Search

In Saccharomyces cerevisiae, Pah1 phosphatidate (PA) phosphatase, which catalyzes the Mg2+-dependent dephosphorylation of PA to produce diacylglycerol, plays a key role in utilizing PA for the synthesis of the neutral lipid triacylglycerol and thereby controlling the PA-derived membrane phospholipids. The enzyme function is controlled by its subcellular location as regulated by phosphorylation and dephosphorylation. Pah1 is initially inactivated in the cytosol through phosphorylation by multiple protein kinases and then activated via its recruitment and dephosphorylation by the protein phosphatase Nem1-Spo7 at the nuclear/endoplasmic reticulum membrane where the PA phosphatase reaction occurs. Many of the protein kinases that phosphorylate Pah1 have yet to be characterized with the identification of the target residues. Here, we established Pah1 as a bona fide substrate of septinassociated Hsl1, a protein kinase involved in mitotic morphogenesis checkpoint signaling. The Hsl1 activity on Pah1 was dependent on reaction time and the amounts of protein kinase, Pah1, and ATP. The Hsl1 phosphorylation of Pah1 occurred on Ser-748 and Ser-773, and the phosphorylated protein exhibited a 5-fold reduction in PA phosphatase catalytic efficiency. Analysis of cells expressing the S748A and S773A mutant forms of Pah1 indicated that Hsl1-mediated phosphorylation of Pah1 promotes membrane phospholipid synthesis at the expense of triacylglycerol, and ensures the dependence of Pah1 function on the Nem1-Spo7 protein phosphatase. This work advances the understanding of how Hsl1 facilitates membrane phospholipid synthesis through the phosphorylation-mediated regulation of Pah1. [ABSTRACT FROM AUTHOR]

Simple Summary: Taurine has a wide range of biological functions in vertebrates but does not participate in protein synthesis. Recent studies showed taurine exhibits an important role in the regulation of fat metabolism in fish and has the effect of reducing liver fat accumulation in high-fed fish. However, it is still unclear how taurine exerts the effect and the underlying metabolic mechanism of taurine intervention. In this study, we conducted an experiment to investigate the fat-lowering effect of taurine on orange-spotted groupers at the proteomic level. Furthermore, we performed an integrated analysis of transcriptomics and proteomics and excavated the key genes and key proteins involved in the regulation of liver fat metabolism, in an attempt to better understand the intrinsic connection between the transcriptional and translational levels and interpret the trajectories in terms of the biological phenomena in a more comprehensive way after taurine intervention on high-fat fed orange-spotted groupers. In order to understand the intervention effect of taurine on liver fat deposition induced by high fat intake in the orange-spotted grouper (Epinephelus coioides), we performed proteomic analysis and association analysis with previously obtained transcriptomic data. Three isoproteic (47% crude protein) diets were designed to contain two levels of fat and were named as the 10% fat diet (10F), 15% fat diet (15F), and 15% fat with 1% taurine (15FT). The 10F diet was used as the control diet. After 8 weeks of feeding, the 15F diet exhibited comparable weight gain, feed conversion ratio, and hepatosomatic index as the 10F diet, but the former increased liver fat content vs. the latter. Feeding with the 15FT diet resulted in an improvement in weight gain and a reduction in feed conversion ratio, hepatosomatic index, and liver fat content compared with feeding the 15F diet. When comparing liver proteomic data between the 15F and 15FT groups, a total of 133 differentially expressed proteins (DEPs) were identified, of which 51 were upregulated DEPs and 82 were downregulated DEPs. Among these DEPs, cholesterol 27-hydroxylase, phosphatidate phosphatase LPIN, phosphatidylinositol phospholipase C, and 6-phosphofructo-2-kinase were further screened out and were involved in primary bile acid biosynthesis, glycerolipid metabolism, the phosphatidylinositol signaling system, and the AMPK signaling pathway as key DEPs in terms of alleviating liver fat deposition of taurine in high-fat fed fish. With the association analysis of transcriptomic and proteomic data through KEGG, three differentially expressed genes (atp1a, arf1_2, and plcd) and four DEPs (CYP27α1, LPIN, PLCD, and PTK2B) were co-enriched into five pathways related to fat metabolism including primary bile acid synthesis, bile secretion, glycerolipid metabolism, phospholipid D signaling, or/and phosphatidylinositol signaling. The results showed that dietary taurine intervention could trigger activation of bile acid biosynthesis and inhibition of triglyceride biosynthesis, thereby mediating the liver fat-lowering effects in high-fat fed orange-spotted grouper. The present study contributes some novel insight into the liver fat-lowering effects of dietary taurine in high-fat fed groupers. [ABSTRACT FROM AUTHOR]

The article explores the impact of knocking out phosphatidate phosphohydrolase (PAH) genes in plants, specifically rice and Arabidopsis. The researchers discovered that this knockout resulted in the accumulation of phosphatidic acid (PA), which in turn enhanced the plants' resistance to various pathogens. The study also revealed that PAHs are present in a wide range of plants and play a role in plant defense. The findings suggest that targeting PAH genes could be a promising approach for developing disease-resistant crops. Additionally, the researchers found that while the knockout of PAH genes improved resistance to pathogens, it also hindered plant growth. They identified changes in gene expression and lipid metabolism associated with the knockout, indicating that manipulating PAH genes could potentially enhance plant resistance without compromising yield. [Extracted from the article]

The Saccharomyces cerevisiae Nem1-Spo7 protein phosphatase complex dephosphorylates and thereby activates Pah1 at the nuclear/endoplasmic reticulum membrane. Pah1, a phosphatidate phosphatase catalyzing the dephosphorylation of phosphatidate to produce diacylglycerol, is one of the most highly regulated enzymes in lipid metabolism. The diacylglycerol produced in the lipid phosphatase reaction is utilized for the synthesis of triacylglycerol that is stored in lipid droplets. Disruptions of the Nem1-Spo7/Pah1 phosphatase cascade cause a plethora of physiological defects. Spo7, the regulatory subunit of the Nem1-Spo7 complex, is required for the Nem1 catalytic function and interacts with the acidic tail of Pah1. Spo7 contains three conserved homology regions (CR1-3) that are important for the interaction with Nem1, but its region for the interaction with Pah1 is unknown. Here, by deletion and site-specific mutational analyses of Spo7, we revealed that the C-terminal basic tail (residues 240-259) containing five arginine and two lysine residues is important for the Nem1-Spo7 complex-mediated dephosphorylation of Pah1 and its cellular function (triacylglycerol synthesis, lipid droplet formation, maintenance of nuclear/endoplasmic reticulum membrane morphology, and cell growth at elevated temperatures). The glutaraldehyde cross-linking analysis of synthetic peptides indicated that the Spo7 basic tail interacts with the Pah1 acidic tail. This work advances our understanding of the Spo7 function and the Nem1-Spo7/Pah1 phosphatase cascade in yeast lipid synthesis. [ABSTRACT FROM AUTHOR]

The PAH1-encoded phosphatidate (PA) phosphatase is a major source of diacylglycerol for the production of the storage lipid triacylglycerol and a key regulator for the de novo phospholipid synthesis in Saccharomyces cerevisiae. The catalytic function of Pah1 depends on its membrane localization which is mediated through its phosphorylation by multiple protein kinases and dephosphorylation by the Nem1-Spo7 protein phosphatase complex. The full-length Pah1 is composed of a catalytic core (N-LIP and HAD-like domains, amphipathic helix, and the WRDPLVDID domain) and non-catalytic regulatory sequences (intrinsically disordered regions, RP domain, and acidic tail) for phosphorylation and interaction with Nem1-Spo7. How the catalytic core regulates Pah1 localization and cellular function is not clear. In this work, we analyzed a variant of Pah1 (i.e., Pah1-CC (catalytic core)) that is composed only of the catalytic core. Pah1-CC expressed on a low-copy plasmid complemented the pah1Δ mutant phenotypes (e.g., nuclear/ER membrane expansion, reduced levels of triacylglycerol, and lipid droplet formation) without requiring Nem1-Spo7. The cellular function of Pah1-CC was supported by its PA phosphatase activity mostly associated with the membrane fraction. Although functional, Pah1-CC was distinct from Pah1 in the protein and enzymological properties, which include overexpression toxicity, association with heat shock proteins, and significant reduction of the Vmax value. These findings on the Pah1 catalytic core enhance the understanding of its structural requirements for membrane localization and activity control. [ABSTRACT FROM AUTHOR]

Lipin family members in mammals include lipins 1, 2, and 3. Lipin family proteins play a crucial role in lipid metabolism due to their bifunctionality as both transcriptional coregulators and phosphatidate phosphatase (PAP) enzymes. In this review, we discuss the structural features, expression patterns, and pathophysiologic functions of lipins, emphasizing their direct as well as indirect roles in cardiovascular diseases (CVDs). Elucidating the regulation of lipins facilitates a deeper understanding of the roles of lipins in the processes underlying CVDs. The activity of lipins is modulated at various levels, e.g., in the form of the transcription of genes, post-translational modifications, and subcellular protein localization. Because lipin characteristics are undergoing progressive clarification, further research is necessitated to then actuate the investigation of lipins as viable therapeutic targets in CVDs. [ABSTRACT FROM AUTHOR]

In order to explore the effect of mulberry leaf extract (ELM) on the liver function of spotted sea bass, 360 fish with healthy constitution (average body weight 9.00 ± 0.02 g) were selected and randomly divided into six groups with three repetitions, and six groups of fish were randomly placed into 18 test tanks (200 L) with 20 fish per tank for the 52-day feeding test. Every day, the fish were fed the experimental feed with different concentrations (0, 3, 6, 9, 12, 15 g/kg) to the level of apparent satiation, with a crude protein content of 48.0% and a crude fat content of 8.6%. And the water temperature was maintained at 25–28°C with a salinity of 0.5%–1‰. After feeding, five fish were randomly selected to collect their livers and serum for detection of indicators. The results showed that, compared with the control group, ELM significantly increased the activities of lipase (LPS) and trypsin (TRS) in the liver, and reached the highest level when the amount of ELM added was 6 g/kg P < 0.05 . ELM significantly increased the activities of lactate dehydrogenase (LDH) and glutamic-oxaloacetic transaminase (GOT) involved in the metabolic process in liver tissue, and GOT activity reached the highest when ELM was added at 9 g/kg, and LDH activity reached the highest when ELM was added at 15 g/kg P < 0.05 . ELM had no significant effect on liver antioxidant enzymes P > 0.05 , but the content of malondialdehyde was significantly reduced P < 0.05 . Compared with the control group, ELM significantly increased the activities of AKP and ACP in the liver, and the AKP activity reached the highest when the ELM addition amount was 3 g/kg, and the ACP activity reached the highest when the ELM addition amount was 9 g/kg P < 0.05 . Through comparative transcriptomic analysis, it was indicated that ELM enhanced the hepatic lipids and carbohydrates metabolism ability, as manifested in the upregulation of expression of phosphatidate phosphatase, glucuronosyltransferase, inositol oxygenase, carbonic anhydrase, and cytochrome c oxidase subunit 2. ELM can also increase the expression of signal transducer and activator of transcription 1, ATP-dependent RNA helicase and C-X-C motif chemokine 9 involved in the immune process. The above results show that the ELM can enhance the digestion, metabolism, and immunity of the liver by increasing the activity of digestive enzymes, metabolic enzymes, and the expression of metabolism and immune regulation genes. This study provides a theoretical basis for the application of ELM in the cultivation of spotted sea bass by exploring the effect of ELM on the liver function of spotted sea bass. [ABSTRACT FROM AUTHOR]

Preeclampsia (PE) is a significant threat to all pregnancies that is highly associated with maternal mortality and developmental disorders in infants. However, the etiopathogenesis of this condition remains unclear. This study aims to explore the regulatory roles of long noncoding RNAs (lncRNAs) and the mediated competing endogenous RNAs (ceRNA) in the etiopathogenesis of PE through analysis of lncRNA expression patterns in PE and healthy pregnant women (HPW), as well as the construction of lncRNA-mediated ceRNA regulatory networks using bioinformatics. A total of 896 significant differentially expressed lncRNAs, including 586 upregulated lncRNAs and 310 downregulated lncRNAs, were identified in comparison between PE and HPW. Analysis of these differential expressed lncRNAs revealed their predominant enrichment in molecular functions such as sphingosine-1-phosphate phosphatase activity, lipid phosphatase activity, phosphatidate phosphatase activity, thymidylate kinase activity, and UMP kinase activity. Moreover, these differential expressed lncRNAs were predominantly enriched in KEGG analyses such as fat digestion and absorption, lysine degradation, ether lipid metabolism, glycerolipid metabolism, and sphingolipid metabolism. Two ceRNA regulatory networks were constructed based on ceRNA score, including one that had 31 upregulated lncRNAs, 11 downregulated miRNAs, and 34 upregulated mRNAs, while the other contained 128 downregulated lncRNAs, 40 upregulated miRNAs, and 113 downregulated mRNAs. These results may provide a clue to explore the roles of lncRNAs in the etiopathogenesis of PE. [ABSTRACT FROM AUTHOR]

Lipin 1 regulates cellular lipid homeostasis through roles in glycerolipid synthesis (through phosphatidic acid phosphatase activity) and transcriptional coactivation. Lipin 1-deficient individuals exhibit episodic disease symptoms that are triggered by metabolic stress, such as stress caused by prolonged fasting. We sought to identify critical lipin 1 activities during fasting. We determined that lipin 1 deficiency induces widespread alternative mRNA splicing in liver during fasting, much of which is normalized by refeeding. The role of lipin 1 in mRNA splicing was largely independent of its enzymatic function. We identified interactions between lipin 1 and spliceosome proteins, as well as a requirement for lipin 1 to maintain homeostatic levels of spliceosome small nuclear RNAs and specific RNA splicing factors. In fasted Lpin1-/- liver, we identified a correspondence between alternative splicing of phospholipid biosynthetic enzymes and dysregulated phospholipid levels; splicing patterns and phospholipid levels were partly normalized by feeding. Thus, lipin 1 influences hepatic lipid metabolism through mRNA splicing, as well as through enzymatic and transcriptional activities, and fasting exacerbates the deleterious effects of lipin 1 deficiency on metabolic homeostasis.

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.

FXR agonists are used to treat non-alcoholic fatty liver disease (NAFLD), in part because they reduce hepatic lipids. Here, we show that FXR activation with the FXR agonist GSK2324 controls hepatic lipids via reduced absorption and selective decreases in fatty acid synthesis. Using comprehensive lipidomic analyses, we show that FXR activation in mice or humans specifically reduces hepatic levels of mono- and polyunsaturated fatty acids (MUFA and PUFA). Decreases in MUFA are due to FXR-dependent repression of Scd1, Dgat2, and Lpin1 expression, which is independent of SHP and SREBP1c. FXR-dependent decreases in PUFAs are mediated by decreases in lipid absorption. Replenishing bile acids in the diet prevented decreased lipid absorption in GSK2324-treated mice, suggesting that FXR reduces absorption via decreased bile acids. We used tissue-specific FXR KO mice to show that hepatic FXR controls lipogenic genes, whereas intestinal FXR controls lipid absorption. Together, our studies establish two distinct pathways by which FXR regulates hepatic lipids.

The Saccharomyces cerevisiae PAH1-encoded phosphatidate (PA) phosphatase, which catalyzes the Mg2+-dependent dephosphorylation of PA to produce diacylglycerol, is one of the most highly regulated enzymes in lipid metabolism. The enzyme controls whether cells utilize PA to produce membrane phospholipids or the major storage lipid triacylglycerol. PA levels, which are regulated by the enzyme reaction, also control the expression of UASINO-containing phospholipid synthesis genes via the Henry (Opi1/Ino2-Ino4) regulatory circuit. Pah1 function is largely controlled by its cellular location, which is mediated by phosphorylation and dephosphorylation. Multiple phosphorylations sequester Pah1 in the cytosol and protect it from 20S proteasome-mediated degradation. The endoplasmic reticulum-associated Nem1-Spo7 phosphatase complex recruits and dephosphorylates Pah1 allowing the enzyme to associate with and dephosphorylate its membrane-bound substrate PA. Pah1 contains domains/regions that include the N-LIP and haloacid dehalogenase-like catalytic domains, Nterminal amphipathic helix for membrane binding, C-terminal acidic tail for Nem1-Spo7 interaction, and a conserved tryptophan within the WRDPLVDID domain required for enzyme function. Through bioinformatics, molecular genetics, and biochemical approaches, we identified a novel RP (regulation of phosphorylation) domain that regulates the phosphorylation state of Pah1. We showed that the ΔRP mutation results in a 57% reduction in the endogenous phosphorylation of the enzyme (primarily at Ser-511, Ser-602, and Ser-773/Ser-774), an increase in membrane association and PA phosphatase activity, but reduced cellular abundance. This work not only identifies a novel regulatory domain within Pah1 but emphasizes the importance of the phosphorylation-based regulation of Pah1 abundance, location, and function in yeast lipid synthesis. [ABSTRACT FROM AUTHOR]

Spermatogonial stem cells (SSCs) are essential for the generation of sperm and have potential therapeutic value for treating male infertility, which afflicts >100 million men world-wide. While much has been learned about rodent SSCs, human SSCs remain poorly understood. Here, we molecularly characterize human SSCs and define conditions favoring their culture. To achieve this, we first identified a cell-surface protein, PLPPR3, that allowed purification of human primitive undifferentiated spermatogonia (uSPG) highly enriched for SSCs. Comparative RNA-sequencing analysis of these enriched SSCs with differentiating SPG (KIT+ cells) revealed the full complement of genes that shift expression during this developmental transition, including genes encoding key components in the TGF-β, GDNF, AKT, and JAK-STAT signaling pathways. We examined the effect of manipulating these signaling pathways on cultured human SPG using both conventional approaches and single-cell RNA-sequencing analysis. This revealed that GDNF and BMP8B broadly support human SPG culture, while activin A selectively supports more advanced human SPG. One condition-AKT pathway inhibition-had the unique ability to selectively support the culture of primitive human uSPG. This raises the possibility that supplementation with an AKT inhibitor could be used to culture human SSCs in vitro for therapeutic applications.

Lipin/Pah phosphatidic acid phosphatases (PAPs) generate diacylglycerol to regulate triglyceride synthesis and cellular signaling. Inactivating mutations cause rhabdomyolysis, autoinflammatory disease, and aberrant fat storage. Disease-mutations cluster within the conserved N-Lip and C-Lip regions that are separated by 500-residues in humans. To understand how the N-Lip and C-Lip combine for PAP function, we determined crystal structures of Tetrahymena thermophila Pah2 (Tt Pah2) that directly fuses the N-Lip and C-Lip. Tt Pah2 adopts a two-domain architecture where the N-Lip combines with part of the C-Lip to form an immunoglobulin-like domain and the remaining C-Lip forms a HAD-like catalytic domain. An N-Lip C-Lip fusion of mouse lipin-2 is catalytically active, which suggests mammalian lipins function with the same domain architecture as Tt Pah2. HDX-MS identifies an N-terminal amphipathic helix essential for membrane association. Disease-mutations disrupt catalysis or destabilize the protein fold. This illustrates mechanisms for lipin/Pah PAP function, membrane association, and lipin-related pathologies.

In the yeast Saccharomyces cerevisiae, the Nem1-Spo7 complex is a protein phosphatase that activates Pah1 phosphatidate phosphatase at the nuclear-endoplasmic reticulum membrane for the synthesis of triacylglycerol. The Nem1-Spo7/Pah1 phosphatase cascade largely controls whether phosphatidate is partitioned into the storage lipid triacylglycerol or into membrane phospholipids. The regulated synthesis of the lipids is crucial for diverse physiological processes during cell growth. Spo7 in the protein phosphatase complex is required as a regulatory subunit for the Nem1 catalytic subunit to dephosphorylate Pah1. The regulatory subunit contains three conserved homology regions (CR1, CR2, and CR3). Previous work showed that the hydrophobicity of LLI (residues 54-56) within CR1 is important for Spo7 function in the Nem1-Spo7/Pah1 phosphatase cascade. In this work, by deletion and site-specific mutational analyses, we revealed that CR2 and CR3 are also required for Spo7 function. Mutations in any one of the conserved regions were sufficient to disrupt the function of the Nem1-Spo7 complex. We determined that the uncharged hydrophilicity of STN (residues 141-143) within CR2 was required for Nem1-Spo7 complex formation. In addition, the hydrophobicity of LL (residues 217 and 219) within CR3 was important for Spo7 stability, which indirectly affected complex formation. Finally, we showed the loss of Spo7 CR2 or CR3 function by the phenotypes (e.g., reduced amounts of triacylglycerol and lipid droplets, temperature sensitivity) that are attributed to defects in membrane translocation and dephosphorylation of Pah1 by the Nem1-Spo7 complex. These findings advance knowledge of the Nem1-Spo7 complex and its role in lipid synthesis regulation. [ABSTRACT FROM AUTHOR]

The regulation of cellular lipid storage and membrane lipid composition plays a critical role in metabolic homeostasis, and dysregulation may contribute to disorders such as obesity, fatty liver, type 2 diabetes, and cardiovascular disease. The mammalian lipin proteins (lipin 1, lipin 2, and lipin 3) are phosphatidic acid phosphatase (PAP) enzymes that modulate levels of cellular triacylglycerols and phospholipids, and also regulate lipid intermediates in cellular signaling pathways. Lipin proteins also have the ability to coactivate/corepress transcription. In humans and mice, lipin gene mutations cause severe metabolic phenotypes including rhabdomyolysis (lipin 1), autoinflammatory disease (lipin 2), and impaired intestinal lipoprotein assembly (lipin 2/lipin 3). Characterization of these diseases has revealed roles for lipin PAP activity in fundamental cellular processes such as autophagy, inflammasome activation, and lipoprotein assembly. Lipin protein activity is regulated at pre- and posttranscriptional levels, which suggests a need for their ordered response to specific physiological stimuli. Challenges for the future include better elucidation of the unique biochemical and physiological properties of individual lipin family members and determination of lipin protein structure-function relationships. Further research may propel exploration of lipin proteins as viable therapeutic targets in metabolic or inflammatory disorders.

The lipin phosphatidic acid phosphatase (PAP) enzymes are required for triacylglycerol (TAG) synthesis from glycerol 3-phosphate in most mammalian tissues. The 3 lipin proteins (lipin 1, lipin 2, and lipin 3) each have PAP activity, but have distinct tissue distributions, with lipin 1 being the predominant PAP enzyme in many metabolic tissues. One exception is the small intestine, which is unique in expressing exclusively lipin 2 and lipin 3. TAG synthesis in small intestinal enterocytes utilizes 2-monoacylglycerol and does not require the PAP reaction, making the role of lipin proteins in enterocytes unclear. Enterocyte TAGs are stored transiently as cytosolic lipid droplets or incorporated into lipoproteins (chylomicrons) for secretion. We determined that lipin enzymes are critical for chylomicron biogenesis, through regulation of membrane phospholipid composition and association of apolipoprotein B48 with nascent chylomicron particles. Lipin 2/3 deficiency caused phosphatidic acid accumulation and mammalian target of rapamycin complex 1 (mTORC1) activation, which were associated with enhanced protein levels of a key phospholipid biosynthetic enzyme (CTP:phosphocholine cytidylyltransferase α) and altered membrane phospholipid composition. Impaired chylomicron synthesis in lipin 2/3 deficiency could be rescued by normalizing phospholipid synthesis levels. These data implicate lipin 2/3 as a control point for enterocyte phospholipid homeostasis and chylomicron biogenesis.

In the yeast Saccharomyces cerevisiae, the PAH1-encoded Mg2+-dependent phosphatidate (PA) phosphatase Pah1 regulates the bifurcation of PA to diacylglycerol (DAG) for triacylglycerol (TAG) synthesis and to CDP-DAG for phospholipid synthesis. Pah1 function is mainly regulated via control of its cellular location by phosphorylation and dephosphorylation. Pah1 phosphorylated by multiple protein kinases is sequestered in the cytosol apart from its substrate PA in the membrane. The phosphorylated Pah1 is then recruited and dephosphorylated by the protein phosphatase complex Nem1 (catalytic subunit)-Spo7 (regulatory subunit) in the endoplasmic reticulum. The dephosphorylated Pah1 hops onto and scoots along the membrane to recognize PA for its dephosphorylation to DAG. Here, we developed a proteoliposome model system that mimics the Nem1-Spo7/Pah1 phosphatase cascade to provide a tool for studying Pah1 regulation. Purified Nem1-Spo7 was reconstituted into phospholipid vesicles prepared in accordance with the phospholipid composition of the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7 phosphatase reconstituted in the proteoliposomes, which were measured 60 nm in an average diameter, was catalytically active on Pah1 phosphorylated by Pho85-Pho80, and its active site was located at the external side of the phospholipid bilayer. Moreover, we determined that PA stimulated the Nem1-Spo7 activity, and the regulatory effect was governed by the nature of the phosphate headgroup but not by the fatty acyl moiety of PA. The reconstitution system for the Nem1-Spo7/Pah1 phosphatase cascade, which starts with the phosphorylation of Pah1 by Pho85-Pho80 and ends with the production of DAG, is a significant advance to understand a regulatory cascade in yeast lipid synthesis.

Pah1 phosphatidate (PA) phosphatase plays a major role in triacylglycerol synthesis in Saccharomyces cerevisiae by producing its precursor diacylglycerol and concurrently regulates de novo phospholipid synthesis by consuming its precursor PA. The function of Pah1 requires its membrane localization, which is controlled by its phosphorylation state. Pah1 is dephosphorylated by the Nem1-Spo7 protein phosphatase, whereas its phosphorylation occurs by multiple known and unknown protein kinases. In this work, we show that Rim11, a yeast homolog of mammalian glycogen synthase kinase-3β, is a protein kinase that phosphorylates Pah1 on serine (Ser12, Ser602, and Ser818) and threonine (Thr163, Thr164, Thr522) residues. Enzymological characterization of Rim11 showed that its Km for Pah1 (0.4 μM) is similar to those of other Pah1-phosphorylating protein kinases, but its Km for ATP (30 μM) is significantly higher than those of these same kinases. Furthermore, we demonstrate Rim11 phosphorylation of Pah1 does not require substrate prephosphorylation but was increased ~2-fold upon its prephosphorylation by the Pho85-Pho80 protein kinase. In addition, we show Rim11-phosphorylated Pah1 was a substrate for dephosphorylation by Nem1-Spo7. Finally, we demonstrate the Rim11 phosphorylation of Pah1 exerted an inhibitory effect on its PAphosphatase activity by reduction of its catalytic efficiency. Mutational analysis of the major phosphorylation sites (Thr163, Thr164, and Ser602) indicated that Rim11-mediated phosphorylation at these sites was required to ensure Nem1-Spo7-dependent localization of the enzyme to the membrane. Overall, these findings advance our understanding of the phosphorylation-mediated regulation of Pah1 function in lipid synthesis. [ABSTRACT FROM AUTHOR]

Triacylglycerols (TAG) serve as the predominant form of energy storage in mammalian cells, and TAG synthesis influences conditions such as obesity, fatty liver, and insulin resistance. In most tissues, the glycerol 3-phosphate pathway enzymes are responsible for TAG synthesis, and the regulation and function of these enzymes is therefore important for metabolic homeostasis. Here we review the sites and regulation of glycerol-3-phosphate acyltransferase (GPAT), acylglycerol-3-phosphate acyltransferase (AGPAT), lipin phosphatidic acid phosphatase (PAP), and diacylglycerol acyltransferase (DGAT) enzyme action. We highlight the critical roles that these enzymes play in human health by reviewing Mendelian disorders that result from mutation in the corresponding genes. We also summarize the valuable insights that genetically engineered mouse models have provided into the cellular and physiological roles of GPATs, AGPATs, lipins and DGATs. Finally, we comment on the status and feasibility of therapeutic approaches to metabolic disease that target enzymes of the glycerol 3-phosphate pathway. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink.

The regulation of glycerolipid biosynthesis is critical for homeostasis of cellular lipid stores and membranes. Here we review the role of lipin phosphatidic acid phosphatase enzymes in glycerolipid synthesis. Lipin proteins are unique among glycerolipid biosynthetic enzymes in their ability to transit among cellular membranes, rather than remain membrane tethered. We focus on the mechanisms that underlie lipin protein interactions with membranes and the versatile roles of lipins in several organelles, including the endoplasmic reticulum, mitochondria, endolysosomes, lipid droplets, and nucleus. We also review the corresponding physiological roles of lipins, which have been uncovered by the study of genetic lipin deficiencies. We propose that the growing body of knowledge concerning the biochemical and cellular activities of lipin proteins will be valuable for understanding the physiological functions of lipin proteins in health and disease. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

The Toxoplasma inner membrane complex (IMC) is a specialized organelle underlying the parasite's plasma membrane that consists of flattened rectangular membrane sacs that are sutured together and positioned atop a supportive cytoskeleton. We have previously identified a novel class of proteins localizing to the transverse and longitudinal sutures of the IMC, which we named IMC sutures components (ISCs). Here, we have used proximity-dependent biotin identification at the sutures to better define the composition of this IMC subcompartment. Using ISC4 as bait, we demonstrate biotin-dependent labeling of the sutures and have uncovered two new ISCs. We also identified five new proteins that exclusively localize to the transverse sutures that we named transverse sutures components (TSCs), demonstrating that components of the IMC sutures consist of two groups: those that localize to the transverse and longitudinal sutures (ISCs) and those residing only in the transverse sutures (TSCs). In addition, we functionally analyze the ISC protein ISC3 and demonstrate that ISC3-null parasites have morphological defects and reduced fitness in vitro. Most importantly, Δisc3 parasites exhibit a complete loss of virulence in vivo. These studies expand the known composition of the IMC sutures and highlight the contribution of ISCs to the ability of the parasite to proliferate and cause disease.

Leishmaniasis is a parasitic vector-borne disease caused by the protistan flagellates of the genus Leishmania. Leishmania (Viannia) guyanensis is one of the most common causative agents of the American tegumentary leishmaniasis. It has previously been shown that L. guyanensis strains that carry the endosymbiotic Leishmania RNA virus 1 (LRV1) cause more severe form of the disease in a mouse model than those that do not. The presence of the virus was implicated into the parasite's replication and spreading. In this respect, studying the molecular mechanisms of cellular control of viral infection is of great medical importance. Here, we report ~30.5 Mb high-quality genome assembly of the LRV1-positive L. guyanensis M4147. This strain was turned into a model by establishing the CRISPR-Cas9 system and ablating the gene encoding phosphatidate phosphatase 2-like (PAP2L) protein. The orthologue of this gene is conspicuously absent from the genome of an unusual member of the family Trypanosomatidae, Vickermania ingenoplastis, a species with mostly bi-flagellated cells. Our analysis of the PAP2L-null L. guyanensis showed an increase in the number of cells strikingly resembling the bi-flagellated V. ingenoplastis, likely as a result of the disruption of the cell cycle, significant accumulation of phosphatidic acid, and increased virulence compared to the wild type cells. Author summary: Worldwide, over one million people are getting infected by the parasitic flagellates of the genus Leishmania annually leading to ~30,000 deaths. Notably, there is still no approved vaccine against human leishmaniases. A range of methods of forward and reverse genetics has recently been developed for several model Leishmania species. Unfortunately, these methods are often not transferrable to non-model species, which may be of even greater medical importance. Leishmania guyanensis is one of such cases. It frequently carries a symbiotic RNA virus that contributes to the development of a more aggressive form of leishmaniasis in an experimental murine model. In order to establish and optimize the system for genetic manipulations in L. guyanensis, we sequenced and annotated its genome. Next, we applied the CRISPR-Cas9 technology to target a gene of interest. This approach was validated by ablating a gene encoding a protein involved in lipid metabolism. In this work, we document that deletion of this gene leads to the disturbance of cell cycle and affects the ratio of critical intracellular lipids. We believe that our study will facilitate research into more effective treatment of leishmaniases. [ABSTRACT FROM AUTHOR]

As with the majority of the hemibiotrophic fungal pathogens, the rice blast fungus Magnaporthe oryzae uses highly specialized infection structures called appressoria for plant penetration. Appressoria differentiated from germ tubes rely on enormous turgor pressure to directly penetrate the plant cell, in which process lipid metabolism plays a critical role. In this study, we characterized the MoPAH1 gene in M. oryzae, encoding a putative highly conserved phosphatidate phosphatase. The expression of MoPAH1 was up‐regulated during plant infection. The MoPah1 protein is expressed at all developmental and infection stages, and is localized to the cytoplasm. Disruption of MoPAH1 causes pleiotropic defects in vegetative growth, sporulation, and heat tolerance. The lipid profile is significantly altered in the Mopah1 mutant. Lipidomics assays showed that the level of phosphatidic acid (PA) was increased in the mutant, which had reduced levels of diacylglycerol and triacylglycerol. Using a PA biosensor, we showed that the increased level of PA in the Mopah1 mutant was primarily accumulated in the vacuole. The Mopah1 mutant was blocked in both conidiation and the formation of appressorium‐like structures at hyphal tips. It was nonpathogenic and failed to cause any blast lesions on rice and barley seedlings. RNA sequencing analysis revealed that MoPah1 regulates the expression of transcription factors critical for various developmental and infection‐related processes. The Mopah1 mutant was reduced in the expression and phosphorylation of Pmk1 MAP kinase and delayed in autophagy. Our study demonstrates that MoPah1 is necessary for lipid metabolism, fungal development, and pathogenicity in M. oryzae. [ABSTRACT FROM AUTHOR]

Oleaginous microorganisms have considerable potential for biofuel and commodity chemical production. Under nitrogen-limitation, Rhodococcus jostii RHA1 grown on benzoate, an analog of lignin depolymerization products, accumulated triacylglycerols (TAGs) to 55% of its dry weight during transition to stationary phase, with the predominant fatty acids being C16:0 and C17:0. Transcriptomic analyses of RHA1 grown under conditions of N-limitation and N-excess revealed 1,826 dysregulated genes. Genes whose transcripts were more abundant under N-limitation included those involved in ammonium assimilation, benzoate catabolism, fatty acid biosynthesis and the methylmalonyl-CoA pathway. Of the 16 atf genes potentially encoding diacylglycerol O-acyltransferases, atf8 transcripts were the most abundant during N-limitation (~50-fold more abundant than during N-excess). Consistent with Atf8 being a physiological determinant of TAG accumulation, a Δatf8 mutant accumulated 70% less TAG than wild-type RHA1 while atf8 overexpression increased TAG accumulation 20%. Genes encoding type-2 phosphatidic acid phosphatases were not significantly expressed. By contrast, three genes potentially encoding phosphatases of the haloacid dehalogenase superfamily and that cluster with, or are fused with other Kennedy pathway genes were dysregulated. Overall, these findings advance our understanding of TAG metabolism in mycolic acid-containing bacteria and provide a framework to engineer strains for increased TAG production.

Background: Rice (Oryza sativa L.) is one of the most widely grown food crops, and its yield and quality are particularly important for a warm-saturated diet. Cold stress restricts rice growth, development, and yield; however, the specific mechanism of cold tolerance in rice remains unknown. Results: The analysis of leaf physiological and photosynthetic characteristics showed that the two rice varieties were significantly affected by cold stress, but the cold-tolerant variety KY131 had more stable physiological characteristics, maintaining relatively good photosynthetic capacity. To better explore the transcriptional regulation mechanism and biological basis of rice response to cold stress, a comprehensive analysis of the rice transcriptome and lipidome under low temperature and control temperature conditions was carried out. The transcriptomic analysis revealed that lipid metabolism, including membrane lipid and fatty acid metabolism, may be an important factor in rice cold tolerance, and 397 lipid metabolism related genes have been identified. Lipidomics data confirmed the importance of membrane lipid remodeling and fatty acid unsaturation for rice adaptation to cold stress. This indicates that the changes in the fluidity and integrity of the photosynthetic membrane under cold stress lead to the reduction of photosynthetic capacity, which could be relieved by increased levels of monogalactosyldiacylglycerol that mainly caused by markedly increased expression of levels of 1,2-diacylglycerol 3-beta-galactosyltransferase (MGD). The upregulation of phosphatidate phosphatase (PAP2) inhibited the excessive accumulation of phosphatidate (PA) to produce more phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG), thereby preventing of membrane phase transition under cold stress. In addition, fatty acid β-oxidation is worth further study in rice cold tolerance. Finally, we constructed a metabolic model for the regulatory mechanism of cold tolerance in rice, in which the advanced lipid metabolism system plays a central role. Conclusions: Lipidome analysis showed that membrane lipid composition and unsaturation were significantly affected, especially phospholipids and galactolipids. Our study provides new information to further understand the response of rice to cold stress. [ABSTRACT FROM AUTHOR]

The Saccharomyces cerevisiae PAH1-encoded phosphatidate (PA) phosphatase, which catalyzes the dephosphorylation of PA to produce diacylglycerol, controls the bifurcation of PA into triacylglycerol synthesis and phospholipid synthesis. Pah1 is inactive in the cytosol as a phosphorylated form and becomes active on the membrane as a dephosphorylated form by the Nem1-Spo7 protein phosphatase. We show that the conserved Trp-637 residue of Pah1, located in the intrinsically disordered region, is required for normal synthesis of membrane phospholipids, sterols, triacylglycerol, and the formation of lipid droplets. Analysis of mutant Pah1-W637A showed that the tryptophan residue is involved in the phosphorylation-mediated/dephosphorylation-mediated membrane association of the enzyme and its catalytic activity. The endogenous phosphorylation of Pah1-W637A was increased at the sites of the N-terminal region but was decreased at the sites of the C-terminal region. The altered phosphorylation correlated with an increase in its membrane association. In addition, membrane-associated PA phosphatase activity in vitro was elevated in cells expressing Pah1-W637A as a result of the increased membrane association of the mutant enzyme. However, the inherent catalytic function of Pah1 was not affected by the W637A mutation. Prediction of Pah1 structure by Alpha-Fold shows that Trp-637 and the catalytic residues Asp-398 and Asp-400 in the haloacid dehalogenase-like domain almost lie in the same plane, suggesting that these residues are important to properly position the enzyme for substrate recognition at the membrane surface. These findings underscore the importance of Trp-637 in Pah1 regulation by phosphorylation, membrane association of the enzyme, and its function in lipid synthesis. [ABSTRACT FROM AUTHOR]

RationalePhosPhatidic Acid Phosphatase type 2B (PPAP2B), an integral membrane protein known as lipid phosphate phosphatase (LPP3) that inactivates lysophosphatidic acid, was implicated in coronary artery disease (CAD) by genome-wide association studies. However, it is unclear whether genome-wide association studies-identified coronary artery disease genes, including PPAP2B, participate in mechanotransduction mechanisms by which vascular endothelia respond to local atherorelevant hemodynamics that contribute to the regional nature of atherosclerosis.ObjectiveTo establish the critical role of PPAP2B in endothelial responses to hemodynamics.Methods and resultsReduced PPAP2B was detected in vivo in mouse and swine aortic arch (AA) endothelia exposed to chronic disturbed flow, and in mouse carotid artery endothelia subjected to surgically induced acute disturbed flow. In humans, PPAP2B was reduced in the downstream part of carotid plaques where low shear stress prevails. In culture, reduced PPAP2B was measured in human aortic endothelial cells under atherosusceptible waveform mimicking flow in human carotid sinus. Flow-sensitive microRNA-92a and transcription factor KLF2 were identified as upstream inhibitor and activator of endothelial PPAP2B, respectively. PPAP2B suppression abrogated atheroprotection of unidirectional flow; inhibition of lysophosphatidic acid receptor 1 restored the flow-dependent, anti-inflammatory phenotype in PPAP2B-deficient cells. PPAP2B inhibition resulted in myosin light-chain phosphorylation and intercellular gaps, which were abolished by lysophosphatidic acid receptor 1/2 inhibition. Expression quantitative trait locus mapping demonstrated PPAP2B coronary artery disease risk allele is not linked to PPAP2B expression in various human tissues but significantly associated with reduced PPAP2B in human aortic endothelial cells.ConclusionsAtherorelevant flows dynamically modulate endothelial PPAP2B expression through miR-92a and KLF2. Mechanosensitive PPAP2B plays a critical role in promoting anti-inflammatory phenotype and maintaining vascular integrity of endothelial monolayer under atheroprotective flow.

Genome-wide association studies (GWAS) have identified over 40 loci that affect risk of coronary artery disease (CAD) and the causal mechanisms at the majority of loci are unknown. Recent studies have suggested that many causal GWAS variants influence disease through altered transcriptional regulation in disease-relevant cell types. We explored changes in transcriptional regulation during a key pathophysiological event in CAD, the environmental lipid-induced transformation of macrophages to lipid-laden foam cells. We used a combination of open chromatin mapping with formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) and enhancer and transcription factor mapping using chromatin immuno-precipitation (ChIP-seq) in primary human macrophages before and after exposure to atherogenic oxidized low-density lipoprotein (oxLDL), with resultant foam cell formation. OxLDL-induced foam cell formation was associated with changes in a subset of open chromatin and active enhancer sites that strongly correlated with expression changes of nearby genes. OxLDL-regulated enhancers were enriched for several transcription factors including C/EBP-beta, which has no previously documented role in foam cell formation. OxLDL exposure up-regulated C/EBP-beta expression and increased genomic binding events, most prominently around genes involved in inflammatory response pathways. Variants at CAD-associated loci were significantly and specifically enriched in the subset of chromatin sites altered by oxLDL exposure, including rs72664324 in an oxLDL-induced enhancer at the PPAP2B locus. OxLDL increased C/EBP beta binding to this site and C/EBP beta binding and enhancer activity were stronger with the protective A allele of rs72664324. In addition, expression of the PPAP2B protein product LPP3 was present in foam cells in human atherosclerotic plaques and oxLDL exposure up-regulated LPP3 in macrophages resulting in increased degradation of pro-inflammatory mediators. Our results demonstrate a genetic mechanism contributing to CAD risk at the PPAP2B locus and highlight the value of studying epigenetic changes in disease processes involving pathogenic environmental stimuli.

The gene encoding a putative phosphatidate phosphatase (PAP) from tolerant saline-alkali (TSA) Chlorella , ChPAP , was identified from a yeast cDNA library constructed from TSA Chlorella after a NaCl treatment. ChPAP expressed in yeast enhanced its tolerance to NaCl and sorbitol. The ChPAP protein from a GFP-tagged construct localized to the plasma membrane and the lumen of vacuoles. The relative transcript levels of ChPAP in Chlorella cells were strongly induced by NaCl and sorbitol as assessed by northern blot analyses. Thus, ChPAP may play important roles in promoting Na-ion movement into the cell and maintaining the cytoplasmic ion balance. In addition, ChPAP may catalyze diacylglycerol pyrophosphate to phosphatidate in vacuoles. [ABSTRACT FROM AUTHOR]

LPIN1 encodes lipin-1, a phosphatidic acid phosphatase (PAP) enzyme that catalyzes the dephosphorylation of phosphatidic acid to form diacylglycerol. Homozygous LPIN1 gene mutations cause severe rhabdomyolysis, and heterozygous LPIN1 missense mutations may promote statin-induced myopathy. We demonstrate that lipin-1-related myopathy in the mouse is associated with a blockade in autophagic flux and accumulation of aberrant mitochondria. Lipin-1 PAP activity is required for maturation of autolysosomes, through its activation of the protein kinase D (PKD)-Vps34 phosphatidylinositol 3-kinase signaling cascade. Statin treatment also reduces PKD activation and autophagic flux, which are compounded by diminished mammalian target of rapamycin (mTOR) abundance in lipin-1-haploinsufficent and -deficient muscle. Lipin-1 restoration in skeletal muscle prevents myonecrosis and statin toxicity in vivo, and activated PKD rescues autophagic flux in lipin-1-deficient cells. Our findings identify lipin-1 PAP activity as a component of the macroautophagy pathway and define the basis for lipin-1-related myopathies.

The Nem1-Spo7 complex in the yeast Saccharomyces cerevisiae is a protein phosphatase that catalyzes the dephosphory-lation of Pah1 phosphatidate phosphatase, required for its translocation to the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7/Pah1 phosphatase cascade plays a major role in triacylglycerol synthesis and in the regulation of phospholipid synthesis. In this work, we examined Spo7, a regulatory subunit required for Nem1 catalytic function, to identify residues that govern formation of the Nem1-Spo7 complex. By deletion analysis of Spo7, we identified a hydrophobic Leu-Leu-Ile (LLI) sequence comprising residues 54-56 as being required for the protein to complement the temperature-sensitive phenotype of an spo7Δ mutant strain. Mutational analysis of the LLI sequence with alanine and arginine substitutions showed that its overall hydrophobicity is crucial for the formation of the Nem1-Spo7 complex as well as for the Nem1 catalytic function on its substrate, Pah1, in vivo. Consistent with the role of the Nem1-Spo7 complex in activating the function of Pah1, we found that the mutational effects of the Spo7 LLI sequence were on the Nem1-Spo7/Pah1 axis that controls lipid synthesis and related cellular processes (e.g. triacylglycerol/phospholipid synthesis, lipid droplet formation, nuclear/endoplasmic reticulum membrane morphology, vacuole fusion, and growth on glycerol medium). These findings advance the understanding of Nem1-Spo7 complex formation and its role in the phosphatase cascade that regulates the function of Pah1 phosphatidate phosphatase. [ABSTRACT FROM AUTHOR]

Cold stress restricts peanut (Arachis hypogaea L.) growth, development, and yield. However, the specific mechanism of cold tolerance in peanut remains unknown. Here, the comparative physiological, transcriptomic, and lipidomic analyses of cold tolerant variety NH5 and cold sensitive variety FH18 at different time points of cold stress were conducted to fill this gap. Transcriptomic analysis revealed lipid metabolism including membrane lipid and fatty acid metabolism may be a significant contributor in peanut cold tolerance, and 59 cold-tolerant genes involved in lipid metabolism were identified. Lipidomic data corroborated the importance of membrane lipid remodeling and fatty acid unsaturation. It indicated that photosynthetic damage, resulted from the alteration in fluidity and integrity of photosynthetic membranes under cold stress, were mainly caused by markedly decreased monogalactosyldiacylglycerol (MGDG) levels and could be relieved by increased digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG) levels. The upregulation of phosphatidate phosphatase (PAP1) and phosphatidate cytidylyltransferase (CDS1) inhibited the excessive accumulation of PA, thus may prevent the peroxidation of membrane lipids. In addition, fatty acid elongation and fatty acid β-oxidation were also worth further studied in peanut cold tolerance. Finally, we constructed a metabolic model for the regulatory mechanism of peanut cold tolerance, in which the advanced lipid metabolism system plays a central role. This study lays the foundation for deeply analyzing the molecular mechanism and realizing the genetic improvement of peanut cold tolerance. [ABSTRACT FROM AUTHOR]

Group IVA phospholipase A(2) (GIVA PLA(2)) catalyzes the release of arachidonic acid (AA) from the sn-2 position of glycerophospholipids. AA is then further metabolized into terminal signaling molecules including numerous prostaglandins. We have now demonstrated the involvement of phosphatidic acid phosphohydrolase 1 (PAP-1) and protein kinase C (PKC) in the Toll-like receptor-4 (TLR-4) activation of GIVA PLA(2). We also studied the effect of PAP-1 and PKC on Ca+2 induced and synergy enhanced GIVA PLA(2) activation. We observed that the AA release induced by exposure of RAW 264.7 macrophages to the TLR-4 specific agonist Kdo(2)-Lipid A is blocked by the PAP-1 inhibitors bromoenol lactone (BEL) and propranolol as well as the PKC inhibitor Ro 31-8220; however these inhibitors did not reduce AA release stimulated by Ca+2 influx induced by the P2X7 purinergic receptor agonist ATP. Additionally, stimulation of cells with diacylglycerol (DAG), the product of PAP-1 mediated hydrolysis, initiated AA release from unstimulated cells as well as restored normal AA release from cells treated with PAP-1 inhibitors. Finally, neither PAP-1 nor PKC inhibition reduced GIVA PLA(2) synergistic activation by stimulation with Kdo(2)-Lipid A and ATP.

Cyclooxygenase (COX) has two isoforms, COX-1 and -2, which catalyze the key step in the conversion of cellular arachidonic acid into prostaglandins. In recent years, interest in COX-2 has significantly increased since it has been a target for the development of specific non-steroidal anti-inflammatory drugs. We report that COX-2 expression is up-regulated in phorbol ester (phorbol myristate acetate, PMA)-differentiated human U937 macrophage-like cells stimulated with lipopolysaccharide (LPS), whereas COX-1 is not up-regulated. We show that the LPS-induced up-regulation of COX-2 depends on the activity of the Mg(+2)-dependent phosphatidic acid phosphohydrolase 1 (PAP-1). Inhibition of PAP-1 by bromoenol lactone, propranolol, or ethanol resulted in a decrease in LPS-induced COX-2 mRNA transcript production, COX-2 protein expression, and prostaglandin E(2) release from U937 macrophages. To ensure that these results did not arise because of PMA treatment of the U937 cells, similar experiments were conducted with the P388D(1) cell line, which does not require PMA differentiation. LPS increased the levels of endogenous cellular diacylglycerol (DAG) within 2 min of stimulation. This increase was observed to be sensitive to the PAP-1 inhibitors. Furthermore, phosphatidic acid phosphohydrolase activity assays showed that the bromoenol lactone-sensitive PAP-1 activity was translocated from the cytosolic fraction to the membrane fraction within 2 min of LPS exposure. Finally, DAG add-back experiments demonstrate that LPS-induced COX-2 expression is enhanced by the addition of exogenous DAG.

Metabolic-dysfunction-associated steatotic liver disease (MASLD) is a growing health problem for which no therapy exists to date. The modulation of the gut microbiome may have treatment potential for MASLD. Here, we investigated Anaerobutyricum soehngenii , a butyrate-producing anaerobic bacterium with beneficial effects in metabolic syndrome, in a diet-induced MASLD mouse model. Male C57BL/6J mice received a Western-type high-fat diet and water with 15% fructose (WDF) to induce MASLD and were gavaged with A. soehngenii (10 8 or 10 9 colony-forming units (CFU) 3 times per week) or a placebo for 6 weeks. The A. soehngenii gavage increased the cecal butyrate concentrations. Although there was no effect on histological MASLD scores, A. soehngenii improved the glycemic response to insulin. In the liver, the WDF-associated altered expression of three genes relevant to the MASLD pathophysiology was reversed upon treatment with A. soehngenii : Lipin-1 ( Lpin1 ), insulin-like growth factor binding protein 1 ( Igfbp1 ) and Interleukin 1 Receptor Type 1 ( Il1r1 ). A. soehngenii administration also increased the intestinal expression of gluconeogenesis and fructolysis genes. Although these effects did not translate into significant histological improvements in MASLD, these results provide a basis for combined gut microbial approaches to induce histological improvements in MASLD.

Background: Poor oocyte quality remains one of the major challenges for polycystic ovary syndrome (PCOS) patients during in vitro fertilization (IVF) treatment. Granulosa cells (GCs) in PCOS display altered functions and could cause an unfavorable microenvironment for oocyte growth and maturation. Ferroptosis is a new form of programmed cell death, but its role in PCOS has been largely unclarified., Methods: Ferroptosis-related differentially expressed genes (DEGs) of GCs in women with PCOS were identified by bioinformatic analyses of GSE155489 and GSE168404 datasets. Functional enrichment analyses were conducted using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Core ferroptosis-related genes were further screened by random forest, and evaluated for diagnostic value by receiver operating characteristic curve analyses. Gene expression was validated by real-time quantitative polymerase chain reaction of collected GC samples, and analyzed for association with oocyte quality. In addition, gene regulatory network was constructed based on predicted RNA interactions and transcription factors, while potential therapeutic compounds were screened through molecular docking with crystallographic protein structures., Results: A total of 14 ferroptosis-related DEGs were identified. These DEGs were mainly enriched in reactive oxygen species metabolic process, mitochondrial outer membrane, antioxidant activity as well as ferroptosis and adipocytokine signaling pathways. Eight core ferroptosis-related genes (ATF3, BNIP3, DDIT4, LPIN1, NOS2, NQO1, SLC2A1 and SLC2A6) were further selected in random forest model, which showed high diagnostic performance for PCOS. Seven of them were validated in GC samples, and five were found to be significantly and positively correlated with one or more oocyte quality parameters in PCOS patients, including oocyte retrieval rate, mature oocyte rate, normal fertilization rate, and good-quality embryo rate. Gene regulatory network revealed JUN and HMGA1 as two important transcription factors, while dicoumarol and flavin adenine dinucleotide were predicted as small molecules with therapeutic potential., Conclusions: This is the first comprehensive report to study the differential expression of ferroptosis-related genes in GCs of PCOS and their clinical relevance with oocyte quality. Our findings could provide novel insights on the potential role of GC ferroptosis in PCOS pathogenesis, diagnosis, and targeted treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Huang, Fan, Li, Yang, Xiong, Xiong, Feng, Chen, Wang, Su, Xu, Yang, Wang and Zhu.)

Sepsis is a dysregulated response to infection that can result in life-threatening organ failure, and septic cardiomyopathy is a serious complication involving ferroptosis. Olaparib, a classic targeted drug used in oncology, has demonstrated potential protective effects against sepsis. However, the exact mechanisms underlying its action remain to be elucidated. In our study, we meticulously screened ferroptosis genes associated with sepsis, and conducted comprehensive functional enrichment analyses to delineate the relationship between ferroptosis and mitochondrial damage. Eight sepsis-characterized ferroptosis genes were identified in sepsis patients, including DPP4, LPIN1, PGD, HP, MAPK14, POR, GCLM, and SLC38A1, which were significantly correlated with mitochondrial quality imbalance. Utilizing DrugBank and molecular docking, we demonstrated a robust interaction of Olaparib with these genes. Lipopolysaccharide (LPS)-stimulated HL-1 cells and monocytes were used to establish an in vitro sepsis model. Additionally, an in vivo model was developed using mice subjected to cecal ligation and perforation (CLP). Intriguingly, low-dose Olaparib (5 mg/kg) effectively targeted and mitigated markers associated with ferroptosis, concurrently improving mitochondrial quality. This led to a marked enhancement in cardiac function and a significant increase in survival rates in septic mice (p < 0.05). The mechanism through which Olaparib ameliorates ferroptosis in cardiac and leukocyte cells post-sepsis is attributed to its facilitation of mitophagy, thus favoring mitochondrial integrity. In conclusion, our findings suggest that low-dose Olaparib can improve mitochondrial quality by accelerating mitophagy flux, consequently inhibiting ferroptosis and preserving cardiac function after sepsis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Prostaglandins are known to play a key role in the initiation of labor in humans, but the mechanisms governing their synthesis in amnion are largely unknown. In this study, we have examined the regulatory pathways for prostaglandin E(2) (PGE(2)) production during protein kinase C-dependent activation of human WISH cells. In these cells, PGE(2) synthesis appears to be limited not by free arachidonic acid availability but by the expression levels of cyclooxygenase-2 (COX-2). Concomitant with the cells being able to synthesize and secrete PGE(2), we detected significant elevations of both COX-2 protein and mRNA levels. Specific inhibition of COX-2 by NS-398 totally ablated PGE(2) synthesis. All of these responses were found to be strikingly dependent on an active phosphatidate phosphohydrolase 1 (PAP-1). Inhibition of PAP-1 activity by three different strategies (i.e. use of bromoenol lactone, propranolol, and ethanol) resulted in inhibition of COX-2 expression and hence of PGE(2) production. These data unveil a novel signaling mechanism for the regulation of PGE(2) production via regulation of COX-2 expression and implicate phosphatidate phosphohydrolase 1 as a key regulatory component of eicosanoid metabolic pathways in the amnion.

The PAH1-encoded phosphatidate phosphatase in Saccharomyces cerevisiae plays a major role in triacylglycerol synthesis and the control of phospholipid synthesis. For its catalytic function on the nuclear/endoplasmic reticulum membrane, Pah1 translocates to the membrane through its phosphorylation/dephosphorylation. Pah1 phosphorylation on multiple serine/threonine residues is complex and catalyzed by diverse protein kinases. In this work, we demonstrate that Pah1 is phosphorylated by the YCK1-encoded casein kinase I (CKI), regulating Pah1 catalytic activity and phosphorylation. Phosphoamino acid analysis coupled with phosphopeptide mapping of the CKIphosphorylated Pah1 indicated that it is phosphorylated mainly on multiple serine residues. Using site-directed mutagenesis and phosphorylation analysis of Pah1, we identified eight serine residues (i.e. Ser-114, Ser-475, Ser-511, Ser-602, Ser-677, Ser- 705, Ser-748, and Ser-774) as the target sites of CKI. Of these residues, Ser-475 and Ser-511 were specific for CKI, whereas the others were shared by casein kinase II (Ser-705), Cdc28-cyclin B(Ser-602), Pho85-Pho80 (Ser-114, Ser-602, and Ser-748), protein kinase A (Ser-667 and Ser-774), and protein kinase C (Ser- 677). CKI-mediated phosphorylation of Pah1 stimulated both its phosphatidate phosphatase activity and its subsequent phosphorylation by casein kinase II. However, the CKI-mediated phosphorylation of Pah1 strongly inhibited its subsequent phosphorylation by Pho85-Pho80, protein kinase A, and protein kinase C. In a reciprocal analysis, Pah1 phosphorylation by Pho85-Pho80 inhibited subsequent phosphorylation by CKI. CKI-mediated Pah1 phosphorylation was also inhibited by a peptide containing the Pah1 residues 506-517, including the kinase-specific Ser-511 residue. These findings advance our understanding of how Pah1 catalytic activity and phosphorylation are regulated by multiple protein kinases. [ABSTRACT FROM AUTHOR]

The Nem1-Spo7 complex in the yeast Saccharomyces cerevisiae is a protein phosphatase required for the nuclear/endoplasmic reticulum membrane localization of Pah1, a phosphatidate phosphatase that produces diacylglycerol for triacylglycerol synthesis at the expense of phospholipid synthesis. In a previous study, we showed that the protein phosphatase is subject to phosphorylation by protein kinase A (PKA). Here, we demonstrate that Nem1-Spo7 is regulated through its phosphorylation by protein kinase C (PKC), which plays multiple roles, including the regulation of lipid synthesis and cell wall integrity. Phosphorylation analyses of Nem1-Spo7 and its synthetic peptides indicate that both subunits of the complex are bona fide PKC substrates. Site-directed mutagenesis of NEM1 and SPO7, coupled with phosphopeptide mapping and immunoblotting with a phosphoserine-specific PKC substrate antibody, revealed that Ser-201 in Nem1 and Ser-22/Ser-28 in Spo7 are major PKC target sites of phosphorylation. Activity analysis of mutant Nem1-Spo7 complexes indicates that the PKC phosphorylation of Nem1 exerts a stimulatory effect, but the phosphorylation of Spo7 has no effect. Lipid-labeling analysis of cells expressing the phosphorylation-deficient alleles of NEM1 and SPO7 indicates that the stimulation of the Nem1-Spo7 activity has the effect of increasing triacylglycerol synthesis. Prephosphorylation of Nem1-Spo7 by PKC inhibits the PKA phosphorylation of Nem1, whereas prephosphorylation of the phosphatase complex by PKA inhibits the PKC phosphorylation of Spo7. Collectively, this work advances the understanding of the Nem1-Spo7 regulation by phosphorylation and its impact on lipid synthesis. [ABSTRACT FROM AUTHOR]

The mammalian lipin 1 phosphatidate phosphatase is a key regulatory enzyme in lipid metabolism. By catalyzing phosphatidate dephosphorylation, which produces diacylglycerol, the enzyme plays a major role in the synthesis of triacylglycerol and membrane phospholipids. The importance of lipin 1 to lipid metabolism is exemplified by cellular defects and lipid-based diseases associated with its loss or overexpression. Phosphorylation of lipin 1 governs whether it is associated with the cytoplasm apart from its substrate or with the endoplasmic reticulum membrane where its enzyme reaction occurs. Lipin 1β is phosphorylated on multiple sites, but less than 10% of them are ascribed to a specific protein kinase. Here, we demonstrate that lipin 1β is a bona fide substrate for casein kinase II (CKII), a protein kinase that is essential to viability and cell cycle progression. Phosphoamino acid analysis and phosphopeptide mapping revealed that lipin 1β is phosphorylated by CKII on multiple serine and threonine residues, with the former being major sites. Mutational analysis of lipin 1β and its peptides indicated that Ser-285 and Ser-287 are both phosphorylated by CKII. Substitutions of Ser-285 and Ser-287 with nonphosphorylatable alanine attenuated the interaction of lipin 1β with 14-3-3β protein, a regulatory hub that facilitates the cytoplasmic localization of phosphorylated lipin 1. These findings advance our understanding of how phosphorylation of lipin 1β phosphatidate phosphatase regulates its interaction with 14-3-3β protein and intracellular localization and uncover a mechanism by which CKII regulates cellular physiology. [ABSTRACT FROM AUTHOR]

This JBC Review on the discoveries of yeast phosphatidate (PA) phosphatase genes is dedicated to Dr. Herbert Tabor, Editor-in-Chief of the Journal of Biological Chemistry (JBC) for 40 years, on the occasion of his 100th birthday. Here, I reflect on the discoveries of the APP1, DPP1, LPP1, and PAH1 genes encoding all the PA phosphatase enzymes in yeast. PA phosphatase catalyzes PA dephosphorylation to generate diacylglycerol; both substrate and product are key intermediates in the synthesis of membrane phospholipids and triacylglycerol. App1 and Pah1 are peripheral membrane proteins catalyzing an Mg2+-dependent reaction governed by the DXDX(T/V) phosphatase motif. Dpp1 and Lpp1 are integral membrane proteins that catalyze an Mg2+-independent reaction governed by the KX6RP--PSGH--SRX5HX3D phosphatase motif. Pah1 is PA-specific and is the onlyPAphosphatase responsible for lipid synthesis at the nuclear/ endoplasmic reticulum membrane. App1, Dpp1, and Lpp1, respectively, are localized to cortical actin patches and the vacuole and Golgi membranes; they utilize several lipid phosphate substrates, including PA, lyso-PA, and diacylglycerol pyrophosphate. App1 is postulated to be involved in endocytosis, whereas Dpp1 and Lpp1 may be involved in lipid signaling. Pah1 is the yeast lipin homolog of mice and humans. A host of cellular defects and lipid-based diseases associated with loss or overexpression of PA phosphatase in yeast, mice, and humans, highlights its importance to cell physiology. [ABSTRACT FROM AUTHOR]

Prostaglandins are known to play a central role in the initiation of labor in humans, and amnionic cells constitute a major source of these compounds. Prostaglandin synthesis and release by amnion cells in response to hormones and ligands takes place after a characteristic 4-5 h lag. However, we report herein that free arachidonic acid (AA), the metabolic precursor of prostaglandins, can be induced at much shorter times (1 h) in human amnionic WISH cells by phorbol 12-myristate 13-acetate (PMA) through activation of protein kinase Calpha (PKCalpha). WISH cells were found to possess both cytosolic group IV phospholipase A2 (cPLA2) and Group VI Ca2+-independent phospholipase A2 (iPLA2). Of these, the cPLA2 was found to be the likely mediator of AA mobilization in PMA-activated WISH cells. PMA also activates phospholipase D (PLD) in these cells and ethanol, a compound that inhibits PLD-mediated phosphatidic acid (PA) formation, blocked AA release. Moreover, prevention of PA dephosphorylation by the PA phosphohydrolase inhibitors propranolol and bromoenol lactone, resulted in inhibition of AA release by PMA-treated WISH cells. Collectively, these data suggest that activation of cPLA2 and attendant AA release by phorbol esters in WISH cells requires prior generation of DAG by phosphatidate phosphohydrolase.

The silkworm (Bombyx mori) is a model species of lepidopteran insect. Microsporidium spp. are obligate intracellular eukaryotic parasites. Infection by the microsporidian Nosema bombycis (Nb) results in an outbreak of Pébrine disease in silkworms and causes substantial losses to the sericulture industry. It has been suggested that Nb depends on nutrients from host cells for spore growth. However, little is known about changes in lipid levels after Nb infection. In this study, ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was performed to analyze the effect of Nb infection on lipid metabolism in the midgut of silkworms. A total of 1601 individual lipid molecules were detected in the midgut of silkworms, of which 15 were significantly decreased after Nb challenge. Classification, chain length, and chain saturation analysis revealed that these 15 differential lipids can be classified into different lipid subclasses, of which 13 belong to glycerol phospholipid lipids and two belong to glyceride esters. The results indicated that Nb uses the host lipids to complete its own replication, and the acquisition of host lipid subclasses is selective; not all lipid subclasses are required for microsporidium growth or proliferation. Based on lipid metabolism data, phosphatidylcholine (PC) was found to be an important nutrient for Nb replication. Diet supplementation with lecithin substantially promoted the replication of Nb. Knockdown and overexpression of the key enzyme phosphatidate phosphatase (PAP) and phosphatidylcholine (Bbc) for PC synthesis also confirmed that PC is necessary for Nb replication. Our results showed that most lipids in the host midgut decreased when silkworms were infected with Nb. Reduction of or supplementation with PC may be a strategy to suppress or promote microsporidial replication. • Infection of Nosema bombycis alters the lipid metabolism of its host silkworm. • Nosema bombycis depends on host's lipids for effective proliferation. • Lecithin (phosphatidylcholine; PC) acts as host metabolite for the proliferation of Nb in silkworm. [ABSTRACT FROM AUTHOR]

Bromoenol lactone (BEL) has previously been identified as a potent, irreversible, mechanism-based phospholipase A2 (PLA2) inhibitor that possesses greater than 1000-fold selectivity for inhibition of Ca2+-independent PLA2 (iPLA2) versus the Ca2+-dependent ones. Thus, this compound has been used as a selective tool for studies aimed at elucidating the role of iPLA2 in certain cellular functions. Herein we report that BEL also inhibits cellular phosphatidic acid phosphohydrolase (PAP) activity in intact P388D1 macrophages with an IC50 of about 8 microM, which is very similar to that previously found for inhibition of iPLA2 under the same experimental conditions. This results in the blockage of the incorporation of exogenous arachidonate and palmitate into diacylglycerol and triacylglycerol. Thus, inhibition of PAP by BEL blocks triacylglycerol biosynthesis in P388D1 cells due to decreased diacylglycerol availability. Because two forms of PAP activity exist in mammalian cells, differential assays were performed to identify which of these forms was inhibited by BEL. The results of these experiments revealed that BEL selectively inhibits the cytosolic, Mg2+-dependent enzyme. No apparent effect of BEL on the membrane-bound Mg2+-independent PAP form could be detected. Collectively, the results reported herein establish that BEL inhibits two cellular phospholipases, namely iPLA2 and Mg2+-dependent PAP, with similar potency. Therefore, the inhibitory effect of BEL on Mg2+-dependent PAP might explain several cellular functions previously attributed to iPLA2.

Many receptors, in response to their specific ligands, trigger activation of phospholipase D (PLD), resulting in the production of phosphatidic acid which, in turn, is acted upon by a specific phosphatase, phosphatidate phosphohydrolase, to produce diacylglycerol. We report here that isolated nuclei from Madin-Darby canine kidneys (MDCK)-D1 cells exhibit a PLD activity that is enhanced by the presence of ATP. PLD activity was measured in the presence of ethanol, by quantitating the production of phosphatidylethanol. Non-phosphorylating ATP analogs were unable to substitute for ATP in activating PLD, indicating that ATP acts as a phosphoryl group donor in a kinase-mediated phosphorylation reaction. The protein kinase C inhibitors chelerythrine and calphostin completely suppressed the ATP-induced nuclear PLD, implicating protein kinase C as the kinase involved in ATP-dependent PLD activity in nuclei from MDCK-D1 cells. In the absence of ethanol, phosphatidic acid was detected in ATP-treated nuclei. Accumulation of phosphatidic acid preceded or closely paralleled that of diacylglycerol, suggesting a precursor-product relationship. Consistent with those results, we detected phosphatidate phosphohydrolase activity in MDCK-D1 cell nuclei. Measurements of phosphatidic acid and diacylglycerol levels at increasing amounts of ethanol demonstrated that PLD and phosphatidate phosphohydrolase are responsible for generating the majority of the diacylglycerol accumulating in MDCK-D1 cell nuclei. The ability of nuclei to generate diacylglycerol from the concerted action of those two enzymes provides a means to regulate nuclear lipid synthesis as well as protein kinase C activity.