Summary
Although several proteases have been identified in homogenates of cultured epithelial cells of the eye lens and in lens tissues, there is little information regarding intracellular protein degradation in intact lens cells in vitro. Cultured lens cells may be useful in the study of intracellular protein degradation in the lens, a tissue with a wide range of protein half-lives. This is of interest because alterations in protein turnover in the lens have been implicated in cataract formation. This study examines intracellular protein degradation in cultured bovine lens epithelial cells (BLEC). Cell cultures were incubated with radiolabeled leucine to label intracellular proteins. Protein degradation was measured by monitoring the release of trichloroacetic-acid-soluble radioactivity into the culture medium. The average half-life of long-lived proteins (half-life >50 h) was typically about 57 h in serum-supplemented medium. Average rates of degradation of long-lived proteins increased by up to 73% when fetal bovine serum was withdrawn from the culture medium. Serum had no effect on the degradation of short-lived proteins (half-life <10 h). Degradation of long-lived proteins in the presence and absence of serum was further studied in cultured BLEC from population doubling level (PDL) 2 to 43. Average half-life of proteins in serum-supplemented medium was 52 to 58 h and did not vary significantly as a function of PDL. Degradation rates in serum-free medium increased approximately twofold up to PDL 7, but returned by PDL 25 to original levels, which were maintained through PDL 43.
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Airhart, J.; Arnold, J. A.; Bulman, C. A., et al. Protein synthesis in pulmonary alveolar macrophages: source of amino acids for leucyl-tRNA. Biochim. Biophys. Acta. 653:108–117; 1981.
Amenta, J. S.; Baccino, F. M.; Sargus, M. F. Cell protein degradation in cultured rat embryo fibroblasts. Biochim. Biophys. Acta. 451:511–516; 1976.
Amenta, J. S.; Brocher, S. C. Mechanisms of protein turnover in cultured cells. Life Sci. 28:1195–1208; 1981.
Amenta, J. S.; Sargus, M. J.; Brocher, S. C. Protein synthesis and degradation in growth regulation in rat embryo fibroblasts: role of fast-turnover and slow-turnover protein. J. Cell. Physiol. 105:51–61; 1980.
Auteri, J. S.; Okada, A.; Bochaki, V., et al. Regulation of intracellular protein degradation in IMR-90 human diploid fibroblasts. J. Cell. Physiol. 115:167–174; 1983.
Blondin, J.; Baragi, V. K.; Schwartz, E. R., et al. Delay of UV-induced eye lens protein damage in guinea pigs by dietary ascorbate. J. Free Rad. Biol. Med. 2:275–281; 1986.
Blondin, J.; Taylor, A. Measures of leucine aminopeptidase can be used to anticipate UV-induced age-related damage to lens proteins. Mech. Ageing Dev. 41:39–46; 1987.
Blow, A. M. J. Proteolysis in the lens. In: Barrett, A. J., ed. Proteinases in mammalian cells and tissues. Amsterdam: North-Holland Publishing; 1979: 501–514.
Courtois, Y.; Simmoneau, L.; Tassin, J., et al. Spontaneous transformation of bovine lens epithelial cells. Differentiation 10:23–30; 1978.
Eisenhauer, D. A.; Berger, J. J.; Peltier, C. Z., et al. Protease activities in cultured bovine lens epithelial cells peak and then decline upon progressive passage. Exp. Eye Res. 46:579–590; 1988.
Epstein, D.; Elias-Bishko, S.; Hershko, A. Requirement for protein synthesis in the regulation of protein breakdown in cultured hepatoma cells. Biochemistry 14:5199–5204; 1975.
Fulks, R. M.; Li, J. B.; Goldberg, A. L. Effects of insulin, glucose, and amino acids on protein turnover in rat diaphragm. J. Biol. Chem. 250:290–298; 1975.
Goldberg, A. L.; Dice, J. F. Intracellular protein degradation in mammalian and bacterial cells. Annu. Rev. Biochem. 43:835–869; 1974.
Goldberg, A. L.; St. John, A. C. Intracellular protein degradation in mammalian and bacterial cells, part 2. Annu. Rev. Biochem. 45: 747–803; 1976.
Harding, J. J. Changes in lens proteins in cataract. In: Bloemendal, H., ed. Molecular and cellular biology of the eye lens. New York: John Wiley & Sons; 1981:327–365.
Hendil, K. Intracellular protein degradation in growing, in density inhibited, and in serum-restricted fibroblast cultures. J. Cell. Physiol. 92:353–364; 1977.
Hershko, A. Ubiquitin: roles in protein modification and breakdown. Cell 34:11–12; 1983.
Hershko, A.; Ciechanover, A. Intracellular protein breakdown. Annu. Rev. Biochem. 51:335–364; 1982.
Hershko, A.; Mamont, P.; Sheilds, P., et al. Pleiotypic response. Nature 232:206–211; 1971.
Hoenders, H. J.; Bloemendal, H. Aging of lens proteins. In: Bloemendal, H. ed. Molecular and cellular biology of the eye lens. New York: John Wiley & Sons; 1981: 279–326.
Jahngen, J. H.; Haas, A. L.; Ciechanover, A., et al. The eye lens has an active ubiquitin-protein conjugation system. J. Biol. Chem. 261:13760–13767; 1986.
Okada, A.; Dice, J. F. Altered degradation of intracellular proteins in aging human fibroblasts. Mech. Ageing Devel. 26:341–356; 1984.
Poole, B.; Wibo, M. Protein degradation in cultured cells. J. Biol. Chem. 248:6221–6226; 1973.
Reddan, J. R.; Friedman, T. B.; Mostafapour, M. K., et al. Donor age influences the growth of rabbit lens epithelial cells in vitro. Vis. Res. 21:11–23; 1981.
Seglen, P. O.; Gordon, P. B.; Poli, A. Amino acid inhibition of the autophagic/lysosomal pathway of protein degradation in isolated rat hepatocytes. Biochim. Biophys. Acta. 630:103–118; 1980.
Taylor, A.; Davies, K. Protein oxidation and loss of protease activity may cause cataract formation in the aged lens. Free Rad. Biol. Med. 3:371–377; 1987.
Thomas, J. A.; Hum, T. P.; Augusteyn, R. C. Reconstructing normal crystallin from modified cataractous protein. Aust. J. Exp. Biol. Med. Sci. 63:563–571; 1985.
Vandenburgh, H.; Kaufman, S. Protein degradation in embryonic skeletal muscle. Effect of medium, cell type, inhibitors, and passive stretch. J. Biol. Chem. 255:5826–5833; 1980.
Varma, S. D.; Chand, D.; Sharma, Y. R., et al. Oxidative stress on lens and cataract formation: role of light and oxygen. Curr. Eye Res. 3:35–56; 1984.
Warburton, M. J.; Poole, B. Effect of medium composition on protein degradation and DNA synthesis in rat embryo fibroblasts. Proc. Natl. Acad. Sci. USA 74:2427–2431; 1977.
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This work was supported in part by grants from U. S. Department of Agriculture contract 53-3K06-5-10, Massachusetts Lions Eye Research Fund, Inc., and the Daniel and Florence Guggenheim Foundation. D. A. E. is a recipient of a National Eye Institute postdoctoral fellowship.
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Berger, J.J., Eisenhauer, D.A. & Taylor, A. Intracellular protein degradation in cultured bovine lens epithelial cells. In Vitro Cell Dev Biol 24, 990–994 (1988). https://doi.org/10.1007/BF02620871
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DOI: https://doi.org/10.1007/BF02620871