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J. Biol. Chem., Vol. 283, Issue 18, 11887-11896, May 2, 2008

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

DIRECT ANALYSIS BY FLUORESCENCE-DETECTED ANALYTICAL ULTRACENTRIFUGATION^*

Jonathan S. Kingsbury¹, Thomas M. Laue^¶, Elena S. Klimtchuk, Roger Théberge^||, Catherine E. Costello^||, and Lawreen H. Connors²

From the Department of Biochemistry, Alan and Sandra Gerry Amyloid Research Laboratory, and ^||Center for Biological Mass Spectrometry, Boston University School of Medicine, Boston, Massachusetts 02118 and the ^¶Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, New Hampshire, 03824

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 (Cys¹⁰) 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.

Received for publication, November 27, 2007 , and in revised form, March 5, 2008.

^* This work was supported by American Heart Association Grant AHA0060149T (to L. H. C.), National Institutes of Health Grants P41 RR10888 and S10 RR10493 (to C. E. C.) and S10 RR020946 (to J. Zaia), the Gerry Foundation, the Young Family Amyloid Research Fund, the Eileen Cochran Amyloid Research Fund, and the David S. Levine Amyloid Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Genzyme Corp., Framingham, MA 01701.

² To whom correspondence should be addressed: Amyloid Treatment and Research Program, Boston University School of Medicine, K-507, 715 Albany Street, Boston, MA 02118. Tel.: 617-638-4313; Fax: 617-638-4493; E-mail: lconnors{at}bu.edu.

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