Ecdysone-induced protein 75B : Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - Ecdysone-induced protein 75B

Synonyms - E75, E75A, E75B and E75C

Cytological map position - E75A8--B2

Function - Transcription factor

Keywords - molting

Symbol - Eip75B

FlyBase ID: FBgn0000568

Genetic map position -

Classification - hormone receptor

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene | UniGene |
Recent literature
Jaumouille, E., Machado Almeida, P., Stahli, P., Koch, R. and Nagoshi, E. (2015). Transcriptional regulation via nuclear receptor crosstalk required for the Drosophila circadian clock. Curr Biol 25: 1502-1508. PubMed ID: 26004759
Summary:
Circadian clocks in large part rely on transcriptional feedback loops. At the core of the clock machinery, the transcriptional activators CLOCK/BMAL1 (in mammals) and Clock/Cycle (Clk/Cyc) (in Drosophila) drive the expression of the period (per) family genes. The Per-containing complexes inhibit the activity of CLOCK/BMAL1 or Clk/Cyc, thereby forming a negative feedback loop. In mammals, the ROR and REV-ERB family nuclear receptors add positive and negative transcriptional regulation to this core negative feedback loop to ensure the generation of robust circadian molecular oscillation. Despite the overall similarities between mammalian and Drosophila clocks, whether comparable mechanisms via nuclear receptors are required for the Drosophila clock remains unknown. This study shows that the nuclear receptor E75, the fly homolog of REV-ERB α and REV-ERB β, and the NR2E3 subfamily nuclear receptor Unfulfilled (Hr51) are components of the molecular clocks in the Drosophila pacemaker neurons. In vivo assays in conjunction with the in vitro experiments demonstrate that E75 and Unf bind to per regulatory sequences and act together to enhance the Clk/Cyc-mediated transcription of the per gene, thereby completing the core transcriptional feedback loop necessary for the free-running clockwork. These results identify a missing link in the Drosophila clock and highlight the significance of the transcriptional regulation via nuclear receptors in metazoan circadian clocks (Jaumouille, 2015).

Rabinovich, D., Yaniv, S.P., Alyagor, I. and Schuldiner, O. (2016). Nitric oxide as a switching mechanism between axon degeneration and regrowth during developmental remodeling. Cell 164: 170-182. PubMed ID: 26771490
Summary:
During development, neurons switch among growth states, such as initial axon outgrowth, axon pruning, and regrowth. By studying the stereotypic remodeling of the Drosophila mushroom body (MB), this study found that the heme-binding nuclear receptor E75 is dispensable for initial axon outgrowth of MB γ neurons but is required for their developmental regrowth. Genetic experiments and pharmacological manipulations on ex-vivo-cultured brains indicate that neuronally generated nitric oxide (NO) promotes pruning but inhibits regrowth. It was found that high NO levels inhibit the physical interaction between the E75 and UNF nuclear receptors, likely accounting for its repression of regrowth. Additionally, NO synthase (NOS) activity is downregulated at the onset of regrowth, at least partially, by short inhibitory NOS isoforms encoded within the NOS locus, indicating how NO production could be developmentally regulated. Taken together, these results suggest that NO signaling provides a switching mechanism between the degenerative and regenerative states of neuronal remodeling.

Xiong, X.P., Kurthkoti, K., Chang, K.Y., Li, J.L., Ren, X., Ni, J.Q., Rana, T.M. and Zhou, R. (2016). miR-34 modulates innate immunity and ecdysone signaling in Drosophila. PLoS Pathog 12: e1006034. PubMed ID: 27893816
Summary:
microRNAs are endogenous small regulatory RNAs that modulate myriad biological processes by repressing target gene expression in a sequence-specific manner. This study shows that the conserved miRNA miR-34 regulates innate immunity and ecdysone signaling in Drosophila. miR-34 over-expression activates antibacterial innate immunity signaling both in cultured cells and in vivo, and flies over-expressing miR-34 display improved survival and pathogen clearance upon Gram-negative bacterial infection; whereas miR-34 knockout animals are defective in antibacterial defense. In particular, miR-34 achieves its immune-stimulatory function, at least in part, by repressing the two novel target genes Dlg1 and Eip75B. In addition, there exists a mutual repression between miR-34 expression and ecdysone signaling, and miR-34 acts as a node in the intricate interplay between ecdysone signaling and innate immunity. Lastly, the cis-regulatory genomic elements and trans-acting transcription factors required for optimal ecdysone-mediated repression of miR-34 were identified. Taken together, these data enrich the repertoire of immune-modulating miRNAs in animals, and provide new insights into the interplay between steroid hormone signaling and innate immunity.


BIOLOGICAL OVERVIEW

Isoform-specific null mutations were used to define the functions of three orphan members of the nuclear receptor superfamily: E75A, E75B, and E75C. These proteins are encoded by the E75 early ecdysteroid-inducible gene (officially termed Ecdysone-induced protein 75B). Mutants for the isoform E75B are viable and fertile, while E75C mutants die as adults. In contrast, E75A mutants have a reduced ecdysteroid titer during larval development, resulting in developmental delays, developmental arrests, and molting defects. Remarkably, some E75A mutant second instar larvae display a heterochronic phenotype in which they induce genes specific to the third instar and pupariate without undergoing a molt. It is proposed that ecdysteroid-induced E75A expression defines a feed-forward pathway that amplifies or maintains the ecdysteroid titer during larval development, ensuring proper temporal progression through the life cycle (Bialecki, 2002).

Ecdysteroids function as key temporal signals in Drosophila, directing each postembryonic transition in the life cycle. Ecdysteroid pulses at 1 day intervals during the first and second larval instars trigger molting of the cuticle, accommodating the growth that occurs during these stages. A high-titer ecdysteroid pulse 2 days after the molt to the third instar triggers puparium formation, signaling the onset of prepupal development and metamorphosis. This is followed by another ecdysteroid pulse, approximately 10 hr after puparium formation, which triggers adult head eversion and the prepupal-pupal transition. A neuropeptide signal from the brain to the endocrine organ of the insect, the ring gland, triggers the release of relatively inactive ecdysteroids into the hemolymph that are converted by peripheral tissues into more active forms of the hormone, primarily 20-hydroxyecdysone, also known as 20E (Bialecki, 2002).

The ecdysteroid signal is transduced by a heterodimer of two members of the nuclear receptor superfamily, Ecdysone receptor and the RXR ortholog, Ultraspiracle. This hormone/receptor complex activates cascades of gene expression, as first defined by studies of the puffing patterns of the giant larval salivary gland polytene chromosomes. Ecdysteroids directly induce the formation of about a half dozen early puffs. The protein products of these puffs induce more than 100 late puffs scattered throughout the genome. The late puff products, in turn, are thought to act as effectors that direct appropriate biological responses to each ecdysteroid pulse during development (Bialecki, 2002).

Molecular characterization of three early puff genes has shown that they encode transcription factors, fulfilling a central prediction of the hierarchical model of ecdysteroid action. The Broad-Complex (BR-C), responsible for the 2B5 early puff, is a complex genetic locus that encodes a family of zinc finger proteins. Null mutations that inactivate all three essential BR-C subfunctions leads to prolonged third instar larvae that fail to pupariate, while mutations that affect only a single subfunction result in defects in imaginal disc morphogenesis, larval tissue cell death, and lethality during prepupal and pupal stages. BR-C mutations also have widespread effects on early and late ecdysteroid-inducible gene expression, consistent with a central role for this gene in transducing the ecdysteroid signal (Bialecki, 2002).

The two best-characterized early puffs, at 74EF and 75B, also encode ecdysteroid-inducible transcription factors. Ecdysone-induced protein 74EF (E74), from the 74EF puff, consists of two overlapping transcription units, E74A and E74B, that encode proteins containing an identical ETS DNA binding domain. E74 mutants display lethality during prepupal and pupal stages, with defects in adult head eversion and leg morphogenesis as well as defects in ecdysteroid-regulated gene expression (Bialecki, 2002).

The E75 ecdysteroid-inducible gene from the 75B early puff encodes three protein isoforms designated E75A, E75B, and E75C (Segraves, 1990). These proteins contain both a canonical DNA binding domain and a ligand binding domain that define members of the nuclear receptor superfamily, although these proteins are referred to as orphan nuclear receptors because a corresponding hormonal ligand has not yet been identified. Each E75 isoform is characterized by a unique N-terminal sequence encoded by a distinct 5' exon (Segraves, 1990). These 5' exons splice to a common set of five 3' exons for E75A and E75C, while E75B shares only the last four 3' exons. As a result of this arrangement, E75B contains only one of the two E75 zinc fingers and is thus incapable of binding DNA. E75B can, however, heterodimerize with the DHR3 ecdysteroid-inducible orphan nuclear receptor and has been detected on salivary gland polytene chromosomes by antibody stains (White, 1997), indicating that it may function at the level of target gene regulation (Bialecki, 2002).

Relatively little is known about E75 functions during the Drosophila life cycle. Evidence from biochemical and ectopic expression studies indicates that E75B can act as a repressor of the ßFTZ-F1 competence factor during metamorphosis (White, 1997). Germline clones of E75 null mutants, missing all three isoforms, led to arrest during mid-oogenesis, similar to the phenotype of EcR mutant germline clones. A zygotic loss of E75 function results in midgut morphogenesis defects during embryogenesis (Bilder, 1995). Genetic studies of individual E75 isoforms, however, and more detailed phenotypic studies at other stages of the life cycle, have not been reported. This study examined the phenotypes of null mutations specific to E75A, E75B, and E75C, as well as a mutant in which none of the three E75 isoforms was expressed (Bialecki, 2002).

E75B mutants are shown to be viable and fertile, suggesting that this gene functions in a redundant pathway during development, while E75C mutants die as adults. In contrast, most E75A mutants die as delayed second instar larvae with a reduced ecdysteroid titer, or arrest during the molt to the third instar. Remarkably, some E75A mutant second instar larvae express genes characteristic of the third instar and pupariate without progressing through a molt, indicating that molting can be uncoupled from the onset of metamorphosis. This study provides a new direction for understanding the functions of early ecdysteroid-inducible regulatory genes, positioning the E75A orphan nuclear receptor upstream from the signal that induces its expression, defining its action in a feed-forward pathway to amplify or maintain ecdysteroid titers during Drosophila larval development (Bialecki, 2002).

E75B null mutants show no detectable phenotypes. In addition, the expression of 16 ecdysteroid-regulated genes at the onset of metamorphosis is normal in these mutants. Interestingly, ßFTZ-F1 is among the genes that remain unaffected by the E75B mutation. Ectopic expression and biochemical studies have shown that E75B can directly interact with the DHR3 orphan nuclear receptor and thereby block its ability to induce ßFTZ-F1 in mid-prepupae (White, 1997). These observations led to the proposal that the timing of ßFTZ-F1 expression is dependent on appropriate decay of the E75B repressor. No reproducible temporal shift, however, was found in ßFTZ-F1 expression in E75B mutant prepupae, indicating that, while E75B may be sufficient to repress ßFTZ-F1, it is not necessary for this response. This conclusion, combined with the absence of obvious phenotypic effects caused by the E75B mutation, raises the possibility that E75B acts in a functionally redundant pathway. E78B is an ideal candidate for exerting this redundant activity. Like E75B, E78B encodes a homolog of the vertebrate Rev-Erb orphan nuclear receptor (NR1D1), and is a truncated isoform that lacks a DNA binding domain (Stone, 1993). E78B is expressed in synchrony with E75B in early prepupae (Karim, 1992; Stone, 1993). In addition, E78B null mutants display no detectable phenotypes, like E75B mutants, suggesting that its function is complemented by another factor. An effort is currently underway to inactivate both E75B and E78B in order to determine whether they act in a redundant manner during development (Bialecki, 2002).

E75C mutants die as pharate adults or within a few days following eclosion. These animals are morphologically normal except for black spots that cover up to one quarter of the eye, but are weak, unable to fly, and severely uncoordinated. Earlier development in these mutants appears to proceed normally. Consistent with this observation, most of the 16 ecdysteroid-regulated transcripts examined in E75C mutant late third instar larvae and prepupae displayed normal temporal patterns of expression. The expression of four transcripts, however, fails to be maintained through the prepupal-pupal transition in E75C mutants: E75B, Fbp-1, L71-1, and L71-3. This coordinate misregulation suggests that the brief peak of ecdysteroid-induced E75C expression in ~10 hr prepupae is required for the continued expression of a subset of ecdysteroid target genes. It is unclear, however, whether these relatively subtle effects on gene expression might be causally related to the late developmental defects observed in E75C mutants (Bialecki, 2002).

Similarities between the E75C adult phenotype and the phenotype exhibited by hypomorphic dare alleles raises the possibility that the adult lethality of E75C mutants may result from an ecdysteroid deficiency. dare plays a pivotal role in Drosophila ecdysteroid biosynthesis. Dare is an ortholog of adrenodoxin reductase, a mammalian enzyme that plays a central role in vertebrate steroid hormone biosynthesis by transferring electrons to all known mitochondrial cytochrome P450s. dare mutant adults are unable to walk or fly, exhibiting twitching of the legs and wings, and die within a week following eclosion (Freeman, 1999). These dare mutant phenotypes appear to arise from progressive degeneration of the CNS, starting at adult eclosion. Future studies could provide a basis for determining whether E75C and dare might function together to control ecdysteroid titers during pupal and adult development (Bialecki, 2002).

Most E75A mutants display defects during the second larval instar, either failing to molt to the third instar, arresting at the molt, or forming prepupae directly from the second instar. E75A mutant larvae develop asynchronously and can molt up to 1 day late. Larvae that do not molt can live for up to a week before dying. These phenotypes resemble those seen with a temperature-sensitive dre-4 mutant as well as hypomorphic alleles of itpr and null alleles of dare. The dre4 gene has not yet been isolated, although its function is required for ecdysteroid pulses throughout the life cycle (Sliter, 1992). itpr encodes an intracellular calcium channel, the inositol 1,4,5-triphosphate (IP3) receptor, that is expressed in the ring gland and appears to be required for ecdysteroid biosynthesis. Consistent with their proposed functions, the molting defects in itpr and dare mutant larvae can be rescued by providing ecdysteroids in the culture medium. Similarly, E75A mutant second instar larvae can molt to the third instar when fed ecdysteroids, suggesting that an ecdysteroid deficiency is the primary cause of the observed developmental defects at this stage. More direct evidence for this conclusion is provided by an enzyme immunoassay which indicates that the ecdysteroid pulse is essentially eliminated in E75A mutant second instar larvae. The ring gland, as well as other larval tissues, appears normal in E75A mutants, indicating that, like other early genes, E75A does not play a role in their growth or development. Rather, it is concluded that E75A is required for appropriate ecdysteroid biosynthesis or release during larval development (Bialecki, 2002).

While these studies define an essential role for E75A in directing ecdysteroid pulses during larval development, they do not preclude additional functions for this gene during the life cycle -- functions that could be masked by the early lethality of E75A mutants. Like other early genes, E75A is expressed widely in third instar larvae, in contrast to itpr and dare, which are expressed primarily in the ring gland. E75A transcripts have been detected in the salivary glands, gut, Malpighian tubules, fat bodies, and imaginal discs (Huet, 1993; Segraves, 1988), reflecting the widespread expression of E75A protein detected by antibody stains, including abundant expression in the ring gland. E75A protein is also bound to multiple sites in the larval salivary gland polytene chromosomes (Hill, 1993). These observations suggest that E75A may play additional roles beyond those revealed by this study, possibly in a redundant manner with other E75 isoforms or in combination with other early ecdysteroid-inducible genes (Bialecki, 2002).

The E75 mutant that is missing all three E75 isoforms dies after a prolonged first instar, failing to molt to the second instar, similar to dre4 and itpr null mutants. In addition, the earliest phenotypes observed in E75 common region mutants -- embryonic lethality with head involution and midgut morphogenesis defects -- resemble the lethal phenotypes of disembodied (dib) mutant embryos, mutants that are missing a key cytochrome P450 required for ecdysteroid biosynthesis (Chavez, 2000). These observations indicate that the E75 locus may play an essential role in maintaining ecdysteroid titers through embryonic and larval development. The individual E75 isoforms, however, appear to contribute to this regulatory function in a redundant manner because the highly penetrant early lethality associated with the E75 common region mutation is not seen with mutations in any of the individual E75 isoforms. It is concluded that E75A and E75C are good candidates for exerting this redundant activity by virtue of their identical DNA binding domain (Bialecki, 2002).

Redundant interactions between E75A and E75C may explain the apparent stage specificity of the E75A mutant phenotypes. E75A and E75C mutants display no defects at puparium formation or head eversion, key developmental transitions triggered by ecdysteroid pulses at the onset of metamorphosis. Indeed, most E75A mutants progress through to pupal stages once they have passed beyond the second instar. Similarly, exposure of E75A mutant second instar larvae to a single 6 hr treatment with ecdysteroids is sufficient to rescue many animals through to pupal and pharate adult stages. In addition, E75A mutant L2 prepupae appear to execute relatively normal changes in ecdysteroid titer at the onset of metamorphosis, as indicated by the patterns of E74 and ßFTZ-F1 transcription. It is thus proposed that E75A and E75C act in a redundant manner to maintain ecdysteroid titers during the onset of metamorphosis, explaining why mutations in either function survive these stages in the life cycle (Bialecki, 2002).

E75A mutant second instar larvae that fail to molt continue to grow, approaching the size of a wild-type late third instar larva. Remarkably, these delayed second instar larvae express markers that are specific to the latter half of the third instar -- the fat body-specific Fbp-1 larval serum protein receptor gene and the Sgs-4 salivary gland glue protein gene. These changes in gene expression, in apparent preparation for metamorphosis, are consistent with the ability of these second instar larvae to pupariate and progress through head eversion, dying as early pupae. E75A mutant L2 prepupae display normal temporal patterns of E74 and ßFTZ-F1 transcription through prepupal and early pupal stages, with an ~2 hr delay relative to control animals. These observations suggest that these mutants can reset their endocrinological clock to execute the proper ecdysteroid pulses that trigger puparium formation and pupation (Bialecki, 2002).

The ability of E75A mutant animals to pupariate directly from the second instar indicates that molting can be uncoupled from progression through the onset of metamorphosis. This appears to be a manifestation of the reduced ecdysteroid titer in these mutants, since pupariating second instar larvae have also been reported in dre4 and itpr mutants, although they were not characterized in any detail. The reduced ecdysteroid titer in E75A mutant second instar larvae apparently causes these animals to miss this cue that would normally trigger the molt to the third instar. In spite of this, however, these animals can still acquire third instar identity and progress relatively normally, albeit with a significant developmental delay that is evident both in terms of E74A induction in staged larvae, as well as the time to puparium formation, which is at least 16 hr later than puparium formation in wild-type animals. This conclusion suggests that larval molting is an epidermal response that has few, if any, consequences for temporal progression throughout the rest of the organism. Moreover, the observation that a reduced ecdysteroid titer can lead to a heterochronic phenotype at the onset of metamorphosis defines a critical role for ecdysteroid pulses in defining not just timing but also the character of the major developmental transitions in the fly life cycle (Bialecki, 2002).

The observation that E75A expression is induced directly by ecdysteroids (Segraves, 1990), combined with its requirement for appropriate ecdysteroid titers during larval development, leads to the proposal that E75A functions in a feed-forward pathway to maintain or amplify ecdysteroid pulses during Drosophila larval development. The most direct means by which E75A could exert this regulatory function would be through transcriptional control of genes that encode steroidogenic enzymes. The transcription of dare and dib in E75A mutant second instar larvae was examined as a means of testing this hypothesis, but no effects on their expression were detected. Although dare and dib are the only members of the ecdysteroid biosynthetic pathway that have been characterized at the molecular level, there are other genes in this pathway that could be dependent on E75A function. These include members of the so-called 'Halloween' class of genes, such as spook, shroud, and phantom (Chavez, 2000), which display lethal mutant phenotypes similar to those of dib (Bialecki, 2002).

The reduced level of ßFTZ-F1 expression in E75A mutant second instar larvae provides a functional link to explain the molting defects in these animals. ßFTZ-F1 has been shown to be required for larval molting. Moreover, ßFTZ-F1 can directly regulate the Edg84A pupal cuticle gene, and ectopic overexpression of ßFTZ-F1 leads to an abnormal larval cuticle structure. Taken together, these observations suggest that ßFTZ-F1 plays a key role in controlling larval molts, directly regulating larval cuticle gene expression. The severe reduction in ßFTZ-F1 expression in E75A mutant larvae is thus consistent with the inability of these animals to molt. It is also concluded that E75A does not directly regulate ßFTZ-F1, since molting can be rescued by feeding ecdysteroids to E75A mutant larvae. This experiment places ßFTZ-F1 downstream from ecdysteroid signaling and E75A expression, indirectly dependent on E75A activity (Bialecki, 2002).

These studies of E75 provide a new direction for understanding early gene function within the ecdysteroid-triggered regulatory cascades. Previous work has indicated that early ecdysteroid-inducible genes operate downstream from the ecdysteroid receptor, coordinating the expression of secondary response late genes that, in turn, execute the appropriate biological responses to each hormone pulse during development. This characterization of E75 functions indicates that early genes not only transduce the ecdysteroid signal but can also affect the signal itself, through feedback regulation. Further studies of E75 should provide a molecular framework for understanding the genetic control of steroidogenesis in insects. In addition, the heterochronic phenotypes in E75A mutants provide a basis for understanding how key developmental landmarks, such as molting, are linked to temporal progression through the insect life cycle (Bialecki, 2002).

Nitric oxide coordinates metabolism, growth, and development via the nuclear receptor E75

Nitric oxide gas acts as a short-range signaling molecule in a vast array of important physiological processes, many of which include major changes in gene expression. How these genomic responses are induced, however, is poorly understood. Previous work showed that ecdysone-induced protein 75 (E75, also known as Eip75B; NR1D3) contains heme constitutively bound to its ligand-binding domain (LBD), and that amino acids coordinately bound to the heme iron can be displaced in vitro by changes in redox state or the presence of nitric oxide (NO) gas (Reinking, 2005; Marvin, 2009). In turn, these structural changes negate the ability of E75 to repress transcription and to reverse the positive transcriptional activity of its heterodimer partner, Drosophila hormone receptor 3 (DHR3; also known as DHR46, NR1F4). This study examined whether these interactions are relevant in vivo and, if so, what their roles are. Using genetic and chemical manipulations, it was shown that nitric oxide is produced in the Drosophila prothoracic gland (PG), where it acts via the nuclear receptor ecdysone-induced protein 75 (E75), reversing its ability to interfere with its heterodimer partner, Drosophila hormone receptor 3 (DHR3). Manipulation of these interactions leads to gross alterations in feeding behavior, fat deposition, and developmental timing. These neuroendocrine interactions and consequences appear to be conserved in vertebrates (Cáceres, 2011).

Although previously documented effects of NO on cells and tissues are numerous, and many of the cytoplasmic mechanisms of action are well documented, this is the first demonstration of a direct effect via a transcription factor in vivo. In the PG, the findings indicate that E75 is the major nuclear mediator of NO function, as evidenced by the similarity and epistatic nature of E75, DHR3, and NOS phenotypes. A review of the literature also shows that these genes tend to be coexpressed in many other Drosophila tissues, where limited analyses also suggest shared functions. Thus, the interactions described in this study are likely to be relevant to numerous other tissues and processes (Cáceres, 2011).

This overlap in expression and functions is also true for the vertebrate NOS and Rev-erb/ROR orthologs. Examples of overlapping functions in vertebrates include their similar roles in lipid metabolism, gluconeogenesis, muscle differentiation, inflammation, circadian rhythm, PGC1α regulation, hypertension, and atherosclerosis. Strikingly, Nos triple-knockout mice that survive gestation are morbidly obese, exhibiting all aspects of metabolic syndrome (e.g., diabetes, hypertension, atherosclerosis), resulting in a maximum life span of 10 mo (Tsutsui, 2006; Tsutsui, 2009). Conversely, NO up-regulation via arginine supplementation yields a reciprocal phenotype, which includes increased lipolysis, fatty acid oxidation, mitochondrial biogenesis, glucose metabolism, and life span. These effects are similar to those seen upon genetic manipulation of the Rev-erbs and RORs (Duez, 2009a; Duez, 2009; Cáceres, 2011 and references therein).

In the case of E75, NO appears to act in two ways. NO blocks the ability of E75 to interfere directly with DHR3-mediated transcriptional activation. However, it also appears to block the ability of E75 to repress target genes independently of DHR3. In vertebrates, no direct interaction has yet been observed between Rev-erbs and RORs. NO does, however, block the ability of the Rev-erbs to repress target genes by preventing the recruitment of coactivators (Pardee, 2009). It is expected that this aspect of NO action is conserved in flies (Cáceres, 2011).

The achievement of critical weight at the end of the third larval instar coincides with a reduction in circulating levels of insulin-like peptides (ILPs) and consequential production of prothoracicotropic hormone (PTTH) peptide by neurons located within the larval brain. The axons of these PTTH-producing neurons extend to the surface of the PG, where binding of the secreted PTTH peptide to Torso receptor results in intracellular signaling (Rewitz, 2009). Intracellular outcomes include activation of Ras/Raf, MEK/Erk, and PKA, and, notably, the production of Calmodulin, NADH, and cytoplasmic Ca2+ influx. The latter three molecules are required cofactors for dNOS enzymatic activity. Hence, it is proposed that PTTH acts, in large part, through NOS activation (Cáceres, 2011).

The remarkable endoreduplication and growth of the PG due to loss of NOS expression is consistent with previous studies showing that NO has negative effects on cell growth. This activity has important implications and potential use in the control of oncogenic growth (Cáceres, 2011).

Interestingly, these effects of NO on PG size and the timing of metamorphosis contradict previous studies that had suggested that PG size is a key determinant of metamorphosis timing. In these studies, metamorphosis appeared to occur when full PG size was achieved. Here, PG size was up to six times larger than normal, with no metamorphosis. Similarly, Colombani (2005) found that altering PG size by manipulation of DMyc or Cyclin D expression could also increase PG size without triggering premature ecdysone production. Furthermore, metamorphosis can be induced prematurely by down-regulation of DHR4 without affecting PG size. These seemingly contradictory results are likely due to cross-talk within the insulin and ecdysone signaling pathways (Cáceres, 2011).

The bright-red color of these enlarged PGs is consistent with previous studies suggesting that E75 and the Rev-erbs also function as heme sensors (Cáceres, 2011).

NOS manipulation in the PG had major effects on lipid uptake and storage, leading to a nearly 20-fold increase in larval lipid content in the case of knockdown, or a nearly fivefold decrease when expressed prematurely. As disruption of EcR activity within fat body cells can also result in lipid overaccumulation (Colombani, 2005), the nonautonomous effects of PG NOS manipulation appear to be ecdysone-mediated. However, it is quite possible that the nearly 100-fold overall changes observed in lipid content may also be due to additional effects on feeding behavior, nutrient uptake, and other aspects of lipid metabolism (Cáceres, 2011).

The extended eating and fat deposition phenotype, caused by failure to produce ecdysone, may equate on many levels to processes underlying obesity and diabetes. Indeed, much as seen with Nos up-regulation, ecdysone supplementation in vertebrates results in decreased appetite, cholesterol synthesis, and weight gain, while at the same time increasing muscle mass and endurance. Given these apparent abilities to switch fat cell activity from lipid storage to lipid mobilization, a better understanding of the signals and mechanisms underlying NO and ecdysone-mediated effects should provide new insights and possible uses in metabolism based disorders (Cáceres, 2011).

Given the previously demonstrated roles for NO, Rev-erbs, and RORs in the control of circadian clocks, and those shown in this study for developmental timing, it is quite possible that NO, Rev-erbs, and RORs also serve general roles in the matching of diurnal, lunar, and seasonal inputs (i.e., light, temperature, and food availability) with appropriate feeding behaviors, metabolism, and developmental progression. Disruption of these functions leads to metabolic, sleep, stress, immune, and hypertension disorders. Hence, further elucidation of ROR, Rev-erb, and NOS interactions in neuroendocrine tissues should provide new insights into how these temporal and metabolic processes are linked and coordinated, and new ways to prevent or treat their related disorders (Cáceres, 2011).


GENE STRUCTURE

cDNA clone length - 5074 (isoform A)

Bases in 5' UTR - 690

Bases in 3' UTR - 1466

A 50-kb ecdysone-inducible gene, E75, occupies the early puff locus at 75B. E75 contains two overlapping transcription units. The E75 A unit is a coextensive with the E75 gene and contains six exons: two 5'-proximal exons, A0 and A1, which are specific to this unit, and exons 2-5, which are common to both units. The E75 B unit is 20 kb long and contains five exons, a 5'-terminal exon, B1, located within the second intron of E75 A, and the common exons 2-5. Large open reading frames start within the first exon of each unit and continue into the last exon and therefore encode two different proteins (Segraves, 1990).


PROTEIN STRUCTURE

Amino Acids - 1237 (isoform A), 1394 (isoform B) and 1443 (isoform C)

Structural Domains

Both E75A and E75B exhibit sequence similarity to the conserved DNA-binding and hormone-binding domains of proteins in the steroid receptor superfamily. The two putative zinc fingers that characterize the DNA-binding domain are encoded by exon A1 and exon 2, so that the E75 A protein contains both fingers, whereas the E75 B protein contains only the second. Both proteins contain the same putative hormone-binding domain encoded by exon 4 (Segraves, 1990).

Drosophila E75 is a member of the nuclear receptor superfamily. These eukaryotic transcription factors are involved in almost all physiological processes. They regulate transcription in response to binding of rigid hydrophobic hormone ligands. As it is the case for many nuclear receptors, the E75 hormone ligand was originally unknown. Recently, however, it was shown that the ligand binding domain (LBD) of E75 contains a tightly bound heme prosthetic group and is gas responsive. This study used site-directed mutagenesis along with UV-visible and electron paramagnetic resonance (EPR) spectroscopies to characterize and assign the heme iron axial ligands in E75. The F370Y mutation and addition of hemin to the growth medium during expression of the protein in Escherichia coli are necessary to produce good yields of heme-enriched E75 LBD. EPR studies revealed the presence of several species containing a strongly iron bound thiolate. The involvement of cysteines 396 and 468 in heme binding was subsequently shown by single and double mutations. Using a similar approach, it has also been established that the sixth iron ligand of a well-defined coordination conformation, which accounts for approximately half of the total species, is histidine 574. The other iron coordination pairs are discussed. It is concluded that E75 is a new example of a thiolate hemoprotein and that it may be involved in hormone synthesis regulation (de Rosny, 2006).

Nuclear receptors E75, which regulates development in Drosophila, and Rev-erbβ, which regulates circadian rhythm in humans, bind heme within their ligand binding domains (LBD). The heme-bound ligand binding domains of E75 and Rev-erbβ were studied using electronic absorption, MCD, resonance Raman, and EPR spectroscopies. Both proteins undergo redox-dependent ligand switching and CO- and NO-induced ligand displacement. In the Fe(III) oxidation state, the nuclear receptor hemes are low spin and 6-coordinate with cysteine(thiolate) as one of the two axial heme ligands. The sixth ligand is a neutral donor, presumably histidine. When the heme is reduced to the Fe(II) oxidation state, the cysteine(thiolate) is replaced by a different neutral donor ligand, whose identity is not known. CO binds to the Fe(II) heme in both E75(LBD) and Rev-erbβ(LBD) opposite a sixth neutral ligand, plausibly the same histidine that served as the sixth ligand in the Fe(III) state. NO binds to the heme of both proteins; however, the NO-heme is 5-coordinate in E75 and 6-coordinate in Rev-erbβ. These nuclear receptors exhibit coordination characteristics that are similar to other known redox and gas sensors, suggesting that E75 and Rev-erbβ may function in heme-, redox-, or gas-regulated control of cellular function (Marvin, 2009; full text of article).


Ecdysone-induced protein 75B : Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 25 October 2002

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