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1.  Exploring metabolic engineering design principles for the photosynthetic production of lactic acid by Synechocystis sp. PCC6803 
Molecular engineering of the intermediary physiology of cyanobacteria has become important for the sustainable production of biofuels and commodity compounds from CO2 and sunlight by “designer microbes.” The chemical commodity product L-lactic acid can be synthesized in one step from a key intermediary metabolite of these organisms, pyruvate, catalyzed by a lactate dehydrogenase. Synthetic biology engineering to make “designer microbes” includes the introduction and overexpression of the product-forming biochemical pathway. For further optimization of product formation, modifications in the surrounding biochemical network of intermediary metabolism have to be made.
To improve light-driven L-lactic acid production from CO2, we explored several metabolic engineering design principles, using a previously engineered L-lactic acid producing mutant strain of Synechocystis sp. PCC6803 as the benchmark. These strategies included: (i) increasing the expression level of the relevant product-forming enzyme, lactate dehydrogenase (LDH), for example, via expression from a replicative plasmid; (ii) co-expression of a heterologous pyruvate kinase to increase the flux towards pyruvate; and (iii) knockdown of phosphoenolpyruvate carboxylase to decrease the flux through a competing pathway (from phosphoenolpyruvate to oxaloacetate). In addition, we tested selected lactate dehydrogenases, some of which were further optimized through site-directed mutagenesis to improve the enzyme’s affinity for the co-factor nicotinamide adenine dinucleotide phosphate (NADPH). The carbon partitioning between biomass and lactic acid was increased from about 5% to over 50% by strain optimization.
An efficient photosynthetic microbial cell factory will display a high rate and extent of conversion of substrate (CO2) into product (here: L-lactic acid). In the existing CO2-based cyanobacterial cell factories that have been described in the literature, by far most of the control over product formation resides in the genetically introduced fermentative pathway. Here we show that a strong promoter, in combination with increased gene expression, can take away a significant part of the control of this step in lactic acid production from CO2. Under these premises, modulation of the intracellular precursor, pyruvate, can significantly increase productivity. Additionally, production enhancement is achieved by protein engineering to increase co-factor specificity of the heterologously expressed LDH.
PMCID: PMC4078008  PMID: 24991233
Cyanobacteria; L-lactic acid production; Bioplastic; Metabolic engineering; Microbial cell factory; Lactate dehydrogenase; Pyruvate kinase; Control coefficient
2.  Integration of molecular genetics and proteomics with cell metabolism: How to proceed; How not to proceed! 
Gene  2011;486(0):88-93.
There now exists a resurgence of interest in the role of intermediary metabolism in medicine; especially in relation to medical disorders. Coupled with this is the contemporary focus on molecular biology, genetics and proteomics and their integration into studies of regulation and alterations in cellular metabolism in health and disease. This is a marriage that has vast potential for elucidation of the factors and conditions that are involved in cellular metabolic and functional changes, which heretofore could not be addressed by the earlier generations of biochemists who established the major pathways of intermediary metabolism. The achievement of this present potential requires the appropriate application and interpretation of genetic and proteomic studies relating to cell metabolism and cell function. This requires knowledge and understanding of the principles, relationships, and methodology, such as biochemistry and enzymology, which are involved in the elucidation of cellular regulatory enzymes and metabolic pathways. Unfortunately, many and possibly most contemporary molecular biologists are not adequately trained and knowledgeable in these areas of cell metabolism. This has resulted in much too common inappropriate application and misinformation from genetic/proteomic studies of cell metabolism and function. This presentation describes important relationships of cellular intermediary metabolism, and provides examples of the appropriate and inappropriate application of genetics and proteomics. It calls for the inclusion of biochemistry, enzymology, cell metabolism and cell physiology in the graduate and postgraduate training of molecular biology and other biomedical researchers.
PMCID: PMC4041378  PMID: 21782907
Cellular intermediary metabolism; Genetics and proteomics; Geneticist and biochemist approach; Medicine and medical disorders; Regulatory enzymes and transporters
3.  Tumor cell metabolism: the marriage of molecular genetics and proteomics with cellular intermediary metabolism; proceed with caution! 
Molecular Cancer  2006;5:59.
Metabolic transformations of malignant cells are essential to the development and progression of all cancers. The understanding of the pathogenesis and progression of cancer requires the establishment of the altered genetic/metabolic factors that are essential to the development, growth, and proliferation of the malignant cells. Recognition of this important relationship has resulted in a resurgence of interest in the intermediary metabolism of tumor cells. The role of molecular genetics and proteomics and the application of molecular technology in assessing altered cellular metabolism has become a major area of biomedical research. The contemporary generation of biomedical scientists is exceptionally well trained in all areas of molecular biology and molecular technology, which are now important tools to be applied to the regulation of cellular intermediary metabolism. Simultaneously, the didactic and methodological training associated with the principles and operation of metabolic pathways, enzymology, cellular enzyme activity, and associated biochemical implications has been diminished and often eliminated from the pre- and post-doctoral programs. Interpretations and conclusions of alterations in cellular enzyme activity and associated metabolic pathways based on genetic/proteomic changes can and will result in misrepresentation of important metabolic implications in malignancy and other diseases. It is essential that the genetic/proteomic studies be coupled to biochemical/metabolic cellular events to satisfy the axiom: "genetic transformations and proteomic alterations will have little relevancy to disease processes if the genetic/proteomic alterations are not manifested in altered and impaired cellular and metabolic function". The appropriate marriage of molecular genetics/proteomics with the regulation of cellular intermediary metabolism will provide new revelations and understanding of malignancy that could not be achieved in earlier generations.
PMCID: PMC1636067  PMID: 17090311
4.  A review of the important central role of altered citrate metabolism during the process of stem cell differentiation 
Stem cells are highly proliferating cells that have the potential for differentiation leading to the development of specialized functional cell types. The process of stem cell differentiation requires an increase in the recruitment and population of the undifferentiated stem cells, which are then differentiated to specific functional cell types. Genetic/metabolic transformations in the cellular intermediary energy metabolism are required to provide the bioenergetic, synthetic, and catabolic requirements of the stem cells during this process. However, the identification of the intermediary energy metabolism pathways and their alterations during the proliferation and differentiation of stem cells remain largely unknown; mainly due to the lack of attention and/or required research that focuses on this relationship. In the absence of such information, a full understanding of the factors and conditions required to promote stem cell differentiation leading to development of normal functional metabolic specialized cells cannot be achieved. The purpose of this review is to provide the background and bring attention to the essential relationship of altered cellular intermediary metabolism in the context of the process of stem cell proliferation and differentiation. Citrate metabolism is central to the genetic and metabolic transformation leading to the development of the specialized functional cells. This review identifies the involvement of altered citrate metabolism and the associated genetic alterations of key pathways, enzymes, and transporters; as well as the bioenergetic implications. The importance is emphasized for identification and employment of required conditions to insure that the process of experimental stem cell differentiation results in the development of specialized cells that represent the functional metabolic characteristics and capabilities of their native specialized cells. This is an essential requirement for the successful application of stem cell therapy and regenerative medicine for many pathological conditions.
PMCID: PMC3815687  PMID: 24194979
Citrate metabolism; stem cells; differentiation and proliferation; osteogenesis; osteoblast and bone formation adipogenesis; krebs cycle; glycolysis
5.  Integrated physiology and systems biology of PPARα 
Molecular Metabolism  2014;3(4):354-371.
The Peroxisome Proliferator Activated Receptor alpha (PPARα) is a transcription factor that plays a major role in metabolic regulation. This review addresses the functional role of PPARα in intermediary metabolism and provides a detailed overview of metabolic genes targeted by PPARα, with a focus on liver. A distinction is made between the impact of PPARα on metabolism upon physiological, pharmacological, and nutritional activation. Low and high throughput gene expression analyses have allowed the creation of a comprehensive map illustrating the role of PPARα as master regulator of lipid metabolism via regulation of numerous genes. The map puts PPARα at the center of a regulatory hub impacting fatty acid uptake, fatty acid activation, intracellular fatty acid binding, mitochondrial and peroxisomal fatty acid oxidation, ketogenesis, triglyceride turnover, lipid droplet biology, gluconeogenesis, and bile synthesis/secretion. In addition, PPARα governs the expression of several secreted proteins that exert local and endocrine functions.
PMCID: PMC4060217  PMID: 24944896
PPARα; Liver; Transcriptional networks; Lipid metabolism; Expression profiling; Metabolic homeostasis; Systems biology
6.  The Evolution of Connectivity in Metabolic Networks 
PLoS Biology  2005;3(7):e228.
Processes in living cells are the result of interactions between biochemical compounds in highly complex biochemical networks. It is a major challenge in biology to understand causes and consequences of the specific design of these networks. A characteristic design feature of metabolic networks is the presence of hub metabolites such as ATP or NADH that are involved in a high number of reactions. To study the emergence of hub metabolites, we implemented computer simulations of a widely accepted scenario for the evolution of metabolic networks. Our simulations indicate that metabolic networks with a large number of highly specialized enzymes may evolve from a few multifunctional enzymes. During this process, enzymes duplicate and specialize, leading to a loss of biochemical reactions and intermediary metabolites. Complex features of metabolic networks such as the presence of hubs may result from selection of growth rate if essential biochemical mechanisms are considered. Specifically, our simulations indicate that group transfer reactions are essential for the emergence of hubs.
Computer simulations show how the complex organization of metabolic networks can arise from selection for a simple trait such as growth rate.
PMCID: PMC1157096  PMID: 16000019
7.  User interactions with the PDQ cancer information system. 
Searches by end users and intermediaries on the online PDQ (Physician Data Query) cancer information system were observed. With the National Library of Medicine (NLM) menu-based interface, end users (physicians) averaged fewer steps per question, while with the BRS command-drive interface, intermediaries appeared to be more efficient. Cancer Information Service (CIS) searchers, who have more PDQ experience than end users or intermediaries, made greater use of command stacking to anticipate menu selections. Retrieval was more complete in the NLM system, where both the menus and predefined print formats assisted the searchers.
PMCID: PMC225612  PMID: 1537014
8.  Caste- and development-associated gene expression in a lower termite 
Genome Biology  2003;4(10):R62.
Social insects such as termites express dramatic polyphenism (the occurrence of multiple forms in a species on the basis of differential gene expression) both in association with caste differentiation and between castes after differentiation. We have used cDNA macroarrays to compare gene expression between polyphenic castes and intermediary developmental stages of the termite Reticulitermes flavipes.
Social insects such as termites express dramatic polyphenism (the occurrence of multiple forms in a species on the basis of differential gene expression) both in association with caste differentiation and between castes after differentiation. We have used cDNA macroarrays to compare gene expression between polyphenic castes and intermediary developmental stages of the termite Reticulitermes flavipes.
We identified differentially expressed genes from nine ontogenic categories. Quantitative PCR was used to quantify precise differences in gene expression between castes and between intermediary developmental stages. We found worker and nymph-biased expression of transcripts encoding termite and endosymbiont cellulases; presoldier-biased expression of transcripts encoding the storage/hormone-binding protein vitellogenin; and soldier-biased expression of gene transcripts encoding two transcription/translation factors, two signal transduction factors and four cytoskeletal/muscle proteins. The two transcription/translation factors showed significant homology to the bicaudal and bric-a-brac developmental genes of Drosophila.
Our results show differential expression of regulatory, structural and enzyme-coding genes in association with termite castes and their developmental precursor stages. They also provide the first glimpse into how insect endosymbiont cellulase gene expression can vary in association with the caste of a host. These findings shed light on molecular processes associated with termite biology, polyphenism, caste differentiation and development and highlight potentially interesting variations in developmental themes between termites, other insects, and higher animals.
PMCID: PMC328451  PMID: 14519197
9.  Emerging Perspectives on Essential Amino Acid Metabolism in Obesity and the Insulin-Resistant State12 
Advances in Nutrition  2011;2(6):445-456.
Dysregulation of insulin action is most often considered in the context of impaired glucose homeostasis, with the defining feature of diabetes mellitus being elevated blood glucose concentration. Complications arising from the hyperglycemia accompanying frank diabetes are well known and epidemiological studies point to higher risk toward development of metabolic disease in persons with impaired glucose tolerance. Although the central role of proper blood sugar control in maintaining metabolic health is well established, recent developments have begun to shed light on associations between compromised insulin action [obesity, prediabetes, and type 2 diabetes mellitus (T2DM)] and altered intermediary metabolism of fats and amino acids. For amino acids, changes in blood concentrations of select essential amino acids and their derivatives, in particular BCAA, sulfur amino acids, tyrosine, and phenylalanine, are apparent with obesity and insulin resistance, often before the onset of clinically diagnosed T2DM. This review provides an overview of these changes and places recent observations from metabolomics research into the context of historical reports in the areas of biochemistry and nutritional biology. Based on this synthesis, a model is proposed that links the FFA-rich environment of obesity/insulin resistance and T2DM with diminution of BCAA catabolic enzyme activity, changes in methionine oxidation and cysteine/cystine generation, and tissue redox balance (NADH/NAD+).
PMCID: PMC3226382  PMID: 22332087
10.  Comparative Genomics of Insect-Symbiotic Bacteria: Influence of Host Environment on Microbial Genome Composition 
Applied and Environmental Microbiology  2003;69(11):6825-6832.
Commensal symbionts, thought to be intermediary amid obligate mutualists and facultative parasites, offer insight into forces driving the evolutionary transition into mutualism. Using macroarrays developed for a close relative, Escherichia coli, we utilized a heterologous array hybridization approach to infer the genomic compositions of a clade of bacteria that have recently established symbiotic associations: Sodalis glossinidius with the tsetse fly (Diptera, Glossina spp.) and Sitophilus oryzae primary endosymbiont (SOPE) with the rice weevil (Coleoptera, Sitophilus oryzae). Functional biologies within their hosts currently reflect different forms of symbiotic associations. Their hosts, members of distant insect taxa, occupy distinct ecological niches and have evolved to survive on restricted diets of blood for tsetse and cereal for the rice weevil. Comparison of genome contents between the two microbes indicates statistically significant differences in the retention of genes involved in carbon compound catabolism, energy metabolism, fatty acid metabolism, and transport. The greatest reductions have occurred in carbon catabolism, membrane proteins, and cell structure-related genes for Sodalis and in genes involved in cellular processes (i.e., adaptations towards cellular conditions) for SOPE. Modifications in metabolic pathways, in the form of functional losses complementing particularities in host physiology and ecology, may have occurred upon initial entry from a free-living to a symbiotic state. It is possible that these adaptations, streamlining genomes, act to make a free-living state no longer feasible for the harnessed microbe.
PMCID: PMC262273  PMID: 14602646
11.  Pyrosequencing of Mytilus galloprovincialis cDNAs: Tissue-Specific Expression Patterns 
PLoS ONE  2010;5(1):e8875.
Mytilus species are important in marine ecology and in environmental quality assessment, yet their molecular biology is poorly understood. Molecular aspects of their reproduction, hybridisation between species, mitochondrial inheritance, skewed sex ratios of offspring and adaptation to climatic and pollution factors are priority areas.
Methodology/Principal Findings
To start to address this situation, expressed genetic transcripts from M. galloprovincialis were pyrosequenced. Transcripts were isolated from the digestive gland, foot, gill and mantle of both male and female mussels. In total, 175,547 sequences were obtained and for foot and mantle, 90% of the sequences could be assembled into contiguous fragments but this reduced to 75% for the digestive gland and gill. Transcripts relating to protein metabolism and respiration dominated including ribosomal proteins, cytochrome oxidases and NADH dehydrogenase subunits. Tissue specific variation was identified in transcripts associated with mitochondrial energy metabolism, with the digestive gland and gill having the greatest transcript abundance. Using fragment recruitment it was also possible to identify sites of potential small RNAs involved in mitochondrial transcriptional regulation. Sex ratios based on Vitelline Envelop Receptor for Lysin and Vitelline Coat Lysin transcript abundances, indicated that an equal sex distribution was maintained. Taxonomic profiling of the M. galloprovincialis tissues highlighted an abundant microbial flora associated with the digestive gland. Profiling of the tissues for genes involved in intermediary metabolism demonstrated that the gill and digestive gland were more similar to each other than to the other two tissues, and specifically the foot transcriptome was most dissimilar.
Pyrosequencing has provided extensive genomic information for M. galloprovincialis and generated novel observations on expression of different tissues, mitochondria and associated microorganisms. It will also facilitate the much needed production of an oligonucleotide microarray for the organism.
PMCID: PMC2810337  PMID: 20111607
12.  Dependence on dose of the acute effects of ethanol on liver metabolism in vivo. 
Journal of Clinical Investigation  1975;56(6):1411-1419.
The dose dependence of the acute effects of ethanol upon liver intermediary metabolism in vivo has been demonstrated in rats. Ethanol was given i.p. in doses of 0.69, 1.7, and 3.0 g/kg in equal volumes (20 ml/kg). The liver was freeze-clamped 120 min after injection, and multiple metabolites were measured in the perchloric acid extract of the tissue. Each group showed a significantly different pattern of metabolites, redox states, and phosphorylation potentials although the rate of ethanol disappearance, at least between the two highest dose groups, was not significantly different. The mitochondrial free [NAD+]/[NADH] ratios and the cytoplasmic free [NADP+]/[NADPH] ratio were paradoxically most reduced with the lowest dose of ethanol and became progressively more oxidized with increasing dose. Once established, the differences in these ratios between the groups tended to persist with time, relatively independent of the concentration of ethanol. In a somewhat different pattern, the phosphorylation potential ([ATP]/[ADP][P1]) remained at the control level in the low-dose group but was significantly elevated in the two higher-dose groups. The results, therefore, show distinct and complicated dose-dependent patterns of intermediary metabolism that cannot be explained completely by any one hypothesis but that imply significant dose-dependent effects of ethanol upon intermediary metabolism not directly related to NADH production.
PMCID: PMC333119  PMID: 422
13.  Potential for Direct Interspecies Electron Transfer in Methanogenic Wastewater Digester Aggregates 
mBio  2011;2(4):e00159-11.
Mechanisms for electron transfer within microbial aggregates derived from an upflow anaerobic sludge blanket reactor converting brewery waste to methane were investigated in order to better understand the function of methanogenic consortia. The aggregates were electrically conductive, with conductivities 3-fold higher than the conductivities previously reported for dual-species aggregates of Geobacter species in which the two species appeared to exchange electrons via interspecies electron transfer. The temperature dependence response of the aggregate conductance was characteristic of the organic metallic-like conductance previously described for the conductive pili of Geobacter sulfurreducens and was inconsistent with electron conduction through minerals. Studies in which aggregates were incubated with high concentrations of potential electron donors demonstrated that the aggregates had no significant capacity for conversion of hydrogen to methane. The aggregates converted formate to methane but at rates too low to account for the rates at which that the aggregates syntrophically metabolized ethanol, an important component of the reactor influent. Geobacter species comprised 25% of 16S rRNA gene sequences recovered from the aggregates, suggesting that Geobacter species may have contributed to some but probably not all of the aggregate conductivity. Microorganisms most closely related to the acetate-utilizing Methanosaeta concilii accounted for more than 90% of the sequences that could be assigned to methane producers, consistent with the poor capacity for hydrogen and formate utilization. These results demonstrate for the first time that methanogenic wastewater aggregates can be electrically conductive and suggest that direct interspecies electron transfer could be an important mechanism for electron exchange in some methanogenic systems.
The conversion of waste organic matter to methane is an important bioenergy strategy, and a similar microbial metabolism of complex organic matter in anaerobic soils and sediments plays an important role in the global carbon cycle. Studies with laboratory cultures have demonstrated that hydrogen or formate can serve as an electron shuttle between the microorganisms degrading organic compounds and methanogens. However, the importance of hydrogen and formate as intermediates in the conversion of organic matter to methane in natural communities is less clear. The possibility that microorganisms within some natural methanogenic aggregates may directly exchange electrons, rather than producing hydrogen or formate as an intermediary electron carrier, is a significant paradigm shift with implications for the modeling and design of anaerobic wastewater reactors and for understanding how methanogenic communities will respond to environmental perturbations.
PMCID: PMC3157894  PMID: 21862629
14.  Comparative Analysis of Predicted Plastid-Targeted Proteomes of Sequenced Higher Plant Genomes 
PLoS ONE  2014;9(11):e112870.
Plastids are actively involved in numerous plant processes critical to growth, development and adaptation. They play a primary role in photosynthesis, pigment and monoterpene synthesis, gravity sensing, starch and fatty acid synthesis, as well as oil, and protein storage. We applied two complementary methods to analyze the recently published apple genome (Malus × domestica) to identify putative plastid-targeted proteins, the first using TargetP and the second using a custom workflow utilizing a set of predictive programs. Apple shares roughly 40% of its 10,492 putative plastid-targeted proteins with that of the Arabidopsis (Arabidopsis thaliana) plastid-targeted proteome as identified by the Chloroplast 2010 project and ∼57% of its entire proteome with Arabidopsis. This suggests that the plastid-targeted proteomes between apple and Arabidopsis are different, and interestingly alludes to the presence of differential targeting of homologs between the two species. Co-expression analysis of 2,224 genes encoding putative plastid-targeted apple proteins suggests that they play a role in plant developmental and intermediary metabolism. Further, an inter-specific comparison of Arabidopsis, Prunus persica (Peach), Malus × domestica (Apple), Populus trichocarpa (Black cottonwood), Fragaria vesca (Woodland Strawberry), Solanum lycopersicum (Tomato) and Vitis vinifera (Grapevine) also identified a large number of novel species-specific plastid-targeted proteins. This analysis also revealed the presence of alternatively targeted homologs across species. Two separate analyses revealed that a small subset of proteins, one representing 289 protein clusters and the other 737 unique protein sequences, are conserved between seven plastid-targeted angiosperm proteomes. Majority of the novel proteins were annotated to play roles in stress response, transport, catabolic processes, and cellular component organization. Our results suggest that the current state of knowledge regarding plastid biology, preferentially based on model systems is deficient. New plant genomes are expected to enable the identification of potentially new plastid-targeted proteins that will aid in studying novel roles of plastids.
PMCID: PMC4231079  PMID: 25393533
15.  LC–MS Proteomics Analysis of the Insulin/IGF-1-Deficient Caenorhabditis elegans daf-2(e1370) Mutant Reveals Extensive Restructuring of Intermediary Metabolism 
Journal of Proteome Research  2014;13(4):1938-1956.
The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity, and metabolism in Caenorhabditis elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including the expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass spectrometry (LC–MS)-based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2);daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the upregulation of many core intermediary metabolic pathways. These include glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid β-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complexes I, II, III, and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative of spatiotemporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. This restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves and possibly also shunting metabolites through alternative energy-generating pathways to sustain longevity.
PMCID: PMC3993954  PMID: 24555535
Caenorhabditis elegans; gene expression; mass spectrometry; metabolism; physiology; aging
16.  Stratus Not Altocumulus: A New View of the Yeast Protein Interaction Network 
PLoS Biology  2006;4(10):e317.
Systems biology approaches can reveal intermediary levels of organization between genotype and phenotype that often underlie biological phenomena such as polygenic effects and protein dispensability. An important conceptualization is the module, which is loosely defined as a cohort of proteins that perform a dedicated cellular task. Based on a computational analysis of limited interaction datasets in the budding yeast Saccharomyces cerevisiae, it has been suggested that the global protein interaction network is segregated such that highly connected proteins, called hubs, tend not to link to each other. Moreover, it has been suggested that hubs fall into two distinct classes: “party” hubs are co-expressed and co-localized with their partners, whereas “date” hubs interact with incoherently expressed and diversely localized partners, and thereby cohere disparate parts of the global network. This structure may be compared with altocumulus clouds, i.e., cotton ball–like structures sparsely connected by thin wisps. However, this organization might reflect a small and/or biased sample set of interactions. In a multi-validated high-confidence (HC) interaction network, assembled from all extant S. cerevisiae interaction data, including recently available proteome-wide interaction data and a large set of reliable literature-derived interactions, we find that hub–hub interactions are not suppressed. In fact, the number of interactions a hub has with other hubs is a good predictor of whether a hub protein is essential or not. We find that date hubs are neither required for network tolerance to node deletion, nor do date hubs have distinct biological attributes compared to other hubs. Date and party hubs do not, for example, evolve at different rates. Our analysis suggests that the organization of global protein interaction network is highly interconnected and hence interdependent, more like the continuous dense aggregations of stratus clouds than the segregated configuration of altocumulus clouds. If the network is configured in a stratus format, cross-talk between proteins is potentially a major source of noise. In turn, control of the activity of the most highly connected proteins may be vital. Indeed, we find that a fluctuation in steady-state levels of the most connected proteins is minimized.
Analysis of multi-validated protein interaction data reveals networks with greater interconnectivity than the more segregated structures seen in previously available data. To help visualize this, the authors draw comparisons between continuous stratus clouds and altocumulus clouds.
PMCID: PMC1569888  PMID: 16984220
17.  Human holocarboxylase synthetase with a start site at methionine-58 is the predominant nuclear variant of this protein and has catalytic activity 
Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of biotin to both carboxylases in extranuclear structures and histones in cell nuclei, thereby mediating important roles in intermediary metabolism, gene regulation, and genome stability. HLCS has three putative translational start sites (methionine-1, -7, and -58), but lacks a strong nuclear localization sequence that would explain its participation in epigenetic events in the cell nucleus. Recent evidence suggests that small quantities of HLCS with a start site in methionine-58 (HLCS58) might be able to enter the nuclear compartment. We generated the following novel insights into HLCS biology. First, we generated a novel HLCS fusion protein vector to demonstrate that methionine-58 is a functional translation start site in human cells. Second, we used confocal microscopy and western blots to demonstrate that HLCS58 enters the cell nucleus in meaningful quantities, and that full-length HLCS localizes predominantly in the cytoplasm but may also enter the nucleus. Third, we produced recombinant HLCS58 to demonstrate its biological activity toward catalyzing the biotinylation of both carboxylases and histones. Collectively, these observations are consistent with roles of HLCS58 and full-length HLCS in nuclear events. We conclude this report by proposing a novel role for HLCS in epigenetic events, mediated by physical interactions between HLCS and other chromatin proteins as part of a larger multiprotein complex that mediates gene repression.
PMCID: PMC3159853  PMID: 21802411
biotin; carboxylases; holocarboxylase synthetase; nucleus; translation
18.  Chorismic acid, a key metabolite in modification of tRNA. 
Journal of Bacteriology  1990;172(1):252-259.
Chorismic acid is the common precursor for the biosynthesis of the three aromatic amino acids as well as for four vitamins. Mutants of Escherichia coli defective in any of the genes involved in the synthesis of chorismic acid are also unable to synthesize uridine 5-oxyacetic acid (cmo5U) and its methyl ester (mcmo5U). Both modified nucleosides are normally present in the wobble position of some tRNA species. Mutants defective in any of the specific pathways leading to phenylalanine, tyrosine, tryptophan, folate, enterochelin, ubiquinone, and menaquinone have normal levels of cmo5U and mcmo5U in their tRNA. The presence of shikimic acid in the growth medium restores the ability of an aroD mutant to synthesize cmo5U, while O-succinylbenzoate, which is an early intermediate in the synthesis of menaquinone, does not. Thus, chorismic acid is a key metabolite in the synthesis of these two modified nucleosides in tRNA. The absence of chorismic acid blocks the formation of cmo5U and mcmo5U at the first step, which might be the formation of 5-hydroxyuridine. This results in an unmodified U in the wobble position of tRNA(1Val) and in most of the tRNAs normally containing cmo5U and mcmo5U. Since cmo5U and mcmo5U are synthesized under anaerobic conditions, the formation of these nucleosides does not require molecular oxygen. One of the carbon atoms of the side chain, --O--CH2--COOH, originates from the methyl group of methionine. The other carbon atom does not originate directly from the C-1 pool, from the carboxyl group methionine, or from bicarbonate. This metabolic link between intermediary metabolism and translation also exists for another member of the family Enterobacteriaceae, Salmonella typhimurium, as well as for the distantly related gram-positive organism Bacillus subtilis.
PMCID: PMC208425  PMID: 2104604
19.  Temporal Expression-based Analysis of Metabolism 
PLoS Computational Biology  2012;8(11):e1002781.
Metabolic flux is frequently rerouted through cellular metabolism in response to dynamic changes in the intra- and extra-cellular environment. Capturing the mechanisms underlying these metabolic transitions in quantitative and predictive models is a prominent challenge in systems biology. Progress in this regard has been made by integrating high-throughput gene expression data into genome-scale stoichiometric models of metabolism. Here, we extend previous approaches to perform a Temporal Expression-based Analysis of Metabolism (TEAM). We apply TEAM to understanding the complex metabolic dynamics of the respiratorily versatile bacterium Shewanella oneidensis grown under aerobic, lactate-limited conditions. TEAM predicts temporal metabolic flux distributions using time-series gene expression data. Increased predictive power is achieved by supplementing these data with a large reference compendium of gene expression, which allows us to take into account the unique character of the distribution of expression of each individual gene. We further propose a straightforward method for studying the sensitivity of TEAM to changes in its fundamental free threshold parameter θ, and reveal that discrete zones of distinct metabolic behavior arise as this parameter is changed. By comparing the qualitative characteristics of these zones to additional experimental data, we are able to constrain the range of θ to a small, well-defined interval. In parallel, the sensitivity analysis reveals the inherently difficult nature of dynamic metabolic flux modeling: small errors early in the simulation propagate to relatively large changes later in the simulation. We expect that handling such “history-dependent” sensitivities will be a major challenge in the future development of dynamic metabolic-modeling techniques.
Author Summary
Understanding the dynamic response of microorganisms to environmental changes is a major challenge in systems biology. In many cases, these responses manifest themselves through changes in gene transcription, which then propagate to adjust flow through metabolism. Here, we implement a Temporal Expression-based Analysis of Metabolism (TEAM) by dynamically integrating a genome-scale model of the metabolism of S. oneidensis with high-throughput measurements of gene expression and growth data. TEAM recapitulates the complex cascade of secretion and re-uptake of intermediary carbon sources that S. oneidensis exhibits in the experimental data. We show that these complicated metabolic behaviors are best captured when TEAM explicitly accounts for each gene's unique transcriptional signature. Furthermore, by way of a newly proposed sensitivity analysis, we reveal and study the inherent difficulty of dynamic metabolic flux modeling: small changes early in a simulation can easily spread and lead to significant changes towards the end of it. We expect that further development of robust dynamic flux balance methods will need to overcome such “history-dependent” sensitivities in order to achieve increased predictive accuracy.
PMCID: PMC3510039  PMID: 23209390
20.  Activation of CAR and PXR by Dietary, Environmental and Occupational Chemicals Alters Drug Metabolism, Intermediary Metabolism, and Cell Proliferation 
The constitutive androstane receptor (CAR) and the pregnane × receptor (PXR) are activated by a variety of endogenous and exogenous ligands, such as steroid hormones, bile acids, pharmaceuticals, and environmental, dietary, and occupational chemicals. In turn, they induce phase I–III detoxification enzymes and transporters that help eliminate these chemicals. Because many of the chemicals that activate CAR and PXR are environmentally-relevant (dietary and anthropogenic), studies need to address whether these chemicals or mixtures of these chemicals may increase the susceptibility to adverse drug interactions. In addition, CAR and PXR are involved in hepatic proliferation, intermediary metabolism, and protection from cholestasis. Therefore, activation of CAR and PXR may have a wide variety of implications for personalized medicine through physiological effects on metabolism and cell proliferation; some beneficial and others adverse. Identifying the chemicals that activate these promiscuous nuclear receptors and understanding how these chemicals may act in concert will help us predict adverse drug reactions (ADRs), predict cholestasis and steatosis, and regulate intermediary metabolism. This review summarizes the available data on CAR and PXR, including the environmental chemicals that activate these receptors, the genes they control, and the physiological processes that are perturbed or depend on CAR and PXR action. This knowledge contributes to a foundation that will be necessary to discern interindividual differences in the downstream biological pathways regulated by these key nuclear receptors.
PMCID: PMC2944248  PMID: 20871735
Constitutive androstane receptor; pregnane × receptor; orphan nuclear receptors; drug targets; gene-environment interaction
21.  Control of Interspecies Electron Flow during Anaerobic Digestion: Role of Floc Formation in Syntrophic Methanogenesis 
The flora of an anaerobic whey-processing chemostat was separated by anaerobic sedimentation techniques into a free-living bacterial fraction and a bacterial floc fraction. The floc fraction constituted a major part (i.e., 57% total protein) of the total microbial population in the digestor, and it accounted for 87% of the total CO2-dependent methanogenic activity and 76% of the total ethanol-consuming acetogenic activity. Lactose was degraded by both cellular fractions, but in the free flora fraction it was associated with higher intermediary levels of H2, ethanol, butyrate, and propionate production. Electron microscopic analysis of flocs showed bacterial diversity and juxtapositioning of tentative Desulfovibrio and Methanobacterium species without significant microcolony formation. Ethanol, an intermediary product of lactose-hydrolyzing bacteria, was converted to acetate and methane within the flocs by interspecies electron transfer. Ethanol-dependent methane formation was compartmentalized and closely coupled kinetically within the flocs but without significant formation of H2 gas. Physical disruption of flocs into fragments of 10- to 20-μm diameter initially increased the H2 partial pressure but did not change the carbon transformation kinetic patterns of ethanol metabolism or demonstrate a significant role for H2 in CO2 reduction to methane. The data demonstrate that floc formation in a whey-processing anaerobic digestor functions in juxtapositioning cells for interspecies electron transfer during syntrophic ethanol conversion into acetate and methane but by a mechanism which was independent of the available dissolved H2 gas pool in the ecosystem.
PMCID: PMC202390  PMID: 16347517
22.  A Genetically Hard-Wired Metabolic Transcriptome in Plasmodium falciparum Fails to Mount Protective Responses to Lethal Antifolates 
PLoS Pathogens  2008;4(11):e1000214.
Genome sequences of Plasmodium falciparum allow for global analysis of drug responses to antimalarial agents. It was of interest to learn how DNA microarrays may be used to study drug action in malaria parasites. In one large, tightly controlled study involving 123 microarray hybridizations between cDNA from isogenic drug-sensitive and drug-resistant parasites, a lethal antifolate (WR99210) failed to over-produce RNA for the genetically proven principal target, dihydrofolate reductase-thymidylate synthase (DHFR-TS). This transcriptional rigidity carried over to metabolically related RNA encoding folate and pyrimidine biosynthesis, as well as to the rest of the parasite genome. No genes were reproducibly up-regulated by more than 2-fold until 24 h after initial drug exposure, even though clonal viability decreased by 50% within 6 h. We predicted and showed that while the parasites do not mount protective transcriptional responses to antifolates in real time, P. falciparum cells transfected with human DHFR gene, and adapted to long-term WR99210 exposure, adjusted the hard-wired transcriptome itself to thrive in the presence of the drug. A system-wide incapacity for changing RNA levels in response to specific metabolic perturbations may contribute to selective vulnerabilities of Plasmodium falciparum to lethal antimetabolites. In addition, such regulation affects how DNA microarrays are used to understand the mode of action of antimetabolites.
Author Summary
Traditional knowledge of gene regulation, learned largely from non-pathogenic model organisms such as E. coli, yeast, and mice, suggests that RNA for metabolic pathways are regulated in large part by DNA-binding transcriptional factors that are responsive to cellular metabolic needs. We demonstrate that the malaria-causing Plasmodium falciparum parasites, under lethal drug pressure from an antifolate with a known mechanism of action, are incapable of large reproducible changes in RNA levels for the target pathways, or for any other gene throughout the genome. Small RNA changes, possibly informative of perturbed pathways, can be detected in dying parasites. In addition, significant RNA changes are seen when the hard-wired program, governing RNA levels, itself is altered. Our data formally proves that RNA levels for intermediary metabolism in malaria parasites are largely predetermined. We propose that as a parasite with a complex life cycle travels from one largely predictable intracellular biochemical environment to another, such hard-wiring may be sufficient to manage transcript levels for intermediary metabolism without employing sensory functions. Such a system-wide host–parasite difference in gene regulation may create unexpected pharmacological opportunities when important target pathways are rigid in the parasite but dynamically regulated in host cells.
PMCID: PMC2581438  PMID: 19023412
23.  Interplay of Factors Leading to Adverse Drug Reactions in the Liver, A Personal Viewpoint 1 
Adverse drug reactions in liver involve formation of a reactive metabolic intermediary of the drug, binding of the intermediary to macromolecules in the cell, notably proteins in the plasma membrane, immunological response to these altered proteins and attack against hepatocytes bearing these altered proteins by immune mechanisms. At each step in this complex process many factors act to enhance or depress drug metabolism, metabolite disposition, macromolecular binding, neoantigen formation, and the cell mediated and humoral immune attack. The extent and direction of each step may be dose dependent but the complexity of the overall mechanism is so immense that predictability of hepatic drug reactions is unlikely in most instances.
PMCID: PMC2595561  PMID: 602258
24.  The Role of Protease Inhibitors in the Pathogenesis of HIV-Associated Lipodystrophy: Cellular Mechanisms and Clinical Implications 
Toxicologic pathology  2009;37(1):65-77.
Metabolic complications associated with HIV infection and treatment frequently present as a relative lack of peripheral adipose tissue associated with dyslipidemia and insulin resistance. In this review we explain the connection between abnormalities of intermediary metabolism, observed either in vitro or in vivo, and this group of metabolic effects. We review molecular mechanisms by which the HIV protease inhibitor (PI) class of drugs may affect the normal stimulatory effect of insulin on glucose and fat storage. We then propose that both chronic inflammation from HIV infection and treatment with some drugs in this class trigger cellular homeostatic stress responses with adverse effects on intermediary metabolism. The physiologic outcome is such that total adipocyte storage capacity is decreased, and the remaining adipocytes resist further fat storage. The excess circulating and dietary lipid metabolites, normally “absorbed” by adipose tissue, are deposited ectopically in lean (muscle and liver) tissue, where they impair insulin action. This process leads to a pathologic cycle of lipotoxicity and lipoatrophy and a clinical phenotype of body fat distribution with elevated waist-to-hip ratio similar to the metabolic syndrome.
PMCID: PMC3170409  PMID: 19171928
protease inhibitors; lipodystrophy; dyslipidemia; insulin resistance; cardiovascular disease; mechanisms of toxicity; molecular pathology; clinical pathology; in vitro toxicology; endocrine system; pharmaceutical development/products
The Journal of Cell Biology  1968;39(3):501-525.
Ultrastructural study of the prothoracic glands of silkworms (Antheraea pernyi and Bombyx mori) at the last two larval stages has shown that the essential modifications which take place during each intermolt affect the chondriome of secretory cells. A description is given of the differentiation of macromitochondria from typical mitochondria by a general swelling, a clearing of the matrix, and the formation of a complex tubular network. The hypothesis of fixation or anaesthesia artifacts has been dismissed because of the persistence of these transformations after different fixations and because of the existence of numerous intermediary stages between these two types of chondriosomes which imply the progressiveness of differentiation. The cytochemical demonstration of mitochondrial DNA fibers suggests that the genetic information, probably present in this type of nucleic acid, controls the differentiation and the specific metabolic activity of these organelles. The frequent relationships observed in Antheraea between the tubules of agranular reticulum and the macromitochondria which are reminiscent of the vacuoles-mitochondria associations of the adrenal cortex, may be related to the transfer of cholesterol and other precursors of steroidogenesis. In the last stages, the macromitochondria become transformed into vacuoles by a disappearance of the tubular network. The correlation revealed between mitochondrial transformations and the cyclical release of ecdysone (65) leads to the conclusion that a prominent fraction of chondriosomes is involved, in relation to the agranular reticulum, in the elaboration of steroid hormones such as ecdysone.
PMCID: PMC2107545  PMID: 5699930

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