Search

The tumor microenvironment is a dynamic network of stromal, cancer, and immune cells that interact and compete for resources. We have previously identified the Vanin1 pathway as a tumor suppressor of sarcoma development via vitamin B5 and coenzyme A regeneration. Using an aggressive sarcoma cell line that lacks Vnn1 expression, we showed that the administration of pantethine, a vitamin B5 precursor, attenuates tumor growth in immunocompetent but not nude mice. Pantethine boosts antitumor immunity, including the polarization of myeloid and dendritic cells towards enhanced IFNγ-driven antigen presentation pathways and improved the development of hypermetabolic effector CD8 + T cells endowed with potential antitumor activity. At later stages of treatment, the effect of pantethine was limited by the development of immune cell exhaustion. Nevertheless, its activity was comparable with that of anti-PD1 treatment in sensitive tumors. In humans, VNN1 expression correlates with improved survival and immune cell infiltration in soft-tissue sarcomas, but not in osteosarcomas. Pantethine could be a potential therapeutic immunoadjuvant for the development of antitumor immunity., (© 2023 Miallot et al.)

Purpose: Ferroptosis is a form of regulated cell death that has the potential to be targeted as a cancer therapeutic strategy. But cancer cells have a wide range of sensitivities to ferroptosis, which limits its therapeutic potential. Accumulation of lipid peroxides determines the occurrence of ferroptosis. However, the type of lipid involved in peroxidation and the mechanism of lipid peroxide accumulation are less studied., Methods: The effects of fatty acids (10 μM) with different carbon chain length and unsaturation on ferroptosis were evaluated by MTT and LDH release assay in cell lines derived from prostate cancer (PC3, 22RV1, DU145 and LNCaP), colorectal cancer (HT-29), cervical cancer (HeLa) and liver cancer (HepG2). Inhibitors of apoptosis, necroptosis, autophagy and ferroptosis were used to determine the type of cell death. Then the regulation of reactive oxygen species (ROS) and lipid peroxidation by docosahexaenoic acid (DHA) was measured by HPLC-MS and flow cytometry. The avtive form of DHA was determined by siRNA mediated gene silencing. The role of lipoxygenases was checked by inhibitors and gene silencing. Finally, the effect of DHA on ferroptosis-mediated tumor killing was verified in xenografts., Results: The sensitivity of ferroptosis was positively correlated with the unsaturation of exogenously added fatty acid. DHA (22:6 n-3) sensitized cancer cells to ferroptosis-inducing reagents (FINs) at the highest level in vitro and in vivo. In this process, DHA increased ROS accumulation, lipid peroxidation and protein oxidation independent of its membrane receptor, GPR120. Inhibition of long chain fatty acid-CoA ligases and lysophosphatidylcholine acyltransferases didn't affect the role of DHA. DHA-involved ferroptosis can be induced in both arachidonate lipoxygenase 5 (ALOX5) negative and positive cells. Down regulation of ALOX5 inhibited ferroptosis, while overexpression of ALOX5 promoted ferroptosis., Conclusion: DHA can effectively promote ferroptosis-mediated tumor killing by increasing intracellular lipid peroxidation. Both ALOX5 dependent and independent pathways are involved in DHA-FIN induced ferroptosis. And during this process, free DHA plays an important role., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

Aims: Metabolic switching during heart development contributes to postnatal cardiomyocyte (CM) cell cycle exit and loss of regenerative capacity in the mammalian heart. Metabolic control has potential for developing effective CM proliferation strategies. We sought to determine whether lactate dehydrogenase A (LDHA) regulated CM proliferation by inducing metabolic reprogramming., Methods and Results: LDHA expression was high in P1 hearts and significantly decreased during postnatal heart development. CM-specific LDHA knockout mice were generated using CRISPR/Cas9 technology. CM-specific LDHA knockout inhibited CM proliferation, leading to worse cardiac function and a lower survival rate in the neonatal apical resection model. In contrast, CM-specific overexpression of LDHA promoted CM proliferation and cardiac repair post-MI. The α-MHC-H2B-mCh/CAG-eGFP-anillin system was used to confirm the proliferative effect triggered by LDHA on P7 CMs and adult hearts. Metabolomics, proteomics and Co-IP experiments indicated that LDHA-mediated succinyl coenzyme A reduction inhibited succinylation-dependent ubiquitination of thioredoxin reductase 1 (Txnrd1), which alleviated ROS and thereby promoted CM proliferation. In addition, flow cytometry and western blotting showed that LDHA-driven lactate production created a beneficial cardiac regenerative microenvironment by inducing M2 macrophage polarization., Conclusions: LDHA-mediated metabolic reprogramming promoted CM proliferation by alleviating ROS and inducing M2 macrophage polarization, indicating that LDHA might be an effective target for promoting cardiac repair post-MI., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Efferocytosis (clearance of apoptotic cells by phagocytosis without inducing inflammation and autoimmunity) is an important mechanism in the resolution of inflammatory processes. Efficient efferocytosis inhibits the accumulation of apoptotic cells/debris and maintains homeostasis before the onset of necrosis (secondary necrosis), which promotes inflammation or injury. Moreover, the detection and clearance of apoptotic cells can promote anti-inflammatory responses. Defective efferocytosis is involved in the pathogenesis of several diseases, such as atherosclerosis, chronic inflammation, autoimmunity and cancer. Statins are 3-hydroxy-3-methylglutaryl coenzyme A-reductase inhibitors which exert cholesterol-lowering effects plus multiple pleiotropic properties, such as inhibition of inflammation and macrophage proliferation. Statins exhibit anti-inflammatory properties by reducing both the prenylation of signaling molecules with downregulation of gene expression and the expression of adhesion molecules, as well as the levels of cytokines and chemokines. Additionally, statins suppress the prenylation of GTPases, such as Rac-1, as a positive regulator of efferocytosis, and RhoA, as a negative regulator of efferocytosis. However, statins alter the membrane balance of Rho GTPases in efferocytosis toward Rac-1. Efferocytosis has modifiable targets, which can be exploited for the treatment of several diseases, although limited attention has been given to the mechanisms by which statins regulate efferocytosis and the resulting therapeutic implications. In this review, we will elaborate on the mechanisms underlying the modulation of apoptotic cell clearance by statins, which, in turn, inhibits uncontrolled inflammation and ensuing diseases., Competing Interests: Declaration of Competing Interest We have no conflicts of interest to declare., (Copyright © 2022. Published by Elsevier Inc.)

Previously, our group found that the dietary trace mineral element selenium and vitamin B 6 (VitB 6 ) alone was involved in lipid metabolism. However, the effects of selenium combined with VitB 6 on hyperlipidemia and lipid metabolism have not been reported until now. We hypothesized that selenium and VitB 6 cosupplementation would alleviate the hyperlipidemic and hepatic dysfunction and with minimum side effects in a Sprague-Dawley rat model of hyperlipidemia induced by a high-fat diet. Our results showed that selenium combined with VitB 6 could improve dyslipidemia and displayed better in vivo hypocholesterolemic abilities at early intervention. Moreover, cosupplementation reduced atherogenic indexes (atherogenic index and atherogenic index of plasm) and the ratio of ApoB/ApoA1. The liver function index aspartate aminotransferase in serum was reduced, as was and total cholesterol, triacylglycerol, and low-density lipoprotein cholesterol in liver. The intervention also increased the levels of ApoA1 in serum and high-density lipoprotein cholesterol of liver. In addition, the combination of selenium and VitB 6 decreased liver lipid deposition and alleviated steatosis, reduced adipocyte size of white adipose tissue, increased the activities of hepatic lipase and total lipase and the hepatic 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) level, decreased the hepatic mRNA transcription of lipogenic and regulatory genes including Srebf1 and downstream fat synthesis-related enzymes (Acc and Fasn) and cholesterol synthesis speed limiting enzyme Hmgr, increased the mRNA abundance of Lcat and Cyp7a1, increased the protein expression of SIRT1 and PPARα, and up-regulated the protein expression of sterol regulatory element-binding protein 1c in the livers of hyperlipidemia rats. We first demonstrated that oral selenium and VitB 6 cosupplementation exerted synergism in lowering blood and liver lipid profiles and antiatherosclerotic effects in hyperlipidemic rats by reducing endogenous cholesterol and lipid synthesis, enhancing the transport of cholesterol to hepatocytes and promoting fatty acid beta oxidation., Competing Interests: Author Declarations The authors declare no conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Dihydromyricetin (DHM) exerts protective effects in various brain diseases. The aim of this research was to investigate the biological role of DHM in cerebral ischemia reperfusion (I/R) injury., Methods: We generated a rat model of cerebral I/R injury by performing middle cerebral artery occlusion/reperfusion (MCAO/R). The neurological score and brain water content of the experimental rats was then evaluated. The infarct volume and extent of apoptosis in brain tissues was then assessed by 2,3,5-triphenyltetrazolium (TTC) and TdT-mediated dUTP nick end labeling (TUNEL) staining. Hippocampal neuronal cells (HT22) were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) and cell counting kit-8 (CCK-8) assays and flow cytometry were performed to detect cell viability and apoptosis. The levels of lipid reactive oxygen species (ROS) and iron were detected and the expression levels of key proteins were assessed by Western blotting., Results: DHM obviously reduced neurological deficits, brain water content, infarct volume and cell apoptosis in the brain tissues of MCAO/R rats. DHM repressed ferroptosis and inhibited the sphingosine kinase 1 (SPHK1)/mammalian target of rapamycin (mTOR) pathway in MCAO/R rats. In addition, DHM promoted cell viability and repressed apoptosis in OGD/R-treated HT22 cells. DHM also suppressed the levels of lipid ROS and intracellular iron in OGD/R-treated HT22 cells. The expression levels of glutathione peroxidase 4 (GPX4) was enhanced while the levels of acyl-CoA synthetase long-chain family member 4 (ACSL4) and phosphatidylethanolamine binding protein 1 (PEBP1) were reduced in OGD/R-treated HT22 cells in the presence of DHM. Moreover, the influence conferred by DHM was abrogated by the overexpression of SPHK1 or treatment with MHY1485 (an activator of mTOR)., Conclusion: This research demonstrated that DHM repressed ferroptosis by inhibiting the SPHK1/mTOR signaling pathway, thereby alleviating cerebral I/R injury. Our findings suggest that DHM may be a candidate drug for cerebral I/R injury treatment., Competing Interests: The authors report no conflicts of interest in this work., (© 2022 Xie et al.)

Background and Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) isolates are associated with various diseases ranged from mild superficial impairments to invasive infections. This study aimed to evaluate the ability of polymerase chain reaction (PCR) based methods namely, restriction fragment length polymorphism (RFLP) of the coa gene and random amplified polymorphic DNA (RAPD), to determine the genetic diversity of MRSA isolates. Materials and Methods: A total of 37 MRSA isolates were conventionally identified depending on their biochemical and microbiological culture characteristics. Genotypic confirmation was based on detection of the associated mecA gene. The genetic variation amongst MRSA isolates was evaluated following the coa gene-based RFLP and RAPD fingerprints. Results: Results illustrated that, the species specific coa gene was detected in all MRSA isolates. The irregular bands intensity, number, and molecular sizes of the PCR amplicons demonstrated the coa gene polymorphism. The incompatible AluI digestion patterns of these amplicons classified the tested MRSA isolates into 20 RFLP patterns which confirm the coa gene polymorphism. Additionally, the PCR-based RAPD analysis showed variable bands number with size range of approximately 130 bp to 4 kbp, which indicated the genetic variation of the tested MRSA isolates as it created 36 variable RAPD banding profiles. Conclusions: coa gene AluI enzymatic restriction sites, amongst the tested MRSA isolates, certify their genetic variation on the basis of the accurate but complicated and relatively expensive coa gene-based RFLP. Conversely, the results verified the excellent ability of the simple and cost-effective PCR-based RAPD analysis to discriminate between MRSA isolates without any preface data about the genome.

Objective: To explore the effects of hydroxyacyl-CoA dehydrogenase alpha subunit (HADHA) on the migration and invasion of HTR-8/SVneo cells, a human trophoblast cell line, and its potential mechanism of action., Methods: Immunofluorescence staining was done to evaluate the expression levels of HADHA in samples of normal villi and recurrent spontaneous abortion (RSA) villi at 6-8 weeks. Lentiviral infection system was used to construct stable HTR-8/SVneo cell lines with HADHA overexpression and knockdown. Western blot, qRT-PCR, Wound-healing assay, and Transwell assay were used to determine the effect of HADHA on the migration and invasion of HTR-8/SVneo cells and the expression of relevant genes. Transcriptome sequencing and bioinformatics analysis were done to screen for the potential target genes and signaling pathways regulated by HADHA. The specific molecular mechanism of how HADHA regulates the migration and invasion of HTR-8/SVneo cells was examined by adding the inhibitor of protein kinase B (PKB/AKT)., Results: HADHA was highly expressed in extravillous trophoblasts (EVT) of RSA villus samples as compared with samples from the normal control group. In HTR-8/SVneo cells overexpressing HADHA , the expression levels of migration and invasion-related genes, including HLA - G , MMP 2, MMP 9, and NCAD , were decreased ( P <0.01, P <0.05), and the migration and invasion abilities of HTR-8/SVneo cells were weakened ( P <0.05). HADHA knockdown increased the expression levels of HLA - G , MMP 2, MMP 9, and NCAD ( P <0.01, P <0.05), and promoted the migration and invasion of HTR-8/SVneo cells ( P <0.05). In addition, HADHA overexpression decreased the phosphorylation levels of PI3K and AKT ( P <0.05) and inhibited the PI3K/AKT signaling pathway. HADHA knockdown activated the PI3K/AKT signaling pathway. When MK-2206, an AKT inhibitor, was added to stable HTR-8/SVneo cell lines with HADHA knockdown, the migration and invasion of the cells were significantly reduced., Conclusion: HADHA inhibits the migration and invasion of HTR-8/SVneo cells by inhibiting the PI3K/AKT signaling pathway., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).)

The resistance of glioblastoma to chemotherapy remains a significant clinical problem. Targeting alternative pathways such as protein prenylation is known to be effective against many cancers. Fluvastatin is a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl- CoA (HMG-CoA) reductase, thereby inhibits prenylation. We demonstrate that fluvastatin alone effectively inhibits proliferation and induces apoptosis in multiple human glioblastoma cell lines. The combination index analysis shows that fluvastatin acts synergistically with common chemotherapy drugs for glioblastoma: temozolomide and irinotecan. We further show that fluvastatin acts on glioblastoma through inhibiting prenylation-dependent Ras activation. The combination of fluvastatin and low dose temozolomide resulted in remarkable inhibition of glioblastoma tumor in mice throughout the whole treatment duration without causing toxicity. Such combinatorial effects provide the basis for utilizing these FDA-approved drugs as a potential clinical approach in overcoming resistance and improving glioblastoma treatment.

Spinal cord injury (SCI) induces severe motor and sensory dysfunction. We previously showed the neuroprotective effects of COA-Cl, a novel synthesized adenosine analog, in a rat stroke model. In this study, we evaluated the neuroprotective effects of COA-Cl in acute phase of SCI. SCI was induced in rats at the T9 vertebra by using a drop device. Rats were divided into acute and subacute groups. A 5-day dose of 6 mg/kg COA-Cl in saline was given to the acute group immediately after SCI and the subacute group 4 days after SCI. Motor function assessed by Basso-Beattie-Bresnahan scoring and inclined plane test improved significantly in the acute group while the subacute group did not. Histological evaluation and TUNEL staining revealed that both the cavity volume and apoptosis were significantly decreased in the acute group compared with the subacute group. In addition, pERK/ERK was increased in the acute group 7 days after SCI. These results suggest that COA-Cl exerts neuroprotective effects via the ERK pathway when administered in the acute phase after SCI, resulting in the recovery of motor function. COA-Cl could be a novel therapeutic agent for the acute phase of SCI., (Copyright © 2020 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.)

Protein N-terminal acetyltransferase D (NatD, NAA40) that specifically acetylates the alpha-N-terminus of histone H4 and H2A has been implicated in various diseases, but no inhibitor has been reported for this important enzyme. Based on the acetyl transfer mechanism of NatD, we designed and prepared a series of highly potent NatD bisubstrate inhibitors by covalently linking coenzyme A to different peptide substrates via an acetyl or propionyl spacer. The most potent bisubstrate inhibitor displayed an apparent K i value of 1.0 nM. Biochemical studies indicated that bisubstrate inhibitors are competitive to the peptide substrate and noncompetitive to the cofactor, suggesting that NatD undergoes an ordered Bi-Bi mechanism. We also demonstrated that these inhibitors are highly specific toward NatD, displaying about 1000-fold selectivity over other closely related acetyltransferases. High-resolution crystal structures of NatD bound to two of these inhibitors revealed the molecular basis for their selectivity and inhibition mechanism, providing a rational path for future inhibitor development.

Coenzyme A (CoA) is a highly selective inhibitor of the mitotic regulatory enzyme Aurora A kinase, with a novel mode of action. Herein we report the design and synthesis of analogues of CoA as inhibitors of Aurora A kinase. We have designed and synthesised modified CoA structures as potential inhibitors, combining dicarbonyl mimics of the pyrophosphate group with a conserved adenosine headgroup and different length pantetheine-based tail groups. An analogue with a -SH group at the end of the pantotheinate tail showed the best IC50, probably due to the formation of a covalent bond with Aurora A kinase Cys290., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mutations in pank2 gene encoding pantothenate kinase 2 determine a pantothenate kinase-associated neurodegeneration, a rare disorder characterized by iron deposition in the globus pallidus. To extend our previous work, we performed microinjections of a new pank2-specific morpholino to zebrafish embryos and thoroughly analyzed vasculature development. Vessels development was severely perturbed in the head, trunk, and tail, where blood accumulation was remarkable and associated with dilation of the posterior cardinal vein. This phenotype was specific as confirmed by p53 expression analysis and injection of the same morpholino in pank2-mutant embryos. We can conclude that pank2 gene is involved in vasculature development in zebrafish embryos. The comprehension of the underlining mechanisms could be of relevance for understanding of pantothenate kinase-associated neurodegeneration.

Aurora A kinase is a master mitotic regulator whose functions are controlled by several regulatory interactions and post-translational modifications. It is frequently dysregulated in cancer, making Aurora A inhibition a very attractive antitumor target. However, recently uncovered links between Aurora A, cellular metabolism and redox regulation are not well understood. In this study, we report a novel mechanism of Aurora A regulation in the cellular response to oxidative stress through CoAlation. A combination of biochemical, biophysical, crystallographic and cell biology approaches revealed a new and, to our knowledge, unique mode of Aurora A inhibition by CoA, involving selective binding of the ADP moiety of CoA to the ATP binding pocket and covalent modification of Cys290 in the activation loop by the thiol group of the pantetheine tail. We provide evidence that covalent CoA modification (CoAlation) of Aurora A is specific, and that it can be induced by oxidative stress in human cells. Oxidising agents, such as diamide, hydrogen peroxide and menadione were found to induce Thr 288 phosphorylation and DTT-dependent dimerization of Aurora A. Moreover, microinjection of CoA into fertilized mouse embryos disrupts bipolar spindle formation and the alignment of chromosomes, consistent with Aurora A inhibition. Altogether, our data reveal CoA as a new, rather selective, inhibitor of Aurora A, which locks this kinase in an inactive state via a "dual anchor" mechanism of inhibition that might also operate in cellular response to oxidative stress. Finally and most importantly, we believe that these novel findings provide a new rationale for developing effective and irreversible inhibitors of Aurora A, and perhaps other protein kinases containing appropriately conserved Cys residues., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a genetic and early-onset neurodegenerative disorder characterized by iron accumulation in the basal ganglia. It is due to mutations in Pantothenate Kinase 2 (PANK2), an enzyme that catalyzes the phosphorylation of vitamin B5, first and essential step in coenzyme A (CoA) biosynthesis. Most likely, an unbalance of the neuronal levels of this important cofactor represents the initial trigger of the neurodegenerative process, yet a complete understanding of the connection between PANK2 malfunctioning and neuronal death is lacking. Most PKAN patients carry mutations in both alleles and a loss of function mechanism is proposed to explain the pathology. When PANK2 mutants were analyzed for stability, dimerization capacity, and enzymatic activity in vitro, many of them showed properties like the wild-type form. To further explore this aspect, we overexpressed the wild-type protein, two mutant forms with reduced kinase activity and two retaining the catalytic activity in zebrafish embryos and analyzed the morpho-functional consequences. While the wild-type protein had no effects, all mutant proteins generated phenotypes that partially resembled those observed in pank2 and coasy morphants and were rescued by CoA and vitamin B5 supplementation. The overexpression of PANK2 mutant forms appears to be associated with perturbation in CoA availability, irrespective of their catalytic activity.

The aim of this study was to use functional and morphological analyses to evaluate the protective effect of coenzyme A (CoA) in cisplatin-induced toxicity in outer hair cells (OHC). Three groups of 8 guinea pigs were used: control (group I), cisplatin-treated (group II) and cisplatin + CoA-treated (group III). In groups II and III, a single ototoxic dose of cisplatin (10 mg/kg) was injected intraperitoneally. Group III was co-treated with CoA (900 μg/kg per day for 7 consecutive days). Electrocochleography (ECoG) recordings were made before and after the 7-day treatment period in all groups. After ECoG on day 7, all animals were anesthetized and the cochleae were removed and fixed for ultrastructural analysis. Cell damage in OHC was observed with transmission electron microscopy. Cisplatin induced a significant increase in auditory thresholds (p<0.001) compared to group I (control). In contrast, group III (cisplatin + CoA) had significantly reduced thresholds (p<0.001) compared to the group treated with cisplatin alone (group II).We found no significant differences between the control group and animals co-treated with cisplatin and CoA. The electron microscopy findings in OHC were consistent with these results. Ultrastructural analysis of OHC in group II showed morphological indications of necrosis, i.e. cytoplasmic swelling and vacuolation, and mitochondrial swelling. In group III the cell morphology of OHC was preserved, with ultrastructural characteristics similar to the control group. In conclusion, co-treatment with cisplatin with CoA inhibited antineoplastic-induced cytotoxicity in OHC in a guinea pig model.

The metabolic cofactor coenzyme A (CoA) gained renewed attention because of its roles in neurodegeneration, protein acetylation, autophagy and signal transduction. The long-standing dogma is that eukaryotic cells obtain CoA exclusively via the uptake of extracellular precursors, especially vitamin B5, which is intracellularly converted through five conserved enzymatic reactions into CoA. This study demonstrates an alternative mechanism that allows cells and organisms to adjust intracellular CoA levels by using exogenous CoA. Here CoA was hydrolyzed extracellularly by ectonucleotide pyrophosphatases to 4'-phosphopantetheine, a biologically stable molecule able to translocate through membranes via passive diffusion. Inside the cell, 4'-phosphopantetheine was enzymatically converted back to CoA by the bifunctional enzyme CoA synthase. Phenotypes induced by intracellular CoA deprivation were reversed when exogenous CoA was provided. Our findings answer long-standing questions in fundamental cell biology and have major implications for the understanding of CoA-related diseases and therapies.

Background: New, safer, and more effective agents to treat hyperlipidemia and thereby prevent cardiovascular events are under research., Objective: To evaluate the lipid-lowering effects and safety of a natural hypolipidemic compound, coenzyme A (CoA) capsule, in Chinese patients with moderate dyslipidemia, compared with pantethine., Methods: Overall, 216 subjects (124 males and 92 females; age, 18-75 years) with moderate dyslipidemia (triglyceride [TG], 2.3-6.5 mmol/L) were randomly divided into 2 groups administered CoA 400 U/d (n = 111) or pantethine 600 U/d (n = 105). Blood lipoproteins, liver and renal function, blood glucose, and complete blood count were measured at baseline and after 4- and 8-week treatment., Results: TG reduction was 26.0% with CoA and 17.4% with pantethine after 4 weeks and 33.3% and 16.5% after 8 weeks; compared with baseline, the reduction was significant (P < .01) in both groups. The difference between the 2 groups was significant at both 4 weeks (P = .0413) and 8 weeks (P < .001). Compared with baseline, total cholesterol and non-high-density lipoprotein cholesterol (non-HDL-C) were reduced, whereas HDL-C was increased with CoA after 8 weeks (all P < .05). Compared with pantethine, total cholesterol (P = .026) and non-HDL-C (P = .005) were significantly reduced after 8 weeks of CoA treatment. There was no statistical difference in low-density lipoprotein cholesterol or HDL-C between the 2 groups (P > .05) and no difference in blood glucose, hepatic or renal function, myopathy, or gastrointestinal tract symptoms., Conclusions: CoA can improve TG and other lipoprotein parameters to a greater extent than pantethine in moderate dyslipidemia, with no obvious adverse effects., (Copyright © 2015 National Lipid Association. Published by Elsevier Inc. All rights reserved.)

Our previous observation that NADP-dependent secondary alcohol dehydrogenase (ADH-1) is down-regulated in metronidazole-resistant Trichomonas vaginalis isolates prompted us to further characterise the enzyme. In addition to its canonical enzyme activity as a secondary alcohol dehydrogenase, a pronounced, so far unknown, background NADPH-oxidising activity in absence of any added substrate was observed when the recombinant enzyme or T. vaginalis extract were used. This activity was strongly enhanced at low oxygen concentrations. Unexpectedly, all functions of ADH-1 were efficiently inhibited by coenzyme A which is a cofactor of a number of key enzymes in T. vaginalis metabolism, i.e. pyruvate:ferredoxin oxidoreductase (PFOR). These observations could be extended to Entamoeba histolytica and Tritrichomonas foetus, both of which have a homologue of ADH-1, but not to Giardia lamblia which lacks an NADP-dependent secondary alcohol dehydrogenase. Although we could not identify the substrate of the observed background activity, we propose that ADH-1 functions as a major sink for NADPH in microaerophilic parasites at low oxygen tension., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objectives: The aim of the study was to evaluate the lipid-lowering effects and clinical safety of a natural hypolipidemic compound, coenzyme A (CoA) capsule, in Chinese patients with moderate dyslipidemia., Methods: A total of 244 subjects (170 males and 74 females; aged 18-75 y) having moderate dyslipidemia (triglyceride [TG], 2.3-6.5 mmol · L(-1)) were randomly divided into 3 groups, to which placebo (group A, n = 81), CoA 200 U/d (group B, n = 79), and CoA 400 U/d (group C, n = 84) were administered, respectively. Blood lipoproteins, liver and renal functions, blood glucose, and complete blood count were measured at the baseline and after 4 or 8 weeks of treatment., Results: After treatment for 4 weeks, TG was reduced by 5.1, 15.7, and 14.4% in groups A, B, and C, respectively. After treatment for 8 weeks, TG decreased .9, 21.7, and 36.1%, respectively. Compared with group A, the primary efficacy outcome TG in groups B and C significantly decreased (P < .01), and the difference between groups B and C was also significant (P < .01). Plasma total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol were not significantly different. Furthermore, there was no difference in blood glucose, hepatic and renal function test parameters, incidence of myopathy, or gastrointestinal tract symptoms among the 3 groups., Conclusion: CoA can effectively reduce plasma TG levels in subjects with moderate dyslipidemia and has no obvious adverse effect.

Disruption of the unusual thiol-based redox homeostasis mechanisms in Staphylococcus aureus represents a unique opportunity to identify new metabolic processes and new targets for intervention. Targeting uncommon aspects of CoASH biosynthetic and redox functions in S. aureus, the antibiotic CJ-15,801 has recently been demonstrated to be an antimetabolite of the CoASH biosynthetic pathway in this organism; CoAS-mimetics containing α,β-unsaturated sulfone and carboxyl moieties have also been exploited as irreversible inhibitors of S. aureus coenzyme A-disulfide reductase (SaCoADR). In this work we have determined the crystal structures of three of these covalent SaCoADR-inhibitor complexes, prepared by inactivation of wild-type enzyme during turnover. The structures reveal the covalent linkage between the active-site Cys43-S(γ) and C(β) of the vinyl sulfone or carboxyl moiety. The full occupancy of two inhibitor molecules per enzyme dimer, together with kinetic analyses of the wild-type/C43S heterodimer, indicates that half-sites-reactivity is not a factor during normal catalytic turnover. Further, we provide the structures of SaCoADR active-site mutants; in particular, Tyr419'-OH plays dramatic roles in directing intramolecular reduction of the Cys43-SSCoA redox center, in the redox asymmetry observed for the two FAD per dimer in NADPH titrations, and in catalysis. The two conformations observed for the Ser43 side chain in the C43S mutant structure lend support to a conformational switch for Cys43-S(γ) during its catalytic Cys43-SSCoA/Cys43-SH redox cycle. Finally, the structures of the three inhibitor complexes provide a framework for design of more effective inhibitors with therapeutic potential against several major bacterial pathogens.

1,4-Dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) synthase, or MenB, catalyzes an intramolecular Claisen condensation involving two oxyanion intermediates in the biosynthetic pathway of menaquinone, an essential respiration electron transporter in many microorganisms. Here we report the finding that the DHNA-CoA product and its analogues bind and inhibit the synthase from Escherichia coli with significant ultraviolet--visible spectral changes, which are similar to the changes induced by deprotonation of the free inhibitors in a basic solution. Dissection of the structure--affinity relationships of the inhibitors identifies the hydroxyl groups at positions 1 (C1-OH) and 4 (C4-OH) of DHNA-CoA or their equivalents as the dominant and minor sites, respectively, for the enzyme--ligand interaction that polarizes or deprotonates the bound ligands to cause the observed spectral changes. In the meantime, spectroscopic studies with active site mutants indicate that C4-OH of the enzyme-bound DHNA-CoA interacts with conserved polar residues Arg-91, Tyr-97, and Tyr-258 likely through a hydrogen bonding network that also includes Ser-161. In addition, site-directed mutation of the conserved Asp-163 to alanine causes a complete loss of the ligand binding ability of the protein, suggesting that the Asp-163 side chain is most likely hydrogen-bonded to C1-OH of DHNA-CoA to provide the dominant polarizing effect. Moreover, this mutation also completely eliminates the enzyme activity, strongly supporting the possibility that the Asp-163 side chain provides a strong stabilizing hydrogen bond to the tetrahedral oxyanion, which takes a position similar to that of C1-OH of the enzyme-bound DHNA-CoA and is the second high-energy intermediate in the intracellular Claisen condensation reaction. Interestingly, both Arg-91 and Tyr-97 are located in a disordered loop forming part of the active site of all available DHNA-CoA synthase structures. Their involvement in the interaction with the small molecule ligands suggests that the disordered loop is folded in interaction with the substrates or reaction intermediates, supporting an induced-fit catalytic mechanism for the enzyme.

Histone and protein acetylation catalyzed by p300/CBP transcriptional coactivator regulates a variety of key biological pathways. This study investigates the proposed Theorell-Chance or "hit-and-run" catalytic mechanism of p300/CBP histone acetyltransferase (HAT) using bisubstrate analogs. A range of histone peptide tail peptide-CoA conjugates with different length linkers were synthesized and evaluated as inhibitors of p300 HAT. We show that longer linkers between the histone tail peptide and the CoA substrate moieties appear to allow for dual engagement of the two binding surfaces. Results with D1625R/D1628R double mutant p300 HAT further confirm the requirement for a negatively charged surface on the enzyme to interact with the histone tail., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Coenzyme A (CoA) analogues containing α,β-unsaturated ester, ketone, and sulfone moieties were prepared by chemo-enzymatic synthesis as inhibitors of coenzyme A disulfide reductase (CoADR), a proven and as yet unexploited drug target in Staphylococcus aureus. Among these Michael acceptor-containing CoA analogues, which were designed to target CoADR's single essential active site cysteine for conjugate addition, a phenyl vinyl sulfone-containing analogue showed the most potent inhibition with a competitive K(i) of ∼40 nM, and time-dependent inactivation with a second-order rate of inactivation constant of ∼40,000 s(-1)·M(-1). Our results suggest that electrophilic substrate analogues should be considered as potential inhibitors of other medicinally relevant disulfide reductase enzymes.

In the aerobic metabolism of aromatic substrates, oxygenases use molecular oxygen to hydroxylate and finally cleave the aromatic ring. In the case of the common intermediate benzoate, the ring cleavage substrates are either catechol (in bacteria) or 3,4-dihydroxybenzoate (protocatechuate, mainly in fungi). We have shown before that many bacteria, e.g. Azoarcus evansii, the organism studied here, use a completely different mechanism. This elaborate pathway requires formation of benzoyl-CoA, followed by an oxygenase reaction and a nonoxygenolytic ring cleavage. Benzoyl-CoA transformation is catalyzed by the iron-containing benzoyl-CoA oxygenase (BoxB) in conjunction with an FAD and iron-sulfur centers containing reductase (BoxA), which donates electrons from NADPH. Here we show that benzoyl-CoA oxygenase actually does not form the 2,3-dihydrodiol of benzoyl-CoA, as formerly postulated, but the 2,3-epoxide. An enoyl-CoA hydratase (BoxC) uses two molecules of water to first hydrolytically open the ring of 2,3-epoxybenzoyl-CoA, which may proceed via its tautomeric seven-membered oxepin ring form. Then ring C2 is hydrolyzed off as formic acid, yielding 3,4-dehydroadipyl-CoA semialdehyde. The semialdehyde is oxidized by a NADP(+)-dependent aldehyde dehydrogenase (BoxD) to 3,4-dehydroadipyl-CoA. Final products of the pathway are formic acid, acetyl-CoA, and succinyl-CoA. This overlooked pathway occurs in 4-5% of all bacteria whose genomes have been sequenced and represents an elegant strategy to cope with the high resonance energy of aromatic substrates by forming a nonaromatic epoxide.

The Arabidopsis thaliana genome contains two genes encoding the mitochondrial NAD-malic enzyme (NAD-ME), NAD-ME1 (At2g13560) and NAD-ME2 (At4g00570). The characterization of recombinant NAD-ME1 and -2 indicated that both enzymes assemble as active homodimers; however, a heterodimeric enzyme (NAD-MEH) can also be detected by electrophoretic studies. To analyze the metabolic contribution of each enzymatic entity, NAD-MEH was obtained by a co-expression-based recombinant approach, and its kinetic and regulatory properties were analyzed. The three NAD-MEs show similar kinetic properties, although they differ in the regulation by several metabolic effectors. In this regard, whereas fumarate activates NAD-ME1 and CoA activates NAD-ME2, both compounds act synergistically on NAD-MEH activity. The characterization of two chimeric enzymes between NAD-ME1 and -2 allowed specific domains of the primary structure, which are involved in the differential allosteric regulation, to be identified. NAD-ME1 and -2 subunits showed a distinct pattern of accumulation in the separate components of the floral organ. In sepals, the NAD-ME1 subunit is present at a slightly higher proportion than the NAD-ME2 subunit, and thus, NAD-MEH and NAD-ME1 act in concert in this tissue. On the other hand, NAD-ME2 is the only isoform present in anthers. In view of the different properties of NAD-ME1, -2, and -H, we suggest that mitochondrial NAD-ME activity may be regulated by varying native association in vivo, rendering enzymatic entities with distinct allosteric regulation to fulfill specific roles. The presence of three different NAD-ME entities, which originate by alternative associations of two subunits, is suggested to be a novel phenomenon unique to plant mitochondria.

Background: 5-Hydroxydecanoate (5-HD) inhibits preconditioning, and it is assumed to be a selective inhibitor of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels. However, 5-HD is a substrate for mitochondrial outer membrane acyl-CoA synthetase, which catalyzes the reaction: 5HD + CoA + ATP --> 5-HD-CoA (5-hydroxydecanoyl-CoA) + AMP + pyrophosphate. We aimed to determine whether the reactants or principal product of this reaction modulate sarcolemmal K(ATP) (sarcK(ATP)) channel activity., Methods: Single sarcK(ATP) channel currents were measured in inside-out patches excised from rat ventricular myocytes. In addition, sarcK(ATP) channel activity was recorded in whole-cell configuration or in giant inside-out patches excised from oocytes expressing Kir6.2/SUR2A., Results: 5-HD inhibited (IC(50) approximately 30 microM) K(ATP) channel activity, albeit only in the presence of (non-inhibitory) concentrations of ATP. Similarly, when the inhibitory effect of 0.2 mM ATP was reversed by 1 microM oleoyl-CoA, subsequent application of 5-HD blocked channel activity, but no effect was seen in the absence of ATP. Furthermore, we found that 1 microM coenzyme A (CoA) inhibited sarcK(ATP) channels. Using giant inside-out patches, which are weakly sensitive to "contaminating" CoA, we found that Kir6.2/SUR2A channels were insensitive to 5-HD-CoA. In intact myocytes, 5-HD failed to reverse sarcK(ATP) channel activation by either metabolic inhibition or rilmakalim., General Significance: SarcK(ATP) channels are inhibited by 5-HD (provided that ATP is present) and CoA but insensitive to 5-HD-CoA. 5-HD is equally potent at "directly" inhibiting sarcK(ATP) and mitoK(ATP) channels. However, in intact cells, 5-HD fails to inhibit sarcK(ATP) channels, suggesting that mitochondria are the preconditioning-relevant targets of 5-HD., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

The adenine nucleotide translocator (ANT) accomplishes the exchange of ATP from the mitochondrial matrix with cytoplasmic ADP. While investigating the biochemical mechanism of retinoic acid (RA) on the ANT via retinoylation, we have found and subsequently demonstrated a positive influence of Coenzyme A (CoA) on the transport of ATP across the membranes of rat liver mitochondria. CoA enhances ANT activity in a dose-dependent manner modifying the V(max) (673.3+/-20.7 nmol ATP/mgprotein/min versus 155.0+/-1.9 nmol ATP/mgprotein/min), the IC(50) for the specific inhibitor carboxyatractyloside (CATR) (0.142+/-0.012 microM versus 0.198+/-0.011 microM) but not the K(m) (22.50+/-0.52 microM versus 22.19+/-0.98 microM). Data suggest a likely enzymatic involvement in the interaction between ANT and CoA. The effect of CoA is observed in mitochondria from several different tissues.

The Arabidopsis thaliana genome contains four genes encoding NADP-malic enzymes (NADP-ME1-4). Two isoenzymes, NADP-ME2 and NADP-ME3, which are shown to be located in the cytosol, share a remarkably high degree of identity (90%). However, they display different expression patterns and show distinct kinetic properties, especially with regard to their regulation by effectors, in both the forward (malate oxidative decarboxylation) and reverse (pyruvate reductive carboxylation) reactions. In order to identify the domains in the primary structure that could be responsible for the regulatory differences, four chimeras between these isoenzymes were constructed and analysed. All chimeric versions exhibited the same native structures as the parental proteins. Analysis of the chimeras constructed indicated that the region from amino acid residue 303 to the C-terminal end of NADP-ME2 is critical for fumarate activation. However, the region flanked by amino acid residues 303 and 500 of NADP-ME3 is involved in the pH-dependent inhibition by high malate concentration. Furthermore, the N-terminal region of NADP-ME2 is necessary for the activation by succinate of the reverse reaction. Overall, the results show that NADP-ME2 and NADP-ME3 are able to distinguish and interact differently with similar C(4) acids as a result of minimal structural differences. Therefore, although the active sites of NADP-ME2 and NADP-ME3 are highly conserved, both isoenzymes acquire different allosteric sites, leading to the creation of proteins with unique regulatory mechanisms, probably best suited to the specific organ and developmental pattern of expression of each isoenzyme.

Acetyl group turnover on specific lysine epsilon-amino groups of the core chromosomal histones regulates DNA accessibility function, and the acetylating and deacetylating enzymes that govern the turnover provide important targets for the development of anti-cancer drugs. Histone deacetylase (HDAC) inhibitors have been developed and evaluated extensively in clinical trials, while the development of inhibitors of histone acetyltransferase (HAT) has proceeded more slowly. Here we have examined the cellular effects of an S-substituted coenzyme A (CoA) inhibitor of histone acetylation, consisting of spermidine (Spd) linked to the S-terminus of CoA through a thioglycolic acid linkage (adduct abbreviated as Spd-CoA), as well as the effects of a truncated Spd-CoA derivative lacking the negatively charged portion of the CoA moiety. While exposure of cancer cells to Spd-CoA has little effect on cell viability, it causes a rapid inhibition of histone acetylation that correlates with a transient arrest of DNA synthesis, a transient delay in S-phase progression, and an inhibition of nucleotide excision repair and DNA double strand break repair. These effects correlate with increased cellular sensitivity to the DNA-targeted chemotherapeutic drugs, cisplatin (Platinol()) and 5-fluorouracil, to the DNA damaging drug, camptothecin, and to UV-C irradiation. The sensitization effects of Spd-CoA are not observed in normal cells due to a barrier to uptake. The truncated Spd-CoA derivative displays similar but enhanced chemosensitization effects, suggesting that further modifications of the Spd-CoA structure could further improve potency. The results demonstrate that Spd-CoA and its truncated version are efficiently and selectively internalized into cancer cells, and suggest that the resulting inhibition of acetylation-dependent DNA repair enhances cellular sensitivity to DNA damage. These and related inhibitors of histone acetylation could therefore constitute a novel class of potent therapy sensitizers applicable to a broad range of conventional cancer treatments.

Block copolymers, poly(N-vinylprrolidone)-block-poly(styrene-alter-maleic anhydride) (PVP-b-PSMA) and poly(N-vinylprrolidone)-block-poly(N,N-dimethylaminoethyl methacrylate) (PVP-b-PDMAEMA), were synthesized by reversible addition- fragmentation chain transfer (RAFT) polymerization. In aqueous media, this a pair of oppositely-charged diblock copolymers could self-assemble into stable and narrow distribution polyion complex micelles (PICMs). Transmission electron micrographs (TEM) and dynamic light scattering (DLS) analysis showed that the micelles to be spherically shaped with mean hydrodynamic diameter around 70nm. In addition, the PICMs display ability to response to external stimuli. All of theses features are quite feasible for utilizing it as a novel intelligent drug delivery system. In order to assess its application in biomedical area, release profiles of coenzyme A (Co A) from PICMs were studied under both simulated gastric and intestinal pH conditions. The release was much quicker in pH 7.4 buffer than in pH 2.0 solution. Based on these results, these PICMs could be a potential pH-sensitive carrier for colon-specific drug delivery system.

The pyruvate uptake rate in inverted submitochondrial vesicles prepared from rat liver was optimized and further characterized; the potential inhibitory effects of the anticonvulsive drug valproic acid or 2-n-propyl-pentanoic acid (VPA), Delta4-valproic acid or 2-n-propyl-4-pentenoic acid and the respective coenzyme A (CoA) conjugates were studied in the presence of a proton gradient. All tested VPA metabolites inhibited the pyruvate uptake, but the CoA esters were stronger inhibitors (40% and 60% inhibition, respectively, for valproyl-CoA and Delta4-valproyl-CoA, at 1mM). At the same concentration, the specific inhibitor 2-cyano-4-hydroxycinnamate decreased the pyruvate uptake rate by 70%. The reported inhibition of the mitochondrial pyruvate uptake may explain the significant impairment of the pyruvate-driven oxidative phosphorylation induced by VPA.

Membrane permeability transition (MPT) of mitochondria has an important role in apoptosis of various cells. The classic type of MPT is characterized by increased Ca(2+) transport, membrane depolarization, swelling, and sensitivity to cyclosporin A. In this study, we investigated whether L-carnitine suppresses oleic acid-induced MPT using isolated mitochondria from rat liver. Oleic acid-induced MPT in isolated mitochondria, inhibited endogenous respiration, caused membrane depolarization, and increased large amplitude swelling, and cytochrome c (Cyt. c) release from mitochondria. L-Carnitine was indispensable to beta-oxidation of oleic acid in the mitochondria, and this reaction required ATP and coenzyme A (CoA). In the presence of ATP and CoA, L-carnitine stimulated oleic acid oxidation and suppressed the oleic acid-induced depolarization, swelling, and Cyt. c release. L-Carnitine also contributed to maintaining mitochondrial function, which was decreased by the generation of free fatty acids with the passage of time after isolation. These results suggest that L-carnitine acts to maintain mitochondrial function and suppresses oleic acid-mediated MPT through acceleration of beta-oxidation., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

This study was designed to determine whether the supplement of energy compound could attenuate strain-induced damage to skeletal muscle in rats. Energy compound is a saline mixture of the following ingredients: ATP (10mg), Coenzyme-A (50 units), Coenzyme-Q(10) (50mg), Cytochrome C (30 mg) and Vitamin B(6) (50mg). Experimental animals were injured in right gastrocnemius muscles by a strain injury model. Energy compound groups were given energy compound 10 ml/kg body weight per day since injured, while saline groups were given saline at the same dose. And a sham operation was performed on the right hindlimb of control group. Plasma was centrifuged to measure lactate dehydrogenase (LDH), lactic acid (La) and creatine kinase (CK) on 3, 7 and 14 d post injury. Muscles were removed and fixed for histology observation and immunohistochemistry assay of desmin and vimentin. The results showed a similar tendency of plasma CK, La and LDH in saline and energy compound groups, while the lower level was found in the energy-compound group. The histological examination of muscle sections revealed a lower degree of damage in the energy compound group in which the expression levels of desmin and vimentin were higher than in the saline group. It is suggested that energy compound supplement may attenuate strain-induced muscle damage and facilitate its regeneration.

We have performed a detailed investigation of the effects on platelet function of coenzyme A (CoA) and several acyl-CoAs. Platelet aggregation was measured by turbidimetry and by platelet counting; platelet shape change was measured using light scattering; P-selectin, Ca2+ mobilization and vasodilator-stimulated phosphoprotein (VASP) phosphorylation were measured by flow cytometry. The compounds investigated inhibited ADP-induced platelet aggregation; those with saturated acyl groups containing 16-18 carbons were most effective. The effects of palmitoyl-CoA (16:0) were studied in depth. It inhibited platelet shape change and Ca2+ mobilization brought about by ADP (but not other agonists) indicating antagonism at P2Y(1) receptors, and also inhibited ADP-induced P-selectin expression. Effects of palmitoyl-CoA on the platelet aggregation and Ca2+ mobilization induced by several different agonists and agonist combinations were compared with those of MRS 2179 (a P2Y(1) antagonist) and AR-C69931 (a P2Y(12) antagonist), and were consistent with palmitoyl-CoA acting mainly at P2Y(1) but also with partial antagonism at P2Y(12) receptors. Antagonism at P2Y(12) receptors was confirmed in studies of VASP-phosphorylation. Palmitoyl-CoA did not act as an antagonist at P2X(1) receptors. The results are discussed in relation to the possibility that acyl-CoAs may contribute as endogenous modulators of platelet function and might serve as lead compounds for the design of novel antithrombotics.

Oxidative damage by non-steroidal anti-inflammatory drugs (NSAIDs) has been considered relevant to the occurrence of gastro-intestinal side-effects. In the case of chiral arylpropionate derivatives like ketoprofen (KPF), this mechanism has been evidenced for the R-enantiomer, especially when chiral inversion was observed, and lets us suppose the involvement of CoA conjugates. Glucose-6-phosphate dehydrogenase (G6PD) is the crucial enzyme to regenerate the GSH pool and maintain the intracellular redox potential. This enzyme is known to be down-regulated by palmitoyl-CoA thioester. We hypothesised then that G6PD is the target of carboxylic NSAIDs, via their CoA metabolites. We used molecular docking to localise a putative site in the human G6PD then we chose the Yeast orthologue, as the most suitable species to study experimentally the precise molecular interaction. KPF-CoA was effectively shown to bind covalently to the unique cysteine residue of the yeast enzyme. Binding was found to occur in the same site as palmitoyl-CoA. It was decreased in the presence of an allosteric inhibitor of G6PD, phospho(enol)pyruvate, and was not detected with G6PD of Leuconostoc mesenteroides, which does not possess the allosteric site. This site is distinct from the catalytic site, and probably allosteric, explaining the observed non-competitive inhibition of its activity by KPF-CoA. KPF-CoA was shown to induce the production of reactive oxygen species in Caco-2 cells, where its inhibition of G6PD activity was observed.

Objective: This study compared the hemodynamic responses elicited by the endothelium-derived relaxing factor (EDRF), L-S-nitrosocysteine (L-SNC), the non-prostanoid EDRF released by acetylcholine (ACh) and nitric oxide (NO)-donors such as MAHMA NONOate, in conscious spontaneously hypertensive (SH) and normotensive Wistar-Kyoto (WKY) rats., Methods: The depressor and/or vasodilator responses elicited by intravenous injections of ACh, L-SNC and MAHMA NONOate were determined in adult WKY and SH rats before and after intravenous injection of the NO synthesis inhibitor, N(G)-nitro-L-arginine methylester (L-NAME), or the cyclooxygenase inhibitor, indomethacin., Results: The responses elicited by ACh and L-SNC were smaller in SH than in WKY rats whereas the responses elicited by MAHMA NONOate were augmented in SH rats. The ACh-induced responses were not diminished after injection of L-NAME in WKY or SH rats. Indomethacin did not affect the responses to any of the vasodilator agents in WKY or SH rats. Addition of L-SNC to whole blood or thoracic aortae from SH rats yielded similar amounts of NO to those of WKY rats., Conclusions: The vasodilator potencies of ACh and L-SNC were diminished whereas that of NO was augmented in SH rats. The loss of potency of L-SNC in SH rats was not obviously due to differences in decomposition to NO or the overactivity of cyclooxygenase factors. This study provides the first evidence that diminished endothelium-dependent vasodilation in SH rats may involve a loss of vasodilator potency of endogenous L-SNC.

Mitochondrial medium-chain acyl-CoA dehydrogenase is a key enzyme for the beta-oxidation of fatty acids, which catalyzes the FAD-dependent oxidation of a variety of acyl-CoA substrates to the corresponding trans-2-enoyl-CoA thioesters. Oct-4-en-2-ynoyl-CoA was identified as a new irreversible inhibitor of acyl-CoA dehydrogenase, and kinetic parameters K(I) and k(inact) were determined to be 11 microM and 0.025 min(-1), respectively. Triple bond between C2 and C3 of the inhibitor was identified as the functional group responsible for enzyme inactivation, and Michael addition is proposed as the mechanism for this inactivation, which is a new pathway for inactivation of MCAD by inhibitors. The inhibitor may become a lead for further development for treating non-insulin-dependent diabetes mellitus.

Activation of the AMP-activated protein kinase (AMPK) results in acute changes in cellular metabolism and transcriptional events that make the cell more robust when encountering an energy challenge. AMPK is thought to be inhibited by glycogen, the major storage form of intracellular carbohydrate. We hypothesized that long-chain acyl-CoA esters (LCACEs) might also inhibit AMPK signaling. Cytosolic LCACEs are available for immediate transport and oxidation within the mitochondria and accordingly may be representative of the lipid energy charge of the cell. We found that LCACEs inhibited phosphorylation of AMPK by the recombinant AMPK kinase (AMPKK) LKB1/STRAD/MO25 in a concentration-dependent manner. Palmitoyl-CoA (PCoA) did not affect the activity of phosphothreonine-172 AMPK. PCoA potently inhibited AMPKK purified from liver. Conversely, PCoA stimulated the kinase activity of LKB1/STRAD/MO25 toward the peptide substrate LKB1tide. Octanoyl-CoA, palmitate, and palmitoylcarnitine did not inhibit AMPKK activity. Removal of AMP from the reaction mixture resulted in reduced AMPKK activity in the presence of PCoA. In conclusion, these results demonstrate that the AMPKK activity of LKB1/STRAD/MO25 is substrate specific and distinct from the kinase activity of LKB1/STRAD/MO25 toward the peptide substrate LKB1tide. They also demonstrate that LCACEs inhibit the AMPKK activity of LKB1/STRAD/MO25 in a specific manner with a dependence on both a long fatty chain and a CoA moiety. These results suggest that the AMPK signaling cascade may directly sense and respond to the lipid energy charge of the cell.

Pantothenate kinase (PanK) catalyzes the first step in the biosynthesis of the essential and ubiquitous cofactor coenzyme A (CoA) in all organisms. Two well characterized isoforms of the enzyme are known: a prokaryotic PanK that predominates in eubacteria and a eukaryotic isoform that has primarily been characterized from mammalian and plant sources. Curiously, the genomes of certain pathogenic bacteria, including Helicobacter pylori and Pseudomonas aeruginosa, do not contain a PanK similar to either isoform, although these organisms possess all the other biosynthetic machinery required for CoA production. In this study we cloned, overexpressed and characterized an enzyme from Bacillus subtilis and its homologue from H. pylori and show that they catalyze the ATP-dependent phosphorylation of pantothenate. These enzymes do not share sequence homology with any known PanK, and unlike the bacterial and eukaryotic PanK isoforms their activity is not regulated by either CoA or acetyl-CoA. They also do not accept the pantothenic acid antimetabolite N-pentylpantothenamide as a substrate or are inhibited by it. Taken together, these results point to the identification of a third distinct isoform of PanK that accounts for the only known activity of the enzyme in pathogens such as H. pylori and P. aeruginosa.

Direct desaturation of free myristic acid by hen liver microsomal Delta(9)-desaturase without prior activation to myristoyl-CoA by the addition of adenosine triphosphate (ATP) and CoA was observed when the incubation medium was mixed at mixing speeds of >250 rpm in the presence of fatty acid-binding proteins (FABP). Desaturation was linear with time and proportional to the microsomal protein concentration. Desaturation was maximal at pH 7.9. The greatest desaturation rate was observed at a mixing speed of 500 rpm in the presence of FABP. Desaturation decreased at mixing speeds of >500 rpm. Data suggest that when myristic acid is bound to FABP in the form of protein-monomer complexes, its activation to the CoA derivative is not necessary for it to be desaturated by the Delta(9)-desaturase when using mixing rates of >250 rpm. Myristic acid-FABP complexes serve as substrates for the Delta(9)-desaturase at mixing rates of >250 rpm. Desaturation was reduced by bovine serum albumin and alpha-bromohexadecanoate, and no desaturation was observed in the absence of FABP. These findings suggest that FABP may regulate the accessibility of fatty acids in the desaturation reaction to the active site of the desaturase rather than just protect the membrane-bound desaturase from the cytotoxic effect of free fatty acids.

Propionyl-coenzyme A synthetases (PrpE) of Salmonella choleraesuis and Ralstonia solanacearum sharing 62% identity in amino acid sequence to each other were cloned, expressed in Escherichia coli and purified. Both enzymes catalyzed acetyl-, propionyl-, butyryl- and acrylyl-coenzyme A formation with the highest k(cat)/K(m) values for propionate. They displayed sigmoidal homotrophic autoactivation kinetics for propionate but not for the other acyl substrates tested. Besides, substrate inhibition kinetics was observed for co-substrates, i.e. ATP and CoA. Based on the kinetic data reported herein, the reaction mechanisms of the enzyme are discussed.

We have recently uncovered the full expression of novel cutaneous serotoninergic and melatoninergic systems in the human and hamster skin. In this work, we have characterized serotonin metabolism in the rat skin using liquid chromatography-mass spectrometry and found that serotonin undergoes acetylation in the presence of acetyl coenzyme A. Inhibition of serotonin acetylation with Cole bisubstrate inhibitor shows that rat skin expresses both arylalkylamine and arylamine N-acetyltransferase activities. The serotonin degradation product-5-hydroxyindole acetic acid is also detected and pargyline (monoaminooxidase inhibitor) suppresses almost completely 5-hydroxyindole acetic acid accumulation. Together with previous data, the present study clearly demonstrates that biotransformation of serotonin in mammalian skin follows two alternate pathways. In the first pathway, serotonin is acetylated by arylalkylamine and arylamine N-acetyltransferases to generate the precursor of melatonin. Alternately, serotonin may undergo oxidative deamination by monoaminooxidase followed by enzymatic degradation by aldehyde dehydrogenase into 5-hydroxyindole acetic acid, which is presumably devoid of biological activity. Thus, the current methodological development of a liquid chromatography-mass spectrometry-based assay allows rapid resolution of the cutaneous metabolism of serotonin.

Lys-CoA (1) is a selective inhibitor of p300 histone acetyltransferase (HAT) but shows poor pharmacokinetic properties because of its multiply charged phosphates. In an effort to overcome this limitation, truncated derivatives of 1 were designed, synthesized and tested as p300HAT inhibitors as well as substrates for the CoA biosynthetic bifunctional enzyme phosphopantetheine adenylyltransferase-dephospho-CoA kinase (PPAT/DPCK). Lys-pantetheine (3) and Lys-phosphopantetheine (2) showed no detectable p300HAT inhibition whereas 3'-dephospho-Lys-CoA (5) was a modest p300 inhibitor with IC(50) of 1.6 microM (compared to IC(50) of approximately 50 nM for 1 blocking p300). Compound 2 was shown to be an efficient substrate for PPAT whereas 5 was a very poor DPCK substrate. Further analysis with 3'-dephospho-Me-SCoA (7) indicated that DPCK shows relatively narrow capacity to accept substrates with sulfur substitution. While these results suggest that truncated derivatives of 1 will be of limited value as lead agents for p300 blockade in vivo, they augur well for prodrug versions of CoA analogues that do not require 3'-phosphate substitution for efficient binding to their targets, such as the GCN-5 related N-acetyltransferases.

Pantothenate kinase (PanK) is a rate-determining enzyme in coenzyme A (CoA) biosynthesis. The mouse murine pantothenate kinase (Pank1) gene consists of seven introns and eight exons and is located on chromosome 19 (19C2-3). Two biochemically distinct PanK1 protein isoforms, PanK1 alpha and PanK1 beta, are encoded by the Pank1 gene. Both proteins have the same 363 amino acid catalytic domain encoded by exons 2 through 7. The PanK1 beta transcript begins with exon 1 beta and translates into a ten-residue amino terminus plus the catalytic domain. The PanK1 alpha transcript initiates at an alternate upstream site at exon 1 alpha which is spliced with exon 2, excluding exon 1 beta. Exon 1 alpha encodes a 184-residue regulatory domain at the amino terminus of the PanK1 alpha protein that confers feedback inhibition by free CoA and long-chain acyl-CoA, and increases the regulation of PanK enzyme activity by acetyl-CoA and malonyl-CoA. Differential expression of the PanK1 alpha and PanK1 beta transcripts would alter the amount of CoA produced in cells as a function of the ratio of free CoA to acetyl-CoA, a reflection of the metabolic status of the tissue.

Coenzyme A, a cofactor in enzymatic acetyl transfer reactions, stimulates collagen production in cultured fibroblasts. The mechanisms involved in this collagen stimulation were investigated. Enzymatic studies using radiolabeled procollagen as substrate revealed that coenzyme A enhanced prolyl hydroxylase activity. Prolyl hydroxylase is a key enzyme in collagen synthesis acting by hydroxylation of proline residues in procollagen peptide, which is necessary for stabilizing collagen. Northern blot analysis demonstrated that coenzyme A also enhanced mRNA levels of both the alpha subunit of prolyl hydroxylase and the alpha1 chain of type I collagen. The levels of protein production of prolyl hydroxylase and type I collagen were also increased in cultured fibroblasts by coenzyme A, which correlated well with observations from Northern blotting. On the other hand, coenzyme A did not stimulate the activity nor the gene expression of two other processing enzymes: lysyl hydroxylase, which provides the sites for glycosylation and crosslinking between collagen peptides, and lysyl oxidase, a fundamental enzyme in intermolecular crosslinking. These results indicate that coenzyme A stimulates collagen production by at least two separate mechanisms: by enhancing prolyl hydroxylase activity as well as stimulating gene expression of the alpha subunit of this enzyme, and by stimulating gene expression of alpha1 chain of type I collagen.

Treatment of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme in carrot cells, with phenylglyoxal yielded a chemically modified protein in which approximately two moles of the reagent were covalently attached to each subunit of the enzyme. Only NADH- but not NADPH-associated form of native 6-hydroxymellein synthase was inhibited by cerulenin; however, the NADPH-synthase complex lost the insensitivity by the chemical modification of the enzyme protein with phenylglyoxal. Appreciable differences in K(m) values observed between the NADPH- and NADH-associated enzymes were greatly reduced by the treatment with phenylglyoxal. Although the catalytic activity of the NADPH-associated synthase was enhanced by the addition of free CoA, the compound exhibited a significant inhibitory activity to the phenylglyoxal-modified enzyme. A marked deuterium isotope effect in the catalytic reaction of the native synthase-NADPH complex was appreciably decreased in the chemically modified enzyme. These results strongly suggest that an electrostatic interaction between the phosphate group attached to the 2'-position of adenosyl moiety of NADPH and the phenylglyoxal-sensitive amino acid residue, probably arginine, at the reaction center of 6-hydroxymellein synthase regulates several biochemical properties of this multifunctional enzyme.

The peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-activated transcription factor and a key regulator of lipid homeostasis. Numerous fatty acids and eicosanoids serve as ligands and activators for PPARalpha. Here we demonstrate that S-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog, antagonizes the effects of agonists on PPARalpha conformation and function in vitro. In electrophoretic mobility shift assays, S-hexadecyl-CoA prevented agonist-induced binding of the PPARalpha-retinoid X receptor alpha heterodimer to the acyl-CoA oxidase peroxisome proliferator response element. PPARalpha bound specifically to immobilized palmitoyl-CoA and Wy14643, but not BRL49653, abolished binding. S-Hexadecyl-CoA increased in a dose-dependent and reversible manner the sensitivity of PPARalpha to chymotrypsin digestion, and the S-hexadecyl-CoA-induced sensitivity required a functional PPARalpha ligand-binding pocket. S-Hexadecyl-CoA prevented ligand-induced interaction between the co-activator SRC-1 and PPARalpha but increased recruitment of the nuclear receptor co-repressor NCoR. In cells, the concentration of free acyl-CoA esters is kept in the low nanomolar range due to the buffering effect of high affinity acyl-CoA-binding proteins, especially the acyl-CoA-binding protein. By using PPARalpha expressed in Sf21 cells for electrophoretic mobility shift assays, we demonstrate that S-hexadecyl-CoA was able to increase the mobility of the PPARalpha-containing heterodimer even in the presence of a molar excess of acyl-CoA-binding protein, mimicking the conditions found in vivo.

all-trans-Retinoic acid, a highly active form of vitamin A in inducing cellular differentiation, is incorporated covalently into proteins both in vivo and in vitro. The relative rates of incorporation of all-trans-11,12-(3)H-retinoic acid into rat tissue homogenates in the presence of ATP and coenzyme A were testes>>lung> or =brain> or =kidney>liver. Although all studied cellular organelles of the testes incorporated (3)H-retinoic acid into protein, mitochondria were by far the most active; indeed, up to 25% of the added tritiated retinoic acid (RA) became covalently bound to protein in a 90 min incubation period. In the absence of ATP, coenzyme A, or both cofactors, the amount of RA incorporated into the proteins of testes mitochondria fell to 37%, 16%, and 11%, respectively, of that incorporated in their presence. N-Ethylmaleimide (5 mM) strongly inhibited the reaction. Boiled mitochondria were inactive. After extensive extraction with CHCl(3)-CH(3)OH, the protein-bound radioactivity, which proved largely to be retinoic acid, was released by treatment with proteinase K, hydroxylamine, and dilute base. Thus, retinoic acid is most probably linked to protein as a thiol ester. By SDS-polyacrylamide gel electrophoresis, four protein fractions with molecular masses of approx. 20, 24, 29, and 45 kDa, as well as smaller amounts of larger entities, were labeled in testes mitochondria. The possible identities and roles of these retinoylated proteins are currently being explored.