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Kinetic analysis of the multistep aggregation pathway of human transthyretin.

Aggregation of transthyretin (TTR) is the causative agent for TTR cardiomyopathy and polyneuropathy amyloidoses. Aggregation is initiated by dissociation of the TTR tetramer into a monomeric intermediate, which self-assembles into amyloid. The coupled multiple-step equilibria and low-concentration,... Full description

Journal Title: Proceedings of the National Academy of Sciences of the United States of America July 3, 2018, Vol.115(27), pp.E6201-E6208
Main Author: Sun, Xun
Other Authors: Dyson, H Jane , Wright, Peter E
Format: Electronic Article Electronic Article
Language: English
Subjects:
ID: E-ISSN: 1091-6490 ; DOI: 10.1073/pnas.1807024115
Link: http://search.proquest.com/docview/2057116702/?pq-origsite=primo
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recordid: proquest2057116702
title: Kinetic analysis of the multistep aggregation pathway of human transthyretin.
format: Article
creator:
  • Sun, Xun
  • Dyson, H Jane
  • Wright, Peter E
subjects:
  • Amino Acid Substitution–Genetics
  • Cardiomyopathies–Metabolism
  • Humans–Pathology
  • Mutation, Missense–Chemistry
  • Nuclear Magnetic Resonance, Biomolecular–Genetics
  • Prealbumin–Metabolism
  • Protein Aggregation, Pathological–Metabolism
  • Protein Multimerization–Metabolism
  • Protein Structure, Quaternary–Metabolism
  • Prealbumin
  • Aggregation Kinetics
  • Amyloidogenic Protein
  • Hydrophobic Interaction
  • Low-Population Intermediate
  • Real-Time NMR
ispartof: Proceedings of the National Academy of Sciences of the United States of America, July 3, 2018, Vol.115(27), pp.E6201-E6208
description: Aggregation of transthyretin (TTR) is the causative agent for TTR cardiomyopathy and polyneuropathy amyloidoses. Aggregation is initiated by dissociation of the TTR tetramer into a monomeric intermediate, which self-assembles into amyloid. The coupled multiple-step equilibria and low-concentration, aggregation-prone intermediates are challenging to probe using conventional assays. We report a [.sup.19]F-NMR assay that leverages a highly sensitive trifluoroacetyl probe at a strategic site that gives distinct [.sup.19]F chemical shifts for the TTR tetramer and monomeric intermediate and enables direct quantification of their populations during the aggregation process. Integration of real-time [.sup.19]F-NMR and turbidity measurements as a function of temperature allows kinetic and mechanistic dissection of the aggregation pathway of both wild-type and mutant TTR. At physiological temperature, the monomeric intermediate formed by wild-type TTR under mildly acidic conditions rapidly aggregates into species that are invisible to NMR, leading to loss of the NMR signal at the same rate as the turbidity increase. Lower temperature accelerates tetramer dissociation and decelerates monomer tetramerization and oligomerization via reduced hydrophobic interactions associated with packing of a phenylalanine (F87) into a neighboring protomer. As a result, the intermediate accumulates to a higher level, and formation of higher-order aggregates is delayed. Application of this assay to pathogenic (V30M, L55P, and V122I) and protective (T119[MU]) mutants revealed significant differences in behavior. A monomeric intermediate was observed only for V122I: aggregation of V30M and L55P proceeds without an observable monomeric intermediate, whereas the protective mutant T119[MU] remains resistant to tetramer dissociation and aggregation. amyloidogenic protein | real-time NMR | aggregation kinetics | low-population intermediate | hydrophobic interaction
language: eng
source:
identifier: E-ISSN: 1091-6490 ; DOI: 10.1073/pnas.1807024115
fulltext: fulltext
issn:
  • 10916490
  • 1091-6490
url: Link


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titleKinetic analysis of the multistep aggregation pathway of human transthyretin.
creatorSun, Xun ; Dyson, H Jane ; Wright, Peter E
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ispartofProceedings of the National Academy of Sciences of the United States of America, July 3, 2018, Vol.115(27), pp.E6201-E6208
identifierE-ISSN: 1091-6490 ; DOI: 10.1073/pnas.1807024115
subjectAmino Acid Substitution–Genetics ; Cardiomyopathies–Metabolism ; Humans–Pathology ; Mutation, Missense–Chemistry ; Nuclear Magnetic Resonance, Biomolecular–Genetics ; Prealbumin–Metabolism ; Protein Aggregation, Pathological–Metabolism ; Protein Multimerization–Metabolism ; Protein Structure, Quaternary–Metabolism ; Prealbumin ; Aggregation Kinetics ; Amyloidogenic Protein ; Hydrophobic Interaction ; Low-Population Intermediate ; Real-Time NMR
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descriptionAggregation of transthyretin (TTR) is the causative agent for TTR cardiomyopathy and polyneuropathy amyloidoses. Aggregation is initiated by dissociation of the TTR tetramer into a monomeric intermediate, which self-assembles into amyloid. The coupled multiple-step equilibria and low-concentration, aggregation-prone intermediates are challenging to probe using conventional assays. We report a [.sup.19]F-NMR assay that leverages a highly sensitive trifluoroacetyl probe at a strategic site that gives distinct [.sup.19]F chemical shifts for the TTR tetramer and monomeric intermediate and enables direct quantification of their populations during the aggregation process. Integration of real-time [.sup.19]F-NMR and turbidity measurements as a function of temperature allows kinetic and mechanistic dissection of the aggregation pathway of both wild-type and mutant TTR. At physiological temperature, the monomeric intermediate formed by wild-type TTR under mildly acidic conditions rapidly aggregates into species that are invisible to NMR, leading to loss of the NMR signal at the same rate as the turbidity increase. Lower temperature accelerates tetramer dissociation and decelerates monomer tetramerization and oligomerization via reduced hydrophobic interactions associated with packing of a phenylalanine (F87) into a neighboring protomer. As a result, the intermediate accumulates to a higher level, and formation of higher-order aggregates is delayed. Application of this assay to pathogenic (V30M, L55P, and V122I) and protective (T119[MU]) mutants revealed significant differences in behavior. A monomeric intermediate was observed only for V122I: aggregation of V30M and L55P proceeds without an observable monomeric intermediate, whereas the protective mutant T119[MU] remains resistant to tetramer dissociation and aggregation. amyloidogenic protein | real-time NMR | aggregation kinetics | low-population intermediate | hydrophobic interaction
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