Proteins In Chinese Hamster Ovary Cells Research Articles

Inhibition of degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in vivo by cysteine protease inhibitors

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is a key regulatory enzyme of cholesterol biosynthesis and is located in the endoplasmic reticulum (ER). A fusion protein, HMGal, consisting of the membrane domain of HMG-CoA reductase fused to Escherichia coli beta-galactosidase and expressed in Chinese hamster ovary (CHO) cells from the SV40 promoter, was previously constructed and was found to respond to regulatory signals for degradation in a similar fashion to the intact HMG-CoA reductase. Degradation of both HMG-CoA reductase and HMGal in CHO cells was enhanced by addition of mevalonate or low density lipoprotein (LDL). In this report we show that 2 cysteine protease inhibitors, N-acetyl-leucyl-leucyl-norleucinal (ALLN) and N-acetyl-leucyl-leucyl-methioninal (ALLM), completely inhibit the mevalonate- or LDL-accelerated degradation of HMG-CoA reductase and HMGal and also block the basal degradation of these enzymes. It has been shown that in vitro these protease inhibitors inhibit the activities of Ca(2+)-dependent neutral proteases as well as lysosomal proteases, including cathepsin L, cathepsin b, and cathepsin D. However, the mevalonate-accelerated degradation of HMG-CoA reductase and HMGal is not affected by lysosomotropic agents, suggesting that the site of action of these inhibitor peptides in preventing the degradation is not the cathepsins. In brefeldin A-treated cells, where protein export from the ER is blocked, ALLN is still effective in inhibiting the degradation of HMG-CoA reductase and HMGal. These results indicate the involvement of non-lysosomal Ca(2+)-dependent proteases in the basal and the accelerated degradation of HMG-CoA reductase and HMGal. Enzymatic assays in vitro and immunoblot analyses have revealed calpain- and calpastatin-like proteins in CHO cells. The activities and the amount of these proteins do not change under conditions of enhanced degradation, indicating that the levels of these proteins are not subject to mevalonate regulation.

Legionella pneumophila inhibits protein synthesis in Chinese hamster ovary cells

Legionella pneumophila is a gram-negative facultative intracellular parasite that causes Legionnaires disease. To explore the interactions between L. pneumophila and host cells, we have developed a continuous cell line model of infection. We show that about 80% of Chinese hamster ovary (CHO) cells were associated with L. pneumophila after incubation for 3 h at a multiplicity of infection of 20 bacteria per cell. Within 3 to 4 h of incubation with L. pneumophila, protein synthesis of CHO cells was markedly inhibited, as shown by the reduction of incorporation of radiolabeled amino acids into proteins. L. pneumophila did not inhibit transport of amino acids or cause degradation of newly synthesized proteins in CHO cells. Cytochalasin D blocked internalization of L. pneumophila by CHO cells, yet CHO cell protein synthesis was inhibited. These results indicated that L. pneumophila could inhibit host protein synthesis from the cell exterior. L. pneumophila that had been killed with antibiotics prior to incubation with CHO cells still inhibited protein synthesis, indicating that the inhibition of CHO cell protein synthesis occurred in the absence of de novo protein synthesis by L. pneumophila.

Cloning of a Chinese hamster protein homologous to the mouse t-complex protein TCP-1: Structural similarity to the ubiquitous ‘chaperonin’ family of heat-shock proteins

The complete cDNA sequence of a Chinese hamster ovary cell protein, homologous to a mouse t-complex protein, TCP-1, has been determined. The deduced amino acid sequences of the mouse / Chinese hamster TCP- 1 proteins exhibit significant identity to the 60–65 kDa heat shock ‘chaperonin’ family of proteins present in prokaryotes and in the eukaryotic cell organelles.

Induction of spermidine/spermine N1-acetyltransferase activity in Chinese-hamster ovary cells by N1N11-bis(ethyl)norspermine (corrected) and related compounds

Treatment of Chinese-hamster ovary (CHO) cells with N1N11-bis(ethyl)norspermine (BENSM) led to a very large increase in the activity of spermidine/spermine N1-acetyltransferase (SAT), which rose by about 600-fold within 48 h. Smaller, but still very large increases, were also produced in decreasing order of potency by 3,7,11,15,19-penta-azaheneicosane, N1N12-bis(ethyl)spermine and by N1N14-bis(ethyl)homospermine. The rise in acetyltransferase activity was due to an increase in enzyme protein, as indicated by immunoblotting using antibodies directed against rat liver SAT. There was an increase in the content of mRNA for SAT, indicating that BENSM regulates the level of enzyme protein partly by means of a change in transcription or stability of the mRNA. There was also a decreased rate of degradation of the protein in CHO cells trated with the drug. This may be due to the binding of BENSM, which is a competitive inhibitor of the enzyme with a Ki of 120 microM. Exposure to BENSM led to an increased conversion of spermidine into N1-acetylspermidine and putrescine, a rapid fall in the content of intracellular polyamines and the excretion from the cell of putrescine, N1-acetylspermidine and spermidine. When polyamine oxidase activity in the treated cells was blocked, increases in N1-acetylspermidine and N1-acetylspermine were much greater, and the formation of putrescine was prevented. These results indicate that the induction of SAT facilities the degradation of spermine and spermidine to putrescine and the subsequent excretion of putrescine from the cell. When the degradation of the N1-acetyl derivatives by polyamine oxidase is blocked, the cells excrete N1-acetylspermidine instead of putrescine. CHO cells also contained and excreted N8-acetylspermidine, but its synthesis was not increased in cells treated with BENSM, confirming data obtained in vitro that SAT does not produce this derivative.

Perturbation of N-linked oligosaccharide structure results in an altered incorporation of [3H]palmitate into specific proteins in Chinese hamster ovary cells.

Increased [3H]palmitate incorporation into specific cellular proteins has been reported to occur in Chinese hamster ovary (Wellner, R. B., Ray, B., Ghosh, P. C., and Wu, H. C. (1984) J. Biol. Chem. 259, 12788-12793) and yeast (Wen, D., and Schlesinger, M. J. (1984) Mol. Cell. Biol. 4, 688-694) mutant cells. In this paper we report studies concerning the relationship between N-linked oligosaccharide structure and [3H]palmitate incorporation into proteins of Chinese hamster ovary (CHO) cells. We have compared the incorporation of [3H]palmitate into proteins of wild-type and four different mutant CHO cell lines defective in various steps of N-linked protein glycosylation. Sodium dodecyl sulfate-gel electrophoretic analysis showed that three of the mutants exhibited increased [3H]palmitate incorporation into several CHO cellular proteins (approximately 30,000-38,000 molecular weight) as compared to the wild-type cells. One of the affected mutants which accumulates the Man5Gn2Asn intermediate structure was examined in detail. In agreement with earlier reports, virtually all of the [3H] palmitate-labeled proteins of both wild-type and mutant cell lines are membrane-bound. Pretreatment of the mutant cell line with tunicamycin blocked the increased [3H]palmitate incorporation into the two specific proteins (both of approximately 30,000 molecular weight) observed in untreated cells; the decreased incorporation of [3H]palmitate into the 30,000 molecular weight species was accompanied by a concomitant increase in the incorporation of [3H]palmitate into two proteins of approximately 20,000 molecular weight. Pretreatment of wild-type cells with tunicamycin also caused increased [3H]palmitate incorporation into the 20,000 molecular weight species. Endoglycosidase H treatment of [3H]palmitate-labeled extracts from the mutant cell line resulted in the disappearance of the heavily labeled 30,000 molecular weight species and the appearance of intensely labeled 20,000 molecular weight species. Pretreatment of the mutant cell line with either castanospermine or deoxynojirimycin reduced the [3H]palmitate incorporation in to the 30,000 molecular weight species increased in untreated cells, but did not cause increased [3H]palmitate incorporation into the 20,000 molecular weight species. Our results indicate that perturbation of N-linked oligosaccharide structure results in altered incorporation of [3H]palmitate into specific proteins in CHO cells.