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Selective control of the degradation of normal and aberrant proteins in Reuber H35 hepatoma cells.

ABSTRACT: 1. Rates of degradation of normal and abnormal protein were measured in hepatoma cells after labelling first for 16h with [14C]leucine plus L-arginine and then for 3h with [3H]-leucine plus the arginine analogue, L-canavanine. 2. Over the first 2h of the degradation period, canavanine-containing proteins were degraded at approximately 5 times the average degradation rate of normal proteins. 3. Degradation of normal proteins was inhibited by about 30% by insulin, cycloheximide, puromycin, leupeptin, antipain and foetal calf serum, whereas these agents had a negligible effect on the breakdown of canavanine-containing proteins. 4. Other compounds inhibited degradation of both classes of protein to equal extents. 5. Combination experiments showed no additional inhibitory effects on the degradation of normal proteins over degradation measured in the presence of a single selective inhibitor. 6. In contrast with the results with a 16 h labelling period, the degradation of normal proteins labelled for only 3 h was not inhibited by insulin. 7. These results are explained by a model with two distinct pathways of protein turnover. The first of these pathways involves the formation of autophagic vacuoles and would be completely inhibited by each of the selective inhibitors. Normal and canavanine-containing proteins would be catabolized by this pathway at equal rates. We propose that degradation by a second pathway is not regulated by the agents tested, but by the inherent stability of each protein.

SUBMITTER: Knowles SE

PROVIDER: S-EPMC1163795 | biostudies-other | 1976 Jun

REPOSITORIES: biostudies-other

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Project description:BackgroundThe liver plays a major role in metabolism and performs a number of vital functions in the body. Therefore, the determination of hepatic metabolite dynamics and the analysis of the control of the respective biochemical pathways are of great pharmacological and medical importance. Extra- and intracellular time-series data from stimulus-response experiments are gaining in importance in the identification of in vivo metabolite dynamics, while dynamic network models are excellent tools for analyzing complex metabolic control patterns. This is the first study that has been undertaken on the data-driven identification of a dynamic liver central carbon metabolism model and its application in the analysis of the distribution of metabolic control in hepatoma cells.ResultsDynamic metabolite data were collected from HepG2 cells after they had been deprived of extracellular glucose. The concentration of 25 extra- and intracellular intermediates was quantified using HPLC, LC-MS-MS, and GC-MS. The in silico metabolite dynamics were in accordance with the experimental data. The central carbon metabolism of hepatomas was further analyzed with a particular focus on the control of metabolite concentrations and metabolic fluxes. It was observed that the enzyme glucose-6-phosphate dehydrogenase exerted substantial negative control over the glycolytic flux, whereas oxidative phosphorylation had a significant positive control. The control over the rate of NADPH consumption was found to be shared between the NADPH-demand itself (0.65) and the NADPH supply (0.38).ConclusionsBased on time-series data, a dynamic central carbon metabolism model was developed for the investigation of new and complex metabolic control patterns in hepatoma cells. The control patterns found support the hypotheses that the glucose-6-phosphate dehydrogenase and the Warburg effect are promising targets for tumor treatment. The systems-oriented identification of metabolite dynamics is a first step towards the genome-based assessment of potential risks posed by nutrients and drugs.

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Selective control of the degradation of normal and aberrant proteins in Reuber H35 hepatoma cells.

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