Advertisement

Cytotechnology

, Volume 68, Issue 4, pp 1103–1113 | Cite as

Endoplasmic reticulium protein profiling of heat-stressed Jurkat cells by one dimensional electrophoresis and liquid chromatography tandem mass spectrometry

  • Xiulian Zhang
  • Yasuhiro Kuramitsu
  • Aiguo Ma
  • Hui Zhang
  • Kazuyuki Nakamura
Original Research
  • 137 Downloads

Abstract

Proteomic study on membrane-integrated proteins in endoplasmic reticulum (ER) fractions was performed. In this study, we examined the effects of heat stress on Jurkat cells. The ER fractions were highly purified by differential centrifugation with sodium carbonate washing and acetone methanol precipitations. The ER membrane proteins were separated by one dimensional electrophoresis (1-DE), and some of the protein bands changed their abundance by heat stress, 12 of the 14 bands containing 40 and 60 ribosomal proteins whose expression level were decreased, on the contrary, 2 of the 14 bands containing ubiquitin and eukaryotic translation initiation factor 3 were increased. Heat treatment of human Jurkat cells led to an increase in the phosphorylation of PERK and eIF2α within 30 min of exposure. This was followed by an increase in the expression of the GRP78. Protein ubiquitination and subsequent degradation by the proteasome are important mechanisms regulating cell cycle, growth and differentiation, the result showed that heat stress enhanced ubiquitination modification of the microsomal proteins. The data of this study strongly suggest that heat treatment led to a significant reduction in protein expression and activated UPR, concomitant with protein hyperubiqutination in ER.

Keywords

Endoplasmic reticulium Heat stress SDS-PAGE LC–MS/MS 

Abbreviations

ER

Endoplasmic reticulium

RPL19

60S ribosomal protein L19

CBB R-250

Coomassie brilliant blue R-250

SDS

Sodium lauryl sulfate

PAGE

Polyacrylamide gel electrophoresis

ACN

Acetonitrile

TBS

Tris buffered saline

Notes

Acknowledgments

MPI preparation was supported by Prof. Bill Jordan (Center for Biodiscovery and School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand).

Conflict of interest

The authors declared that they have no competing interests.

References

  1. Balch WE, Morimoto RI, Dillin A, Kelly JW (2008) Adapting proteostasis for disease intervention. Science 319:916–919CrossRefGoogle Scholar
  2. Chung M, Nakamura K, Jordan TW (2008) The AOHUPO membrane proteomics initiative, fourth workshop 22 June 2008, Cairns, Australia. Proteomics 8:3920–3923CrossRefGoogle Scholar
  3. Ferella M, Nilsson D, Darban H, Rodrigues C, Bontempi EJ, Docampo R, Andersson B (2008) Proteomics in Trypanosoma cruzi–localization of novel proteins to various organelles. Proteomics 8:2735–2749CrossRefGoogle Scholar
  4. Finley D, Ciechanover A, Varshavsky A (1984) Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85. Cell 37:43–55CrossRefGoogle Scholar
  5. Fujimoto M, Nagasaka Y, Tanaka T, Nakamura K (1998) Analysis of heat shock-induced monophosphorylation of stathmin in human T lymphoblastic cell line JURKAT by two-dimensional gel electrophoresis. Electrophoresis 19:2515–2520Google Scholar
  6. Goldfinger M, Shmuel M, Benhamron S, Tirosh B (2011) Protein synthesis in plasma cells is regulated by crosstalk between endoplasmic reticulum stress and mTOR signaling. Eur J Immunol 41:491–502CrossRefGoogle Scholar
  7. Han J, Back SH, Hur J, Lin YH, Robert Gildersleeve R, Shan JX, Yuan CL, Krokowski D, Wang SY, Hatzoglou M, Kilberg MS, Sartor MA, Kaufman RJ (2013) ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death. Nat Cell Biol 15:481–490CrossRefGoogle Scholar
  8. Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425–479CrossRefGoogle Scholar
  9. Hetz C (2012) The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol 13:89–102Google Scholar
  10. Kelly SM, Vanslyke JK, Musil LS (2007) Regulation of ubiquitin-proteasome system-mediated degradation by cytosolic stress. Mol Bio Cell 18:4279–4291CrossRefGoogle Scholar
  11. Kondo S, Murakami T, Tatsumi K, Ogata M, Kanemoto S, Otori K, Iseki K, Wanaka A, Imaizumi K (2005) OASIS, a CREB/ATF-family member, modulates UPR signalling in astrocytes. Nat Cell Biol 7:186–194CrossRefGoogle Scholar
  12. Malzer E, Daly ML, Moloney A, Sendall TJ, Thomas SE, Ryder E, Ryoo HD, Crowther DC, Lomas DA, Marciniak SJ (2010) Impaired tissue growth is mediated by checkpoint kinase 1 (CHK1) in the integrated stress response. J Cell Sci 123:2892–2900CrossRefGoogle Scholar
  13. Nakamura K, Zhang XL, Kuramitsu Y, Fujimoto M, Yuan XQ, Akada J, Aoshima-Okuda M, Mitani N, Itoh Y, Katoh T, Morita Y, Nagasaka Y, Yamazaki Y, Kuriki T, Sobel A (2006) Analysis on heat stress-induced hyperphosphorylation of stathmin at serine 37 in Jurkat cells by means of two-dimensional gel electrophoresis and tandem mass spectrometry. J Chromatogr A 1106:181–189CrossRefGoogle Scholar
  14. Neutzner M, Neutzner A (2012) Enzymes of ubiquitination and deubiquitination. Essays Biochem 52:37–50CrossRefGoogle Scholar
  15. Nomura S, Kashiwagi S, Ito H, Mimura Y, Nakamura K (1993) Degradation of fibrinogen and fibrin by plasmin and nonplasmin proteases in the chronic subdural hematoma: evaluation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot. Electrophoresis 14:1318–1321CrossRefGoogle Scholar
  16. Park HG, Han SI, Oh SY, Kang HS (2005) Cellular responses to mild heat stress. Cell Mol Life Sci 62:10–23CrossRefGoogle Scholar
  17. Paschen W (2003) Shutdown of translation: lethal or protective? Unfolded protein response versus apoptosis. J Cereb Blood Flow Metab 23:773–779CrossRefGoogle Scholar
  18. Peng LF, Rawson P, Mclauchlan D, Lehnert K, Snell R, Jordan TW (2008) Proteomic analysis of microsomes from lactating bovine mammary. J Proteome Res 7:1427–1432CrossRefGoogle Scholar
  19. Rebecca M, Michael H (1999) Protein delipidation and precipitation by tri-n-butylphosphate, acetone, and methanol treatment for isoelectric focusing and two-dimensional gel electrophoresis. Anal Biochem 273:313–315CrossRefGoogle Scholar
  20. Ron D, Walter P (2007) Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 8:519–529CrossRefGoogle Scholar
  21. Saito A, Ochiai K, Kondo S, Tsumagari K, Murakami T, Cavener DR, Imaizumi K (2011) Endoplasmic reticulum stress response mediated by the PERK-eIF2α-ATF4 pathway is involved in osteoblast differentiation induced by BMP2. J Biol Chem 286(6):4809–4818Google Scholar
  22. Sarkars R, Mukherjee S, Roy M (2013) Targeting heat shock proteins by phenethyl isothiocyanate results in cell-cycle arrest and apoptosis of human breast cancer cells. Nutr Cancer 65:480–493CrossRefGoogle Scholar
  23. Tamura T, Cormier JH, Hebert DN (2008) Sweet bays of ERAD. Trends Biochem Sci 33:298–300CrossRefGoogle Scholar
  24. Taylor SW, Fahy E, Ghosh SS (2003) Global orgallar proteomics. Trends Biotechnol 21:82–88CrossRefGoogle Scholar
  25. Varshavsky A (2012) The ubiquitin system, an immense realm. Annu Rev Biochem 81:167–176CrossRefGoogle Scholar
  26. Wek RC, Jiang HY, Anthony TG (2006) Coping with stress: eIF2 kinases and translational control. Biochem Soc Trans 34:7–11CrossRefGoogle Scholar
  27. Xu GQ, Jaffrey SR (2011) The new landscape of protein ubiquitination. Nat Biotechnol 29:1098–1100CrossRefGoogle Scholar
  28. Yuan XQ, Kuramitsu Y, Furumoto H, Zhang XL, Hayashi E, Fujimoto M, Nakamura K (2007) Nuclear protein profiling of Jurkat cells during heat stress-induced apoptosis by 2-DE and MS/MS. Electrophoresis 28:2018–2026CrossRefGoogle Scholar
  29. Zhu G, Lee AS (2014) Role of the unfolded protein response, GRP78 and GRP94 in organ homeostasis. J Cell Physiol. doi: 10.1002/jcp.24923

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Xiulian Zhang
    • 1
    • 2
  • Yasuhiro Kuramitsu
    • 3
  • Aiguo Ma
    • 1
  • Hui Zhang
    • 4
  • Kazuyuki Nakamura
    • 3
  1. 1.The Institute of Human NutritionMedical College of Qingdao UniversityQingdaoPeople’s Republic of China
  2. 2.Qingdao Center for Disease Control and PreventionQingdaoPeople’s Republic of China
  3. 3.Department of Biochemistry and Functional ProteomicsYamaguchi University Graduate School of MedicineUbeJapan
  4. 4.Zaozhuang University HospitalZaozhuangPeople’s Republic of China

Personalised recommendations