lipid changes, which were detectable at bulk levels by mass spectrometry. A rapid and transient increase in DAG 38:4 and a longer increase in PA 38:4 were detected. These changes in DAG and PA were localized to the plasma membrane by bioluminescence resonance energy transfer (BRET). These data demonstrated that DAG 38:4, generated from PI(4,5)P₂ 38:4, was being locally converted into PA 38:4 by DGKs. Using radioisotope labeling to monitor metabolic fluxes, they then found that PLC activation stimulates CDP‐DAG and PI synthesis, which could be blocked by either DGK inhibition or CDS2 knockdown. Finally, they showed that the recovery of PIP₂ levels after PLC activation was highest for PIP₂ 38:4 species. Thus, Kim et al (2022) showed that DAG generated by PLC is efficiently recycled via the PI cycle. Although BRET‐based assays or radiolabeling did not discriminate lipid acyl chains, the authors persuasively used bulk lipidomics to suggest strongly the role of 38:4 acyl chains for efficient recycling. In addition, the authors provided additional information about the localization of metabolic conversions with the BRET‐based assays, and biochemical evidence that arachidonic acid‐containing substrates are efficiently used for PI synthesis.

These two studies therefore reveal how cells maintain lipid pools even after PLC activation while preventing the biochemical dispersal of PI cycle lipids (Fig. 1). Here, both selectivity for 38:4 acyl chains and spatial segregation of metabolism appear to play key roles. An elegant enzymatic specialization occurs here too: the selectivity for 38:4 species is strongest for DAG and PA, the substrates that are most likely to be lost to other pathways. By contrast, CDP‐DAG to PI conversion is not specific for 38:4 species, probably not leading to substrate losses as outside the mitochondria, CDP‐DAG is focused on the PI cycle (Fig. 1).

In summary, 38:4 acyl chains enable a rapid and selective regeneration of PIPs after PLC activation by tagging them to the PI cycle. We still do not know how 38:4 acyl chains accumulate in the first place, since PI and PIPs remained rich in 38:4 in the absence of MBOAT7 or CDS2. In addition, the pathophysiological importance of 38:4 acyl chains remains to be found. For example, is repetitive PLC signaling impaired when 38:4 acyl chains are depleted, and when might this cause a problem? Nevertheless, these studies demonstrate the importance of focusing on acyl chains. Due to recent breakthroughs, it is now possible to analyze PIP head groups and acyl chains simultaneously (Morioka et al, 2022; Shimanaka et al, 2022), now might therefore be the time to understand how acyl chains and head groups cooperate to regulate some of our most fundamental biological processes.

Disclosure and competing interests statement

The authors declare that they have no conflict of interest.

The EMBO Journal (2022) 41: e112163.