The tetrameric protein transthyretin dissociates to a non-native monomer in solution. A novel model for amyloidogenesis.
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In amyloidosis, normally innocuous soluble proteins polymerize to form insoluble fibrils. Amyloid fibril formation and deposition have been associated with a wide range of diseases, including spongiform encephalopathies, Alzheimer's disease, and familial amyloid polyneuropathies (FAP). In certain forms of FAP, the amyloid fibrils are mostly constituted by variants of transthyretin (TTR), a homotetrameric plasma protein implicated in the transport of thyroxine and retinol. The most common amyloidogenic TTR variant is V30M-TTR, and L55P-TTR is the variant associated with the most aggressive form of FAP. Recently, we reported that TTR dissociates to a monomeric species at pH 7.0 and nearly physiological ionic strengths (Quintas, A., Saraiva, M. J., and Brito, R. M. (1997) FEBS Lett. 418, 297-300). Here, we show that the tetramer dissociation is apparently irreversible; and based on intrinsic tryptophan fluorescence and fluorescence quenching experiments, we show that the monomeric species formed upon tetramer dissociation is non-native. We also show, based on 1-anilino-8-naph-thalenesulfonate binding studies, that this monomeric species appears not to behave like a molten globule. These data allowed us to propose a model for TTR amyloidogenesis based on tetramer dissociation occurring naturally under commonly observed physiological solution conditions. (+info)
Transthyretin in high density lipoproteins: association with apolipoprotein A-I.
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Previous studies have revealed the presence of transthyretin (TTR) on lipoproteins. To further address this issue, we fractionated plasma lipoproteins from 9 normal individuals, 10 familial amyloidotic polyneuropathy (FAP) patients, and 19 hyperlipidemic subjects using gel filtration. In the majority of the subjects, as well as in 9 of the 10 FAP patients and 14 of the 19 patients with hyperlipidemia, TTR was detected by ELISA in the high density lipoprotein (HDL) fraction. The presence of TTR in HDL was confirmed by direct sequencing and by immunoblotting; using non-reducing conditions, TTR was found by immunoblotting in a high molecular weight complex, which reacted also for apolipoprotein A-I (apoA-I). The amount of TTR present in HDL (HDL-TTR), as quantified by ELISA corresponded to 1;-2% of total plasma TTR. However, no detectable TTR levels were found in HDL fraction from 6 of the hyperlipidemic subjects. No correlation was found between the lack of TTR in HDL and plasma levels of total, LDL-, or HDL-associated cholesterol as well as levels of apoA-I and total plasma TTR. Ligand binding experiments showed that radiolabeled TTR binds to the HDL fraction of individuals with HDL-TTR but not to the corresponding fractions of individuals devoid of HDL-TTR, suggesting that HDL composition may interfere with TTR binding. The component(s) to which TTR binds in the HDL fraction were investigated. Polyclonal antibody against apoA-I was able to block the interaction of TTR with HDL, suggesting that the interaction of TTR with the HDL particle occurs via apoA-I. This hypothesis was further demonstrated by showing the formation of a complex of TTR with HDL and apoA-I by crosslinking experiments. Furthermore, anti-apoA-I immunoblot under native conditions suggested the existence of differences in HDL particle properties and/or stability between individuals with and without HDL-TTR. (+info)
Designing transthyretin mutants affecting tetrameric structure: implications in amyloidogenicity.
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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. (+info)
Liver transplantation for hereditary transthyretin amyloidosis.
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Transthyretin (TTR) amyloidosis is the most common form of hereditary amyloidosis. It is a systemic amyloidosis caused by an amyloidogenic variant TTR (ATTR), of which the methionine for valine at position 30 (ATTR Val30Met) gives rise to a fatal neuropathic amyloidosis. Because more than 95% of TTR is produced by the liver, a liver transplantation should abolish the liver's production of amyloidogenic mutant TTR and thereby halt amyloid formation. The first liver transplantation for hereditary TTR amyloidosis was performed in Sweden in 1990 on a patient with ATTR Val30Met amyloidosis, and the result was encouraging. Today, liver transplantation for TTR amyloidosis is an established treatment. However, the disease is rarely seen except in a few endemic areas; therefore, most transplantation centers only receive a few cases. Because the disease phenotype varies with different TTR mutations and variability is even encountered for the same mutation, an evaluation of patients for transplantation must include an investigation of all organs that may be affected by the disease and may impact on the morbidity and mortality of the procedure. The aim of this review is to present the results of liver transplantation for TTR amyloidosis and give recommendations for patient evaluation and selection based on the literature and our experience with the disease. (+info)
The course and prognostic factors of familial amyloid polyneuropathy after liver transplantation.
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Familial amyloid polyneuropathy (FAP) associated with mutations of the transthyretin (TTR) gene is the most common type of FAP, a devastating disease causing death within 10 years after the first symptoms. Because most of the amyloidogenic mutated TTR is secreted by the liver, transplantation is widely used to treat these patients, but long-term quantitative evaluation of the effects of liver transplantation on the progression of the neuropathy are not available. We have treated 45 patients with symptomatic TTR-FAP, including 43 with the Met30 TTR gene mutation, and report on the results of periodic evaluation of markers of neuropathy in 25 of them, who have been followed for more than 2 years after liver transplantation (mean follow-up 4 years). The overall survival rates at 1 and 5 years were 82 and 60%, respectively. Urinary incontinence and a low Norris score at liver transplantation were associated with poorer outcome. The motor score stabilized in seven of 11 patients (64%) with mild sensorimotor neuropathy (walking unaided) and in two of the eight patients (25%) with severe sensorimotor deficit (walking with aid) at liver transplantation. In five other patients, deterioration of motor deficit occurred only within the first year after liver transplantation, but was progressive after this interval in two patients. None of the six patients with pure sensory neuropathy developed motor loss and superficial sensory loss remained unchanged. Two years after liver transplantation, the rate of myelinated axon loss in nerve biopsy specimens was markedly lower in seven transplanted patients (0.9/mm(2) of endoneurial area/month) than in non-transplanted patients (70/mm(2) of endoneurial area/month). Symptoms of dysautonomia and quantitated cardiocirculatory autonomic tests remained unchanged. In all patients, serum mutated TTR decreased to 2.5% of pre-liver transplantation values and remained at this level during follow-up. We presently recommend liver transplantation in FAP patients at onset of first symptoms and exclusion of those with a Norris score below 55 and/or with urinary incontinence. (+info)
Rapid method to characterize mutations in transthyretin in cerebrospinal fluid from familial amyloidotic polyneuropathy patients by use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
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BACKGROUND: Familial amyloidotic polyneuropathy (FAP) type I, the most common dominantly inherited form of amyloidosis, is caused by a Val-to-Met point mutation at position 30 (Val(30)-->Met) in the protein transthyretin. Mass spectrometric analysis can identify modification of proteins, such as point mutations, acetylation, phosphorylation, sulfation, oxidation, and glycosylation. METHODS: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra from cerebrospinal fluid (CSF) drawn from a patient with FAP were compared with CSF from controls. We also isolated transthyretin with a Centrisart molecular size cutoff filter and performed high-accuracy peptide mass mapping to localize the site of the amino acid substitution (Val(30)-->Met). RESULTS: Mass spectra of transthyretin were produced directly from human CSF as well as from CSF after a simple prepurification method without immunoprecipitation. On-target tryptic digestion and MALDI-MS verified mass spectrometric peak identification. The point mutation was still detectable in CSF after hepatic transplantation. CONCLUSIONS: It is possible to diagnose FAP by a rapid MALDI-TOF MS analysis using only 100 microL of CSF, with only 250 nL actually consumed on target. The approach may also be useful to monitor production of mutated transthyretin by choroid plexus, especially after liver transplantation. (+info)
Expression of macrophage colony-stimulating factor receptor is increased in the AbetaPP(V717F) transgenic mouse model of Alzheimer's disease.
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Inflammation is an important neuropathological change in Alzheimer's disease (AD). However, the pathophysiological factors that initiate and maintain the inflammatory response in AD are unknown. We examined AbetaPP(V717F) transgenic mice, which show numerous brain amyloid-beta (Abeta) deposits, for expression of the macrophage colony-stimulating factor (M-CSF) and its receptor (M-CSFR). M-CSF is increased in the brain in AD and dramatically augments the effects of Abeta on cultured microglia. AbetaPP(V717F) animals 12 months of age showed large numbers of microglia strongly labeled with an M-CSFR antibody near Abeta deposits. M-CSFR mRNA and protein levels were also increased in brain homogenates from AbetaPP(V717F) animals. Dystrophic neurites and astroglia showed no M-CSFR labeling in the transgenic animals. A M-CSF antibody decorated neuritic structures near hippocampal Abeta deposits in transgenic animals. M-CSF mRNA was also increased in AbetaPP(V717F) animals in comparison with wild-type controls. Simultaneous overexpression of M-CSFR and its ligand in AbetaPP(V717F) animals could result in augmentation of Abeta-induced activation of microglia. Because chronic activation of microglia is thought to result in neuronal injury, the M-CSF system may be a potential target for therapeutic intervention in AD. (+info)
Elucidating the mechanism of familial amyloidosis- Finnish type: NMR studies of human gelsolin domain 2.
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Familial amyloidosis-Finnish type (FAF) results from a single mutation at residue 187 (D187N or D187Y) within domain 2 of the actin-regulating protein gelsolin. The mutation somehow allows a masked cleavage site to be exposed, leading to the first step in the formation of an amyloidogenic fragment. We have performed NMR experiments investigating structural and dynamic changes between wild-type (WT) and D187N gelsolin domain 2 (D2). On mutation, no significant structural or dynamic changes occur at or near the cleavage site. Areas in conformational exchange are observed between beta-strand 4 and alpha-helix 1 and within the loop region following beta-strand 5. Chemical shift differences are noted along the face of alpha-helix 1 that packs onto the beta-sheet, suggesting an altered conformation. Conformational changes within these areas can have an effect on actin binding and may explain why D187N gelsolin is inactive. [(1)H-(15)N] nuclear Overhauser effect and chemical shift data suggest that the C-terminal tail of D187N gelsolin D2 is less structured than WT by up to six residues. In the crystal structure of equine gelsolin, the C-terminal tail of D2 lies across a large cleft between domains 1 and 2 where the masked cleavage site sits. We propose that the D187N mutation destabilizes the C-terminal tail of D2 resulting in a more exposed cleavage site leading to the first proteolysis step in the formation of the amyloidogenic fragment. (+info)