The modulation of transthyretin tetramer stability by cysteine 10 adducts and the drug diflunisal. Direct analysis by fluorescence-detected analytical ultracentrifugation.
Transthyretin (TTR) is normally a stable plasma protein. However, in cases of familial TTR-related amyloidosis and senile systemic amyloidosis (SSA), TTR is deposited as amyloid fibrils, leading to organ dysfunction and possibly death. The mechanism by which TTR undergoes the transition from stable, soluble precursor to insoluble amyloid fibril and the factors that promote this process are largely undetermined. Most models involve the dissociation of the native TTR tetramer as the initial step. It is largely accepted that the TTR gene mutations associated with TTR-related amyloidosis lead to the expression of variant proteins that are intrinsically unstable and prone to aggregation. It has been suggested that amyloidogenicity may be conferred to wild-type TTR (the form deposited in SSA) by chemical modification of the lone cysteine residue (Cys(10)) through mixed disulfide bonds. S-Sulfonation and S-cysteinylation are prevalent TTR modifications physiologically, and studies have suggested their ability to modulate the structure of TTR under denaturing conditions. In the present study, we have used fluorescence-detected sedimentation velocity to determine the effect of S-sulfonate and S-cysteine on the quaternary structural stability of fluorophore-conjugated recombinant TTR under nondenaturing conditions. We determined that S-sulfonation stabilized TTR tetramer stability by a factor of 7, whereas S-cysteinylation enhanced dissociation by 2-fold with respect to the unmodified form. In addition, we report the direct observation of tetramer stabilization by the potential therapeutic compound diflunisal. Finally, as proof of concept, we report the sedimentation of TTR in serum and the qualitative assessment of the resulting data.
Kingsbury
Jonathan S
JS
Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Laue
Thomas M
TM
Klimtchuk
Elena S
ES
Théberge
Roger
R
Costello
Catherine E
CE
Connors
Lawreen H
LH
D003545:Cysteine / Q000737:chemistry*; D004061:Diflunisal / Q000494:pharmacology*; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011228:Prealbumin / Q000737:chemistry*; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013816:Thermodynamics; D014461:Ultracentrifugation
D011228:Prealbumin; D011994:Recombinant Proteins; D004061:Diflunisal; D016650:Fluorescein-5-isothiocyanate; D003545:Cysteine