| 1. |
- Gielnik, Maciej, et al.
(author)
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Prion Protein Octarepeat Domain Forms Transient β-Sheet Structures upon Residue-Specific Binding to Cu(II) and Zn(II) Ions
- 2023
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In: Biochemistry. - 0006-2960 .- 1520-4995. ; 62:11, s. 1689-1705
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Journal article (peer-reviewed)abstract
- Misfolding of the cellular prion protein (PrPC) is associated with the development of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs). Metal ions appear to play a crucial role in PrPC misfolding. PrPC is a combined Cu(II) and Zn(II) metal-binding protein, where the main metal-binding site is located in the octarepeat (OR) region. Thus, the biological function of PrPC may involve the transport of divalent metal ions across membranes or buffering concentrations of divalent metal ions in the synaptic cleft. Recent studies have shown that an excess of Cu(II) ions can result in PrPC instability, oligomerization, and/or neuroinflammation. Here, we have used biophysical methods to characterize Cu(II) and Zn(II) binding to the isolated OR region of PrPC. Circular dichroism (CD) spectroscopy data suggest that the OR domain binds up to four Cu(II) ions or two Zn(II) ions. Binding of the first metal ion results in a structural transition from the polyproline II helix to the β-turn structure, while the binding of additional metal ions induces the formation of β-sheet structures. Fluorescence spectroscopy data indicate that the OR region can bind both Cu(II) and Zn(II) ions at neutral pH, but under acidic conditions, it binds only Cu(II) ions. Molecular dynamics simulations suggest that binding of either metal ion to the OR region results in the formation of β-hairpin structures. As the formation of β-sheet structures can be a first step toward amyloid formation, we propose that high concentrations of either Cu(II) or Zn(II) ions may have a pro-amyloid effect in TSE diseases.
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| 2. |
- Pollak, Joanna, et al.
(author)
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Production of Cystatin C Wild Type and Stabilized Mutants
- 2010
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In: EJIFCC. - 1650-3414. ; 20:4, s. 70-166
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Journal article (peer-reviewed)abstract
- Cystatin C is produced in all nucleated cells. It has various functions and biological activities. Researchers are focused on its role in kidney diseases as a marker of glomerular filtration but also as a very important link in development of amyloid diseases. This work describes expression and purification of both wild type (wt) and stabilized form (stab 1 and 2) of wt cystatin C and amyloid-forming L68Q mutant of cystatin C. The recombinant cystatin C can be used in projects requiring pure cystatin C to examine models of dimerization and fibrils formation as well as a standard in clinical tests.
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| 3. |
- Spodzieja, Marta, et al.
(author)
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Interaction of serum amyloid A with human cystatin Cuidentification of binding sites
- 2012
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In: Journal of Molecular Recognition. - : Wiley. - 1099-1352 .- 0952-3499. ; 25:10, s. 513-524
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Journal article (peer-reviewed)abstract
- Serum amyloid A (SAA) is a multifunctional acute-phase protein whose natural role seems to be participation in many physiologic and pathological processes. Prolonged increased SAA level in a number of chronic inflammatory and neoplastic diseases gives rise to reactive systemic amyloid A amyloidosis, where the N-terminal 76-amino acid residue-long segment of SAA is deposited as amyloid fibrils. Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteaseshuman cystatin C (hCC)has been described. Here, we report further evidence corroborating this interaction, and the identification of the SAA and hCC binding sites in the SAAhCC complex, using a combination of selective proteolytic excision and high-resolution mass spectrometry. The shortest binding site in the SAA sequence was determined as SAA(86104), whereas the binding site in hCC sequence was identified as hCC(96102). Binding specificities of both interacting sequences were ascertained by affinity experiments (ELISA) and by registration of mass spectrum of SAAhCC complex. Copyright (c) 2012 John Wiley & Sons, Ltd.
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| 4. |
- Szymańska, Aneta, et al.
(author)
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Governing the monomer-dimer ratio of human cystatin C by single amino acid substitution in the hinge region
- 2009
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In: Acta Biochimica Polonica. - 0001-527X. ; 56:3, s. 455-463
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Journal article (peer-reviewed)abstract
- Three dimensional domain swapping is one of the mechanisms involved in formation of insoluble aggregates of some amyloidogenic proteins. It has been proposed that proteins able to swap domains may share some common structural elements like conformationally constrained flexible turns/loops. We studied the role of loop L1 in the dimerization of human cystatin C using mutational analysis. Introduction of turn-favoring residues such as Asp or Asn into the loop sequence (in position 57) leads to a significant reduction of the dimer fraction in comparison with the wild type protein. On the other hand, introduction of a proline residue in position 57 leads to efficient dimer formation. Our results confirm the important role of the loop L1 in the dimerization process of human cystatin C and show that this process can be to some extent governed by single amino acid substitution.
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| 5. |
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