Ukr.Biochem.J. 2017; Volume 89, Issue 5, Sep-Oct, pp. 52-61


Expression of ubiquitin specific peptidase and ATG7 genes in U87 glioma cells upon glutamine deprivation

O. V. Halkin1, D. O. Minchenko1,2, О. O. Riabovol1,
V. V. Telychko1, О. O. Ratushna1, O. H. Minchenko1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Bohomolets National Medical University, Kyiv, Ukraine

We have studied the effect of glutamine deprivation on the expression of genes encoding for ubiquitin specific peptidases (USP) and ubiquitin activating enzyme E1-like protein/autophagy related 7 (GSA7/ATG7) in U87 glioma cells in relation to inhibition of inositol requiring enzyme-1 (IRE1). It was shown that exposure of control glioma cells (transfected by empty vector) upon glutamine deprivation led to suppression of USP1 and ATG7 mRNA expression and up-regulated USP25 mRNA. At the same time, glutamine deprivation did not significantly change USP4, USP10, USP14, and USP22 gene expressions in these cells. Inhibition of ІRE1 signaling enzyme function in U87 glioma cells increased effect of glutamine deprivation on the expression of USP1 gene and introduced sensitivity of USP4 and USP14 genes to this condition. Therefore, glutamine deprivation affected the expression level of most studied genes in gene specific manner in relation to the functional activity of IRE1 enzyme, a central mediator of endoplasmic reticulum stress, which controls cell proliferation and tumor growth.

Keywords: , , , , ,


  1. Colombo SL, Palacios-Callender M, Frakich N, Carcamo S, Kovacs I, Tudzarova S, Moncada S. Molecular basis for the differential use of glucose and glutamine in cell proliferation as revealed by synchronized HeLa cells. Proc Natl Acad Sci USA. 2011 Dec 27;108(52):21069-74. PubMed, PubMedCentral, CrossRef
  2. Daye D, Wellen KE. Metabolic reprogramming in cancer: unraveling the role of glutamine in tumorigenesis. Semin Cell Dev Biol. 2012 Jun;23(4):362-9. PubMed, CrossRef
  3. Hensley CT, Wasti AT, DeBerardinis RJ. Glutamine and cancer: cell biology, physiology, and clinical opportunities. J Clin Invest. 2013 Sep;123(9):3678-84. PubMed, PubMedCentral, CrossRef
  4. Li Y, Erickson JW, Stalnecker CA, Katt WP, Huang Q, Cerione RA, Ramachandran S. Mechanistic basis of glutaminase activation: a key enzyme that promotes glutamine metabolism in cancer cells. J Biol Chem. 2016 Sep 30;291(40):20900-20910. PubMed, PubMedCentral, CrossRef
  5. Szeliga M, Albrecht J. Opposing roles of glutaminase isoforms in determining glioblastoma cell phenotype. Neurochem Int. 2015 Sep;88:6-9. PubMed, CrossRef
  6. Zhang C, Liu J, Zhao Y, Yue X, Zhu Y, Wang X, Wu H, Blanco F, Li S, Bhanot G, Haffty BG, Hu W, Feng Z. Glutaminase 2 is a novel negative regulator of small GTPase Rac1 and mediates p53 function in suppressing metastasis. Elife. 2016 Jan 11;5:e10727. PubMed, PubMedCentral, CrossRef
  7. Satija YK, Bhardwaj A, Das S. A portrayal of E3 ubiquitin ligases and deubiquitylases in cancer. Int J Cancer. 2013 Dec 15;133(12):2759-68. PubMed, CrossRef
  8. Kessler BM, Edelmann MJ. PTMs in conversation: activity and function of deubiquitinating enzymes regulated via post-translational modifications. Cell Biochem Biophys. 2011 Jun;60(1-2):21-38. PubMed, PubMedCentral, CrossRef
  9. Li J, Tan Q, Yan M, Liu L, Lin H, Zhao F, Bao G, Kong H, Ge C, Zhang F, Yu T, Li J, He X, Yao M. miRNA-200c inhibits invasion and metastasis of human non-small cell lung cancer by directly targeting ubiquitin specific peptidase 25. Mol Cancer. 2014 Jul 6;13:166. PubMed, PubMedCentral, CrossRef
  10. Mojsa B, Lassot I, Desagher S. Mcl-1 ubiquitination: unique regulation of an essential survival protein. Cells. 2014 May 8;3(2):418-37. PubMed, PubMedCentral, CrossRef
  11. Zhang J, Zhang X, Xie F, Zhang Z, van Dam H, Zhang L, Zhou F. The regulation of TGF-β/SMAD signaling by protein deubiquitination. Protein Cell. 2014 Jul;5(7):503-17. PubMed, PubMedCentral, CrossRef
  12. Danilovskyi SV, Minchenko DO, Moliavko OS, Kovalevska OV, Karbovskyi LL, Minchenko OH.  ERN1 knockdown modifies the hypoxic regulation of TP53, MDM2, USP7 and PERP gene expressions in U87 glioma cells. Ukr Biochem J. 2014 Jul-Aug;86(4):90-102. PubMed, CrossRef
  13. Kashiwaba S, Kanao R, Masuda Y, Kusumoto-Matsuo R, Hanaoka F, Masutani C. USP7 is a suppressor of PCNA ubiquitination and oxidative-stress-induced mutagenesis in human Cells. Cell Rep. 2015 Dec 15;13(10):2072-80. PubMed, CrossRef
  14. Zhiqiang Z, Qinghui Y, Yongqiang Z, Jian Z, Xin Z, Haiying M, Yuepeng G. USP1 regulates AKT phosphorylation by modulating the stability of PHLPP1 in lung cancer cells. J Cancer Res Clin Oncol. 2012 Jul;138(7):1231-8. PubMedCrossRef
  15. Lévy J, Cacheux W, Bara MA, L’Hermitte A, Lepage P, Fraudeau M, Trentesaux C, Lemarchand J, Durand A, Crain AM, Marchiol C, Renault G, Dumont F, Letourneur F, Delacre M, Schmitt A, Terris B, Perret C, Chamaillard M, Couty JP, Romagnolo B. Intestinal inhibition of Atg7 prevents tumour initiation through a microbiome-influenced immune response and suppresses tumour growth. Nat Cell Biol. 2015 Aug;17(8):1062-73. PubMed, CrossRef
  16. Lee JK, Chang N, Yoon Y, Yang H, Cho H, Kim E, Shin Y, Kang W, Oh YT, Mun GI, Joo KM, Nam DH, Lee J. USP1 targeting impedes GBM growth by inhibiting stem cell maintenance and radioresistance. Neuro Oncol. 2016 Jan;18(1):37-47. PubMed, PubMedCentral, CrossRef
  17. Villamil MA, Liang Q, Chen J, Choi YS, Hou S, Lee KH, Zhuang Z. Serine phosphorylation is critical for the activation of ubiquitin-specific protease 1 and its interaction with WD40-repeat protein UAF1. Biochemistry. 2012 Nov 13;51(45):9112-23. PubMed, PubMedCentral, CrossRef
  18. Li Z Hao Q, Luo J, Xiong J, Zhang S, Wang T, Bai L, Wang W, Chen M, Wang W, Gu L, Lv K, Chen J. USP4 inhibits p53 and NF-κB through deubiquitinating and stabilizing HDAC2. Oncogene. 2016 Jun 2;35(22):2902-12. PubMed, PubMedCentral, CrossRef
  19. Yun SI, Kim HH, Yoon JH, Park WS, Hahn MJ, Kim HC, Chung CH, Kim KK. Ubiquitin specific protease 4 positively regulates the WNT/β-catenin signaling in colorectal cancer. Mol Oncol. 2015 Nov;9(9):1834-51. PubMed, PubMedCentral, CrossRef
  20. Zhang J, Zhang X, Xie F, Zhang Z, van Dam H, Zhang L, Zhou F. The regulation of TGF-β/SMAD signaling by protein deubiquitination. Protein Cell. 2014 Jul;5(7):503-17. PubMed, PubMedCentral, CrossRef
  21. Liu H, Xu XF, Zhao Y, Tang MC, Zhou YQ, Lu J, Gao FH. MicroRNA-191 promotes pancreatic cancer progression by targeting USP10. Tumour Biol. 2014 Dec;35(12):12157-63. PubMed, CrossRef
  22. Lin Z, Yang H, Tan C, Li J, Liu Z, Quan Q, Kong S, Ye J, Gao B, Fang D. USP10 antagonizes c-Myc transcriptional activation through SIRT6 stabilization to suppress tumor formation. Cell Rep. 2013 Dec 26;5(6):1639-49. PubMed, PubMedCentral, CrossRef
  23. Wang Y, Wang J, Zhong J, Deng Y, Xi Q, He S, Yang S, Jiang L, Huang M, Tang C, Liu R. Ubiquitin-specific protease 14 (USP14) regulates cellular proliferation and apoptosis in epithelial ovarian cancer. Med Oncol. 2015 Jan;32(1):379. PubMed, CrossRef
  24. Tang B, Tang F, Li B, Yuan S, Xu Q, Tomlinson S, Jin J, Hu W, He S. High USP22 expression indicates poor prognosis in hepatocellular carcinoma. Oncotarget. 2015 May 20;6(14):12654-67. PubMed, PubMedCentral, CrossRef
  25. Liu YL, Zheng J, Tang LJ, Han W, Wang JM, Liu DW, Tian QB. The deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth. Gene. 2015 Nov 1;572(1):49-56. PubMed, CrossRef
  26. 26. Ao N, Liu Y, Bian X, Feng H, Liu Y. Ubiquitin-specific peptidase 22 inhibits colon cancer cell invasion by suppressing the signal transducer and activator of transcription 3/matrix metalloproteinase 9 pathway. Mol Med Rep. 2015 Aug;12(2):2107-13. PubMed, CrossRef
  27. Blount JR, Burr AA, Denuc A, Marfany G, Todi SV. Ubiquitin-specific protease 25 functions in Endoplasmic Reticulum-associated degradation. PLoS One. 2012;7(5):e36542. PubMed, PubMedCentral, CrossRef
  28. Kim S, Lee D, Lee J, Song H, Kim HJ, Kim KT. Vaccinia-Related Kinase 2 Controls the Stability of the Eukaryotic Chaperonin TRiC/CCT by Inhibiting the Deubiquitinating Enzyme USP25. Mol Cell Biol. 2015 May;35(10):1754-62. PubMed, PubMedCentral, CrossRef
  29. Galluzzi L, Bravo-San Pedro JM, Kroemer G. Autophagy Mediates Tumor Suppression via Cellular Senescence. Trends Cell Biol. 2016 Jan;26(1):1-3. PubMedCrossRef
  30. Catalano M, D’Alessandro G, Lepore F, Corazzari M, Caldarola S, Valacca C, Faienza F, Esposito V, Limatola C, Cecconi F, Di Bartolomeo S. Autophagy induction impairs migration and invasion by reversing EMT in glioblastoma cells. Mol Oncol. 2015 Oct;9(8):1612-25. PubMed, PubMedCentral, CrossRef
  31. Mortensen M, Soilleux EJ, Djordjevic G, Tripp R, Lutteropp M, Sadighi-Akha E, Stranks AJ, Glanville J, Knight S, Jacobsen SE, Kranc KR, Simon AK. The autophagy protein Atg7 is essential for hematopoietic stem cell maintenance. J Exp Med. 2011 Mar 14;208(3):455-67. PubMed, PubMedCentral, CrossRef
  32. Ye Y, Tan S, Zhou X, Li X, Jundt MC, Lichter N, Hidebrand A, Dhasarathy A, Wu M. Inhibition of p-IκBα Ubiquitylation by autophagy-related Gene 7 to regulate inflammatory responses to bacterial infection. J Infect Dis. 2015 Dec 1;212(11):1816-26. PubMed, PubMedCentral, CrossRef
  33. Antonucci L, Fagman JB, Kim JY, Todoric J, Gukovsky I, Mackey M, Ellisman MH, Karin M. Basal autophagy maintains pancreatic acinar cell homeostasis and protein synthesis and prevents ER stress. Proc Natl Acad Sci USA. 2015 Nov 10;112(45):E6166-74. PubMed, PubMedCentral, CrossRef
  34. Deegan S, Saveljeva S, Logue SE, Pakos-Zebrucka K, Gupta S, Vandenabeele P, Bertrand MJ, Samali A. Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions. Autophagy. 2014;10(11):1921-36. PubMed, PubMedCentral,CrossRef
  35. Malhotra JD, Kaufman RJ. ER stress and its functional link to mitochondria: role in cell survival and death. Cold Spring Harb Perspect Biol. 2011 Sep 1;3(9):a004424. PubMed, PubMedCentral, CrossRef
  36. Auf G, Jabouille A, Guérit S, Pineau R, Delugin M, Bouchecareilh M, Magnin N, Favereaux A, Maitre M, Gaiser T, von Deimling A, Czabanka M, Vajkoczy P, Chevet E, Bikfalvi A, Moenner M. Inositol-requiring enzyme 1alpha is a key regulator of angiogenesis and invasion in malignant glioma. Proc Natl Acad Sci USA. 2010 Aug 31;107(35):15553-8. PubMed, PubMedCentral, CrossRef
  37. Lenihan CR, Taylor CT. The impact of hypoxia on cell death pathways. Biochem Soc Trans. 2013 Apr;41(2):657-63. PubMed, CrossRef
  38. Hetz C, Chevet E, Harding HP. Targeting the unfolded protein response in disease. Nat Rev Drug Discov. 2013 Sep;12(9):703-19. PubMed, CrossRef
  39. Minchenko OH, Tsymbal DO, Minchenko DO, Moenner M, Kovalevska OV, Lypova NM. Inhibition of kinase and endoribonuclease activity of ERN1/IRE1α affects expression of proliferation related genes in U87 glioma cells. Endoplasm Reticul Stress Dis. 2015; 2(1): 18-29.  CrossRef
  40. Manié SN, Lebeau J, Chevet E. Cellular mechanisms of endoplasmic reticulum stress signaling in health and disease. 3. Orchestrating the unfolded protein response in oncogenesis: an update. Am J Physiol Cell Physiol. 2014 Nov 15;307(10):C901-7. PubMed, CrossRef
  41. Tsymbal DO, Minchenko DO, Riabovol OO, Ratushna OO, Minchenko OH.  IRE1 knockdown modifies glucose and glutamine deprivation effects on the expression of proliferation related genes in U87 glioma cells. Biotechnol Acta. 2016; 9(1): 26-37. CrossRef
  42. Minchenko DO, Danilovskyi SV, Kryvdiuk IV, Bakalets TV, Lypova NM, Karbovsky LL, Minchenko OH. Inhibition of ERN1 modifies the hypoxic regulation of the expression of TP53-related genes in U87 glioma cells. Endoplasm Reticul Stress Dis. 2014; 1(1): 18-26.  CrossRef
  43. Minchenko OH,  Tsymbal DO, Minchenko DO, Riabovol OO, Halkin OV, Ratushna OO.  IRE-1α regulates expression of ubiquitin specific peptidases during hypoxic response in U87 glioma cells. Endoplasm Reticul Stress Dis. 2016; 3(1): 50-62. CrossRef
  44. Minchenko OH, Riabovol OO, Halkin OV, Danilovskyi SV, Minchenko DO,  Ratushna OO. Expression of ubiquitin specific peptidase genes in IRE1 knockdown U87 glioma cells upon glucose deprivation. Biotechnol Acta. 2016; 9(5): 7-17. CrossRef
  45. Bochkov VN, Philippova M, Oskolkova O, Kadl A, Furnkranz A, Karabeg E, Afonyushkin T, Gruber F, Breuss J, Minchenko A, Mechtcheriakova D, Hohensinner P, Rychli K, Wojta J, Resink T, Erne P, Binder BR, Leitinger N. Oxidized phospholipids stimulate angiogenesis via autocrine mechanisms, implicating a novel role for lipid oxidation in the evolution of atherosclerotic lesions. Circ Res. 2006; 99(8): 900-908. CrossRef
  46. Han J, Hou W, Goldstein LA, Stolz DB, Watkins SC, Rabinowich H. A complex between Atg7 and caspase-9: a novel mechanism of cross-regulation between autophagy and apoptosis. J Biol Chem. 2014 Mar 7;289(10):6485-97. PubMed, PubMedCentral, CrossRef

Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License.