The Most Pathogenic Transthyretin Variant, L55P, Forms Amyloid Fibrils under Acidic Conditions and Protofilaments under Physiological Conditions

Abstract

The L55P transthyretin (TTR) familial amyloid polyneuropathy-associated variant is distinct from the other TTR variants studied to date and the wild-type protein in that the L55P tetramer can dissociate to the monomeric amyloidogenic intermediate and form fibril precursors under physiological conditions (pH 7.0, 37°C). The activation barrier associated with L55P-TTR tetramer dissociation is lower than the barrier for wild-type transthyretin dissociation, which does not form fibrils under physiological conditions. The L55P-TTR tetramer is also very sensitive to acidic conditions, readily dissociating to form the monomeric amyloidogenic intermediate between pH 5.5-5.0 where the wildtype TTR adopts a nonamyloidogenic tetrameric structure. The formation of the L55P monomeric amyloidogenic intermediate involves subtle tertiary structural changes within the -sheet rich subunit as discerned from Trp fluorescence, circular dichroism analysis, and ANS binding studies. The assembly of the L55P-TTR amyloidogenic intermediate at physiological pH (pH 7.5) affords protofilaments that elongate with time. TEM studies suggest that the entropic barrier associated with filament assembly (amyloid fibril formation) is high in vitro, amyloid being defined by the laterally assembled four filament structure observed by Blake upon isolation of "fibrils" from the eye of a FAP patient. The L55P-TTR protofilaments formed in vitro bind Congo red and thioflavin T (albeit more weakly than the fibrils produced at acidic pH), suggesting that the structure observed probably represents an amyloid precursor. The structural continuum from misfolded monomer through protofilaments, filaments, and ultimately fibrils must be considered as a possible source of pathology associated with these diseases.There are several human neurodegenerative diseases putatively caused by extracellular protein deposition (1). The most familiar of these include the amyloid diseases, e.g. Alzheimer's disease and senile systemic amyloidosis (2-7). In all amyloid diseases, proteins undergo conformational changes either before or coincident with their self-assembly into highly ordered amyloid fibrils. Amyloid fibrils having a cross -sheet quaternary structure are typically deposited in the brain and/or in peripheral tissues and have been linked to neurodegeneration and/or organ dysfunction by genetic evidence (2,(8)(9)(10)(11). The amyloid hypothesis implicates amyloid fibrils as the causative agent in amyloid diseases through a gain of function phenotype. (1,7,9,(12)(13)(14). Recent results from several laboratories, including our own, demonstrate the existence of preamyloid quaternary structural intermediates (protofilaments), implying that the whole process of amyloid fibril formation must be considered as a possible cause of pathology, including the first step in the process, which is protein misfolding in the case of structurally well-defined amyloid proteins (14-21).Understanding the mechanism of how a folded protein is converted into an insoluble amyloid fibr...

Quantification of the Thermodynamically Linked Quaternary and Tertiary Structural Stabilities of Transthyretin and Its Disease-Associated Variants: The Relationship between Stability and Amyloidosis

“…The value of ΔG diss H2O for L55P TTR derived from the fit is similar to that of WT TTR, even though a plethora of other data suggests that the L55P tetramer is less stable (e.g., the dissociation rate of L55P tetramer is ~9-fold faster than that of WT TTR tetramer (7) and L55P TTR tetramers dissociate at higher pH values than WT TTR tetramers (10,42)). This observation and the large error in the parameter estimates from the global fit raise the suspicion that the two-step model used in the global analysis does not describe the denaturation pathway of L55P TTR well.…”

Probing Solvent Accessibility of Transthyretin Amyloid by Solution NMR Spectroscopy

“…It combines a pulsed H/D experiment to trap solvent exchange patterns of the fibrils followed by a rapid dissociation into its monomeric constituents and analysis using solution NMR spectroscopy. The method was applied successfully to the highly amyloidogenic A␤ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) derived from the Alzheimer ␤-peptide (10). Within this work we have challenged our technique and probed the fibrillar core structure of transthyretin amyloid, a comparatively large protein associated with both inherited and idiopathic amyloidosis.…”

Native state stabilization by NSAIDs inhibits transthyretin amyloidogenesis from the most common familial disease variants

“…Expression and purification were performed as described in detail previously. 31 Protein identification was verified through mass spectrometry.…”