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1.
Curr Drug Targets CNS Neurol Disord ; 4(5): 587-96, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16266291

RESUMEN

In transthyretin (TTR) amyloidosis TTR variants deposit as amyloid fibrils giving origin, in most cases, to peripheral polyneuropathy, cardiomyopathy, carpal tunnel syndrome and/or amyloid deposition in the eye. More than eighty TTR variants are known, most of them being pathogenic. The mechanism of TTR fibril formation is still not completely elucidated. However it is widely accepted that the amino acid substitutions in the TTR variants contribute to a destabilizing effect on the TTR tetramer molecule, which in particular conditions dissociate into non native monomeric intermediates that aggregate and polymerize in amyloid fibrils that further elongate. Since this is a multi-step process there is the possibility to impair TTR amyloid fibril formation at different stages of the process namely by tetramer stabilization, inhibition of fibril formation or fibril disruption. Till now the only efficient therapy available is liver transplant when performed in an early phase of the onset of the disease symptoms. Since this is a very invasive therapy alternatives are desirable. In that sense, several compounds have been proposed to impair amyloid formation or disruption. Based on the proposed mechanism for TTR amyloid fibril formation we discuss the action of some of the proposed TTR stabilizers such as derivatives of some NSAIDs (diflunisal, diclofenac, flufenamic acid, and derivatives) and the action of amyloid disrupters such as 4'-iodo-4'-deoxydoxorubicin (I-DOX) and tetracyclines. Among all these compounds, TTR stabilizers seem to be the most interesting since they would impair very early the process of amyloid formation and could also have a prophylactic effect.


Asunto(s)
Amiloide/metabolismo , Amiloidosis/tratamiento farmacológico , Prealbúmina/metabolismo , Pliegue de Proteína , Amiloidosis/metabolismo , Animales , Humanos , Prealbúmina/farmacología , Isoformas de Proteínas
2.
Curr Med Chem ; 12(21): 2499-515, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-16250874

RESUMEN

Xanthones, synthesized or isolated from a natural source, display a wide range of biological and pharmacological activities. In a few cases, their chemical characterization has involved the structure elucidation by single crystal X-ray diffraction. The purpose of this review is to assess in detail this three-dimensional structural data, and thus contribute to a better understanding of the molecular mechanisms involved in the different biological activities presented by xanthones.


Asunto(s)
Xantonas/química , Cristalografía , Modelos Moleculares , Estructura Molecular
3.
J Mol Biol ; 317(5): 683-95, 2002 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-11955017

RESUMEN

Extracellular accumulation of transthyretin (TTR) variants in the form of fibrillar amyloid deposits is the pathological hallmark of familial amyloidotic polyneuropathy (FAP). The TTR Leu55Pro variant occurs in the most aggressive forms of this disease. Inhibition of TTR wild-type (WT) and particularly TTR Leu55Pro fibril formation is of interest as a potential therapeutic strategy and requires a thorough understanding of the fibril assembly mechanism. To this end, we report on the in vitro assembly properties as observed by transmission electron microscopy (TEM), atomic force microscopy (AFM) and quantitative scanning transmission electron microscopy (STEM) for both TTR WT fibrils produced by acidification, and TTR Leu55Pro fibrils assembled at physiological pH. The morphological features and dimensions of TTR WT and TTR Leu55Pro fibrils were similar, with up to 300 nm long, 8 nm wide fibrils being the most prominent species in both cases. Other species were evident; 4-5 nm wide fibrils, 9-10 nm wide fibrils and oligomers of various sizes. STEM mass-per-length (MPL) measurements revealed discrete fibril types with masses of 9.5 and 14.0(+/-1.4) KDa/nm for TTR WT fibrils and 13.7, 18.5 and 23.2(+/-1.5) kDa/nm for TTR Leu55Pro fibrils. These MPL values are consistent with a model in which fibrillar TTR structures are composed of two, three, four or five elementary protofilaments, with each protofilament being a vertical stack of structurally modified TTR monomers assembled with the 2.9 nm axial monomer-monomer spacing indicated by X-ray fibre diffraction data. Ex vivo TTR amyloid fibrils were examined. From their morphological appearance compared to these, the in vitro assembled TTR WT and Leu55Pro fibrils examined may represent immature fibrillar species. The in vitro system operating at physiological pH for TTR Leu55Pro and the model presented for the molecular arrangement of TTR monomers within fibrils may, therefore, describe early fibril assembly events in vivo.


Asunto(s)
Modelos Moleculares , Placa Amiloide/química , Placa Amiloide/ultraestructura , Prealbúmina/química , Prealbúmina/metabolismo , Neuropatías Amiloides Familiares/metabolismo , Humanos , Leucina/química , Microscopía de Fuerza Atómica , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Peso Molecular , Prealbúmina/ultraestructura , Prolina/química , Unión Proteica , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Estructura Cuaternaria de Proteína , Factores de Tiempo
4.
Acta Crystallogr C ; 57(Pt 11): 1319-23, 2001 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-11706262

RESUMEN

In order to study structure-activity relationships, a series of mono-, di- and trioxygenated xanthones has been synthesized and the structures of methyl 2-(3,4-dimethoxyphenoxy)benzoate, C(16)H(16)O(5), 2-(3,4-dimethoxyphenoxy)benzoic acid, C(15)H(14)O(5), 1,2-dimethoxy-9H-xanthen-9-one, C(15)H(12)O(4), and 1,2,8-trimethoxy-9H-xanthen-9-one, C(16)H(14)O(5), have been determined. The first two compounds both assume skew conformations, the dihedral angles between the two phenyl rings being 80.04 (8) and 83.0 (1) degrees, respectively. The latter two compounds are essentially planar and their methoxy substituents assume orientations consistent with minimum steric interactions.


Asunto(s)
Compuestos de Bifenilo/química , Éteres/química , Plantas/química , Xantenos/química , Xantonas , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Conformación Molecular
5.
J Nat Prod ; 64(8): 1056-8, 2001 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-11520226

RESUMEN

Extraction of the marine sponge Tetilla japonica from the Bay of Thailand furnished tetillapyrone and nortetillapyrone, two unusual tetrahydrofurylhydroxypyran-2-ones, whose structures were established by NMR spectrometry and an X-ray analysis of tetillapyrone.


Asunto(s)
Poríferos/química , Pironas/aislamiento & purificación , Animales , Espectroscopía de Resonancia Magnética , Conformación Molecular , Estructura Molecular , Pironas/química , Espectrofotometría Ultravioleta , Tailandia , Difracción de Rayos X
6.
J Mol Biol ; 306(4): 733-44, 2001 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-11243784

RESUMEN

Transthyretin (TTR) amyloidosis is a conformational disturbance, which, like other amyloidoses, represents a life threat. Here, we report a TTR variant, TTR Thr119Met, that has been shown to have a protective role in the development of clinical symptoms in carriers of TTR Val30Met, one of the most frequent variants among TTR amyloidosis patients. In order to understand this effect, we have determined the structures of the TTR Val30Met/Thr119Met double mutant isolated from the serum of one patient and of both the native and thyroxine complex of TTR Thr119Met. Major conclusions are: (i) new H-bonds within each monomer and monomer-monomer inter-subunit contacts, e.g. Ser117-Ser117 and Met119-Tyr114, increase protein stability, possibly leading to the protective effect of the TTR Val30Met/Thr119Met variant when compared to the single variant TTR Val30Met. (ii) The mutated residue (Met119) extends across the thyroxine binding channel inducing conformational changes that lead to closer contacts between different dimers within the tetramer. The data, at atomic resolution, were essential to detect, for the first time, the subtle changes in the inter-subunit contacts of TTR, and explain the non-amyloidogenic potential of the TTR Thr119Met variant, improving considerably current research on the TTR amyloid fibril formation pathway.


Asunto(s)
Amiloidosis/metabolismo , Prealbúmina/química , Prealbúmina/metabolismo , Sustitución de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Mutación , Prealbúmina/genética , Unión Proteica , Conformación Proteica , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tiroxina/química , Tiroxina/metabolismo
7.
J Mol Biol ; 304(3): 461-70, 2000 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-11090287

RESUMEN

Familial Amyloidotic Polyneuropathy (FAP) is caused by the assembly of TTR into an insoluble beta-sheet. The TTR tetramer is thought to dissociate into monomeric intermediates and subsequently polymerise into the pathogenic amyloid form. The biochemical mechanism behind this transformation is unknown. We characterised intermediate TTR structures in the in vitro amyloidogenesis pathway by destabilising the AB loop through substitution of residue 78. Changes at this residue, should destabilise the TTR tetrameric fold, based on the known crystallographic structure of a Leu55Pro transthyretin variant. We generated a soluble tetrameric form of TTR that is recognised by a monoclonal antibody, previously reported to react only with highly amyloidogenic mutant proteins lacking the tetrameric native fold and with amyloid fibrils. BIAcore system analysis showed that Tyr78Phe had similar binding properties as synthetic fibrils. The affinity of this interaction was 10(7) M(-1). We suggest that the tetrameric structure of Tyr78Phe is altered due to the loosening of the AB loops of the tetramer, leading to a structure that might represent an early intermediate in the fibrillogenesis pathway.


Asunto(s)
Epítopos/inmunología , Placa Amiloide/química , Placa Amiloide/inmunología , Prealbúmina/química , Prealbúmina/inmunología , Sustitución de Aminoácidos/genética , Anticuerpos Monoclonales/inmunología , Cromatografía Líquida de Alta Presión , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Humanos , Enlace de Hidrógeno , Sueros Inmunes/inmunología , Cinética , Modelos Moleculares , Mutación/genética , Placa Amiloide/genética , Placa Amiloide/metabolismo , Prealbúmina/genética , Prealbúmina/metabolismo , Pruebas de Precipitina , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Solubilidad , Resonancia por Plasmón de Superficie
8.
Biochem J ; 351(Pt 1): 273-9, 2000 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-10998371

RESUMEN

The crystal structure of the amyloidogenic Leu-55Pro transthyretin (TTR) variant has revealed an oligomer structure that may represent a putative amyloid protofibril [Sebastião, Saraiva and Damas (1998) J. Biol. Chem. 273, 24715-24722]. Here we report biochemical evidence that corroborates the isolation of an intermediate structure, an 'amyloid-like' oligomer, which is most probably present in the biochemical pathway that leads to amyloid deposition and that was isolated by the crystallization of the Leu-55Pro TTR variant. 4'-Iodo-4'-deoxydoxorubicin (IDOX) is a compound that interacts with amyloid fibrils of various compositions and it has been reported to reduce the amyloid load in immunoglobulin light chain amyloidosis [Merlini, Ascari, Amboldi, Bellotti, Arbustini, Perfetti, Ferrari, Zorzoli, Marinone, Garini et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 2959-2963]. In this work, we observed that the monoclinic Leu-55Pro TTR crystals, soaked with IDOX, undergo rapid dissociation. Moreover, under the same conditions, the orthorhombic wild-type TTR crystals are quite stable. This is explained by the different TTR conformations isolated upon crystallization of the two proteins; while the Leu-55Pro TTR exhibits the necessary conformation for IDOX binding, the same structure is not present in the crystallized wild-type protein. A theoretical model concerning the interaction of Leu-55Pro TTR with IDOX, which is consistent with the dissociation of the amyloid-like oligomer, is presented. In this model the IDOX iodine atom is buried in a pocket located between the two beta-sheets of the Leu-55Pro TTR monomer with the IDOX aromatic-moiety long axis nearly perpendicular to the direction of the beta-sheets.


Asunto(s)
Sustitución de Aminoácidos/genética , Amiloidosis/metabolismo , Doxorrubicina/análogos & derivados , Doxorrubicina/metabolismo , Leucina/metabolismo , Prealbúmina/química , Prealbúmina/metabolismo , Prolina/metabolismo , Benzotiazoles , Cristalización , Doxorrubicina/química , Doxorrubicina/farmacología , Humanos , Yodo/metabolismo , Leucina/genética , Modelos Moleculares , Prealbúmina/antagonistas & inhibidores , Prealbúmina/genética , Prolina/genética , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacos , Tiazoles/metabolismo , Difracción de Rayos X
9.
J Struct Biol ; 130(2-3): 290-9, 2000 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-10940233

RESUMEN

Transthyretin amyloidosis represents a spectrum of clinical syndromes that, in all cases except senile systemic amyloidosis, are dependent on the mutation present in the transthyretin (TTR) protein. Although the role of amyloid deposits in the pathogenesis of the disease is not clear, preventing their formation or promoting their disaggregation is necessary to control the development of clinical symptoms. The design of therapies aiming at preventing amyloid formation or promoting its dissociation requires detailed knowledge of the fibrils' molecular structure and a complete view about the factors responsible for protein aggregation. This review is focused on the structural studies, performed on amyloid fibrils and amyloidogenic TTR variants, aiming at understanding the aggregation mechanism as well as the atomic structure of the fibril assembly. Based on the available information possible therapies are also surveyed.


Asunto(s)
Amiloidosis/metabolismo , Prealbúmina/química , Amiloidosis/terapia , Dimerización , Variación Genética , Humanos , Modelos Moleculares , Prealbúmina/genética , Prealbúmina/ultraestructura , Conformación Proteica
10.
Biochem J ; 348 Pt 1: 167-72, 2000 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-10794728

RESUMEN

The molecular mechanisms that convert soluble transthyretin (TTR) tetramers into insoluble amyloid fibrils are still unknown; dissociation of the TTR tetramer is a pre-requisite for amyloid formation in vitro and involvement of monomers and/or dimers in fibril formation has been suggested by structural studies. We have designed four mutated proteins with the purpose of stabilizing [Ser(117)-->Cys (S117C) and Glu(92)-->Cys (E92C)] or destabilizing [Asp(18)-->Asn (D18N) and Leu(110)-->Ala (D110A)] the dimer/tetramer interactions in TTR, aiming at elucidating structural determinants in amyloidogenesis. The resistance of the mutated proteins to dissociation was analysed by HPLC studies of diluted TTR preparations. Both 'stabilized' mutants migrated as tetramers and, upon dilution, no other TTR species was observed, confirming the increased resistance to dissociation. For the 'destabilized' mutants, a mixture of tetrameric and monomeric forms co-existed at low dilution and the latter increased upon 10-fold dilution. Both of the destabilizing mutants formed amyloid in vitro when acidified. This result indicated that both the AB loop of TTR, destabilized in D18N, and the hydrophobic interactions affecting the dimer-dimer interfaces in L110A are implicated in the stability of the tetrameric structure. The stabilized mutants, which were dimeric in nature through disulphide bonding, were unable to polymerize into amyloid, even at pH 3.2. When the amyloid formation assay was repeated in the presence of 2-mercaptoethanol, upon disruption of the S-S bridges of these stable dimers, amyloid fibril formation was observed. This experimental evidence suggests that monomers, rather than dimers, are the repeating structural subunit comprising the amyloid fibrils.


Asunto(s)
Prealbúmina/química , Estructura Cuaternaria de Proteína , Neuropatías Amiloides/genética , Biopolímeros/química , Dimerización , Escherichia coli , Modelos Moleculares , Prealbúmina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
11.
Eur J Biochem ; 267(8): 2307-11, 2000 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-10759855

RESUMEN

Different low molecular mass ligands have been used to identify amyloid deposits. Among these markers, the dyes Thioflavin T and Congo Red interact specifically with the beta-sheet structure arranged in a cross-beta conformation, which is characteristic of amyloid. However, the molecular details of this interaction remain unknown. When labelled with technetium-99m, the proteinase inhibitor aprotinin has been shown to represent a very important radiopharmaceutical agent for in vivo imaging of extra-abdominal deposition of amyloid in amyloidosis of the immunoglobulin type. However, no information is available as to whether aprotinin binds other types of amyloid fibrils and on the nature and characteristics of the interaction. The present work shows aprotinin binding to insulin, transthyretin, beta-amyloid peptide and immunoglobulin synthetic amyloid fibrils by a specific dot-blot ligand-binding assay. Aprotinin did not bind amorphous precipitates and/or the soluble fibril precursors. A Ka of 2.9 microM-1 for the binding of aprotinin to insulin amyloid fibrils was determined by Scatchard analysis. In competition experiments, analogues such as an aprotinin variant, a spermadhesin and the soybean trypsin inhibitor were tested and results suggest that both aprotinin and the spermadhesin interact with amyloid fibrils through pairing of beta-sheets of the ligands with exposed structures of the same type at the surface of amyloid deposits. An electrostatic component may also be involved in the binding of aprotinin to amyloid fibrils because important differences in binding constants are observed when substitutions V15L17E52 are introduced in aprotinin; on the other hand beta-sheet containing acidic proteins, such as the soybean trypsin inhibitor, are unable to bind amyloid fibrils.


Asunto(s)
Amiloide/metabolismo , Aprotinina/metabolismo , Microfibrillas/metabolismo , Proteínas de Secreción de la Vesícula Seminal , Animales , Aprotinina/química , Unión Competitiva , Biomarcadores/química , Biotinilación , Glicoproteínas/química , Glicoproteínas/metabolismo , Insulina/metabolismo , Radioisótopos de Yodo , Microfibrillas/química , Modelos Moleculares , Mutación , Prealbúmina/química , Prealbúmina/genética , Unión Proteica , Estructura Secundaria de Proteína
12.
Amyloid ; 5(3): 163-74, 1998 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-9818053

RESUMEN

Familial amyloidotic polyneuropathy (FAP) is characterized by deposits of amyloid fibers in which the major protein component is transthyretin (TTR). Nearly fifty mutations have been reported for the TTR in hereditary FAP. Protein crystallography of mutant TTRs has shown that the molecular structures of the variant molecules are similar to those found in the wild type. On this basis, the FAP fibers were initially proposed to consist of native-like TTR tetramers. In the current paper, we used x-ray fiber diffraction to study the structure of FAP fibers from biopsy samples of vitreous humor and kidney. The reflections of the vitreous sample showed a cross-beta diffraction pattern. All the meridional reflections were indexed by a one-dimensional, 29 A-period lattice, and the equatorial reflections were indexed by an apparent one-dimensional 67 A-period lattice. The x-ray intensity distribution indicated that the unit structure, which is similar to a TTR monomer, is composed of a pair of beta-sheets consisting of four hydrogen-bonded beta-chains per sheet, with the beta-chains oriented approximately normal to the fiber axis. The axial disposition of these units, with a 29 A-period, constitutes the protofilament; and a tetrameric lateral assembly of the protofilaments containing the core domain of the approximately 20 A-wide beta-sheet structure constitutes the FAP amyloid fiber. An inter-fiber separation of 75 A in these concentrated samples accounts for the apparent one-dimensional lattice perpendicular to the fiber axis. In the delipidated kidney FAP sample, the diffraction pattern indicated a pair of beta-sheets, suggesting that the protofilament structure in kidney is similar to that in vitreous humor. In the non-delipidated sample the successive sharp reflections indexed to a one-dimensional, 48.9 A-lattice, and the electron density projection showed a density elevation at the center of a lipid bilayer. This suggests that lipid may be associated with the monomeric TTR in the kidney FAP protofilament.


Asunto(s)
Neuropatías Amiloides , Amiloide/química , Riñón/química , Prealbúmina/química , Cuerpo Vítreo/química , Humanos , Modelos Estructurales , Conformación Proteica , Difracción de Rayos X
13.
J Biol Chem ; 273(38): 24715-22, 1998 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-9733771

RESUMEN

The x-ray crystal structure of the amyloidogenic Leu55 --> Pro transthyretin (TTR) variant, implicated as the causative agent in early-onset familial amyloidotic polyneuropathy (Jacobson, D. R., McFarlin, D. E., Kane, I., and Buxbaum, J. N. (1992) Hum. Genet. 89, 353-356), has been solved by molecular replacement, refined at 2.7 A to a Rcryst value of 0.190 (Fobs > 2.0sigma), and compared with wild-type transthyretin to understand the molecular mechanism(s) involved in amyloidogenesis. Leu55 --> Pro TTR crystallizes in space group C2, with eight monomers in the asymmetric unit, and the observed packing contacts are considerably different from those described for the wild-type protein. Refinement of the crystal structure shows that the proline for leucine substitution disrupts the hydrogen bonds between strands D and A, resulting in different interface contacts. Based on the assumption that the observed packing contacts may be significant for amyloidogenesis, a model for the TTR amyloid is proposed. It consists of a tubular structure with inner and outer diameters approximately of 30 and 100 A and four monomers per cross-section.


Asunto(s)
Variación Genética , Leucina , Prealbúmina/química , Prolina , Conformación Proteica , Secuencia de Aminoácidos , Neuropatías Amiloides/genética , Simulación por Computador , Cristalografía por Rayos X/métodos , Humanos , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Puntual , Prealbúmina/genética , Prealbúmina/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes/química
14.
Endocrine ; 6(3): 309-15, 1997 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-9368688

RESUMEN

The majority of the known transthyretin (TTR) variants are associated with amyloidosis, but there are also variants associated with euthyroid hyperthyroxinemia and others are apparently nonpathogenic. TTR Met 119 is a nonpathogenic variant found to be frequent in the Portuguese population. Previous studies on thyroxine (T4) binding to semi-purified TTR from heterozygotic carriers of TTR Met 119, reported by us and other groups, revealed different results. Therefore, to further characterize T4 binding to TTR Met 119 we performed T4-TTR binding studies in homotetrameric-recombinant TTR Met 119 variant and normal TTR. We also studied T4 binding to TTR purified from serum of different heterozygotic carriers of TTR Met 119 including compound heterozygotic individuals carriers of a TTR mutation in the other allele. We observed an increased T4 binding affinity to TTR Met 119 from heterozygotic individuals and compound heterozygotes and this effect of increasing T4 binding affinity was consistent and independent from the mutation present in the other allele. Recombinant homotetrameric TTR Met 119 and heterotetrameric protein from heterozygotic carriers of TTR Met 119 presented similar T4 binding affinity demonstrating the increased T4 binding affinity of TTR Met 119. X-ray crystallography studies performed on the recombinant TTR Met 119 variant revealed structural alterations mainly at the level of residue Leu 110 allowing a closer contact between the hormone and the mutant protein. These results are consistent with the observed T4 binding results.


Asunto(s)
Heterocigoto , Mutación/genética , Prealbúmina/metabolismo , Tiroxina/metabolismo , Unión Competitiva , Cristalografía por Rayos X , Humanos , Radioisótopos de Yodo , Metionina/química , Metionina/genética , Prealbúmina/química , Prealbúmina/genética , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiroxina/análisis
15.
Acta Crystallogr D Biol Crystallogr ; 52(Pt 5): 966-72, 1996 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-15299606

RESUMEN

The Val122Ile mutant transthyretin (TTR Ile122) is an amyloidogenic protein which has been described as the major protein component of amyloid fibrils isolated from patients with familial amyloidotic cardiomyopathy (FAC), a disease characterized by cardiac failure and amyloid deposits in the heart. The reasons for the deposition of TTR are still unknown and it is conceivable that a conformational alteration, resulting from the mutation, is fundamental for amyloid formation. The three-dimensional structure of TTR Ile122 was determined and refined to a crystallographic R factor of 15.8% at 1.9 A resolution. The r.m.s. deviation from ideality in bond distances is 0.019 A and in angle-bonded distances is 0.027 A. The presence of two crystallographically independent monomers in the asymmetric unit allowed additional means of estimation of atomic coordinate error. The structure of the mutant is essentially identical to that of the wild-type transthyretin (TTR). The largest deviations occur in surface loops and in the region of the substitution. The protein is a tetramer composed of identical subunits; each monomer has two four-stranded beta-sheets which are extended to eight-stranded beta-sheets when two monomers associate through hydrogen bonds forming a dimer, which is the crystallographic asymmetric unit. The replacement of valine for isoleucine introduces very small alterations in relation to the wild-type protein; nevertheless they seem to confirm a tendency for a less stable tetrameric structure. This would support the idea that the tetrameric structure might be disrupted in amyloid fibrils.

16.
Acta Crystallogr D Biol Crystallogr ; 52(Pt 3): 566-8, 1996 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-15299680

RESUMEN

The amyloidogenic Leu55Pro variant of transthyretin has been expressed, purified and crystallized in space group C2. The cell constants are a = 149.99, b = 78.74, c = 98.95 A, beta = 100.5 degrees and the crystals diffract to 2.7 A resolution. There are eight monomers in the asymmetric unit giving a V(M) = 2.6 A(3) Da(-1) and 53% solvent content. In the wild-type protein, the crystals are orthorhombic with two monomers in the asymmetric unit. The wild-type protein is a tetramer composed of four identical subunits [Blake, Geisow, Oatley, Rerat & Rerat (1978). J. Mol. Biol. 121, 339-356.] and a molecular-replacement solution for the Leu55Pro variant was obtained using one monomer of the wild-type protein as a model. Rigid-body refinement of the eight monomers in the asymmetric unit and subsequent refinement using molecular dynamics were performed with X-PLOR, leading to a current R factor of 20.3% for all the data. The crystallographic packing of the molecules is different from the one presented by the wild-type protein, opening new perspectives for understanding how this protein aggregates to form amyloid fibrils.

17.
Ciba Found Symp ; 199: 47-52; discussion 52-7, 1996.
Artículo en Inglés | MEDLINE | ID: mdl-8915603

RESUMEN

We have analysed the structure, binding properties, stability and amyloidogenicity of particular transthyretin (TTR) mutations-TTR Met30 and TTR Pro55, both associated with familial amyloid polyneuropathy, and TTR Met119, a non-pathogenic TTR mutation with apparent protective effects on the amyloidogenicity of the Met30 mutation. Our results show that in contrast to the Met30 mutation, the Met119 mutation increases the stability of the tetramer towards dissociation into monomers and confers a higher affinity to thyroxine, which binds on the channel that runs through the tetramer. This variant also shows a greater resistance to amyloid formation in vitro, in contrast to the Pro55 variant, which is more susceptible to amyloid formation. Crystallographic studies of the structure of the Pro55 variant are underway and reveal major conformational changes. Interestingly, these changes affect the D strand of TTR, which when deleted or modified in vitro leads to accelerated rates of amyloid formation. The conformational changes observed in these "aggressive' mutations may resemble intermediate forms in the process of amyloidogenesis.


Asunto(s)
Amiloide/biosíntesis , Amiloide/genética , Mutación , Prealbúmina/genética , Amiloide/química , Amiloidosis/etiología , Amiloidosis/metabolismo , Humanos , Prealbúmina/química , Prealbúmina/metabolismo , Conformación Proteica
18.
EMBO J ; 12(2): 735-41, 1993 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-8382610

RESUMEN

Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant hereditary type of lethal amyloidosis involving single (or double) amino acid substitutions in the amyloidogenic protein transthyretin (TTR). The most common type of FAP (Type I, or Portuguese) is characterized by a Val-->Met substitution at position 30. The Met30 variant of TTR has been produced by recombinant methods, crystallized in a form isomorphous with native TTR, subjected to X-ray analysis and compared structurally with the wild-type protein. The comparison shows that the effect of the substitution at position 30 is transmitted through the protein core to Cys10, the only thiol group in the TTR subunit, which becomes slightly more exposed. The variant TTR molecule is otherwise in a near-native state. Use of computer graphics has shown that it is possible to model a linear aggregate of TTR molecules, each linked to the next by a pair of disulphide bonds involving Cys10 residues. Formation of these disulphide bonds involves a small number of slightly short molecular contacts with native TTR molecules, most of which are relieved in the Met30 variant. We propose this model as a possible basis for a molecular description of the FAP amyloid fibrils.


Asunto(s)
Amiloidosis/genética , Metionina/genética , Enfermedades del Sistema Nervioso Periférico/genética , Prealbúmina/química , Aminoácidos/química , Simulación por Computador , Disulfuros/química , Modelos Moleculares , Prealbúmina/genética , Conformación Proteica , Difracción de Rayos X
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