liu.seSearch for publications in DiVA
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • oxford
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Early events in disease associated protein misfolding
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The scope of this thesis is to unravel some of the mysteries concerning events takingplace early in the amyloid cascade. In vitro studies of early misfolded states ofamyloidogenic proteins are important since the use of recombinant proteins allow us to monitor slight changes in environmental conditions as well as in amino acid composition and thereby illuminate the problem at near atomic resolution.

Human prion protein (HuPrP) (associated with e.g. Creutzfeldt-Jakob disease) andthe Aβ1-42 peptide (associated with Alzheimer’s disease) recombinantly expressed in Escherichia coli have been used as model systems for these studies.

A new protocol for amyloid fibril formation of human prion protein under native conditions was developed. This revealed an unusual pathway of conformational conversion from early formed disordered aggregates that later matured into amyloidfibrils.

The polymorphism 129M/V in HuPrP has a large impact on susceptibility both to sporadic and infectious prion diseases. Some features of this polymorphism havebeen elucidated, employing a mutational study in position 129 (M, A, L, V, P, M, W,E, and K). These investigations have rendered new knowledge about the impact ofsize, charge and β-carbon branching in position 129 upon early intermolecular interactions and the effects of fibril seeding.

Investigations of the interactions between different assembly forms of HuPrP and components of the innate immune system revealed that both native, oligomeric and fibrillar forms of HuPrP activate both the classical and alternative pathways of the Complement System. Most efficient activation is achieved upon binding of oligomeric HuPrP to the complement component C1q.

We have developed a system for recombinant expression of human A,1-42. The monomeric peptides are assembled into various sized soluble oligomers (trimer, hexamer, nonamer, dodecamer). The oligomeric forms were stable in 8 M urea, 6 MGuHCl and SDS suggesting that these were covalently cross-linked. Some mechanistic features in the assembly process have been investigated and we have shown that cupric ions facilitates formation of stable oligomers in our system.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2009. , 116 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1270
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:liu:diva-52743ISBN: 978-91-7393-544-9 (print)OAI: oai:DiVA.org:liu-52743DiVA: diva2:285459
Public defence
2009-11-20, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 10:15 (English)
Opponent
Supervisors
Available from: 2010-01-18 Created: 2010-01-12 Last updated: 2012-11-15Bibliographically approved
List of papers
1. Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates
Open this publication in new window or tab >>Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates
2009 (English)In: Prion, ISSN 1933-6896, Vol. 3, no 4, 224-235 p.Article in journal (Refereed) Published
Abstract [en]

Propagation and infectivity of prions in human prionopathies are likely associated with conversion of the mainly α-helical human prion protein, HuPrP, into an aggregated form with amyloid-like properties. Previous reports on efficient conversion of recombinant HuPrP have used mild to harsh denaturing conditions to generate amyloid fibrils in vitro. Herein we report on the in vitro conversion of four forms of truncated HuPrP (sequences 90-231 and 121-231 with and without an N-terminal hexa histidine tag) into amyloid-like fibrils within a few hours by using a protocol (phosphate buffered saline solutions at neutral pH with intense agitation) close to physiological conditions. The conversion process monitored by thioflavin T, ThT, revealed a three stage process with lag, growth and equilibrium phases. Seeding with preformed fibrils shortened the lag phase demonstrating the classic nucleated polymerization mechanism for the reaction. Interestingly, comparing thioflavin T kinetics with solubility and turbidity kinetics it was found that the protein initially formed non-thioflavionophilic, morphologically disordered aggregates that over time matured into amyloid fibrils. By transmission electron microscopy and by fluorescence microscopy of aggregates stained with luminescent conjugated polythiophenes (LCPs); we demonstrated that HuPrP undergoes a conformational conversion where spun and woven fibrils protruded from morphologically disordered aggregates. The initial aggregation functioned as a kinetic trap that decelerated nucleation into a fibrillation competent nucleus, but at the same time without aggregation there was no onset of amyloid fibril formation. The agitation, which was necessary for fibril formation to be induced, transiently exposes the protein to the air-water interface suggests a hitherto largely unexplored denaturing environment for prion conversion.

Place, publisher, year, edition, pages
Austin: Landes Bioscience Journals, 2009
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-21064 (URN)10.4161/pri.3.4.10112 (DOI)000280061100009 ()
Available from: 2009-09-28 Created: 2009-09-28 Last updated: 2014-09-09
2. Multiple substitutions of methionine 129 in human prion protein reveal its importance in the amyloid fibrillation pathway
Open this publication in new window or tab >>Multiple substitutions of methionine 129 in human prion protein reveal its importance in the amyloid fibrillation pathway
2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 31, 25975-25984 p.Article in journal (Refereed) Published
Abstract [en]

The role of the polymorphism Met or Val in position 129 in the human prion protein is well documented regarding disease susceptibility and clinical manifestations. However, little is known about the molecular background to this phenomenon. We investigated herein the conformational stability, amyloid fibrillation kinetics, and seeding propensity of different 129 mutants, located in β-strand 1 of PrP (Met129 (WT), M129A, M129V, M129L, M129W, M129P, M129E, M129K, and M129C) in HuPrP(90–231). The mutations M129V, M129L, M129K, and M129C did not affect stability (midpoints of thermal denaturation, Tm = 65–66 °C), whereas the mutants M129A and M129E and the largest side chain M129W were destabilized by 3–4 °C. The most destabilizing substitution was M129P, which lowered the Tm by 7.2 °C. All mutants, except for M129C, formed amyloid-like fibrils within hours during fibril formation under near physiological conditions. Fibril-forming mutants showed a sigmoidal kinetic profile and showed shorter lag times during seeding with preformed amyloid fibrils implicating a nucleated polymerization reaction. In the spontaneous reactions, the lag time of fibril formation was rather uniform for the mutants M129A, M129V, and M129L resembling the wild type. When the substituted amino acid had a distinct feature discriminating it from the wild type, such as size (M129W), charge (M129E, M129K), or rotational constraint (M129P), the fibrillation was impeded. M129C did not form ThT/Congo red-positive fibrils, and non-reducing SDS-PAGE of M129C during fibrillation conditions at different time points revealed covalent dimer formation already 15 min after fibrillation reaction initiation. Position 129 appears to be a key site for dictating PrP receptiveness toward recruitment into the amyloid state.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology, 2012
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-53174 (URN)10.1074/jbc.M112.372136 (DOI)000306916300025 ()
Note

funding agencies|EU-FP7 Health Programme Project LUPAS||Swedish Research Council||Knut and Alice Wallenberg Foundation||Swedish Foundation for Strategic Research||Linkoping University Center for Neuroscience||

Available from: 2010-01-18 Created: 2010-01-18 Last updated: 2017-12-12
3. Native, amyloid fibrils and β-oligomers of the C-terminal domain of human prion protein display differential activation of complement and bind C1q, factor H and C4b-binding protein directly
Open this publication in new window or tab >>Native, amyloid fibrils and β-oligomers of the C-terminal domain of human prion protein display differential activation of complement and bind C1q, factor H and C4b-binding protein directly
2008 (English)In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 45, no 11, 3213-3221 p.Article in journal (Refereed) Published
Abstract [en]

Prion protein (PrP) is an endogenous protein involved in the pathogenesis of bovine spongiform encephalopathy and Creutzfeldt–Jakob disease. Murine PrP has been reported to bind C1q and activate the classical pathway of complement in a copper-dependent manner. Here we show that various conformational isoforms (native, amyloid fibrils, and β-oligomers) of recombinant human PrP (90–231 and 121–231) bind C1q and activate complement. PrP binds both the globular head and collagenous stalk domains of C1q. Native, β-oligomeric and amyloid fibrils of PrP all activate the classical and alternative pathways of complement to different extent. However, they do not trigger the lectin pathway. Of the tested PrP conformational isoforms we find that β-oligomers bind C1q and activate complement most strongly. Membrane attack complex formation initiated by PrP is subdued in comparison to deposition of early complement components. This is most likely attributed to the interaction between human PrP and complement inhibitors factor H and C4b-binding protein. Accordingly, PrP-triggered complement activation in the terminal pathway was increased in serum lacking C4b-binding protein. Taken together the present study indicates that complement activation may be an important factor in human prion diseases, suggesting that complement induced activities may prove relevant therapeutic targets.

Keyword
C1q, C4b-binding protein, Complement activation, Factor H, Human prion protein, Transmissible spongiform encephalopathies
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-45977 (URN)10.1016/j.molimm.2008.02.023 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13
4. Properties of defined recombinant oligomeric forms of Aβ1‐42
Open this publication in new window or tab >>Properties of defined recombinant oligomeric forms of Aβ1‐42
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Oligomers of Aβ1-42 have been identified in human Alzheimer´s disease (AD) patients and in mouse models of AD. These species have attracted intense interest as possible neurological pathogens in AD. In our hands, expression of recombinant human Aβ1-42 in Escherichia coli followed by purification in the presence of cupric ions (CuCl2) afforded recovery of high quantities (>5 mg/L of culture) of well defined trimeric, hexameric, nonameric and dodecameric Aβ1-42. Strong denaturing conditions such as 6 M GuHCI, 8 M urea or boiling in 6.5 M urea supplemented with 2.5 % SDS all failed to separate the oligomers into smaller building blocks implicating that the oligomers are composed of covalently cross-linked Aβ1-42 monomers. Purification in the absence of cupric ions resulted in monomeric Aβ1-42. The Aβ1-42 oligomers were toxic and induced apoptosis when administered to neuroblastoma cells in culture. The described method producing oligomeric Aβ1-42 from a recombinant expression system paves the way for mechanistic studies, structural analysis, drug screening and opens up for vaccine development.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-53175 (URN)
Available from: 2010-01-18 Created: 2010-01-18 Last updated: 2012-11-15

Open Access in DiVA

No full text

Authority records BETA

Nyström, Sofie

Search in DiVA

By author/editor
Nyström, Sofie
By organisation
BiochemistryThe Institute of Technology
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

Search outside of DiVA

GoogleGoogle Scholar

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 812 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • oxford
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf