The amyloidoses are diseases of protein conformation, in which a particular soluble innocuous protein transforms and aggregates into an insoluble fibrillar structure that deposits in extracellular spaces of specific organs (reviewed in refs. 1–4). Organ dysfunction accompanies fibrillar deposition, and the amyloid hypothesis proposes a cause and effect relationship between deposition and dysfunction (reviewed in refs. 4–7). Transthyretin (TTR) is one of 20 proteins that are known currently to form fibrillar deposits in human amyloidoses (1, 8). TTR is a tetrameric protein comprised of four identical subunits that contain two antiparallel β-sheets packed together in a β-sandwich architecture. It is synthesized primarily in the liver and is the primary transport protein for thyroid hormone (l-thyroxine or T4) in cerebrospinal fluid and the secondary l-thyroxine transporter in blood plasma. With the assistance of the retinol binding protein (RBP), TTR also transports vitamin A (all-trans-retinol) in plasma. RBP bound to vitamin A forms multimolecular complexes with TTR, and under physiological conditions, dissociation of vitamin A causes the complexes to disassemble. Both natural sequence TTR and mutated variants of TTR are involved in amyloid disease. In certain elderly individuals, natural sequence TTR is known to transform into amyloid fibrils that deposit in cardiac and other tissues, giving rise to the condition known as senile systemic amyloidosis. The occurrence of mutations in TTR accelerates the process of TTR fibrillogenesis and is the most important risk factor for TTR amyloidosis. Whereas deposition of amyloid fibrils of variant TTR in cardiac tissue produces the condition familial amyloidotic cardiomyopathy, deposition in peripheral nerve tissue produces familial amyloid polyneuropathy. There are more than 80 TTR variants that are known currently to give rise to TTR amyloidoses (8). The involvement of TTR in the pathology of amyloid disease is well established; however, the cause and effect relationship between TTR amyloid deposition and organ dysfunction has not yet been proven. In three papers published recently (9–11), one of which appears in this issue of PNAS (9), Jeffery Kelly and his colleagues from The Scripps Research Institute report results from in vitro studies that provide a biophysical explanation of how disease-associated mutations in TTR affect the course of TTR amyloidoses, and thus strengthen the hypothesis that amyloid fibril deposition is the causative agent in these diseases.
Jeffery Kelly and his colleagues report results from in vitro studies that provide a biophysical explanation of how disease-associated mutations in TTR affect the course of TTR amyloidoses.
Although TTR amyloid deposition is known to occur in extracellular spaces, fibrillogenesis may initiate in acidic environments of endosomes or lysosomes (12, 13). In 1992, Colon and Kelly (12) reported that incubation of TTR in low-pH environments is all that is required to initiate the fibrillogenesis reaction. Since then the acid-induced denaturation/fibrillogenesis pathway of TTR has been mapped out in great detail with a variety of biophysical and biochemical techniques (Fig. 1). Under mild acidic conditions (pH 5.75), tetrameric wild-type TTR can be induced to partially dissociate into monomers by dilution (14, 15). Hydrogen-deuterium exchange experiments indicate that the dissociated monomers at pH 5.75 retain a native-like structure that is present in the tetramer (15). Further acidification to pH 4.5 induces greater monomer formation; however, the structures of the pH 4.5 monomers show conformational changes indicative of partial unfolding (12, 14, 16). Hydrogen-deuterium exchange experiments indicate that the conformational instability is localized to 13 residues of the CBEF β-sheet of TTR; the other β-sheet (DAGH) is stable and shows similar protection from hydrogen-deuterium exchange as tetrameric TTR (15). Amyloid fibril formation ensues at this moderately low pH if the temperature and protein concentration are sufficiently high (12, 14, 16). Further reductions in pH reduce the rate of fibril formation and favor the formation of molten globule-like acid-denatured states (A-states) that form low molecular weight aggregates that are not amyloid fibrils (14, 16). The partially denatured monomeric state of TTR that is populated at pH 4.5 appears to be the critical precursor to amyloid fibril formation, and it has been named the amyloidogenic intermediate. The presence of mutations in TTR associated with amyloidosis greatly affects the acid-induced denaturation/fibrillogenesis pathway (13, 14, 16), the major effect being an increased tendency to form the amyloidogenic intermediate at higher pH values.
Figure 1.
Acid-induced denaturation/fibrillogenesis pathway of TTR.
In their most recent work on TTR amyloidosis, Kelly and colleagues investigate the V122I variant of TTR (9). This variant is the most common TTR mutation producing familial amyloidotic cardiomyopathy (17). It originated in West Africa and is carried by 3.9% of African Americans and >5% of individuals in some areas of West Africa. The major effect of this mutation, which in chemical terms represents the addition of a single methylene group, is to destabilize the quaternary structure of TTR while leaving the tertiary structure unaffected. Demonstration of this differential destabilization required the engineering of a TTR variant that was monomeric but otherwise behaved similarly to natural sequence TTR. Two conservative mutations in the subunit interfaces proved sufficient to generate a normally folded monomeric variant of TTR (18). The x-ray crystal structure of monomeric TTR revealed that it had near-identical tertiary structure to natural sequence TTR (18). Furthermore, the monomeric variant was similar to its tetrameric counterpart in its ability to bind retinol binding protein and show acid-induced fibrillogenesis. Introduction of the V122I mutation into the monomeric variant had no detectable effect on either conformational stability or the propensity to form amyloid fibrils. These results indicate that the effects of this mutation are manifested at the level of the tetramer (9). Comparison of the tetrameric V122I variant with tetrameric natural sequence TTR, on the other hand, revealed many differences. Relative to the natural sequence protein, the V122I variant displayed greater rates of tetramer to monomer dissociation and a lower level of quaternary structural stability. These alterations lead to monomeric species being populated over a broader pH range. The end result of the increases in the rate and extent of tetramer dissociation of the V122I variant is a greater rate of fibrillogenesis and a broader pH range where fibrillogenesis ensues. These in vitro results support the amyloid hypothesis, which in this case would predict that the V122I mutation causes familial amyloidotic cardiomyopathy by facilitating amyloid fibril production.
Using a similar in vitro approach, White and Kelly (11) have recently examined another genetic aspect of TTR amyloidosis. Familial TTR amyloidoses are autosomal dominant diseases; however, they show large variations in penetrance. Individuals harboring amyloidosis-associated TTR mutations show large variations in the age of onset of symptoms, which depend on mutation type as well as other factors such as geographical location and sex. These findings have led to the suggestion that the product of several different genes influences the pathology of TTR amyloidosis. White and Kelly (11) proposed the hypothesis that genes that control the concentrations of molecules that interact with TTR affect the size of the pool of TTR molecules that are prone to form amyloid, thus accounting for the multigenic nature of TTR amyloidosis. In support of this hypothesis, they show that retinol binding protein, vitamin A, and l-thyroxine can act synergistically to inhibit the rate and extent of fibrillogenesis in vitro. Approximately 70 genes were estimated to control the levels of these TTR interacting molecules. Perhaps studies examining the expression levels of these candidate genes will show correlations with age of symptom onset and provide further support for the multigenic hypothesis.
The V30M variant of TTR was the first amyloidogenic mutant discovered and it is also the most common. The T119M variant, on the other hand, is nonamyloidogenic. However, compound heterozygotic individuals who express both V30M and T119M variants show a much more benign form of the disease with delayed onset, indicating that coexpression of the T119M variant provides a protective effect (19). TTR purified from the serum of a compound heterozygotic individual expressing both V30M and T119M variants displays stability comparable to natural sequence TTR (20). The x-ray crystal structure of TTR from the serum of the compound heterozygote has been reported recently (21). The crystal was composed of a heterogeneous mixture of mixed tetramers composed of both variants. The structure of these mixed tetramers possessed greater numbers of noncovalent interactions both between and within monomers, consistent with their increased stability. Kelly and coworkers have recently investigated the fibrillogenesis potential of different hetero-tetrameric forms of V30M and T119M variants (10). Using a procedure they developed to form, separate, and purify different hetero-tetramers of TTR (22), Kelly and coworkers produced all combinations of hetero- and homo-tetramers of V30M and T119M variants. Although homo-tetramers of V30M variant exhibited the greatest extent of fibril formation, homo-tetramers of T119M variant were the least fibrillogenic, and the mixed tetramers displayed a decreasing gradation of intermediate values that were proportional to the mole fraction of T119M variant present. The unexpected result was that the mixed tetramer containing three subunits of V30M variant and a single subunit of T119M variant possessed the same fibrillogenic potential as homo-tetrameric natural sequence TTR. In addition to providing a biophysical explanation of the protective effects of the T119M mutation, this result suggests a potential therapeutic strategy in which T119M variant could be delivered to TTR amyloidosis patients through either gene transfer methods or direct administration (10).
In addition to providing much evidence for the amyloid hypothesis, the in vitro studies of TTR fibrillogenesis have helped us better understand the mechanism of amyloid fibril formation. Recent work has demonstrated that in addition to the 20 proteins associated with amyloid diseases, many other proteins can be induced to form amyloid fibrils (23, 24). The conversions of myoglobin (25) and cytochrome c552 (26) into amyloid fibrils provide excellent examples of this phenomenon. Although the sequences and native structures of the amyloid proteins are vastly different, the morphology and properties of all amyloid fibrils are remarkably similar. Furthermore, amyloid fibrils composed of different component proteins give similar high-resolution x-ray fiber diffraction patterns that are consistent with a helical array of β-sheets, in which the hydrogen bonding direction is parallel and the strands are perpendicular to the major axis of the fiber (27). These striking similarities have led to the proposal that all amyloid fibrils share a common core structure, irrespective of the sequence or size of their constituent proteins (27). Given that all amyloid fibrils have the same core structure, are the fibrillogenesis pathways conserved as well? Current evidence suggests they are not. TTR fibrillogenesis proceeds through the formation of a monomeric amyloidogenic intermediate, the structure of which appears to contain native-like subdomains as well as non-native dynamic regions (15). Amyloid fibril formation by acylphosphatase initiates from an ensemble of denatured conformations in environments that favor noncovalent interactions (28). Before fibril formation, the Alzheimer amyloid peptide (Aβ) associates into small oligomers ranging from dimers to tetramers (29, 30). Other intermediates of Aβ fibrillogenesis include soluble spherical aggregates (29–32) and protofibrils (32, 33). Transient formation of spherical aggregates has also been observed during fibrillogenesis of α-synuclein (34) and islet amyloid polypeptide (35). Certain short amyloidogenic peptides that are unstructured in their nonaggregated states form amyloid through all-or-none processes (36, 37). This diversity in fibril formation pathways implies that strategies to inhibit one form of amyloid may not work for others, thus making it necessary for inhibitors of individual amyloidosis to be tailor made.
Footnotes
See companion article on page 14943.
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