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Human Transthyretin (human + transthyretin)
Selected AbstractsGenetic microheterogeneity of human transthyretin detected by IEFELECTROPHORESIS, Issue 12 2007Klaus Altland Professor Dr. Abstract Mutations of the human transthyretin (TTR) gene have attracted medical interest as a cause of amyloidosis. Recently, we have described in detail an electrophoretic procedure with PAGE followed by IEF in urea gradients for the study of the microheterogeneity of TTR monomers (Altland, K., Winter, P., Sauerborn, M. K., Electrophoresis 1999, 20, 1349,1364). In this paper, we present a study on 49 different mutations of TTR including 33 that result in electrically neutral amino acid substitutions. The aims of the investigation were to test the sensitivity of the procedure to detect TTR variants in patients with TTR amyloidosis and their relatives and to identify some common characteristics that could explain the amyloidogenicity of these variants. We found that all tested amyloidogenic mutations could be detected by our method with the exception of those for which the corresponding variant was absent in plasma samples. Most of the electrically neutral amyloidogenic TTR variants had in common a reduced conformational stability of monomers by the activity of protons and urea. For three variants, e.g. TTR,F64L, TTR,I107V and TTR,V122I, the monomers had a conformational stability close to that of normal monomers but we found experimental and structural arguments for a weakening of the monomer-monomer contact. All types of amyloidogenic mutations affected the stability of TTR tetramers. [source] Polyacrylamide gel electrophoresis followed by sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis for the study of the dimer to monomer transition of human transthyretinELECTROPHORESIS, Issue 14 2003Klaus Altland Abstract Familial amyloidotic polyneuropathy (FAP) is caused by mutations which destabilize transthyretin (TTR) and facilitate the aggregation into extracellular amyloid fibrils preferentially in peripheral nerve and heart tissues. Therapeutic and preventive trials for FAP at the plasma TTR level require a careful study of the destabilization of TTR under variable conditions. We have developed a simple double one-dimensional (D1-D) electrophoretic procedure with polyacrylamide gel electrophoresis (PAGE) followed by sodium dodecylsulfate (SDS) gradient PAGE to study the dimer to monomer transition. TTR is first isolated by PAGE from other plasma proteins. The gel strip containing the TTR fraction is incubated in 2% SDS under varying conditions of temperature, buffer composition, pH, and additives like urea and/or a sulfhydryl-reactive agent, followed by SDS-gradient PAGE for the separation of TTR dimers and monomers. We demonstrate that an unidirectional dimer to monomer transition of normal TTR is achieved at 70,80°C in neutral to mild alkaline buffers or at 37°C and slightly acidic pH (6,7). Addition of urea favors the transition into monomers. Amyloidogenic mutations like amyloidogenic TTR (ATTR)-V30M or ATTR-I107V favor the transition into monomers in buffer systems close to the physiological pH of human plasma. We conclude that this finding has to be considered by any hypothesis on ATTR-derived amyloidogenesis. [source] A non-invasive method based on saliva to characterize transthyretin in familial amyloidotic polyneuropathy patients using FT-ICR high-resolution MSPROTEOMICS - CLINICAL APPLICATIONS, Issue 6-7 2010Gonçalo da Costa Abstract Purpose: To identify, characterize and perform a relative quantification of human transthyretin (TTR) variants in human saliva. Experimental design: Serum and saliva samples were collected from healthy and familial amyloidotic polyneuropathy (FAP) patients, proteins separated by SDS-PAGE, TTR bands excised, in-gel digested and analyzed by MALDI-FTICR. Results: We identified and performed a relative quantification of mutated and native TTR forms in human saliva, based on FTICR-MS. The results are quantitatively identical to the ones obtained with human serum. In FAP patients subjected to cadaveric liver transplant, the TTR mutant form is no longer detected in saliva, while in patients receiving a domino liver from a FAP donor the mutant form of TTR becomes detectable in saliva, thus demonstrating the serum origin of TTR in saliva. Conclusions and clinical relevance: Saliva TTR originates in serum and the ratio of mutant to native TTR is preserved. The method provides a non-invasive detection of mutated TTR and a relative quantification of TTR forms. Diagnostic and disease prognosis of FAP is crucial at early stages of the disease and after liver transplantation, the only curative therapy. A suitable non-invasive method was developed for monitoring the most important FAP biomarker in human saliva. [source] Ligand binding at the transthyretin dimer,dimer interface: structure of the transthyretin,T4Ac complex at 2.2,Å resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2005Vivian Cody The crystal structure of the complex of human transthyretin (hTTR) with 3,3,,5,5,-tetraiodothyroacetic acid (T4Ac) has been determined to 2.2,Å resolution. The complex crystallizes in the orthorhombic space group P21212, with unit-cell parameters a = 43.46, b = 85.85, c = 65.44,Å. The structure was refined to R = 17.3% and Rfree = 21.9% for reflections without any ,-cutoff. T4Ac is bound in both the forward and the reverse mode in the two binding sites of hTTR. In the forward orientation, T4Ac binds in a position similar to that described for thyroxine (T4) in the orthorhombic hTTR,T4 complex. In this orientation, the iodine substituents of the phenolic ring are bound in the P3,/P2 halogen pockets. In the reverse orientation, which is the major binding mode of T4Ac, the ligand is bound deep in the TTR channel, with the carboxylic group bound in the P3, pocket and forming simultaneous polar interactions with the residues constituting the two hormone-binding sites. Such interactions of a thyroxine-analogue ligand bound in the reverse mode have never been observed in TTR complexes previously. [source] Structure of human transthyretin complexed with bromophenols: a new mode of bindingACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2000Minakshi Ghosh The binding of two organohalogen substances, pentabromophenol (PBP) and 2,4,6-tribromophenol (TBP), to human transthyretin (TTR), a thyroid hormone transport protein, has been studied by in vitro competitive binding assays and by X-ray crystallography. Both compounds bind to TTR with high affinity, in competition with the natural ligand thyroxine (T4). The crystal structures of the TTR,PBP and TTR,TBP complexes show some unusual binding patterns for the ligands. They bind exclusively in the `reversed' mode, with their hydroxyl group pointing towards the mouth of the binding channel and in planes approximately perpendicular to that adopted by the T4 phenolic ring in a TTR,T4 complex, a feature not observed before. The hydroxyl group in the ligands, which was previously thought to be a key ingredient for a strong binding to TTR, does not seem to play an important role in the binding of these compounds to TTR. In the TTR,PBP complex, it is primarily the halogens which interact with the TTR molecule and therefore must account for the strong affinity of binding. The interactions with the halogens are smaller in number in TTR,TBP and there is a decrease in affinity, even though the interaction with the hydroxyl group is stronger than that in the TTR,PBP complex. [source] |