Skip to main content
SpringerLink
Log in

Conformational changes during amyloid fibril formation of pancreatic thiol proteinase inhibitor: effect of copper and zinc

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Pancreatic thiol proteinase inhibitor (PTPI), a variant of cystatin superfamily of cysteine protease inhibitors, has been isolated from pancreas of Capra hircus. In the present study, we examined the effects of acid denaturation and a co-solvent on PTPI with a focus on protein conformational changes and amyloid fibril formation. The results demonstrate that PTPI can form amyloid like fibrils. Acid denaturation as studied by CD and fluorescence spectroscopy showed that PTPI populates three partly unfolded species, a native like state at pH 3.0, a structured molten globule at pH 1.0 and partly unfolded species at pH 2.0, from each of which amyloid like fibrils grow as assessed by Thioflavin T (ThT) spectroscopy. Effect of trifluoroethanol (TFE) on acid induced states of PTPI was analyzed. TFE stabilized each of the three acid-induced intermediates at predenaturational concentrations (10%) and accelerated fibril formation. Morphology of the protein species at the beginning and end of reactions was observed using transmission electron microscopy. Solvent conditions were decisive for final fibril morphology. Biometals, Cu2+ and Zn2+ produced a concentration dependent decline in ThT fluorescence suggesting deaggregation of the fibrils. When added prior to amyloid fibril initiation 50 μM Cu2+ or 10 μM Zn2+ prevented any amyloid aggregation. Implications for therapeutics in view of Cu2+ and Zn2+ as essential micronutrients are suggested.

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
Fig. 8
Scheme 1

Similar content being viewed by others

References

  1. Rashid F, Sharma S, Baig MA, Bano B (2006) Molten globule state of human placental cystatin (HPC) at low pH conditions and the effects of trifluoroethanol (TFE) and methanol. Biochem Cell Biol 84:126–134

    Article  PubMed  CAS  Google Scholar 

  2. Ahmad B, Khan RH (2006) Studies on the acid unfolded and molten globule states of catalytically active stem bromelain: a comparison with catalytically inactive form. J Biochem 140:501–508

    Article  PubMed  CAS  Google Scholar 

  3. Zerovnik E, Jerala R, Kroon-Zitko L et al (1992) Intermediates in denaturation of a small globular protein, recombinant human stefin B. J Biol Chem 267:9041–9046

    PubMed  CAS  Google Scholar 

  4. Luo P, Baldwin RL (1997) Mechanism of helix induction by trifluoroethanol: a framework for extrapolating the helix-forming properties of peptides from trifluoroethanol/water mixtures back to water. Biochemistry 36:8413–8421

    Article  PubMed  CAS  Google Scholar 

  5. Khurana R, Ionescu-Zanetti C, Pope M et al (2003) A general model for amyloid fibril assembly based on morphological studies using atomic force microscopy. Biophys J 85:1135–1144

    Article  PubMed  CAS  Google Scholar 

  6. Chiti F, Stefani M, Taddei N et al (2003) Rationalization of the effects of mutations on peptide and protein aggregation rates. Nature 424:805–808

    Article  PubMed  CAS  Google Scholar 

  7. Williams AD, Portelius E, Kheterpal I et al (2004) Mapping a beta amyloid fibril secondary structure using scanning proline mutagenesis. J Mol Biol 335:833–842

    Article  PubMed  CAS  Google Scholar 

  8. Priyadarshini M, Bano B (2010) Cystatin like thiol proteinase inhibitor from pancreas of Capra hircus: purification and detailed biochemical characterization. Amino Acids 38(4):1001–1010

    Article  PubMed  CAS  Google Scholar 

  9. Turk V, Stoka V, Turk D (2008) Cystatins: biochemical and structural properties, and medical relevance. Front Biosci 13:5406–5420

    Article  PubMed  CAS  Google Scholar 

  10. Skerget K, Vilfan A, Pompe-Novak M et al (2009) The mechanism of amyloid-fibril formation by stefin B: temperature and protein concentration dependence of the rates. Proteins 74:425–436

    Article  PubMed  CAS  Google Scholar 

  11. Zerovnik E, Skerget K, Tusek-Znidaric M et al (2006) High affinity copper binding by stefin B (cystatin B) and its role in the inhibition of amyloid fibrillation. FEBS J 273:4250–4263

    Article  PubMed  CAS  Google Scholar 

  12. Lowry OH, Rosebrough NJ, Farr AL et al (1951) Protein determination with the Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  13. Mulqueen PM, Kronamn MJ (1982) Binding of naphthalene dye to N and A conformers of bovine α-lactalbumin. Arch Biochem Biophys 215:28–39

    Article  PubMed  CAS  Google Scholar 

  14. Jenko S, Skarabot M, Kenig M et al (2004) Different propensity to form amyloid fibrils by two homologous proteins-human stefins A and B: searching for an explanation. Proteins 55:417–425

    Article  PubMed  CAS  Google Scholar 

  15. Lai Z, Colón W, Kelly JW (1996) The acid-mediated denaturation pathway of transthyretin yields a conformational intermediate that can self-assemble into amyloid. Biochemistry 35:6470–6482

    Article  PubMed  CAS  Google Scholar 

  16. Chiti F, Webster P, Taddei N et al (1999) Designing conditions for in vitro formation of amyloid protofilaments and fibrils. Proc Natl Acad Sci USA 96:3590–3594

    Article  PubMed  CAS  Google Scholar 

  17. Rabzelj S, Turk V, Zerovnik E (2005) In vitro study of stability and amyloid-fibril formation of two mutants of human stefin B (cystatin B) occurring in patients with EPM1. Protein Sci 14:2713–2722

    Article  PubMed  CAS  Google Scholar 

  18. Haq SK, Rasheedi S, Khan RH (2002) Characterization of a partially folded intermediate of stem bromelain at low pH. Eur J Biochem 269:47–52

    Article  PubMed  CAS  Google Scholar 

  19. Gupta P, Khan RH, Saleemuddin M (2003) Trifluoroethanol-induced “molten globule” state in stem bromelain. Arch Biochem Biophys 413:199–206

    Article  PubMed  CAS  Google Scholar 

  20. Artigues A, Iriarte A, Martinez-Carrion M (1994) Acid-induced reversible unfolding of mitochondrial aspartate aminotransferase. J Biol Chem 269:21990–21999

    PubMed  CAS  Google Scholar 

  21. LeVine H 3rd (1999) Quantification of beta-sheet amyloid fibril structures with thioflavin T. Methods Enzymol 309:274–284

    Article  PubMed  CAS  Google Scholar 

  22. Harper JD, Lansbury PT Jr (1997) Models of amyloid seeding in Alzheimer’s disease and scrapie: mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins. Annu Rev Biochem 66:385–407

    Article  PubMed  CAS  Google Scholar 

  23. Holm NK, Jespersen SK, Thomassen LV et al (2007) Aggregation and fibrillation of bovine serum albumin. Biochim Biophys Acta 1774:1128–1138

    PubMed  CAS  Google Scholar 

  24. Buck M, Radford SE, Dobson CM (1993) A partially folded state of hen egg white lysozyme in trifluoroethanol: structural characterization and implications for protein folding. Biochemistry 32:669–678

    Article  PubMed  CAS  Google Scholar 

  25. Zerovnik E, Skarabot M, Skerget K et al (2007) Amyloid fibril formation by human stefin B: influence of pH and TFE on fibril growth and morphology. Amyloid 14:237–247

    Article  PubMed  CAS  Google Scholar 

  26. Ahmad A, Uversky VN, Hong D et al (2005) Early events in the fibrillation of monomeric insulin. J Biol Chem 280:42669–42675

    Article  PubMed  CAS  Google Scholar 

  27. He Y, Zhou H, Tang H et al (2006) Deficiency of disulfide bonds facilitating fibrillogenesis of endostatin. J Biol Chem 281:1048–1057

    Article  PubMed  CAS  Google Scholar 

  28. Zerovnik E, Pompe-Novak M, Skarabot M et al (2002) Human stefin B readily forms amyloid fibrils in vitro. Biochim Biophys Acta 1594:1–5

    Article  PubMed  CAS  Google Scholar 

  29. Bhakuni V (1998) Alcohol-induced molten globule intermediates of proteins: are they real folding intermediates or off pathway products? Arch Biochem Biophys 357:274–284

    Article  PubMed  CAS  Google Scholar 

  30. Chiti F, Bucciantini M, Capanni C et al (2001) Solution conditions can promote formation of either amyloid protofilaments or mature fibrils from the HypF N-terminal domain. Protein Sci 10:2541–2547

    Article  PubMed  CAS  Google Scholar 

  31. Fändrich M, Dobson CM (2002) The behaviour of polyamino acids reveals an inverse side chain effect in amyloid structure formation. EMBO J 21:5682–5690

    Article  PubMed  Google Scholar 

  32. Grudzielanek S, Smirnovas V, Winter R (2006) Solvation-assisted pressure tuning of insulin fibrillation: from novel aggregation pathways to biotechnological applications. J Mol Biol 356:497–509

    Article  PubMed  CAS  Google Scholar 

  33. Hong DP, Gozu M, Hasegawa K et al (2002) Conformation of beta2-microglobulin amyloid fibrils analyzed by reduction of the disulfide bond. J Biol Chem 277:21554–21560

    Article  PubMed  CAS  Google Scholar 

  34. Findeis MA (2000) Approaches to discovery and characterization of inhibitors of amyloid b-peptide polymerization. Biochim Biophys Acta 1502:76–84

    PubMed  CAS  Google Scholar 

  35. Rivière C, Richard T, Vitrac X et al (2008) New polyphenols active on beta-amyloid aggregation. Bioorg Med Chem Lett 18:828–831

    Article  PubMed  Google Scholar 

  36. Ward B, Walker K, Exley C (2008) Copper (II) inhibits the formation of amylin amyloid in vitro. J Inorg Biochem 102:371–375

    Article  PubMed  CAS  Google Scholar 

  37. Tõugu V, Karafin A, Zovo K, Chung RS, Howells C, West AK, Palumaa P (2009) Zn(II)- and Cu(II)-induced non-fibrillar aggregates of amyloid-beta (1–42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators. J Neurochem 110:1784–1795

    Article  PubMed  Google Scholar 

  38. Cherny RA, Legg JT, McLean CA, Fairlie DP, Huang X, Atwood CS, Beyreuther K, Tanzi RE, Masters CL, Bush AI (1999) Aqueous dissolution of Alzheimer’s disease Ab amyloid deposits by biometal depletion. J Biol Chem 274:23223–23228

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

Financial support to Medha Priyadarshini as a Senior Research Fellow of the Council of Scientific and Industrial Research (CSIR), New Delhi, India is acknowledged. We are grateful to SAP-DRS and UGC-FIST programmes for their generous research support.

Author information

Authors and Affiliations

  1. Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202002, UP, India

    Medha Priyadarshini & Bilqees Bano

Authors
  1. Medha Priyadarshini
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bilqees Bano
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bilqees Bano.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Priyadarshini, M., Bano, B. Conformational changes during amyloid fibril formation of pancreatic thiol proteinase inhibitor: effect of copper and zinc. Mol Biol Rep 39, 2945–2955 (2012). https://doi.org/10.1007/s11033-011-1056-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-011-1056-z

Keywords

Search

Navigation