Abstract
Transthyretin is a tetrameric protein associated with the commonest form of systemic amyloid disease. Using isotopically labeled proteins and mass spectrometry, we compared subunit exchange in wild-type transthyretin with that of the variant associated with the most aggressive form of the disease, L55P. Wild-type subunit exchange occurs via both monomers and dimers, whereas exchange via dimers is the dominant mechanism for the L55P variant. Because patients with the L55P mutation are heterozygous, expressing both proteins simultaneously, we also analyzed the subunit exchange reaction between wild-type and L55P tetramers. We found that hybrid tetramers containing two or three L55P subunits dominate in the early stages of the reaction. Surprisingly, we also found that, in the presence of L55P transthyretin, the rate of dissociation of wild-type transthyretin is increased. This implies interactions between the two proteins that accelerate the formation of hybrid tetramers, a result with important implications for transthyretin amyloidosis.
Highlights
Transthyretin is a 55-kDa tetrameric protein found in blood plasma and cerebrospinal fluid that transports the hormone thyroxine and, via interactions with retinol-binding protein, vitamin A
The role of dimers is suggested by the numerous x-ray studies of transthyretin and its disease-associated variants [14], and it has been hypothesized that the extended -sheet structure, which comprises the -sheet interface, is a starting point for amyloid fibril formation [15, 16]
It is not clear whether a universal mechanism for transthyretin fibril formation will emerge for the various transthyretin variants, the situation is further complicated in vivo, because individuals suffering from familial amyloidotic polyneuropathy are heterozygous, co-expressing both wild-type and variant proteins in the liver [17,18,19]
Summary
Transthyretin is a 55-kDa tetrameric protein found in blood plasma and cerebrospinal fluid that transports the hormone thyroxine and, via interactions with retinol-binding protein, vitamin A (retinol). The role of dimers is suggested by the numerous x-ray studies of transthyretin and its disease-associated variants [14], and it has been hypothesized that the extended -sheet structure, which comprises the -sheet interface, is a starting point for amyloid fibril formation [15, 16] It is not clear whether a universal mechanism for transthyretin fibril formation will emerge for the various transthyretin variants, the situation is further complicated in vivo, because individuals suffering from familial amyloidotic polyneuropathy are (in general) heterozygous, co-expressing both wild-type and variant proteins in the liver [17,18,19]. We describe a novel mass spectrometry (MS) approach whereby the subunit composition of intact wild-type and L55P tetramers was monitored as a function of time by incubating solutions in which one set of homotetramers possessed only natural abundance isotopes and the other was uniformly labeled with
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