Phosphatidic Acid Phosphatase Research Articles

Normal human adipose tissue functions and differentiation in patients with biallelic LPIN1 inactivating mutations

Lipin-1 is a Mg2+-dependent phosphatidic acid phosphatase (PAP) that in mice is necessary for normal glycerolipid biosynthesis, controlling adipocyte metabolism, and adipogenic differentiation. Mice carrying inactivating mutations in the Lpin1 gene display the characteristic features of human familial lipodystrophy. Very little is known about the roles of lipin-1 in human adipocyte physiology. Apparently, fat distribution and weight is normal in humans carrying LPIN1 inactivating mutations, but a detailed analysis of adipose tissue appearance and functions in these patients has not been available so far. In this study, we performed a systematic histopathological, biochemical, and gene expression analysis of adipose tissue biopsies from human patients harboring LPIN1 biallelic inactivating mutations and affected by recurrent episodes of severe rhabdomyolysis. We also explored the adipogenic differentiation potential of human mesenchymal cell populations derived from lipin-1 defective patients. White adipose tissue from human LPIN1 mutant patients displayed a dramatic decrease in lipin-1 protein levels and PAP activity, with a concomitant moderate reduction of adipocyte size. Nevertheless, the adipose tissue develops without obvious histological signs of lipodystrophy and with normal qualitative composition of storage lipids. The increased expression of key adipogenic determinants such as SREBP1, PPARG, and PGC1A shows that specific compensatory phenomena can be activated in vivo in human adipocytes with deficiency of functional lipin-1.

The phosphatidic acid–binding, polybasic domain is responsible for the differences in the phosphoregulation of lipins 1 and 3

Lipins 1, 2, and 3 are Mg2+-dependent phosphatidic acid phosphatases and catalyze the penultimate step of triacylglycerol synthesis. We have previously investigated the biochemistry of lipins 1 and 2 and shown that di-anionic phosphatidic acid (PA) augments their activity and lipid binding and that lipin 1 activity is negatively regulated by phosphorylation. In the present study, we show that phosphorylation does not affect the catalytic activity of lipin 3 or its ability to associate with PA in vitro The lipin proteins each contain a conserved polybasic domain (PBD) composed of nine lysine and arginine residues located between the conserved N- and C-terminal domains. In lipin 1, the PBD is the site of PA binding and sensing of the PA electrostatic charge. The specific arrangement and number of the lysines and arginines of the PBD vary among the lipins. We show that the different PBDs of lipins 1 and 3 are responsible for the presence of phosphoregulation on the former but not the latter enzyme. To do so, we generated lipin 1 that contained the PBD of lipin 3 and vice versa. The lipin 1 enzyme with the lipin 3 PBD lost its ability to be regulated by phosphorylation but remained downstream of phosphorylation by mammalian target of rapamycin. Conversely, the presence of the lipin 1 PBD in lipin 3 subjected the enzyme to negative intramolecular control by phosphorylation. These results indicate a mechanism for the observed differences in lipin phosphoregulation in vitro.

"Prokaryotic Pathway" Is Not Prokaryotic: Noncyanobacterial Origin of the Chloroplast Lipid Biosynthetic Pathway Revealed by Comprehensive Phylogenomic Analysis.

Lipid biosynthesis within the chloroplast, or more generally plastids, was conventionally called “prokaryotic pathway,” which produces glycerolipids bearing C18 acids at the sn-1 position and C16 acids at the sn-2 position, as in cyanobacteria such as Anabaena and Synechocystis. This positional specificity is determined during the synthesis of phosphatidate, which is a precursor to diacylglycerol, the acceptor of galactose for the synthesis of galactolipids. The first acylation at sn-1 is catalyzed by glycerol-3-phosphate acyltransferase (GPAT or GPT), whereas the second acylation at sn-2 is performed by lysophosphatidate acyltransferase (LPAAT, AGPAT, or PlsC). Here we present comprehensive phylogenomic analysis of the origins of various acyltransferases involved in the synthesis of phosphatidate, as well as phosphatidate phosphatases in the chloroplasts. The results showed that the enzymes involved in the two steps of acylation in cyanobacteria and chloroplasts are entirely phylogenetically unrelated despite a previous report stating that the chloroplast LPAAT (ATS2) and cyanobacterial PlsC were sister groups. Phosphatidate phosphatases were separated into eukaryotic and prokaryotic clades, and the chloroplast enzymes were not of cyanobacterial origin, in contrast with another previous report. These results indicate that the lipid biosynthetic pathway in the chloroplasts or plastids did not originate from the cyanobacterial endosymbiont and is not “prokaryotic” in the context of endosymbiotic theory of plastid origin. This is another line of evidence for the discontinuity of plastids and cyanobacteria, which has been suggested in the glycolipid biosynthesis.

Small phosphatidate phosphatase (TtPAH2) of Tetrahymena complements respiratory function and not membrane biogenesis function of yeast PAH1.

Phosphatidate phosphatases (PAH) play a central role in lipid metabolism and intracellular signaling. Herein, we report the presence of a low-molecular-weight PAH homolog in the single-celled ciliate Tetrahymena thermophila. In vitro phosphatase assay showed that TtPAH2 belongs to the magnesium-dependent phosphatidate phosphatase (PAP1) family. Loss of function of TtPAH2 did not affect the growth of Tetrahymena. Unlike other known PAH homologs, TtPAH2 did not regulate lipid droplet number and ER morphology. TtPAH2 did not rescue growth and ER/nuclear membrane defects of the pah1Δ yeast cells, suggesting that the phosphatidate phosphatase activity of the protein is not sufficient to perform these cellular functions. Surprisingly, TtPAH2 complemented the respiratory defect in the pah1Δ yeast cells indicating a specific role of TtPAH2 in respiration. Overall, our results indicate that TtPAH2 possesses the minimal function of PAH protein family in respiration. We suggest that the amino acid sequences absent from TtPAH2 but present in all other known PAH homologs are critical for lipid homeostasis and membrane biogenesis.

Growth arrest and DNA damage-inducible 45α protects against nonalcoholic steatohepatitis induced by methionine- and choline-deficient diet

Growth arrest and DNA damage-inducible 45 α (Gadd45α) is a stress-inducible protein that plays an important role in cell survival/death and DNA repair, but its contribution to the development of nonalcoholic steatohepatitis (NASH) has not been investigated. C57BL/6 Gadd45a-null and wild-type (WT) mice were treated with a methionine and choline-deficient diet (MCD) for eight weeks and phenotypic changes examined. Gadd45a-null mice had more severe hepatic inflammation and fibrosis, higher levels of mRNAs encoding pro-inflammatory, pro-fibrotic, and pro-apoptotic proteins, and greater oxidative and endoplasmic reticulum (ER) stress compared with WT mice. Indeed, Gadd45a mRNA was induced in response to ER stress in primary hepatocytes. Lipidomic analysis of NASH livers demonstrated decreased triacylglycerol (TG) in MCD-treated Gadd45a-null mice, which was associated with increased mRNAs encoding phospholipase D (Pld1/2), phosphatidic acid phosphatase type 2A, and choline/ethanolamine phosphotransferase 1 (Cept1), involved in the phosphatidylcholine-phosphatidic acid-diacylglycerol cycle, and decreased mRNAs encoding fatty acid (FA)-binding protein 1 (Fabp1) and FA transport protein 5. Treatment of cultured primary hepatocytes with tumor necrosis factor α, transforming growth factor β, and hydrogen peroxide led to the corresponding induction of Fabp1, Pld1/2, and Cept1 mRNAs. Collectively, Gadd45α plays protective roles against MCD-induced NASH likely due to attenuating cellular stress and ensuing inflammatory signaling. These results also suggest an interconnection between hepatocyte injury, inflammation and disrupted glycerophospholipid/FA metabolism that yields a possible mechanism for decreased TG accumulation with NASH progression (i.e., “burned-out” NASH).

Phosphatidate phosphatase regulates membrane phospholipid synthesis via phosphatidylserine synthase

The yeast Saccharomyces cerevisiae serves as a model eukaryote to elucidate the regulation of lipid metabolism. In exponentially growing yeast, a diverse set of membrane lipids are synthesized from the precursor phosphatidate via the liponucleotide intermediate CDP-diacylglycerol. As cells exhaust nutrients and progress into the stationary phase, phosphatidate is channeled via diacylglycerol to the synthesis of triacylglycerol. The CHO1-encoded phosphatidylserine synthase, which catalyzes the committed step in membrane phospholipid synthesis via CDP-diacylglycerol, and the PAH1-encoded phosphatidate phosphatase, which catalyzes the committed step in triacylglycerol synthesis are regulated throughout cell growth by genetic and biochemical mechanisms to control the balanced synthesis of membrane phospholipids and triacylglycerol. The loss of phosphatidate phosphatase activity (e.g., pah1Δ mutation) increases the level of phosphatidate and its conversion to membrane phospholipids by inducing Cho1 expression and phosphatidylserine synthase activity. The regulation of the CHO1 expression is mediated through the inositol-sensitive upstream activation sequence (UASINO), a cis-acting element for the phosphatidate-controlled Henry (Ino2-Ino4/Opi1) regulatory circuit. Consequently, phosphatidate phosphatase activity regulates phospholipid synthesis through the transcriptional regulation of the phosphatidylserine synthase enzyme.

Yeast PAH1-encoded phosphatidate phosphatase controls the expression of CHO1-encoded phosphatidylserine synthase for membrane phospholipid synthesis

The PAH1-encoded phosphatidate phosphatase (PAP), which catalyzes the committed step for the synthesis of triacylglycerol in Saccharomyces cerevisiae, exerts a negative regulatory effect on the level of phosphatidate used for the de novo synthesis of membrane phospholipids. This raises the question whether PAP thereby affects the expression and activity of enzymes involved in phospholipid synthesis. Here, we examined the PAP-mediated regulation of CHO1-encoded phosphatidylserine synthase (PSS), which catalyzes the committed step for the synthesis of major phospholipids via the CDP-diacylglycerol pathway. The lack of PAP in the pah1Δ mutant highly elevated PSS activity, exhibiting a growth-dependent up-regulation from the exponential to the stationary phase of growth. Immunoblot analysis showed that the elevation of PSS activity results from an increase in the level of the enzyme encoded by CHO1 Truncation analysis and site-directed mutagenesis of the CHO1 promoter indicated that Cho1 expression in the pah1Δ mutant is induced through the inositol-sensitive upstream activation sequence (UASINO), a cis-acting element for the phosphatidate-controlled Henry (Ino2-Ino4/Opi1) regulatory circuit. The abrogation of Cho1 induction and PSS activity by a CHO1 UASINO mutation suppressed pah1Δ effects on lipid synthesis, nuclear/endoplasmic reticulum membrane morphology, and lipid droplet formation, but not on growth at elevated temperature. Loss of the DGK1-encoded diacylglycerol kinase, which converts diacylglycerol to phosphatidate, partially suppressed the pah1Δ-mediated induction of Cho1 and PSS activity. Collectively, these data showed that PAP activity controls the expression of PSS for membrane phospholipid synthesis.

Induction of Lipin1 by ROS-Dependent SREBP-2 Activation

Lipin1 was identified as a phosphatidate phosphatase enzyme, and it plays a key role in lipid metabolism. Since free radicals contribute to metabolic diseases in the liver, this study investigated the effects of free radicals on the regulation of Lipin1 expression in Huh7 and AML12 cells. Hydrogen peroxide induced mRNA and protein expression of Lipin1 in Huh7 cells, which was assayed by quantitative RT-PCR and immunoblotting, respectively. Induction of Lipin1 by hydrogen peroxide was confirmed in AML12 cells. Hydrogen peroxide treatment significantly increased expression of sterol regulatory element-binding protein (SREBP)-2, but not SREBP-1. Moreover, nuclear translocation of SREBP-2 was detected after hydrogen peroxide treatment. Hydrogen peroxide-induced Lipin1 or SREBP-2 expression was significantly reduced by N-acetyl-L-cysteine treatment, indicating that reactive oxygen species (ROS) were implicated in Lipin1 expression. Next, we investigated whether the hypoxic environments that cause endogenous ROS production in mitochondria in metabolic diseases affect the expression of Lipin1. Exposure to hypoxia also increased Lipin1 expression. In contrast, pretreatment with antioxidants attenuated hypoxia-induced Lipin1 expression. Collectively, our results show that ROS activate SREBP-2, which induces Lipin1 expression.

Regulation Involved in Colonization of Intercellular Spaces of Host Plants in Ralstonia solanacearum.

A soil-borne bacterium Ralstonia solanacearum invading plant roots first colonizes the intercellular spaces of the root, and eventually enters xylem vessels, where it replicates at high levels leading to wilting symptoms. After invasion into intercellular spaces, R. solanacearum strain OE1-1 attaches to host cells and expression of the hrp genes encoding components of the type III secretion system (T3SS). OE1-1 then constructs T3SS and secrets effectors into host cells, inducing expression of the host gene encoding phosphatidic acid phosphatase. This leads to suppressing plant innate immunity. Then, OE1-1 grows on host cells, inducing quorum sensing (QS). The QS contributes to regulation of OE1-1 colonization of intercellular spaces including mushroom-type biofilm formation on host cells, leading to its virulence. R. solanacearum strains AW1 and K60 produce methyl 3-hydroxypalmitate (3-OH PAME) as a QS signal. The methyltransferase PhcB synthesizes 3-OH PAME. When 3-OH PAME reaches a threshold level, it increases the ability of the histidine kinase PhcS to phosphorylate the response regulator PhcR. This results in elevated levels of functional PhcA, the global virulence regulator. On the other hand, strains OE1-1 and GMI1000 produce methyl 3-hydroxymyristate (3-OH MAME) as a QS signal. Among R. solanacearum strains, the deduced PhcB and PhcS amino acid sequences are related to the production of QS signals. R. solanacearum produces aryl-furanone secondary metabolites, ralfuranones, which are extracellularly secreted and required for its virulence, dependent on the QS. Interestingly, ralfuranones affect the QS feedback loop. Taken together, integrated signaling via ralfuranones influences the QS, contributing to pathogen virulence.

Signal Transduction Mechanisms of Alcoholic Fatty Liver Disease: Emer ging Role of Lipin-1.

Lipin-1, a mammalian phosphatidic acid phosphatase (PAP), is a bi-functional molecule involved in various signaling pathways via its function as a PAP enzyme in the triglyceride synthesis pathway and in the nucleus as a transcriptional co-regulator. In the liver, lipin-1 is known to play a vital role in controlling the lipid metabolism and inflammation process at multiple regulatory levels. Alcoholic fatty liver disease (AFLD) is one of the earliest forms of liver injury and approximately 8-20% of patients with simple steatosis can develop into more severe forms of liver injury, including steatohepatitis, fibrosis/ cirrhosis, and eventually hepatocellular carcinoma (HCC). The signal transduction mechanisms for alcohol-induced detrimental effects in liver involves alteration of complex and multiple signaling pathways largely governed by a central and upstream signaling system, namely, sirtuin 1 (SIRT1)-AMP activated kinase (AMPK) axis. Emerging evidence suggests a pivotal role of lipin-1 as a crucial downstream regulator of SIRT1-AMPK signaling system that is likely to be ultimately responsible for development and progression of AFLD. Several lines of evidence demonstrate that ethanol exposure significantly induces lipin-1 gene and protein expression levels in cultured hepatocytes and in the livers of rodents, induces lipin-1-PAP activity, impairs the functional activity of nuclear lipin-1, disrupts lipin-1 mRNA alternative splicing and induces lipin-1 nucleocytoplasmic shuttling. Such impairment in response to ethanol leads to derangement of hepatic lipid metabolism, and excessive production of inflammatory cytokines in the livers of the rodents and human alcoholics. This review summarizes current knowledge about the role of lipin-1 in the pathogenesis of AFLD and its potential signal transduction mechanisms.

A survey of proteomic biomarkers for heterotopic ossification in blood serum

BackgroundHeterotopic ossification (HO) is a significant problem for wounded warriors surviving high-energy blast injuries; however, currently, there is no biomarker panel capable of globally characterizing, diagnosing, and monitoring HO progression. The aim of this study was to identify biomarkers for HO using proteomic techniques and blood serum.MethodsIsobaric tags for relative and absolute quantitation (iTRAQ) was used to generate a semi-quantitative global proteomics survey of serum from patients with and without heterotopic ossification. Leveraging the iTRAQ data, a targeted selection reaction monitoring mass spectrometry (SRM-MS) assay was developed for 10 protein candidates: alkaline phosphatase, osteocalcin, alpha-2 type I collagen, collagen alpha-1(V) chain isoform 2 preprotein, bone sialoprotein 2, phosphatidate phosphatase LPIN2, osteomodulin, protein phosphatase 1J, and RRP12-like protein.ResultsThe proteomic survey of serum from both healthy and disease patients includes 1220 proteins and was enriched for proteins involved in the response to elevated platelet Ca+2, wound healing, and extracellular matrix organization. Proteolytic peptides from three of the ten SRM-MS proteins, osteocalcin preprotein, osteomodulin precursor, and collagen alpha-1(v) chain isoform 2 preprotein from serum, are potential clinical biomarkers for HO.ConclusionsThis study is the first reported SRM-MS analysis of serum from individuals with and without heterotopic ossification, and differences in the serum proteomic profile between healthy and diseased subjects were identified. Furthermore, our results indicate that normal wound healing signals can impact the ability to identify biomarkers, and a multi-protein panel assay, including osteocalcin preproprotein, osteomodulin precursor, and collagen alpha-1(v) chain isoform 2 preprotein, may provide a solution for HO detection and monitoring.

Deleting the DAG kinase Dgk1 augments yeast vacuole fusion through increased Ypt7 activity and altered membrane fluidity.

Diacylglycerol (DAG) is a fusogenic lipid that can be produced through phospholipase C activity on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2 ], or through phosphatidic acid (PA) phosphatase activity. The fusion of Saccharomyces cerevisiae vacuoles requires DAG, PA and PI(4,5)P2 , and the production of these lipids is thought to provide temporally specific stoichiometries that are critical for each stage of fusion. Furthermore, DAG and PA can be interconverted by the DAG kinase Dgk1 and the PA phosphatase Pah1. Previously we found that pah1 Δ vacuoles were fragmented, blocked in SNARE priming and showed arrested endosomal maturation. In other pathways the effects of deleting PAH1 can be compensated for by additionally deleting DGK1 ; however, deleting both genes did not rescue the pah1 Δ vacuolar defects. Deleting DGK1 alone caused a marked increase in vacuole fusion that was attributed to elevated DAG levels. This was accompanied by a gain in resistance to the inhibitory effects of PA as well as inhibitors of Ypt7 activity. Together these data show that Dgk1 function can act as a negative regulator of vacuole fusion through the production of PA at the cost of depleting DAG and reducing Ypt7 activity.

Regulation of Phosphatidic Acid Phosphatase by High Glucose in the Oleaginous Yeast Yarrowia lipolytica

Phosphatidic acid phosphatase (PAP) catalyzes the conversion of phosphatidic acid to diacylglycerol, which is considered the committed step in triacylglycerol (TAG) biosynthesis. TAG biosynthesis in oleaginous microorganisms like Yarrowia lipolytica is typically triggered by high glucose amounts and results in cells accumulating lipids made mostly of TAGs. The objective of this work was to study the regulation of PAP during lipid accumulation induced by high glucose amounts. We hypothesized that the induction of TAG biosynthesis under these conditions should be mediated by PAP. In this work, we grew Y. lipolytica in a high glucose media (HGM) that favors lipid accumulation and in a low glucose media (LGM) where lipid accumulation is minimal. We measured PAP activity and lipid amounts throughout the cultivation period. Our results showed that, over time, the TAG amounts increased eight‐fold in the HGM cultures, while there was no increase in the LGM cultures. PAP activity was induced during TAG accumulation in HGM, which resulted in a two‐fold increase in activity. In contrast, PAP activity was down‐regulated in the LGM cultures, and that decrease coincided with the complete consumption of glucose in the medium. These results indicate that PAP plays a role in the regulation of lipid accumulation in Y. lipolytica.Support or Funding InformationThis project was supported by the Evans‐Allen Grant no.1005735 and Capacity BuildingGrants Program Grant no.1007503 from the USDA National Institute of Food and Agriculture.

Structure‐function Analysis of Yeast Pah1 Phosphatidate Phosphatase

Mg2+‐dependent phosphatidate phosphatase (PAP), encoded by PAH1 in Saccharomyces cerevisiae, catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol. The diacylglycerol produced in the reaction is utilized for the synthesis of triacylglycerol or the major membrane phospholipids phosphatidylcholine and phosphatidylethanolamine. The PAP enzyme from yeast contains two conserved domains; the NLIP domain is found at the N‐terminus and the haloacid dehalogenase (HAD)‐like domain is found in the middle region of Pah1. The G80R mutation in the NLIP domain of Pah1 causes a 98% reduction in PAP activity along with other phenotypes characteristic of the pah1Δ mutation. The HAD‐like domain of Pah1 contains the DXDX (T/V) motif that is essential for catalytic activity. In this study, we carried out a structure‐function analysis of the conserved and non‐conserved regions of yeast Pah1. For constructs expressed and purified from Escherichia coli, G80R and G80A mutations, as well as deletion of the NLIP domain caused > 99% loss of PAP activity; the deletion of the non‐conserved region at the N‐terminus had little effect on PAP activity; and the deletion of the non‐conserved region at the C‐terminus caused a 3‐fold increase in PAP activity. The effects of the mutations on the in vivo function of Pah1 were examined by the ability to complement cold or warm sensitivities exhibited by the pah1Δ mutant. Cells expressing the deletion of the non‐conserved region at the N‐terminus complemented both the cold and warm sensitive phenotypes of the pah1Δ mutant, whereas the mutations in the conserved NLIP domain and the deletion of the non‐conserved region at the C‐terminus did not rescue the temperature sensitive phenotypes. The effects of the mutations on Pah1 PAP functions such as the synthesis of triacylglycerol and lipid droplet formation are being investigated.Support or Funding InformationSupported by NIH grant GM028140

Phosphorylation of lipid metabolic enzymes by yeast protein kinase C requires phosphatidylserine and diacylglycerol

Protein kinase C in Saccharomyces cerevisiae, i.e., Pkc1, is an enzyme that plays an important role in signal transduction and the regulation of lipid metabolic enzymes. Pkc1 is structurally similar to its counterparts in higher eukaryotes, but its requirement of phosphatidylserine (PS) and diacylglycerol (DAG) for catalytic activity has been unclear. In this work, we examined the role of these lipids in Pkc1 activity with protein and peptide substrates. In agreement with previous findings, yeast Pkc1 did not require PS and DAG for its activity on the peptide substrates derived from lipid metabolic proteins such as Pah1 [phosphatidate (PA) phosphatase], Nem1 (PA phosphatase phosphatase), and Spo7 (protein phosphatase regulatory subunit). However, the lipids were required for Pkc1 activity on the protein substrates Pah1, Nem1, and Spo7. Compared with DAG, PS had a greater effect on Pkc1 activity, and its dose-dependent interaction with the protein kinase was shown by the liposome binding assay. The Pkc1-mediated degradation of Pah1 was attenuated in the cho1Δ mutant, which is deficient in PS synthase, supporting the notion that the phospholipid regulates Pkc1 activity in vivo.

LPIN1 promotes epithelial cell transformation and mammary tumourigenesis via enhancing insulin receptor substrate 1 stability

LPIN1, a phosphatidic acid phosphatase enzyme, dramatically influence tumor progression, however, their mechanism of action is still poorly understood. Here, we demonstrate that LPIN1 enhances the tumour‐promoting function of IRS1 by controlling IRS1 stability. LPIN1 interacts with IRS1 in an IGF‐1‐dependent signalling pathway and inhibits its serine phosphorylation, thereby eliminating ubiquitin‐dependent degradation of IRS1 via proteasomal and lysosomal pathways. Consequently, LPIN1 overexpression increases IRS1 abundance and enhances IRS1's ability to induce epithelial cell proliferation and mammary tumourigenesis. By contrast, depletion or inhibition of LPIN1 in breast cancer cells leads to a decreased IRS1 level, which subsequently inhibits the RAF1‐mediated signalling pathway as well as AP‐1 transcriptional activity. In the syngeneic 4T1 breast cancer model, LPIN1 overexpression increased tumour development. Consistent with these observations, LPIN1 levels were positively correlated with IRS1 expression in human breast cancer. Thus, our results indicate that LPIN1 may be a promising drug target for anticancer therapy.Support or Funding InformationThis research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI14C2004).

Yeast Pah1 Phosphatidate Phosphatase Regulates the Expression of Phosphatidylserine Synthase for Membrane Phospholipid Synthesis

Pah1 phosphatidate phosphatase (PAP), which catalyzes a committed step for the synthesis of triacylglycerol in Saccharomyces cerevisiae, exerts a negative regulatory effect on the level of PA required for de novo phospholipid synthesis. In this study, we examined the Pah1 PAP‐mediated regulation of phosphatidylserine (PS) synthase, which catalyzes a committed step for the synthesis of major membrane phospholipids. PS synthase activity was highly increased by the lack of Pah1 PAP with a growth‐dependent upregulation from the exponential to the stationary phase. Immunoblot analysis showed that the elevation of PS synthase activity results from an increase in the level of the enzyme encoded by CHO1. By promoter truncation analysis and site‐directed mutagenesis, the induced expression of CHO1 in the pah1Δ mutant was shown to occur through the inositol‐sensitive upstream activation sequence (UASINO, −163 ~ −154), a cis‐acting element for the PA‐controlled Ino2‐Ino4/Opi1 regulatory circuit. The abrogation of CHO1 induction by the UASINO mutation suppressed the pah1Δ phenotypes related to the increase of PA levels (e.g., increased phospholipid synthesis, nuclear/ER membrane expansion, and reduced lipid droplet formation) with its effect stronger in the exponential phase than in the stationary phase. These results demonstrate that Pah1 PAP regulates the expression of PS synthase through its control on the PA level.Support or Funding InformationSupported by NIH grant GM028140

Lipin-1 regulation of phospholipid synthesis maintains endoplasmic reticulum homeostasis and is critical for triple-negative breast cancer cell survival.

Cancer cells reprogram their metabolism to increase the synthesis of macromolecules for rapid proliferation. Compared to fatty acids, much less is known about the synthesis of phospholipids, which is essential for membrane biogenesis in cancer cells. We found that LPIN1, which encodes lipin-1, a phosphatidic acid phosphatase (PAP) controlling the rate-limiting step in the phospholipid synthesis pathway, is highly up-regulated in basal-like triple-negative breast cancer (TNBC). Moreover, high LPIN1 expression correlates with the poor prognosis of these patients. Knockdown of LPIN1 increases apoptosis in basal-like TNBC cell lines, whereas it has minimal or less effect on normal human mammary gland epithelial cells (HMECs) and estrogen receptor-positive breast cancer cell lines. Fatty acid incorporation and lipidomics analyses showed that LPIN1 knockdown blocks phospholipid synthesis and changes membrane lipid compositions that ultimately induce the activation of 1 of the 3 branches of unfolded protein responses, the inositol-requiring enzyme-1α pathway. We also show for the first time, to our knowledge, that lipin-1 knockdown significantly inhibits tumor growth in vivo using an orthotopic xenograft breast mouse model. Our results suggest that lipin-1 is a potential target for cancer therapy.-He, J., Zhang, F., Tay, L. W. R., Boroda, S., Nian, W., Levental, K. R., Levental, I., Harris, T. E., Chang, J. T., Du, G. Lipin-1 regulation of phospholipid synthesis maintains endoplasmic reticulum homeostasis and is critical for triple-negative breast cancer cell survival.

Bioinformatics analysis on the differentiation of bone mesenchymal stem cells into osteoblasts and adipocytes.

The present study aimed to screen several differentially expressed genes (DEGs) and differentially expressed microRNAs (miRNAs) for two types of mesenchymal stem cell (MSC) differentiation. Bone morphogenetic protein 6 (BMP-6) and dexamethasone were used to induce MSCs towards osteoblastic differentiation or adipocytic differentiation. The t-test in the Bioconductor bioinformatics software tool was used to screen DEGs and differentially expressed miRNAs in the two samples. Subsequent gene ontology (GO) and pathway analyses on the DEGs were performed using the GO and Kyoto Encyclopedia of Genes and Genomes databases, respectively; potential target genes for the screened miRNAs were predicted using the TargetScan database. In addition, an interaction network between the DEGs and miRNAs was constructed. Numerous DEGs and miRNAs were screened during osteoblastic and adipocytic differentiation of MSCs. Important pathways, such as glutathione metabolism, pathogenic Escherichia coli infection and Parkinson's disease, and GO terms, including cytoskeletal protein binding and phospholipase inhibitor activity, were enriched in the screened DEGs from MSCs undergoing osteogenic differentiation and adipocytic differentiation. miRNAs, including miRNA (miR)-382 and miR-203, and DEGs, including neuronal growth regulator 1 (NEGR1), phosphatidic acid phosphatase 2B (PPAP2B), platelet-derived growth factor receptor alpha (PDGFRA), interleukin 6 signal transducer (IL6ST) and sortilin 1 (SORT1), were demonstrated to be involved in osteoblastic differentiation. In addition, the downregulated miRNAs (including miR-495, miR-376a and miR-543), the upregulated miR-106a, the upregulated DEGs, including enabled homolog (ENAH), polypeptide N-acetylgalactosaminyltransferase 1 and acyl-CoA synthetase long-chain family member 1, and the downregulated repulsive guidance molecule family member B and semaphorin SEMA7A were demonstrated to be involved in adipocytic differentiation. The results of the present study suggested that miRNAs (miR-203 and miR-382) and DEGs (NEGR1, PPAP2B, PDGFRA, IL6ST and SORT1) may serve pivotal functions in the osteoblastic differentiation of MSCs, whereas miR-495, which is also involved in osteoblast differentiation and had four targets, including NEGR1, miR-376a, miR-543 and ENAH may have crucial roles in adipocytic differentiation of MSCs.

Crystal structure and biochemical characterization of the transmembrane PAP2 type phosphatidylglycerol phosphate phosphatase from Bacillus subtilis.

Type 2 phosphatidic acid phosphatases (PAP2s) can be either soluble or integral membrane enzymes. In bacteria, integral membrane PAP2s play major roles in the metabolisms of glycerophospholipids, undecaprenyl-phosphate (C55-P) lipid carrier and lipopolysaccharides. By in vivo functional experiments and biochemical characterization we show that the membrane PAP2 coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate (PGP) phosphatase of B. subtilis. We also confirm that this enzyme, renamed bsPgpB, has a weaker activity on C55-PP. Moreover, we solved the crystal structure of bsPgpB at 2.25Å resolution, with tungstate (a phosphate analog) in the active site. The structure reveals two lipid chains in the active site vicinity, allowing for PGP substrate modeling and molecular dynamic simulation. Site-directed mutagenesis confirmed the residues important for substrate specificity, providing a basis for predicting the lipids preferentially dephosphorylated by membrane PAP2s.