The Modulation of Transthyretin Tetramer Stability by Cysteine 10 Adducts and the Drug Diflunisal

Abstract

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 fluorescencedetected 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.

Endoplasmic Reticulum Proteostasis Influences the Oligomeric State of an Amyloidogenic Protein Secreted from Mammalian Cells

“…These modified subunits integrate into the homo- and hetero-tetramers that comprise the 8 tetramer peaks observed by our UPLC assay. These modifications are in agreement with what has been seen before for TTR and other FLAG-tagged proteins (Kingsbury et al, 2008; Schmidt et al, 2012; Zhang and Kelly, 2003). While these modifications could have modest effects on TTR stability (Kingsbury et al, 2008; Schneider et al, 2001; Zhang and Kelly, 2003), these modifications are not predicted to globally impact the stability of secreted TTR tetramers.…”

How I treat amyloidosis

“…44 The drugs diflunisal and tafamidis stabilize the tetramer in vitro and in vivo and are in clinical testing for ATTR. 45,46 In Portugal and Japan, Val30Met ATTR can present as polyneuropathy in patients 20 to 40 years old, leading to disability and early death unless treated with orthotopic liver transplantation. 47 In blacks, Val122Ile ATTR often presents in the sixth or seventh decade with progressive heart failure and/or neuropathy but not with proteinuria, although glomerulosclerosis can mimic AL renal involvement.…”

Transthyretin microheterogeneity and molecular interactions: implications for amyloid formation

“…Hence, the altered aggregation propensity of the S-sulfonated TTR could in this case not be ascribed solely to the sulfonation (44). Instead, several studies have identified that S-sulfonation of WT and V30M TTR increases the stability of the tetrameric form by providing additional intramonomeric contacts and in this manner prevents amyloid formation (41,45,46). This raises the possibility that increased S-sulfonation of Cys10, for example, through the dietary supplementation of sulfites, may have a protective effect against TTR amyloidosis (47).…”

“…Although it was reported that the presence of mixed disulfides predominantly affects the aggregation propensity of monomeric TTR at mildly acidic pH (39), analytical ultracentrifugation experiments have shown that S-cysteinylation also enhances tetramer dissociation at physiological pH (41). The heterogeneous oxidative modifications produced by carbonylation of TTR destabilize its monomeric form and interfere with the binding of tetramer-stabilizing compounds (42).…”