Mitochondrial Substrate-Level Phosphorylation as Energy Source for Glioblastoma: Review and Hypothesis

Glioblastoma multiforme (GBM) is the most common and malignant of the primary adult brain cancers. Ultrastructural and biochemical evidence shows that GBM cells exhibit mitochondrial abnormalities incompatible with energy production through oxidative phosphorylation (OxPhos). Under such conditions, the mitochondrial F0-F1 ATP synthase operates in reverse at the expense of ATP hydrolysis to maintain a moderate membrane potential. Moreover, expression of the dimeric M2 isoform of pyruvate kinase in GBM results in diminished ATP output, precluding a significant ATP production from glycolysis. If ATP synthesis through both glycolysis and OxPhos was impeded, then where would GBM cells obtain high-energy phosphates for growth and invasion? Literature is reviewed suggesting that the succinate-CoA ligase reaction in the tricarboxylic acid cycle can substantiate sufficient ATP through mitochondrial substrate-level phosphorylation (mSLP) to maintain GBM growth when OxPhos is impaired. Production of high-energy phosphates would be supported by glutaminolysis-a hallmark of GBM metabolism-through the sequential conversion of glutamine -> glutamate -> alpha-ketoglutarate -> succinyl CoA -> succinate. Equally important, provision of ATP through mSLP would maintain the adenine nucleotide translocase in forward mode, thus preventing the reverse-operating F0-F1 ATP synthase from depleting cytosolic ATP reserves. Because glucose and glutamine are the primary fuels driving the rapid growth of GBM and most tumors for that matter, simultaneous restriction of these two substrates or inhibition of mSLP should diminish cancer viability, growth, and invasion

Functional Assessment of EnvZ/OmpR Two-Component System in Shewanella oneidensis

EnvZ and OmpR constitute the bacterial two-component signal transduction system known to mediate osmotic stress response in a number of Gram-negative bacteria. In an effort to understand the mechanism through which Shewanella oneidensis senses and responds to environmental osmolarity changes, structure of the ompR-envZ operon was determined with Northern blotting assay and roles of the EnvZ/OmpR two-component system in response to various stresses were investigated with mutational analysis, quantitative reverse transcriptase PCR (qRT-PCR), and phenotype microarrays. Results from the mutational analysis and qRT-PCR suggested that the EnvZ/OmpR system contributed to osmotic stress response of S. oneidensis and very likely engaged a similar strategy employed by E. coli, which involved reciprocal regulation of two major porin coding genes. Additionally, the ompR-envZ system was also found related to cell motility. We further showed that the ompR-envZ dependent regulation of porin genes and motility resided almost completely on ompR and only partially on envZ, indicating additional mechanisms for OmpR phosphorylation. In contrast to E. coli lacking ompR-envZ, however, growth of S. oneidensis did not show a significant dependence on ompR-envZ even under osmotic stress. Further analysis with phenotype microarrays revealed that the S. oneidensis strains lacking a complete ompR-envZ system displayed hypersensitivities to a number of agents, especially in alkaline environment. Taken together, our results suggest that the function of the ompR-envZ system in S. oneidensis, although still connected with osmoregulation, has diverged considerably from that of E. coli. Additional mechanism must exist to support growth of S. oneidensis under osmotic stress

Genome-wide analysis of the role of GlnR in Streptomyces venezuelae provides new insights into global nitrogen regulation in actinomycetes

<p>Abstract</p> <p>Background</p> <p>GlnR is an atypical response regulator found in actinomycetes that modulates the transcription of genes in response to changes in nitrogen availability. We applied a global <it>in vivo </it>approach to identify the GlnR regulon of <it>Streptomyces venezuelae</it>, which, unlike many actinomycetes, grows in a diffuse manner that is suitable for physiological studies. Conditions were defined that facilitated analysis of GlnR-dependent induction of gene expression in response to rapid nitrogen starvation. Microarray analysis identified global transcriptional differences between <it>glnR</it>⁺and <it>glnR </it>mutant strains under varying nitrogen conditions. To differentiate between direct and indirect regulatory effects of GlnR, chromatin immuno-precipitation (ChIP) using antibodies specific to a FLAG-tagged GlnR protein, coupled with microarray analysis (ChIP-chip), was used to identify GlnR binding sites throughout the <it>S. venezuelae </it>genome.</p> <p>Results</p> <p>GlnR bound to its target sites in both transcriptionally active and apparently inactive forms. Thirty-six GlnR binding sites were identified by ChIP-chip analysis allowing derivation of a consensus GlnR-binding site for <it>S. venezuelae</it>. GlnR-binding regions were associated with genes involved in primary nitrogen metabolism, secondary metabolism, the synthesis of catabolic enzymes and a number of transport-related functions.</p> <p>Conclusions</p> <p>The GlnR regulon of <it>S. venezuelae </it>is extensive and impacts on many facets of the organism's biology. GlnR can apparently bind to its target sites in both transcriptionally active and inactive forms.</p

Polymorphisms in the Mitochondrial Ribosome Recycling Factor EF-G2mt/MEF2 Compromise Cell Respiratory Function and Increase Atorvastatin Toxicity

Mitochondrial translation, essential for synthesis of the electron transport chain complexes in the mitochondria, is governed by nuclear encoded genes. Polymorphisms within these genes are increasingly being implicated in disease and may also trigger adverse drug reactions. Statins, a class of HMG-CoA reductase inhibitors used to treat hypercholesterolemia, are among the most widely prescribed drugs in the world. However, a significant proportion of users suffer side effects of varying severity that commonly affect skeletal muscle. The mitochondria are one of the molecular targets of statins, and these drugs have been known to uncover otherwise silent mitochondrial mutations. Based on yeast genetic studies, we identify the mitochondrial translation factor MEF2 as a mediator of atorvastatin toxicity. The human ortholog of MEF2 is the Elongation Factor Gene (EF-G) 2, which has previously been shown to play a specific role in mitochondrial ribosome recycling. Using small interfering RNA (siRNA) silencing of expression in human cell lines, we demonstrate that the EF-G2mt gene is required for cell growth on galactose medium, signifying an essential role for this gene in aerobic respiration. Furthermore, EF-G2mt silenced cell lines have increased susceptibility to cell death in the presence of atorvastatin. Using yeast as a model, conserved amino acid variants, which arise from non-synonymous single nucleotide polymorphisms (SNPs) in the EF-G2mt gene, were generated in the yeast MEF2 gene. Although these mutations do not produce an obvious growth phenotype, three mutations reveal an atorvastatin-sensitive phenotype and further analysis uncovers a decreased respiratory capacity. These findings constitute the first reported phenotype associated with SNPs in the EF-G2mt gene and implicate the human EF-G2mt gene as a pharmacogenetic candidate gene for statin toxicity in humans

Metatranscriptomics reveal differences in in situ energy and nitrogen metabolism among hydrothermal vent snail symbionts

Despite the ubiquity of chemoautotrophic symbioses at hydrothermal vents, our understanding of the influence of environmental chemistry on symbiont metabolism is limited. Transcriptomic analyses are useful for linking physiological poise to environmental conditions, but recovering samples from the deep sea is challenging, as the long recovery times can change expression profiles before preservation. Here, we present a novel, in situ RNA sampling and preservation device, which we used to compare the symbiont metatranscriptomes associated with Alviniconcha, a genus of vent snail, in which specific host–symbiont combinations are predictably distributed across a regional geochemical gradient. Metatranscriptomes of these symbionts reveal key differences in energy and nitrogen metabolism relating to both environmental chemistry (that is, the relative expression of genes) and symbiont phylogeny (that is, the specific pathways employed). Unexpectedly, dramatic differences in expression of transposases and flagellar genes suggest that different symbiont types may also have distinct life histories. These data further our understanding of these symbionts' metabolic capabilities and their expression in situ, and suggest an important role for symbionts in mediating their hosts' interaction with regional-scale differences in geochemistry

Alanine Racemase Mutants of Burkholderia pseudomallei and Burkholderia mallei and Use of Alanine Racemase as a Non-Antibiotic-Based Selectable Marker

Burkholderia pseudomallei and Burkholderia mallei are category B select agents and must be studied under BSL3 containment in the United States. They are typically resistant to multiple antibiotics, and the antibiotics used to treat B. pseudomallei or B. mallei infections may not be used as selective agents with the corresponding Burkholderia species. Here, we investigated alanine racemase deficient mutants of B. pseudomallei and B. mallei for development of non-antibiotic-based genetic selection methods and for attenuation of virulence. The genome of B. pseudomallei K96243 has two annotated alanine racemase genes (bpsl2179 and bpss0711), and B. mallei ATCC 23344 has one (bma1575). Each of these genes encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain ALA1. Herein, we show that B. pseudomallei with in-frame deletions in both bpsl2179 and bpss0711, or B. mallei with an in-frame deletion in bma1575, requires exogenous d-alanine for growth. Introduction of bpsl2179 on a multicopy plasmid into alanine racemase deficient variants of either Burkholderia species eliminated the requirement for d-alanine. During log phase growth without d-alanine, the viable counts of alanine racemase deficient mutants of B. pseudomallei and B. mallei decreased within 2 hours by about 1000-fold and 10-fold, respectively, and no viable bacteria were present at 24 hours. We constructed several genetic tools with bpsl2179 as a selectable genetic marker, and we used them without any antibiotic selection to construct an in-frame ΔflgK mutant in the alanine racemase deficient variant of B. pseudomallei K96243. In murine peritoneal macrophages, wild type B. mallei ATCC 23344 was killed much more rapidly than wild type B. pseudomallei K96243. In addition, the alanine racemase deficient mutant of B. pseudomallei K96243 exhibited attenuation versus its isogenic parental strain with respect to growth and survival in murine peritoneal macrophages

Cooperative Adaptation to Establishment of a Synthetic Bacterial Mutualism

To understand how two organisms that have not previously been in contact can establish mutualism, it is first necessary to examine temporal changes in their phenotypes during the establishment of mutualism. Instead of tracing back the history of known, well-established, natural mutualisms, we experimentally simulated the development of mutualism using two genetically-engineered auxotrophic strains of Escherichia coli, which mimic two organisms that have never met before but later establish mutualism. In the development of this synthetic mutualism, one strain, approximately 10 hours after meeting the partner strain, started oversupplying a metabolite essential for the partner's growth, eventually leading to the successive growth of both strains. This cooperative phenotype adaptively appeared only after encountering the partner strain but before the growth of the strain itself. By transcriptome analysis, we found that the cooperative phenotype of the strain was not accompanied by the local activation of the biosynthesis and transport of the oversupplied metabolite but rather by the global activation of anabolic metabolism. This study demonstrates that an organism has the potential to adapt its phenotype after the first encounter with another organism to establish mutualism before its extinction. As diverse organisms inevitably encounter each other in nature, this potential would play an important role in the establishment of a nascent mutualism in nature

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

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

Comparative analyses imply that the enigmatic sigma factor 54 is a central controller of the bacterial exterior

Contains fulltext : 95738.pdf (publisher's version ) (Open Access)BACKGROUND: Sigma-54 is a central regulator in many pathogenic bacteria and has been linked to a multitude of cellular processes like nitrogen assimilation and important functional traits such as motility, virulence, and biofilm formation. Until now it has remained obscure whether these phenomena and the control by Sigma-54 share an underlying theme. RESULTS: We have uncovered the commonality by performing a range of comparative genome analyses. A) The presence of Sigma-54 and its associated activators was determined for all sequenced prokaryotes. We observed a phylum-dependent distribution that is suggestive of an evolutionary relationship between Sigma-54 and lipopolysaccharide and flagellar biosynthesis. B) All Sigma-54 activators were identified and annotated. The relation with phosphotransfer-mediated signaling (TCS and PTS) and the transport and assimilation of carboxylates and nitrogen containing metabolites was substantiated. C) The function annotations, that were represented within the genomic context of all genes encoding Sigma-54, its activators and its promoters, were analyzed for intra-phylum representation and inter-phylum conservation. Promoters were localized using a straightforward scoring strategy that was formulated to identify similar motifs. We found clear highly-represented and conserved genetic associations with genes that concern the transport and biosynthesis of the metabolic intermediates of exopolysaccharides, flagella, lipids, lipopolysaccharides, lipoproteins and peptidoglycan. CONCLUSION: Our analyses directly implicate Sigma-54 as a central player in the control over the processes that involve the physical interaction of an organism with its environment like in the colonization of a host (virulence) or the formation of biofilm