2-oxoglutarate Levels Research Articles

Abstract 5391: MUC1 and HIF-1α signaling interactions modulate glucose flux in pancreatic cancer.

Abstract MUC1, a type-I transmembrane protein, is significantly overexpressed in pancreatic cancer. High expression of MUC1 in pancreatic cancer correlates with poor prognosis. Pancreatic tumors demonstrate significant hypoxia, which causes stabilization of HIF-1α, a key modulator of glycolytic gene expression. Recently, we observed that MUC1 occupies multiple HIF-1α–regulated glycolytic gene promoters. By performing in vitro and in vivo studies we observed that MUC1 overexpression causes up-regulation of glucose uptake, lactate release and expression of genes involved in glucose metabolism in pancreatic cancer cell lines. Based on these data we hypothesized that MUC1 regulates the stabilization/activity of HIF-1α to facilitate the glycolytic phenotype in pancreatic cancer. To investigate the regulation of HIF-1α by MUC1, we performed lentiviral shRNA-mediated knock down of HIF-1α in MUC1-overexpressing S2-013 and Capan1 pancreatic cancer cells and assayed for glucose uptake, lactate release and glycolytic gene expression. We observed that knocking down HIF-1α abrogated the MUC1-induced up-regulation of glucose metabolism. To investigate if MUC1 physically interacts with HIF-1α, we performed co-immunoprecipitation assays by utilizing a mAb against the cytoplasmic tail of MUC1. Our results indicated significant interaction between MUC1 and HIF-1α. Additionally, by performing chromatin immunoprecipitation we observed that MUC1 co-occupies the promoter regions of ENO1 and PGM2 glycolytic genes along with HIF-1α. MUC1 also increased HIF-1α activity by facilitating P300 recruitment and resultantly enhancing histone3 lysine9 acetylation on the MUC1-occupied promoters. By performing 1H-13C HSQC NMR experiments on the methanol extracts from MUC1-overexpressing or control S2-013 cells cultured with 13C-glucose, we observed an increased glycolytic flux and faster glucose turnover in pancreatic cancer cells. Furthermore, MUC1 overexpression caused a reduction in the levels of 2-oxoglutarate, a substrate for prolyl hydroxylases that regulate HIF stability. Thus, MUC1-mediated reduction in 2-oxoglutarate levels indirectly stabilizes HIF-1α by reducing the activity of prolyl hydroxylases. Overall, our results demonstrate that MUC1 serves as a novel metabolic regulator in pancreatic cancer that facilitates glycolytic flux by increasing HIF-1α stability and activity. Citation Format: Vinee Purohit, Nina V. Chaika, Teklab Gebregiworgis, Prakash Radhakrishnan, Bo Zhang, Kamiya Mehla, Fang Yu, Keith R. Johnson, Robert Powers, Michael A. Hollingsworth, Pankaj K. Singh. MUC1 and HIF-1α signaling interactions modulate glucose flux in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5391. doi:10.1158/1538-7445.AM2013-5391

Mutational Analysis of the Cyanobacterial Nitrogen Regulator PipX

PipX provides a functional link between the cyanobacterial global transcriptional regulator NtcA and the signal transduction protein PII, a protein found in all three domains of life as integrators of signals of the nitrogen and carbon balance. PipX, which is toxic in the absence of PII, can form alternative complexes with NtcA and PII and these interactions are respectively stimulated and inhibited by 2-oxoglutarate, providing a mechanism by which PII can modulate expression at the NtcA regulon. Structural information on PipX-NtcA complexes suggests that PipX coactivates NtcA controlled genes by stabilizing the active conformation of NtcA bound to 2-oxoglutarate and by possibly helping recruit RNA polymerase. To get insights into PipX functions, we perform here a mutational analysis of pipX informed by the structures of PipX-PII and PipX-NtcA complexes and evaluate the impact of point mutations on toxicity and gene expression. Two amino acid substitutions (Y32A and E4A) were of particular interest, since they increased PipX toxicity and activated NtcA dependent genes in vivo at lower 2-oxoglutarate levels than wild type PipX. While both mutations impaired complex formation with PII, only Y32A had a negative impact on PipX-NtcA interactions.

2-oxoglutarate enhances NtcA binding activity to promoter regions of the microcystin synthesis gene cluster

The binding affinity of NtcA towards promoter regions of the microcystin gene cluster from Microcystis aeruginosa PCC 7806 has been analyzed by band-shift assay (EMSA). The key nitrogen transcriptional regulator exhibits affinity for two fragments of the bidirectional mcyDA promoter, as well as for promoter regions of mcyE and mcyH. The presence of 2-oxoglutarate increased by 2.5 fold the affinity of NtcA for the mcyA promoter region. The 2-oxoglutarate effect peaked at 0.8 mM, a physiological concentration for this compound under nitrogen-limiting conditions. The results suggest that the 2-oxoglutarate level, as a signal of the C to N balance of the cells, regulates the microcystin gene cluster.

Overcoming Fluctuation and Leakage Problems in the Quantification of Intracellular 2-Oxoglutarate Levels in Escherichia coli

2-Oxoglutarate is located at the junction between central carbon and nitrogen metabolism, serving as an intermediate for both. In nitrogen metabolism, 2-oxoglutarate acts as both a carbon skeletal carrier and an effector molecule. There have been only sporadic reports of its internal concentrations. Here we describe a sensitive and accurate method for determination of the 2-oxoglutarate pool concentration in Escherichia coli. The detection was based on fluorescence derivatization followed by reversed-phase high-pressure liquid chromatography separation. Two alternative cell sampling strategies, both of which were based on a fast filtration protocol, were sequentially developed to overcome both its fast metabolism and contamination from 2-oxoglutarate that leaks into the medium. We observed rapid changes in the 2-oxoglutarate pool concentration upon sudden depletion of nutrients: decreasing upon carbon depletion and increasing upon nitrogen depletion. The latter was studied in mutants lacking either of the two enzymes using 2-oxoglutarate as the carbon substrate for glutamate biosynthesis. The results suggest that flux restriction on either reaction greatly influences the internal 2-oxoglutarate level. Additional study indicates that KgtP, a 2-oxoglutarate proton symporter, functions to recover the leakage loss of 2-oxoglutarate. This recovery mechanism benefits the measurement of cellular 2-oxoglutarate level in practice by limiting contamination from 2-oxoglutarate leakage.

Endogenous 2-oxoglutarate levels impact potencies of competitive HIF prolyl hydroxylase inhibitors

The stability and transcriptional activity of the hypoxia-inducible factors (HIFs) are regulated by oxygen-dependent hydroxylation that is catalyzed by three HIF prolyl 4-hydroxylases (HPHs). Use of HPH inhibition as a mean for HIF-upregulation has recently gained interest as a potential treatment paradigm against neurodegenerative diseases like ischemia and Parkinson's disease. In the present investigation we report the development of a new and robust assay to measure HPH activity. The assay is based on capture of hydroxylated peptide product by the von Hippel–Lindau protein which is directly measured in a scintillation proximity assay. In addition we describe the determination of HPH subtype potencies of HPH inhibitors which either directly or indirectly inhibit the HPH enzyme. The potencies of the HPH inhibitors displayed almost identical IC50 values toward the HPH1 and HPH2 subtype while the potency against the HPH3 subtype was increased for several of the compounds. For the most potent compound, a hydroxyl thiazole derivative, the potency against HPH2 and HPH3 was 7nM and 0.49nM, respectively corresponding to a 14-fold difference. These results suggest that HPH subtype-selective compounds may be developed. In addition we determined the 2-oxoglutarate concentration in brain tissue and neuronal cell lines as 2-oxoglutarate is an important co-factor used by the HPH enzyme during the hydroxylation reaction. The high intracellular 2-oxoglutarate concentration provides an explanation for the diminished cellular HIF activating potency of a competitive HPH inhibitor compared to its orders of magnitude higher HPH inhibiting potency. The present reported data suggest that in the development of specific Hif prolyl hydroxylase inhibitors the high 2-oxoglutarate tissue level should be taken into account as this might affect the cellular potency. Thus to specifically inhibit the intracellular HPH enzymatic reaction a competitive inhibitor with a low Ki should be developed.

Control of AmtB-GlnK Complex Formation by Intracellular Levels of ATP, ADP, and 2-Oxoglutarate

P(II) proteins are one of the most widespread families of signal transduction proteins in nature, being ubiquitous throughout bacteria, archaea, and plants. They play a major role in coordinating nitrogen metabolism by interacting with, and regulating the activities of, a variety of enzymes, transcription factors, and membrane transport proteins. The regulatory properties of P(II) proteins derive from their ability to bind three effectors: ATP, ADP, and 2-oxoglutarate. However, a clear model to integrate physiological changes with the consequential structural changes that mediate P(II) interaction with a target protein has so far not been developed. In this study, we analyzed the fluctuations in intracellular effector pools in Escherichia coli during association and dissociation of the P(II) protein GlnK with the ammonia channel AmtB. We determined that key features promoting AmtB-GlnK complex formation are the rapid drop in the 2-oxoglutarate pool upon ammonium influx and a simultaneous, but transient, change in the ATP/ADP ratio. We were also able to replicate AmtB-GlnK interactions in vitro using the same effector combinations that we observed in vivo. This comprehensive data set allows us to propose a model that explains the way in which interactions between GlnK and its effectors influence the conformation of GlnK and thereby regulate its interaction with AmtB.

Metabolic Response to Iron Deficiency in Saccharomyces cerevisiae

Iron is an essential cofactor for enzymes involved in numerous cellular processes, yet little is known about the impact of iron deficiency on cellular metabolism or iron proteins. Previous studies have focused on changes in transcript and proteins levels in iron-deficient cells, yet these changes may not reflect changes in transport activity or flux through a metabolic pathway. We analyzed the metabolomes and transcriptomes of yeast grown in iron-rich and iron-poor media to determine which biosynthetic processes are altered when iron availability falls. Iron deficiency led to changes in glucose metabolism, amino acid biosynthesis, and lipid biosynthesis that were due to deficiencies in specific iron-dependent enzymes. Iron-sulfur proteins exhibited loss of iron cofactors, yet amino acid synthesis was maintained. Ergosterol and sphingolipid biosynthetic pathways had blocks at points where heme and diiron enzymes function, whereas Ole1, the essential fatty acid desaturase, was resistant to iron depletion. Iron-deficient cells exhibited depletion of most iron enzyme activities, but loss of activity during iron deficiency did not consistently disrupt metabolism. Amino acid homeostasis was robust, but iron deficiency impaired lipid synthesis, altering the properties and functions of cellular membranes.

Prolyl hydroxylases as regulators of cell metabolism

Cellular response to oxygen depletion is mediated by HIF (hypoxia-inducible factor). HIF is a heterodimer consisting of a constitutively expressed subunit (HIFbeta) and an oxygen-regulated subunit (HIFalpha). HIFalpha stability is regulated by prolyl hydroxylation by PHD (prolyl hydroxylase domain-containing protein) family members. PHD activity depends on the availability of molecular oxygen, making PHDs the oxygen-sensing system in animal cells. However, PHDs have recently been shown to respond to stimuli other than oxygen, such as 2-oxoglutarate (alpha-ketoglutarate), succinate or fumarate, as illustrated by the pseudo-hypoxic response in succinate dehydrogenase- or fumarate dehydrogenase-deficient tumours. Moreover, HIFalpha is not the sole PHD effector, suggesting that PHDs have functions that extend beyond oxygen sensing. Currently, we are investigating the role of PHDs in the cellular response to amino acid deprivation, a process regulated by mTOR (mammalian target of rapamycin). The precise mechanism whereby amino acids are signalling to mTOR is not fully understood. Given that 2-oxoglutarate is a limiting co-substrate for PHD activity during normoxia and that 2-oxoglutarate levels depend on amino acid availability, it is possible that PHD activity depends not only on oxygen, but also on amino acid availability, suggesting a global metabolic sensor function for PHDs which could be signalling not only to HIF, but also to mTOR.

Inactivation of the general transcription factor TnrA in Bacillus subtilis by proteolysis

Under conditions of nitrogen limitation, the general transcription factor TnrA in Bacillus subtilis activates the expression of genes involved in assimilation of various nitrogen sources. Previously, TnrA activity has been shown to be controlled by protein-protein interaction with glutamine synthetase, the key enzyme of ammonia assimilation. Furthermore, depending on ATP and 2-oxoglutarate levels, TnrA can bind to the GlnK-AmtB complex. Here, we report that upon transfer of nitrate-grown cells to combined nitrogen-depleted medium, TnrA is rapidly eliminated from the cells by proteolysis. As long as TnrA is membrane-bound through GlnK-AmtB interaction it seems to be protected from degradation. Upon removal of nitrogen sources, the localization of TnrA becomes cytosolic and degradation occurs. The proteolytic activity against TnrA was detected in the cytosolic fraction but not in the membrane, and its presence does not depend on the nitrogen regime of cell growth. The proteolytic degradation of TnrA as a response to complete nitrogen starvation might represent a novel mechanism of TnrA control in B. subtilis.

Altered Metabolism of Growth Hormone Receptor Mutant Mice: A Combined NMR Metabonomics and Microarray Study

BackgroundGrowth hormone is an important regulator of post-natal growth and metabolism. We have investigated the metabolic consequences of altered growth hormone signalling in mutant mice that have truncations at position 569 and 391 of the intracellular domain of the growth hormone receptor, and thus exhibit either low (around 30% maximum) or no growth hormone-dependent STAT5 signalling respectively. These mutations result in altered liver metabolism, obesity and insulin resistance.Methodology/Principal FindingsThe analysis of metabolic changes was performed using microarray analysis of liver tissue and NMR metabonomics of urine and liver tissue. Data were analyzed using multivariate statistics and Gene Ontology tools. The metabolic profiles characteristic for each of the two mutant groups and wild-type mice were identified with NMR metabonomics. We found decreased urinary levels of taurine, citrate and 2-oxoglutarate, and increased levels of trimethylamine, creatine and creatinine when compared to wild-type mice. These results indicate significant changes in lipid and choline metabolism, and were coupled with increased fat deposition, leading to obesity. The microarray analysis identified changes in expression of metabolic enzymes correlating with alterations in metabolite concentration both in urine and liver. Similarity of mutant 569 to the wild-type was seen in young mice, but the pattern of metabolites shifted to that of the 391 mutant as the 569 mice became obese after six months age.Conclusions/SignificanceThe metabonomic observations were consistent with the parallel analysis of gene expression and pathway mapping using microarray data, identifying metabolites and gene transcripts involved in hepatic metabolism, especially for taurine, choline and creatinine metabolism. The systems biology approach applied in this study provides a coherent picture of metabolic changes resulting from impaired STAT5 signalling by the growth hormone receptor, and supports a potentially important role for taurine in enhancing β-oxidation.

Inactivation of spkD, encoding a Ser/Thr kinase, affects the pool of the TCA cycle metabolites in Synechocystis sp. strain PCC 6803

The inactivation of sll0776 (spkD), a gene encoding a protein Ser/Thr kinase in Synechocystis PCC 6803, led to a pleiotropic phenotype of the SpkD null mutant. This mutant is impaired in its growth ability under low concentration of inorganic carbon (C(i)), though its C(i)-uptake system is not affected. Addition of glucose, phosphoglyceraldehyde or pyruvate does not allow the mutant to grow under low-C(i) conditions. In contrast, this growth defect can be restored when the low-C(i) culture medium is supplemented with metabolites of the TCA cycle. Growth of the mutant is also inhibited when ammonium is provided as nitrogen source, whatever the carbon regime of the cells, due to the high demand for 2-oxoglutarate, which is the carbon skeleton for ammonium assimilation. When mutant cells are cultured under standard growth conditions, the intracellular concentration of 2-oxoglutarate is 20 % lower than is observed in the wild-type strain. However, this decrease of 2-oxoglutarate level only slightly affects the phosphorylation state of PII, a protein that regulates nitrogen and carbon metabolism according to the intracellular levels of 2-oxoglutarate. Properties of the SpkD mutant suggest that the Ser/Thr kinase SpkD could be involved in adjusting the pool of the TCA cycle metabolites according to C(i) supply in the culture medium.

Metabonomic study on the toxicity of Hei-Shun-Pian, the processed lateral root of Aconitum carmichaelii Debx. (Ranunculaceae)

The purpose of this paper was to study the effects of Hei-Shun-Pian, the processed lateral root of Aconitum carmichaelii Debx. (Ranunculaceae), on the metabolic profile of rats, to discuss the mechanism of toxicology and to find out the potential biomarkers of the toxic effects. Twenty male Wistar rats were divided into four groups ( n = 5) and each group were administered orally with the decoction of Hei-Shun-Pian (88.1 g/kg per day, 35.6 g/kg per day, 17.6 g/kg per day) or equal volume of drinking water respectively for 14 days. Urine of every 24-h and the plasma of the last day were collected for NMR experiments, and then analyzed by multivariate analysis methods. Decreases in urinary excretion of taurine and trimethylamine- N-oxide (TMAO) and increases in urinary levels of citrate, 2-oxoglutarate (2-OG), succinate and hippurate were observed in the high and medium dosed groups at the early stage of the dosing period. Taurine level increased at the later stage of the dosing period to the normal value, and then even to a value higher than that of the control group at the end of the experiment. No metabolic differences were observed between low dosed and control groups until the later stage of the dosing period when a slight increase in urinary taurine level was observed, suggesting a cumulative effect. These results suggest the toxic effect of Hei-Shun-Pian on rat heart in a dose dependent manner.

Prolyl 4-hydroxylase activity-responsive transcription factors: from hydroxylation to gene expression and neuroprotection.

Most homeostatic processes including gene transcription occur as a result of deviations in physiological tone that threatens the survival of the organism. A prototypical homeostatic stress response includes changes in gene expression following alterations in oxygen, iron or 2-oxoglutarate levels. Each of these cofactors plays an important role in cellular metabolism. Accordingly, a family of enzymes known as the Prolyl 4-hydroxylase (PHD) enzymes are a group of dioxygenases that have evolved to sense changes in 2-oxoglutarate, oxygen and iron via changes in enzyme activity. Indeed, PHDs are a part of an established oxygen sensor system that regulates transcriptional regulation of hypoxia/stress-regulated genes and thus are an important component of events leading to cellular rescue from oxygen, iron or 2-oxoglutarate deprivations. The ability of PHD activity to regulate homeostatic responses to oxygen, iron or 2-oxoglutarate metabolism has led to the development of small molecule inhibitors of the PHDs as a strategy for activating or augmenting cellular stress responses. These small molecules are proving effective in preclinical models of stroke and Parkinson's disease. However the precise protective pathways engaged by PHD inhibition are only beginning to be defined. In the current review, we summarize the role of iron, 2-oxoglutarate and oxygen in the PHD catalyzed hydroxylation reaction and provide a brief discussion of some of the transcription factors that play an effective role in neuroprotection against oxidative stress as a result of changes in PHD activity.

NAD-Dependent Isocitrate Dehydrogenase Mutants of Arabidopsis Suggest the Enzyme Is Not Limiting for Nitrogen Assimilation

NAD-dependent isocitrate dehydrogenase (IDH) is a tricarboxylic acid cycle enzyme that produces 2-oxoglutarate, an organic acid required by the glutamine synthetase/glutamate synthase cycle to assimilate ammonium. Three Arabidopsis (Arabidopsis thaliana) IDH mutants have been characterized, corresponding to an insertion into a different IDH gene (At5g03290, idhv; At4g35260, idhi; At2g17130, idhii). Analysis of IDH mRNA and protein show that each mutant lacks the corresponding gene products. Leaf IDH activity is reduced by 92%, 60%, and 43% for idhv, idhi, and idhii, respectively. These mutants do not have any developmental or growth phenotype and the reduction of IDH activity does not impact on NADP-dependent isocitrate dehydrogenase activity. Soil-grown mutants do not exhibit any alterations in daytime sucrose, glucose, fructose, citrate, ammonium, and total soluble amino acid levels. However, gas chromatography-mass spectrometry metabolic profiling analyses indicate that certain free amino acids are reduced in comparison to the wild type. These data suggest that IDH activity is not limiting for tricarboxylic acid cycle functioning and nitrogen assimilation. On the other hand, liquid culture-grown mutants give a reduced growth phenotype, a large increase in organic acid (citrate is increased 35-fold), hexose-phosphate, and sugar content, whereas ammonium and free amino acids are moderately increased with respect to wild-type cultures. However, no significant changes in 2-oxoglutarate levels were observed. Under these nonphysiological growth conditions, pyridine nucleotide levels remained relatively constant between the wild-type and the idhv line, although some small, but significant, alterations were measured in idhii (lower NADH and higher NADPH levels). On the other hand, soil-grown idhv plants exhibited a reduction in NAD and NADPH content.

Biochemical Studies of Klebsiella pneumoniae NifL Reduction Using Reconstituted Partial Anaerobic Respiratory Chains of Wolinella succinogenes

In the diazotroph Klebsiella pneumoniae the flavoprotein NifL inhibits the activity of the nif-specific transcriptional activator NifA in response to molecular oxygen and combined nitrogen. Sequestration of reduced NifL to the cytoplasmic membrane under anaerobic and nitrogen-limited conditions impairs inhibition of cytoplasmic NifA by NifL. To analyze whether NifL is reduced by electrons directly derived from the reduced menaquinone pool, we studied NifL reduction using artificial membrane systems containing purified components of the anaerobic respiratory chain of Wolinella succinogenes. In this in vitro assay using proteoliposomes containing purified formate dehydrogenase and purified menaquinone (MK(6)) or 8-methylmenaquinone (MMK(6)) from W. succinogenes, reduction of purified NifL was achieved by formate oxidation. Furthermore, the respective reduction rates, which were determined using equal amounts of NifL, have been shown to be directly dependent on the concentration of both formate dehydrogenase and menaquinones incorporated into the proteoliposomes, demonstrating a direct electron transfer from menaquinone to NifL. When purified hydrogenase and MK(6) from W. succinogenes were inserted into the proteoliposomes, NifL was reduced with nearly the same rate by hydrogen oxidation. In both cases reduced NifL was found to be highly associated to the proteoliposomes, which is in accordance with our previous findings in vivo. On the bases of these experiments, we propose that the redox state of the menaquinone pool is the redox signal for nif regulation in K. pneumoniae by directly transferring electrons onto NifL under anaerobic conditions.

Interaction network in cyanobacterial nitrogen regulation: PipX, a protein that interacts in a 2‐oxoglutarate dependent manner with PII and NtcA

Cyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate levels. The global nitrogen transcription factor NtcA and the signal transduction protein PII are both involved in 2-oxoglutarate sensing. PII proteins, probably the most conserved signal transduction proteins in nature, are remarkable for their ability to interact with very diverse protein targets in different systems. Despite widespread efforts to understand nitrogen signalling in cyanobacteria, the involvement of PII in the regulation of transcription activation by NtcA remains enigmatic. Here we show that PipX, a protein only present in cyanobacteria, interacts with both PII and NtcA and provides a mechanistic link between these two factors. A variety of in vivo and in vitro approaches were used to study PipX and its interactions with PII and NtcA. 2-Oxoglutarate favours complex formation between PipX and NtcA, but impairs binding to PII, suggesting that partner swapping between these nitrogen regulators is driven by the 2-oxoglutarate concentration. PipX is required for NtcA-dependent transcriptional activation in vivo, thus implying that PipX may function as a prokaryotic transcriptional coactivator.

An increase in the level of 2-oxoglutarate promotes heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120.

In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, a starvation of combined nitrogen induces differentiation of heterocysts, cells specialized in nitrogen fixation. How do filaments perceive the limitation of the source of combined nitrogen, and what determines the proportion of heterocysts? In cyanobacteria, 2-oxoglutarate provides a carbon skeleton for the incorporation of inorganic nitrogen. Recently, it has been proposed that the concentration of 2-oxoglutarate reflects the nitrogen status in cyanobacteria. To investigate the effect of 2-oxoglutarate on heterocyst development, a heterologous gene encoding a 2-oxoglutarate permease under the control of a regulated promoter was expressed in Anabaena sp. PCC 7120. The increase of 2-oxoglutarate within cells can trigger heterocyst differentiation in a subpopulation of filaments even in the presence of nitrate. In the absence of a source of combined nitrogen, it can increase heterocyst frequency, advance the timing of commitment to heterocyst development and further increase the proportion of heterocysts in a patS mutant. Here, it is proposed that the intracellular concentration of 2-oxoglutarate is involved in the determination of the proportion of the two cell types according to the carbon/nitrogen status of the filament.

Sensitivity of the 2-oxoglutarate carrier to alcohol intake contributes to mitochondrial glutathione depletion

The mitochondrial pool of reduced glutathione (mGSH) is known to play a protective role against liver injury and cytokine-mediated cell death. However, the identification of the mitochondrial carriers involved in its transport in hepatocellular mitochondria remains unestablished. In this study, we show that the functional expression of the 2-oxoglutarate carrier from HepG2 cells in mitochondria from Xenopus laevis oocytes conferred a reduced glutathione (GSH) transport activity that was inhibited by phenylsuccinate, a specific inhibitor of the carrier. In addition, the mitochondrial transport of GSH and 2-oxoglutarate in isolated mitochondria from rat liver exhibited mutual competition and sensitivity to glutamate and phenylsuccinate. Interestingly, the kinetics of 2-oxoglutarate transport in rat liver mitochondria displayed a single Michaelis-Menten component with a Michaelis constant of 3.1 ± 0.3 mmol/L and maximum velocity of 1.9 ± 0.1 nmol/mg protein/25 seconds. Furthermore, the initial rate of 2-oxoglutarate was reduced in mitochondria from alcohol-fed rat livers, an effect that was not accompanied by an alcohol-induced decrease in the 2-oxoglutarate messenger RNA levels but rather by changes in mitochondrial membrane dynamics induced by alcohol. The fluidization of mitochondria by the fluidizing agent 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl) (A 2C) restored the initial transport rate of both GSH and 2-oxoglutarate. Finally, these changes were reproduced in normal liver mitochondria enriched in cholesterol where the fluidization of cholesterol-enriched mitochondria with A 2C restored the order membrane parameter and the mitochondrial 2-oxoglutarate uptake. In conclusion, these findings provide unequivocal evidence for 2-oxoglutarate as a GSH carrier and its sensitivity to membrane dynamics perturbation contributes in part to the alcohol-induced mGSH depletion. (H epatology 2003;38:692-702.)

Polymorphism of the vitamin D3 receptor in patients with psoriasis

1,25-Dihydroxyvitamin D is the biologically active form of vitamin D for the treatment of skin eruptions in patients with psoriasis. 1,25-(OH)(2)D(3) elicits its action on skin eruptions through the vitamin D receptor (VDR). Allelic frequencies of VDR were studied in 86 normal subjects and 50 patients with psoriasis. Genomic DNA was extracted from peripheral blood leukocytes and the VDR gene was amplified using a heminested polymerase chain reaction (PCR). The products were digested with respective restriction enzymes ApaI, TaqI and BsmI. The restriction fragment length polymorphisms (RFLP) were coded as Aa, Tt or Bb. The frequencies of ApaI, BsmI and TaqI RFLP genotypes in psoriasis patients showed no significant differences compared with normal controls. The frequency of the AA genotype was significantly higher in pustulosis palmaris et plantaris patients than in psoriasis vulgaris patients ( P<0.05), and in psoriasis vulgaris patients than in psoriasis pustulosa patients ( P<0.01). In patients with psoriasis, the levels of serum alanine 2-oxoglutarate aminotransferase (ALT) were significantly higher in patients with the AA genotype (54.0+/-22.0 IU/l, n=4) than in those with the aa genotype (24.0+/-15.9 IU/l, n=27; P<0.02). The distribution of ApaI, BsmI, TaqI RFLP VDR genotypes showed no significant relationship to the PASI score, serum aspartate 2-oxoglutarate aminotransferase or triglyceride levels, or age at onset. These results show that the VDR genotype contributes to the liver dysfunction in patients with psoriasis, although no correlation was found between VDR genotype and the skin eruptions of psoriasis.

The novel protein phosphatase PphA from Synechocystis PCC 6803 controls dephosphorylation of the signalling protein PII.

The family of PII signal transduction proteins consists of one of the most highly conserved signalling proteins in nature. The cyanobacterial PII homologue transmits signals on the nitrogen and carbon status of the cells through phosphorylation of a seryl residue. Recently, we identified a protein phosphatase 2C (PP2C) homologue from the cyanobacterium Synechocystis PCC 6803, termed PphA, to be the cellular phospho-PII (PII-P) phosphatase. In this investigation, we characterized the enzymatic properties of PphA and investigated the regulation of its catalytic activity towards PII-P. PphA dephosphorylates phosphocasein and PII-P with similar efficiency in a strictly Mg2+- or Mn2+-dependent reaction. Low-molecular-weight phosphorylated molecules are poor substrates for PphA. Its reactivity towards PII-P, but not towards phosphocasein, is inhibited by various nucleotides, suggesting that this effect is based on specific properties of the PII protein. The inhibitory effect of ATP can be strongly enhanced by the addition of 2-oxoglutarate or oxaloacetate. At low concentrations of 2-oxoglutarate, changes in the ATP levels within the physiological range affect the degree of PII-Pase inhibition, whereas at 2-oxoglutarate levels beyond 0.1 mM, inhibition is almost complete at very low ATP levels. This suggests that PII dephosphorylation is not only sensitive to 2-oxoglutarate and oxaloacetate levels, it also integrates signals from the energy charge of the cells under specific cellular conditions.