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Prefibrillar Amyloid Aggregates and Cold Shocked Tetrameric Wild Type Transthyretin are Cytotoxic
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
Department of Physics, Norwegian University of Science and Technology, 7491 Trondheim, Norway.
Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology . Linköping University, Faculty of Health Sciences.
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(English)Manuscript (Other academic)
Abstract [en]

Recent studies suggest that soluble, oligomeric species, which are intermediates in the fibril formation process in amyloid disease, might be the key species in amyloid pathogenesis. Soluble oligomers of TTR were produced by kinetic sampling from a TTR fibrillation reaction (A-state TTR, pH 2, 100 mM NaCl). The reaction was terminated at different time points, and different states in the aggregation process were captured and analyzed to elucidate the oligomer properties followed by sampling for cytotoxicity using exposure towards human SH-SYY5 neuroblastoma cells. Employing ThT fluorescence, time-resolved fluorescence anisotropy of pyrenelabeled TTR, chemical cross-linking and electron microscopy we demonstrated that early formed oligomers from A-state TTR were soluble and comprised on the average 20-30 TTR monomers. Early oligomers were highly cytotoxic and induced apoptosis as indicated by the MTT assay and caspase-3 activation, whereas mature fibrils were non-toxic. We also indicate an activated unfolded protein response in cells exposed to oligomers as evidenced by an increased expression of the endoplasmic reticulum located molecular chaperone BiP. Following exposure, BiP appeared relocalized to the cytoplasm. Surprisingly, we also found that native tetrameric TTR purified and stored under cold conditions (4 °C) was highly cytotoxic. The effect could be partially restored by increasing the temperature of the protein. The molecular basis for this pathogenicity is rather unclear but likely stems from previously reported increased sensitivity towards dissociation and denaturation of TTR at low temperatures and opens the possibility that rearranged tetrameric TTR is cytotoxic towards neuroblastoma cells.

Keyword [en]
Amyloid, apoptosis, transthyretin, chaperone, misfolding, oligomer
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:liu:diva-12562OAI: diva2:1712
Available from: 2008-09-15 Created: 2008-09-15 Last updated: 2010-01-14
In thesis
1. Molecular Aspects of Transthyretin Amyloid Disease
Open this publication in new window or tab >>Molecular Aspects of Transthyretin Amyloid Disease
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis was made to get a deeper understanding of how chaperones interact with unstable, aggregation prone, misfolded proteins involved in human disease. Over the last two decades, there has been much focus on misfolding diseases within the fields of biochemistry and molecular biotechnology research. It has become obvious that proteins that misfold (as a consequence of a mutation or outer factors), are the cause of many diseases. Molecular chaperones are proteins that have been defined as agents that help other proteins to fold correctly and to prevent aggregation. Their role in the misfolding disease process has been the subject for this thesis.

Transthyretin (TTR) is a protein found in human plasma and in cerebrospinal fluid. It works as a transport protein, transporting thyroxin and holo-retinol binding protein. The structure of TTR consists of four identical subunits connected through hydrogen bonds and hydrophobic interactions. Over 100 point mutations in the TTR gene are associated with amyloidosis often involving peripheral neurodegeneration (familial amyloidotic polyneuropathy (FAP)). Amyloidosis represents a group of diseases leading to extra cellular deposition of fibrillar protein known as amyloid. We used human SH-SY5Y neuroblastoma cells as a model for neurodegeneration. Various conformers of TTR were incubated with the cells for different amounts of time. The experiments showed that early prefibrillar oligomers of TTR induced apoptosis when neuroblastoma cells were exposed to these species whereas mature fibrils were not cytotoxic. We also found increased expression of the molecular chaperone BiP in cells challenged with TTR oligomers.

Point mutations destabilize TTR and result in monomers that are unstable and prone to aggregate. TTR D18G is naturally occurring and the most destabilized TTR mutant found to date. It leads to central nervous system (CNS) amyloidosis. The CNS phenotype is rare for TTR amyloid disease. Most proteins associated with amyloid disease are secreted proteins and secreted proteins must pass the quality control check within the endoplasmic reticulum (ER). BiP is a Hsp70 molecular chaperone situated in the ER. BiP is one of the most important components of the quality control system in the cell. We have used TTR D18G as a model for understanding how an extremely aggregation prone protein is handled by BiP. We have shown that BiP can selectively capture TTR D18G during co-expression in both E. coli and during over expression in human 293T cells and collects the mutant in oligomeric states. We have also shown that degradation of TTR D18G in human 293T cells occurs slower in presence of BiP, that BiP is present in amyloid deposition in human brain and mitigates cytotoxicity of TTR D18G oligomers.

Abstract [sv]

Denna avhandling handlar om proteiner. Särskilt de som inte fungerar som de ska utan har blivit vad man kallar ”felveckade”. Anledningen till att proteiner veckas fel beror ofta (men inte alltid) på mutationer i arvsmassan. Felveckade proteiner kan leda till sjukdomar hos människor och djur (man brukar tala om amyloidsjukdomar), ofta av neurologisk karaktär. Exempel på amyloidsjukdomar är polyneuropati, där perifera nervsystemet är drabbat, vilket leder till begränsad rörelseförmåga och senare till förlamning; och Alzheimer´s sjukdom, där centrala nervsystemet är drabbat och leder till begränsad tankeförmåga och minnesförluster.

Studierna som presenteras i denna avhandling har gått ut på att få en bättre förståelse för hur felveckade proteiner interagerar med det som vi har naturligt i cellerna och som fungerar som skyddande, hjälpande proteiner, så kallade chaperoner.

Transtyretin (TTR) är ett protein som cirkulerar i blodet och transporterar tyroxin (som är ett hormon som bland annat har betydelse för ämnesomsättningen) samt retinol-bindande protein (vitamin A). I TTR genen har man funnit över 100 punktmutationer, vilka har kopplats samman med amyloidsjukdomar, bland annat ”Skellefteåsjukan”. Mutationer i TTR genen leder ofta till att proteinet blir instabilt vilket leder till upplösning av TTR tetrameren till monomerer. Dessa monomerer kan därefter sammanfogas på nytt men denna gång på ett sätt som är farligt för organismen. I denna avhandling har fokus legat på en mutation som kallas TTR D18G, vilken har identifierats i olika delar av världen och leder till en dödlig form av amyloidos i centrala nervsystemet.

Det chaperon som vi har studerat benämns BiP och är beläget i en cellkomponent som kallas för det endoplasmatiska retiklet (ER). I ER finns cellens kontrollsystem i vilket det ses till att felveckade proteiner inte släpps ut utan istället bryts ned.

Denna avhandling har visat att BiP kan fånga upp TTR D18G inuti celler och där samla mutanten i lösliga partiklar som i detta fall är ofarliga för cellen. Avhandligen har också visat att nedbrytningen av TTR D18G sker mycket långsammare när BiP finns i riklig mängd.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2008. 61 p.
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1179
Amyloid, apoptosis, BiP, chaperone, misfolding, oligomer, transthyretin
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
urn:nbn:se:liu:diva-12566 (URN)978-91-7393-906-5 (ISBN)
Public defence
2008-05-30, Planck, Campus Valla, Linköpings universitet, Linköping, 14:15 (English)
Available from: 2008-09-15 Created: 2008-09-15 Last updated: 2009-05-15Bibliographically approved

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