What is transthyretin?

Transthyretin (TTR), previously called “prealbumin,” is a protein that transports thyroxine and retinol. It is synthesized in the liver, found mainly in plasma, and makes up a major component (25%) of cerebrospinal fluid (CSF).¹ It is also found in the vitreous humor of the eye, but does not cross the blood-brain barrier.²

Transthyretin Structure

Transthyretin is a 55 kDa protein that is a native homotetramer with two thyroxine-binding sites in

Transthyretin Tetramer with T4 Thyroxine Binding Channel

a 50 Å channel located at the center of the molecule.³ Two monomers join edge-to-edge via hydrogen bonding to form a dimer.² Two dimers then join to form a tetramer via hydrophobic interactions.³

Transthyretin in Diseases

Transthyretin can aggregate into fibrils and get deposited in the heart (cardiomyopathy) and nervous system (neuropathy). This is known as transthyretin amyloidosis. There are several variations, depending on where the TTR gets deposited and whether the disease is familial.

• Familial Amyloid Polyneuropathy (FAP) is associated with the V30M mutation and is inherited in an autosomal dominant pattern. It is caused by the accumulation of TTR amyloid around peripheral nerves.² The average age of onset is in the fourth decade of life.²
• Familial Amyloid Cardiomyopathy (FAC) is caused by the accumulation of TTR in the heart, and is associated with a variety of mutations.
• Senile Systemic Amyloidosis (SSA) is similar to FAC, but is caused by the accumulation of WT TTR rather than mutant TTR.⁴ The age of onset is usually over 60, and over 25% of people over the age of 80 have cardiac deposition of WT TTR.⁵ This deposition leads to heart failure.

Mechanisms of TTR Aggregation

TTR aggregation is thought to begin with the tetramer dissociating into monomers, which are prone to misfolding and self-aggregation.⁶ This aggregation occurs in acidic conditions similar to those found in lysosomes.^7,8 The misfolded monomers assemble into fibrils via oligomers and protofibrils.⁹ The rate-limiting step is the tetramer disassembly; therefore, fibril formation rates are determined by tetramer dissociation rates.¹⁰

Transthyretin Misfolding and Aggregation Mechanism

Not all monomers continue on the path to aggregation; they can also refold properly and combine to form dimers, which then associate to form tetramers.¹¹

TTR Mutations and Aggregation

Factors that promote TTR tetramer dissociation and monomer aggregation can be better understood by investigating the effects of both disease-causing and protective mutations. Over 100 TTR mutations are known to be amyloidogenic.¹² There is a “hot spot region” between amino acid residues 42 and 58, but amyloidogenic mutations can span the entire protein.^13,14 The V30M mutation, common in Portuguese, Swedish, and Japanese populations, is associated with autosomal dominant familial amyloid polyneuropathy. The V30M TTR tetramer is less stable than the WT TTR tetramer, but dissociates into monomers more slowly.¹⁰  This slow dissociation rate is associated with low disease penetrance that increases with age.¹⁵ V30M TTR aggregation is also influenced by the rate at which unfolded monomers can refold into dimers and tetramers. The rate of refolding is slow, which leads to the accumulation of monomers than then form fibrils.¹⁶

The L55P mutation is rare, but produces aggressive amyloidosis with severe pathology. This is because the L55P mutation lowers the activation barrier for the dissociation of a tetramer to monomer, which allows for dissociation under physiological conditions.¹⁷  This lowering of the activation barrier occurs because the starting tetrameric state is destabilized to a greater extent than the transition state.¹⁰

Conversely, the T119M mutation is protective against amyloidosis; individuals with both the V30M and T119M mutations do not develop FAP.¹⁸ This is mirrored by T119M TTR’s reluctance to aggregate in solution when combined with V30M TTR.¹⁸ T119M tetramers are very slow to dissociate due to an increased activation barrier that prevents tetramers from dissociating into monomers.¹⁹ This activation barrier is the result of a stabilized quaternary structure of the tetramer and a destabilized dissociative transition state.²⁰ 

The T119M mutation prevents aggregation by increasing the activation energy required for the tetramer to dissociate into monomers.

Disease-preventing mutations, such as T119M and R104H add hydrophobic residues to the thyroxine (T₄)-binding cavity, which establishes contact between amino acid residues of different dimers and prevents dissociation.²¹

TTR is present in the body throughout the human lifespan, but, in the absence of mutations, does not aggregate until later in life. There are a variety of aging-associated processes that could contribute to its aggregation, including oxidative stress, mitochondrial dysfunction, inflammation, and metabolic dysregulation.²²

Transthyretin Toxicity

Like other diseases, the smaller TTR oligomers are thought to be more toxic than fibrils.² These toxic oligomers are thought to be less than 100 kDa, consisting of 6-10 monomers.^4,23,24 These small species can bind to lipid membranes, disrupting lipid rafts and activating voltage-gated calcium channels.²⁴ The subsequent calcium influx can induce cytotoxicity.²⁴

Structure of WT TTR complexed with tafamidis. Source: PDB ID: 3TCT.³¹

Transthyretin Fibril Seeding

Ex-vivo transthyretin fibrils have been shown to recruit WT transthyretin into aggregates.²⁵  This seeded polymerization mechanism could provide an alternate route for TTR aggregation, and could explain why in some cases stabilizing tetramers is insufficient to prevent disease progression.²⁵

Therapeutics Targeting Transthyretin

Because mutant TTR is synthesized by the liver, liver transplantation is used to treat familial amyloid diseases by replacing mutant TTR with WT TTR.  However, sometimes TTR continues to accumulate in the heart²⁶ and eyes.²⁷ Tafamidis and diflunisal are TTR kinetic stabilizers that have been associated with improved survival in cardiac amyloidosis patients and function by binding in the T₄ binding channel.^28-30

Other therapeutic approaches include gene silencing therapies to prevent the expression of amyloidogenic mutations. Certain flavonoids and other small molecules inhibit TTR oligomerization,^32-34 and doxycycline combined with Tauroursodeoxycholic acid (TUDCA) has been shown to disrupt TTR fibrils in mouse models.³⁵ Immunization with Y78F mutant TTR has also been shown to reduce TTR deposition in transgenic mice.³⁶

Transthyretin Fibrils

StressMarq offers L55P and Y78F mutant TTR monomers and filaments for researchers to study the mechanisms of TTR aggregation and methods to inhibit fibrillization and reduce toxicity.

TEM of Human Recombinant Transthyretin L55P Variant Protein Filaments (SPR-464)

References

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