Biomedical subjects

Samantha J Richardson

Publications and source records attributed to Samantha J Richardson.

14 recordsLinked to original sources

PubMed2006

Structural and functional evolution of transthyretin and transthyretin-like proteins.

Transthyretin (TTR) is a tetrameric protein involved in the distribution of thyroid hormones in vertebrates. The amino acid sequence of TTR is highly conserved across vertebrates. Hypothetical TTR-like proteins (TLPs) were inferred from the identification of genes in nonvertebrate species. Here, we identified five motifs defining TLPs and three motifs defining both TTRs and TLPs. These motifs were mapped onto structurally conserved and functionally important regions of TTRs. These motifs were used to build hidden Markov models for accurate identification of TLPs in other organisms. TLPs were divided into three main groups based on their N-terminal regions. Most TLPs are cytosolic, but in plants and slime mold, we predict they are peroxisomal. We verified that the TLPs from enterobacteria were periplasmic. We demonstrated that TLP genes are expressed in a bacterium (E. coli), an invertebrate animal (C. elegans), and a plant (A. thaliana). These TLPs have similar subunit molecular weights to TTRs, are tetramers, and are predicted to have similar three-dimensional (3D) structures to TTRs, but do not bind thyroid hormones or similar ligands. We suggest that like TTRs, the N-terminal and C-terminal regions of TLPs are integral in defining the function of TLPs in nonvertebrate species and that the TLP gene duplicated in primitive vertebrates to produce the TTR gene. TLP/TTR has retained its overall structure, but changed function and localization during evolution in bacteria, invertebrates, plants, and vertebrates.

Amino Acid Motifs↗

PubMed2006

Change in structure of the N-terminal region of transthyretin produces change in affinity of transthyretin to T4 and T3.

The relationship between the structure of the N-terminal sequence of transthyretin (TTR) and the binding of thyroid hormone was studied. A recombinant human TTR and two derivatives of Crocodylus porosus TTRs, one with the N-terminal sequence replaced by that of human TTR (human/crocTTR), the other with the N-terminal segment removed (truncated crocTTR), were synthesized in Pichia pastoris. Subunit mass, native molecular weight, tetramer formation, cross-reactivity to TTR antibodies and binding to retinol-binding protein of these recombinant TTRs were similar to TTRs found in nature. Analysis of the binding affinity to thyroid hormones of recombinant human TTR showed a dissociation constant (Kd) for triiodothyronine (T3) of 53.26+/-3.97 nM and for thyroxine (T4) of 19.73+/-0.13 nM. These values are similar to those found for TTR purified from human serum, and gave a Kd T3/T4 ratio of 2.70. The affinity for T4 of human/crocTTR (Kd=22.75+/-1.89 nM) was higher than those of both human TTR and C. porosus TTR, but the affinity for T3 (Kd=5.40+/-0.25 nM) was similar to C. porosus TTR, giving a Kd T3/T4 ratio of 0.24. A similar affinity for both T3 (Kd=57.78+/-5.65 nM) and T4 (Kd=59.72+/-3.38 nM), with a Kd T3/T4 ratio of 0.97, was observed for truncated crocTTR. The obtained results strongly confirm the hypothesis that the unstructured N-terminal region of TTR critically influences the specificity and affinity of thyroid hormone binding to TTR.

Alligators and Crocodiles↗

PubMed2006

The crystal structure of the transthyretin-like protein from Salmonella dublin, a prokaryote 5-hydroxyisourate hydrolase.

The mechanism of binding of thyroid hormones by the transport protein transthyretin (TTR) in vertebrates is structurally well characterised. However, a homologous family of transthyretin-like proteins (TLPs) present in bacteria as well as eukaryotes do not bind thyroid hormones, instead they are postulated to perform a role in the purine degradation pathway and function as 5-hydroxyisourate hydrolases. Here we describe the 2.5 Angstroms X-ray crystal structure of the TLP from the Gram-negative bacterium Salmonella dublin, and compare and contrast its structure with vertebrate TTRs. The overall architecture of the homotetramer is conserved and, despite low sequence homology with vertebrate TTRs, structural differences within the monomer are restricted to flexible loop regions. However, sequence variation at the dimer-dimer interface has profound consequences for the ligand binding site and provides a structural rationalisation for the absence of thyroid hormone binding affinity in bacterial TLPs: the deep, negatively charged thyroxine-binding pocket that characterises vertebrate TTR contrasts with a shallow and elongated, positively charged cleft in S. dublin TLP. We have demonstrated that Sdu_TLP is a 5-hydroxyisourate hydrolase. Furthermore, using site-directed mutagenesis, we have identified three conserved residues located in this cleft that are critical to the enzyme activity. Together our data reveal that the active site of Sdu_TLP corresponds to the thyroxine binding site in TTRs.

Amidohydrolases↗

PubMed2005

Binding of amyloidogenic transthyretin to the plasma membrane alters membrane fluidity and induces neurotoxicity.

Transthyretin (TTR) can deposit as amyloid in the peripheral nervous system and induce a peripheral neuropathy. We examined the mechanism of TTR amyloid neurotoxicity on SH-SY5Y neuroblastoma cells. Wild-type (WT) TTR and two amyloidogenic mutants (V30M and L55P) were expressed in Escherichia coli. Incubation (aging) of WT TTR at 37 degrees C for 1 week caused no significant aggregation. However, there was a significant increase in the extent of amyloid fibril formation after the amyloidogenic mutants had been aged. L55P TTR aggregated more readily than V30M TTR. Both amyloidogenic mutants were neurotoxic after aging. The order of neurotoxicity was as follows: L55P > V30M > WT. As binding of amyloid proteins to the plasma membrane may cause cytotoxicity, we studied the binding of TTR to a plasma membrane-enriched preparation from SH-SY5Y cells by surface plasmon resonance. All three forms bound to the plasma membrane through electrostatic interactions. The binding of the amyloidogenic mutants was increased by aging. The amount of binding correlated closely with the amount of aggregation and with the cytotoxicity of each form. As membrane fluidity can influence cell viability, we also examined the effect of TTR on membrane fluidity using a fluorescence anisotropy method. Binding of the amyloidogenic TTR mutants increased membrane fluidity, and once again, the order of potency was as follows: L55P > V30M > WT. These results demonstrate that TTR can bind to the plasma membrane and cause a change in membrane fluidity. Altered membrane fluidity may be the cause of the neurotoxicity.

Amino Acid Substitution↗

PubMed2005

Developmentally regulated thyroid hormone distributor proteins in marsupials, a reptile, and fish.

Thyroid hormones are essential for vertebrate development. There is a characteristic rise in thyroid hormone levels in blood during critical periods of thyroid hormone-regulated development. Thyroid hormones are lipophilic compounds, which readily partition from an aqueous environment into a lipid environment. Thyroid hormone distributor proteins are required to ensure adequate distribution of thyroid hormones, throughout the aqueous environment of the blood, and to counteract the avid partitioning of thyroid hormones into the lipid environment of cell membranes. In human blood, these proteins are albumin, transthyretin and thyroxine-binding globulin. We analyzed the developmental profile of thyroid hormone distributor proteins in serum from a representative of each order of marsupials (M. eugenii; S.crassicaudata), a reptile (C. porosus), in two species of salmonoid fishes (S. salar; O. tshawytsch), and throughout a calendar year for sea bream (S. aurata). We demonstrated that during development, these animals have a thyroid hormone distributor protein present in their blood which is not present in the adult blood. At least in mammals, this additional protein has higher affinity for thyroid hormones than the thyroid hormone distributor proteins in the blood of the adult. In fish, reptile and polyprotodont marsupial, this protein was transthyretin. In a diprotodont marsupial, it was thyroxine-binding globulin. We propose an hypothesis that an augmented thyroid hormone distributor protein network contributes to the rise in total thyroid hormone levels in the blood during development.

Alligators and Crocodiles↗

PubMed2005

Evolution of the thyroid hormone distributor protein transthyretin in microbes, C. elegans, and vertebrates.

Transthyretin (TTR) is an extracellular thyroid hormone distributor protein in vertebrates, whose structure has been highly conserved between fish and humans. However, the ligand preferentially bound by TTR has changed during evolution from 3',3,5-L-triiodothyronine (T3) to 3',5',3,5-l-tetraiodothyronine (T4). We identified genes in the genomes of >50 species of nonvertebrates, which could code for TTR-like proteins. Molecular modeling suggested most would have similar 3D structures and electrostatic surface potentials to vertebrate TTRs. We amplified TTR-like genes from a C. elegans cDNA library, demonstrating that it is transcribed. We synthesized recombinant TTR-like proteins from S. dublin and C. elegans. These proteins form tetramers similarly to vertebrate TTRs, but their ligands remain elusive.

Animals↗

PubMed2004

Expression of the glucocorticoid receptor from the 1A promoter correlates with T lymphocyte sensitivity to glucocorticoid-induced cell death.

Glucocorticoid (GC) hormones cause pronounced T cell apoptosis, particularly in immature thymic T cells. This is possibly due to tissue-specific regulation of the glucocorticoid receptor (GR) gene. In mice the GR gene is transcribed from five separate promoters designated: 1A, 1B, 1C, 1D, and 1E. Nearly all cells express GR from promoters 1B-1E, but the activity of the 1A promoter has only been reported in the whole thymus or lymphocyte cell lines. To directly assess the role of GR promoter use in sensitivity to glucocorticoid-induced cell death, we have compared the activity of the GR 1A promoter with GC sensitivity in different mouse lymphocyte populations. We report that GR 1A promoter activity is restricted to thymocyte and peripheral lymphocyte populations and the cortex of the brain. The relative level of expression of the 1A promoter to the 1B-1E promoters within a lymphocyte population was found to directly correlate with susceptibility to GC-induced cell death, with the extremely GC-sensitive CD4+CD8+ thymocytes having the highest levels of GR 1A promoter activity, and the relatively GC-resistant alphabetaTCR+CD24(int/low) thymocytes and peripheral T cells having the lowest levels. DNA sequencing of the mouse GR 1A promoter revealed a putative glucocorticoid-response element. Furthermore, GR 1A promoter use and GR protein levels were increased by GC treatment in thymocytes, but not in splenocytes. These data suggest that tissue-specific differences in GR promoter use determine T cell sensitivity to glucocorticoid-induced cell death.

Animals↗

PubMed2003

Altered epithelial cell proportions in the fetal lung of glucocorticoid receptor null mice.

Glucocorticoids provide important signals for maturation of the fetal lung and antenatal glucocorticoids are used to reduce the respiratory insufficiency suffered by preterm infants. To further understand the role of glucocorticoids in fetal lung maturation, we have analyzed mice with a targeted null mutation for the glucocorticoid receptor (GR) gene, which severely retards lung development. The lungs of fetal GR-null mice have increased lung weight and DNA content, are condensed and hypercellular, with reduced septal thinning leading to a 6-fold increase in the airway to capillary diffusion distance. In fetal GR-null mice, mRNA levels of the type II epithelial cell surfactant protein genes A and C were reduced by approximately 50%. Analysis of epithelial cell types by electron microscopy revealed that the proportions of type II cells were increased by approximately 30%, whereas the proportions of type-I cells were markedly reduced (by approximately 50%). Similarly, we found a 50% reduction in mRNA levels for T1alpha and aquaporin-5, two type I cell-specific markers, and a 20% reduction in aquaporin-1 mRNA levels. This demonstrates that during murine embryonic development, receptor-mediated glucocorticoid signaling facilitates the differentiation of epithelial cells into type I cells, but is not obligatory for type II cell differentiation.

Animals↗

PubMed2002

Characterization of outer ring iodothyronine deiodinases in tissues of the saltwater crocodile (Crocodylus porosus).

The distribution and characterization of outer ring deiodination (ORD) using reverse triiodothyronine (rT3) and thyroxine (T4) as substrates is reported in microsomes of liver, kidney, lung, heart, gut, and brain tissues from juvenile saltwater crocodiles (Crocodylus porosus). In lung and heart only small amounts of rT3 ORD and T4 ORD were detected, while in brain only a small amount of T4 ORD was detected. More detailed characterization studies could be performed on liver, kidney, and gut microsomes. Reverse T3 outer ring deiodination (rT3 ORD) was the predominant activity in liver and kidney microsomes. The properties of crocodile liver and kidney rT3 ORD, such as preference for rT3 as substrate, a dithiothreitol (DTT) requirement of 10 mM, inhibition by propylthiouracil (PTU), and Michaelis-Menten (Km) constant in the micromolar range, correspond to the properties previously reported for a type I deiodinase. The temperature optimum for rT3 ORD was between 30 and 35 degrees. There was also rT3 ORD activity in gut microsomes, along with what appeared to be a type II-like, low-Km deiodinase with a substrate preference for T4. There was also a small amount of T4 ORD activity in liver and kidney microsomes. Liver T4 ORD, like a type II deiodinase, had a preference for T4 as substrate at low substrate concentrations and a DTT requirement of 15 mM and was insensitive to PTU. However, at high substrate concentrations the predominant activity was of the type I deiodinase nature. T4 ORD in liver had an optimal incubation temperature of 30 to 35 degrees. Gut microsomal T4 ORD was also type II-like at low substrate concentrations and type I-like at high substrate concentrations. Gut T4 ORD had an optimal incubation temperature of 25 to 30 degrees and a DTT requirement of 20 mM DTT. Kidney microsomal T4 ORD had the same optimal temperature and DTT requirement as that in gut microsomes; however, there was no competition by low substrate concentrations. These results suggest that ORD in juvenile saltwater crocodile kidney is most likely exclusively catalyzed by a type I-like deiodinase. Liver and gut ORD, in contrast, is catalyzed by two enzymes, with a predominance of a type I-like deiodinase in liver and a type II-like deiodinase in gut. Low-Km T3 IRD activity could not be detected in any tissues of the juvenile saltwater crocodile.

Alligators and Crocodiles↗

PubMed2002

Developmental profile of thyroid hormone distributor proteins in a marsupial, the tammar wallaby Macropus eugenii.

The ontogeny of thyroxine distributor proteins in serum of the marsupial Macropus eugenii (tammar wallaby) was investigated from day 3 after birth until adulthood. The thyroxine distributor proteins in the serum of adult M. eugenii are transthyretin and albumin. Northern analysis of RNA prepared from liver showed that transthyretin mRNA levels were initially high (about adult levels at the earliest ages tested), reduced to about 60% adult levels (between days 50 and 150), and then steadily increased to adult levels (by days 200 to 250). Albumin mRNA levels were initially about 50% of adult levels (day 3) and steadily rose to 90% of adult levels by days 175 to 220. A globulin, "wallaby thyroxine-binding protein" (W-TBP), bound [(125)I]thyroxine from day 3 until about day 200. Of the protein-bound thyroxine, the proportion bound by transthyretin had a similar pattern to the transthyretin mRNA levels. From day 26 onward, about half of the protein-bound thyroxine was bound to albumin. On day 3, less than 10% was bound to W-TBP and the proportion steadily increased to a maximum of about 46% by about day 120 and then reduced to undetectable levels by around day 250. The developmentally regulated W-TBP was present throughout pouch life, when the pouch young is dependent on obtaining thyroxine required for normal growth and development from the mother. After the young tammar wallaby leaves its mother's pouch, a time when it has reached a level of physiological development approximately equivalent to that at the time of birth in precocious eutherian mammals such as cattle and sheep, W-TBP was no longer detected as a thyroxine distributor protein in serum.

Aging↗

PubMed2002

Thyroid hormone deiodinases during embryonic development of the saltwater crocodile (Crocodylus porosus).

All tissues of the embryonic saltwater crocodile (Crocodylus porosus) gradually increased in weight during development except for lung tissue, which had a peak weight of 1.09 g at day 67, thereafter decreasing in weight. The brain was a relatively large organ. Deiodinase activities in liver, kidney, lung, heart, gut, and brain from day 29 to day 77 of development of the saltwater crocodile were investigated. High-K(m) reverse triiodothyronine (rT(3)) outer ring deiodination (ORD) activity was present in all tissues except the brain. Activity ranged from 559 +/- 51.3 pmol rT(3) deiodinated/mg protein/min in the liver at day 77 to below 10 pmol rT(3) deiodinated/mg protein/min in gut, lung, and heart tissue. rT(3) ORD increased during development in the liver and kidney but decreased in the gut and lung. Activity in the heart was very low (less than 2 pmol rT(3) deiodinated/mg protein/min) and did not change during development. Low-K(m) thyroxine (T(4)) ORD in liver and kidney tissue had peaks of activity around day 49 of incubation (0.52 and 0.09 fmol T(4) deiodinated/mg protein/min, respectively). After day 49, T(4) ORD activity in these tissues decreased. T(4) ORD activity in gut, lung, and heart was very low (less than 0.04 fmol T(4) deiodinated/mg protein/min), with activity in lung increasing slightly during the rest of development. T(4) ORD activity in the brain increased toward day 77 (0.14 +/- 0.03 fmol T(4) deiodinated/mg protein/min), illustrating its importance in local triiodothyronine (T(3)) production during brain development. T(3) inner ring deiodination activity was present only in the embryonic liver and peaked at day 49 (10.1 fmol T(3) deiodinated/mg protein/min), after which activity decreased.

Alligators and Crocodiles↗

PubMed2002

Glucocorticoids, thyroid hormones, and iodothyronine deiodinases in embryonic saltwater crocodiles.

We investigated the relationship between glucocorticoids, thyroid hormones, and outer ring and inner ring deiodinases (ORD and IRD) during embryonic development in the saltwater crocodile (Crocodylus porosus). We treated the embryos with the synthetic glucocorticoid dexamethasone (Dex), 3,3',5-triiodothyronine (T(3)), and a combination of these two hormones (Dex + T(3)). The effects of these treatments were specific in different tissues and at different stages of development and also brought about changes in plasma concentrations of free thyroid hormones and corticosterone. Administration of Dex to crocodile eggs resulted in a decrease in 3,3',5,5'-tetraiodothyronine (T(4)) ORD activities in liver and kidney microsomes, and a decrease in the high-K(m) rT(3) ORD activity in kidney microsomes, on day 60 of incubation. Dex treatment increased the T(4) ORD activity in liver microsomes, but not kidney microsomes, on day 75 of incubation. Dex administration decreased T(3) IRD activity in liver microsomes. However, this decrease did not change plasma-free T(3) concentrations, which suggests that free thyroid hormone levels are likely to be tightly regulated during development.

Alligators and Crocodiles↗

PubMed2002

Crocodile transthyretin: structure, function, and evolution.

Structure and function were studied for Crocodylus porosus transthyretin (crocTTR), an important intermediate in TTR evolution. The cDNA for crocTTR mRNA was cloned and sequenced and the amino acid sequence of crocTTR was deduced. In contrast to mammalian TTRs, but similar to avian and lizard TTRs, the subunit of crocTTR had a long and hydrophobic NH(2)-terminal region. Different from the situation in mammals, triiodothyronine (T(3)) was bound by crocTTR with higher affinity than thyroxine (T(4)). Recombinant crocTTR and a chimeric construct, with the NH(2)-terminal region of crocTTR being replaced by that of Xenopus laevis TTR, were synthesized in the yeast Pichia pastoris. Analysis of the affinity of the chimeric TTRs showed that the NH(2)-terminal region modulates T(4) and T(3) binding characteristics of TTR. The structural differences of the NH(2)-terminal regions of reptilian and amphibian TTRs were caused by a shift in splice sites at the 5' end of exon 2. The comparison of crocodile and other vertebrate TTRs shows that TTR evolution is an example for positive Darwinian evolution and identifies its molecular mechanism.

Alligators and Crocodiles↗

PubMed2002

The evolution of transthyretin synthesis in vertebrate liver, in primitive eukaryotes and in bacteria.

Thyroid hormones are evolutionarily old signal molecules, which can partition between compartments by partitioning into lipid membranes. The role of thyroid hormone distributor proteins is to ensure that sufficient thyroid hormone remains in the bloodstream. Of particular interest is the role of transthyretin, synthesised by the liver and secreted into the blood. In this review, three hypotheses are presented, suggesting the selection pressures leading to the onset of transthyretin synthesis in the liver during evolution. A thyroid hormone distribution network would be a selection advantage over a single protein performing this function. Similarly to the situation in eutherians, hepatic transthyretin synthesis in marsupials is under negative acute phase regulation. The overall three-dimensional structure of transthyretin did not change appreciably during vertebrate evolution. The region of the primary sequence which evolved most was the N-terminal region of the subunit. The N-termini of transthyretin changed from longer and more hydrophobic in reptiles/birds, to shorter and more hydrophilic in eutherians. These changes are correlated with a change in preference from binding of triiodothyronine, to binding thyroxine. As the rest of the molecule had not changed significantly during vertebrate evolution, the gene coding for transthyretin must have evolved prior to the divergence of the vertebrates from the non-vertebrates. Five open reading frames in the genomes of C. elegans (2), S. dublin, S. pombe and E. coli were identified. The protein products are predicted to form tetramers similar to transthyretins. Two possible functions of these proteins are suggested.

Animals↗