Acyl Coenzyme Research Articles

Systematic Mutational Analysis of Histidine Kinase Genes in the Nosocomial Pathogen Stenotrophomonas maltophilia Identifies BfmAK System Control of Biofilm Development

The Gram-negative bacterium Stenotrophomonas maltophilialives in diverse ecological niches. As a result of its formidable capabilities of forming biofilm and its resistance to multiple antibiotic agents, the bacterium is also a nosocomial pathogen of serious threat to the health of patients whose immune systems are suppressed or compromised. Besides the histidine kinase RpfC, the two-component signal transduction system (TCS), which is the canonical regulatory machinery used by most bacterial pathogens, has never been experimentally investigated inS. maltophilia Here, we annotated 62 putative histidine kinase genes in the S. maltophilia genome and successfully obtained 51 mutants by systematical insertional inactivation. Phenotypic characterization identified a series of mutants with deficiencies in bacterial growth, swimming motility, and biofilm development. A TCS, named here BfmA-BfmK (Smlt4209-Smlt4208), was genetically confirmed to regulate biofilm formation inS. maltophilia Together with interacting partner prediction and chromatin immunoprecipitation screens, six candidate promoter regions bound by BfmA in vivo were identified. We demonstrated that, among them, BfmA acts as a transcription factor that binds directly to the promoter regions of bfmA-bfmK and Smlt0800(acoT), a gene encoding an acyl coenzyme A thioesterase that is associated with biofilm development, and positively controls their transcription. Genome-scale mutational analyses of histidine kinase genes and functional dissection of BfmK-BfmA regulation in biofilm provide genetic information to support more in-depth studies on cellular signaling inS. maltophilia, in the context of developing novel approaches to fight this important bacterial pathogen.

Memprotmd: Protein-Lipid Interactions of Phospholipid Biosynthetic Enzymes and Development of the Web Database

Membrane bound proteins play an important role in many biological processes, therefore understanding their structure and function represents an important scientific challenge. Whilst improvements in experimental techniques are revealing high resolution structures for an increasing number of membrane proteins, these structures are rarely resolved in complex with membrane lipids. The MemProtMD pipeline uses Coarse-Grained molecular dynamics simulations to allow a lipid bilayer to self-assemble around a membrane protein, and can be used to study the structure and dynamics of membrane proteins and lipids at both Coarse-Grained and atomistic representations.A web database (http://sbcb.bioch.ox.ac.uk/memprotmd/beta/) has been developed to make data generated by the MemProtMD pipeline available to the scientific community. Simulations and the results of subsequent analysis can be viewed using a web browser, including interactive 3D visualisations of the assembled bilayer and 2D visualisations of membrane protein topology and lipid contact data. All files required to run further coarse-grained or atomistic simulations of proteins in the database are provided. Proteins may be searched using keywords or browsed using classification systems such as MPStruc or the Transporter Classification Database.Phospholipid biosynthetic enzymes catalyse reactions involving membrane lipids and often interact with membrane lipids in unusual ways. Many of these interactions are important to the biological function of the enzyme and can be identified using the MemProtMD pipeline. Tools provided through the MemProtMD web database were used to examine the properties of the phospholipid biosynthetic enzymes diacylglycerol kinase, phosphatidate cytidylyltransferase and acyl-CoA desaturase when simulated in a membrane environment.

Alisol A 24-Acetate Prevents Hepatic Steatosis and Metabolic Disorders in HepG2 Cells

Background: Non-alcoholic fatty liver disease (NAFLD) is closely associated with metabolic disorders including hepatic lipid accumulation and inflammation. Alisol A 24-acetate, a triterpene from Alismatis rhizome, has multiple biologic activities such as hypolipidemic, anti-inflammatory and anti-diabetic. Thus we hypothesized that Alisol A 24 -acetate would have effect on NAFLD. The present study was conducted to investigate the therapeutic effects and potential mechanisms of Alisol A 24-acetate against hepatic steatosis in a free fatty acids (FFAs) induced NAFLD cell model. Methods: This study was divided into four groups including Control group, Model group (FFA group), Alisol A 24-acetate (FFA+A) group, Fenofibrate (FFA+F) group. Preventive role of Alisol A 24-acetate was evaluated using 10µM Alisol A 24-acetate plus 1 mM FFA (oleate:palmitate=2:1) incubated with HepG2 cells for 24 h, which was determined by Oil Red O Staining, Oil Red O based colorimetric assay and intracellular triglyceride (TG) content. Besides, the inflammatory cytokines tumor necrosis factor (TNF)- α, interleukin (IL)-6 levels as well as the protein and mRNA expressions that were involved in fatty acid synthesis and oxidation including Adiponectin, AMP-activated protein kinase (AMPK) α, peroxisome proliferator-activated receptor (PPAR) α, sterol regulatory element binding protein 1c (SREBP-1c), acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), carnitine palmitoyltransferase 1 (CPT1) and acyl coenzyme A oxidase 1 (ACOX1) were detected. Results: Alisol A 24-acetate significantly decreased the numbers of lipid droplets, Oil Red O lipid content, and intracellular TG content. Besides, inflammatory cytokines TNF-α, IL-6 levels were markedly inhibited by Alisol A 24-acetate. Furthermore, Alisol A 24-acetate effectively increased the protein and mRNA expressions of Adiponectin, the phosphorylation of AMPKα, CPT1 and ACOX1, whereas decreased SREBP-1c, the phosphorylation of ACC and FAS at both protein and mRNA levels. However, there was no significant effect on the protein and mRNA expressions of PPARα by Alisol A 24-acetate. Conclusions: These results demonstrated that Alisol A 24-acetate effectively ameliorated hepatic steatosis likely through Adiponectin, which activated AMPKα signaling pathways via down-regulating SREBP-1c, ACC, FAS and up-regulating CPT1 and ACOX1, and inhibited inflammation. Thereby, Alisol A 24-acetate could be a promising candidate for the treatment of NAFLD.

Lipid Regulation Effects of Raw and Processed Notoginseng Radix Et Rhizome on Steatotic Hepatocyte L02 Cell.

Introduction. Raw and processed Notoginseng Radix Et Rhizome (NRR) have been widely used in treatment of metabolic syndromes and related disease, including nonalcoholic fatty liver disease (NAFLD). This study was designed to investigate lipid regulation effects of raw and processed NRR in steatotic L02 cell. Materials and Methods. Steatotic L02 cells were obtained after being cultured with 5% fat emulsion-10% FBS-RPMI 1640 medium for 48 h. Contents of total cholesterol (TC), triglyceride (TG), free fatty acid (FFA), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in steatotic L02 cells were evaluated after treatment. Furthermore, the lipid metabolism regulation mechanism of Panax notoginseng saponins (PNS) and its monomers were evaluated by detecting the expressions of hydroxymethyl glutaric acyl coenzyme A reductase (HMG-CoAR), sterol regulating element binding protein-2 (SREBP-2), and cholesterol 7α-hydroxylase (CYP7α). Results. TG and TC contents were doubled in model group compared to those in normal L02 cells group. Raw NRR and NRR heated with sand (NRR-B) showed much remarkable lipid-lowering effects in steatotic L02 cells. PNS, notoginsenoside R1, ginsenoside Rg1, and ginsenoside Rb1 displayed the best TG and TC regulation activity, which could significantly reduce contents of SREBP-2 and HMG-CoAR and increase the content of CYP7α. Conclusions. Our results may support the fact that both raw NRR and NRR-B might have more satisfactory effects in the treatment of NAFLD.

VY6, a β-lactoglobulin-derived peptide, altered metabolic lipid pathways in the zebra fish liver.

Today enormous research efforts are being focused on alleviating the massive, adverse effects of obesity. Short peptides are key targets for research as they can be generated from natural proteins, like milk. Here we conducted trypsinogen digestion of beta-lactoglobulin (β-lg), the major mammalian milk protein, to release the hexamer VY6. It was assayed in vivo for its activities on lipid metabolism using zebra fish as a vertebrate model. Zebra fish juveniles were injected with two different doses of the peptide: 100 and 800 μg per g fish and left for 5 days before sacrificing. Lipid measurements showed significant reduction in liver triglycerides and free cholesterol, as well as increased liver HDL cholesterol. Dose-dependent increases of the mRNA levels of the genes coding for the enzymes acyl coenzyme A oxidase 1 (acox1) and lipoprotein lipase (lpl) were also found. The complete results suggest significant anti-obesity activity of the β-lg-derived VY6 peptide. Its use as a nutraceutical has been discussed.

Important Metabolic Pathways and Biological Processes Expressed by Chicken Cecal Microbiota.

The gut microbiota plays important roles in its host. However, how each microbiota member contributes to the behavior of the whole population is not known. In this study, we therefore determined protein expression in the cecal microbiota in chickens of selected ages and in 7-day-old chickens inoculated with different cecal extracts on the day of hatching. Campylobacter, Helicobacter, Mucispirillum, and Megamonas overgrew in the ceca of 7-day-old chickens inoculated with cecal extracts from donor hens. Firmicutes were characterized by ABC and phosphotransferase system (PTS) transporters, extensive acyl coenzyme A (acyl-CoA) metabolism, and expression of l-fucose isomerase. Anaerostipes, Anaerotruncus, Pseudoflavonifractor, Dorea, Blautia, and Subdoligranulum expressed spore proteins. Firmicutes (Faecalibacterium, Butyrivibrio, Megasphaera, Subdoligranulum, Oscillibacter, Anaerostipes, and Anaerotruncus) expressed enzymes required for butyrate production. Megamonas, Phascolarctobacterium, and Blautia (exceptions from the phylum Firmicutes) and all Bacteroidetes expressed enzymes for propionate production pathways. Representatives of Bacteroidetes also expressed xylose isomerase, enzymes required for polysaccharide degradation, and ExbBD, TonB, and outer membrane receptors likely to be involved in oligosaccharide transport. Based on our data, Anaerostipes, Anaerotruncus, and Subdoligranulum might be optimal probiotic strains, since these represent spore-forming butyrate producers. However, certain care should be taken during microbiota transplantation because the microbiota may behave differently in the intestinal tract of a recipient depending on how well the existing communities are established.

Stimulatory effect of ethanol on libertellenone H biosynthesis by Arctic fungus Eutypella sp. D-1.

Libertellenone H (1) was a promising antitumor diterpenoid isolated from Arctic fungus Eutypella sp. D-1, however, its production was very limited. In this study, we investigated the effects of ethanol on cell growth and libertellenone H production. The mycelium in ethanol-feeding cultures was fragmented and dispersed, and the titer of libertellenone H was remarkably increased to 4.88 mg l(-1) in an optimal feeding manner, which was 16.4-fold higher than the control group. To provide an insight into the cell response to ethanol, genes critical to the libertellenone H biosynthesis were successfully cloned and their transcription levels were determined. The results suggested that the gene transcription levels of 3-hydroxy-3-methyl glutaric acyl coenzyme A reductase and geranylgeranyl diphosphate synthase were up-regulated by ethanol stimulation. The results from this study were helpful for further understanding of the ethanol function on diterpenes biosynthesis as well as developing more effective strategies for over-production of these desired secondary metabolites.

Secreted Acb1 Contributes to the Yeast-to-Hypha Transition in Cryptococcus neoformans.

Adaptation to stress by eukaryotic pathogens is often accompanied by a transition in cellular morphology. The human fungal pathogen Cryptococcus neoformans is known to switch between the yeast and the filamentous form in response to amoebic predation or during mating. As in the classic dimorphic fungal pathogens, the morphotype is associated with the ability of cryptococci to infect various hosts. Many cryptococcal factors and environmental stimuli, including pheromones (small peptides) and nutrient limitation, are known to induce the yeast-to-hypha transition. We recently discovered that secreted matricellular proteins could also act as intercellular signals to promote the yeast-to-hypha transition. Here we show that the secreted acyl coenzyme A (acyl-CoA)-binding protein Acb1 plays an important role in enhancing this morphotype transition. Acb1 does not possess a signal peptide. Its extracellular secretion and, consequently, its function in filamentation are dependent on an unconventional GRASP (Golgi reassembly stacking protein)-dependent secretion pathway. Surprisingly, intracellular recruitment of Acb1 to the secretory vesicles is independent of Grasp. In addition to Acb1, Grasp possibly controls the secretion of other cargos, because the graspΔ mutant, but not the acb1Δ mutant, is defective in capsule production and macrophage phagocytosis. Nonetheless, Acb1 is likely the major or the sole effector of Grasp in terms of filamentation. Furthermore, we found that the key residue of Acb1 for acyl binding, Y80, is critical for the proper subcellular localization and secretion of Acb1 and for cryptococcal morphogenesis.

Toxicity of Carboxylic Acid-Containing Drugs: The Role of Acyl Migration and CoA Conjugation Investigated.

Many carboxylic acid-containing drugs are associated with idiosyncratic drug toxicity (IDT), which may be caused by reactive acyl glucuronide metabolites. The rate of acyl migration has been earlier suggested as a predictor of acyl glucuronide reactivity. Additionally, acyl Coenzyme A (CoA) conjugates are known to be reactive. Here, 13 drugs with a carboxylic acid moiety were incubated with human liver microsomes to produce acyl glucuronide conjugates for the determination of acyl glucuronide half-lives by acyl migration and with HepaRG cells to monitor the formation of acyl CoA conjugates, their further conjugate metabolites, and trans-acylation products with glutathione. Additionally, in vitro cytotoxicity and mitochondrial toxicity experiments were performed with HepaRG cells to compare the predictability of toxicity. Clearly, longer acyl glucuronide half-lives were observed for safe drugs compared to drugs that can cause IDT. Correlation between half-lives and toxicity classification increased when "relative half-lives," taking into account the formation of isomeric AG-forms due to acyl migration and eliminating the effect of hydrolysis, were used instead of plain disappearance of the initial 1-O-β-AG-form. Correlation was improved further when a daily dose of the drug was taken into account. CoA and related conjugates were detected primarily for the drugs that have the capability to cause IDT, although some exceptions to this were observed. Cytotoxicity and mitochondrial toxicity did not correlate to drug safety. On the basis of the results, the short relative half-life of the acyl glucuronide (high acyl migration rate), high daily dose and detection of acyl CoA conjugates, or further metabolites derived from acyl CoA together seem to indicate that carboxylic acid-containing drugs have a higher probability to cause drug-induced liver injury (DILI).

Macrophage-mediated cholesterol handling in atherosclerosis.

Formation of foam cells is a hallmark at the initial stages of atherosclerosis. Monocytes attracted by pro-inflammatory stimuli attach to the inflamed vascular endothelium and penetrate to the arterial intima where they differentiate to macrophages. Intimal macrophages phagocytize oxidized low-density lipoproteins (oxLDL). Several scavenger receptors (SR), including CD36, SR-A1 and lectin-like oxLDL receptor-1 (LOX-1), mediate oxLDL uptake. In late endosomes/lysosomes of macrophages, oxLDL are catabolysed. Lysosomal acid lipase (LAL) hydrolyses cholesterol esters that are enriched in LDL to free cholesterol and free fatty acids. In the endoplasmic reticulum (ER), acyl coenzyme A: cholesterol acyltransferase-1 (ACAT1) in turn catalyses esterification of cholesterol to store cholesterol esters as lipid droplets in the ER of macrophages. Neutral cholesteryl ester hydrolases nCEH and NCEH1 are involved in a secondary hydrolysis of cholesterol esters to liberate free cholesterol that could be then out-flowed from macrophages by cholesterol ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 and SR-BI. In atherosclerosis, disruption of lipid homoeostasis in macrophages leads to cholesterol accumulation and formation of foam cells.

Antihyperlipidaemic effects of oxyresveratrol-containing Ramulus mori ethanol extract in rats fed a high-cholesterol diet

The antihyperlipidaemic effect of an ethanol extract of Ramulus mori containing a high concentration of oxyresveratrol (ERMO) was investigated in a high-cholesterol diet (HCD)-fed rat model. Cholesterol metabolism-related genes and proteins, such as lecithin cholesterol acyltransferase (LCAT), low-density lipoprotein receptor (LDLR), cholesterol 7α-hydroxylase (CYP7A1), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and acyl coenzyme A cholesterol acyltransferase-2 (ACAT2) were analysed by quantitative real-time reverse-transcription polymerase chain reaction and western blot analysis. ERMO-treated HCD-fed rats had decreased body and liver weights and reduced serum lipid levels, atherogenic index (AI) and coronary artery risk index (CRI). ERMO treatment lowered serum cholesterol levels by increasing the expression of LCAT, CYP7A1 and LDLR and by decreasing HMGCR and ACAT2. Histological analysis of liver and aortic tissues revealed a significant decrease in lipid droplets and foam cell formation in HCD-induced hyperlipidaemic rats treated with ERMO. ERMO is therefore a potentially potent functional food for preventing hyperlipidaemia.

Chemoenzymatic Synthesis of Acyl Coenzyme A Substrates Enables in Situ Labeling of Small Molecules and Proteins.

A chemoenzymatic approach to generate fully functional acyl coenzyme A molecules that are then used as substrates to drive in situ acyl transfer reactions is described. Mass spectrometry based assays to verify the identity of acyl coenzyme A enzymatic products are also illustrated. The approach is responsive to a diverse array of carboxylic acids that can be elaborated to their corresponding coenzyme A thioesters, with potential applications in wide-ranging chemical biology studies that utilize acyl coenzyme A substrates.

Targeting Mycobacterium tuberculosis Biotin Protein Ligase (MtBPL) with Nucleoside-Based Bisubstrate Adenylation Inhibitors.

Mycobacterium tuberculosis (Mtb), responsible for both latent and symptomatic tuberculosis (TB), remains the second leading cause of mortality among infectious diseases worldwide. Mycobacterial biotin protein ligase (MtBPL) is an essential enzyme in Mtb and regulates lipid metabolism through the post-translational biotinylation of acyl coenzyme A carboxylases. We report the synthesis and evaluation of a systematic series of potent nucleoside-based inhibitors of MtBPL that contain modifications to the ribofuranosyl ring of the nucleoside. All compounds were characterized by isothermal titration calorimetry (ITC) and shown to bind potently with K(D)s ≤ 2 nM. Additionally, we obtained high-resolution cocrystal structures for a majority of the compounds. Despite fairly uniform biochemical potency, the whole-cell Mtb activity varied greatly with minimum inhibitory concentrations (MIC) ranging from 0.78 to >100 μM. Cellular accumulation studies showed a nearly 10-fold enhancement in accumulation of a C-2'-α analogue over the corresponding C-2'-β analogue, consistent with their differential whole-cell activity.

An Extended Cyclic Di-GMP Network in the Predatory Bacterium Bdellovibrio bacteriovorus.

Over the course of the last 3 decades the role of the second messenger cyclic di-GMP (c-di-GMP) as a master regulator of bacterial physiology was determined. Although the control over c-di-GMP levels via synthesis and breakdown and the allosteric regulation of c-di-GMP over receptor proteins (effectors) and riboswitches have been extensively studied, relatively few effectors have been identified and most are of unknown functions. The obligate predatory bacterium Bdellovibrio bacteriovorus has a peculiar dimorphic life cycle, in which a phenotypic transition from a free-living attack phase (AP) to a sessile, intracellular predatory growth phase (GP) is tightly regulated by specific c-di-GMP diguanylate cyclases. B. bacteriovorus also bears one of the largest complement of defined effectors, almost none of known functions, suggesting that additional proteins may be involved in c-di-GMP signaling. In order to uncover novel c-di-GMP effectors, a c-di-GMP capture-compound mass-spectroscopy experiment was performed on wild-type AP and host-independent (HI) mutant cultures, the latter serving as a proxy for wild-type GP cells. Eighty-four proteins were identified as candidate c-di-GMP binders. Of these proteins, 65 did not include any recognized c-di-GMP binding site, and 3 carried known unorthodox binding sites. Putative functions could be assigned to 59 proteins. These proteins are included in metabolic pathways, regulatory circuits, cell transport, and motility, thereby creating a potentially large c-di-GMP network. False candidate effectors may include members of protein complexes, as well as proteins binding nucleotides or other cofactors that were, respectively, carried over or unspecifically interacted with the capture compound during the pulldown. Of the 84 candidates, 62 were found to specifically bind the c-di-GMP capture compound in AP or in HI cultures, suggesting c-di-GMP control over the whole-cell cycle of the bacterium. High affinity and specificity to c-di-GMP binding were confirmed using microscale thermophoresis with a hypothetical protein bearing a PilZ domain, an acyl coenzyme A dehydrogenase, and a two-component system response regulator, indicating that additional c-di-GMP binding candidates may be bona fide novel effectors. In this study, 84 putative c-di-GMP binding proteins were identified in B. bacteriovorus, an obligate predatory bacterium whose lifestyle and reproduction are dependent on c-di-GMP signaling, using a c-di-GMP capture compound precipitation approach. This predicted complement covers metabolic, energy, transport, motility and regulatory pathways, and most of it is phase specific, i.e., 62 candidates bind the capture compound at defined modes of B. bacteriovorus lifestyle. Three of the putative binders further demonstrated specificity and high affinity to c-di-GMP via microscale thermophoresis, lending support for the presence of additional bona fide c-di-GMP effectors among the pulled-down protein repertoire.

Molecular mechanism of a new Laminaria japonica polysaccharide on the suppression of macrophage foam cell formation via regulating cellular lipid metabolism and suppressing cellular inflammation.

Laminaria japonica is an important marine vegetable with great health benefits for preventing atherosclerosis. Since the foam cell formation is an important hallmark for the initiation of atherosclerosis, we examined the effect and underlying mechanism of a purified L. japonica polysaccharide (LJP61A) on the suppression of macrophage foam cell formation in this study. The chemical structure was further characterized. Using oxidized low-density lipoprotein (ox-LDL)-induced foam cell model, we found that the cellular lipid accumulation was significantly attenuated by 25 μg/mL LJP61A. Meanwhile, LJP61A caused a remarkable decrease in mRNA expression of peroxisome proliferator-activated receptor γ that was accompanied by the reduction of CD36 and Acyl coenzyme A: cholesterol acyltransferase-1 mRNA levels, and the enhancement of ATP-binding cassette transporters A1 and scavenger receptor B1 mRNA levels. Besides these, the ox-LDL-induced cellular inflammation was also restricted by LJP61A treatment via mammalian target of rapamycin-mediated Toll-like receptor 2/4-Mitogen-activated protein kinases/nuclear factor kappa-B pathways. The structure of LJP61A was characterized as a repeating unit consisting of →3,6)-α-d-Manp-(1→, →4)-α-d-Manp-(1→, →4)-2-O-acetyl-β-d-Glcp-(1→, →4)-β-d-Glcp-(1→, →6)-4-O-SO3 -β-d-Galp-(1→, →6)-β-d-Galp-(1→, →3)-β-d-Galp-(1→, and a terminal residue of α-d-Glcp-(1→. Our findings suggest that LJP61A inhibits the conversion of macrophage into foam cell via regulating cellular lipid metabolism and suppressing cellular inflammation.

Transcriptional Regulation by the Short-Chain Fatty Acyl Coenzyme A Regulator (ScfR) PccR Controls Propionyl Coenzyme A Assimilation by Rhodobacter sphaeroides.

Propionyl coenzyme A (propionyl-CoA) assimilation by Rhodobacter sphaeroides proceeds via the methylmalonyl-CoA pathway. The activity of the key enzyme of the pathway, propionyl-CoA carboxylase (PCC), was upregulated 20-fold during growth with propionate compared to growth with succinate. Because propionyl-CoA is an intermediate in acetyl-CoA assimilation via the ethylmalonyl-CoA pathway, acetate growth also requires the methylmalonyl-CoA pathway. PCC activities were upregulated 8-fold in extracts of acetate-grown cells compared to extracts of succinate-grown cells. The upregulation of PCC activities during growth with propionate or acetate corresponded to increased expression of the pccB gene, which encodes a subunit of PCC. PccR (RSP_2186) was identified to be a transcriptional regulator required for the upregulation of pccB transcript levels and, consequently, PCC activity: growth substrate-dependent regulation was lost when pccR was inactivated by an in-frame deletion. In the pccR mutant, lacZ expression from a 215-bp plasmid-borne pccB upstream fragment including 27 bp of the pccB coding region was also deregulated. A loss of regulation as a result of mutations in the conserved motifs TTTGCAAA-X4-TTTGCAAA in the presence of PccR allowed the prediction of a possible operator site. PccR, together with homologs from other organisms, formed a distinct clade within the family of short-chain fatty acyl coenzyme A regulators (ScfRs) defined here. Some members from other clades within the ScfR family have previously been shown to be involved in regulating acetyl-CoA assimilation by the glyoxylate bypass (RamB) or propionyl-CoA assimilation by the methylcitrate cycle (MccR). Short-chain acyl-CoAs are intermediates in essential biosynthetic and degradative pathways. The regulation of their accumulation is crucial for appropriate cellular function. This work identifies a regulator (PccR) that prevents the accumulation of propionyl-CoA by controlling expression of the gene encoding propionyl-CoA carboxylase, which is responsible for propionyl-CoA consumption by Rhodobacter sphaeroides. Many other Proteobacteria and Actinomycetales contain one or several PccR homologs that group into distinct clades on the basis of the pathway of acyl-CoA metabolism that they control. Furthermore, an upstream analysis of genes encoding PccR homologs allows the prediction of conserved binding motifs for these regulators. Overall, this study evaluates a single regulator of propionyl-CoA assimilation while expanding the knowledge of the regulation of short-chain acyl-CoAs in many bacterial species.

Sirtuins Function as the Modulators in Aging-related Diseases in Common or Respectively.

Sirtuins Function as the Modulators in Aging-related Diseases in Common or Respectively.

ATR-101 phase 1 clinical study for adrenocortical carcinoma.

TPS4585 Background: ATR-101 (Atterocor, Inc., Ann Arbor, MI, USA) is a selective inhibitor of ACAT1 (acyl coenzyme A:cholesterol acyltransferase) in clinical development for the treatment of adrenocortical carcinoma (ACC). ACAT1 catalyzes cholesterol ester formation from cholesterol and long-chain fatty acyl-CoA and, in the adrenals, is particularly important in creating a reservoir of substrate for steroid biosynthesis. ATR-101 selectively distributes to the adrenals, inhibits steroidogenesis and causes apoptosis of cells of the adrenal cortex, as well as in H295R ACC cells. ACC is an ultra-rare malignancy, occurring in about 2 per million population annually. ACC is frequently discovered in Stage 4 and the overall disease survival is approximately 17 months. Tumors often overproduce steroids normally produced in the adrenal cortex and cause therapy-resistant Cushing’s syndrome. Current therapies are toxic, difficult to administer, and poorly effective. Methods: ATR-101-001 is a phase 1 study being conducted at four centers in the United States and one in Germany. It is a “3+3” design: 3 subjects with advanced ACC who have failed or declined existing therapies are enrolled at each dose level for 28 days. If no Dose Limiting Toxicity is observed, three additional subjects are enrolled at a higher dose. An expansion cohort of 20 subjects will be enrolled after the MTD has been determined. Subjects who appear to be deriving benefit may stay on ATR-101 indefinitely. ATR-101 is taken by mouth. The primary objective is to determine the safety and tolerability of ATR-101 in subjects with advanced ACC. Secondary objectives include efficacy by RECIST, measurement of production of steroid hormones and intermediates, determination of pharmacokinetics, the Maximum Tolerated Dose and the recommended phase 2 dose. The study is open to patients age 18 and over with advanced ACC. Patient’s mitotane level must be 5 µg/ml or less; the QTcF 470 ms or less; and if present, CNS metastases must be treated and inactive. Full study information is available on ClinicalTrials.gov, Identifier NCT01898715. Clinical trial information: NCT01898715.

An acyltransferase domain of FK506 polyketide synthase recognizing both an acyl carrier protein and coenzyme A as acyl donors to transfer allylmalonyl and ethylmalonyl units.

Acyltransferase (AT) domains of polyketide synthases (PKSs) usually use coenzyme A (CoA) as an acyl donor to transfer common acyl units to acyl carrier protein (ACP) domains, initiating incorporation of acyl units into polyketides. Two clinical immunosuppressive agents, FK506 and FK520, are biosynthesized by the same PKSs in several Streptomyces strains. In this study, characterization of AT4FkbB (the AT domain of the fourth module of FK506 PKS) in transacylation reactions showed that AT4FkbB recognizes both an ACP domain (ACPT csA) and CoA as acyl donors for transfer of a unique allylmalonyl (AM) unit to an acyl acceptor ACP domain (ACP4FkbB), resulting in FK506 production. In addition, AT4FkbB uses CoA as an acyl donor to transfer an unusual ethylmalonyl (EM) unit to ACP4FkbB, resulting in FK520 production, and transfers AM units to non-native ACP acceptors. Characterization of AT4FkbB in self-acylation reactions suggests that AT4FkbB controls acyl unit specificity in transacylation reactions but not in self-acylation reactions. Generally, AT domains of PKSs only recognize one acyl donor; however, we report here that AT4FkbB recognizes two acyl donors for the transfer of different acyl units. Nucleotide sequence data have been submitted to the GenBank database under accession numbers KJ000382 and KJ000383.

PPARα Protein Expression Was Increased by Four Weeks of Intermittent Hypoxic Training via AMPKα2-Dependent Manner in Mouse Skeletal Muscle.

Peroxisome proliferator-activated receptor α (PPARα) is critical for muscle endurance due to its role in the regulation of fatty acid oxidation. The 5’-AMP-activated protein kinase (AMPK) is an energy sensor in cells, but its role in PPARα regulation in vivo remains unknown. In this study, we examined PPARα expression in the skeletal muscle of AMPKα2 overexpression (OE), knockout (KO) and wild-type (WT) mice after four weeks of exercise under intermittent hypoxia. WT, OE and KO mice were used at 40 mice/strain and randomly subdivided into four subgroups: control (C), running (R), hypoxia (H), and running plus hypoxia (R+H) at 10 mice/group. The treadmill running was performed at the speed of 12 m/min, 60 min/day with a slope of 0 degree for four weeks. The hypoxia treatment was performed in daytime with normobaric hypoxia (11.20% oxygen, 8 hours/day). In the R+H group, the treadmill running was conducted in the hypoxic condition. AMPKα2, phosphor-AMPKα (p-AMPKα) (Thr172), nuclear PPARα proteins were measured by Western blot and the medium chain acyl coenzyme A dehydrogenase (MCAD) mRNA, the key enzyme for fatty acid oxidation and one of the PPARα target genes, was also measured in skeletal muscles after the interventions. The results showed that nuclear PPARα protein was significantly increased by R+H in WT muscles, the increase was enhanced by 41% (p<0.01) in OE mice, but was reduced by 33% (p<0.01) in KO mice. The MCAD mRNA expression was increased after four weeks of R+H intervention. The change in MCAD mRNA followed a similar trend as that of PPARα protein in OE and KO mice. Our data suggest that the increase in nuclear PPARα protein by four-week exercise training under the intermittent hypoxia was dependent on AMPK activation.