Metabolism is the consortium of reactions within a cell which directs a variety of processes incl... more Metabolism is the consortium of reactions within a cell which directs a variety of processes including energy synthesis, signalling and the behaviour of a biological system. Metabolic networks, and more specifically the activity of enzymes within them, provide an accurate status of how cellular information is being executed. The performance of these networks and their ability to siphon metabolites in a number of directions may be the difference between a healthy and diseased state. Blue native polyacrylamide gel electrophoresis (BN-PAGE), owing to its simplicity and wide-ranging applications, permits the inspection of these nodules. The separation of proteins and enzyme complexes in their native format enables the exploration of enzymatic activity in metabolic networks via in-gel assays. These are quick, specific, and amenable to further studies. This electrophoretic technology not only enables the visualization of enzymatic efficacy but reveals the crosstalk among enzymes and their interactions with other organellar partners.
Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, Jun 13, 2014
Although nitrosative stress is known to severely impede the ability of living systems to generate... more Although nitrosative stress is known to severely impede the ability of living systems to generate adenosine triphosphate (ATP) via oxidative phosphorylation, there is limited information on how microorganisms fulfill their energy needs in order to survive reactive nitrogen species (RNS). In this study we demonstrate an elaborate strategy involving substrate-level phosphorylation that enables the soil microbe Pseudomonas fluorescens to synthesize ATP in a defined medium with fumarate as the sole carbon source. The enhanced activities of such enzymes as phosphoenolpyruvate carboxylase and pyruvate phosphate dikinase coupled with the increased activities of phospho-transfer enzymes like adenylate kinase and nucleoside diphophate kinase provide an effective strategy to produce high energy nucleosides in an O 2-independent manner. The alternate ATP producing machinery is fuelled by the precursors derived from fumarate with the aid of fumarase C and fumarate reductase. This metabolic reconfiguration is key to the survival of P. fluorescens and reveals potential targets against RNS-resistant organisms.
It is well-established that mitochondria are the powerhouses of the cell, producing adenosine tri... more It is well-established that mitochondria are the powerhouses of the cell, producing adenosine triphosphate (ATP), the universal energy currency. However, the most significant strengths of the electron transport chain (ETC), its intricacy and efficiency, are also its greatest downfalls. A reliance on metal complexes (FeS clusters, hemes), lipid moities such as cardiolipin, and cofactors including alpha-lipoic acid and quinones render oxidative phosphorylation vulnerable to environmental toxins, intracellular reactive oxygen species (ROS) and fluctuations in diet. To that effect, it is of interest to note that temporal disruptions in ETC activity in most organisms are rarely fatal, and often a redundant number of failsafes are in place to permit continued ATP production when needed. Here, we highlight the metabolic reconfigurations discovered in organisms ranging from parasitic Entamoeba to bacteria such as pseudomonads and then complex eukaryotic systems that allow these species to adapt to and occasionally thrive in harsh environments. The overarching aim of this review is to demonstrate the plasticity of metabolic networks and recognize that in times of duress, life finds a way.
The significance of metabolic networks in guiding the fate of the stem cell differentiation is on... more The significance of metabolic networks in guiding the fate of the stem cell differentiation is only beginning to emerge. Oxidative metabolism has been suggested to play a major role during this process. Therefore, it is critical to understand the underlying mechanisms of metabolic alterations occurring in stem cells to manipulate the ultimate outcome of these pluripotent cells. Here, using P19 murine embryonal carcinoma cells as a model system, the role of mitochondrial biogenesis and the modulation of metabolic networks during dimethyl sulfoxide (DMSO)-induced differentiation are revealed. Blue native polyacrylamide gel electrophoresis (BN-PAGE) technology aided in profiling key enzymes, such as hexokinase (HK) [EC 2.7.1.1], glucose-6-phosphate isomerase (GPI) [EC 5.3.1.9], pyruvate kinase (PK) [EC 2.7.1.40], Complex I [EC 1.6.5.3], and Complex IV [EC 1.9.3.1], that are involved in the energy budget of the differentiated cells. Mitochondrial adenosine triphosphate (ATP) production was shown to be increased in DMSO-treated cells upon exposure to the tricarboxylic acid (TCA) cycle substrates, such as succinate and malate. The increased mitochondrial activity and biogenesis were further confirmed by immunofluorescence microscopy. Collectively, the results indicate that oxidative energy metabolism and mitochondrial biogenesis were sharply upregulated in DMSO-differentiated P19 cells. This functional metabolic and proteomic study provides further evidence that modulation of mitochondrial energy metabolism is a pivotal component of the cellular differentiation process and may dictate the final destiny of stem cells.
Phosphoenolpyruvate (PEP)-dependent kinases are central to numerous metabolic processes and media... more Phosphoenolpyruvate (PEP)-dependent kinases are central to numerous metabolic processes and mediate the production of adenosine triphosphate (ATP) by substrate-level phosphorylation (SLP). While pyruvate kinase (PK, EC: 2.7.1.40), the final enzyme of the glycolytic pathway is critical in the anaerobic synthesis of ATP from ADP, pyruvate phosphate dikinase (PPDK, EC: 2.7.9.1), and phosphoenolpyruvate synthase (PEPS, EC: 2.7.9.2) help generate ATP from AMP coupled to PEP as a substrate. Here we demonstrate an inexpensive and effective electrophoretic technology to determine the activities of these enzymes by blue-native polyacrylamide gel electrophoresis (BN-PAGE). The generation of pyruvate is linked to exogenous lactate dehydrogenase (LDH), and the oxidation of reduced nicotinamide adenine dinucleotide (NADH) coupled to 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium chloride (INT) results in a formazan precipitate which is easily quantifiable. The selectivity of the enzymes is ensured by including either AMP or ADP and pyrophosphate (PP i) or inorganic phosphate (P i). Activity bands were readily obtained after incubation in the respective reaction mixtures for 20-30 min. Cell-free extract concentrations as low as 20 g protein equivalent yielded activity bands and substrate levels were manipulated to optimize sensitivity of this analytical technique. High-pressure liquid chromatography (HPLC), two-dimensional (2-D) SDS-PAGE (where SDS is sodium dodecyl sulfate), and immunoblot studies of the excised activity band help further characterize these PEP-dependent kinases. Furthermore, these enzymes were readily identified on the same gel by incubating it sequentially in the respective reaction mixtures. This technique provides a facile method to elucidate these kinases in biological systems.
Although the ability of microbial systems to adapt to the toxic challenge posed by numerous metal... more Although the ability of microbial systems to adapt to the toxic challenge posed by numerous metal pollutants individually has been well documented, there is little detailed information on how bacteria survive in a multiple-metal environment. Here we describe the metabolic reconfiguration invoked by the soil microbe Pseudomonas fluorescens in a medium with millimolar amounts of aluminum (Al), iron (Fe), gallium (Ga), calcium (Ca), and zinc (Zn). While enzymes involved in the production of NADH were decreased, there was a marked increase in enzymatic activities dedicated to NADPH formation. A modified tricarboxylic acid (TCA) cycle coupled to an alternate glyoxylate shunt mediated the synthesis of adenosine triphosphate (ATP) with the concomitant generation of oxalate. This dicarboxylic acid was a key ingredient in the sequestration of the metals that were detoxified as a lipid complex. It appears that the microbe favors this strategy as opposed to a detoxification process aimed at each metal separately. These findings have interesting implications for bioremediation technologies.
There is mounting evidence that metabolic reprogramming is critical for the survival of organisms... more There is mounting evidence that metabolic reprogramming is critical for the survival of organisms exposed to changing and stressed environments. Using the soil microbe Pseudomonas fluorescens as a model system, we demonstrate that the metabolic networks aimed at the conversion of aspartate into pyruvate are enhanced in the presence of hydrogen peroxide (H2O2). The metabolites pyruvate, oxaloacetate and acetate were increased in the treated cultures as measured by HPLC. Enzymes such as aspartate transaminase and phosphoenolpyruvate carboxylase (PEPC) that mediate the conversion of aspartate to phosphoenolpyruvate (PEP) were up-regulated. This high-energy phosphate was readily converted into ATP, a process facilitated by the increased activity of pyruvate orthophosphate dikinase (PPDK) and phosphoenolpyruvate synthase (PEPS) as oxidative phosphorylation was severely compromised. The ensuing formation of pyruvate readily detoxified reactive oxygen species with the concomitant formation of acetate. This H2O2-induced metabolic reconfiguration not only helps generate the antioxidants necessary to thwart oxidative stress but also powers the formation of energy.
Although lactate has traditionally been known to be an end product of anaerobic metabolism, recen... more Although lactate has traditionally been known to be an end product of anaerobic metabolism, recent studies have revealed its disparate biological functions. Oxidative energy production and cell signaling are two important roles assigned to this monocarboxylic acid. Here we demonstrate that mitochondrial lactate metabolism to pyruvate mediated by lactate dehydrogenase (LDH) in a human astrocytic cell line is involved in antioxidative defense. The pooling of this α-ketoacid helps to detoxify reactive oxygen species, with the concomitant formation of acetate. In-gel activity assays following blue native PAGE electrophoresis were utilized to demonstrate the increase in mitochondrial LDH activity coupled to the decrease in pyruvate dehydrogenase activity in the cells challenged by oxidative stress. The enhanced production of pyruvate with the concomitant formation of acetate in astrocytoma cells was monitored by high-performance liquid chromatography. The ability of pyruvate to fend off oxidative stress was visualized by fluorescence microscopy with the aid of the dye 2',7'-dichlorodihydrofluorescein diacetate. Immunoblotting helped confirm the presence of elevated levels of LDH in cells exposed to oxidative stress, and recovery experiments were performed with pyruvate to diminish the oxidative burden on the astrocytoma. The acetate, generated as a consequence of the antioxidative attribute of pyruvate, was subsequently channeled toward the production of lipids, a process facilitated by the upregulation in activity of acetyl-CoA synthetase and acetyl-CoA carboxylase, as demonstrated by in-gel activity assays. The mitochondrial lactate metabolism mediated by LDH appears to play an important role in antioxidative defence in this astrocytic system.
Biochimica Et Biophysica Acta - General Subjects, 2015
Background: It is well-known that elevated amounts of nitric oxide and other reactive nitrogen sp... more Background: It is well-known that elevated amounts of nitric oxide and other reactive nitrogen species (RNS) impact negatively on the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. These perturbations severely compromise O 2-dependent energy production. While bacteria are known to adapt to RNS, a key tool employed by macrophages to combat infections, the exact mechanisms are unknown. Methods: The bacterium was cultured in a defined mineral medium and cell-free extracts obtained at the same growth phase were utilized for various biochemical studies Blue native polyacrylamide gel electrophoresis followed by in-gel activity assays, high performance liquid chromatography and co-immunoprecipitaton are applied to investigate the effects of RNS on the model microbe Pseudomonas fluorescens. Results: Citrate is channeled away from the tricarboxylic acid cycle using a novel metabolon consisting of citrate lyase (CL), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK). This metabolic engine comprising three disparate enzymes appears to transiently assemble as a supercomplex aimed at ATP synthesis. The up-regulation in the activities of adenylate kinase (AK) and nucleoside diphosphate kinase (NDPK) ensured the efficacy of this ATP-making machine. Conclusion: Microbes may escape the effects of nitrosative stress by re-engineering metabolic networks in order to generate and store ATP anaerobically when the electron transport chain is defective. General significance: The molecular configuration described herein provides further understanding of how metabolism plays a key role in the adaptation to nitrosative stress and reveals novel targets that will inform the development of antimicrobial agents to counter RNS-resistant pathogens.
Biochemical and Biophysical Research Communications, Nov 1, 2014
It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genet... more It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD +), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H 2 O 2) in the culture medium. Under oxidative stress, the NAD + generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD + reveals an intricate link between metabolism and the processing of genetic information.
The brain is one of the most energy-demanding organs in the body. It has evolved intricate metabo... more The brain is one of the most energy-demanding organs in the body. It has evolved intricate metabolic networks to fulfill this need and utilizes a variety of substrates to generate ATP, the universal energy currency. Any disruption in the supply of energy results in various abnormalities including Alzheimer's disease (AD), a condition with markedly diminished cognitive ability. Astrocytes are an important participant in maintaining the cerebral ATP budget. However, under oxidative stress induced by numerous factors including aluminum toxicity, the ability of astroctyes to generate ATP is impaired due to dysfunctional mitochondria. This leads to globular, glycolytic, lipogenic and ATP-deficient astrocytes, cerebral characteristics common in AD patients. The reversal of these perturbations by such natural metabolites as pyruvate, α-ketoglutarate, acetoacetate and L-carnitine provides valuable therapeutic cues against AD.
Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, Dec 1, 2015
As all aerobic organisms are exposed to oxidative stress, they are known to devise intricate mech... more As all aerobic organisms are exposed to oxidative stress, they are known to devise intricate mechanisms to counter reactive oxygen species (ROS). Metabolic networks contributing to the production of ketoacids are prominent in alleviating the oxidative burden. When glyoxylate detoxifies ROS, formate is the principal by-product generated. In this study the contribution of formate in enabling the survival of the microbe Pseudomonas fluorescens challenged by hydrogen peroxide (H2O2) has been elucidated. When grown in the presence of H2O2 (stressed culture), the levels of formate were higher in the spent fluid and the soluble cell-free extracts compared to the controls. Formate was subsequently utilized as a reducing factor to produce NADPH and succinate. The former is mediated by formate dehydrogenase (FDH-NADP), whose activity was enhanced in the stressed cells. Fumarate reductase (FRD) that catalyzes the conversion of fumarate into succinate was also markedly increased in the stressed cells. Metabolic adaptation is a pivotal tool in combatting oxidative stress. Formate, a by-product of glyoxylate-mediated detoxification of ROS is recuperated as a potent reductive fuel. It is becoming quite evident that this simple metabolite has other biological roles that have not been fully appreciated.
Aims: To identify the molecular networks in Pseudomonas fluorescens that convey resistance to tox... more Aims: To identify the molecular networks in Pseudomonas fluorescens that convey resistance to toxic concentrations of Zn, a common pollutant and hazard to biological systems. Methods and Results: Pseudomonas fluorescens strain ATCC 13525 was cultured in growth medium with millimolar concentrations of Zn. Enzymatic activities and metabolite levels were monitored with the aid of in-gel activity assays and high-performance liquid chromatography, respectively. As oxidative phosphorylation was rendered ineffective, the assimilation of citric acid mediated sequentially by citrate lyase (CL), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK) appeared to play a key role in ATP synthesis via substrate-level phosphorylation (SLP). Enzymes generating the antioxidant, reduced nicotinamide adenine dinucleotide phosphate (NADPH) were enhanced, while metabolic modules mediating the formation of the pro-oxidant, reduced nicotinamide adenine dinucleotide (NADH) were downregulated. Conclusions: Pseudomonas fluorescens reengineers its metabolic networks to generate ATP via SLP, a stratagem that allows the microbe to compensate for an ineffective electron transport chain provoked by excess Zn. Significance and Impact of the Study: The molecular insights described here are critical in devising strategies to bioremediate Zn-polluted environments.
The role of metabolism in anti-oxidative defence is only now beginning to emerge. Here, we show t... more The role of metabolism in anti-oxidative defence is only now beginning to emerge. Here, we show that the nutritionally-versatile microbe, Pseudomonas fluorescens, reconfigures its metabolism in an effort to generate NADPH, ATP and glyoxylate in order to fend off oxidative stress. Glyoxylate was produced predominantly via the enhanced activities of glycine dehydrogenase-NADP + (GDH), glycine transaminase (GTA) and isocitrate lyase (ICL) in a medium exposed to hydrogen peroxide (H 2 O 2). This ketoacid was utilized to produce ATP by substrate-level phosphorylation and to neutralize reactive oxygen species with the concomitant formation of formate. The latter was also a source of NADPH, a process mediated by formate dehydrogenase-NADP + (FDH). The increased activities of phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) worked in tandem to synthesize ATP in the H 2 O 2challenged cells that had markedly diminished capacity for oxidative phosphorylation. These metabolic networks provide an effective means of combating ROS and reveal therapeutic targets against microbes resistant to oxidative stress.
l-Carnitine is a critical metabolite indispensable for the metabolism of lipids as it facilitates... more l-Carnitine is a critical metabolite indispensable for the metabolism of lipids as it facilitates fatty acid transport into the mitochondrion where -oxidation occurs. Human astrocytes (CCF-STTG1 cells) and hepatocytes (HepG2 cells) exposed to aluminum (Al) and hydrogen peroxide (H 2 O 2), were characterized with lower levels of l-carnitine, diminished -oxidation, and increased lipid accumulation compared to the controls. ␥-Butyrobetainealdehyde dehydrogenase (BADH) and butyrobetaine dioxygenase (BBDOX), two key enzymes mediating the biogenesis of l-carnitine, were sharply reduced during Al and H 2 O 2 challenge. Exposure of the Al and H 2 O 2-treated cells to ␣-ketoglutarate (KG), led to the recovery of lcarnitine production with the concomitant reduction in ROS levels. It appears that the channeling of KG to combat oxidative stress results in decreased l-carnitine synthesis, an event that contributes to the dyslipidemia observed during Al and H 2 O 2 insults in these mammalian cells. Hence, KG may help alleviate pathological conditions induced by oxidative stress.
Pseudomonas fluorescens invoked a metabolic reconfiguration that resulted in enhanced production ... more Pseudomonas fluorescens invoked a metabolic reconfiguration that resulted in enhanced production of pyruvate under the challenge of hydrogen peroxide (H 2 O 2). Although this stress led to a sharp reduction in the activities of numerous tricarboxylic acid (TCA) cycle enzymes, there was a marked increase in the activities of catalase and various NADPH-generating enzymes to counter the oxidative burden. The upregulation of phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK) coupled with the reduction of pyruvate dehydrogenase (PDH) in the H 2 O 2-challenged cells appear to be important contributors to the elevated levels of pyruvate found in these bacteria. Increased pyruvate synthesis was evident in the presence of a variety of carbon sources including d-glucose. Intact cells rapidly consumed d-glucose with the concomitant formation of this monocarboxylic acid. At least a 12-fold increase in pyruvate production within 1 h was observed in the stressed cells. These findings may be exploited in the development of technologies aimed at the conversion of carbohydrates into pyruvate.
Metal pollutants are a global health risk due to their ability to contribute to a variety of dise... more Metal pollutants are a global health risk due to their ability to contribute to a variety of diseases. Aluminum (Al), a ubiquitous environmental contaminant is implicated in anemia, osteomalacia, hepatic disorder, and neurological disorder. In this review, we outline how this intracellular generator of reactive oxygen species (ROS) triggers a metabolic shift towards lipogenesis in astrocytes and hepatocytes. This Alevoked phenomenon is coupled to diminished mitochondrial activity, anerobiosis, and the channeling of α-ketoacids towards anti-oxidant defense. The resulting metabolic reconfiguration leads to fat accumulation and a reduction in ATP synthesis, characteristics that are common to numerous medical disorders. Hence, the ability of Al toxicity to create an oxidative environment promotes dysfunctional metabolic processes in astrocytes and hepatocytes. These molecular events triggered by Al-induced ROS production are the potential mediators of brain and liver disorders.
Metabolism is the consortium of reactions within a cell which directs a variety of processes incl... more Metabolism is the consortium of reactions within a cell which directs a variety of processes including energy synthesis, signalling and the behaviour of a biological system. Metabolic networks, and more specifically the activity of enzymes within them, provide an accurate status of how cellular information is being executed. The performance of these networks and their ability to siphon metabolites in a number of directions may be the difference between a healthy and diseased state. Blue native polyacrylamide gel electrophoresis (BN-PAGE), owing to its simplicity and wide-ranging applications, permits the inspection of these nodules. The separation of proteins and enzyme complexes in their native format enables the exploration of enzymatic activity in metabolic networks via in-gel assays. These are quick, specific, and amenable to further studies. This electrophoretic technology not only enables the visualization of enzymatic efficacy but reveals the crosstalk among enzymes and their interactions with other organellar partners.
Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, Jun 13, 2014
Although nitrosative stress is known to severely impede the ability of living systems to generate... more Although nitrosative stress is known to severely impede the ability of living systems to generate adenosine triphosphate (ATP) via oxidative phosphorylation, there is limited information on how microorganisms fulfill their energy needs in order to survive reactive nitrogen species (RNS). In this study we demonstrate an elaborate strategy involving substrate-level phosphorylation that enables the soil microbe Pseudomonas fluorescens to synthesize ATP in a defined medium with fumarate as the sole carbon source. The enhanced activities of such enzymes as phosphoenolpyruvate carboxylase and pyruvate phosphate dikinase coupled with the increased activities of phospho-transfer enzymes like adenylate kinase and nucleoside diphophate kinase provide an effective strategy to produce high energy nucleosides in an O 2-independent manner. The alternate ATP producing machinery is fuelled by the precursors derived from fumarate with the aid of fumarase C and fumarate reductase. This metabolic reconfiguration is key to the survival of P. fluorescens and reveals potential targets against RNS-resistant organisms.
It is well-established that mitochondria are the powerhouses of the cell, producing adenosine tri... more It is well-established that mitochondria are the powerhouses of the cell, producing adenosine triphosphate (ATP), the universal energy currency. However, the most significant strengths of the electron transport chain (ETC), its intricacy and efficiency, are also its greatest downfalls. A reliance on metal complexes (FeS clusters, hemes), lipid moities such as cardiolipin, and cofactors including alpha-lipoic acid and quinones render oxidative phosphorylation vulnerable to environmental toxins, intracellular reactive oxygen species (ROS) and fluctuations in diet. To that effect, it is of interest to note that temporal disruptions in ETC activity in most organisms are rarely fatal, and often a redundant number of failsafes are in place to permit continued ATP production when needed. Here, we highlight the metabolic reconfigurations discovered in organisms ranging from parasitic Entamoeba to bacteria such as pseudomonads and then complex eukaryotic systems that allow these species to adapt to and occasionally thrive in harsh environments. The overarching aim of this review is to demonstrate the plasticity of metabolic networks and recognize that in times of duress, life finds a way.
The significance of metabolic networks in guiding the fate of the stem cell differentiation is on... more The significance of metabolic networks in guiding the fate of the stem cell differentiation is only beginning to emerge. Oxidative metabolism has been suggested to play a major role during this process. Therefore, it is critical to understand the underlying mechanisms of metabolic alterations occurring in stem cells to manipulate the ultimate outcome of these pluripotent cells. Here, using P19 murine embryonal carcinoma cells as a model system, the role of mitochondrial biogenesis and the modulation of metabolic networks during dimethyl sulfoxide (DMSO)-induced differentiation are revealed. Blue native polyacrylamide gel electrophoresis (BN-PAGE) technology aided in profiling key enzymes, such as hexokinase (HK) [EC 2.7.1.1], glucose-6-phosphate isomerase (GPI) [EC 5.3.1.9], pyruvate kinase (PK) [EC 2.7.1.40], Complex I [EC 1.6.5.3], and Complex IV [EC 1.9.3.1], that are involved in the energy budget of the differentiated cells. Mitochondrial adenosine triphosphate (ATP) production was shown to be increased in DMSO-treated cells upon exposure to the tricarboxylic acid (TCA) cycle substrates, such as succinate and malate. The increased mitochondrial activity and biogenesis were further confirmed by immunofluorescence microscopy. Collectively, the results indicate that oxidative energy metabolism and mitochondrial biogenesis were sharply upregulated in DMSO-differentiated P19 cells. This functional metabolic and proteomic study provides further evidence that modulation of mitochondrial energy metabolism is a pivotal component of the cellular differentiation process and may dictate the final destiny of stem cells.
Phosphoenolpyruvate (PEP)-dependent kinases are central to numerous metabolic processes and media... more Phosphoenolpyruvate (PEP)-dependent kinases are central to numerous metabolic processes and mediate the production of adenosine triphosphate (ATP) by substrate-level phosphorylation (SLP). While pyruvate kinase (PK, EC: 2.7.1.40), the final enzyme of the glycolytic pathway is critical in the anaerobic synthesis of ATP from ADP, pyruvate phosphate dikinase (PPDK, EC: 2.7.9.1), and phosphoenolpyruvate synthase (PEPS, EC: 2.7.9.2) help generate ATP from AMP coupled to PEP as a substrate. Here we demonstrate an inexpensive and effective electrophoretic technology to determine the activities of these enzymes by blue-native polyacrylamide gel electrophoresis (BN-PAGE). The generation of pyruvate is linked to exogenous lactate dehydrogenase (LDH), and the oxidation of reduced nicotinamide adenine dinucleotide (NADH) coupled to 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium chloride (INT) results in a formazan precipitate which is easily quantifiable. The selectivity of the enzymes is ensured by including either AMP or ADP and pyrophosphate (PP i) or inorganic phosphate (P i). Activity bands were readily obtained after incubation in the respective reaction mixtures for 20-30 min. Cell-free extract concentrations as low as 20 g protein equivalent yielded activity bands and substrate levels were manipulated to optimize sensitivity of this analytical technique. High-pressure liquid chromatography (HPLC), two-dimensional (2-D) SDS-PAGE (where SDS is sodium dodecyl sulfate), and immunoblot studies of the excised activity band help further characterize these PEP-dependent kinases. Furthermore, these enzymes were readily identified on the same gel by incubating it sequentially in the respective reaction mixtures. This technique provides a facile method to elucidate these kinases in biological systems.
Although the ability of microbial systems to adapt to the toxic challenge posed by numerous metal... more Although the ability of microbial systems to adapt to the toxic challenge posed by numerous metal pollutants individually has been well documented, there is little detailed information on how bacteria survive in a multiple-metal environment. Here we describe the metabolic reconfiguration invoked by the soil microbe Pseudomonas fluorescens in a medium with millimolar amounts of aluminum (Al), iron (Fe), gallium (Ga), calcium (Ca), and zinc (Zn). While enzymes involved in the production of NADH were decreased, there was a marked increase in enzymatic activities dedicated to NADPH formation. A modified tricarboxylic acid (TCA) cycle coupled to an alternate glyoxylate shunt mediated the synthesis of adenosine triphosphate (ATP) with the concomitant generation of oxalate. This dicarboxylic acid was a key ingredient in the sequestration of the metals that were detoxified as a lipid complex. It appears that the microbe favors this strategy as opposed to a detoxification process aimed at each metal separately. These findings have interesting implications for bioremediation technologies.
There is mounting evidence that metabolic reprogramming is critical for the survival of organisms... more There is mounting evidence that metabolic reprogramming is critical for the survival of organisms exposed to changing and stressed environments. Using the soil microbe Pseudomonas fluorescens as a model system, we demonstrate that the metabolic networks aimed at the conversion of aspartate into pyruvate are enhanced in the presence of hydrogen peroxide (H2O2). The metabolites pyruvate, oxaloacetate and acetate were increased in the treated cultures as measured by HPLC. Enzymes such as aspartate transaminase and phosphoenolpyruvate carboxylase (PEPC) that mediate the conversion of aspartate to phosphoenolpyruvate (PEP) were up-regulated. This high-energy phosphate was readily converted into ATP, a process facilitated by the increased activity of pyruvate orthophosphate dikinase (PPDK) and phosphoenolpyruvate synthase (PEPS) as oxidative phosphorylation was severely compromised. The ensuing formation of pyruvate readily detoxified reactive oxygen species with the concomitant formation of acetate. This H2O2-induced metabolic reconfiguration not only helps generate the antioxidants necessary to thwart oxidative stress but also powers the formation of energy.
Although lactate has traditionally been known to be an end product of anaerobic metabolism, recen... more Although lactate has traditionally been known to be an end product of anaerobic metabolism, recent studies have revealed its disparate biological functions. Oxidative energy production and cell signaling are two important roles assigned to this monocarboxylic acid. Here we demonstrate that mitochondrial lactate metabolism to pyruvate mediated by lactate dehydrogenase (LDH) in a human astrocytic cell line is involved in antioxidative defense. The pooling of this α-ketoacid helps to detoxify reactive oxygen species, with the concomitant formation of acetate. In-gel activity assays following blue native PAGE electrophoresis were utilized to demonstrate the increase in mitochondrial LDH activity coupled to the decrease in pyruvate dehydrogenase activity in the cells challenged by oxidative stress. The enhanced production of pyruvate with the concomitant formation of acetate in astrocytoma cells was monitored by high-performance liquid chromatography. The ability of pyruvate to fend off oxidative stress was visualized by fluorescence microscopy with the aid of the dye 2',7'-dichlorodihydrofluorescein diacetate. Immunoblotting helped confirm the presence of elevated levels of LDH in cells exposed to oxidative stress, and recovery experiments were performed with pyruvate to diminish the oxidative burden on the astrocytoma. The acetate, generated as a consequence of the antioxidative attribute of pyruvate, was subsequently channeled toward the production of lipids, a process facilitated by the upregulation in activity of acetyl-CoA synthetase and acetyl-CoA carboxylase, as demonstrated by in-gel activity assays. The mitochondrial lactate metabolism mediated by LDH appears to play an important role in antioxidative defence in this astrocytic system.
Biochimica Et Biophysica Acta - General Subjects, 2015
Background: It is well-known that elevated amounts of nitric oxide and other reactive nitrogen sp... more Background: It is well-known that elevated amounts of nitric oxide and other reactive nitrogen species (RNS) impact negatively on the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. These perturbations severely compromise O 2-dependent energy production. While bacteria are known to adapt to RNS, a key tool employed by macrophages to combat infections, the exact mechanisms are unknown. Methods: The bacterium was cultured in a defined mineral medium and cell-free extracts obtained at the same growth phase were utilized for various biochemical studies Blue native polyacrylamide gel electrophoresis followed by in-gel activity assays, high performance liquid chromatography and co-immunoprecipitaton are applied to investigate the effects of RNS on the model microbe Pseudomonas fluorescens. Results: Citrate is channeled away from the tricarboxylic acid cycle using a novel metabolon consisting of citrate lyase (CL), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK). This metabolic engine comprising three disparate enzymes appears to transiently assemble as a supercomplex aimed at ATP synthesis. The up-regulation in the activities of adenylate kinase (AK) and nucleoside diphosphate kinase (NDPK) ensured the efficacy of this ATP-making machine. Conclusion: Microbes may escape the effects of nitrosative stress by re-engineering metabolic networks in order to generate and store ATP anaerobically when the electron transport chain is defective. General significance: The molecular configuration described herein provides further understanding of how metabolism plays a key role in the adaptation to nitrosative stress and reveals novel targets that will inform the development of antimicrobial agents to counter RNS-resistant pathogens.
Biochemical and Biophysical Research Communications, Nov 1, 2014
It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genet... more It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD +), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H 2 O 2) in the culture medium. Under oxidative stress, the NAD + generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD + reveals an intricate link between metabolism and the processing of genetic information.
The brain is one of the most energy-demanding organs in the body. It has evolved intricate metabo... more The brain is one of the most energy-demanding organs in the body. It has evolved intricate metabolic networks to fulfill this need and utilizes a variety of substrates to generate ATP, the universal energy currency. Any disruption in the supply of energy results in various abnormalities including Alzheimer's disease (AD), a condition with markedly diminished cognitive ability. Astrocytes are an important participant in maintaining the cerebral ATP budget. However, under oxidative stress induced by numerous factors including aluminum toxicity, the ability of astroctyes to generate ATP is impaired due to dysfunctional mitochondria. This leads to globular, glycolytic, lipogenic and ATP-deficient astrocytes, cerebral characteristics common in AD patients. The reversal of these perturbations by such natural metabolites as pyruvate, α-ketoglutarate, acetoacetate and L-carnitine provides valuable therapeutic cues against AD.
Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, Dec 1, 2015
As all aerobic organisms are exposed to oxidative stress, they are known to devise intricate mech... more As all aerobic organisms are exposed to oxidative stress, they are known to devise intricate mechanisms to counter reactive oxygen species (ROS). Metabolic networks contributing to the production of ketoacids are prominent in alleviating the oxidative burden. When glyoxylate detoxifies ROS, formate is the principal by-product generated. In this study the contribution of formate in enabling the survival of the microbe Pseudomonas fluorescens challenged by hydrogen peroxide (H2O2) has been elucidated. When grown in the presence of H2O2 (stressed culture), the levels of formate were higher in the spent fluid and the soluble cell-free extracts compared to the controls. Formate was subsequently utilized as a reducing factor to produce NADPH and succinate. The former is mediated by formate dehydrogenase (FDH-NADP), whose activity was enhanced in the stressed cells. Fumarate reductase (FRD) that catalyzes the conversion of fumarate into succinate was also markedly increased in the stressed cells. Metabolic adaptation is a pivotal tool in combatting oxidative stress. Formate, a by-product of glyoxylate-mediated detoxification of ROS is recuperated as a potent reductive fuel. It is becoming quite evident that this simple metabolite has other biological roles that have not been fully appreciated.
Aims: To identify the molecular networks in Pseudomonas fluorescens that convey resistance to tox... more Aims: To identify the molecular networks in Pseudomonas fluorescens that convey resistance to toxic concentrations of Zn, a common pollutant and hazard to biological systems. Methods and Results: Pseudomonas fluorescens strain ATCC 13525 was cultured in growth medium with millimolar concentrations of Zn. Enzymatic activities and metabolite levels were monitored with the aid of in-gel activity assays and high-performance liquid chromatography, respectively. As oxidative phosphorylation was rendered ineffective, the assimilation of citric acid mediated sequentially by citrate lyase (CL), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK) appeared to play a key role in ATP synthesis via substrate-level phosphorylation (SLP). Enzymes generating the antioxidant, reduced nicotinamide adenine dinucleotide phosphate (NADPH) were enhanced, while metabolic modules mediating the formation of the pro-oxidant, reduced nicotinamide adenine dinucleotide (NADH) were downregulated. Conclusions: Pseudomonas fluorescens reengineers its metabolic networks to generate ATP via SLP, a stratagem that allows the microbe to compensate for an ineffective electron transport chain provoked by excess Zn. Significance and Impact of the Study: The molecular insights described here are critical in devising strategies to bioremediate Zn-polluted environments.
The role of metabolism in anti-oxidative defence is only now beginning to emerge. Here, we show t... more The role of metabolism in anti-oxidative defence is only now beginning to emerge. Here, we show that the nutritionally-versatile microbe, Pseudomonas fluorescens, reconfigures its metabolism in an effort to generate NADPH, ATP and glyoxylate in order to fend off oxidative stress. Glyoxylate was produced predominantly via the enhanced activities of glycine dehydrogenase-NADP + (GDH), glycine transaminase (GTA) and isocitrate lyase (ICL) in a medium exposed to hydrogen peroxide (H 2 O 2). This ketoacid was utilized to produce ATP by substrate-level phosphorylation and to neutralize reactive oxygen species with the concomitant formation of formate. The latter was also a source of NADPH, a process mediated by formate dehydrogenase-NADP + (FDH). The increased activities of phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) worked in tandem to synthesize ATP in the H 2 O 2challenged cells that had markedly diminished capacity for oxidative phosphorylation. These metabolic networks provide an effective means of combating ROS and reveal therapeutic targets against microbes resistant to oxidative stress.
l-Carnitine is a critical metabolite indispensable for the metabolism of lipids as it facilitates... more l-Carnitine is a critical metabolite indispensable for the metabolism of lipids as it facilitates fatty acid transport into the mitochondrion where -oxidation occurs. Human astrocytes (CCF-STTG1 cells) and hepatocytes (HepG2 cells) exposed to aluminum (Al) and hydrogen peroxide (H 2 O 2), were characterized with lower levels of l-carnitine, diminished -oxidation, and increased lipid accumulation compared to the controls. ␥-Butyrobetainealdehyde dehydrogenase (BADH) and butyrobetaine dioxygenase (BBDOX), two key enzymes mediating the biogenesis of l-carnitine, were sharply reduced during Al and H 2 O 2 challenge. Exposure of the Al and H 2 O 2-treated cells to ␣-ketoglutarate (KG), led to the recovery of lcarnitine production with the concomitant reduction in ROS levels. It appears that the channeling of KG to combat oxidative stress results in decreased l-carnitine synthesis, an event that contributes to the dyslipidemia observed during Al and H 2 O 2 insults in these mammalian cells. Hence, KG may help alleviate pathological conditions induced by oxidative stress.
Pseudomonas fluorescens invoked a metabolic reconfiguration that resulted in enhanced production ... more Pseudomonas fluorescens invoked a metabolic reconfiguration that resulted in enhanced production of pyruvate under the challenge of hydrogen peroxide (H 2 O 2). Although this stress led to a sharp reduction in the activities of numerous tricarboxylic acid (TCA) cycle enzymes, there was a marked increase in the activities of catalase and various NADPH-generating enzymes to counter the oxidative burden. The upregulation of phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK) coupled with the reduction of pyruvate dehydrogenase (PDH) in the H 2 O 2-challenged cells appear to be important contributors to the elevated levels of pyruvate found in these bacteria. Increased pyruvate synthesis was evident in the presence of a variety of carbon sources including d-glucose. Intact cells rapidly consumed d-glucose with the concomitant formation of this monocarboxylic acid. At least a 12-fold increase in pyruvate production within 1 h was observed in the stressed cells. These findings may be exploited in the development of technologies aimed at the conversion of carbohydrates into pyruvate.
Metal pollutants are a global health risk due to their ability to contribute to a variety of dise... more Metal pollutants are a global health risk due to their ability to contribute to a variety of diseases. Aluminum (Al), a ubiquitous environmental contaminant is implicated in anemia, osteomalacia, hepatic disorder, and neurological disorder. In this review, we outline how this intracellular generator of reactive oxygen species (ROS) triggers a metabolic shift towards lipogenesis in astrocytes and hepatocytes. This Alevoked phenomenon is coupled to diminished mitochondrial activity, anerobiosis, and the channeling of α-ketoacids towards anti-oxidant defense. The resulting metabolic reconfiguration leads to fat accumulation and a reduction in ATP synthesis, characteristics that are common to numerous medical disorders. Hence, the ability of Al toxicity to create an oxidative environment promotes dysfunctional metabolic processes in astrocytes and hepatocytes. These molecular events triggered by Al-induced ROS production are the potential mediators of brain and liver disorders.
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