Skip to main content

Advertisement

SpringerLink
Log in

Anchorless 23–230 PrPC Interactomics for Elucidation of PrPC Protective Role

  • Published:
Molecular Neurobiology Aims and scope Submit manuscript

Abstract

Accumulation of conformationally altered cellular proteins (i.e., prion protein) is the common feature of prions and other neurodegenerative diseases. Previous studies demonstrated that the lack of terminal sequence of cellular prion protein (PrPC), necessary for the addition of glycosylphosphatidylinositol lipid anchor, leads to a protease-resistant conformation that resembles scrapie-associated isoform of prion protein. Moreover, mice overexpressing the truncated form of PrPC showed late-onset, amyloid deposition, and the presence of a short protease-resistant PrP fragment in the brain similar to those found in Gerstmann–Sträussler–Scheinker disease patients. Therefore, the physiopathological function of truncated_/anchorless 23–230 PrPC (Δ23–230 PrPC) has come into focus of attention. The present study aims at revealing the physiopathological function of the anchorless PrPC form by identifying its interacting proteins. The truncated_/anchorless Δ23–230 PrPC along with its interacting proteins was affinity purified using STrEP-Tactin chromatography, in-gel digested, and identified by quadrupole time-of-flight tandem mass spectrometry analysis in prion protein-deficient murine hippocampus (HpL3-4) neuronal cell line. Twenty-three proteins appeared to interact with anchorless Δ23–230 PrPC in HpL3-4 cells. Out of the 23 proteins, one novel protein, pyruvate kinase isozymes M1/M2 (PKM2), exhibited a potential interaction with the anchorless Δ23–230 form of PrPC. Both reverse co-immunoprecipitation and confocal laser-scanning microscopic analysis confirmed an interaction of PKM2 with the anchorless Δ23–230 form of PrPC. Furthermore, we provide the first evidence for co-localization of PKM2 and PrPC as well as PrPC-dependent PKM2 expression regulation. In addition, given the involvement of PrPC in the regulation of apoptosis, we exposed HpL3-4 cells to staurosporine (STS)-mediated apoptotic stress. In response to STS-mediated apoptotic stress, HpL3-4 cells transiently expressing 23–230-truncated PrPC were markedly less viable, were more prone to apoptosis and exhibited significantly higher PKM2 expressional regulation as compared with HpL3-4 cells transiently expressing full-length PrPC (1–253 PrPC). The enhanced STS-induced apoptosis was shown by increased caspase-3 cleavage. Together, our data suggest that the misbalance or over expression of anchorless Δ23–230 form of PrPC in association with the expressional regulation of interacting proteins could render cells more prone to cellular insults-stress response, formation of aggregates and may ultimately be linked to the cell death.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Telling G (2005) Anchors away–of plaques and pathology in prion disease. N Engl J Med 353(11):1177–1179

    Article  CAS  PubMed  Google Scholar 

  2. Hsiao KK, Groth D, Scott M, Yang SL, Serban H, Rapp D, Foster D, Torchia M, DeArmond SJ, Prusiner SB (1994) Serial transmission in rodents of neurodegeneration from transgenic mice expressing mutant prion protein. Proc Natl Acad Sci U S A 91(19):9126–9130

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Solforosi L, Criado JR, McGavern DB, Wirz S, Sanchez-Alavez M, Sugama S, DeGiorgio LA, Volpe BT, Wiseman E, Abalos G, Masliah E, Gilden D, Oldstone MB, Conti B, Williamson RA (2004) Cross-linking cellular prion protein triggers neuronal apoptosis in vivo. Science 303(5663):1514–1516

    Article  CAS  PubMed  Google Scholar 

  4. Chesebro B, Trifilo M, Race R, Meade-White K, Teng C, Lacasse R, Raymond L, Favara C, Baron G, Priola S, Caughey B, Masliah E, Oldstone M (2005) Anchorless prion protein results in infectious amyloid disease without clinical scrapie. Science 308(5727):1435–1439

    Article  CAS  PubMed  Google Scholar 

  5. Hornemann S, Korth C, Oesch B, Riek R, Wider G, Wuthrich K, Glockshuber R (1997) Recombinant full-length murine prion protein, mPrP(23–231): purification and spectroscopic characterization. FEBS Lett 413(2):277–281

    Article  CAS  PubMed  Google Scholar 

  6. Linden R, Martins VR, Prado MA, Cammarota M, Izquierdo I, Brentani RR (2008) Physiology of the prion protein. Physiol Rev 88(2):673–728

    Article  CAS  PubMed  Google Scholar 

  7. Riek R, Hornemann S, Wider G, Billeter M, Glockshuber R, Wuthrich K (1996) NMR structure of the mouse prion protein domain PrP(121–231). Nature 382(6587):180–182

    Article  CAS  PubMed  Google Scholar 

  8. Harris DA (1999) Cellular biology of prion diseases. Clin Microbiol Rev 12(3):429–444

    CAS  PubMed Central  PubMed  Google Scholar 

  9. Hegde RS, Voigt S, Lingappa VR (1998) Regulation of protein topology by trans-acting factors at the endoplasmic reticulum. Mol Cell 2(1):85–91

    Article  CAS  PubMed  Google Scholar 

  10. Westergard L, Christensen HM, Harris DA (2007) The cellular prion protein (PrP(C)): its physiological function and role in disease. Biochim Biophys Acta 1772(6):629–644

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Stahl N, Borchelt DR, Hsiao K, Prusiner SB (1987) Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell 51(2):229–240

    Article  CAS  PubMed  Google Scholar 

  12. Taraboulos A, Raeber AJ, Borchelt DR, Serban D, Prusiner SB (1992) Synthesis and trafficking of prion proteins in cultured cells. Mol Biol Cell 3(8):851–863

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Laine J, Marc ME, Sy MS, Axelrad H (2001) Cellular and subcellular morphological localization of normal prion protein in rodent cerebellum. Eur J Neurosci 14(1):47–56

    Article  CAS  PubMed  Google Scholar 

  14. Shyng SL, Heuser JE, Harris DA (1994) A glycolipid-anchored prion protein is endocytosed via clathrin-coated pits. J Cell Biol 125(6):1239–1250

    Article  CAS  PubMed  Google Scholar 

  15. Shyng SL, Moulder KL, Lesko A, Harris DA (1995) The N-terminal domain of a glycolipid-anchored prion protein is essential for its endocytosis via clathrin-coated pits. J Biol Chem 270(24):14793–14800

    Article  CAS  PubMed  Google Scholar 

  16. Caughey B, Raymond GJ, Ernst D, Race RE (1991) N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state. J Virol 65(12):6597–6603

    CAS  PubMed Central  PubMed  Google Scholar 

  17. Prusiner SB (1998) Prions. Proc Natl Acad Sci U S A 95(23):13363–13383

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Borchelt DR, Taraboulos A, Prusiner SB (1992) Evidence for synthesis of scrapie prion proteins in the endocytic pathway. J Biol Chem 267(23):16188–16199

    CAS  PubMed  Google Scholar 

  19. Bertuchi FR, Bourgeon DM, Landemberger MC, Martins VR, Cerchiaro G (2012) PrPC displays an essential protective role from oxidative stress in an astrocyte cell line derived from PrPC knockout mice. Biochem Biophys Res Commun 418(1):27–32

    Article  CAS  PubMed  Google Scholar 

  20. Sakudo A, Nakamura I, Tsuji S, Ikuta K (2008) GPI-anchorless human prion protein is secreted and glycosylated but lacks superoxide dismutase activity. Int J Mol Med 21(2):217–222

    CAS  PubMed  Google Scholar 

  21. Sakudo A, Onodera T, Suganuma Y, Kobayashi T, Saeki K, Ikuta K (2006) Recent advances in clarifying prion protein functions using knockout mice and derived cell lines. Mini Rev Med Chem 6(5):589–601

    Article  CAS  PubMed  Google Scholar 

  22. Elmallah MI, Borgmeyer U, Betzel C, Redecke L (2013) Impact of methionine oxidation as an initial event on the pathway of human prion protein conversion. Prion 7(5)

  23. Llorens F, Del Rio JA (2012) Unraveling the neuroprotective mechanisms of PrP(C) in excitotoxicity. Prion 6(3):245–251

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Suzuki G, Tanaka M (2013) Active conversion to the prion state as a molecular switch for cellular adaptation to environmental stress. Bioessays 35(1):12–16

    Article  CAS  PubMed  Google Scholar 

  25. Yuan F, Yang L, Zhang Z, Wu W, Zhou X, Yin X, Zhao D (2013) Cellular prion protein (PrPC) of the neuron cell transformed to a PK-resistant protein under oxidative stress, comprising main mitochondrial damage in prion diseases. J Mol Neurosci 51(1):219–224

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Chatterjee B, Lee CY, Lin C, Chen EH, Huang CL, Yang CC, Chen RP (2013) Amyloid core formed of full-length recombinant mouse prion protein involves sequence 127–143 but not sequence 107–126. PLoS One 8(7):e67967

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Sakudo A, Lee DC, Saeki K, Nakamura Y, Inoue K, Matsumoto Y, Itohara S, Onodera T (2003) Impairment of superoxide dismutase activation by N-terminally truncated prion protein (PrP) in PrP-deficient neuronal cell line. Biochem Biophys Res Commun 308(3):660–667

    Article  CAS  PubMed  Google Scholar 

  28. Zafar S, von Ahsen N, Oellerich M, Zerr I, Schulz-Schaeffer WJ, Armstrong VW, Asif AR (2011) Proteomics approach to identify the interacting partners of cellular prion protein and characterization of Rab7a interaction in neuronal cells. J Proteome Res 10:3123–3135

    Google Scholar 

  29. Wessel D, Flugge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138(1):141–143

    Article  CAS  PubMed  Google Scholar 

  30. Asif AR, Armstrong VW, Voland A, Wieland E, Oellerich M, Shipkova M (2007) Proteins identified as targets of the acyl glucuronide metabolite of mycophenolic acid in kidney tissue from mycophenolate mofetil treated rats. Biochimie 89(3):393–402

    Article  CAS  PubMed  Google Scholar 

  31. Cory AH, Owen TC, Barltrop JA, Cory JG (1991) Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun 3(7):207–212

    CAS  PubMed  Google Scholar 

  32. Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35(4):495–516

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Goel R, Muthusamy B, Pandey A, Prasad TS (2011) Human protein reference database and human proteinpedia as discovery resources for molecular biotechnology. Mol Biotechnol 48(1):87–95

    Article  CAS  PubMed  Google Scholar 

  34. Keller A, Nesvizhskii AI, Kolker E, Aebersold R (2002) Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 74(20):5383–5392

    Article  CAS  PubMed  Google Scholar 

  35. Nesvizhskii AI, Keller A, Kolker E, Aebersold R (2003) A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 75(17):4646–4658

    Article  CAS  PubMed  Google Scholar 

  36. Gupta V, Bamezai RN (2010) Human pyruvate kinase M2: a multifunctional protein. Protein Sci 19(11):2031–2044

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. Gavrilovic M, Wahlby C (2009) Quantification of colocalization and cross-talk based on spectral angles. J Microsc 234(3):311–324

    Article  CAS  PubMed  Google Scholar 

  38. Wu G, Nakajima K, Takeyama N, Yukawa M, Taniuchi Y, Sakudo A, Onodera T (2008) Species-specific anti-apoptotic activity of cellular prion protein in a mouse PrP-deficient neuronal cell line transfected with mouse, hamster, and bovine Prnp. Neurosci Lett 446(1):11–15

    Article  CAS  PubMed  Google Scholar 

  39. Ma J, Wollmann R, Lindquist S (2002) Neurotoxicity and neurodegeneration when PrP accumulates in the cytosol. Science 298(5599):1781–1785

    Article  CAS  PubMed  Google Scholar 

  40. Fioriti L, Dossena S, Stewart LR, Stewart RS, Harris DA, Forloni G, Chiesa R (2005) Cytosolic prion protein (PrP) is not toxic in N2a cells and primary neurons expressing pathogenic PrP mutations. J Biol Chem 280(12):11320–11328

    Article  CAS  PubMed  Google Scholar 

  41. Junttila MR, Saarinen S, Schmidt T, Kast J, Westermarck J (2005) Single-step STrEP-tag purification for the isolation and identification of protein complexes from mammalian cells. Proteomics 5(5):1199–1203

    Article  CAS  PubMed  Google Scholar 

  42. Satoh J, Onoue H, Arima K, Yamamura T (2005) The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein. J Neuropathol Exp Neurol 64(10):858–868

    Article  CAS  PubMed  Google Scholar 

  43. Jang B, Kim E, Choi JK, Jin JK, Kim JI, Ishigami A, Maruyama N, Carp RI, Kim YS, Choi EK (2008) Accumulation of citrullinated proteins by up-regulated peptidylarginine deiminase 2 in brains of scrapie-infected mice: a possible role in pathogenesis. Am J Pathol 173(4):1129–1142

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  44. Giorgi A, Di FL, Principe S, Mignogna G, Sennels L, Mancone C, Alonzi T, Sbriccoli M, De PA, Rappsilber J, Cardone F, Pocchiari M, Maras B, Schinina ME (2009) Proteomic profiling of PrP27-30-enriched preparations extracted from the brain of hamsters with experimental scrapie. Proteomics 9(15):3802–3814

    Article  CAS  PubMed  Google Scholar 

  45. Oakley AJ (2005) Glutathione transferases: new functions. Curr Opin Struct Biol 15(6):716–723

    Article  CAS  PubMed  Google Scholar 

  46. Weiss E, Ramljak S, Asif AR, Ciesielczyk B, Schmitz M, Gawinecka J, Schulz-Schaeffer W, Behrens C, Zerr I (2010) Cellular prion protein overexpression disturbs cellular homeostasis in SH-SY5Y neuroblastoma cells but does not alter p53 expression: a proteomic study. Neuroscience 169(4):1640–1650

    Article  CAS  PubMed  Google Scholar 

  47. Liu GP, Wei W, Zhou X, Zhang Y, Shi HH, Yin J, Yao XQ, Peng CX, Hu J, Wang Q, Li HL, Wang JZ (2012) I(2)(PP2A) regulates p53 and Akt correlatively and leads the neurons to abort apoptosis. Neurobiol Aging 33:254–264

    Article  PubMed  Google Scholar 

  48. Spisni E, Valerii MC, Manerba M, Strillacci A, Polazzi E, Mattia T, Griffoni C, Tomasi V (2009) Effect of copper on extracellular levels of key pro-inflammatory molecules in hypothalamic GN11 and primary neurons. Neurotoxicology 30(4):605–612

    Article  CAS  PubMed  Google Scholar 

  49. Ramljak S, Asif AR, Armstrong VW, Wrede A, Groschup MH, Buschmann A, Schulz-Schaeffer W, Bodemer W, Zerr I (2008) Physiological role of the cellular prion protein (PrPc): protein profiling study in two cell culture systems. J Proteome Res 7(7):2681–2695

    Article  CAS  PubMed  Google Scholar 

  50. Gawinecka J, Dieks J, Asif AR, Carimalo J, Heinemann U, Streich JH, Dihazi H, Schulz-Schaeffer W, Zerr I (2010) Codon 129 polymorphism specific cerebrospinal fluid proteome pattern in sporadic Creutzfeldt–Jakob disease and the implication of glycolytic enzymes in prion-induced pathology. J Proteome Res 9(11):5646–5657

    Article  CAS  PubMed  Google Scholar 

  51. Kayne FJ, Price NC (1973) Amino acid effector binding to rabbit muscle pyruvate kinase. Arch Biochem Biophys 159(1):292–296

    Article  CAS  PubMed  Google Scholar 

  52. Valentini G, Chiarelli L, Fortin R, Speranza ML, Galizzi A, Mattevi A (2000) The allosteric regulation of pyruvate kinase. J Biol Chem 275(24):18145–18152

    Article  CAS  PubMed  Google Scholar 

  53. Spoden GA, Rostek U, Lechner S, Mitterberger M, Mazurek S, Zwerschke W (2009) Pyruvate kinase isoenzyme M2 is a glycolytic sensor differentially regulating cell proliferation, cell size and apoptotic cell death dependent on glucose supply. Exp Cell Res 315(16):2765–2774

    Article  CAS  PubMed  Google Scholar 

  54. Yamada K, Noguchi T (1999) Regulation of pyruvate kinase M gene expression. Biochem Biophys Res Commun 256(2):257–262

    Article  CAS  PubMed  Google Scholar 

  55. Yamada K, Noguchi T, Matsuda T, Takenaka M, Monaci P, Nicosia A, Tanaka T (1990) Identification and characterization of hepatocyte-specific regulatory regions of the rat pyruvate kinase L gene. The synergistic effect of multiple elements. J Biol Chem 265(32):19885–19891

    CAS  PubMed  Google Scholar 

  56. van Veelen CW, Staal GE, Verbiest H, Vlug AM (1977) Alanine inhibition of pyruvate kinase in gliomas and meningiomas. A diagnostic tool in surgery for gliomas? Lancet 2(8034):384–385

    Article  PubMed  Google Scholar 

  57. van Veelen CW, Verbiest H, Zulch KJ, van Ketel B, van der Vlist MJ, Vlug AM, Rijksen G, Staal GE (1980) Pyruvate kinase as a marker of human brain tumors. Ned Tijdschr Geneeskd 124(40):1678–1685

    PubMed  Google Scholar 

  58. Noguchi T, Inoue H, Tanaka T (1986) The M1- and M2-type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing. J Biol Chem 261(29):13807–13812

    CAS  PubMed  Google Scholar 

  59. Lee J, Kim HK, Han YM, Kim J (2008) Pyruvate kinase isozyme type M2 (PKM2) interacts and cooperates with Oct-4 in regulating transcription. Int J Biochem Cell Biol 40(5):1043–1054

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This study was supported by a European Commission Grant (Priority-222887). We are indebted to Mrs. Christina Wiese for technical assistance at various stages of this investigation. Special thanks to Prof. Victor W. Armstrong (deceased in 2010), Prof. Michael Oellerich, PD Walter J. Schulz-Schaeffer, and Dr. Joachim Bertram (IBA, Goettingen) for their support.

Disclosure Statement

Authors declared no actual or potential conflicts of interest.

Author information

Authors and Affiliations

  1. Department of Neurology, University Medical Center Göttingen (UMG), Robert-Koch-Str. 40, 37075, Göttingen, Germany

    Saima Zafar, Sanja Ramljak, Waqas Tahir, Matthias Schmitz & Inga Zerr

  2. Department of Clinical Chemistry, University Medical Center Göttingen (UMG), Robert-Koch-Str. 40, 37075, Göttingen, Germany

    Saima Zafar & Abdul R. Asif

  3. Institute for Clinical Research and Development (ikfe) Services, Mainz, Germany

    Sanja Ramljak

Authors
  1. Saima Zafar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Abdul R. Asif
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sanja Ramljak
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Waqas Tahir
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Matthias Schmitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Inga Zerr
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Saima Zafar.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary Fig. 1

(JPEG 20 kb)

High resolution image (TIFF 2825 kb)

Supplementary Table 1

(DOCX 85 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zafar, S., Asif, A.R., Ramljak, S. et al. Anchorless 23–230 PrPC Interactomics for Elucidation of PrPC Protective Role. Mol Neurobiol 49, 1385–1399 (2014). https://doi.org/10.1007/s12035-013-8616-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12035-013-8616-2

Keywords

Search

Navigation