Amyloidogenic Potential of Transthyretin Variants

Abstract

Human transthyretin (TTR) is an amyloidogenic protein whose mild amyloidogenicity is enhanced by many point mutations affecting considerably the amyloid disease phenotype. To ascertain whether the high amyloidogenic potential of TTR variants may be explained on the basis of the conformational change hypothesis, an aim of this work was to determine structural alterations for five amyloidogenic TTR variants crystallized under native and/or destabilizing (moderately acidic pH) conditions. While at acidic pH structural changes may be more significant because of a higher local protein flexibility, only limited alterations, possibly representing early events associated with protein destabilization, are generally induced by mutations. This study was also aimed at establishing to what extent wildtype TTR and its amyloidogenic variants are intrinsically prone to ␤-aggregation. We report the results of a computational analysis predicting that wild-type TTR possesses a very high intrinsic ␤-aggregation propensity which is on average not enhanced by amyloidogenic mutations. However, when located in ␤-strands, most of these mutations are predicted to destabilize the native ␤-structure. The analysis also shows that rat and murine TTR have a lower intrinsic ␤-aggregation propensity and a similar native ␤-structure stability compared with human TTR. This result is consistent with the lack of in vitro amyloidogenicity found for both murine and rat TTR. Collectively, the results of this study support the notion that the high amyloidogenic potential of human pathogenic TTR variants is determined by the destabilization of their native structures, rather than by a higher intrinsic ␤-aggregation propensity.Protein misfolding and aggregation are involved in the pathogenesis of particularly relevant human deposition diseases, known as amyloidoses. In such diseases, normally soluble proteins undergo misfolding and become insoluble, causing the extracellular deposition of fibrillar aggregates (for reviews, see Ref. 1, 2). To date, more than 40 distinct human proteins have been associated with amyloidoses. For some of such proteins, including transthyretin (TTR), 4 lysozyme, gelsolin, ApoAI, and ApoAII, fibrinogen A ␣-chain and cystatin C, the amyloidogenic potential is induced, or is enhanced as in the case of TTR (see below), by specific mutations. The most frequent hereditary amyloidoses are caused by the genetic variants of human TTR (2).TTR is a homotetramer of about 55 kDa involved in the transport of thyroxine in the extracellular fluids and in the cotransport of vitamin A, by forming a macromolecular complex with retinol-binding protein, the specific plasma carrier of retinol (3-5). Its three-dimensional structure is known at high resolution (6, 7). The structure is characterized by a large predominance of ␤-strands, and its four monomers are arranged according to a 222 symmetry, where one of the 2-fold symmetry axes of the molecule coincides with a long channel that transverses the entire tetramer and harbors two symmetrical bind...

Localized Structural Fluctuations Promote Amyloidogenic Conformations in Transthyretin

“…6 Computational studies also showed that the general effect of these mutations is to increase local flexibility at the site of the mutations. 35,36 The links between local flexibility and amyloid formation in TTR are further demonstrated by our observation that the protective T119M mutant shows consistently less dynamic behavior than WT under both physiological and acidic conditions.…”

Conformational Flexibility Tunes the Propensity of Transthyretin to Form Fibrils Through Non‐Native Intermediate States

“…Residues where S 2 could not be obtained, including unassigned resonances, are shown in gray. [11] V122I (E), [13] V30M (F), [14] L55P (G), [15] and T119M (H) [16] TTR are shown for 14.1 T (solid circles) and 18.78 T (empty circles) magnetic fields (see Table S12). R ex values from the 14.1 T field were mapped onto the dimer of the respective crystal structures, with rigid residues shown in blue (R ex % 0 Hz) and dynamic ones in red (R ex > 30 Hz).…”

“…Although protein dynamics have been shown to be key to the misfolding of many proteins, [1] this has not been explicitly studied before with the TTR tetramer and its mutants on a residue-specific basis. 15 N relaxation rates (R 1 and R 2 ) and NOE are shaped by fast motions on the picosecond-nanosecond timescale. Fitting relaxation rates [7] to various motional models [8] enables the estimation of rotational diffusion tensor and order parame-…”

Conformational Flexibility Tunes the Propensity of Transthyretin to Form Fibrils Through Non‐Native Intermediate States

“…B) Representation of a plot of R 2,eff against ν CPMG , depicting the R ex value for a typical residue. Plots of the R ex values from 15 N relaxation dispersion experiments on WTTR (C),11 V122I (E),13 V30M (F),14 L55P (G),15 and T119M (H)16 TTR are shown for 14.1 T (solid circles) and 18.78 T (empty circles) magnetic fields (see Table S12). R ex values from the 14.1 T field were mapped onto the dimer of the respective crystal structures, with rigid residues shown in blue ( R ex ≈0 Hz) and dynamic ones in red ( R ex >30 Hz).…”