Steroid Receptors and Vertebrate Evolution

Considering that life on earth evolved about 3.7 billion years ago, vertebrates are young, appearing in the fossil record during the Cambrian explosion about 542 to 515 million years ago. Results from sequence analyses of genomes from bacteria, yeast, plants, invertebrates and vertebrates indicate that receptors for adrenal steroids (aldosterone, cortisol), and sex steroids (estrogen, progesterone, testosterone) also are young, with receptors for estrogens and 3-ketosteroids first appearing in basal chordates (cephalochordates: amphioxus), which are close ancestors of vertebrates. An ancestral progesterone receptor and an ancestral corticoid receptor, the common ancestor of the glucocorticoid and mineralocorticoid receptors, evolved in jawless vertebrates (cyclostomes: lampreys, hagfish). This was followed by evolution of an androgen receptor and distinct glucocorticoid and mineralocorticoid receptors in cartilaginous fishes (gnathostomes: sharks). Adrenal and sex steroid receptors are not found in echinoderms: and hemichordates, which are ancestors in the lineage of cephalochordates and vertebrates. The presence of steroid receptors in vertebrates, in which these steroid receptors act as master switches to regulate differentiation, development, reproduction, immune responses, electrolyte homeostasis and stress responses, argues for an important role for steroid receptors in the evolutionary success of vertebrates, considering that the human genome contains about 22,000 genes, which is not much larger than genomes of invertebrates, such as Caenorhabditis elegans (~18,000 genes) and Drosophila (~14,000 genes).Comment: 18 pages, 5 figure

Insights from the Structure of Estrogen Receptor into the Evolution of Estrogens: Implications for Endocrine Disruption

In the last decade, there has been important progress in understanding the origins and evolution of receptors for adrenal steroids (aldosterone, cortisol) and sex steroids (estradiol, progesterone, testosterone) due to the sequencing of genomes from animals that are at key sites in vertebrate evolution. Although the estrogen receptor [ER] appears to be the ancestral vertebrate steroid receptor and estradiol [E2] is the physiological ligand for vertebrate ERs, the identity of the ancestral ligand(s) for the ER remains unknown. Here, using an analysis of crystal structures of human ER[alpha] with E2 and other chemicals and 3D models of human ER[alpha] with 27-hydroxycholesterol and 5-androsten-3[beta],17[beta]-diol, we propose that one or more [DELTA]5 steroids were the ancestral ligands for the ER, with E2 evolving later as the canonical estrogen. The evidence that chemicals with a [beta]-hydroxy at C3 in a saturated A ring can act as estrogens and the conformational flexibility of the vertebrate ER can explain the diversity of synthetic chemicals that disrupt estrogen responses by binding to vertebrate ERs

Trichoplax, the simplest known animal, contains an estrogen-related receptor: Implications for the evolution of vertebrate and invertebrate estrogen receptors

Although, as their names imply, vertebrate and invertebrate estrogen receptors [ERs] and estrogen-related receptors [ERRs] are related transcription factors, their evolutionary relationships to each other are not fully understood. We searched recently sequenced genome of _Trichoplax_, the simplest known animal, and genomes from three lophotrochozoans: _Capitella_, a worm, _Helobdella robusta_, a leech, and _Lottia gigantea_, a snail, to elucidate the origins and evolution of ERs and ERRs. BLAST found an ERR in _Trichoplax_, but no ER. BLAST searches of the lophotrochozaons found ERRs in all three and invertebrate ERs in _Capitella_ and _Lottia_, but not in _Helobdella_. These database searches and a phylogenetic analyses indicate that invertebrate ERs arose in a protostome, and vertebrate ERs arose later in deuterostome

Origin and diversification of steroids: Co-evolution of enzymes and nuclear receptors

Recent sequencing of amphioxus and sea urchin genomes has provided important data for understanding the origins of enzymes that synthesize adrenal and sex steroids and the receptors that mediate physiological response to these vertebrate steroids. Phylogenetic analyses reveal that CYP11A and CYP19, which are involved in the synthesis of adrenal and sex steroids, first appear in the common ancestor of amphioxus and vertebrates. This correlates with recent evidence for the first appearance in amphioxus of receptors with close similarity to vertebrate steroid receptors. Other CYP450 enzymes involved in steroid synthesis can be traced back to invertebrates, in which they have at least two functions: detoxifying xenobiotics and catalyzing the synthesis of sterols that activate nuclear receptors. CYP450 metabolism of hydrophobic xenobiotics may have been a key event in the origin of ligand-activated steroid receptors from constitutively active nuclear receptors

Motif analysis of amphioxus, lamprey and invertebrate estrogen receptors and amphioxus and human estrogen-related receptors: Towards a better understanding of estrogen receptor evolution

*Background.* The origins of steroid-dependent regulation of the vertebrate estrogen receptor (ER) are poorly understood. Genes with statistically significant sequence similarity to vertebrate ERs have been found in lamprey, a basal vertebrate, and amphioxus, a basal chordate. Motif analysis of these sequences provides an opportunity to investigate early events in the evolution of the ER.
*Results.* We used artificial intelligence-based software to construct twelve motifs specific to the estrogen-binding domain of ER[alpha] and ER[beta] in land vertebrates and teleosts. We mapped these ER-specific motifs onto the sequences of lamprey, amphioxus, invertebrate and selected vertebrate ERs and amphioxus, Ciona and human estrogen-related receptor (ERR). We find that lamprey ER contains eleven motifs common to ERs in the training set. In contrast, amphioxus ER contains only six motifs. Various invertebrate ERs contain either six or seven motifs. Unexpectedly, human and amphioxus ERRs contain nine of the twelve motifs, despite extensive sequence divergence during the descent of chordate ERs and ERRs from a common ancestor. We mapped the twelve motifs onto a multiple alignment of human, lamprey and amphioxus ERs, which depicted residues in human ER[alpha] that are known to bind estradiol. There is excellent conservation of these key residues in lamprey ER and poor conservation in amphioxus ER. Out of seventeen residues on human ER[alpha] that bind estradiol, sixteen and six are identical in lamprey and amphioxus ER, respectively. A phylogenetic tree of ERs and ERRs reveals a long branch for amphioxus ER, which is in agreement with the low sequence and motif similarity between amphioxus ER and other ERs.
*Conclusions.* There are significant differences between _B. floridae_ ER and vertebrate ERs in the steroid-binding domain as measured by motif analysis and percent of amino acids that are known to stabilize estradiol in human ER[alpha]. The absence in lamprey ER of motif 10, which maps to the C-terminus half of [alpha]-helix 9, may be important in recognition of novel estrogens, such as 15[alpha]-hydroxy-estradiol. 
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3D model of amphioxus steroid receptor complexed with estradiol

The origins of signaling by vertebrate steroids are not fully understood. An important advance was the report that an estrogen-binding steroid receptor [SR] is present in amphioxus, a basal chordate with a similar body plan as vertebrates. To investigate the evolution of estrogen binding to steroid receptors, we constructed a 3D model of amphioxus SR complexed with estradiol. This 3D model indicates that although the SR is activated by estradiol, some interactions between estradiol and human ER[alpha] are not conserved in the SR, which can explain the low affinity of estradiol for the SR. These differences between the SR and ER[alpha] in the steroid-binding domain are sufficient to suggest that another steroid is the physiological regulator of the SR. The 3D model predicts that mutation of Glu-346 to Gln will increase the affinity of testosterone for amphioxus SR and elucidate the evolution of steroid binding to nuclear receptors

3D models of lamprey corticoid receptor complexed with 11-deoxycortisol and deoxycorticosterone

The serum of Atlantic sea lamprey, a basal vertebrate, contains two corticosteroids, 11-deoxycortisol and deoxycorticosterone. Only 11-deoxycortisol has high affinity [Kd~3 nM] for the corticoid receptor [CR] in lamprey gill cytosol. To investigate the binding of 11-deoxycortisol to the CR, we constructed 3D models of lamprey CR complexed with 11-deoxycortisol and deoxycorticosterone. These 3D models reveal that Leu-220 and Met-299 in lamprey CR have contacts with the 17[alpha]-hydroxyl on 11-deoxycortisol. Lamprey CR is the ancestor of the mineralocorticoid receptor [MR] and glucocorticoid receptor [GR]. Unlike human MR and human GR, the 3D model of lamprey CR finds a van der Waals contact between Cys-227 in helix 3 and Met-264 in helix 5. Mutant human MR and GR containing a van der Waals contact between helix 3 and helix 5 display enhanced responses to progesterone and glucocorticoids, respectively. We propose that this interaction was present in the CR and lost during the evolution of the MR and GR, leading to changes in their response to progesterone and corticosteroids, respectively

An athletic approach to studying perception-action integration: Does sport-specific training, and the impact of injury, influence how individuals visually guide navigation?

The objective of this thesis was to investigate perception-action integration capabilities of individuals during a choice navigation task. This task assessed navigation strategies in open space while individuals avoided colliding with two vertical obstacles that created a body-scaled, horizontal gap, at three varying obstacle distances from the starting location (3m, 5m, 7m). The two studies completed in this thesis employed the same paradigm to assess the hypothesized group differences. Gaze behaviours and kinematics of navigation strategies were compared between: 1) athletes specifically trained in navigating in open space versus non-athletes; and 2) athletes with post-concussion syndrome (PCS) versus non-concussed, specifically trained athletes. Specifically trained athletes have been identified as demonstrating more successful perception-action integration in discrete motor tasks related to their sport (Mann et al., 2007; Vickers, 2007). However, whether these abilities translate to the continuous motor task of obstacle avoidance in open space was unknown. The purpose of Study 1 was to identify the influence of sport-specific training on navigating in open space (i.e. navigational strategies of large field sport athletes) compared to age-matched, non-athletes. It was hypothesized that specifically-trained athletes would demonstrate fewer, longer fixations, suggesting a more successful perception-action integration strategy (as defined by Mann et al., 2007), and would employ more sport-specific navigation strategies than non-athletes by maintaining their straight trajectory toward the goal (Fajen & Warren, 2003). Athletes were found to make fewer, longer fixations than non-athletes. However, no differences were observed between navigation strategies of the two groups, nor were any kinematic measures found to differ between groups. It can be concluded that athletes and non-athletes differentially obtain visual information to perform the same actions, suggesting that athletes and non-athletes differentially perform perception-action integration when navigating in open space. Future studies are required to identify sport-specific nuances of navigation (moving obstacles, running) to better identify athletic-related navigation strategies. Although athletic training can enhance perception-action integration strategies, sport-related injuries can hinder this process. Following a concussion, individuals experience deficits of perception-action integration that persist well beyond 30 days of recovery, post-concussion (Baker and Cinelli, 2014; Slobounov et al., 2006). These perception-action integration deficits may also exist in individual with postconcussion syndrome (PCS). The purpose of the Study 2 was to identify whether perception-action integration deficits persist with the persistent physical symptoms of concussion characteristic of PCS. The current study revealed that athletes with PCS did not differ from non-concussed athletes on any measure of visual fixation strategy, nor were they found to differ on any kinematic measure assessed. These findings suggest that in the context of the current paradigm, athletes with PCS have no perception-action integration deficit. In that, athletes with PCS may have adapted perception-action integration strategies to navigate with equal efficiency as a specifically-trained group of athletes or that the paradigm was not sensitive enough to identify these differences. Such findings suggest that more research is required to assess what, if any, perception-action integration deficits persist with persisting physical symptoms of PCS to better benefit rehabilitative procedures and outcomes for these individuals. Together, these studies add to what was previously known about perception-action integration, as it relates to navigation. Both studies assessed perception-action integration in unique populations that add to understanding of behavioural dynamics in the sport setting. Study 1 builds on a line of research assessing affordance theory and behavioural dynamics in sport (Fajen, Riley, & Turvey, 2008). The findings of this study suggest that although navigation strategies did not differ between specifically trained athletes and non-athletes, visual search strategies employed in task did. Such findings add to the understanding that sport-specific training influences perception-action integration, through our understanding of how athletes obtain visual information to perform actions. This thesis did not identify perception-action integration deficits in athletes with PCS. These findings suggest that the individuals in the present study likely adapted to their injury as they demonstrated equal ability in gaze and navigation strategies to specifically-trained athletes. As such, further research is required to assess the cognitive, motor, and sensory-motor deficits that may persist with the persisting physical symptoms of PCS. As individuals with PCS do not demonstrate similar visuomotor integration deficits as individuals with acute concussions (Baker & Cinelli, 2014), such individuals must be assessed and researched as a separate population

Cysteine-10 on 17 β-Hydroxysteroid dehydrogenase 1 has stabilizing interactions in the cofactor binding region and renders sensitivity to sulfhydryl modifying chemicals

17 β-Hydroxysteroid dehydrogenase type 1 (17 β -HSD1) catalyzes the conversion of estrone to the potent estrogen estradiol. 17 β -HSD1 is highly expressed in breast and ovary tissues and represents a prognostic marker for the tumor progression and survival of patients with breast cancer and other estrogen-dependent tumors. Therefore, the enzyme is considered a promising drug target against estrogen-dependent cancers. For the development of novel inhibitors, an improved understanding of the structure-function relationships is essential. In the present study, we examined the role of a cysteine residue, Cys(10), in the Rossmann-fold NADPH binding region, for 17 β -HSD1 function and tested the sensitivity towards sulfhydryl modifying chemicals. 3D structure modeling revealed important interactions of Cys(10) with residues involved in the stabilization of amino acids of the NADPH binding pocket. Analysis of enzyme activity revealed that 17 β -HSD1 was irreversibly inhibited by the sulfhydryl modifying agents N-ethylmaleimide (NEM) and dithiocarbamates. Preincubation with increasing concentrations of NADPH protected 17 β -HSD1 from inhibition by these chemicals. Cys(10)Ser mutant 17 β -HSD1 was partially protected from inhibition by NEM and dithiocarbamates, emphasizing the importance of Cys(10) in the cofactor binding region. Substitution of Cys(10) with serine resulted in a decreased protein half-life, without significantly altering kinetic properties. Despite the fact that Cys(10) on 17 β -HSD1 seems to have limited potential as a target for new enzyme inhibitors, the present study provides new insight into the structure-function relationships of this enzyme