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Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.ORCID iD: 0000-0002-5582-140X
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
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. Vol. 3, no 4, 224-235 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-21064DOI: 10.4161/pri.3.4.10112ISI: 000280061100009OAI: oai:DiVA.org:liu-21064DiVA: diva2:240500
Available from: 2009-09-28 Created: 2009-09-28 Last updated: 2014-09-09
In thesis
1. Protein Misfolding in Human Diseases
Open this publication in new window or tab >>Protein Misfolding in Human Diseases
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

There are several diseases well known that are due to aberrant protein folding. These types of diseases can be divided into three main categories:

  1. Loss-of-function diseases
  2. Gain-of-toxic-function diseases
  3. Infectious misfolding diseases

 

Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to inherited mutations. The rare disease marble brain disease (MBD) also known as carbonic anhydrase II deficiency syndrome (CADS) can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. We have over the past 10-15 years studied the folding, misfolding and aggregation of the enzyme human carbonic anhydrase II. In summary our HCA II folding studies have shown that the protein folds via an intermediate of molten-globule type, which lacks enzyme activity and the molten globule state of HCA II is prone to aggregation. One mutation associated with MBD entails the His107Tyr (H107Y) substitution. We have demonstrated that the H107Y mutation is a remarkably destabilizing mutation influencing the folding behavior of HCA II. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II towards populating ensembles of intermediates of molten globule type under physiological conditions. The enormously destabilizing effects of the H107Y mutation is not due to loss of specific interactions of H107 with residue E117, instead it is caused by long range sterical destabilizing effects of the bulky tyrosine residue. We also showed that the folding equilibrium can be shifted towards the native state by binding of the small-molecule drug acetazolamide, and we present a small molecule inhibitor assessment with select sulfonamide inhibitors of varying potency to investigate the effectiveness of these molecules to inhibit the misfolding of HCA II H107Y. We also demonstrate that high concentration of the activator compound L-His increases the enzyme activity of the mutant but without stabilizing the folded protein.

 

The infectious misfolding diseases is the smallest group of misfolding diseases. The only protein known to have the ability to be infectious is the prion protein. The human prion diseases Kuru, Gerstmann-Sträussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob are characterized by depositions of amyloid plaque from misfolded prion protein (HuPrP) in various regions of the brain depending on disease. Amyloidogenesis of HuPrP is hence strongly correlated with prion disease.

Our results show that amyloid formation of recHuPrP90-231 can be achieved starting from the native protein under gentle conditions without addition of denaturant or altered pH. The process is efficiently catalyzed by addition of preformed recHuPrP90-231 amyloid seeds. It is plausible that amyloid seeding reflect the mechanism of transmissibility of prion diseases. Elucidating the mechanism of PrP amyloidogenesis is therefore of interest for strategic prevention of prion infection.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2009. 103 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1239
Keyword
Misfolding, carbonic anhydrase, prion protein, protein stability
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-21077 (URN)978-91-7393-698-9 (ISBN)
Public defence
2009-02-26, Planck, Campus Valla, Linköpings universitet, Linköping, 13:15 (English)
Opponent
Supervisors
Available from: 2009-10-16 Created: 2009-09-28 Last updated: 2009-10-16Bibliographically approved
2. Early events in disease associated protein misfolding
Open this publication in new window or tab >>Early events in disease associated protein misfolding
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:nbn:se:liu:diva-52743 (URN)978-91-7393-544-9 (ISBN)
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

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Almstedt, KarinNyström, SofieNilsson, PeterHammarström, Per

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