人類葡萄糖六磷酸

The effects of N- and C- terminal domains of human glucose-6-phosphatase β on protein stability, protein sorting and phosphohydrolase activity

吳宗翰(Tzeng-Han Wu)

指導教授 ： 謝政哲

長榮大學/健康科學學院/醫學研究所/碩士(2006年)

https://doi.org/10.6833/CJCU.2006.00111

摘要

肝醣經分解所產生的葡萄糖是人類於維持血糖平衡重要機制之ㄧ，而肝醣分解最後一個步驟為葡萄糖六磷酸

關鍵字

葡萄糖六磷酸α ； 葡萄糖六磷酸?﹝] ； 突變 ； 肝醣儲積症 ； 蛋白質穩定性 ； 磷酸水解活性

並列摘要

Interprandial blood glucose homeostasis is maintained by endogeneous hepatic and renal glucose production via glycogenolysis and gluconeogenesis. In the final step of both pathways, intracellular glucose-6-phosphate (G6P) is transported into the endoplasmic reticulum (ER) where it is hydrolyzed to glucose by glucose-6-phosphatase (G6Pase, now renamed as G6Pase-α), which embedded in ER membrane. Unlike G6Pase-α which is expressed only in the liver, kidney, and intestine, a second G6Pase isoform, G6Pase-β (previously known as UGRP, ubiquitously expressed G6Pase catalytic subunit-related protein), has been recently identified, which is expressed ubiquitously with a high expression amount in skeleton muscle, kidney, heart, brain and testis. Both enzymes share similar kinetics and structures and couple with the G6P transporter (G6PT) to form an active G6Pase complex. It has been shown that the N- and C- terminal end of G6Pase-α are critical for maintenances of protein stability, sorting and enzyme activity. However the same effects on G6Pase-β are still not clear. The purpose of this study was to construct serially deleted mutations at the N- and C- terminal regions of G6Pase-β and evaluate their effects on the protein stability, ER membrane sorting and G6P phosphohydrolase activity by using PCR, transient transfection, indirected- immunofluorescence staining, phosphohydrolase assays, western blotting, and semi-quantitative PCR. Here, we found that the N- terminus was more significant than the C- terminus with the reduction of protein stability and enzyme activity. Nevertheless, it seemed no difference on their ER membrane sublocalization. Furthermore, we showed that degradation of both wild-type and mutant G6Pase-β was inhibited by lactacystin, a potent proteasome inhibitor. Base on this study, we should further understand the molecular basis of G6Pase family, especially G6Pase-β.

並列關鍵字

G6Pase-α ； G6Pase-β ； mutation ； glycogen storage disease ； protein stability ； phosphohydrolase activity

參考文獻

2. Snyder, W. S., Cook, M. J., Nasset, E. S., Karhausen, L. R. Howells, G. P., and Tipton, I. H. (1975) in Report of the Task Group on Reference Man (International Commission on Radiological Protection, ed) Pergamon Press, Oxford.

3. Janssen, I., Heymsfield, S. B., Baumgartner, R. N., Ross, R. (2000) Estimation of skeletal muscle mass by bioelectrical impedance analysis. J Appl Physiol. Aug;89(2):465-471.

5. Chou, J. Y., Matern, D., Mansfield, B. C., Chen, Y. T. (2001) Type I glycogen storage diseases: disorders of the glucose-6-phosphatase complex. Curr. Mol. Med. Mar;2(2):121-143.

6. Lei, K. J., Shelly, L. L., Pan, C. J., Sidbury, J. B., Chou J. Y. (1993) Mutations in the glucose-6-phosphatase gene that cause glycogen storage disease type 1a. Science Oct 22;262(5133):580-583.

8. Lei, K. J., Pan, C. J., Shelly, L. L., Liu, J. L., Chou, J. Y. (1994) Identification of mutations in the gene for glucose-6-phosphatase, the enzyme deficient in glycogen storage disease type 1a. J Clin Invest. May;93(5):1994-1999.