| 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 wild-type TTR and its amyloidogenic variants are intrinsically prone to β-aggregation. We report the results of a computational analysis predicting that wild-type TTR possesses a very high intrinsic β-aggregation propensity which is on average not enhanced by amyloidogenic mutations. However, when located in β-strands, most of these mutations are predicted to destabilize the native β-structure. The analysis also shows that rat and murine TTR have a lower intrinsic β-aggregation propensity and a similar native β-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 β-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 α-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 co-transport 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 β-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 binding sites for the thyroid hormone thyroxine. Each monomer contains eight β-strands (A-H), arranged in a β-sandwich of two four-stranded β-sheets, with a short α-helix connecting two of the eight β-strands. In the tetramer, the four monomers are organized as a dimer of dimers. Two monomers are held together, forming a stable dimer through a net of H-bond interactions involving the two external β-strands H and F. The two dimers associate back to back and form the tetramer, by interacting mostly through hydrophobic contacts between residues of the AB and GH loops.Normal TTR possesses an inherent potential, albeit low, to generate amyloid fibrils, giving rise to Senile Systemic Amyloidosis (SSA) in ∼25% of the population aged over 80 years (8). More than 100 point mutations are described for human TTR. Most of them are involved in the hereditary amyloidoses known as familial amyloidotic polyneuropathy (FAP) or cardiomyopathy (FAC) (9). Single point mutations enhance the amyloidogenicity of TTR, so that patients show an earlier age of onset and a faster disease progression compared with SSA patients. The observation that single point mutations can drastically influence the disease phenotype is particularly relevant. In fact, the study of pathogenic TTR variants may provide clues to the mechanism of their abnormal behavior leading to amyloid formation. Although amyloidogenic proteins in general may be structurally unrelated to each other, and lead to various pathological phenotypes in humans, the amyloid fibrils originating from different proteins share the common cross-β structure, consisting in continuous β-sheets lying parallel to the longitudinal axis of the fibril, with the constituent β-strands running perpendicular to this axis. Therefore, the amyloidogenic proteins have to undergo structural alterations to be able to generate the cross-β structure, i.e. new β-pairing interactions have to be established on the way to fibril formation. However, the molecular mechanisms underlying protein misfolding and aggregation into highly ordered fibrillar structures are not clarified definitely, although significant progress is recently been made toward their elucidation (1, 10, 11).Based on the seminal observation that the rates of aggregation into amyloid fibrils in vitro correlate with simple physico-chemical amino acid features (12), several algorithms were introduced in recent years to predict, with good success, the intrinsic β-aggregation propensities of protein and peptide sequences (for a review, see Ref. 13). The intrinsic β-aggregation propensity is a measure of the tendency polypeptide chains may have to aggregate into the amyloid structure, provided that aggregation proceeds from unstructured monomers. The prediction of intrinsic propensities to β-aggregation for amyloidogenic or non-amyloidogenic variants of the same sequence was used to explain in several instances their relative ability to speed up/slow down in vitro fibrillogenesis or the enhancement/reduction of their amyloidogenic potential in vivo (14). However, a high intrinsic aggregation propensity may not result in an actual aggregation, due to the protecting role of the ordered native structure (15, 16). Therefore, the amyloidogenic potential in the TTR variants may depend further on the change of stability in the native TTR tetramer induced by mutations. In particular, it remains to be clarified to what extent human TTR possesses an intrinsic propensity to β-aggregation, and whether amyloidogenic mutations enhance such a propensity, or only destabilize the TTR tetramer, thereby facilitating the misfolding and misassembly of a protein which is in itself prone to β-aggregation.With regard to the pathway from native to misfolded TTR and to amyloid aggregation, the results of a number of in vitro studies are consistent with the rate-limiting dissociation of the TTR tetramer, followed by misfolding of TTR monomers and their downhill polymerization to generate pathological aggregates (17–25). The crystal structures of amyloidogenic TTR variants are generally well conserved (26–30). Accordingly, the functional properties of the variants, such as the ability to interact with retinol-binding protein (5), are maintained, being consistent with the fact that large conformational changes are not induced by amyloidogenic mutations, at least under native-like conditions (11). In vitro studies have shown that a moderately acidic medium (pH 4–5) facilitates TTR fibrillogenesis (17) and that the extent of fibril formation is remarkably enhanced for amyloidogenic TTR variants in comparison to wild-type TTR (31). Recently, it has been shown by x-ray analysis that an acidic pH (4.6) causes a large local conformational change in an amyloidogenic TTR variant (I84S) affecting two subunits within the tetramer, which probably destabilizes the TTR tetramer (32). In contrast, no significant structural changes for wild-type TTR at pH 4.6 and for I84S TTR at neutral pH were found, suggesting that conformational changes associated with a destabilization of the TTR native state may be induced or enhanced in amyloidogenic TTR variants by partially denaturing conditions (32). Pursuing these observations, we extend here our investigation to include other amyloidogenic TTR variants in comparison to the wild-type protein, with the aim to unravel structural alterations that are possibly associated with an enhanced amyloidogenic potential, according to the conformational change hypothesis (11). In addition, we report the results of a computational analysis of the mutational effects on both the intrinsic propensity to β-aggregation and the stability of the native β-structure. The same analysis is performed on murine and rat TTRs, whose structural organizations are very similar to that of the human protein (33, 34). |
