Dihydroxyacetone Phosphate Pathway Research Articles

Targeting the plasma membrane of neoplastic cells through alkylation: a novel approach to cancer chemotherapy

SummaryBackground Although DNA-directed alkylating agents and related compounds have been a mainstay in chemotherapeutic protocols due to their ability to readily interfere with the rapid mitotic progression of malignant cells, their clinical utility is limited by DNA repair mechanisms and immunosuppression. However, the same destructive nature of alkylation can be reciprocated at the cell surface using novel plasma membrane alkylating agents. Results Plasma membrane alkylating agents have elicited long term survival in mammalian models challenged with carcinomas, sarcomas, and leukemias. Further, a specialized group of plasma membrane alkylating agents known as tetra-O-acetate haloacetamido carbohydrate analogs (Tet-OAHCs) potentiates a substantial leukocyte influx at the administration and primary tumor site, indicative of a potent immune response. The effects of plasma membrane alkylating agents may be further potentiated through the use of another novel class of chemotherapeutic agents, known as dihydroxyacetone phosphate (DHAP) inhibitors, since many cancer types are known to rely on the DHAP pathway for lipid synthesis. Conclusion Despite these compelling data, preliminary clinical trials for plasma membrane-directed agents have yet to be considered. Therefore, this review is intended for academics and clinicians to postulate a novel approach of chemotherapy; altering critical malignant cell signaling at the plasma membrane surface through alkylation, thereby inducing irreversible changes to functions needed for cell survival.

1-Acyldihydroxyacetone-phosphate Reductase (Ayr1p) of the YeastSaccharomyces cerevisiae Encoded by the Open Reading Frame YIL124w Is a Major Component of Lipid Particles

Biosynthesis of phosphatidic acid through the dihydroxyacetone phosphate pathway requires NADPH-dependent reduction of the intermediate 1-acyldihydroxyacetone phosphate before the second step of acylation. Studies with isolated subcellular fractions of the yeast Saccharomyces cerevisiae revealed that lipid particles and the endoplasmic reticulum harbor 1-acyldihydroxyacetone-phosphate reductase (ADR) activity. Deletion of the open reading frame YIL124w (in the following named AYR1) abolished reduction of 1-acyldihydroxyacetone phosphate in lipid particles, whereas ADR activity in microsomes of the deletion strain was decreased approximately 3-fold as compared with the wild-type level. This result indicates that (i) both lipid particles and microsomes harbor Ayr1p, which was confirmed by immunological detection of the protein in these two cellular compartments, and (ii) microsomes contain at least one additional ADR activity. As a consequence of this redundancy, deletion of AYR1 neither results in an obvious growth phenotype nor affects the lipid composition of a haploid deletion strain. When a heterozygous AYR1(+)/ayr1(-) diploid strain was subjected to sporulation; however, spores bearing the ayr1 defect failed to germinate, suggesting that Ayr1p plays an essential role at this stage. Overexpression of Ayr1p at a 5- to 10-fold level of wild type caused growth arrest. Heterologous expression of Ayr1p in Escherichia coli resulted in gain of ADR activity in the prokaryote, confirming that YIL124w is the structural gene of the enzyme and does not encode a regulatory or auxiliary component required for reduction of 1-acyldihydroxyacetone phosphate. Taken together, these results identified Ayr1p of the yeast as the first ADR from any organism at the molecular level.

Redundant systems of phosphatidic acid biosynthesis via acylation of glycerol-3-phosphate or dihydroxyacetone phosphate in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae lipid particles harbor two acyltransferases, Gat1p and Slc1p, which catalyze subsequent steps of acylation required for the formation of phosphatidic acid. Both enzymes are also components of the endoplasmic reticulum, but this compartment contains additional acyltransferase(s) involved in the biosynthesis of phosphatidic acid (K. Athenstaedt and G. Daum, J. Bacteriol. 179:7611-7616, 1997). Using the gat1 mutant strain TTA1, we show here that Gat1p present in both subcellular fractions accepts glycerol-3-phosphate and dihydroxyacetone phosphate as a substrate. Similarly, the additional acyltransferase(s) present in the endoplasmic reticulum can acylate both precursors. In contrast, yeast mitochondria harbor an enzyme(s) that significantly prefers dihydroxyacetone phosphate as a substrate for acylation, suggesting that at least one additional independent acyltransferase is present in this organelle. Surprisingly, enzymatic activity of 1-acyldihydroxyacetone phosphate reductase, which is required for the conversion of 1-acyldihydroxyacetone phosphate to 1-acylglycerol-3-phosphate (lysophosphatidic acid), is detectable only in lipid particles and the endoplasmic reticulum and not in mitochondria. In vivo labeling of wild-type cells with [2-3H, U-14C]glycerol revealed that both glycerol-3-phosphate and dihydroxyacetone phosphate can be incorporated as a backbone of glycerolipids. In the gat1 mutant and the 1-acylglycerol-3-phosphate acyltransferase slc1 mutant, the dihydroxyacetone phosphate pathway of phosphatidic acid biosynthesis is slightly preferred as compared to the wild type. Thus, mutations of the major acyltransferases Gat1p and Slc1p lead to an increased contribution of mitochondrial acyltransferase(s) to glycerolipid synthesis due to their substrate preference for dihydroxyacetone phosphate.

The dihydroxyacetonephosphate pathway for biosynthesis of ether lipids in Leishmania mexicana promastigotes

Biosynthetic studies using both [ 14C]- and [ 32P]-labelled substrates and a cell-free system to synthesise 1- O-alkyl moieties in glycerolipids, have shown that the three initial steps in ether-lipid biosynthesis in Leishmania mexicana promastigotes resemble those described for mammals and are associated with glycosomes. Purified glycosomes were able to sequentially synthesise the first intermediates of the ether-lipid biosynthetic pathway [acyl-dihydroxyacetonephosphate (DHAP), alkyl-DHAP and acyl/alkyl-glycerol-3-phosphate (G3P)] when incubated in the presence of radiolabelled DHAP, palmitoyl-CoA, hexadecanol and NADPH. However, when glycosomes were incubated under the same conditions in the presence of radiolabelled G3P, a rapid synthesis of acyl-G3P and phosphatidic acid was observed without any formation of alkyl-G3P, suggesting that the enzyme alkyl-synthase recognises only acyl-DHAP as substrate. Both the DHAP acyltransferase (DHAP-AT) and alkyl-DHAP synthase activities were located inside glycosomes whereas the alkyl/acyl-DHAP oxidoreductase activity was associated with the cytoplasmic face of the glycosomal membrane. The G3P acyltransferase (G3P-AT) and lyso-phosphatidic acid acyltransferase activities were not found inside glycosomes. The results suggest that the DHAP-AT and G3P-AT activities are catalysed by two distinct enzymes associated with different sub-cellular compartments.

Ethanol stimulates lipid biosynthesis in the rat reticulocyte by activating glycerol kinase

In the liver, the ability of ethanol to stimulate glycerolipid biosynthesis has been attributed mainly to its oxidation by alcohol dehydrogenase and to the subsequent enhanced production of glycerol 3-phosphate via the dihydroxyacetone phosphate pathway. To explore alternative pathways, rat reticulocytes, which are devoid of alcohol deshydrogenase, have been exposed to ethanol and their glycerolipid metabolism has been investigated using glycerol as a lipid precursor. The results indicate that short-term (< 30 min) cell exposure to ethanol (50–500 mM) stimulated the incorporation of [ 14C]glycerol into glycerol 3-phosphate, and thereby enhanced glycerolipid biosynthesis. The involvment of glycerol kinase in the stimulatory effect was confirmed by the stimulation by ethanol of glycerol kinase activity directly assayed in reticulocyte lysates. This ethanol effect may be important under physiological or pathophysiological conditions associated with increased glycerol to glucose ratio in blood.

Exclusive localization in peroxisomes of dihydroxyacetone phosphate acyltransferase and alkyl-dihydroxyacetone phosphate synthase in rat liver.

Dihydroxyacetone phosphate acyl transferase (DHAP-AT), alkyl dihydroxyacetone phosphate synthase (alkyl-DHAP-synthase), and glycerol-3-phosphate acyltransferase (GPAT) activities were investigated under optimal assay conditions using highly purified organelle preparations. The data presented clearly indicate that GPAT activity was mainly localized in mitochondria and microsomes, whereas DHAP-AT and alkyl-DHAP-synthase activities were exclusively localized in peroxisomes. A small fraction of the total DHAP-AT and alkyl-DHAP-synthase activities observed in purified mitochondrial preparations was due to the presence of intact peroxisomes. DHAP-AT and alkyl-DHAP-synthase activities were very low in purified microsomes (< 1% compared to peroxisomes) and these activities are thought to be due to sedimentation of peroxisomal fragments (generated during homogenization of liver and processing of liver homogenate) with microsomes. The results indicate that the dihydroxyacetone phosphate pathway does not contribute to the synthesis of glycerolipids other than ether lipids in rat liver. The ether bond formation occurs exclusively in peroxisomes, and all the biosynthetic reactions for plasmalogen synthesis may also be operating within peroxisomes in rat liver.

The acyl dihydroxyacetone phosphate pathway enzymes for glycerolipid biosynthesis are present in the yeast Saccharomyces cerevisiae

The presence of the acyl dihydroxyacetone phosphate (acyl DHAP) pathway in yeasts was investigated by examining three key enzyme activities of this pathway in Saccharomyces cerevisiae. In the total membrane fraction of S. cerevisiae, we confirmed the presence of both DHAP acyltransferase (DHAPAT; Km = 1.27 mM; Vmax = 5.9 nmol/min/mg of protein) and sn-glycerol 3-phosphate acyltransferase (GPAT; Km = 0.28 mM; Vmax = 12.6 nmol/min/mg of protein). The properties of these two acyltransferases are similar with respect to thermal stability and optimum temperature of activity but differ with respect to pH optimum (6.5 for GPAT and 7.4 for DHAPAT) and sensitivity toward the sulfhydryl blocking agent N-ethylmaleimide. Total membrane fraction of S. cerevisiae also exhibited acyl/alkyl DHAP reductase (EC 1.1.1.101) activity, which has not been reported previously. The reductase has a Vmax of 3.8 nmol/min/mg of protein for the reduction of hexadecyl DHAP (Km = 15 microM) by NADPH (Km = 20 microM). Both acyl DHAP and alkyl DHAP acted as substrates. NADPH was the specific cofactor. Divalent cations and N-ethylmaleimide inhibited the enzymatic reaction. Reductase activity in the total membrane fraction from aerobically grown yeast cells was twice that from anaerobically grown cells. Similarly, DHAPAT and GPAT activities were also greater in aerobically grown yeast cells. The presence of these enzymes, together with the absence of both ether glycerolipids and the ether lipid-synthesizing enzyme (alkyl DHAP synthase) in S. cerevisiae, indicates that non-ether glycerolipids are synthesized in this organism via the acyl DHAP pathway.

Glycerol as a substrate for phospholipid biosynthesis in type II pneumocytes isolated from streptozotocin-diabetic rats

Glycerol and glucose utilization for phospholipid biosynthesis was examined in type II pneumocytes isolated from normal and streptozotocin-diabetic rats. In cells from diabetic rats, incorporation of [1,3- 14C]glycerol into total phosphatidylcholine (PC), disaturated phosphatidylcholine (DSPC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) occurred to a greater degree by the glycerol 3-phosphate pathway as opposed to the dihydroxyacetone phosphate pathway. Total incorporation of glycerol into each of the major cellular phospholipids was increased up to 6-fold in cells from diabetic rats, while the total incorporation of glucose into the same lipids was decreased 2-fold. While the percentage of both glucose and glycerol carbons incorporated into the backbone of DSPC was increased in cells from diabetic rats, the percentage of carbons from both substrates incorporated into the fatty acid moieties was decreased. As a measure of DSPC synthesis, choline incorporation into DSPC was significantly decreased in type II cells from diabetic animals if the cells were incubated in the presence of glucose, palmitate and choline but not glycerol. Addition of 0.1 or 0.3 mM glycerol to the incubation medium restored choline incorporation to the control value in cells from diabetic rats, but did not affect the rate of choline incorporation into DSPC in cells from normal rats. These results suggest that exogenous glycerol can compensate for reduced glucose metabolism in type II cells of diabetic animals to maintain a constant rate of DSPC synthesis.

In vivo incorporation of [U-14C]glycerol and [2-3H]glycerol into phospholipids of brain subcellular fractions in normal and malnourished animals

A double label design was used to study the in vivo incorporation of [U-(14)C] and [2-(3)H]glycerol into total and individual phospholipids of various brain subcellular fractions isolated from 20-day old normal and undernourished rats. In control animals, synthesis of glycerophospholipids of microsomes, mitochondria and nerve endings seems to occur through the glycerol-3-phosphate (G-3-P) pathway while a large part of the synthesis of myelin glycerophospholipids appears to proceed through the dihydroxyacetone phosphate (DHAP) pathway. In starved animals, on the other hand the incorporation of phospholipid precursors through the DHAP pathway was found to be lower than in controls while synthesis of phospholipids in the other subcellular fractions was unaffected. The possible relationship between the synthesis of glycerophospholipids and especially plasmalogens of the myelin membrane and microperoxisomes of oligodendroglial cells, where the enzymes of the DHAP pathway are located, is discussed.

Rat liver dihydroxyacetone-phosphate acyltransferases and their contribution to glycerolipid synthesis.

Differential and isopycnic centrifugation of rat liver homogenates showed that, besides its established localization in peroxisomes and endoplasmic reticulum, dihydroxyacetone-phosphate acyltransferase is also present in mitochondria. The three activities differed in a number of properties (pH optimum, palmitoyl-CoA and dihydroxyacetone-phosphate dependence, and sensitivity toward N-ethylmaleimide) and are therefore likely associated with three distinct proteins. Glycerol 3-phosphate (5 mM) did not inhibit peroxisomal dihydroxyacetone-phosphate acyltransferase but inhibited the extraperoxisomal activities virtually completely. Peroxisomal dihydroxyacetone-phosphate acyltransferase was located at the inner aspect of the peroxisomal membrane, but the enzyme was not latent. Purified microsomes, from which intact peroxisomes had been removed, were still contaminated with peroxisomal membranes as deduced from the presence of two dihydroxyacetone-phosphate acyltransferase activities: a glycerol 3-phosphate-resistant activity with properties similar to those of peroxisomal dihydroxyacetone-phosphate acyltransferase and a glycerol 3-phosphate-sensitive "true" microsomal dihydroxyacetone-phosphate acyltransferase. We propose that, assayed in the presence of 5mM glycerol 3-phosphate, dihydroxyacetone-phosphate acyltransferase can be used as a marker enzyme for peroxisomal membranes. Such a marker enzyme has not hitherto been available. The differential effect of 5 mM glycerol 3-phosphate on peroxisomal and extraperoxisomal dihydroxyacetone-phosphate acyltransferases enabled us to determine the relative contribution of these activities to overall dihydroxyacetone-phosphate acylation in whole liver homogenates. At near-physiological pH and at near-physiological concentrations of unbound palmitoyl-CoA and of dihydroxyacetone-phosphate plus glycerol 3-phosphate, peroxisomes contributed 50-75%. The remaining percentage was mostly accounted for by the microsomal enzyme. At near-physiological concentrations of glycerol 3-phosphate plus dihydroxyacetone-phosphate, glycerolphosphate acyltransferase contributed 93% and dihydroxyacetone-phosphate acyltransferase 7% to overall glycerolipid synthesis in homogenates. This suggests that the dihydroxyacetone-phosphate pathway is of minor quantitative importance in overall hepatic glycerolipid synthesis but that its main function lies in the synthesis of ether lipids, which have acyldihydroxyacetone-phosphate as obligatory precursor.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of inositol starvation on glycerolipid metabolism in Saccharomyces uvarum

The influence of inositol deprivation on glycerolipid metabolism in the inositol-requiring yeast, Saccharomyces uvarum, was determined by measuring the decrease in radioactivity of the major phospholipids after prelabeling cells with either [2- 3H]glycerol, [1- 14C]palmitic acid, [methyl- 3H]choline or [ 32P]orthophosphate. In another set of experiments incorporation of exogenously supplied [ 3H]oleic acid and [2- 3H]glycerol into cellular lipids was studied. Comparison of data obtained with inositol-deficient cultures and inositol-supplemented controls indicate that inositol starvation leads to elevated rates of phospholipid synthesis and turnover. During incorporation of [2- 3H, U- 14C]glycerol into glycerolipids 3H is retained, suggesting that the dihydroxyacetonephosphate pathway of glycerolipid synthesis is absent in S. uvarum. The hypothesis that triacylglycerols accumulating in inositol-starved cells might (at least in part) be synthesized from diacylglycerols formed during increased phospholipid breakdown could not be verified, since [2- 3H]glycerol was not transferred from phospholipids to triacylglycerols. Essentially all the [2- 3H]glycerol lost from phospholipids during growth of prelabeled cells was excreted into the culture medium in the form of water-soluble deacylation products (mainly glycerophosphoinositol and glycerophosphocholine). Fatty acids set free during degradation of phospholipids in inositol-deficient cells are shifted to triacylglycerols.

Importance of the acyl dihydroxyacetone phosphate pathway in the synthesis of phosphatidylglycerol and phosphatidylcholine in alveolar type II cells.

Importance of the acyl dihydroxyacetone phosphate pathway in the synthesis of phosphatidylglycerol and phosphatidylcholine in alveolar type II cells.

Biosynthesis of dipalmitoyl- sn-glycero-3-phosphocholine by adenoma alveolar type II cells

Adenomas induced in lungs of BALB/c mice by urethane treatment consist almost exclusively of pulmonary type II cells and, like normal type II cells, produce dipalmitoylphosphatidylcholine, the major lipid component of lung surfactant [Snyder, C., Malone, B., Nettesheim, P., and Snyder, F. (1973) Cancer Res. 33, 2437–2443]. In the present investigation, these adenomas were used for studying the biosynthesis of dipalmitoylphosphatidylcholine in the intact cells. Whole adenomas were incubated in Krebs-Ringer phosphate buffer or Medium 199 with [1- 14C]palmitate, [1- 14C]acetate, [U- 14Clglycerol, or [1,3- 14C,2- 3H]glycerol and the synthesis of [ 14C]dipalmitoylphosphatidylcholine was subsequently determined after its isolation by cryochromatography. The [1- 14C]acetate was converted to [ 14C]palmitate, which was incorporated into disaturated phosphatidylcholine in the same manner as exogenous [1- 14C]palmitate. After a 1-h incubation of either [1- 14C]palmitate or [1- 14C]acetate, approximately 60% of the [ 14C]phosphatidylcholine was disaturated while 75–80% of the label in the disaturated species was in the acyl group at the 2 position. The same results were obtained when minced lung was used instead of adenomas, except the incorporation rate was higher in the adenomas. It was concluded from these studies that palmitate, whether synthesized de novo or provided exogenously, enters a common pool before being used by the type II cells for the synthesis of dipalmitoylphosphatidylcholine. When [1,3- 14C,2- 3H]glycerol was used to label phosphatidylcholine in the system, all molecular species, based on the degree of unsaturation, retained the same 3 H 14 C ratio. This indicated that the dihydroxyacetone phosphate pathway is of no more significance for the synthesis of disaturated phosphatidylcholine than for the other molecular species. The study demonstrates the usefulness of the adenoma system as a model for studying surfactant biosynthesis in pulmonary type II cells and indicates that the major pathway by which palmitate enters dipalmitoylphosphatidylcholine is by the deacylation and reacylation of phosphatidylcholine.

Incorporation of D-[3-3H, U-14C] glucose into glycerolipid via acyl dihydroxyacetone phosphate untransformed and viral-transformed BHK-21-c13 fibroblasts.

Untransformed BHK-21-c13 fibroblasts as well as 4 polyoma-transformed strains were incubated with D-[U-14C,3-3H]glucose. This substrate generates intracellular labeled glycerol, and also [4-3H]NADPH via the phosphogluconate oxidative pathway. The latter selectively transfers hydrogen to C-2 of glycerol in glycerolipid via the acyl dihydroxyacetone phosphate pathway. After incubation, the distribution of radioactivity and the ratios of 3H/14C at the three positions of recovered glycerol were determined in sn-glycerol 3-phosphate, saponifiable glycerolipids, alkyl ether glycerolipids, and plasmalogens. In each of the cell types examined, 3H in the sn-1 position of glycerol in the recovered ether-containing glycerolipids was negligible, yet this position contained most of the recovered 3H in sn-glycerol 3-phosphate and saponifiable glycerolipids. The 3H/14C ratio in position 2 of glycerol, measured at various incubation times, was from 5- to 200-fold greater in the saponifiable glycerolipids than in free sn-glycerol 3-phosphate. The ratio in position 2 of ether-containing glycerolipids was the same or greater than that in the saponifiable glycerolipids in all of the cell types employed. A similar pattern in the 3H/14C ratio was observed when BHK-21-c13 cells were incubated with D-[U-14C,1-3H]glucose. These observations demonstrate significant participation of the acyl dihydroxyacetone phosphate pathway in glycerolipid synthesis in BHK cells.

Characteristics of mitochondrial and microsonal monoacyl- and diacylglycerol 3-phosphate biosynthesis in rabbit heart.

Acylation of sn-glycerol 3-phosphate by heart subcellular fractions was characterized. The enzyme kinetics revealed that the rate of reaction of acylation by mitochondria was slower, but constant for a longer period (up to 20min), than that by the microsomal fraction. The range of palmitate, oleate and linoleate concentrations yielding optimal sn-glycerol 3-phosphate acylation was broader for mitochondria than for the microsomal fraction, the latter showing a preference for linoleate. The mitochondrial fraction synthesized a relatively large quantity of monoacyl-sn-glycerol 3-phosphate, reaching 135% of the microsomal biosynthesis during an assay period of 15min. By contrast, the microsomal fraction formed considerably more diacyl- than monoacyl-sn-glycerol 3-phosphate, except with linoleate as the acyl donor, in which case approximately equal quantities of the two products were produced. The biosynthesis of monoacyl-sn-glycerol 3-phosphate was also observed in experiments in which hepatic subcellular fractions were used to provide supporting evidence. Cardiac mitochondrial diacyl-sn-glycerol 3-phosphate formation was less than 17% of the microsomal formation. However, evidence is presented to exclude the possibility that monoacyl-sn-glycerol 3-phosphate in the mitochondrial fraction is formed by deacylation of the contaminating microsomal diacyl-sn-glycerol 3-phosphate. The participation of the dihydroxyacetone phosphate pathway in the biosynthesis of these substances was minimal. The addition of CTP and the fatty acid specificity of the reaction both provided results that reinforced the postulate that mitochondrial differs from microsomal acylation. Thus our findings demonstrate that the characteristics of acyl-CoA-sn-glycerol 3-phosphate O-acyltransferase (EC 2.3.1.15) in rabbit heart mitochondria are distinct from those of cardiac microsomal enzyme and hepatic enzymes.

The acyl dihydroxyacetone phosphate pathway for glycerolipid biosynthesis in mouse liver and Ehrlich ascites tumor cells.

The glycerol portion of lipids may be derived biosynthetically by reduction of dihydroxyacetone phosphate to glycerolphosphate, and then be acylated with fatty acids or, alternatively, dihydroxyacetone phosphate may first be acylated and then reduced to 1-acyl sn glcerol-3-phosphate. Since the former pathway utilizes NADH for reduction of the C-2 carbonyl, while the latter requires NADPH, we were able to compare the relative participation of the two pathways for phospholipid synthesis by measuring the incorporation of radioactivity from tritiumlabeled NADH and NADPH into C-2 of lipid glycerol. The acyl-dihydroxyacetone phosphate pathway plays a significant role in glycerolipid synthesis in mouse liver homogenates and a clearly dominant one in Ehrlich ascites tumor cell homogenates. This finding is related to a reported lack of glycerol-3-phosphate dehydrogenase in tumor cells and to their high glycerol ether lipid content.

A test for the dihydroxyacetone phosphate pathway

A test for the dihydroxyacetone phosphate pathway