Trapping of palindromic ligands within native transthyretin prevents amyloid formation

Kolstoe, Simon; Mangione, Palma; Bellotti, Vittorio; Taylor, Graham W.; Tennent, Glenys A.; Deroo, Stéphanie; Morrison, Angus J.; Cobb, Alexander J. A.; Coyne, Anthony G.; McCammon, Margaret G.; Warner, Timothy D.; Mitchell, Jane A.; Gill, Raj; Smith, Martin D.; Ley, Steven V.; Robinson, Carol V.; Wood, Stephen P.; Pepys, Mark B.

doi:10.1073/pnas.1008255107

Cited by 59 publications

(78 citation statements)

References 43 publications

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“…The subunits of recombinant wild-type and Ser52Pro variant TTR proteins had molecular masses of 13,892.2 ± 0.9 Da and 13,902.1 ± 1.5 Da, respectively, corresponding to the expected theoretical mass values of 13,892.6 Da and 13,902.7 Da, including the additional N-terminal methionine residue, Met0. Correct assembly of the subunits into native homotetrameric wild-type and variant TTR was confirmed by both size-exclusion chromatography and native mass spectrometry, and these intact proteins bound the natural ligand, thyroxine, normally, showing the same IC 50 with mds84 and Tafamidis (13,14) as native wild-type TTR isolated from normal human serum (Table S1). However, the guanidine thiocyanate (Gdn-SCN)-mediated transition from folded tetramer to unfolded monomers ( Fig.…”

Section: Resultsmentioning

confidence: 90%

“…The effect of trypsin also was tested at a 1:200 enzyme:substrate (wt/wt) ratio in the presence of a fivefold molar excess of Tafamidis (14), mds84 (13), and epigallocatechin (19), respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Binding of Tafamidis and mds84 (13,14), which both enter the TTR inner channel, had no significant effect on the products (Fig. 5A) or kinetics (Fig.…”

Section: Limited Proteolysis Of Recombinant Wild-type and Variant Ttrmentioning

confidence: 99%

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Proteolytic cleavage of Ser52Pro variant transthyretin triggers its amyloid fibrillogenesis

Mangione

Porcari²,

Gillmore³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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105

143

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The Ser52Pro variant of transthyretin (TTR) produces aggressive, highly penetrant, autosomal-dominant systemic amyloidosis in persons heterozygous for the causative mutation. Together with a minor quantity of full-length wild-type and variant TTR, the main component of the ex vivo fibrils was the residue 49-127 fragment of the TTR variant, the portion of the TTR sequence that previously has been reported to be the principal constituent of type A, cardiac amyloid fibrils formed from wild-type TTR and other TTR variants [Bergstrom J, et al. (2005) J Pathol 206(2):224-232]. This specific truncation of Ser52Pro TTR was generated readily in vitro by limited proteolysis. In physiological conditions and under agitation the residue 49-127 proteolytic fragment rapidly and completely self-aggregates into typical amyloid fibrils. The remarkable susceptibility to such cleavage is likely caused by localized destabilization of the β-turn linking strands C and D caused by loss of the wildtype hydrogen-bonding network between the side chains of residues Ser52, Glu54, Ser50, and a water molecule, as revealed by the high-resolution crystallographic structure of Ser52Pro TTR. We thus provide a structural basis for the recently hypothesized, crucial pathogenic role of proteolytic cleavage in TTR amyloid fibrillogenesis. Binding of the natural ligands thyroxine or retinol-binding protein (RBP) by Ser52Pro variant TTR stabilizes the native tetrameric assembly, but neither protected the variant from proteolysis. However, binding of RBP, but not thyroxine, inhibited subsequent fibrillogenesis.misfolding | protein aggregation

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Section: Resultsmentioning

confidence: 90%

Section: Methodsmentioning

confidence: 99%

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Proteolytic cleavage of Ser52Pro variant transthyretin triggers its amyloid fibrillogenesis

Mangione

Porcari²,

Gillmore³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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105

143

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“…Of the multiple TTR kinetic stabilizer structures reported (15,(30)(31)(32)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45), the benzoxazoles (32) were pursued to identify an orally bioavailable candidate exhibiting potent and selective TTR binding in blood, while lacking nonsteroidal antiinflammatory (NSAID) activity, which is contraindicated in patients with cardiomyopathy. Tafamidis, or 2-(3,5-dichloro-phenyl)-benzoxazole-6-carboxylic acid, meets all of these criteria and was selected for clinical development.…”

Section: Drug | Aggregation Inhibitionmentioning

confidence: 99%

Tafamidis, a potent and selective transthyretin kinetic stabilizer that inhibits the amyloid cascade

Bulawa

Connelly²,

DeVit³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

624

610

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The transthyretin amyloidoses (ATTR) are invariably fatal diseases characterized by progressive neuropathy and/or cardiomyopathy. ATTR are caused by aggregation of transthyretin (TTR), a natively tetrameric protein involved in the transport of thyroxine and the vitamin A–retinol-binding protein complex. Mutations within TTR that cause autosomal dominant forms of disease facilitate tetramer dissociation, monomer misfolding, and aggregation, although wild-type TTR can also form amyloid fibrils in elderly patients. Because tetramer dissociation is the rate-limiting step in TTR amyloidogenesis, targeted therapies have focused on small molecules that kinetically stabilize the tetramer, inhibiting TTR amyloid fibril formation. One such compound, tafamidis meglumine (Fx-1006A), has recently completed Phase II/III trials for the treatment of Transthyretin Type Familial Amyloid Polyneuropathy (TTR-FAP) and demonstrated a slowing of disease progression in patients heterozygous for the V30M TTR mutation. Herein we describe the molecular and structural basis of TTR tetramer stabilization by tafamidis. Tafamidis binds selectively and with negative cooperativity (K d s ∼2 nM and ∼200 nM) to the two normally unoccupied thyroxine-binding sites of the tetramer, and kinetically stabilizes TTR. Patient-derived amyloidogenic variants of TTR, including kinetically and thermodynamically less stable mutants, are also stabilized by tafamidis binding. The crystal structure of tafamidis-bound TTR suggests that binding stabilizes the weaker dimer-dimer interface against dissociation, the rate-limiting step of amyloidogenesis.

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“…Significant advances in treatment have been made over the past decade, particularly in AL amyloidosis in which the precursor protein is derived from immunoglobulin light chain fragments [2]. Transthyretin (TTR)-derived amyloidosis has also been the subject of intensive investigation, and several new therapeutic approaches have completed, or are approaching, preliminary clinical testing [3][4][5][6][7]. Refinements in immunohistochemical and proteomic techniques have permitted precise definition of the amyloid type, a prerequisite for initiating appropriate (and avoiding inappropriate) therapies [8].…”

mentioning

confidence: 99%

Pursuing an underdiagnosed disease: a simple imaging test for increasing suspicion of cardiac amyloidosis

Falk

Dorbala

2011

Eur J Nucl Med Mol Imaging

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Trapping of palindromic ligands within native transthyretin prevents amyloid formation

Cited by 59 publications

References 43 publications

Proteolytic cleavage of Ser52Pro variant transthyretin triggers its amyloid fibrillogenesis

Proteolytic cleavage of Ser52Pro variant transthyretin triggers its amyloid fibrillogenesis

Tafamidis, a potent and selective transthyretin kinetic stabilizer that inhibits the amyloid cascade

Pursuing an underdiagnosed disease: a simple imaging test for increasing suspicion of cardiac amyloidosis

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