The Ile-84-->Ser amino acid substitution in transthyretin interferes with the interaction with plasma retinol-binding protein.

In plasma the thyroid hormone-binding protein transthyretin (TTR) forms a tight complex with the specific retinol carrier retinol-binding protein (RBP). The Ile-84–>Ser mutation and several other point mutations in TTR are associated with familial amyloidotic polyneuropathy, which is characterized by extracellular depositions of amyloid fibrils mainly consisting of mutated TTRs. The interactions with human RBP of recombinant human normal and Ser-84 TTRs were investigated by monitoring the fluorescence anisotropy of RBP-bound retinol. A nearly negligible affinity of the recombinant Ser-84 TTR for RBP was found. This result indicates the participation of a region on the outer surface of TTR that comprises Ile-84 in the recognition of RBP. In preliminary studies the Ser-84 TTR was the only one among several amyloidogenic variant TTRs to display negligible interaction with RBP. Therefore, in general a substantially altered binding of TTR to RBP is not associated with familial amyloidotic polyneuropathy. Instead, the altered binding of Ser-84 TTR to RBP appears to be responsible for an abnormal plasma transport of RBP. The recombinant normal TTR exhibits binding properties, in its interaction with human RBP, approximately similar to those of TTR purified from human plasma. Two independent and equivalent RBP binding sites on recombinant normal TTR are characterized by a dissociation constant of about 0.4 microM

Characterization of the microheterogeneity of transthyretin in plasma and urine using SELDI-TOF-MS immunoassay

BACKGROUND: It has been shown that transthyretin (TTR) exists in different molecular variants. Besides point mutations associated with different diseases such as amyloidosis, other posttranslational modifications occur that might be of diagnostic interest. RESULTS: TTR levels as determined by ELISA in plasma and urine of healthy individuals were 489 ± 155 μg/ml plasma and 46 ± 24 ng/g creatinine, respectively. Average levels in urine of pregnant women were 45 ± 65 μg/g creatinine. The molecular heterogeneity of TTR was analyzed using a high-throughput mass spectrometric immunoassay system. TTR was extracted from plasma or urine onto an antibody-coated (via protein A) affinity chip surface (PS20) using the surface-enhanced laser desorption/ionization (SELDI) technique. Subsequently samples were subjected to time-of-flight mass spectrometry (TOF-MS). In healthy individuals, TTR in plasma occurred rather consistently in two variants of 13732 ± 12 and 13851 ± 9 Da for the native and S-cysteinylated forms and at a smaller signal of 14043 ± 17 Da for the S-glutathionylated form. In urine of pregnant women, various signals were observed with a dominant signal at 13736 ± 10 Da and a varying number of smaller immunoreactive fragments. These fragments are possibly the consequence of metabolism in plasma or kidney. CONCLUSION: This chip-based approach represents a rapid and accurate method to characterize the molecular variants of TTR including protein or peptide fragments which are either related to TTR or have resulted from its catabolism. These molecular variants may be of diagnostic importance as alternative or novel biomarkers due to their predominant relation to the TTR metabolism both in healthy and diseased individuals

Plasminogen activation triggers transthyretin amyloidogenesis in vitro

Systemic amyloidosis is a usually fatal disease caused by extracellular accumulation of abnormal protein fibers, amyloid fibrils, derived by misfolding and aggregation of soluble globular plasma protein precursors. Both WT and genetic variants of the normal plasma protein transthyretin (TTR) form amyloid, but neither the misfolding leading to fibrillogenesis nor the anatomical localization of TTR amyloid deposition are understood. We have previously shown that, under physiological conditions, trypsin cleaves human TTR in a mechano-enzymatic mechanism that generates abundant amyloid fibrils in vitro. In sharp contrast, the widely used in vitro model of denaturation and aggregation of TTR by prolonged exposure to pH 4.0 yields almost no clearly defined amyloid fibrils. However, the exclusive duodenal location of trypsin means that this enzyme cannot contribute to systemic extracellular TTR amyloid deposition in vivo. Here, we therefore conducted a bioinformatics search for systemically active tryptic proteases with appropriate tissue distribution, which unexpectedly identified plasmin as the leading candidate. We confirmed that plasmin, just as trypsin, selectively cleaves human TTR between residues 48 and 49 under physiological conditions in vitro. Truncated and full-length protomers are then released from the native homotetramer and rapidly aggregate into abundant fibrils indistinguishable from ex vivo TTR amyloid. Our findings suggest that physiological fibrinolysis is likely to play a critical role in TTR amyloid formation in vivo. Identification of this surprising intersection between two hitherto unrelated pathways opens new avenues for elucidating the mechanisms of TTR amyloidosis, for seeking susceptibility risk factors, and for therapeutic innovation

Plasminogen activation triggers transthyretin amyloidogenesis in vitro

Systemic amyloidosis is a usually fatal disease caused by extracellular accumulation of abnormal protein fibers, amyloid fibrils, derived by misfolding and aggregation of soluble globular plasma protein precursors. Both WT and genetic variants of the normal plasma protein transthyretin (TTR) form amyloid, but neither the misfolding leading to fibrillogenesis nor the anatomical localization of TTR amyloid deposition are understood. We have previously shown that, under physiological conditions, trypsin cleaves human TTR in a mechano-enzymatic mechanism that generates abundant amyloid fibrils in vitro. In sharp contrast, the widely used in vitro model of denaturation and aggregation of TTR by prolonged exposure to pH 4.0 yields almost no clearly defined amyloid fibrils. However, the exclusive duodenal location of trypsin means that this enzyme cannot contribute to systemic extracellular TTR amyloid deposition in vivo. Here, we therefore conducted a bioinformatics search for systemically active tryptic proteases with appropriate tissue distribution, which unexpectedly identified plasmin as the leading candidate. We confirmed that plasmin, just as trypsin, selectively cleaves human TTR between residues 48 and 49 under physiological conditions in vitro. Truncated and full-length protomers are then released from the native homotetramer and rapidly aggregate into abundant fibrils indistinguishable from ex vivo TTR amyloid. Our findings suggest that physiological fibrinolysis is likely to play a critical role in TTR amyloid formation in vivo. Identification of this surprising intersection between two hitherto unrelated pathways opens new avenues for elucidating the mechanisms of TTR amyloidosis, for seeking susceptibility risk factors, and for therapeutic innovation

Calcium Binds to Transthyretin with Low Affinity

The plasma protein transthyretin (TTR), a transporter for thyroid hormones and retinol in plasma and cerebrospinal fluid, is responsible for the second most common type of systemic (ATTR) amyloidosis either in its wild type form or as a result of destabilizing genetic mutations that increase its aggregation propensity. The association between free calcium ions (Ca2+) and TTR is still debated, although recent work seems to suggest that calcium induces structural destabilization of TTR and promotes its aggregation at non-physiological low pH in vitro. We apply high-resolution NMR spectroscopy to investigate calcium binding to TTR showing the formation of labile interactions, which leave the native structure of TTR substantially unaltered. The effect of calcium binding on TTR-enhanced aggregation is also assessed at physiological pH through the mechano-enzymatic mechanism. Our results indicate that, even if the binding is weak, about 7% of TTR is likely to be Ca2+-bound in vivo and therefore more aggregation prone as we have shown that this interaction is able to increase the protein susceptibility to the proteolytic cleavage that leads to aggregation at physiological pH. These events, even if involving a minority of circulating TTR, may be relevant for ATTR, a pathology that takes several decades to develop

Microalbuminuria is a major determinant of elevated plasma retinol-binding protein 4 in type 2 diabetic patients

Plasma retinol-binding protein 4 (RBP4) may be a new adipokine linked to obesity-induced insulin resistance and type 2 diabetes. The impact of diabetic nephropathy on plasma RBP4 levels, however, is not known. We tested the hypothesis that microalbuminuria is associated with elevated plasma concentrations of RBP4 in type 2 diabetic subjects. Retinol, its binding protein and transthyretin (TTR) were measured in the plasma and urine of 62 type 2 diabetic subjects, 26 of whom had microalbuminuria. The results were compared to 35 healthy control subjects. Despite no differences in plasma retinol, concentrations of the RBP4 were significantly elevated in plasma of diabetic patients and significantly higher in those with microalbuminuria. The higher plasma levels of the binding protein in subjects with microalbuminuria were accompanied by both significantly elevated plasma TTR and increased urinary levels of RBP4. There were no correlations of plasma-binding protein levels and parameters of insulin resistance. Our study suggests that plasma RBP4 levels in type 2 diabetic patients are affected by incipient nephropathy. Therefore, further studies evaluating RBP4 as a regulator of systemic insulin resistance and type 2 diabetes will need to take renal function into consideration

Natural compounds as inhibitors of transthyretin amyloidosis and neuroprotective agents: analysis of structural data for future drug design

Natural compounds, such as plant and fruit extracts have shown neuroprotective effect against neurodegenerative diseases. It has been reported that several natural compounds binding to transthyretin (TTR) can be useful in amyloidosis prevention. TTR is a transporter protein that under physiological condition carries thyroxine (T4) and retinol in plasma and in cerebrospinal fluid (CSF); it also has a neuroprotective role against Alzheimer’s disease (AD). However, TTR also is an amyloidogenic protein responsible for familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC). The TTR amyloidogenic potential is speeded up by several point mutations. One therapeutic strategy against TTR amyloidosis is the stabilisation of the native tetramer by natural compounds and small molecules. In this review, we examine the natural products that, starting from 2012 to present, have been studied as a stabiliser of TTR tetramer. In particular, we discussed the chemical and structural features which will be helpful for future drug design of new TTR stabilisers

Transthyretin stabilization: An emerging strategy for the treatment of alzheimer’s disease?

Transthyretin (TTR), previously named prealbumin is a plasma protein secreted mainly by the liver and choroid plexus (CP) that is a carrier for thyroid hormones (THs) and retinol (vitamin A). The structure of TTR, with four monomers rich in β-chains in a globular tetrameric protein, accounts for the predisposition of the protein to aggregate in fibrils, leading to a rare and severe disease, namely transthyretin amyloidosis (ATTR). Much effort has been made and still is required to find new therapeutic compounds that can stabilize TTR (“kinetic stabilization”) and prevent the amyloid genetic process. Moreover, TTR is an interesting therapeutic target for neurodegenerative diseases due to its recognized neuroprotective properties in the cognitive impairment context and interestingly in Alzheimer’s disease (AD). Much evidence has been collected regarding the neuroprotective effects in AD, including through in vitro and in vivo studies as well as a wide range of clinical series. Despite this supported hypothesis of neuroprotection for TTR, the mechanisms are still not completely clear. The aim of this review is to highlight the most relevant findings on the neuroprotective role of TTR, and to summarize the recent progress on the development of TTR tetramer stabilizers

Monoaryl derivatives as transthyretin fibril formation inhibitors: Design, synthesis, biological evaluation and structural analysis

Transthyretin (TTR) is a ß-sheet-rich homotetrameric protein that transports thyroxine (T4) and retinol both in plasma and in cerebrospinal fluid. TTR also interacts with amyloid-β, playing a protective role in Alzheimer's disease. Dissociation of the native transthyretin (TTR) tetramer is widely accepted as the critical step in TTR amyloids fibrillogenesis, and is responsible for extracellular deposition of amyloid fibrils. Small molecules, able to bind in T4 binding sites and stabilize the TTR tetramer, are interesting tools to treat and prevent systemic ATTR amyloidosis. We report here the synthesis, in vitro evaluation and three-dimensional crystallographic analyses of new monoaryl-derivatives in complex with TTR. Of the derivatives reported here, the best inhibitor of TTR fibrillogenesis, 1d, exhibits an activity similar to diflunisal

Beyond Genetic Factors in Familial Amyloidotic Polyneuropathy: Protein Glycation and the Loss of Fibrinogen's Chaperone Activity

Familial amyloidotic polyneuropathy (FAP) is a systemic conformational disease characterized by extracellular amyloid fibril formation from plasma transthyretin (TTR). This is a crippling, fatal disease for which liver transplantation is the only effective therapy. More than 80 TTR point mutations are associated with amyloidotic diseases and the most widely accepted disease model relates TTR tetramer instability with TTR point mutations. However, this model fails to explain two observations. First, native TTR also forms amyloid in systemic senile amyloidosis, a geriatric disease. Second, age at disease onset varies by decades for patients bearing the same mutation and some mutation carrier individuals are asymptomatic throughout their lives. Hence, mutations only accelerate the process and non-genetic factors must play a key role in the molecular mechanisms of disease. One of these factors is protein glycation, previously associated with conformational diseases like Alzheimer's and Parkinson's. The glycation hypothesis in FAP is supported by our previous discovery of methylglyoxal-derived glycation of amyloid fibrils in FAP patients. Here we show that plasma proteins are differentially glycated by methylglyoxal in FAP patients and that fibrinogen is the main glycation target. Moreover, we also found that fibrinogen interacts with TTR in plasma. Fibrinogen has chaperone activity which is compromised upon glycation by methylglyoxal. Hence, we propose that methylglyoxal glycation hampers the chaperone activity of fibrinogen, rendering TTR more prone to aggregation, amyloid formation and ultimately, disease