Fumarate Analogs Act as Allosteric Inhibitors of the Human Mitochondrial NAD(P)+-Dependent Malic Enzyme

Human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME) is allosterically activated by the four-carbon trans dicarboxylic acid, fumarate. Previous studies have suggested that the dicarboxylic acid in a trans conformation around the carbon-carbon double bond is required for the allosteric activation of the enzyme. In this paper, the allosteric effects of fumarate analogs on m-NAD(P)-ME are investigated. Two fumarate-insensitive mutants, m-NAD(P)-ME_R67A/R91A and m-NAD(P)-ME_K57S/E59N/K73E/D102S, as well as c-NADP-ME, were used as the negative controls. Among these analogs, mesaconate, trans-aconitate, monomethyl fumarate and monoethyl fumarate were allosteric activators of the enzyme, while oxaloacetate, diethyl oxalacetate, and dimethyl fumarate were found to be allosteric inhibitors of human m-NAD(P)-ME. The IC50 value for diethyl oxalacetate was approximately 2.5 mM. This paper suggests that the allosteric inhibitors may impede the conformational change from open form to closed form and therefore inhibit m-NAD(P)-ME enzyme activity

Glycoprotein Ib activation by thrombin stimulates the energy metabolism in human platelets

<div><p>Thrombin-induced platelet activation requires substantial amounts of ATP. However, the specific contribution of each ATP-generating pathway <i>i</i>.<i>e</i>., oxidative phosphorylation (OxPhos) versus glycolysis and the biochemical mechanisms involved in the thrombin-induced activation of energy metabolism remain unclear. Here we report an integral analysis on the role of both energy pathways in human platelets activated by several agonists, and the signal transducing mechanisms associated with such activation. We found that thrombin, Trap-6, arachidonic acid, collagen, A23187, epinephrine and ADP significantly increased glycolytic flux (3–38 times <i>vs</i>. non-activated platelets) whereas ristocetin was ineffective. OxPhos (33 times) and mitochondrial transmembrane potential (88%) were increased only by thrombin. OxPhos was the main source of ATP in thrombin-activated platelets, whereas in platelets activated by any of the other agonists, glycolysis was the principal ATP supplier. In order to establish the biochemical mechanisms involved in the thrombin-induced OxPhos activation in platelets, several signaling pathways associated with mitochondrial activation were analyzed. Wortmannin and LY294002 (PI3K/Akt pathway inhibitors), ristocetin and heparin (GPIb inhibitors) as well as resveratrol, ATP (calcium-release inhibitors) and PP1 (Tyr-phosphorylation inhibitor) prevented the thrombin-induced platelet activation. These results suggest that thrombin activates OxPhos and glycolysis through GPIb-dependent signaling involving PI3K and Akt activation, calcium mobilization and protein phosphorylation.</p></div

Large-scale genome-wide association studies and meta-analyses of longitudinal change in adult lung function.

BACKGROUND: Genome-wide association studies (GWAS) have identified numerous loci influencing cross-sectional lung function, but less is known about genes influencing longitudinal change in lung function. METHODS: We performed GWAS of the rate of change in forced expiratory volume in the first second (FEV1) in 14 longitudinal, population-based cohort studies comprising 27,249 adults of European ancestry using linear mixed effects model and combined cohort-specific results using fixed effect meta-analysis to identify novel genetic loci associated with longitudinal change in lung function. Gene expression analyses were subsequently performed for identified genetic loci. As a secondary aim, we estimated the mean rate of decline in FEV1 by smoking pattern, irrespective of genotypes, across these 14 studies using meta-analysis. RESULTS: The overall meta-analysis produced suggestive evidence for association at the novel IL16/STARD5/TMC3 locus on chromosome 15 (P  =  5.71 × 10(-7)). In addition, meta-analysis using the five cohorts with ≥3 FEV1 measurements per participant identified the novel ME3 locus on chromosome 11 (P  =  2.18 × 10(-8)) at genome-wide significance. Neither locus was associated with FEV1 decline in two additional cohort studies. We confirmed gene expression of IL16, STARD5, and ME3 in multiple lung tissues. Publicly available microarray data confirmed differential expression of all three genes in lung samples from COPD patients compared with controls. Irrespective of genotypes, the combined estimate for FEV1 decline was 26.9, 29.2 and 35.7 mL/year in never, former, and persistent smokers, respectively. CONCLUSIONS: In this large-scale GWAS, we identified two novel genetic loci in association with the rate of change in FEV1 that harbor candidate genes with biologically plausible functional links to lung function

Exploiting Mitochondrial Dysfunction for Effective Elimination of Imatinib-Resistant Leukemic Cells

Challenges today concern chronic myeloid leukemia (CML) patients resistant to imatinib. There is growing evidence that imatinib-resistant leukemic cells present abnormal glucose metabolism but the impact on mitochondria has been neglected. Our work aimed to better understand and exploit the metabolic alterations of imatinib-resistant leukemic cells. Imatinib-resistant cells presented high glycolysis as compared to sensitive cells. Consistently, expression of key glycolytic enzymes, at least partly mediated by HIF-1α, was modified in imatinib-resistant cells suggesting that imatinib-resistant cells uncouple glycolytic flux from pyruvate oxidation. Interestingly, mitochondria of imatinib-resistant cells exhibited accumulation of TCA cycle intermediates, increased NADH and low oxygen consumption. These mitochondrial alterations due to the partial failure of ETC were further confirmed in leukemic cells isolated from some imatinib-resistant CML patients. As a consequence, mitochondria generated more ROS than those of imatinib-sensitive cells. This, in turn, resulted in increased death of imatinib-resistant leukemic cells following in vitro or in vivo treatment with the pro-oxidants, PEITC and Trisenox, in a syngeneic mouse tumor model. Conversely, inhibition of glycolysis caused derepression of respiration leading to lower cellular ROS. In conclusion, these findings indicate that imatinib-resistant leukemic cells have an unexpected mitochondrial dysfunction that could be exploited for selective therapeutic intervention

Induction of Erythroid Differentiation in Human Erythroleukemia Cells by Depletion of Malic Enzyme 2

Malic enzyme 2 (ME2) is a mitochondrial enzyme that catalyzes the conversion of malate to pyruvate and CO2 and uses NAD as a cofactor. Higher expression of this enzyme correlates with the degree of cell de-differentiation. We found that ME2 is expressed in K562 erythroleukemia cells, in which a number of agents have been found to induce differentiation either along the erythroid or the myeloid lineage. We found that knockdown of ME2 led to diminished proliferation of tumor cells and increased apoptosis in vitro. These findings were accompanied by differentiation of K562 cells along the erythroid lineage, as confirmed by staining for glycophorin A and hemoglobin production. ME2 knockdown also totally abolished growth of K562 cells in nude mice. Increased ROS levels, likely reflecting increased mitochondrial production, and a decreased NADPH/NADP+ ratio were noted but use of a free radical scavenger to decrease inhibition of ROS levels did not reverse the differentiation or apoptotic phenotype, suggesting that ROS production is not causally involved in the resultant phenotype. As might be expected, depletion of ME2 induced an increase in the NAD+/NADH ratio and ATP levels fell significantly. Inhibition of the malate-aspartate shuttle was insufficient to induce K562 differentiation. We also examined several intracellular signaling pathways and expression of transcription factors and intermediate filament proteins whose expression is known to be modulated during erythroid differentiation in K562 cells. We found that silencing of ME2 leads to phospho-ERK1/2 inhibition, phospho-AKT activation, increased GATA-1 expression and diminished vimentin expression. Metabolomic analysis, conducted to gain insight into intermediary metabolic pathways that ME2 knockdown might affect, showed that ME2 depletion resulted in high orotate levels, suggesting potential impairment of pyrimidine metabolism. Collectively our data point to ME2 as a potentially novel metabolic target for leukemia therapy

Drug Off-Target Effects Predicted Using Structural Analysis in the Context of a Metabolic Network Model

Recent advances in structural bioinformatics have enabled the prediction of protein-drug off-targets based on their ligand binding sites. Concurrent developments in systems biology allow for prediction of the functional effects of system perturbations using large-scale network models. Integration of these two capabilities provides a framework for evaluating metabolic drug response phenotypes in silico. This combined approach was applied to investigate the hypertensive side effect of the cholesteryl ester transfer protein inhibitor torcetrapib in the context of human renal function. A metabolic kidney model was generated in which to simulate drug treatment. Causal drug off-targets were predicted that have previously been observed to impact renal function in gene-deficient patients and may play a role in the adverse side effects observed in clinical trials. Genetic risk factors for drug treatment were also predicted that correspond to both characterized and unknown renal metabolic disorders as well as cryptic genetic deficiencies that are not expected to exhibit a renal disorder phenotype except under drug treatment. This study represents a novel integration of structural and systems biology and a first step towards computational systems medicine. The methodology introduced herein has important implications for drug development and personalized medicine

Defining the Molecular Basis of Tumor Metabolism: a Continuing Challenge Since Warburg's Discovery

Cancer cells are the product of genetic disorders that alter crucial intracellular signaling pathways associated with the regulation of cell survival, proliferation, differentiation and death mechanisms. the role of oncogene activation and tumor suppressor inhibition in the onset of cancer is well established. Traditional antitumor therapies target specific molecules, the action/expression of which is altered in cancer cells. However, since the physiology of normal cells involves the same signaling pathways that are disturbed in cancer cells, targeted therapies have to deal with side effects and multidrug resistance, the main causes of therapy failure. Since the pioneering work of Otto Warburg, over 80 years ago, the subversion of normal metabolism displayed by cancer cells has been highlighted by many studies. Recently, the study of tumor metabolism has received much attention because metabolic transformation is a crucial cancer hallmark and a direct consequence of disturbances in the activities of oncogenes and tumor suppressors. in this review we discuss tumor metabolism from the molecular perspective of oncogenes, tumor suppressors and protein signaling pathways relevant to metabolic transformation and tumorigenesis. We also identify the principal unanswered questions surrounding this issue and the attempts to relate these to their potential for future cancer treatment. As will be made clear, tumor metabolism is still only partly understood and the metabolic aspects of transformation constitute a major challenge for science. Nevertheless, cancer metabolism can be exploited to devise novel avenues for the rational treatment of this disease. Copyright (C) 2011 S. Karger AG, BaselFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Fed ABC UFABC, CCNH, Santo Andre, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Ciencias Biol, São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Bioquim, São Paulo, BrazilUniv Fed Sao Carlos UFSCar, DFQM, Sorocaba, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Ciencias Biol, São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Bioquim, São Paulo, BrazilFAPESP: 10/16050-9FAPESP: 10/11475-1FAPESP: 08/51116-0Web of Scienc