Cytosolic Aspartate Aminotransferase Research Articles

MRNA Display Pipeline for Protein Biosensor Construction.

Despite the significant potential of protein biosensors, their construction remains a trial-and-error process. The most obvious approach for addressing this is to utilize modular biosensor architectures where specificity-conferring modalities can be readily generated to recognize new targets. Toward this goal, we established a workflow that uses mRNA display-based selection of hyper-stable monobody domains for the target of choice or ribosome display to select equally stable DARPins. These binders were integrated into a two-component allosteric biosensor architecture based on a calmodulin-reporter chimera. This workflow was tested by developing biosensors for liver toxicity markers such as cytosolic aspartate aminotransferase, mitochondrial aspartate aminotransferase, and alanine aminotransferase 1. We demonstrate that our pipeline consistently produced >103 unique binders for each target within a week. Our analysis revealed that the affinity of the binders for their targets was not a direct predictor of the binder's performance in a biosensor context. The interactions between the binding domains and the reporter module affect the biosensor activity and the dynamic range. We conclude that following binding domain selection, the multiplexed biosensor assembly and prototyping appear to be the most promising approach for identifying biosensors with the desired properties.

Rod photoreceptor-specific deletion of cytosolic aspartate aminotransferase, GOT1, causes retinal degeneration.

Photoreceptor cell death is the cause of vision loss in many forms of retinal disease. Metabolic dysfunction within the outer retina has been shown to be an underlying factor contributing to photoreceptor loss. Therefore, a comprehensive understanding of the metabolic pathways essential to photoreceptor health and function is key to identifying novel neuroprotective strategies. Glutamic-oxaloacetic transaminase 1 (Got1) encodes for a cytosolic aspartate aminotransferase that reversibly catalyzes the transfer of an amino group between glutamate and aspartate and is an important aspect of the malate-aspartate shuttle (MAS), which transfers reducing equivalents from the cytosol to the mitochondrial matrix. Previous work has demonstrated that the activity of this enzyme is highest in photoreceptor inner segments. Furthermore, ex vivo studies have demonstrated that the retina relies on aspartate aminotransferase for amino acid metabolism. Importantly, aspartate aminotransferase has been suggested to be an early biomarker of retinal degeneration in retinitis pigmentosa and a possible target for neuroprotection. In the present study, we characterized the effect of Got1 deletion on photoreceptor metabolism, function, and survival in vivo by using a rod photoreceptor-specific, Got1 knockout mouse model. Loss of the GOT1 enzyme from rod photoreceptors resulted in age-related photoreceptor degeneration with an accumulation of retinal aspartate and NADH and alterations in the expression of genes involved in the MAS, the tricarboxylic acid (TCA) cycle, and redox balance. Hence, GOT1 is critical to in vivo photoreceptor metabolism, function, and survival.

The molecular background of the aspartate aminotransferase polymorphism in Littorina snails maintained by strong selection on small spatial scales

Allozymes present several classical examples of divergent selection, including the variation in the cytosolic aspartate aminotransferase (AAT) in the intertidal snails Littorina saxatilis. AAT is a part of the asparate-malate shuttle, in the interidal molluscs involved in the anaerobic respiration during desiccation. Previous allozyme studies reported the sharp gradient in the frequencies of the AAT100and the AAT120 alleles between the low and high shores in the Northern Europe and the differences in their enzymatic activity, supporting the role of AAT in adaptation to desiccation. However, the populations in the Iberian Peninsula showed the opposite allele cline. Using the mRNA sequencing and the genome pool-seq analyses we characterize DNA sequences of the different AAT alleles, report the amino acid replacements behind the allozyme variation and show that same allozyme alleles in Northern and Southern populations have different protein sequences. Gene phylogeny reveals that the AAT100 and the northern AAT120 alleles represent the old polymorphism, shared among the closely related species of Littorina, while the southern AAT120 allele is more recently derived from AAT100. Further, we show that the Aat gene is expressed at constitutive level in different genotypes and conditions, supporting the role of structural variation in regulation of enzyme activity. Finally, we report the location and the structure of the gene in the L. saxatilis genome and the presence of two additional non-functional gene copies. Altogether, we provide a missing link between the classical allozyme studies and the genome scans and bring together the results produced over decades of the genetic research.

Cytosolic aspartate aminotransferase moonlights as a ribosome-binding modulator of Gcn2 activity during oxidative stress.

Regulation of translation is a fundamental facet of the cellular response to rapidly changing external conditions. Specific RNA-binding proteins (RBPs) co-ordinate the translational regulation of distinct mRNA cohorts during stress. To identify RBPs with previously under-appreciated roles in translational control, we used polysome profiling and mass spectrometry to identify and quantify proteins associated with translating ribosomes in unstressed yeast cells and during oxidative stress and amino acid starvation, which both induce the integrated stress response (ISR). Over 800 proteins were identified across polysome gradient fractions, including ribosomal proteins, translation factors, and many others without previously described translation-related roles, including numerous metabolic enzymes. We identified variations in patterns of PE in both unstressed and stressed cells and identified proteins enriched in heavy polysomes during stress. Genetic screening of polysome-enriched RBPs identified the cytosolic aspartate aminotransferase, Aat2, as a ribosome-associated protein whose deletion conferred growth sensitivity to oxidative stress. Loss of Aat2 caused aberrantly high activation of the ISR via enhanced eIF2α phosphorylation and GCN4 activation. Importantly, non-catalytic AAT2 mutants retained polysome association and did not show heightened stress sensitivity. Aat2 therefore has a separate ribosome-associated translational regulatory or 'moonlighting' function that modulates the ISR independent of its aspartate aminotransferase activity.

Abstract 1213: RNA-based screens in primary human immune cells

Abstract Genetic screens have long been used as an approach to identify and validate new targets for drug discovery. The vast majority of these have been carried out in cell lines: mostly cancer cell lines. However, with improvements in tissue culture techniques, the increasing interest in using the immune system to tackle disease and the discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 mediated genome editing, screening primary cells that have not been subverted by transformation into immortal lines, is both appealing and feasible. We have successfully carried out a CRISPR-Cas9 screen in primary T cells using a combined lentivirus and electroporation protocol. Freshly isolated primary T cells are stimulated with anti-CD3 and anti-CD28 antibodies and then transduced with a pooled sgRNA library. After antibiotic selection, T cells successfully transduced with sgRNAs are electroporated to introduce Cas9 mRNA. We chose to validate this approach by carrying out a screen similar to that published by Birsoy et al., 2015, in which they ran a CRISPR-Cas9 screen in Jurkat T cells in the presence and absence of the electron transport chain inhibitor phenformin. In our screen, we exposed the CRISPR-Cas9 edited pool of primary T cells to a dose of phenformin that resulted in growth inhibition to a similar degree to that used by Birsoy and colleagues. Our data are in agreement with the published screen, showing that loss of the cytosolic aspartate aminotransferase GOT1 sensitises primary T cells to phenformin. We took multiple time points in our primary T cell screen and used T cells isolated from three different donors, allowing for the analysis of guide drop-out kinetics and reproducibility between donors. We anticipate that these data will be useful in building more complex screens that assess T cell biology in the presence of additional cells, such as myeloid derived suppressor cells (MDSCs). With a view to this, we have also carried out an arrayed siRNA screen in MDSCs to look for genes that when knocked down reduce the capacity of MDSCs to inhibit T cell proliferation. The endpoint for this screen is based on co-culture of siRNA transfected MDSCs with proliferating primary T cells. Using this complex data set, we have identified several potential targets, which when validated could provide new therapeutic targets through which the immunosuppressive nature of MDSCs in the tumour microenvironment can be mitigated. Birsoy, K., et al. (2015) http://dx.doi.org/10.1016/j.cell.2015.07.016 Citation Format: Bronwyn Joubert, Cristina Ghirelli, Isabelle Nett, John Prime, Glynn Martin, Jonathan Moore, Benedict Cross, Nicola J. McCarthy. RNA-based screens in primary human immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1213.

Diurnal temperature-related variations in photosynthetic enzyme activities of two C<sub>4</sub> species of Chenopodiaceae grown in natural environment

The effects of the diurnal variations in ambient temperature on some C3 and C4 enzymes in the Salsola dendroides and Suaeda altissima species of Chenopodiaceae family were studied during the intensive vegetation period. Activities of phosphoenolpyruvate carboxylase (PEPC) and cytosolic aspartate aminotransferase (AsAT) were shown to decrease in both species in the afternoon and evening. The activity of the mitochondrial AsAT decreased in S. altissima, remained relatively constant in S. dendroides during the day. The activity of alanine aminotransferase was high in the S. dendroides species in the morning and evening and decreased in the S. altissima species by the evening. Glucose-6-phosphate activated PEPC in both species throughout the day. The study of the redox status-regulated C3 enzymes showed temperature-related increases in NADP-glyceraldehyde 3-phosphate dehydrogenase activity in both plants, in fructose-2,6-bisphosphatase activity in the S. altissima species, and in NADP-MDH activity in the S. dendroides species in the afternoon.

Cytosolic aspartate aminotransferase mediates the mitochondrial membrane potential and cell survival by maintaining the calcium homeostasis of BV2 microglia.

Both mitochondrial aspartate aminotransferase (mAST) and cytosolic aspartate aminotransferase (cAST) are important components in the malate-aspartate shuttle - one of the two types of NADH shuttles in cells. A major goal of our current study was to determine specifically the roles of cAST in maintaining the [Ca]i, mitochondrial membrane potential and the survival of BV2 microglia by applying molecular approach to modulate the cAST levels. Our study found that decreased cAST by cAST siRNA can lead to significant increases in the [Ca]i, mitochondrial depolarization and apoptosis of BV2 microglia. The cAST siRNA-induced mitochondrial depolarization can be significantly attenuated by an inhibitor of calpain. We further found that the cAST siRNA-induced apoptosis can be prevented by the calpain inhibitor. Collectively, our study suggests that decreased cAST induces calpain activation by increasing the [Ca]i of BV2 microglia, resulting in mitochondrial depolarization and cell death. Moreover, our data suggest that decreased cAST may produce these pathological effects by malate-aspartate shuttle-independent pathways.

2‑Deoxy‑D‑glucose initiates hepatocyte differentiation in human induced pluripotent stem cells.

To initiate hepatocyte differentiation in human induced pluripotent stem (iPS) cells, cells are cultured in a medium lacking glucose but supplemented with galactose (hepatocyte selection medium, HSM) or in medium supplemented with oncostatin M and small molecules (hepatocyte differentiation inducer, HDI). In the present study, 2‑Deoxy‑D‑glucose (2DG), an analogue of glucose, was utilized instead of glucose deprivation and the effect of 2DG supplementation on iPS differentiation was examined. First, 201B7 cells, an iPS cell line, were cultured in HSM or HDI media for 2days and then subjected to reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) in order to analyze expression levels of established hepatocyte markers, including cytosolic aspartate aminotransferase (AST), mitochondrial AST, alanine aminotransferase (ALT), and glycerol kinase. mRNA expression levels of mitochondrial AST, ALT, and glycogen synthase significantly increased following culture in HSM and HDI compared with ReproFF media. Cytosolic AST mRNA expression levels significantly increased following culture in HDI compared with ReproFF media, but not in HSM. To test the effect of 2DG on iPS differentiation, 201B7 cells were cultured in ReproFF, a feeder‑free medium that retains pluripotency, supplemented with 2DG. Following 7days of culture, the cells were subjected to RT‑qPCR to analyze expression levels of α‑fetoprotein (AFP), a marker of immature hepatocytes. AFP mRNA expression levels significantly increased with the addition of 0.1µM 2DG in the media, and galactose addition acted synergistically with 2DG to further upregulate AFP expression. In conclusion, the present study demonstrated that hepatocyte differentiation was initiated in iPS cells cultured in HSM and HDI media and that 2DG could be used as a supplement instead of glucose deprivation to initiate hepatocyte differentiation in iPS cells.

Abstract A85: Metabolic wiring dictates GOT1 dependency in pancreatic cancer

Abstract Pancreatic Ductal Adenocarcinoma (PDA) is an extremely aggressive disease. The 5-year survival rate is a shockingly low 8%, a number that has hardly increased in the last 30 years. This owes in large part to the fact that effective treatment options for PDA do not exist. A hallmark of pancreatic cancers, and one that contributes to this resistance, is the metabolic environments that they create locally and systemically. Pancreatic tumors exist in a highly inflammatory environment, are under severe physical and oxidative stress, and do not have functional vasculature to deliver oxygen and nutrients. Despite these unfavorable circumstances for growth, these tumors thrive. Clearly metabolism is rewired in unique ways to facilitate survival and growth in this unusual and hostile environment. Based on this framework, we have been mapping the metabolic pathways in PDA to identify aspects of metabolism that are unique or highly over-utilized. Previously, we defined a novel pathway used by PDA cells to maintain redox balance via NADPH generation. Inhibition of a central enzyme in this pathway, cytosolic aspartate aminotransferase (GOT1), dramatically impairs growth in vitro and in vivo. Importantly, these effects can be completely reversed by supplying PDA cells with antioxidants. We also found that this pathway is regulated by mutant KRAS in PDA and, accordingly, non-transformed pancreatic epithelial cells are unresponsive to GOT1 inhibition. Subsequently, we interrogated GOT1 dependence in another KRAS transformed cancer, colon cancer, and found that they, like non-transformed cells, do not respond to GOT1 inhibition. Through a detailed series of metabolomics and functional assays, we ultimately found that colon cancers bypass GOT1 dependence favoring flux through ATP citrate lyase (ACL), a mechanism dictated not at the transcriptional level but by the metabolic wiring in these cells. Furthermore, ACL knockdown dramatically sensitizes colon cancer cells to GOT1 inhibition mediated redox and growth defects. In addition to revealing novel aspects of tissue specific metabolic pathway regulation, this analysis also illustrated novel metabolic dependencies that can be exploited for therapeutic gain. Citation Format: Costas A. Lyssiotis.{Authors}. Metabolic wiring dictates GOT1 dependency in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A85.

Methanol Expression Regulator 1 (Mxr1p) Is Essential for the Utilization of Amino Acids as the Sole Source of Carbon by the Methylotrophic Yeast, Pichia pastoris

Unlike Saccharomyces cerevisiae, the methylotrophic yeast Pichia pastoris can assimilate amino acids as the sole source of carbon and nitrogen. It can grow in media containing yeast extract and peptone (YP), yeast nitrogen base (YNB) + glutamate (YNB + Glu), or YNB + aspartate (YNB + Asp). Methanol expression regulator 1 (Mxr1p), a zinc finger transcription factor, is essential for growth in these media. Mxr1p regulates the expression of several genes involved in the utilization of amino acids as the sole source of carbon and nitrogen. These include the following: (i) GDH2 encoding NAD-dependent glutamate dehydrogenase; (ii) AAT1 and AAT2 encoding mitochondrial and cytosolic aspartate aminotransferases, respectively; (iii) MDH1 and MDH2 encoding mitochondrial and cytosolic malate dehydrogenases, respectively; and (iv) GLN1 encoding glutamine synthetase. Synthesis of all these enzymes is regulated by Mxr1p at the level of transcription except GDH2, whose synthesis is regulated at the level of translation. Mxr1p activates the transcription of AAT1, AAT2, and GLN1 in cells cultured in YP as well as in YNB + Glu media, whereas transcription of MDH1 and MDH2 is activated in cells cultured in YNB + Glu but not in YP. A truncated Mxr1p composed of 400 N-terminal amino acids activates transcription of target genes in cells cultured in YP but not in YNB + Glu. Mxr1p binds to Mxr1p response elements present in the promoters of AAT2, MDH2, and GLN1 We conclude that Mxr1p is essential for utilization of amino acids as the sole source of carbon and nitrogen, and it is a global regulator of multiple metabolic pathways in P. pastoris.

Abstract 233: Identification of cancer vulnerabilities to metabolic perturbation using genome wide CRISPR screens

Abstract The CRISPR-Cas9 system provides an effective way to introduce targeted loss-of-function mutations in mammalian cells. The advance that the CRISPR-Cas9 technology brings to human genetics sets the stage for identifying cellular fitness genes that operate either globally (core fitness genes) or specifically within a particular genetic background or environmental context (context-specific fitness genes). In tumors, this is the foundation for the concept of synthetic lethality as genes required in tumor cells but not in adjacent normal tissues should make ideal therapeutic targets with high effectiveness and minimal side effects. Towards this goal, we have developed a second-generation CRISPR gRNA library of 176,500 guides targeting 17,661 human protein-coding genes. We used the library to screen five human cell lines, representing a cross-section of wild type and cancer tissues, to identify genes whose knockouts induce significant fitness defects. Our screens accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and identify novel context-specific vulnerabilities. Interestingly, we identified a specific dependency on mitochondrial activity and we validated this using various complex I inhibitors. This strongly supports the idea that oxidative phosphorylation (OXPHOS) dependency - a clear exception to the Warburg effect - is a targetable weakness of some tumors. In order to further understand this metabolic vulnerability, we performed a synthetic lethal screen to identify sensitizers of OXPHOS inhibition. Our screen revealed that inhibition of mitochondrial OXPHOS sensitizes the cells to loss of other metabolic pathways such as glycolysis, pentose phosphate pathway and lipid biosynthesis. Furthermore, loss of the cytosolic aspartate aminotransferase GOT1 was found to be synthetic lethal with perturbation of OXPHOS. This is consistent with the essential role of the electron transport chain in cell proliferation, which is to enable aspartate synthesis. Additionally, we also find novel genes, whose loss of function might alleviate the affect of OXPHOS perturbation. Our findings demonstrate that CRISPR-Cas9 screens enable a high-resolution view of the genetic vulnerabilities of a cell that may represent therapeutic opportunities in cancer. Further, synthetic lethal chemical-genetic screens can reveal novel functional drug combinations, which will enhance the efficacy of targeted therapies. Citation Format: Megha Chandrashekhar, Michael Arreger, Ashwin Seetharaman, Traver Hart, Jason Moffat. Identification of cancer vulnerabilities to metabolic perturbation using genome wide CRISPR screens. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 233.

Cytosolic aspartate aminotransferase, a direct binding protein of kamikihito, regulates axon growth

Aim Alzheimer's disease (AD) is the most common cause of dementia. The aim of this study was to identify the direct binding proteins of kamikihito (KKT), a traditional Japanese Kampo medicine used as an anti-AD drug. Method Using the drug affinity responsive target stability (DARTS) method, cytosolic aspartate aminotransferase (cAST) was identified as the direct binding protein of KKT. Primary cultured cortical neurons were treated with amyloid beta (Aβ)(25–35), and cAST expression and activity were evaluated on western blotting and AST activity assay, respectively. To investigate the effects of AST inhibition and cAST knockdown on KKT activity, cortical neurons were treated with O-(carboxymethyl) hydroxylamine hemihydrochloride (OCHH; an AST inhibitor) or transfected with small interfering ribonucleic acid (siRNA) for cAST. The degree of axon atrophy was then evaluated under those conditions. Results On DARTS analysis a 42 kDa protein underwent decreased proteolysis when KKT was present. Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) indicated that the protein was cAST. cAST in Aβ(25–35)-treated neurons had no change in expression level but the activity was reduced. In contrast, treatment with KKT restored cAST activity to control levels. Treatment of cortical neurons with Aβ(25–35) significantly decreased axonal density. KKT treatment restored axonal density, whereas the KKT-induced increase in axonal density was diminished by OCHH treatment or knockdown of cAST. Conclusion Aβ(25–35)-triggered cAST inactivation may be related to axonal atrophy. KKT upregulates cAST activity, probably via direct binding to cAST, resulting in axonal growth.

Structural basis for the relaxed substrate selectivity of Leishmania mexicana broad specificity aminotransferase

Leishmania species are early branching eukaryotic parasites that cause difficult-to-treat tissue-damaging diseases known as leishmaniases. As a hallmark of their parasitic lifestyle, Leishmaniae express a number of aminotransferases that are involved in important cellular processes and exhibit broader substrate specificity than their mammalian host's counterparts. Here, we have determined the crystal structure of the broad specificity aminotransferase from Leishmania mexicana (LmexBSAT) at 1.91Å resolution. LmexBSAT is a homodimer and belongs to the α-branch of family-I aminotransferases. Despite the fact that the protein was crystallized in the absence of substrates and has lost the pyridoxal-5′-phosphate (PLP) cofactor during crystallization, the structure resembles the closed, ligand-bound form of related enzymes such as chicken cytosolic aspartate aminotransferase. Its broader substrate specificity seems to be rooted in increased flexibility of a substrate-binding arginine (R291) and the interactions of this residue with the N-terminus of the second chain of the dimer.

An Essential Role of the Mitochondrial Electron Transport Chain in Cell Proliferation Is to Enable Aspartate Synthesis

The mitochondrial electron transport chain (ETC) enables many metabolic processes, but why its inhibition suppresses cell proliferation is unclear. It is also not well understood why pyruvate supplementation allows cells lacking ETC function to proliferate. We used a CRISPR-based genetic screen to identify genes whose loss sensitizes human cells to phenformin, a complex I inhibitor. The screen yielded GOT1, the cytosolic aspartate aminotransferase, loss of which kills cells upon ETC inhibition. GOT1 normally consumes aspartate to transfer electrons into mitochondria, but, upon ETC inhibition, it reverses to generate aspartate in the cytosol, which partially compensates for the loss of mitochondrial aspartate synthesis. Pyruvate stimulates aspartate synthesis in a GOT1-dependent fashion, which is required for pyruvate to rescue proliferation of cells with ETC dysfunction. Aspartate supplementation or overexpression of an aspartate transporter allows cells without ETC activity to proliferate. Thus, enabling aspartate synthesis is an essential role of the ETC in cell proliferation.

Abstract B45: Pancreatic cancers depend on a non-canonical glutamine metabolism pathway

Abstract Cancer cells exhibit metabolic dependencies that distinguish them from their normal counterparts. Among these addictions is an increased utilization of the amino acid glutamine (Gln) to fuel anabolic processes. Recently, we reported the identification of a non-canonical pathway of Gln utilization in human pancreatic cancer cells that is required for tumor growth. While most cells utilize glutamate dehydrogenase (GLUD1) to convert Gln-derived glutamate (Glu) into alpha-ketoglutarate (alpha-KG) in the mitochondria to fuel the tricarboxylic acid cycle, pancreatic cancer cells rely on a distinct pathway that integrates the mitochondrial and cytosolic aspartate aminotransferases GOT2 and GOT1. By generating alpha-KG from Glu (in conjunction with the conversion of oxaloacetate into aspartate), GOT2 fuels anaplerosis in place of GLUD1. The Asp created is released into the cytosol and acted on by GOT1. This is subsequently used through a series of reactions to yield cytosolic NADPH from malic enzyme. Importantly, we have demonstrated that pancreatic cancers are strongly dependent on this series of reactions to maintain redox homeostasis which enables proliferation. Herein, we detail the subcellular compartmentalization and consequences of the aforementioned reactions on pancreatic cancer metabolism. We have also investigated the essentiality of this pathway in other contexts and find that pancreatic cancers have a uniform and unique reliance on this pathway, which may provide novel therapeutic approaches to treat these refractory tumors. Citation Format: Costas A. Lyssiotis, Jaekyoung Son, Joesph D. Mancias, Haoqiang Ying, Lewis C. Cantley, Alec C. Kimmelman. Pancreatic cancers depend on a non-canonical glutamine metabolism pathway. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B45.

Chromosomal Localization and Contribution of Three Homoeologous Genes to Biosynthesis of Cytosolic Aspartate Aminotransferase in Common Wheat.

Chromosomal localization of the three homoeologous genes encoding cytosolic aspartate aminotransferase in common wheat (Triticum aestivum cv. Chinese Spring, 2n = 6x = 42, AABBDD) was specified to: 3AL (0.42÷0.61), 3BL (0.38÷0.41) and 3DL (0.23÷0.81) by a comparative zymographic analysis of the enzymatic activities in deletion lines. It was also attempted to precisely explain the nature of the relationship between a number of genes encoding α and β subunits and a distribution of staining intensity of cytosolic aspartate aminotransferase allozyme activity bands using aneuploid lines of common wheat with modified third pair of homoeologous chromosomes from genomes A, B and D, on which the genes encoding subunit α (genome A) and β (genome B and D) are localized. The highest consistency between the experimental results and the theoretical distributions was achieved by substituting values of α = 0.57 and β = 0.43 in a theoretical model. These results demonstrate that the individual participation of the diploid genome A in the biosynthesis of the cytosolic aspartate aminotransferase allozymes subunits is greater than the individual participation of the diploid genomes B and D.

Variation in metabolic enzymatic activity in white muscle and liver of blue tilapia, Oreochromis aureus, in response to long-term thermal acclimatization

The effects of rearing temperature on white muscle and hepatic phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were examined in fingerlings of blue tilapia, Oreochromis aureus. The experiment was conducted for 14 weeks at temperatures of 18, 22, 26, 30, and 34°C. The activity of the glycolytic enzymes PFK, PK, and LDH in white muscle increased significantly with increase in water temperature. A reverse trend was observed for these enzymes in the liver, except for LDH, which behaved in the same manner as in white muscle. Cytosolic AST and ALT activity increased in both white muscle and liver in response to warm thermal acclimatization, while a reduction in mitochondrial AST and ALT activity was noticed at high temperatures in comparison with those at a lower temperature.

Pancreatic cancers depend on a non-canonical glutamine metabolism pathway

Cancer cells exhibit metabolic dependencies that distinguish them from their normal counterparts. Among these addictions is an increased utilization of the amino acid glutamine (Gln) to fuel anabolic processes. Recently, we reported the identification of a non-canonical pathway of Gln utilization in human pancreatic cancer cells that is required for tumor growth. While most cells utilize glutamate dehydrogenase (GLUD1) to convert Gln-derived glutamate (Glu) into a-ketoglutarate (aKG) in the mitochondria to fuel the tricarboxylic acid cycle, pancreatic cancer cells rely on a distinct pathway that integrates the mitochondrial and cytosolic aspartate aminotransferases GOT2 and GOT1. By generating aKG from Glu (in conjunction with the conversion of oxaloacetate into aspartate), GOT2 fuels anaplerosis in place of GLUD1. The Asp created is released into the cytosol and acted on by GOT1. This is subsequently used through as eries of reactions to yield cytosolic NADPH from malic enzyme. Importantly, we have demonstrated that pancreatic cancers are strongly dependent on this series of reactions to maintain redox homeostasis which enables proliferation. Herein, we detail the subcellular compartmentalization and consequences of the aforementioned reactions on pancreatic cancer metabolism. We have also investigated the essentiality of this pathway in other contexts and find that pancreatic cancers have a uniform and unique reliance on this pathway, which may provide novel therapeutic approaches to treat these refractory tumors.

Genome-wide association study identifies genetic variants in GOT1 determining serum aspartate aminotransferase levels

We carried out a genome-wide association study of serum aspartate aminotransferase (AST) activity in 866 Amish participants of the Heredity and Phenotype Intervention Heart Study and identified significant association of AST activity with a cluster of single nucleotide polymorphisms located on chromosome 10q24.1 (peak association was rs17109512; P=2.80E-14), in the vicinity of GOT1, the gene encoding cytosolic AST (cAST). Sequencing of GOT1 revealed an in-frame deletion of three nucleic acids encoding asparagine at position 389 c.1165_1167delAAC (p.Asn389del) in the gene. Deletion carriers had significantly lower AST activity levels compared with homozygotes for the common allele (mean±s.d.: 10.0±2.8 versus 18.8±5.2 U l(-1); P=2.80E-14). Further genotyping of the deletion in other Amish samples (n=1932) identified an additional 20 carriers (minor allele frequency (MAF)=0.0052). The deletion was not detected in 647 outbred Caucasians. Asn at codon 389 is conserved among known mammalian cASTs. In vitro transient transfection of wild-type and mutant cAST indicated that mutant cAST protein was barely detectable in the cells. Furthermore, even after correction for cAST expression, mutant cAST had markedly diminished enzymatic activity. Remarkably, we did not find any association between the deletion and metabolic traits including serum fasting glucose or insulin, fasting and post-meal lipids, inflammatory markers, or sub-clinical markers of cardiovascular disease. In conclusion, we discovered a rare in-frame deletion in GOT1 gene, which inactivates cAST enzyme in the Old Order Amish. This finding will help us to understand structure and function of the enzyme and would be useful for predicting serum AST levels.

Root uv-b sensitive Mutants Are Suppressed by Specific Mutations in ASPARTATE AMINOTRANSFERASE2 and by Exogenous Vitamin B6

Vitamin B6 (vitB6) serves as an essential cofactor for more than 140 enzymes. Pyridoxal 5'-phosphate (PLP), active cofactor form of vitB6, can be photolytically destroyed by trace amounts of ultraviolet-B (UV-B). How sun-exposed organisms cope with PLP photosensitivity and modulate vitB6 homeostasis is currently unknown. We previously reported on two Arabidopsis mutants, rus1 and rus2, that are hypersensitive to trace amounts of UV-B light. We performed mutagenesis screens for second-site suppressors of the rus mutant phenotype and identified mutations in the ASPARTATE AMINOTRANSFERASE2 (ASP2) gene. ASP2 encodes for cytosolic aspartate aminotransferase (AAT), a PLP-dependent enzyme that plays a key role in carbon and nitrogen metabolism. Genetic analyses have shown that specific amino acid substitutions in ASP2 override the phenotypes of rus1 and rus2 single mutants as well as rus1 rus2 double mutant. These substitutions, all shown to reside at specific positions in the PLP-binding pocket, resulted in no PLP binding. Additional asp2 mutants that abolish AAT enzymatic activity, but which alter amino acids outside of the PLP-binding pocket, fail to suppress the rus phenotype. Furthermore, exogenously adding vitB6 in growth media can rescue both rus1 and rus2. Our data suggest that AAT plays a role in vitB6 homeostasis in Arabidopsis.