Glucose-derived acetate and ACSS2 as key players in cisplatin resistance in bladder cancer.

Cisplatin is an important chemotherapeutic agent against metastatic bladder cancer, but resistance often limits its usage. With the recent recognition of lipid metabolic alterations in bladder cancers, we studied the metabolic implications of cisplatin resistance using cisplatin-sensitive (T24S) and resistant (T24R) bladder cancer cells. Real-time live metabolomics revealed that T24R cells consume more glucose, leading to higher production of glucose-derived acetate and fatty acids. Along with the activation of general metabolic regulators, enzymes involved in acetate usage (ACSS2) and fatty acid synthesis (ACC) and a precursor for fatty acid synthesis (acetyl-CoA) were elevated in T24R cells. Consistently, metabolic analysis with 13C isotope revealed that T24R cells preferred glucose to acetate as the exogenous carbon source for the increased fatty acid synthesis, contrary to T24S cells. In addition, ACSS2, rather than the well-established ACLY, was the key enzyme that supplies acetyl-CoA in T24R cells through glucose-derived endogenous acetate. The relevance of ACSS2 in cisplatin resistance was further confirmed by the abrogation of resistance by an ACSS2 inhibitor and, finally, by the higher expression of ACSS2 in the patient tissues with cisplatin resistance. Our results may help improve the treatment options for chemoresistant bladder cancer patients and provide possible vulnerability targets to overcome the resistance.

Roles of acetyl-CoA synthetase (ADP-forming) and acetate kinase (PPi-forming) in ATP and PPi supply in Entamoeba histolytica.

BACKGROUND: Acetate is an end-product of the PPi-dependent fermentative glycolysis in Entamoeba histolytica; it is synthesized from acetyl-CoA by ADP-forming acetyl-CoA synthetase (ACS) with net ATP synthesis or from acetyl-phosphate by a unique PPi-forming acetate kinase (AcK). The relevance of these enzymes to the parasite ATP and PPi supply, respectively, are analyzed here. METHODS: The recombinant enzymes were kinetically characterized and their physiological roles were analyzed by transcriptional gene silencing and further metabolic analyses in amoebae. RESULTS: Recombinant ACS showed higher catalytic efficiencies (Vmax/Km) for acetate formation than for acetyl-CoA formation and high acetyl-CoA levels were found in trophozoites. Gradual ACS gene silencing (49-93%) significantly decreased the acetate flux without affecting the levels of glycolytic metabolites and ATP in trophozoites. However, amoebae lacking ACS activity were unable to reestablish the acetyl-CoA/CoA ratio after an oxidative stress challenge. Recombinant AcK showed activity only in the acetate formation direction; however, its substrate acetyl-phosphate was undetected in axenic parasites. AcK gene silencing did not affect acetate production in the parasites but promoted a slight decrease (10-20%) in the hexose phosphates and PPi levels. CONCLUSIONS: These results indicated that the main role of ACS in the parasite energy metabolism is not ATP production but to recycle CoA for glycolysis to proceed under aerobic conditions. AcK does not contribute to acetate production but might be marginally involved in PPi and hexosephosphate homeostasis. SIGNIFICANCE: The previous, long-standing hypothesis that these enzymes importantly contribute to ATP and PPi supply in amoebae can now be ruled out.

Acetate as a Metabolic and Epigenetic Modifier of Cancer Therapy.

Metabolic networks are significantly altered in neoplastic cells. This altered metabolic program leads to increased glycolysis and lipogenesis and decreased dependence on oxidative phosphorylation and oxygen consumption. Despite their limited mitochondrial respiration, cancer cells, nonetheless, derive sufficient energy from alternative carbon sources and metabolic pathways to maintain cell proliferation. They do so, in part, by utilizing fatty acids, amino acids, ketone bodies, and acetate, in addition to glucose. The alternative pathways used in the metabolism of these carbon sources provide opportunities for therapeutic manipulation. Acetate, in particular, has garnered increased attention in the context of cancer as both an epigenetic regulator of posttranslational protein modification, and as a carbon source for cancer cell biomass accumulation. However, to date, the data have not provided a clear understanding of the precise roles that protein acetylation and acetate oxidation play in carcinogenesis, cancer progression or treatment. This review highlights some of the major issues, discrepancies, and opportunities associated with the manipulation of acetate metabolism and acetylation-based signaling in cancer development and treatment.

Acetyl-coenzyme A synthetase 2 is a nuclear protein required for replicative longevity in Saccharomyces cerevisiae.

Acs2p is one of two acetyl-coenzyme A synthetases in Saccharomyces cerevisiae. We have prepared and characterized a monoclonal antibody specific for Acs2p and find that Acs2p is localized primarily to the nucleus, including the nucleolus, with a minor amount in the cytosol. We find that Acs2p is required for replicative longevity: an acs2 Delta strain has a reduced replicative life span compared to wild-type and acs1 Delta strains. Furthermore, replicatively aged acs2 Delta cells contain elevated levels of extrachromosomal rDNA circles, and silencing at the rDNA locus is impaired in an acs2 Delta strain. These findings indicate that Acs2p-mediated synthesis of acetyl-CoA in the nucleus functions to promote rDNA silencing and replicative longevity in yeast.

Acetate produced in the mitochondrion is the essential precursor for lipid biosynthesis in procyclic trypanosomes.

Acetyl-CoA produced in mitochondria from carbohydrate or amino acid catabolism needs to reach the cytosol to initiate de novo synthesis of fatty acids. All eukaryotes analyzed so far use a citrate/malate shuttle to transfer acetyl group equivalents from the mitochondrial matrix to the cytosol. Here we investigate how this acetyl group transfer occurs in the procyclic life cycle stage of Trypanosoma brucei, a protozoan parasite responsible of human sleeping sickness and economically important livestock diseases. Deletion of the potential citrate lyase gene, a critical cytosolic enzyme of the citrate/malate shuttle, has no effect on de novo biosynthesis of fatty acids from (14)C-labeled glucose, indicating that another route is used for acetyl group transfer. Because acetate is produced from acetyl-CoA in the mitochondrion of this parasite, we considered genes encoding cytosolic enzymes producing acetyl-CoA from acetate. We identified an acetyl-CoA synthetase gene encoding a cytosolic enzyme (AceCS), which is essential for cell viability. Repression of AceCS by inducible RNAi results in a 20-fold reduction of (14)C-incorporation from radiolabeled glucose or acetate into de novo synthesized fatty acids. Thus, we demonstrate that the essential cytosolic enzyme AceCS of T. brucei is responsible for activation of acetate into acetyl-CoA to feed de novo biosynthesis of lipids. To date, Trypanosoma is the only known eukaryotic organism that uses acetate instead of citrate to transfer acetyl groups over the mitochondrial membrane for cytosolic lipid synthesis.

Fasting-induced hypothermia and reduced energy production in mice lacking acetyl-CoA synthetase 2.

Acetate is activated to acetyl-CoA by acetyl-CoA synthetase 2 (AceCS2), a mitochondrial enzyme. Here, we report that the activation of acetate by AceCS2 has a specific and unique role in thermogenesis during fasting. In the skeletal muscle of fasted AceCS2(-/-) mice, ATP levels were reduced by 50% compared to AceCS2(+/+) mice. Fasted AceCS2(-/-) mice were significantly hypothermic and had reduced exercise capacity. Furthermore, when fed a low-carbohydrate diet, 4-week-old weaned AceCS2(-/-) mice also exhibited hypothermia accompanied by sustained hypoglycemia that led to a 50% mortality. Therefore, AceCS2 plays a significant role in acetate oxidation needed to generate ATP and heat. Furthermore, AceCS2(-/-) mice exhibited increased oxygen consumption and reduced weight gain on a low-carbohydrate diet. Our findings demonstrate that activation of acetate by AceCS2 plays a pivotal role in thermogenesis, especially under low-glucose or ketogenic conditions, and is crucially required for survival.

The role of acetyl-coenzyme a synthetase in Arabidopsis.

The acs1 knockout mutant that has a disruption in the plastidic acetyl-coenzyme A (CoA) synthetase (ACS; At5g36880) gene was used to explore the role of this protein and plastidic acetate metabolism in Arabidopsis (Arabidopsis thaliana). Disruption of the ACS gene decreased ACS activity by 90% and largely blocked the incorporation of exogenous (14)C-acetate and (14)C-ethanol into fatty acids. Whereas the disruption had no significant effect on the synthesis of bulk seed triacylglycerols, the acs1 plants were smaller and flowered later. This suggests that the pyruvate dehydrogenase bypass provided by the aerobic fermentation pathway that converts pyruvate to acetate and probably on to fatty acids is important to the plants during normal growth. The role of ACS in destroying fermentative intermediates is supported by the increased sensitivity of the acs1 mutant to exogenous acetate, ethanol, and acetaldehyde compared to wild-type plants. Whereas these observations suggest that flux through the aerobic fermentation pathway is important, the reason for this flux is unclear. Interestingly, acetate is able to support high rates of plant growth on medium and this growth is blocked in the acs1 mutant.

Discovery of amide (peptide) bond synthetic activity in Acyl-CoA synthetase.

Acyl-CoA synthetase, which is one of the acid-thiol ligases (EC 6.2.1), plays key roles in metabolic and regulatory processes. This enzyme forms a carbon-sulfur bond in the presence of ATP and Mg(2+), yielding acyl-CoA thioesters from the corresponding free acids and CoA. This enzyme belongs to the superfamily of adenylate-forming enzymes, whose three-dimensional structures are analogous to one another. We here discovered a new reaction while studying the short-chain acyl-CoA synthetase that we recently reported (Hashimoto, Y., Hosaka, H., Oinuma, K., Goda, M., Higashibata, H., and Kobayashi, M. (2005) J. Biol. Chem. 280, 8660-8667). When l-cysteine was used as a substrate instead of CoA, N-acyl-l-cysteine was surprisingly detected as a reaction product. This finding demonstrated that the enzyme formed a carbon-nitrogen bond (EC 6.3.1 acid-ammonia (or amide) ligase (amide synthase); EC 6.3.2 acid-amino acid ligase (peptide synthase)) comprising the amino group of the cysteine and the carboxyl group of the acid. N-Acyl-d-cysteine, N-acyl-dl-homocysteine, and N-acyl-l-cysteine methyl ester were also synthesized from the corresponding cysteine analog substrates by the enzyme. Furthermore, this unexpected enzyme activity was also observed for acetyl-CoA synthetase and firefly luciferase, indicating the generality of the new reaction in the superfamily of adenylate-forming enzymes.

Nucleocytosolic acetyl-coenzyme a synthetase is required for histone acetylation and global transcription.

Metabolic enzymes rarely regulate informational processes like gene expression. Yeast acetyl-CoA synthetases (Acs1p and 2p) are exceptional, as they are important not only for carbon metabolism but also are shown here to supply the acetyl-CoA for histone acetylation by histone acetyltransferases (HATs). acs2-Ts mutants exhibit global histone deacetylation, transcriptional defects, and synthetic growth defects with HAT mutants at high temperatures. In glycerol with ethanol, Acs1p is an alternate acetyl-CoA source for HATs. Rapid deacetylation after Acs2p inactivation suggests nuclear acetyl-CoA synthesis is rate limiting for histone acetylation. Different histone lysines exhibit distinct deacetylation rates, with N-terminal tail lysines deacetylated rapidly and H3 lysine 56 slowly. Yeast mitochondrial and nucleocytosolic acetyl-CoA pools are biochemically isolated. Thus, acetyl-CoA metabolism is directly linked to chromatin regulation and may affect diverse cellular processes in which acetylation and metabolism intersect, such as disease states and aging.

Acetyl-CoA synthetase from Pseudomonas putida U is the only acyl-CoA activating enzyme induced by acetate in this bacterium.

The gene (acs) encoding the acetyl-CoA synthetase (Acs) in Pseudomonas putida U has been cloned, sequenced and expressed in different microbes. The protein has been purified and characterized from a biochemical, structural and evolutionary point of view. Disruption or deletion of acs handicapped the bacterium for growth in a chemically defined medium containing acetate; this ability was regained when P. putida U was transformed with a plasmid carrying this gene. By contrast, all the acs knock-out mutants could assimilate n-alkanoic acids having a carbon length greater than C2, suggesting that other acyl-CoA activating enzymes (different from Acs) are involved in the catabolism of these compounds. However, these enzymes that can replace the function played by Acs in vivo are not induced by acetate.

Life with carbon monoxide.

This review focuses on how microbes live on CO as a sole source of carbon and energy and with CO by generating carbon monoxide as a metabolic intermediate. The use of CO is a property of organisms that use the Wood-L jungdahl pathway of autotrophic growth. The review discusses when CO metabolism originated, when and how it was discovered, and what properties of CO are ideal for microbial growth. How CO sensing by a heme-containing transcriptional regulatory protein activates the expression of CO metabolism-linked genes is described. Two metalloenzymes are the cornerstones of growth with CO: CO dehydrogenase (CODH) and acetyl-CoA synthase (ACS). CODH oxidizes CO to CO2, providing low-potential electrons for the cell, or alternatively reduces CO2 to CO. The latter reaction, when coupled to ACS, forms a machine for generating acetyl-CoA from CO2 for cell carbon synthesis. The recently solved crystal structures of CODH and ACS along with spectroscopic measurements and computational studies provide insights into novel bio-organometallic catalytic mechanisms and into the nature of a 140 A gas channel that coordinates the generation and utilization of CO. The enzymes that are coupled to CODH/ACS are also described, with a focus on a corrinoid protein, a methyltransferase, and pyruvate ferredoxin oxidoreductase.

Isolation and preliminary characterization of the medium-chain fatty acid:CoA ligase responsible for activation of short- and medium-chain fatty acids in colonic mucosa from swine.

The ATP-dependent activation of short- and medium-chain fatty acids to their respective CoA thioester adducts was investigated in the colonic mucosa from swine. Subcellular fractionation of a homogenate of the mucosa from the entire length of the colon revealed a predominantly mitochondrial localization for activity toward fatty acids ranging from propionate through laurate. These activities could be released from mitochondria in soluble form by freeze-thaw lysis. Purification of these activities revealed that they all appeared to reside with a single enzyme. This suggests that the entire colon contains a single form of medium-chain fatty acid:CoA ligase (MCFA:CoA ligase). The ligase also had activity toward benzoate and salicylate, although this activity was significantly lower than activity toward medium-chain fatty acids. The enzyme had the highest activity at Vmax with butyrate as substrate and had the lowest Km for octanoate. Butyrate and octanoate were mutually inhibitory. Activity toward both substrates was also efficiently inhibited by cyclohexane carboxylate. The molecular weight of the enzyme was estimated by gel filtration chromatography to be ca. 46,500. These data indicate that the colonic MCFA:CoA ligase is significantly different from the hepatic and kidney MCFA:CoA ligases.