Öz

Glikojen sentaz kinaz 3β (GSK3B), hücre proliferasyonu, DNA onarımı, hücre döngüsü, sinyalleşme ve metabolik yolaklar gibi çok sayıda hücresel süreçte işlev gören bir serin/treonin kinazdır. GSK3B, iltihaplanma, nörodejeneratif hastalık, diyabet ve kanser dahil olmak üzere farklı hastalıklarda rol oynamaktadır. Mayalar, insan genlerine homolog genler taşıması nedeniyle çeşitli hücresel süreçlerin araştırılmasında kullanılan uygun modellerdir. Bu çalışmada insan GSK3B geni, bu gen bakımından delesyonlu Schizosaccharomyces pombe (Lindner) (gsk3Δ) hücrelerine transforme edildi. Gsk3 geni bakımından delesyon taşıyan ve insan GSK3B geni aktarılarak delesyonu genetik komplementasyon ile geri döndürülen hücreler, değişen glukoz konsantrasyonu koşullarında hücresel solunumda meydana gelen değişiklikler bakımından karşılaştırmalı olarak gen ifadesi düzeyinde incelendi. Bu amaçla farklı glukoz konsantrasyonu içeren koşullarda üretilen hücrelerde fbp1, pka1 ve gsk3 genlerinin ifadesi analiz edildi. Glukoz açlığı koşullarında GSK3B geninin diğer koşullara göre daha fazla ifade edildiği sonucuna vardık. Ayrıca ortam koşullarından bağımsız olarak gsk3 geni yokluğunda mitokondriyal solunumu tercih eden hücrelerde insan GSK3B geni aktarıldığında mitokondriyal solunumda gen ifadesinde azalma gözlemledik.

Effects of glucose on the cellular respiration in fission yeast expressing human GSK3B gene

Abstract

Glycogen synthase kinase 3β (GSK3B) acts as a signalling and metabolic enzyme and plays a variety of roles in cellular processes such as cell proliferation, DNA repair, cell cycle, signaling, and metabolic processes. GSK3B has been implicated in numerous diseases, including inflammation, neurodegenerative disease, diabetes, and cancer. Yeasts are suitable models for the investigation of various cellular processes because they include homologous genes to human genes. In this study, we transferred the human GSK3B gene to Schizosac-charomyces pombe (Lindner) cells (gsk3Δ) which include a deletion for this gene. Cells with gsk3 gene deletion and transformant cells with the human GSK3B gene that was reversed by genetic complementation were comparatively examined at the level of gene expression for changes in cellular respiration under varying glucose concentration conditions. For this purpose, the expression of fbp1, pka1 and gsk3 genes were analyzed in cells grown under conditions with different glucose concentrations. We revealed that the GSK3B gene was expressed more in glucose starvation conditions than in other conditions. We also observed a decrease in the level of gene expression in mitochondrial respiration when the human GSK3B gene was transferred in cells that preferred mitochondrial respiration in the absence of the gsk3 gene, regardless of ambient conditions.

Keywords

• Schizosaccharomyces pombe
• glucose metabolism
• glycogen synthase kinase 3
• GSK3B gene

References

1. 1. Avila, J. 2008. Tau kinases and phosphatases: commentary. Journal of Cellular and Molecular Medicine, 12(1): 258-259. https://doi.org/10.1111/j.1582-4934.2007.00214.x
2. 2. Brandis, K.A., Holmes, I.F., England, S.J., Sharma, N., Kukreja, L. & DebBurman, S.K. 2006. α-synuclein fission yeast model. Journal of Molecular Neuroscience, 28(2): 179-191. https://doi.org/10.1385/JMN:28:2:179
3. 3. Breitenbach, M., Ralser, M., Perrone, G.G., Iglseder, B., Rinnerthaler, M., & Dawes, I.W. 2013. Oxidative stress and neurodegeneration: the yeast model system. Frontiers in Bioscience, 18: 1174-1193.
4. 4. Byrne, S.M. & Hoffman, C.S. 1993. Six git genes encode a glucose-induced adenylate cyclase activation pathway in the fission yeast Schizosaccharomyces pombe. Journal of Cell Science, 105(4): 1095-1100. https://doi.org/10.1242/jcs.105.4.1095
5. 5. Byun, H.O., Jung, H.J., Seo, Y.H., Lee, Y.K., Hwang, S.C., Hwang, E.S., & Yoon, G. 2012. GSK3 inactivation is involved in mitochondrial complex IV defect in transforming growth factor (TGF) β1-induced senescence. Experimental Cell Research, 318(15): 1808-1819. https://doi.org/10.1016/j.yexcr.2012.04.012
6. 6. Coronas-Serna, J.M., Del Val, E., Kagan, J.C., Molina, M., & Cid, V.J. 2021. Heterologous expression and assembly of human TLR signaling components in Saccharomyces cerevisiae. Biomolecules, 11(11): 1737. https://doi.org/10.3390/biom11111737
7. 7. Duda, P., Wiśniewski, J., Wójtowicz, T., Wójcicka, O., Jaśkiewicz, M., Drulis-Fajdasz, D., Rakus, D., McCubrey J.A. & Gizak, A. 2018. Targeting GSK3 signaling as a potential therapy of neurodegenerative diseases and aging. Expert Opinion on Therapeutic Targets, 22(10): 833-848. https://doi.org/10.1080/14728222.2018.1526925
8. 8. Gutz, H., Heslot, H., Leupold, U. & Loprieno, N. 1974. Schizosaccharomyces pombe, pp. 395-446. In: King, R.C.,(ed.). Handbook of Genetics. Vol 1. Bacteria, bacteriophages, and fungi. Plenum Press, New York, XVI+ 676 pp. https://doi.org/10.1007/978-1-4899-1710-2_25

9. 9. Hoffman, C.S. & Winston, F. 1990. Isolation and characterization of mutants constitutive for expression of the fbp1 gene of Schizosaccharomyces pombe. Genetics, 124(4): 807-816. https://doi.org/10.1093/genetics/124.4.807
10. 10. Hoffman, C.S. & Winston, F. 1991. Glucose repression of transcription of the Schizosaccharomyces pombe fbp1 gene occurs by a cAMP signaling pathway. Genes & Development, 5(4): 561-571. https://doi.org/10.1101/gad.5.4.561
11. 11. Hoffman, C.S. 2005. Glucose sensing via the protein kinase A pathway in Schizosaccharomyces pombe. Biochemical Society Transactions, 33(1): 257-260. https://doi.org/10.1042/BST0330257
12. 12. Hoffman, C.S., Wood, V. & Fantes, P.A. 2015. An ancient yeast for young geneticists: a primer on the Schiz-osaccharomyces pombe model system. Genetics, 201(2): 403-423. https://doi.org/10.1534/genetics.115.181503
13. 13. Inoue, H., Nojima, H., & Okayama, H. 1990. High efficiency transformation of Escherichia coli with plasmids. Gene, 96(1): 23-28. https://doi.org/10.1016/0378-1119(90)90336-P
14. 14. Johnston, M. 1999. Feasting, fasting and fermenting. Glucose sensing in yeast and other cells. Trend in Genet-ics, 15:29-33. https://doi.org/10.1016/S0168-9525(98)01637-0
15. 15. Johnston, M. 2020. Model Organisms: Nature's Gift to Disease Research. Genetics, 214(2): 233-234. https://doi.org/10.1534/genetics.120.303050
16. 16. Kamat, P.K., Rai, S., Swarnkar, S., Shukla, R. & Nath, C. 2014. Molecular and cellular mechanism of okadaic acid (OKA)-induced neurotoxicity: a novel tool for Alzheimer’s Disease therapeutic application. Molecular Neurobiology, 50: 852–865. https://doi.org/10.1007/s12035-014-8699-4
17. 17. Kanter-Smoler, G., Dahlkvist, A. & Sunnerhagen, P. 1994. Improved method for rapid transformation of intact Schizosaccharomyces pombe cells. Biotechniques, 16(5): 798-800.
18. 18. Karaer, S. 2001. Schizosaccharomyces pombe'nin inozin monofostat dehidrogenaz (gua1) geninin klonlanması ve yapısal analizi. Tez No: 105418, İstanbul Üniversitesi, Fen Bilimleri Enstitüsü, Doktora Tezi. YÖK Tez Merkezi. https://tez.yok.gov.tr/UlusalTezMerkezi/
19. 19. Kimura, T., Yamashita, S., Nakao, S., Park, J.M., Mu-rayama, M., Mizoroki, T., Yoshiike, Y., Sahara, N. & Ta-kashima, A. 2008. GSK-3beta is required for memory re-consolidation in adult brain. PLoS One, 3(10): 3540. https://doi.org/10.1371/journal.pone.0003540
20. 20. Lin, J., Song, T., Li, C. & Mao, W. 2020. GSK-3β in DNA repair, apoptosis, and resistance of chemotherapy, radiotherapy of cancer. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1867(5): 118659. https://doi.org/10.1016/j.bbamcr.2020.118659
21. 21. Liu, Y., Bafaro, E.M., & Dempski, R.E. 2022. Heterologous Expression of Full-Length and Truncated Human ZIP4 Zinc Transporter in Saccharomyces cerevisiae. Bio-molecules, 12(5): 726. https://doi.org/10.3390/biom12050726
22. 22. Lunt, S.Y. & Vander Heiden, M.G. 2011. Aerobic glycolysis: meeting the metabolic requirements of cell proliferation. Annual Review of Cell and Developmental Biology, 27: 441-464.
23. 23. Ma, Y.Q., Kato, T. & Furuyashiki, T. 2016. Genetic interactions among AMPK catalytic subunit Ssp2 and glycogen synthase kinases Gsk3 and Gsk31 in Schizosaccha-romyces pombe. Kobe Journal of Medical Sciences, 62(3): 70-78.
24. 24. Malecki, M., Kamrad, S., Ralser, M. & Bähler, J. 2020. Mitochondrial respiration is required to provide amino acids during fermentative proliferation of fission yeast. EM-BO Reports, 21(11): e50845. https://doi.org/10.15252/embr.202050845
25. 25. Martin, S.A., Souder, D.C., Miller, K.N., Clark, J.P., Sagar, A.K., Eliceiri, K.W., Pugliell, L., Beasley, T.M. & Anderson, R.M. 2018. GSK3β regulates brain energy metabolism. Cell Reports, 23(7): 1922-1931. https://doi.org/10.1016/j.celrep.2018.04.045
26. 26. Nidelet, T., Brial, P., Camarasa, C. & Dequin, S. 2016. Diversity of flux distribution in central carbon metabolism of S. cerevisiae strains from diverse environments. Microbial Cell Factories, 15(1): 1-13. https://doi.org/10.1186/s12934-016-0456-0
27. 27. Pfaffl, M.W. 2001. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Research, 29(9): e45-e45. https://doi.org/10.1093/nar/29.9.e45
28. 28. Porzoor, A. & Macreadie, I.G. 2013. Application of yeast to study the tau and amyloid β abnormalities of Alzheimer’s Disease. Journal of Alzheimer's Disease, 35: 217-225. https://doi.org/10.3233/JAD-122035
29. 29. Rallis, C., Townsend, S. & Bähler, J. 2017. Genetic interactions and functional analyses of the fission yeast gsk3 and amk2 single and double mutants defective in TORC1-dependent processes. Scientific Reports, 7: 44257. https://doi.org/10.1038/srep44257
30. 30. Ren, Z., Zhong, H., Song, C., Deng, C., Hsieh, H. T., Liu, W., & Chen, G. 2020. Insulin promotes mitochondrial respiration and survival through PI3K/AKT/GSK3 pathway in human embryonic stem cells. Stem Cell Reports, 15(6): 1362-1376. https://doi.org/10.1016/j.stemcr.2020.10.008
31. 31. Turcotte, B., Liang, X.B., Robert, F. & Soontorngun, N. 2009. Transcriptional regulation of nonfermentable carbon utilization in budding yeast. FEMS Yeast Research, 10(1): 2-13. https://doi.org/10.1111/j.1567-1364.2009.00555.x
32. 32. Vanhelmont, T., Vandebroek, T., De Vos, A., Terwel, D., Lemaire, K., Anandhakumar, J., ... & Winderickx, J. 2010. Serine-409 phosphorylation and oxidative damage define aggregation of human protein tau in yeast. FEMS Yeast Research, 10(8): 992-1005.
33. 33. Vyas, A., Freitas, A.V., Ralston, Z.A. & Tang, Z. 2021. Fission yeast Schizosaccharomyces pombe: A unicellular “micromammal” model organism. Current Protocols, 1(6): e151. https://doi.org/10.1002/cpz1.151
34. 34. Walker, G. 1998. Yeast physiology and biotechnology. John Wiley & Sons, UK. 362 pp.
35. 35. Wang, L., Li, J. & Di, L.J. 2021. Glycogen synthesis and beyond, a comprehensive review of GSK3 as a key regulator of metabolic pathways and a therapeutic target for treating metabolic diseases. Medicinal Research Reviews, 42(2): 946-982. https://doi.org/10.1002/med.21867
36. 36. Wangler, M.F., Yamamoto, S., Chao, H.T., Posey, J.E., Westerfield M., John Postlethwait, Members of the Undiagnosed Diseases Network (UDN), Hieter, P., Boycott, K.M., Campeau, P.M. & Bellen, H.J. 2017. Model organisms facilitate rare disease diagnosis and therapeutic research. Genetics, 207: 9-27. https://doi.org/10.1534/genetics.117.203067
37. 37. Warburg, O. 1956. On the origin of cancer cells. Science, 123(3191): 309-314. https://doi.org/10.1126/science.123.3191.309
38. 38. Winderickx, J., Delay, C., De Vos, A., Klinger, H., Pel-lens, K., Vanhelmont, T., ... & Zabrocki, P. 2008. Protein folding diseases and neurodegeneration: lessons learned from yeast. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1783(7): 1381-1395. https://doi.org/10.1016/j.bbamcr.2008.01.020