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Biological & Pharmaceutical Bulletin 2018Transthyretin (TTR) is a tetrameric beta-sheet-rich protein that is important in the plasma transport of thyroxine and retinol. Mutations in the TTR gene cause TTR...
Transthyretin (TTR) is a tetrameric beta-sheet-rich protein that is important in the plasma transport of thyroxine and retinol. Mutations in the TTR gene cause TTR tetramer protein to dissociate to monomer, which is the rate-limiting step in familial amyloid polyneuropathy. Amyloidogenicity of individual TTR variants depends on the types of mutation that induce significant changes in biophysical, biochemical and/or biological properties. G101S TTR variant was previously identified in a Japanese male without amyloidotic symptom, and was considered as a non-amyloidogenic TTR variant. However, little is known about G101S TTR. Here, we found slight but possibly important biophysical differences between wild-type (WT) and G101S TTR. G101S TTR had slower rate of tetramer dissociation and lower propensity for amyloid fibril formation, especially at mild low pH (4.2 and 4.5), and was likely to have strong hydrophobic interaction among TTR monomers, suggesting relatively higher stability of G101S TTR compared with WT TTR. Cycloheximide (CHX)-based assay in HEK293 cells revealed that intracellular G101S TTR expression level was lower, but extracellular expression was higher than WT TTR, implying enhanced secretion efficiency of G101S TTR protein compared with WT TTR. Moreover, we found that STT3B-dependent posttranslational N-glycosylation at N98 residue occurred in G101S TTR but not in other TTR variants, possibly due to amino acid alterations that increase N-glycosylation preference or accelerate rigid structure formation susceptible to N-glycosylation. Taken together, our study characterizes G101S TTR as a stable and N-glycosylable TTR, which may be linked to its non-amyloidogenic characteristic.
Topics: Amyloid; Amyloid Neuropathies, Familial; Glycosylation; HEK293 Cells; HeLa Cells; Hexosyltransferases; Humans; Membrane Proteins; Prealbumin
PubMed: 29607936
DOI: 10.1248/bpb.b17-01021 -
Biochemistry Mar 2020Recombinant proteins have increased our knowledge regarding the physiological role of proteins; however, affinity purification tags are often not cleaved prior to...
Recombinant proteins have increased our knowledge regarding the physiological role of proteins; however, affinity purification tags are often not cleaved prior to analysis, and their effects on protein structure, stability and assembly are often overlooked. In this study, the stabilizing effects of an N-terminus dual-FLAG (FT) tag fusion to transthyretin (TTR), a construct used in previous studies, are investigated using native ion mobility-mass spectrometry (IM-MS). A combination of collision-induced unfolding and variable-temperature electrospray ionization is used to compare gas- and solution-phase stabilities of FT-TTR to wild-type and C-terminal tagged TTR. Despite an increased stability of both gas- and solution-phase FT-TTR, thermal degradation of FT-TTR was observed at elevated temperatures, viz., backbone cleavage occurring between Lys9 and Cys10. This cleavage reaction is consistent with previously reported metalloprotease activity of TTR [Liz et al. 2009] and is suppressed by either metal chelation or excess zinc. This study brings to the fore the effect of affinity tag stabilization of TTR and emphasizes unprecedented detail afforded by native IM-MS to assess structural discrepancies of recombinant proteins from their wild-type counterparts.
Topics: Humans; Oligopeptides; Prealbumin; Recombinant Proteins; Spectrometry, Mass, Electrospray Ionization; Structure-Activity Relationship
PubMed: 32101399
DOI: 10.1021/acs.biochem.0c00105 -
Journal of Biochemistry Dec 1985In an attempt to construct model systems for familial amyloidotic polyneuropathy, prealbumin cDNA was cloned from a mouse liver cDNA library, using previously cloned... (Comparative Study)
Comparative Study
In an attempt to construct model systems for familial amyloidotic polyneuropathy, prealbumin cDNA was cloned from a mouse liver cDNA library, using previously cloned human prealbumin cDNA as a hybridization probe. The primary structure of mouse prealbumin deduced from the cDNA sequence shows that it consists of 147 amino acids, including a whole prealbumin sequence (127 amino acids) and a putative signal sequence (20 amino acids). These numbers are in complete agreement with those determined for the human prealbumin. Among the 127 amino acid residues of the mature human prealbumin, 25 are replaced by different amino acids in the mouse prealbumin. Interestingly, 24 out of the 25 substituted amino acids are located at the outer surface of the protein, and the regions corresponding to the core and central channel of the protein are almost completely conserved. The cloned cDNA provided essential information for manipulating amyloidosis in mice.
Topics: Amino Acid Sequence; Amyloidosis; Animals; Base Sequence; Cloning, Molecular; DNA; Disease Models, Animal; Humans; Male; Mice; Mice, Inbred C3H; Models, Molecular; Peripheral Nervous System Diseases; Prealbumin; Protein Conformation; Recombinant Proteins; Sequence Homology, Nucleic Acid
PubMed: 3005251
DOI: 10.1093/oxfordjournals.jbchem.a135442 -
The Journal of Biological Chemistry Oct 2020The protective effect of transthyretin (TTR) on cellular toxicity of β-amyloid (Aβ) has been previously reported. TTR is a tetrameric carrier of thyroxine in blood and...
The protective effect of transthyretin (TTR) on cellular toxicity of β-amyloid (Aβ) has been previously reported. TTR is a tetrameric carrier of thyroxine in blood and cerebrospinal fluid, the pathogenic aggregation of which causes systemic amyloidosis. However, studies have documented a protective effect of TTR against cellular toxicity of pathogenic Aβ, a protein associated with Alzheimer's disease. TTR binds Aβ, alters its aggregation, and inhibits its toxicity both and In this study, we investigate whether the amyloidogenic ability of TTR and its antiamyloid inhibitory effect are associated. Using protein aggregation and cytotoxicity assays, we found that the dissociation of the TTR tetramer, required for its amyloid pathogenesis, is also necessary to prevent cellular toxicity from Aβ oligomers. These findings suggest that the Aβ-binding site of TTR may be hidden in its tetrameric form. Aided by computational docking and peptide screening, we identified a TTR segment that is capable of altering Aβ aggregation and toxicity, mimicking TTR cellular protection. EM, immune detection analysis, and assessment of aggregation and cytotoxicity revealed that the TTR segment inhibits Aβ oligomer formation and also promotes the formation of nontoxic, nonamyloid amorphous aggregates, which are more sensitive to protease digestion. Finally, this segment also inhibits seeding of Aβ catalyzed by Aβ fibrils extracted from the brain of an Alzheimer's patient. Together, these findings suggest that mimicking the inhibitory effect of TTR with peptide-based therapeutics represents an additional avenue to explore for the treatment of Alzheimer's disease.
Topics: Aged, 80 and over; Alzheimer Disease; Amyloid beta-Peptides; Binding Sites; Cell Line; Female; Humans; Molecular Docking Simulation; Prealbumin; Protein Aggregation, Pathological
PubMed: 32769117
DOI: 10.1074/jbc.RA120.013440 -
The FEBS Journal Oct 2009
Topics: Animals; Evolution, Molecular; Humans; Prealbumin
PubMed: 19725884
DOI: 10.1111/j.1742-4658.2009.07242.x -
International Journal of Biological... Nov 2021Wild-type human transthyretin (TTR) is a tetrameric protein that transports thyroxine and retinol in the blood and brain. However, a number of mutations or aging leads...
Wild-type human transthyretin (TTR) is a tetrameric protein that transports thyroxine and retinol in the blood and brain. However, a number of mutations or aging leads to destabilization of the quaternary structure of TTR, which results in dissociation of TTR tetramers to monomers, followed by oligomerization and subsequent amyloid formation. TTR amyloid is a pathogenic factor underlying several diseases. It has recently been documented that destabilization of the structure of TTR is driven by Ca. The present work shows that the in vitro redox conditions contribute to the destabilization and formation of the highly unstable substoichiometric population(s) of TTR molecules. Importantly, destabilized TTR forms acquire the ability to emit fluorescence in the blue range of the light spectrum. Dithiothreitol (DTT), in the presence of Ca, enhances the formation of complex autofluorophore which displays maxima at 417 nm and 438 nm in the emission spectrum of TTR.
Topics: Calcium; Dithiothreitol; Fluorescence; Humans; Oxidation-Reduction; Prealbumin; Protein Multimerization; Protein Stability
PubMed: 34562536
DOI: 10.1016/j.ijbiomac.2021.09.107 -
British Journal of Clinical Pharmacology Dec 2022Transthyretin-mediated amyloidosis is a progressive and fatal disease caused by the build-up of misfolded transthyretin (TTR) protein. Eplontersen is a triantennary...
AIMS
Transthyretin-mediated amyloidosis is a progressive and fatal disease caused by the build-up of misfolded transthyretin (TTR) protein. Eplontersen is a triantennary N-acetyl galactosamine (GalNAc3)-conjugated antisense oligonucleotide targeting TTR messenger ribonucleic acid (mRNA) to inhibit production of both variant and wild-type TTR. We aimed to develop a population pharmacokinetic/pharmacodynamic (PK/PD) model for eplontersen and to evaluate the impact of covariates on exposure and response.
METHODS
Plasma eplontersen and serum TTR concentration data were obtained from two phase 1 studies in healthy volunteers (ClinicalTrials.gov: NCT03728634, NCT04302064). Model development was conducted using a nonlinear mixed-effects approach.
RESULTS
Eplontersen PK was well described by a two-compartment model. Evaluation of demographics identified significant covariates of lean body mass on clearance and body weight on intercompartmental clearance and volumes of distribution. Population PK modelling showed the absorption rate was 29.6% greater with injection into the abdomen versus the arm. The typical population terminal elimination half-life was 25.5 days. Serum TTR was well described by an indirect response model with inhibition of TTR production by eplontersen. Maximum fractional inhibition (I ) was 0.970 (0.549%RSE) and the half maximal inhibitory concentration (IC ) was 0.0283 ng/ml (13.3%RSE). Simulations showed subjects with lower weight had higher exposure (AUC, C ), while higher C was observed when comparing site of administration (ratio abdomen/arm = 1.18), but differences in exposure did not significantly impact response at evaluated doses.
CONCLUSION
The exposure-response relationship of eplontersen was well characterised by the PKPD model. Weight and injection site were found to affect systemic exposure, but this effect does not seem to result in clinically relevant variation in response.
Topics: Humans; Prealbumin; Oligonucleotides, Antisense; Amyloid Neuropathies, Familial; Oligonucleotides
PubMed: 35869634
DOI: 10.1111/bcp.15468 -
Physiological Reports Mar 2022Age-related wild-type transthyretin amyloidosis (wtATTR) is characterized by systemic deposition of amyloidogenic fibrils of misfolded transthyretin (TTR) in the...
Age-related wild-type transthyretin amyloidosis (wtATTR) is characterized by systemic deposition of amyloidogenic fibrils of misfolded transthyretin (TTR) in the connective tissue of many organs. In the heart, this leads to age-related heart failure with preserved ejection fraction (HFpEF). The hypothesis tested is that TTR deposited in vitro disrupts cardiac myocyte cell-to-cell and cell-to-matrix adhesion complexes, resulting in altered calcium handling, force generation, and sarcomeric disorganization. Human iPSC-derived cardiomyocytes and neonatal rat ventricular myocytes (NRVMs), when grown on TTR-coated polymeric substrata mimicking the stiffness of the healthy human myocardium (10 kPa), had decreased contraction and relaxation velocities as well as decreased force production measured using traction force microscopy. Both NRVMs and adult mouse atrial cardiomyocytes had altered calcium kinetics with prolonged transients when cultured on TTR fibril-coated substrates. Furthermore, NRVMs grown on stiff (~GPa), flat or microgrooved substrates coated with TTR fibrils exhibited significantly decreased intercellular electrical coupling as shown by FRAP dynamics of cells loaded with the gap junction-permeable dye calcein-AM, along with decreased gap junction content as determined by quantitative connexin 43 staining. Significant sarcomeric disorganization and loss of sarcomere content, with increased ubiquitin localization to the sarcomere, were seen in NRVMs on various TTR fibril-coated substrata. TTR presence decreased intercellular mechanical junctions as evidenced by quantitative immunofluorescence staining of N-cadherin and vinculin. Current therapies for wtATTR are cost-prohibitive and only slow the disease progression; therefore, better understanding of cardiomyocyte maladaptation induced by TTR amyloid may identify novel therapeutic targets.
Topics: Amyloid Neuropathies, Familial; Animals; Calcium; Calcium, Dietary; Heart Failure; Mice; Myocytes, Cardiac; Prealbumin; Rats; Sarcomeres; Stroke Volume
PubMed: 35262277
DOI: 10.14814/phy2.15207 -
Protein Engineering, Design & Selection... Jun 2016Aggregation of β-amyloid (Aβ) is widely believed to cause neuronal dysfunction in Alzheimer's disease. Transthyretin (TTR) binds to Aβ and inhibits its aggregation...
Aggregation of β-amyloid (Aβ) is widely believed to cause neuronal dysfunction in Alzheimer's disease. Transthyretin (TTR) binds to Aβ and inhibits its aggregation and neurotoxicity. TTR is a homotetrameric protein, with each monomer containing a short α-helix and two anti-parallel β-sheets. Dimers pack into tetramers to form a hydrophobic cavity. Here we report the discovery of a TTR mutant, N98A, that was more effective at inhibiting Aβ aggregation than wild-type (WT) TTR, although N98A and WT bound Aβ equally. The N98A mutation is located on a flexible loop distant from the putative Aβ-binding sites and does not alter secondary and tertiary structures nor prevent correct assembly into tetramers. Under non-physiological conditions, N98A tetramers were kinetically and thermodynamically less stable than WT, suggesting a difference in the tetramer folded structure. In vivo, the lone cysteine in TTR is frequently modified by S-cysteinylation or S-sulfonation. Like the N98A mutation, S-cysteinylation of TTR modestly decreased tetramer stability and increased TTR's effectiveness at inhibiting Aβ aggregation. Collectively, these data indicate that a subtle change in TTR tetramer structure measurably increases TTR's ability to inhibit Aβ aggregation.
Topics: Alzheimer Disease; Amyloid beta-Peptides; Binding Sites; Humans; Models, Molecular; Point Mutation; Prealbumin; Protein Aggregation, Pathological; Protein Binding; Protein Conformation; Protein Multimerization
PubMed: 27099354
DOI: 10.1093/protein/gzw008 -
Biochimica Et Biophysica Acta. General... Feb 2019Human transthyretin (TTR) is a homotetramer that transports thyroid hormones and retinol in the serum and cerebrospinal fluid. TTR is also an intracellular protein found...
Human transthyretin (TTR) is a homotetramer that transports thyroid hormones and retinol in the serum and cerebrospinal fluid. TTR is also an intracellular protein found in tissues such as those in the brain, eye and pancreas. TTR is a nutrition marker, reflecting the health of the organism, and TTR levels are linked to the normal and diseased states of the body. The switch from a protective to a pathological role is attributed to the destabilisation of the TTR structure, which leads to tetramer dissociation and amyloid formation. Native and destabilised TTR have been associated with osteoarthritis and bone density in humans. Moreover, TTR is present in eggshell mammillary cones; therefore, we verified the putative TTR engagement in the process of mineral formation. Using an in vitro assay, we found that TTR affected calcium carbonate crystal growth and morphology, producing asymmetric crystals with a complex nanocrystalline composition. The crystals possessed rounded edges and corners and irregular etch pits, suggesting the selective inhibition of crystal growth and/or dissolution imposed by TTR. The occurrence of many porosities, fibrillary inclusions and amorphous precipitates suggested that destabilisation of the TTR structure is an important factor involved in the mineralisation process. Crystals grown in the presence of TTR exhibited the characteristic features of crystals controlled by biomineralisation-active proteins, suggesting novel functions of TTR in the mineral formation process.
Topics: Calcium Carbonate; Crystallization; Humans; Particle Size; Prealbumin; Protein Conformation; Protein Stability
PubMed: 30394286
DOI: 10.1016/j.bbagen.2018.10.017