Biosynthesis and processing of mitochondrial glutaminase in HTC hepatoma cells.
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ABSTRACT:
SUBMITTER: Perera SY
PROVIDER: S-EPMC1149840 | biostudies-other | 1991 Jan
REPOSITORIES: biostudies-other
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Project description:A bisbenzyl polyamine analogue, MDL 27695, rapidly repressed ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (AdoMet DC) activity and depleted polyamines in rat hepatoma (HTC) cells. The suppression of ODC and AdoMet DC activity was temporally related to metabolism of MDL 27695 by intracellular polyamine oxidase to a free-amine analogue, MDL 26752, which, when added directly to HTC cells, suppressed ODC activity and polyamine biosynthesis more rapidly and to a greater extent than did the bisbenzyl analogue. The ODC suppression caused by MDL 27695 was completely blocked by the addition of a polyamine oxidase inhibitor to the HTC-cell cultures along with MDL 27695. These data suggested that MDL 27695 acted as a prodrug, with metabolism to an active analogue being necessary for ODC repression to occur. MDL 27695 and MDL 26752 completely abolished division of HTC cells when added to cultures at 1 microM. This established them as being among the most potent antiproliferative polyamine analogues yet described. MDL 27695 has also been shown to possess significant antimalarial effects both in vitro and in vivo, and it is possible that the marked suppression of polyamine biosynthesis described herein may contribute to its anti-malarial effects as well as its antiproliferative effects in mammalian cells.
| S-EPMC1135654 | biostudies-other
Project description:We have separated and characterized a Ca2+- and calmodulin-insensitive cyclic nucleotide phosphodiesterase from rat liver supernatant as well as an analogous enzyme from HTC hepatoma cells. Chromatography of rat liver supernatant on DEAE-cellulose in the presence and subsequently in the absence of 0.1 mM-CaCl2 resulted in the separation of two distinct phosphodiesterase activities, both of which preferentially hydrolysed cyclic GMP rather than cyclic AMP. One enzyme, E-Ib, was activated in the presence of Ca2+ and calmodulin, and the other, E-Ia, was not. The E-Ia enzyme, which did not bind to calmodulin-Sepharose, had Mr 325 000 and displayed anomalous kinetic behaviour [Km (cyclic GMP) 1.2 microM; Km (cyclic AMP) 15.4 microM]. The E-Ib enzyme, which bound to calmodulin-Sepharose in the presence of Ca2+, had Mr 150 000 and exhibited Michaelis-Menten kinetics for hydrolysis of cyclic GMP [Km (basal) 6.5 microM; Km (activated) 12.0 microM]. E-Ia activity was diminished by incubation with alpha-chymotrypsin and was unaffected by the action of a rat kidney lysosomal proteinase. Partial hydrolysis of E-Ib enzyme by alpha-chymotrypsin or the kidney proteinase resulted in irreversible activation of the enzyme. The E-I enzyme isolated from HTC hepatoma cells was similar to the rat liver E-Ia enzyme in many respects. Its apparent Mr was 325 000. Its activity was unaffected by calmodulin in the presence of Ca2+ or by incubation with the kidney proteinase, and was decreased by digestion with alpha-chymotrypsin. Unlike the liver E-Ia enzyme, however, the hepatoma enzyme exhibited normal kinetic behaviour, with Km (cyclic GMP) 3.2 microM. Although HTC cells contain two other phosphodiesterases analogous to those in rat liver and a calmodulin-like activator of phosphodiesterase, no calmodulin-sensitive phosphodiesterase was detected.
| S-EPMC1152138 | biostudies-other
Project description:AimsGlucocorticoids, such as dexamethasone, enhance hepatic energy metabolism and gluconeogenesis partly through changes in mitochondrial function. Mitochondrial function is influenced by the balance between mitochondrial fusion and fission events. However, whether glucocorticoids modulate mitochondrial function through the regulation of mitochondrial dynamics is currently unknown.ResultsHere, we report that the effects of dexamethasone on mitochondrial function and gluconeogenesis in hepatoma cells are dependent on the mitochondrial fission protein dynamin-related protein 1 (Drp1). Dexamethasone increased routine oxygen consumption, maximal respiratory capacity, superoxide anion, proton leak, and gluconeogenesis in hepatoma cells. Under these conditions, dexamethasone altered mitochondrial morphology, which was paralleled by a large increase in Drp1 expression, and reduced mitofusin 1 (Mfn1) and Mfn2. In vivo dexamethasone treatment also enhanced Drp1 expression in mouse liver. On the basis of these observations, we analyzed the dependence on the Drp1 function of dexamethasone effects on mitochondrial respiration and gluconeogenesis. We show that the increase in mitochondrial respiration and gluconeogenesis induced by dexamethasone are hampered by the inhibition of Drp1 function.InnovationOur findings provide the first evidence that the effects of glucocorticoids on hepatic metabolism require the mitochondrial fission protein Drp1.ConclusionIn summary, we demonstrate that the mitochondrial effects of dexamethasone both on mitochondrial respiration and on the gluconeogenic pathway depend on Drp1.
| S-EPMC3700019 | biostudies-literature
Project description:Nuclear magnetic resonance (NMR) spectroscopy was used to monitor glutathione metabolism in alginate-encapsulated JM-1 hepatoma cells perfused with growth media containing [3,3'-13C2]-cystine. After 20 h of perfusion with labeled medium, the 13C NMR spectrum is dominated by the signal from the 13C-labeled glutathione. Once 13C-labeled, the high intensity of the glutathione resonance allows the acquisition of subsequent spectra in 1.2 min intervals. At this temporal resolution, the detailed kinetics of glutathione metabolism can be monitored as the thiol alkylating agent monobromobimane (mBBr) is added to the perfusate. The addition of a bolus dose of mBBr results in rapid diminution of the resonance for 13C-labeled glutathione due to a loss of this metabolite through alkylation by mBBr. As the glutathione resonance decreases, a new resonance due to the production of intracellular glutathione-bimane conjugate is detectable. After clearance of the mBBr dose from the cells, intracellular glutathione repletion is then observed by a restoration of the 13C-glutathione signal along with wash-out of the conjugate. These data demonstrate that standard NMR techniques can directly monitor intracellular processes such as glutathione depletion with a time resolution of approximately < 2 min.
| S-EPMC9104575 | biostudies-literature
Project description:Rho GTPases impact a number of activities important for oncogenesis. We describe a small molecule inhibitor that blocks oncogenic transformation induced by various Rho GTPases in fibroblasts, and the growth of human breast cancer and B lymphoma cells, without affecting normal cells. We identify the target of this inhibitor to be the metabolic enzyme glutaminase, which catalyzes the hydrolysis of glutamine to glutamate. We show that transformed fibroblasts and breast cancer cells exhibit elevated glutaminase activity that is dependent on Rho GTPases and NF-?B activity, and is blocked by the small molecule inhibitor. These findings highlight a previously unappreciated connection between Rho GTPase activation and cellular metabolism and demonstrate that targeting glutaminase activity can inhibit oncogenic transformation.
| S-EPMC3078749 | biostudies-literature
Project description:BackgroundGlutaminolysis is a critical metabolic process that promotes cancer cell proliferation, including hepatocellular carcinoma (HCC). Delineating the molecular control of glutaminolysis could identify novel targets to ameliorate this oncogenic metabolic pathway. Here, we evaluated the role of general control of amino acid synthesis 5 like 1 (GCN5L1), a regulator of mitochondrial protein acetylation, in modulating the acetylation and activity of glutaminase to regulate HCC development.MethodsCell proliferation was determined by MTT, 2D and soft agar clone formation assays and orthotopic tumour assays in nude mice. GLS1/2 acetylation and activities were measured in cells and tumours to analyse the correlation with GCN5L1 expression and mTORC1 activation.ResultsHepatic GCN5L1 ablation in mice markedly increased diethylnitrosamine (DEN)-induced HCC, and conversely, the transduction of mitochondrial-restricted GCN5L1 protected wild-type mice against HCC progression in response to DEN and carbon tetrachloride (CCl4 ) exposure. GCN5L1-depleted HepG2 hepatocytes enhanced tumour growth in athymic nude mice. Mechanistically, GCN5L1 depletion promoted cell proliferation through mTORC1 activation. Interestingly, liver-enriched glutaminase 2 (GLS2) appears to play a greater role than ubiquitous and canonical tumour-enriched glutaminase 1 (GLS1) in promoting murine HCC. Concurrently, GCN5L1 promotes acetylation and inactivation of both isoforms and increases enzyme oligomerisation. In human HCC tumours compared to adjacent tissue, there were variable levels of mTORC1 activation, GCN5L1 levels and glutaminase activity. Interestingly, the levels of GCN5L1 inversely correlated with mTORC1 activity and glutaminase activity in these tumours.ConclusionsOur study identified that glutaminase activity, rather than GLS1 or GLS2 expression, is the key factor in HCC development that activates mTORC1 and promotes HCC. In the Kaplan-Meier analysis of liver cancer, we found that HCC patients with high GCN5L1 expression survived longer than those with low GCN5L1 expression. Collectively, GCN5L1 functions as a tumour regulator by modulating glutaminase acetylation and activity in the development of HCC.
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Project description:The mitochondrial enzyme glutaminase (GLS) is frequently up-regulated during tumorigenesis and is being evaluated as a target for cancer therapy. GLS catalyzes the hydrolysis of glutamine to glutamate, which then supplies diverse metabolic pathways with carbon and/or nitrogen. Here, we report that SIRT5, a mitochondrial NAD+-dependent lysine deacylase, plays a key role in stabilizing GLS. In transformed cells, SIRT5 regulates glutamine metabolism by desuccinylating GLS and thereby protecting it from ubiquitin-mediated degradation. Moreover, we show that SIRT5 is up-regulated during cellular transformation and supports proliferation and tumorigenesis. Elevated SIRT5 expression in human breast tumors correlates with poor patient prognosis. These findings reveal a mechanism for increasing GLS expression in cancer cells and establish a role for SIRT5 in metabolic reprogramming and mammary tumorigenesis.
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Project description:The processing of polyprotein(s) to form structural and non-structural components remains an enigma due to the non-existence of an efficient and robust Hepatitis E Virus (HEV) culture system. We used the BacMam approach to construct an HEV replication model in which the HEV genome was cloned in the BacMam vector under the CMV promoter. The recombinant BacMam was used to infect Huh7 cells to transfer the HEV genome. HEV replication was authenticated by the presence of RNAs of both the polarity (+) and (-) and formation of hybrid RNA, a replication intermediate. The presence of genes for Papain-like Cysteine Protease (PCP), methyltransferase (MeT), RNA dependent RNA polymerase (RdRp), and ORF2 was confirmed by PCR amplification. Further, the infectious nature of the culture system was established as evidenced by the cross-infection of uninfected cells using the cell lysate from the infected cells. The HEV replication model was validated by detection of the ORF1 (Open Reading Frame1) encoded proteins, identified by Western blotting and Immunofluorescence by using epitope-specific antibodies against each protein. Consequently, discrete bands of 18, 35, 37, and 56 kDa corresponding to PCP, MeT, RdRp, and ORF2, respectively, were seen. Besides demonstrating the presence of non-structural enzymes of HEV along with ORF2, activity of a key enzyme, HEV-methyltransferase has also been observed. A 20% decrease in the replicative forms of RNA could be seen in presence of 100 μM Ribavirin after 48 h of treatment. The inhibition gradually increased from 0 to 24 to 48 h post-treatment. Summarily, infectious HEV culture system has been established, which could demonstrate the presence of HEV replicative RNA forms, the structural and non-structural proteins and the methyltransferase in its active form. The system may also be used to study the mechanism of action of Ribavirin in inhibiting HEV replication and develop a therapy.
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Project description:Glutathione (GSH) is transported into renal mitochondria by the dicarboxylate (DIC; Slc25a10) and 2-oxoglutarate carriers (OGC; Slc25a11). To determine whether these carriers function similarly in liver mitochondria, we assessed the effect of competition with specific substrates or inhibitors on GSH uptake in isolated rat liver mitochondria. GSH uptake was uniphasic, independent of ATP hydrolysis, and exhibited K(m) and V(max) values of 4.08 mM and 3.06 nmol/min per mg protein, respectively. Incubation with butylmalonate and phenylsuccinate inhibited GSH uptake by 45-50%, although the individual inhibitors had no effect, suggesting in rat liver mitochondria, the DIC and OGC are only partially responsible for GSH uptake. H4IIE cells, a rat hepatoma cell line, were stably transfected with the cDNA for the OGC, and exhibited increased uptake of GSH and 2-oxoglutarate and were protected from cytotoxicity induced by H(2)O(2), methyl vinyl ketone, or cisplatin, demonstrating the protective function of increased mitochondrial GSH transport in the liver.
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