DOI QR코드

DOI QR Code

Evidence of Multimeric Forms of HSP70 with Phosphorylation on Serine and Tyrosine Residues - Implications for Roles of HSP70 in Detection of GI Cancers

  • Dutta, Anand (Division of Gastroenterology, Department of Medicine, Johns Hopkins University/Sinai Hospital Program in Internal Medicine) ;
  • Girotra, Mohit (Division of Gastroenterology, Department of Medicine, Johns Hopkins University/Sinai Hospital Program in Internal Medicine) ;
  • Merchant, Nipun (Department of Surgery and Cancer Biology, Vanderbilt University Medical Center) ;
  • Nair, Padmanabhan (Johns Hopkins Bloomberg School of Public Health) ;
  • Dutta, Sudhir Kumar (Division of Gastroenterology, Department of Medicine, Johns Hopkins University/Sinai Hospital Program in Internal Medicine)
  • Published : 2013.10.30

Abstract

Background: Heat-shock protein70 (HSP70) are intracellular protein chaperones, with emerging evidence of their association with various diseases. We have previously reported significantly elevated plasma-HSP70 (pHSP70) in pancreatic cancer. Current methods of pHSP70 isolation are ELISA-based which lack specificity due to cross-reactivity by similarities in the amino-acid sequence in regions of the protein backbone resulting in overestimated HSP70 value. Materials and Methods: This study was undertaken to develop a methodology to capture all isoforms of pHSP70, while further defining their tyrosine and serine phosphorylation status. Results: The methodology included gel electrophoresis on centrifuged supernatant obtained from plasma incubated with HSP70 antibody-coupled beads. After blocking non-specific binding sites, blots were immunostained with monoclonal-antibody specific for human-HSP70, phosphoserine and phosphotyrosine. Conclusions: Our novel immunocapture approach has distinct advantages over the commercially available methods of pHSP70 quantification by allowing isolation of molecular aggregates of HSP70 with additional ability to precisely distinguish phosphorylation state of HSP70 molecules at serine and tyrosine residues.

Keywords

HSP70;multimeric forms;phosphorylation;serine;tyrosine;GI cancer detection

References

  1. Bottoni P, Giardina B, Scatena R (2009). Proteomic profiling of heat shock proteins: an emerging molecular approach with direct pathophysiological and clinical implications. Proteomics Clin Appl, 3, 636-53. https://doi.org/10.1002/prca.200800195
  2. Aghdassi A, Phillips P, Dudeja V, et al (2007). Heat shock protein 70 increases tumorigenicity and inhibits apoptosis in pancreatic adenocarcinoma. Cancer Res, 67, 616-25. https://doi.org/10.1158/0008-5472.CAN-06-1567
  3. Baird TD, Wek RC (2012). Eukaryotic initiation factor 2 phosphorylation and translational control in metabolism. Adv Nutr, 3, 307-21. https://doi.org/10.3945/an.112.002113
  4. Berger JC, Vander Griend DJ, Robinson VL, Hickson JA, Rinker-Schaeffer CW (2005). Metastasis suppressor genes: from gene identification to protein function and regulation. Cancer Biol Ther, 4, 805-12. https://doi.org/10.4161/cbt.4.8.1865
  5. Bhagat L, Singh VP, Hietaranta AJ, et al (2000). Heat shock protein 70 prevents secretagogue-induced cell injury in the pancreas by preventing intracellular trypsinogen activation. J Clin Invest, 106, 81-9. https://doi.org/10.1172/JCI8706
  6. Bhagat L, Singh VP, Song AM, et al (2002). Thermal stress-induced HSP70 mediates protection against intra-pancreatic trypsinogen activation and acute pancreatitis in rats. Gastroenterol, 122, 156-65. https://doi.org/10.1053/gast.2002.30314
  7. Dudeja V, Mujumdar N, Phillips P, et al (2009). Heat shock protein 70 inhibits apoptosis in cancer cells through simultaneous and independent mechanisms. Gastroenterol, 136, 1772-82. https://doi.org/10.1053/j.gastro.2009.01.070
  8. Dutta A, Dutta SK, Nair PP (2000). Evidence for multimeric forms of heat shock protein 70 with varying degrees of phosphorylation on serine and tyrosine residues in human plasma. FASEB J, 14, 1488.
  9. Dutta SK, Girotra M, Singla M, et al (2012). Serum HSP70: a novel biomarker for early detection of pancreatic cancer. Pancreas, 41, 530-4. https://doi.org/10.1097/MPA.0b013e3182374ace
  10. Joly AL, Wettstein G, Mignot G, Ghiringhelli F, Garrido C (2010). Dual role of heat shock proteins as regulators of apoptosis and innate immunity. J Innate Immun, 2, 238-47. https://doi.org/10.1159/000296508
  11. Kaul G, Thippeswamy H (2011). Role of heat shock proteins in diseases and their therapeutic potential. Indian J Microbiol, 51, 124-31 . https://doi.org/10.1007/s12088-011-0147-9
  12. Krepuska M, Szeberin Z, Sotonyi P, et al (2010). Serum level of soluble Hsp70 is associated with vascular calcification. Cell Stress and Chaperones, 16, 257-65 .
  13. Kiang JG, Tsokos GC (1998). Heat shock protein 70 kDa: molecular biology, biochemistry, and physiology. Pharmacol Ther, 80, 183-201. https://doi.org/10.1016/S0163-7258(98)00028-X
  14. Kim D, Lee YJ, Corry PM (1992). Constitutive HSP70: oligomerization and its dependence on ATP binding. J Cellular Physiol, 153, 353-61. https://doi.org/10.1002/jcp.1041530215
  15. Kocsis J, Meszaros T, Madaras B, et al (2010). High levels of acute phase proteins and soluble 70 kD heat shock proteins are independent and additive risk factors for mortality in colorectal cancer. Cell Stress and Chaperones, 16, 49-55.
  16. Lim YP, Wong CY, Ooi LL, Druker BJ, Epstein RJ (2004). Selective tyrosine hyperphosphorylation of cytoskeletal and stress proteins in primary human breast cancers implications for adjuvant use of kinase-inhibitory drugs. Clin Cancer Res, 10, 3980-7. https://doi.org/10.1158/1078-0432.CCR-03-0663
  17. Liu RY, Corry PM, Lee YJ (1994). Regulation of chemical stress-induced HSP70 gene expression in murine L929 cells. J Cell Sci, 107, 2209-14.
  18. Matouschek A, Pfanner N, Voos W (2000). Protein unfolding by mitochondria. The Hsp70 import motor. EMBO Rep, 1, 404-10. https://doi.org/10.1093/embo-reports/kvd093
  19. Murphy ME (2013). The HSP70 family and cancer. Carcinogenesis, 34, 1181-8. https://doi.org/10.1093/carcin/bgt111
  20. Nollen EA, Morimoto RI (2002). Chaperoning signaling pathways: molecular chaperones as stress sensing 'heat shock' proteins. J Cell Sci, 115, 2809-16.
  21. Phillips PA, Dudeja V, McCarroll JA, et al (2007). Triptolide induces pancreatic cancer cell death via inhibition of heat shock protein70. Cancer Res, 67, 9407-16. https://doi.org/10.1158/0008-5472.CAN-07-1077
  22. Rea IM, McNerlan S, Pockley AG (2001). Serum heat shock protein and anti-heat shock protein antibody levels in aging. Exp Gerontol, 36, 341-52. https://doi.org/10.1016/S0531-5565(00)00215-1
  23. Pockley AG, Shepherd J, Corton JM (1998). Detection of heat shock protein70 (Hsp70) and anti-Hsp70 antibodies in serum of normal individuals. Immunol Invest, 27, 367-77. https://doi.org/10.3109/08820139809022710
  24. Rakonczay Z Jr, Takacs T, Boros I, Lonovics J (2003). Heat shock proteins and the pancreas. J Cell Physiol, 195, 383-91. https://doi.org/10.1002/jcp.10268
  25. Rappa F, Farina F, Zummo G, et al (2012). HSP-molecular chaperones in cancer biogenesis and tumor therapy: an overview. Anticancer Res, 32, 5139-50.
  26. Ritossa FM (1962). A new puffing pattern induced by temperature shock and DNP in drosophila. Experientia, 18, 571-3. https://doi.org/10.1007/BF02172188
  27. Robin MA, Prabu SK, Raza H, Anandatheerthavarada HK, Avadhani NG (2003). Phosphorylation enhances mitochondrial targeting of GSTA4-4 through increased affinity for binding to cytoplasmic Hsp70. J Biol Chem, 278, 18960-70. https://doi.org/10.1074/jbc.M301807200
  28. Shamaei-Tousi A, Halcox JP, Henderson B (2007). Stressing the obvious? Cell stress and cell stress proteins in cardiovascular disease. Cardiovasc Res, 74, 19-28 . https://doi.org/10.1016/j.cardiores.2006.10.025
  29. Sharp FR, Bernaudin M, Bartels M, Wagner KR (2001). Glial expression of heat shock proteins (HSPs) and oxygen-regulated proteins (ORPs). Prog Brain Res, 132, 427-40. https://doi.org/10.1016/S0079-6123(01)32093-9
  30. Sikora A, Grzesiuk E (2007). Heat shock response in gastrointestinal tract. J Physiol Pharmacol, 58, 43-62.
  31. Smith DF, Whitesell L, Katsanis E (1998). Molecular chaperones: biology and prospects for pharmacological intervention. Pharmacol Rev, 50, 493-514.
  32. Xie K, Huang S (2003). Regulation of cancer metastasis by stress pathways. Clin Exp Metastasis, 20, 31-43. https://doi.org/10.1023/A:1022590402748
  33. Soti C, Csermely P (2007). Protein stress and stress proteins: implications in aging and disease. J Biosci, 32, 511-5. https://doi.org/10.1007/s12038-007-0050-z
  34. Takashima M, Kuramitsu Y, Yokoyama Y, et al (2003). Proteomic profiling of heat shock protein 70 family members as biomarkers for hepatitis C virus-related hepatocellular carcinoma. Proteomics, 3, 2487-93. https://doi.org/10.1002/pmic.200300621
  35. Udono H, Srivastava PK (1993). Heat shock protein 70-associated peptides elicit specific cancer immunity. J Exp Med, 178, 1391-6. https://doi.org/10.1084/jem.178.4.1391