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Structural and functional properties of transthyretin
Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The hereditary transthyretin (TTR) amyloidoses are rare, and in severe cases, fatal disorders caused by mutations in the TTR gene. The clinical picture is diverse, involving neuropathies and myopathies, and mainly depends on the causative mutation and the sites and rates of amyloid deposition. The ultimate aim of the field of research presented in this thesis is to prevent TTR amyloid disease. To reach this ambitious goal, a thorough understanding of the normal as well as the pathological properties of the protein is essential. Here, comparisons between TTR from humans and other species may provide valuable information.

The three-dimensional structure of TTR from Gilthead sea bream (Sparus aurata) was determined at 1.75 Å resolution by X-ray crystallography, and was found to be structurally similar to human TTR. However, significant differences were observed in the area at and around β-strand D, an area believed to dissociate from the structure prior to amyloid formation, thereby allowing the β-strands A and B to participate in polymerization. During evolution, the preference of TTR for the thyroid hormones, 3,5,3’-triiodo-L-thyronine (T3) and 3,5,3’,5’-tetraiodo-L-thyronine (T4), has shifted. While human TTR has higher affinity for T4, the opposite is true in lower vertebrates, e.g. fish and reptiles, where T3 is the main ligand. We have determined two separate structures of sea bream TTR in complex with T3 and T4, both at 1.9 Å resolution, as well as the complex of human TTR with T3. A significantly wider entrance and narrower thyroid hormone binding channel suggest a structural explanation to the differences in thyroid hormone preference between human and piscine TTR.

The Tyr114Cys substitution in TTR is associated with severe systemic amyloidosis. The mutation introduces a second cysteinyl group in the TTR monomer, and has been shown to inhibit the formation of fibril formation in vitro, promoting the formation of disulfide-bonded amorphous aggregates. To deduce the role of intermolecular disulfide bonds in fibril formation we characterized the TTR Cys10Ala/Tyr114Cys double mutant. Our results suggest that an intermolecular disulfide bond at position 114 enhances the exposure of Cys10, which promotes the formation of additional intermolecular disulfide-linked assemblies. Also, we were able to isolate a disulfide-linked dimeric form of this mutant that formed protofibrils in vitro, suggesting the architecture of TTR amyloid may be the result of different underlying structures rather than that of a highly stringent assembly.

We have also been able to successfully adapt a method of protein pre-heating to enable crystallization, thereby succeeding in a particularly problematic protein crystallization experiment. By heating the protein solution, we succeeded in separating several forms of protein micro-heterogeneities from the properly folded protein species, thereby allowing the growth of well diffracting crystals.

Abstract [sv]

Ärftlig transthyretinamyloidos är en ovanlig och i allvarliga fall dödlig proteininlagringssjukdom som orsakas av mutationer i genen för transthyretin. Den kliniska bilden är huvudsakligen beroende av den bakomliggande genförändringen samt amyloidlokaliseringen och -depositionshastigheten och omfattar vanligen neuropatier och myopatier av varierande grad. Det slutgiltiga målet med forskningsfältet som presenteras i denna avhandling är att förhindra eller bota transthyretinamyloidos. En förutsättning för att lyckas med detta ambitiösa mål är en ingående förståelse för proteinets grundläggande egenskaper, såväl i normalfallet som i de patologiska processerna, bland annat genom jämförande studier av humant och icke-humant transthyretin (TTR).

Den tredimensionella röntgenkristallografiska strukturen av TTR från fisken guldsparid (Sparus aurata) bestämdes till en upplösning på 1,75Å och befanns vara strukturellt snarlik humant TTR. Signifikanta skillnader observerades emellertid i och kring β-sträng D, en region som tros dissociera från huvudstrukturen innan själva bildningen av amyloid. Enligt denna hypotes leder D-strängsdissociationen till exponering av β-strängarna A och B, vilka därmed kan delta i de reaktioner som bildar amyloid. Under evolutionen har bindningspreferenserna för thyroideahormonerna T3 (3,5,3’-trijod-L-thyronin) och T4 (3,5,3’,5’-tetrajod-L-thyronin) hos TTR ändrats. Humant TTR har högre affinitet för T4 än för T3, medan det motsatta förhållandet gäller för lägre vertebrater, t ex fisk, där T3 är den huvudsakliga liganden. Strukturerna bestämdes för guldsparid i komplex med T4 och med T3 till 1,9 Å upplösning, samt för humant TTR i komplex med T3 till 1,7 Å upplösning. Jämförande analyser visade på signifikanta skillnader i thyroideahormonbindningskanalen, vilken var vidare och grundare i fisk än i människa. Dessa strukturella skillnader kan delvis förklara olikheterna i hormonbindning mellan högre och lägre vertebrater.

Substitutionen Tyr114Cys i TTR är kopplad till en allvarlig form av systemisk transthyretinamyloidos. Mutationen introducerar en andra cysteinylgrupp i TTR-monomererna, vilket förhindrar fibrillbildning in vitro, men gynnar bildningen av amorfa disulfidbundna aggregat. För att närmare studera betydelsen av disulfidbindningar vid fibrillbildning av detta protein så karakteriserades dubbelmutanten TTR Cys10Ala/Tyr114Cys. Baserat på våra resultat föreslår vi att intermolekylära disulfidbindningar i position 114 ökar exponeringen av Cys10, vilket förstärker tendensen att bilda ytterligare disulfidbundna aggregat. Vi isolerade även en disulfidbunden dimerisk form av dubbelmutanten som kan bilda protofibriller in vitro. Baserat på denna observation föreslår vi att transthyretinamyloids underliggande arkitektur är sammansatt och kan nås genom sammanfogning av olika substrukturer snarare än genom en strikt ordnad uppbyggnad.

Vi har också modifierat och anpassat en metod för uppvärmning av proteiner för att möjliggöra kristallisation i ett synnerligen problematiskt proteinkristallisations-experiment. Genom uppvärmning av proteinlösningen lyckades vi separera olika former av mikroheterogeniteter från det rättveckade proteinet, som sedan bildade kristaller av god röntgendiffraktiv kvalitet.

Place, publisher, year, edition, pages
Umeå: Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet) , 2008. , 51 p.
Keyword [en]
Transthyretin, Amyloidosis, X-ray crystallography, Protein structure, Protein
National Category
Structural Biology
Identifiers
URN: urn:nbn:se:umu:diva-1507ISBN: 978-91-7264-488-5 (print)OAI: oai:DiVA.org:umu-1507DiVA: diva2:141261
Public defence
2008-02-15, Sal B, 1D, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2008-01-24 Created: 2008-01-24 Last updated: 2009-06-23Bibliographically approved
List of papers
1. High resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxine.
Open this publication in new window or tab >>High resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxine.
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2004 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 279, no 25, 26411-6 p.Article in journal (Refereed) Published
Abstract [en]

Transthyretin (TTR) is an extracellular transport protein involved in the distribution of thyroid hormones and vitamin A. So far, TTR has only been found in vertebrates, of which piscine TTR displays the lowest sequence identity with human TTR (47%). Human and piscine TTR bind both thyroid hormones 3,5,3'-triiodo-l-thyronine (T(3)) and 3,5,3',5'-tetraiodo-l-thyronine (thyroxine, T(4)). Human TTR has higher affinity for T(4) than T(3), whereas the reverse holds for piscine TTR. X-ray structures of Sparus aurata (sea bream) TTR have been determined as the apo-protein at 1.75 A resolution and bound to ligands T(3) and T(4), both at 1.9 A resolution. The apo structure is similar to human TTR with structural changes only at beta-strand D. This strand forms an extended loop conformation similar to the one in chicken TTR. The piscine TTR.T(4) complex shows the T(4)-binding site to be similar but not identical to human TTR, whereas the TTR.T(3) complex shows the I3' halogen situated at the site normally occupied by the hydroxyl group of T(4). The significantly wider entrance of the hormone-binding channel in sea bream TTR, in combination with its narrower cavity, provides a structural explanation for the different binding affinities of human and piscine TTR to T(3) and T(4).

Keyword
Animals, Chickens, Cloning; Molecular, Crystallography; X-Ray, Escherichia coli/metabolism, Humans, Ligands, Models; Molecular, Prealbumin/*chemistry/metabolism, Protein Binding, Protein Conformation, Protein Structure; Tertiary, Sea Bream, Thyroxine/*chemistry, Triiodothyronine/*chemistry
Identifiers
urn:nbn:se:umu:diva-13622 (URN)10.1074/jbc.M313553200 (DOI)15082720 (PubMedID)
Available from: 2007-10-12 Created: 2007-10-12Bibliographically approved
2. Cys114-linked dimers of transthyretin are compatible with amyloid formation.
Open this publication in new window or tab >>Cys114-linked dimers of transthyretin are compatible with amyloid formation.
2005 (English)In: Biochemistry, ISSN 0006-2960, Vol. 44, no 39, 13063-70 p.Article in journal (Refereed) Published
Abstract [en]

The Tyr114Cys substitution in the human plasma protein transthyretin leads to a particularly aggressive form of familial amyloidotic polyneuropathy. In a previous study we demonstrated that ATTR Tyr114Cys forms intermolecular disulfide bonds, which partly impair fibril formation and result in a more amorphous morphology. Apart from the introduced cysteinyl group in position 114, the native sequence contains one cysteine located at position 10. To deduce the role of intermolecular disulfide bridging in fibril formation we generated and characterized the TTR Cys10Ala/Tyr114Cys double mutant. Our results suggest that an intermolecular cysteine bridge at position 114 enhances the exposure of cysteine 10, thereby facilitating additional intermolecular cysteine assemblies. We also purified a disulfide-linked dimeric form of TTR Cys10Ala/Tyr114Cys, which was recognized by the anti-TTR amyloid-specific monoclonal antibody MAb (39-44). Moreover, this dimeric molecule can form protofibrils indistinguishable from the fibrils formed under reducing conditions, as judged by atomic force microscopy. Assuming that both molecules of the dimer are part of the core of the fibril, the assembly is incompatible with a preserved native or near-native dimeric interphase. Our findings raise the question of whether TTR-amyloid architecture is indeed the result of one highly stringent assembly of structures or if different fibrils may be built from different underlying structures.

Keyword
Amino Acid Substitution, Amyloid/*biosynthesis/chemistry/ultrastructure, Amyloid Neuropathies; Familial/etiology, Cysteine/*metabolism, Dimerization, Humans, Microscopy; Atomic Force, Prealbumin/chemistry/*metabolism
Identifiers
urn:nbn:se:umu:diva-13608 (URN)10.1021/bi050795s (DOI)16185074 (PubMedID)
Available from: 2007-10-12 Created: 2007-10-12Bibliographically approved
3. Heating of proteins as a means of improving crystallization: a successful case study on a highly amyloidogenic triple mutant of human transthyretin.
Open this publication in new window or tab >>Heating of proteins as a means of improving crystallization: a successful case study on a highly amyloidogenic triple mutant of human transthyretin.
2007 (English)In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, Vol. 63, no Pt 8, 695-700 p.Article in journal (Refereed) Published
Abstract [en]

The use of high temperatures in the purification procedures of heat-stable proteins is a well established technique. Recently, rapid pre-heat treatment of protein samples prior to crystallization trials was described as a final polishing step to improve the diffraction properties of crystals [Pusey et al. (2005), Prog. Biophys. Mol. Biol. 88, 359-386]. The present study demonstrates that extended high-temperature incubation (328 K for 48 h) of the highly amyloidogenic transthyretin mutant TTR G53S/E54D/L55S successfully removes heterogeneities and allows the reproducible growth of well diffracting crystals. Heat treatment might be applied as an optimization method to other cases in which the protein/biomolecule fails to form diffracting crystals.

Keyword
Amyloid/*biosynthesis/genetics, Crystallization/methods, Crystallography; X-Ray, Genetic Heterogeneity, Heat, Humans, Mutation, Prealbumin/*chemistry/*genetics/isolation & purification
Identifiers
urn:nbn:se:umu:diva-16830 (URN)10.1107/S1744309107033957 (DOI)17671371 (PubMedID)
Available from: 2007-10-12 Created: 2007-10-12Bibliographically approved

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