Functional Annotation of Uncharacterised Proteins Whose Expression Patterns Affect the Lifespan under Metformin Treatment in Fission Yeast

Year 2023, Volume: 82 Issue: 2, 196 - 211, 21.12.2023

Çağatay Tarhan , Sümeyra Zeynep Çalıcı , Buse Özden

https://doi.org/10.26650/EurJBiol.2023.1372233

Abstract

Objective: Metformin, a well-known anti-diabetic drug and a caloric restriction mimetic, seems to attenuate aging through myriad cellular processes, wherein most of its mode of action is still elusive. Thus, bioinformatic analyses that might direct experimental studies are crucial. Moreover, uncharacterised proteins with unknown molecular functions might withhold information regarding metformin’s mode of action. Here, we aimed to elucidate genes encoding uncharacterised proteins that are somehow involved in metformin metabolism and elaborate their involvement through functional annotation to reveal novel cellular processes in which metformin interferes.

Materials and Methods: Total RNA isolation was conducted from Schizosaccharomyces pombe wild-type cells that were grown in standard and overnutrition conditions. Following the gene expression analysis of the uncharacterised proteins, the bioinformatics analysis of the up- and down-regulated uncharacterised proteins upon metformin treatment in both was conducted using the functional annotator called PANNZER2.

Results: Genes that might be related to cellular processes such as meiosis, protein folding, calcium homeostasis, and heme production are up- and down-regulated upon metformin treatment. Moreover, the up-regulation of apoptosis and antioxidationrelated genes and the down-regulation of mitosis, DNA damage, apoptosis, mitochondria, and telomere-capping-related genes were also determined.

Conclusion: We effectively identified associations between metformin and a wide range of cellular processes and genetic mechanisms through the comprehensive annotation of uncharacterised genes. Our findings are consistent with the literature, and many of these uncharacterised proteins could be used as targets for research into aging in the future.

Keywords

Metformin, Aging, Uncharacterized Proteins, Schizosaccharomyces pombe

References

• López-Otín C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hallmarks of aging. Cell. 2013;153(6):1194-1217. doi:10.1016/j.cell.2013.05.039 google scholar
• Carlsen SM, Rossvoll O, Bjerve KS, Folling I. Metformin im-proves blood lipid pattern in nondiabetic patients with coronary heart disease. J Intern Med. 1996;239(3):227-233. google scholar
• Podhorecka M, Ibanez B, Dmoszynska A. Metformin-its poten-tial anti-cancer and anti-aging effects. Postepy Hig Med Dosw (Online). 2017;71(0):170-175. google scholar
• Soukas AA, Hao H, Wu L. Metformin as anti-aging therapy: Is ıt for everyone? Trends Endocrinol Metab. 2019;30(10):745-755. google scholar
• Zhu Z, Jiang T, Suo H, et al. Metformin potentiates the ef-fects of anlotinib in NSCLC via AMPK/mTOR and ROS-mediated signaling pathways. Front Pharmacol. 2021;12:712181. doi:10.3389/fphar.2021.712181 google scholar
• Anisimov VN, Berstein LM, Egormin PA, et al. Metformin slows down aging and extends life span of female SHR mice. Cell Cycle. 2008;7(17):2769-2773. google scholar
• Cabreiro F, Au C, Leung KY, et al. Metformin retards aging in C. elegans by altering microbial folate and methionine metabolism. Cell. 2013;153(1):228-239. google scholar
• De Haes W, Frooninckx L, Van Assche R, et al. Metformin promotes lifespan through mitohormesis via the peroxiredoxin PRDX-2. Proc Natl Acad Sci USA. 2014;111(24):E2501-E2509. doi:10.1073/pnas.1321776111 google scholar
• Suzuta S, Nishida H, Ozaki M, Kohno N, Le TD, Inoue YH. Metformin suppresses progression of muscle aging via activation of the AMP kinase-mediated pathways in Drosophila adults. Eur Rev Med Pharmacol Sci. 2022;26(21):8039-8056. google scholar
• Şeylan C, Tarhan Ç. Metformin extends the chronological lifespan of fission yeast by altering energy metabolism and stress resistance capacity. FEMS Yeast Res. 2023;23:foad018. doi:10.1093/femsyr/foad018 google scholar
• Törönen P, Medlar A, Holm L. PANNZER2: A rapid functional annotation web server. Nucleic Acids Res. 2018;46(W1):W84-W88. doi:10.1093/nar/gky350 google scholar
• Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. (Wilson J, Hunt T, eds.). W.W. Norton & Company; 2017. doi:https://doi.org/10.1201/9781315735368 google scholar
• Konc J, Hodoscek M, Ogrizek M, Trykowska Konc J, Janezic D. Structure-based function prediction of uncharacterized protein using binding sites comparison. PLoS Comput Biol. 2013;9(11):e1003341. doi:10.1371/journal.pcbi.1003341 google scholar
• Rosamond J. Harnessing the power of the genome in the search for new antibiotics. Science. 2000;287(5460):1973-1976. google scholar
• Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. JMol Biol. 1990;215(3):403-410. google scholar
• Buchfink B, Reuter K, Drost HG. Sensitive protein align-ments at tree-of-life scale using DIAMOND. Nat Methods. 2021;18(4):366-368. google scholar
• Zhou N, Jiang Y, Bergquist TR, et al. The CAFA challenge re-ports improved protein function prediction and new functional annotations for hundreds of genes through experimental screens. Genome Biol. 2019;20(1). doi:https://doi.org/10.1186/s13059-019-1835-8 google scholar
• Jensen LJ, Gupta R, Staerfeldt HH, Brunak S. Prediction of hu-man protein function according to Gene Ontology categories. Bioinformatics. 2003;19(5):635-642. google scholar
• Koskinen P, Törönen P, Nokso-Koivisto J, Holm L. PANNZER: High-throughput functional annotation of uncharac-terized proteins in an error-prone environment. Bioinformatics. 2015;31(10):1544-1552. google scholar
• Chen BR, Runge KW. A new Schizosaccharomyces pombe chronological lifespan assay reveals that caloric restriction pro-motes efficient cell cycle exit and extends longevity. Exp Gerontol. 2009;44(8):493-502. google scholar
• Martm-Castellanos C, Blanco M, Rozalen AE, et al. A large-scale screen in S. pombe identifies seven novel genes required for critical meiotic events. Curr Biol. 2005;15(22):2056-2062. google scholar
• Mata J, Lyne R, Burns G, Bahler J. The transcriptional program of meiosis and sporulation in fission yeast. Nat Genet. 2002;32(1):143-147. google scholar
• Yamashita A, Sakuno T, Watanabe Y, Yamamoto M. Syn-chronous ınduction of meiosis in the fission yeast Schizosac-charomyces pombe. Cold Spring Harb Protoc.2017;2017(9). doi:10.1101/pdb.prot091777 google scholar
• Lee SH, Min KJ. Caloric restriction and its mimetics. BMB Rep. 2013;46(4):181-187. google scholar
• Edskes HK, Hanover JA, Wickner RB. Mks1p is a regulator of nitrogen catabolism upstream of Ure2p in Saccharomyces cerevisiae. Genetics. 1999;153(2):585-594. google scholar
• Chitwood PJ, Hegde RS. An intramembrane chaperone complex facilitates membrane protein biogenesis. Nature. 2020;584(7822):630-634. google scholar
• Morimoto M, Waller-Evans H, Ammous Z, et al. Bi-allelic CCDC47 variants cause a disorder characterized by woolly hair, liver dysfunction, dysmorphic features, and global developmental delay. Am J Hum Genet. 2018;103(5):794-807. google scholar
• Yamamoto S, Yamazaki T, Komazaki S, et al. Contribution of calumin to embryogenesis through participation in the en-doplasmic reticulum-associated degradation activity. Dev Biol. 2014;393(1):33-43. google scholar
• Hou C, Tian W, Kleist T, et al. DUF221 proteins are a family of osmosensitive calcium-permeable cation channels conserved across eukaryotes. Cell Res. 2014;24(5):632-635. google scholar
• Hetz C. The unfolded protein response: controlling cell fate de-cisions under ER stress and beyond. Nat Rev Mol Cell Biol. 2012;13(2):89-102. google scholar
• Conza D, Mirra P, Cali G, et al. Metformin dysregulates the unfolded protein response and the WNT/p-catenin pathway in endometrial cancer cells through an AMPK-independent mecha-nism. Cells. 2021;10(5):1067. doi:10.3390/cells10051067 google scholar
• Mönkemeyer L, Klaips CL, Balchin D, Körner R, Hartl FU, Bracher A. Chaperone function of Hgh1 in the biogenesis of eukaryotic elongation factor 2. Mol Cell. 2019;74(1):88-100.e9. doi:https://doi.org/10.1016/j.molcel.2019.01.034 google scholar
• Moldavski O, Amen T, Levin-Zaidman S, et al. Lipid droplets are essential for efficient clearance of cytosolic ınclusion bodies. Dev Cell. 2015;33(5):603-610. google scholar
• Gregan J, Rabitsch PK, Sakem B, et al. Novel genes re-quired for meiotic chromosome segregation are identified by a high-throughput knockout screen in fission yeast. Curr Biol. 2005;15(18):1663-1669. google scholar
• Levine B, Kroemer G. SnapShot: Macroautophagy. Cell. 2008;132(1):162.e1-162.e3. doi:10.1016/j.cell.2007.12.026 google scholar
• Sun LL, Li M, Suo F, et al. Global analysis of fission yeast mating genes reveals new autophagy factors. PLoS Genet. 2013;9(8):e1003715. doi:10.1371/journal.pgen.1003715 google scholar
• Salminen A, Kaarniranta K. AMP-activated protein kinase (AMPK) controls the aging process via an integrated signaling network. Ageing Res Rev. 2012;11(2):230-241. google scholar
• Pallauf K, Rimbach G. Autophagy, polyphenols and healthy age-ing. Ageing Res Rev. 2013;12(1):237-252. google scholar
• Caton PW, Nayuni NK, Kieswich J, Khan NQ, Yaqoob MM, Corder R. Metformin suppresses hepatic gluconeogenesis through induction of SIRT1 and GCN5. J Endocrinol. 2010;205(1):97-106. google scholar
• Sreelatha A, Yee SS, Lopez VA, et al. Protein AMPylation by an evolutionarily conserved pseudokinase. Cell. 2018;175(3):809-821.e19. doi:10.1016/j.cell.2018.08.046 google scholar
• Deng L, Kabeche R, Wang N, Wu JQ, Moseley JB. Megadalton-node assembly by binding of Skb1 to the membrane anchor Slf1. Mol Biol Cell. 2014;25(17):2660-2668. google scholar
• Hou H, Zhou Z, Wang Y, et al. Csi1 links centromeres to the nuclear envelope for centromere clustering. J Cell Biol. 2012;199(5):735-744. google scholar
• Zheng F, Li T, Jin DY, et al. Csi1p recruits alp7p/TACC to the spindle pole bodies for bipolar spindle formation. Mol Biol Cell. 2014;25(18):2750-2760. google scholar
• Strawbridge AB, Elmendorf JS. Phosphatidylinositol 4,5-bisphosphate reverses endothelin-1-induced insulin resistance via an actin-dependent mechanism. Diabetes. 2005;54(6):1698-1705. google scholar
• Polianskyte-Prause Z, Tolvanen TA, Lindfors S, et al. Metformin increases glucose uptake and acts renoprotectively by reducing SHIP2 activity. FASEB J. 2019;33(2):2858-2869. google scholar
• Polakova S, Molnarova L, Hyppa RW, et al. Dbl2 Regulates Rad51 and DNA joint molecule metabolism to ensure proper meiotic chromosome segregation. PLoS Genet. 2016;12(6):e1006102. doi:10.1371/journal.pgen.1006102 google scholar
• Huang L, Khusnutdinova A, Nocek B, et al. A family of metal-dependent phosphatases implicated in metabolite damage-control. Nat Chem Biol. 2016;12(8):621-627. google scholar
• Nishimura A, Yamamoto K, Oyama M, Kozuka-Hata H, Saito H, Tatebayashi K. Scaffold protein Ahk1, which associates with Hkr1, Sho1, Ste11, and Pbs2, inhibits cross talk signaling from the Hkr1 osmosensor to the Kss1 mitogen-activated protein kinase. Mol Cell Biol. 2016;36(7):1109-1123. google scholar
• Yue J, Lopez JM. Understanding MAPK Signaling path-ways in apoptosis. Int J Mol Sci. 2020;21(7):2346. doi:10.3390/ijms21072346 google scholar
• Oscilowska I, Rolkowski K, Baszanowska W, et al. Pro-line dehydrogenase/proline oxidase (PRODH/POX) ıs ın-volved in the mechanism of metformin-ınduced apoptosis in C32 melanoma cell line. Int J Mol Sci. 2022;23(4):2354. doi:10.3390/ijms23042354 google scholar
• Zulkifli M, Neff JK, Timbalia SA, et al. Yeast homologs of hu-man MCUR1 regulate mitochondrial proline metabolism. Nat Commun. 2020;11(1):4866. doi:https://doi.org/10.1038/s41467-020-18704-1 google scholar
• Ragno S, Estrada-Garcia I, Butler R, Colston MJ. Regulation of macrophage gene expression by Mycobacterium tuberculosis: Down-regulation of mitochondrial cytochrome c oxidase. Infect Immun. 1998;66(8):3952-3958. google scholar
• Schüll S, Günther SD, Brodesser S, et al. Cytochrome c ox-idase deficiency accelerates mitochondrial apoptosis by acti-vating ceramide synthase 6. Cell Death Dis. 2015;6(3):e1691. doi:10.1038/cddis.2015.62 google scholar
• Crivellone MD. Characterization of CBP4, a new gene es-sential for the expression of ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae. J Biol Chem. 1994;269(33):21284-21292. google scholar
• Chen S, Gan D, Lin S, et al. Metformin in aging and aging-related diseases: Clinical applications and relevant mechanisms. Theranostics. 2022;12(6):2722-2740 google scholar
• Loubiere C, Clavel S, Gilleron J, et al. The energy disrup-tor metformin targets mitochondrial integrity via modifica-tion of calcium flux in cancer cells. Sci Rep. 2017;7(1):5040. doi:https://doi.org/10.1038/s41598-017-05052-2 google scholar
• Messerschmitt M, Jakobs S, Vogel F, et al. The inner membrane protein Mdm33 controls mitochondrial morphology in yeast. J Cell Biol. 2003;160(4):553-564. google scholar
• Tamura Y, Kawano S, Endo T. Organelle contact zones as sites for lipid transfer. J Biochem. 2019;165(2):115-123. google scholar
• Lang A, John Peter AT, Kornmann B. ER-mitochondria contact sites in yeast: Beyond the myths of ERMES. Curr Opin Cell Biol. 2015;35:7-12. google scholar
• Kawano S, Tamura Y, Kojima R, et al. Structure-function insights into direct lipid transfer between membranes by Mmm 1-Mdm 12 of ERMES. J Cell Biol. 2018;217(3):959-974. google scholar
• Jeong H, Park J, Jun Y, Lee C. Crystal structures of Mmm1 and Mdm12-Mmm1 reveal mechanistic insight into phospholipid trafficking at ER-mitochondria contact sites. Proc Natl Acad Sci USA. 2017;114(45):E9502-E9511. google scholar
• Rasul F, Zheng F, Dong F, et al. Emr1 regulates the number of foci of the endoplasmic reticulum-mitochondria encounter structure complex. Nat Commun. 2021;12(1):521. doi:10.1038/s41467-020-20866-x google scholar
• Fukuda T, Ebi Y, Saigusa T, et al. Atg43 tethers isolation mem-branes to mitochondria to promote starvation-induced mitophagy in fission yeast. Elife. 2020;9:e61245.. doi:10.7554/eLife.61245 google scholar
• de Maranon AM, D^az-Pozo P, Canet F, et al. Metformin modu-lates mitochondrial function and mitophagy in peripheral blood mononuclear cells from type 2 diabetic patients. Redox Biol. 2022;53:102342. doi:10.1016/j.redox.2022.102342 google scholar
• Shenouda SM, Widlansky ME, Chen K, et al. Altered mitochon-drial dynamics contributes to endothelial dysfunction in diabetes mellitus. Circulation. 2011;124(4):444-453. google scholar
• Zhang Q, Wu J, Wu R, et al. DJ-1 promotes the proteasomal degradation of Fis1: Implications of DJ-1 in neuronal protection. Biochem J. 2012;447(2):261-269. google scholar
• Garcia-Martin I, Penketh RJA, Janssen AB, et al. Met-formin and insulin treatment prevent placental telom-ere attrition in boys exposed to maternal diabetes. Rosenfeld CS, ed. PLoS One. 2018;13(12):e0208533. doi:https://doi.org/10.1371/journal.pone.0208533 google scholar