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Quantification of transthyretin kinetic stability in human plasma using subunit exchange

Academic Article
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Overview

related to degree

  • Monteiro, Cecilia, Ph.D. in Chemical Biology, Scripps Research 2014 - 2018
  • Solis, Gregory, Ph.D. in Chemical Biology, Scripps Research 2012 - 2018

authors

  • Rappley, I.
  • Monteiro, Cecilia
  • Novais, M.
  • Baranczak, A.
  • Solis, Gregory
  • Wiseman, R. Luke
  • Helmke, S.
  • Maurer, M. S.
  • Coelho, T.
  • Powers, Evan
  • Kelly, Jeffery

publication date

  • April 2014

journal

  • Biochemistry  Journal

abstract

  • The transthyretin (TTR) amyloidoses are a group of degenerative diseases caused by TTR aggregation, requiring rate-limiting tetramer dissociation. Kinetic stabilization of TTR, by preferential binding of a drug to the native tetramer over the dissociative transition state, dramatically slows the progression of familial amyloid polyneuropathy. An established method for quantifying the kinetic stability of recombinant TTR tetramers in buffer is subunit exchange, in which tagged TTR homotetramers are added to untagged homotetramers at equal concentrations to measure the rate at which the subunits exchange. Herein, we report a subunit exchange method for quantifying the kinetic stability of endogenous TTR in human plasma. The subunit exchange reaction is initiated by the addition of a substoichiometric quantity of FLAG-tagged TTR homotetramers to endogenous TTR in plasma. Aliquots of the subunit exchange reaction, taken as a function of time, are then added to an excess of a fluorogenic small molecule, which immediately arrests further subunit exchange. After binding, the small molecule reacts with the TTR tetramers, rendering them fluorescent and detectable in human plasma after subsequent ion exchange chromatography. The ability to report on the extent of TTR kinetic stabilization resulting from treatment with oral tafamidis is important, especially for selection of the appropriate dose for patients carrying rare mutations. This method could also serve as a surrogate biomarker for the prediction of the clinical outcome. Subunit exchange was used to quantify the stabilization of WT TTR from senile systemic amyloidosis patients currently being treated with tafamidis (20 mg orally, once daily). TTR kinetic stability correlated with the tafamidis plasma concentration.

subject areas

  • Amyloidosis
  • Animals
  • Benzoxazoles
  • Chromatography, Ion Exchange
  • Humans
  • Mice
  • Mice, Knockout
  • Prealbumin
  • Protein Binding
  • Protein Stability
  • Protein Structure, Secondary
  • Protein Subunits
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Identity

PubMed Central ID

  • PMC3977577

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi500171j

PubMed ID

  • 24661308
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Additional Document Info

start page

  • 1993

end page

  • 2006

volume

  • 53

issue

  • 12

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