Cytoplasmic Glutamate-oxaloacetate Transaminase Research Articles

USP16 is an ISG15 cross-reactive deubiquitinase that targets pro-ISG15 and ISGylated proteins involved in metabolism.

Interferon-induced ubiquitin (Ub)-like modifier ISG15 covalently modifies host and viral proteins to restrict viral infections. Its function is counteracted by the canonical deISGylase USP18 or Ub-specific protease 18. Notwithstanding indications for the existence of other ISG15 cross-reactive proteases, these remain to be identified. Here, we identify deubiquitinase USP16 as an ISG15 cross-reactive protease by means of ISG15 activity-based profiling. Recombinant USP16 cleaved pro-ISG15 and ISG15 isopeptide-linked model substrates in vitro, as well as ISGylated substrates from cell lysates. Moreover, interferon-induced stimulation of ISGylation was increased by depletion of USP16. The USP16-dependent ISG15 interactome indicated that the deISGylating function of USP16 may regulate metabolic pathways. Targeted enzymes include malate dehydrogenase, cytoplasmic superoxide dismutase 1, fructose-bisphosphate aldolase A, and cytoplasmic glutamic-oxaloacetic transaminase 1. USP16 may thus contribute to the regulation of a subset of metabolism-related proteins during type-I interferon responses.

Targeting glutamine metabolism sensitizes pancreatic cancer to PARP-driven metabolic catastrophe induced by ß-lapachone.

BackgroundPancreatic ductal adenocarcinomas (PDA) activate a glutamine-dependent pathway of cytosolic nicotinamide adenine dinucleotide phosphate (NADPH) production to maintain redox homeostasis and support proliferation. Enzymes involved in this pathway (GLS1 (mitochondrial glutaminase 1), GOT1 (cytoplasmic glutamate oxaloacetate transaminase 1), and GOT2 (mitochondrial glutamate oxaloacetate transaminase 2)) are highly upregulated in PDA, and among these, inhibitors of GLS1 were recently deployed in clinical trials to target anabolic glutamine metabolism. However, single-agent inhibition of this pathway is cytostatic and unlikely to provide durable benefit in controlling advanced disease.ResultsHere, we report that reducing NADPH pools by genetically or pharmacologically (bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) or CB-839) inhibiting glutamine metabolism in mutant Kirsten rat sarcoma viral oncogene homolog (KRAS) PDA sensitizes cell lines and tumors to ß-lapachone (ß-lap, clinical form ARQ761). ß-Lap is an NADPH:quinone oxidoreductase (NQO1)-bioactivatable drug that leads to NADPH depletion through high levels of reactive oxygen species (ROS) from the futile redox cycling of the drug and subsequently nicotinamide adenine dinucleotide (NAD)+ depletion through poly(ADP ribose) polymerase (PARP) hyperactivation. NQO1 expression is highly activated by mutant KRAS signaling. As such, ß-lap treatment concurrent with inhibition of glutamine metabolism in mutant KRAS, NQO1 overexpressing PDA leads to massive redox imbalance, extensive DNA damage, rapid PARP-mediated NAD+ consumption, and PDA cell death—features not observed in NQO1-low, wild-type KRAS expressing cells.ConclusionsThis treatment strategy illustrates proof of principle that simultaneously decreasing glutamine metabolism-dependent tumor anti-oxidant defenses and inducing supra-physiological ROS formation are tumoricidal and that this rationally designed combination strategy lowers the required doses of both agents in vitro and in vivo. The non-overlapping specificities of GLS1 inhibitors and ß-lap for PDA tumors afford high tumor selectivity, while sparing normal tissue.Electronic supplementary materialThe online version of this article (doi:10.1186/s40170-015-0137-1) contains supplementary material, which is available to authorized users.

Changes in Liver Enzyme Activity in the TeleostSparus auratain Response to Cadmium Intoxication

Enzyme activity modulation by cadmium in the liver of the teleost fishSparus auratawas investigatedin vivofollowing 3 and 6 days of CdCl2administration (2.5 mg/kg body wt). The specific activities of the mitochondrial enzymes NAD-isocitrate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase were stimulated by approximately 20% after 3 days administration and were further increased (by about 40%) after 6 days treatment. In comparison with these enzymes, the activities of glutamate–oxaloacetate transaminase (GOT) and glutamate–pyruvate transaminase (GPT) in mitochondria were less stimulated after the two indicated intervals of treatment. Cadmium significantly reduced the activities of liver cytoplasmic GOT and GPT while a simultaneous increase occurred in the serum activities of these same enzymes. The activity of liver NADPH-cytochrome P450 reductase was stimulated by 25 and 40% after 3 and 6 days cadmium intoxication, respectively. Lastly, the antioxidant enzymes glutathione peroxidase and glutathione reductase in liver and catalase in both liver and blood were strongly reduced after 3 and 6 days cadmium administration. These data suggest that cadmium in fish hepatocytes alters cell membrane structure and concomitantly induces some perturbation in the integrity of the mitochondrial membrane.

Description of a new variant in the gluamateoxaloacetate transaminase system by thin‐layer agarose gel isoelectric focusing

Thin-layer isoelectric focusing in agarose within the pH range of 4.0-6.5 has shown a high resolution of the soluble cytoplasmic glutamate-oxaloacetate transaminase (GOT1) banding pattern. The complex pattern of the common GOT1 phenotype consists of eight bands with different intensities. A genetic variant of GOT1, which has been designated GOT1 Mexico, could tentatively be identified.

Electrophoretic variants of blood proteins in Japanese. VII. Cytoplasmic glutamate-oxaloacetate transaminase (GOT1).

A total of 16,835 children, of whom 11,737 are unrelated, from Hiroshima and Nagasaki were examined for erythrocyte cytoplasmic glutamate-oxaloacetate transaminase (GOT1) by starch gel electrophoresis. A variant allele namedGOT1 * 2HR1 which seems to be identical withGOT1 *2 was encountered in polymorphic frequency. Five kinds of rare variants, 3NG1, 4NG1, 5NG1, 6HR1 and 7NG1 were encountered in a total of 109 children. Except for 7NG1 for which complete family study was unable, family studies confirmed the genetic nature of these rare variants, since for all instances in which both parents could be examined, one of the parents exhibited the same variant as that of their child. Thermostability profiles of these six variants were normal. The enzyme activities of five were decreased, while the value of one was normal compared to that of GOT1 1.

Effect of dose of gene for cytoplasmic glutamate-oxaloacetate transaminase in mouse embryos with trisomy for autosome No. 19

Effect of dose of gene for cytoplasmic glutamate-oxaloacetate transaminase in mouse embryos with trisomy for autosome No. 19

Assignment of the gene for cytoplasmic glutamate-oxaloacetate transaminase to mouse chromosome 19 using chinese hamster x mouse somatic cell hybrids.

Chinese hamster and mouse cytoplasmic glutamate-oxaloacetate transaminase (GOT-1, EC 2.6.1.1) were separated by isoelectric focusing of cell extracts on thin-layer polyacrylamide plates. The expression of mouse GOT-1 was correlated with the retention of mouse chromosomes and the expression of 16 marker isozymes in 77 Chinese hamster x mouse somatic cell hybrids. Mouse GOT-1 expression was discordant to the expression of isozyme genes assigned to mouse chromosomes 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 17, 18, and X. Furthermore it showed no concordant expression with mouse chromosomes 3, 13, and 15. The expression of mouse GOT-1 was highly concordant to mouse chromosome 19 in 21 hybrid clones analyzed karyotypically. Our data confirm by means of somatic cell genetics the chromosomal assignment of the mouse GOT-1 gene based on Mendelian genetics by Eicher, Reynolds, and Southard (3). The appearance of a heteropolymeric band in hybrid cells expressing mouse GOT-1 suggests that this enzyme molecule is probably composed of two subunits.

Assignment of the gene for cytoplasmic glutamic-oxaloacetic transaminase to the region q24-qter of human chromosome 10

Somatic cell hybrids between thymidine kinase-deficient mouse cells and human fibroblasts carrying a translocation of the distal third of the long arm of chromosome 10 to chromosome 17 were studied for the expression of cytoplasmic glutamic-oxaloacetic transaminase. A positive correlation between the expression of human cytoplasmic glutamic-oxaloacetic transaminase and the presence of the distal third of the long arm of chromosome 10 was established.

Regional mapping of human chromosome 10: assignment of the gene for cytoplasmic glutamate-oxaloacetate transaminase to 10q24 leads to qter.

Segregation analysis of human-mouse somatic cell hybrids involving fibroblasts of a 10/17 translocation carrier as human partner allowed to assign the gene for human soluble GOT to 10q24 leads to qter.

Localization of the structural locus for cytoplasmic glutamic-oxaloacetic transaminase to region q24→qter of human chromosome 10

Localization of the structural locus for cytoplasmic glutamic-oxaloacetic transaminase to region q24→qter of human chromosome 10

Assignment of the genes for human peptidase A to chromosome 18 and cytoplasmic glutamic oxaloacetate transaminase to chromosome 10 using somatic-cell hybrids.

Evidence based on analysis of mouse-human somatic-cell hybrids is presented that supports the assignment for the structural loci of peptidase A to human chromosome 18 and of cytoplasmic glutamate oxaloacetate transaminase (GOT) (EC 2.6.1.1) to chromosome 10. The medical and evolutionary significance of these assignments is discussed.