Amyloidogenic Nature of Circulating Transthyretin Variants, V122I and V30A, and the Effect of the NSAID, Diflunisal
MJ Greene, K Laporte, E Klimtchuk, D Seldin, LH Connors. Boston University School of Medicine (BUSM), Boston, MA; BUSM, Boston
Background: The systemic amyloidoses are diseases caused by protein misfolding, aggregation, and deposition in tissues throughout the body. One familial type of amyloid disease is associated with variant forms of transthyretin (TTR), a plasma protein which normally circulates as a homotetramer. More than 80 pathologic TTR point mutations have been identified, each giving rise to an amyloidogenic variant with destabilized quaternary and tertiary structure.
Design: The aim of this study was to investigate the tetrameric stabilities and amyloid fibril forming propensities of TTR-V122I and TTR-V30A, two amyloidogenic variants; V122I/T119M, a double mutant containing a putative protective residue; and wild type (wt) TTR. In addition, we tested the ability of diflunisal, a non-steroidal anti-inflammatory drug (NSAID) currently being tested in a clinical trial at our institution, to inhibit TTR tetramer dissociation. Using recombinantly-generated proteins, we analyzed TTR tetramer dissociation (acid or urea denaturation) by SDS-PAGE and amyloid fibril formation (acid-mediated) by Congo red (CR) binding spectroscopy.
Results: In the acid denaturation studies, our results showed that V122I and V30A had reduced tetramer stabilities compared to wt TTR across a decreasing pH gradient. Incubation of V122I and V30A with 50x molar excess diflunisal prior to acid addition increased tetramer stability of each protein. In the amyloid fibril formation assays, V122I and V30A each demonstrated enhanced fibril formation over 5 days incubation relative to the wt protein as evidenced by red shifts (Amax 512nm-V122I; Amax 502nm-V30A; Amax 491-wt) in the absorbance spectrum of CR (Amax 485nm). The formation of amyloid fibrils was verified by polarized light microscopic examination of the CR-bound samples which exhibited the characteristic green birefringence.
Conclusions: Our data shows that the TTR variants, V122I and V30A, have a greater propensity than wt TTR to form amyloid fibrils and that the addition of diflunisal reduces tetramer dissociation of these variants, thereby inhibiting fibril formation. It is interesting that these variants with conservative amino acid substitutions are destabilized and cause amyloid formation in cardiac tissue, with V122I being almost exclusive to the African American population.
Wednesday, March 11, 2009 9:30 AM
Poster Session V # 201, Wednesday Morning