Trapping the Monomer of a Non-amyloidogenic Variant of Transthyretin

Abstract

Transthyretin (TTR) is a 127-residue homotetrameric ␤-sheetrich protein that transports thyroxine in the blood and cerebrospinal fluid. The deposition of fibrils and amorphous aggregates of TTR in patients' tissues is a hallmark of TTR amyloid disease. Familial amyloidotic polyneuropathy is a hereditary form of TTR amyloidosis that is associated with one among 80 different variants of TTR. The most aggressive variants of TTR are V30M, L55P, and A25T, and the propensity to undergo aggregation seems to be linked to tetramer stability. T119M is a very stable, non-amyloidogenic variant of TTR. Here we show that the combination of high hydrostatic pressure with subdenaturing concentrations of urea (4 M) at 1°C irreversibly dissociates T119M into monomers in less than 30 min in a concentration-dependent fashion. After pressure and urea removal, long lived monomers are the only species present in solution. We took advantage of the slow reassociation kinetics of these monomers into tetramers to produce heterotetramers by mixing the T119M monomers with the tetramers of the aggressive mutants of TTR. Our data show that T119M monomers can be successfully incorporated into all of these tetramers even when the exchange is performed in a more physiological environment such as human plasma; these monomers render the resultant heterotetramers less amyloidogenic. The data presented here are relevant for the understanding of T119M folding and association reactions and provide a protocol for producing T119M monomers that function as inhibitors of TTR aggregation when incorporated into tetramers. This protocol may provide a new strategy for treating TTR diseases for which there is no therapy available other than liver transplantation. Transthyretin (TTR)2 is a 55-kDa homotetrameric protein composed of identical 127-residue subunits with a predominantly ␤-sheet structure (1). TTR is found in human plasma (0.1-0.4 mg/ml) and cerebral spinal fluid (0.017 mg/ml). The plasma form serves as a secondary carrier for thyroxine (T4) and binds to retinol-binding protein (1), whereas the form that resides in the cerebral spinal fluid is the primary T4 transporter (2).Wild-type TTR is responsible for senile systemic amyloidosis, a disease that affects 10% of people over 80 years old and is characterized by heavy amyloid deposits in the heart (3). Around 80 point mutants of TTR have been described thus far that are involved in familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy, and central nervous system amyloidosis (4). In general, patients with the familial form of the disease experience the first symptoms by the time they reach their second or third decade with peripheral neuropathy, cardiomyopathy, and leptomeningeal deposition (5).Among the variants of TTR identified worldwide, V30M and L55P are the most important because of their high frequency of occurrence and the aggressiveness of the symptoms they evoke. A25T is one of the most unstable known tetramer of TTR that is involved in central nervous system amyloid...

Cavity filling mutations at the thyroxine-binding site dramatically increase transthyretin stability and prevent its aggregation

“…A red shift of 18 nm was observed when the sample was incubated in these conditions, indicating tetramer dissociation and monomer denaturation (Fig. 5B)39. HHP was released and the samples were dialyzed in order to remove the urea and allow the re-association of monomers, rendering a heterogeneous population of tetramers constituted by subunits of TTR A108I and TTR V30M in random ratio.…”

“…We next addressed whether the incorporation of one or more subunits of TTR A108I could stabilize the pathogenic TTR V30M variant through the formation of heterotetramers, a phenomenon described before for TTR T119M273039. In order to dissociate the stable TTR A108I tetramers, we used a combination of HHP, 4 M urea and low temperature39.…”

“…In order to dissociate the stable TTR A108I tetramers, we used a combination of HHP, 4 M urea and low temperature39. Figure 5A shows a scheme of the assay.…”

“…Pressure was released and the mixture was dialyzed for 24 h at 4 °C against PBS to remove residual urea and to allow for re-association and subunit exchange39. To probe the oligomeric state of the sample, high performance liquid chromatography was performed in a Superdex 75 10/300 GL column (GE healthcare Life Sciences) at room temperature using an HPLC system at a flow rate used was of 0.5 ml/min.…”

Activated microglia mediate synapse loss and short-term memory deficits in a mouse model of transthyretin-related oculoleptomeningeal amyloidosis

“…42 For internalization assays, A25T was labeled with the fluorescent probe acrylodan as described previously. 43 In all assays, fibrillar and soluble A25T were used at 1  μ M.…”