Kinetic Stabilization of the Native State by Protein Engineering: Implications for Inhibition of Transthyretin Amyloidogenesis

Foss, Ted R.; Kelker, M.S.; Wiseman, R. Luke; Wilson, Ian A.

doi:10.1016/j.jmb.2005.01.050

Cited by 71 publications

(96 citation statements)

References 72 publications

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Section: Nih-pa Author Manuscriptmentioning

confidence: 93%

“…Generally, rate-limiting tetramer dissociation enables monomer misfolding that is required for TTR to form amyloid (5,(16)(17)(18)(19)(20)(21)(22), by a very efficient downhill polymerization process (23).…”

Section: Published In Final Edited Form Asmentioning

confidence: 99%

“…The thermodynamic linkage of the TTR tetramer dissociation and monomer unfolding equilibria make it challenging to extract thermodynamic data from the urea denaturation curves (7). How disease-associated mutations influence quaternary and tertiary structural stability and how folding and tetramerization energetics correlate with disease phenotypes is some of the valuable information sought herein.Generally, rate-limiting tetramer dissociation enables monomer misfolding that is required for TTR to form amyloid (5,(16)(17)(18)(19)(20)(21)(22), by a very efficient downhill polymerization process (23).Most of the disease-associated TTR mutations form normal tetrameric structures based on crystallographic analysis (24) and function normally, including transporting and binding thyroid hormone and holo retinol binding protein in the plasma and cerebrospinal fluid (CSF). Thus, it is the propensity of these variants to misfold, not their inability to fold and function, that results in disease through a gain of toxic function mechanism(s), the details of which remain elusive but appear to be associated with TTR aggregation (7,17,18,25,26).…”

mentioning

confidence: 99%

“…Resveratrol binding is weakly dependent on urea concentration in this range, suggesting that V30M TTR dissociation intermediates may be able to bind resveratrol. Such a dissociation pathway would be in stark contrast to that of WT TTR, which dissociates into dimers that do not contain a small molecule binding site (16,49).V30M is kinetically stable relative to WT TTR (7), consistent with a more robust smallmolecule-binding dimer interface. The possibility that a slow rate of tetramer dissociation (t ½ = 68 h, compared with 41 h for WT TTR) might be responsible for the biphasic denaturation curves was examined.…”

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confidence: 99%

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

2008

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Urea denaturation studies were carried out as a function of transthyretin (TTR) concentration to quantify the thermodynamically linked quaternary and tertiary structural stability and to better understand the relationship between mutant folding energetics and amyloid disease phenotype. Urea denaturation of TTR involves at least two equilibria-dissociation of tetramers into folded monomers, and monomer unfolding. To deal with the thermodynamic linkage of these equilibria, we analyzed concentration-dependent denaturation data by global fitting to an equation that simultaneously accounts for the two-step denaturation process. Using this method, the quaternary and tertiary structural stabilities of well-behaved TTR sequences, wild type (WT) TTR and the disease-associated variant V122I, were scrutinized. The V122I variant is linked to late onset familial amyloid cardiomyopathy, the most common familial TTR amyloid disease. V122I TTR exhibits a destabilized quaternary structure and a stable tertiary structure relative to WT TTR. Three other variants of TTR were also examined, L55P, V30M, and A25T TTR. The L55P mutation is associated with the most aggressive familial TTR amyloid disease. L55P TTR has a complicated denaturation pathway that includes dimers and trimers, and so globally fitting its concentration-dependent urea denaturation data yielded error-laden estimates of stability parameters. Nevertheless, it is clear that L55P TTR is substantially less stable than WT TTR, primarily because its tertiary structure is unstable, although its quaternary structure is destabilized as well. V30M is the most common mutation associated with neuropathic forms of TTR amyloid disease. V30M TTR is certainly destabilized relative to WT TTR, but like L55P TTR it has a complex denaturation pathway that cannot be fit to the aforementioned two-step denaturation model. Literature data suggest that V30M † This research was supported by NIH Grant DK46335, the Skaggs Institute for Chemical Biology, and the Lita Annenberg Hazen Foundation. A.R.H.B. was supported by NIH Grants T32-AG00080 and F32-GM067348. *To whom correspondence should be addressed. Phone: (858) 784-9601; Fax: (858) 784-9610; E-mail: epowers@scripps.edu, jkelly@scripps.edu. 1 Abbreviations. TTR, transthyretin; WT, wild-type; M-TTR, a monomeric variant of transthyretin harboring F87M and L110M mutations; SSA, senile systemic amyloidosis; FAP, familial amyloid polyneuropathy; FAC, familial amyloid cardiomyopathy; Tris, Tris (hydroxymethyl)aminomethane; EDTA, ethylenediamine-N,N,N′,N′-tetraacetic acid; DTT, dithiothreitol; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CSF, cerebrospinal fluid. SUPPORTING INFORMATION AVAILABLEPlots showing the global fit of our three state model (native tetramer, folded monomer, unfolded monomer) to the concentration-dependent urea denaturation data for WT, V122I, and L55P TTR, and a plot showing the relatively poor global fit to a two state model (native tetramer, unfolded monomer) for WT TTR. This material is ...

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Section: Nih-pa Author Manuscriptmentioning

confidence: 93%

Section: Published In Final Edited Form Asmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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

2008

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“…WT, V122I, V30M, and dual-FLAG-tagged TTR (FT) 4 were expressed and purified from Escherichia coli as described in ref. 51. TTR (400 l of 4.5 M tetramer; 0.25 mg͞ml) was preincubated with genistein at either 4.5 or 9.0 M for 18 h at 25°C.…”

Section: Methodsmentioning

confidence: 99%

Genistein, a natural product from soy, is a potent inhibitor of transthyretin amyloidosis

Green

Foss

Kelly

2005

Proc. Natl. Acad. Sci. U.S.A.

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The misfolding of transthyretin (TTR), including rate-limiting tetramer dissociation and partial monomer denaturation, is sufficient for TTR misassembly into amyloid and other abnormal quaternary structures associated with three amyloid diseases: senile systemic amyloidosis, familial amyloid polyneuropathy, and familial amyloid cardiomyopathy. Small molecules can bind to one or both of the unoccupied TTR thyroid hormone-binding sites, stabilizing the native tetramer more than the dissociative transition state, thereby raising the kinetic barrier for tetramer dissociation. Herein we demonstrate that genistein, the major isoflavone natural product in soy, works in this fashion and is an excellent inhibitor of transthyretin tetramer dissociation and amyloidogenesis, reducing acid-mediated fibril formation to <10% of that exhibited by TTR alone. Genistein also inhibits the amyloidogenesis of the most common familial amyloid polyneuropathy and familial amyloid cardiomyopathy mutations in TTR: V30M and V122I, respectively. Genistein additionally inhibits tetramer dissociation under physiological conditions thought to lead to slow amyloidogenesis in humans. Furthermore, this natural product exhibits highly selective binding to TTR in plasma over all of the other plasma proteins. Isothermal titration calorimetry shows that genistein binds to TTR with negative cooperativity (K d1 ‫؍‬ 40 nM, Kd2 ‫؍‬ 1.4 M). The benefits of using a nutraceutical such as genistein to treat orphan diseases such as the TTR amyloidoses include known oral bioavailability and safety data. It is conceivable that some patients could benefit from simply increasing their intake of soy products or supplements.amyloidogenesis inhibitor ͉ kinetic stabilization F or many years nutritionists and dieticians have noted the health benefits of a soy-based diet, citing the much lower incidence of cancer, including breast cancer, in Asian countries (1-4). The isoflavone genistein (compound 1 in Fig. 1), found in various soy foods at concentrations of 1.9-229 g͞g, is the component of soy implicated in cancer chemoprevention (5). An additional 71-968 g͞g of genistein is present as its O-glucoside conjugate, genistin (2), which is rapidly deglycosylated by intestinal bacteria in vivo. Genistein is being evaluated in preliminary trials for treatment of breast, prostate, and uterine cancers (6, 7), as well as for osteoporosis (8), cardiovascular disease (9), and treatment of menopausal symptoms (10). Toxicity studies reveal that this isoflavone does not appear to cause adverse health effects, even at the relatively high concentrations used (11-13). The isoflavone daidzein (3), lacking the hydroxyl group at the 5 position of genistein, is also found in soy foods, but no chemoprotective effects have been attributed to it.Transthyretin (TTR) is a tetrameric protein composed of identical 127-aa ␤-sheet sandwich subunits (14, 15). TTR functions to transport holo-retinol-binding protein and thyroxine (T4) in the blood and cerebrospinal fluid (16,17). Under denaturing con...

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Understanding and Ameliorating the TTR Amyloidoses

Johnson

Wiseman

Paulsson

et al. 2010

Protein Misfolding Diseases

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Kinetic Stabilization of the Native State by Protein Engineering: Implications for Inhibition of Transthyretin Amyloidogenesis

Cited by 71 publications

References 72 publications

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

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

Genistein, a natural product from soy, is a potent inhibitor of transthyretin amyloidosis

Understanding and Ameliorating the TTR Amyloidoses

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