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Molecular Chaperones Activate the Drosophila Ecdysone Receptor, an RXR Heterodimer
Abstract
The steroid hormone 20-hydroxyecdysone coordinates the stages of Drosophila development by activating a nuclear receptor heterodimer consisting of the ecdysone receptor, EcR, and the Drosophila RXR receptor, USP. We show that EcR/USP DNA binding activity requires activation by a chaperone heterocomplex like that required for activation of the vertebrate steroid receptors, but not previously shown to be required for activation of RXR heterodimers. Six proteins normally present in the chaperone complex were individually purified and shown to be sufficient for this activation. We also show that two of the six (Hsp90 and Hsc70) are required in vivo for ecdysone receptor activity, and that EcR is the primary target of the chaperone complex.
... Ecdysone and juvenile hormone are important hormones that participate in insect growth and development. A chaperone is required to activate the DNA-binding activities of the ecdysone receptor (EcR)/ultraspiracle protein (USP) heterocomplex (Arbeitman and Hogness 2000). The activation of the EcR/USP heterocomplex initiates a cascade ecdysone-responsive gene expression that leads to drastic changes in the differentiation of adult tissues and larval organs. ...
... A functional relationship between steroid hormones and HSPs has been reported, and it is known that Hsp70, Hsc70, and Hsp90 play important roles in the folding and maturation of steroid hormone receptors and different transcription factors (Wegele et al. 2004). Studies of Drosophila EcR/USP DNA-binding activity have demonstrated that Hsp90 and Hsc70 act as molecular chaperones associated with EcR/ USP (Arbeitman and Hogness 2000). There are four Hsp90 family members in P. vicina adults (Fig. 5 and Table 3), and based on comparative homologies with other species, Unigene0012302 showed 99% identities to Hsp83 of Camponotus floridanus (XP_011250812.1), and unigene0019217 was 67% identities to heat shock protein 90 of Scylla paramamosain (AGC54636.1). ...
The heat shock protein 90 (Hsp90) and heat shock cognate proteins (Hsc70) have been identified as chaperones of the ecdysone receptor (EcR)/ultraspiracle protein (USP) heterocomplex. However, little is known about the status of Hsp90 and Hsc70 in Polyrhachis vicina Roger. Here, we sequenced the transcriptomes of adult ants in P. vicina for the first time. Clean reads in female, male, and worker ants were annotated into 40,147 transcripts, and 37,488, 28,300, and 33,638 unigenes were assembled in female, male, and worker ants, respectively. According to RPKM, the numbers of differentially expressed genes between female and male ants, between female and worker ants, and between male and worker ants and the common differentially expressed genes were 12,657, 21,630, 15,112 and 3704, respectively. These results reveal that caste differentiation, caste specificity formation, and social divisions of P. vicina ants may be due to gene expression differences. Moreover, PvEcR and PvUSP were also detected as differentially expressed genes in the ants; specifically, PvUSP expression was higher than PvEcR expression in all castes. We speculate that PvUSP may have a role similar to that of juvenile hormone receptor. Four identified PvHsp90 family members and 23 identified PvHsp70 family members were found in the ants, and 2 PvHsp90 genes and 8 PvHsp70 genes were analyzed by qRT-PCR. Among those genes, the expression of 2 PvHsp90 genes and 5 PvHsp70 genes coincided with the expression profiles of PvEcR and PvUSP, which suggest that the characterization of PvHsp90 and PvHsc70 may be as EcR/USP molecular chaperones in P. vicina.
... In general, but not always, cells undergoing heat shock and many other stresses increase levels of Hsps (hence their name) and in the absence of stress express basal low level of Hsps and/or their cognates. The basal expression of Hsps (and/or their cognates) is required for normal protein folding, maintenance of signal transduction and/or normal development (Arbeitman & Hogness, 2000;Karagoz & Rudiger, 2015;King & MacRae, 2015;McClellan et al., 2007). Hsps comprise several families based on their molecular weights (e.g., Hsp90, Hsp70 and small Hsps, among others) (Huang, Wang, & Kang, 2008;Schlesinger, 1990). ...
... As insects enter diapause, for example, they undergo changes in 20-hydroxyecdysone levels. This hormone in turn induces the expression of Hsp70 and Hsp90 (Arbeitman & Hogness, 2000;Denlinger, 2002 hsp mutations affect fecundity, developmental rate and feeding (Chen & Wagner, 2012;Lerman & Feder, 2005). Such effects may even occur through interaction with a second species. ...
Heat‐shock proteins (Hsps) and their cognates are primary mitigators of cell stress. With increasingly severe impacts of climate change and other human modifications of the biosphere, the ability of the heat‐shock system to affect evolutionary fitness in environments outside the laboratory and to evolve in response are topics of growing importance. Since the last major reviews, several advances have occurred. First, demonstrations of the heat‐shock response outside the laboratory now include many additional taxa and environments. Many of these demonstrations are only correlative, however. More importantly, technical advances in “omic” quantification of nucleic acids and proteins, genome‐wide association analysis, and manipulation of genes and their expression have enabled the field to move beyond correlation. Several consequent advances are already evident: The pathway from heat‐shock gene expression to stress tolerance in nature can be extremely complex, mediated through multiple biological processes and systems, and even multiple species. The underlying genes are more numerous, diverse, and variable than previously appreciated, especially with respect to their regulatory variation and epigenetic changes. The impacts and limitations (e.g., due to trade‐offs) of natural selection on these genes have become more obvious and better established. Finally, as evolutionary capacitors Hsps may have distinctive impacts on the evolution of other genes and ecological consequences.
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... doi: bioRxiv preprint first posted online May. 8, 2018;al., 2000;Pflumm and Botchan, 2001;Balasov, et al., 2009) showed strong S-phase defects. Surprisingly, dE2F1, dDP (Royman et al., 1999), dE2F2 (Cayirlioglu et al., 2001Cayirlioglu et al., 2003), Rbf (Basco et al., 2001) and Myb complex (Beall et al., 2002;Beall et al., 2004) are necessary for this process (Cayirlioglu et al., 2003). ...
... Ecdysone works by binding to a nuclear receptor, EcR (Koelle et al., 1991). The EcR heterodimerizes with the retinoid X receptor ortholog Ultraspiracle (USP), which acts as a general heterodomer partner for the class of factors repsentated by EcR (Hall and Thummel, 1998;Arbeitman and Hogness, 2000;Ghbeish et al., 2001;Hu et al., 2003). Both partners are required for binding to ligand or their target DNA. ...
We have revealed that the chorion gene clusters amplify by repeatedly initiating DNA replication from chorion gene amplification origins in the response to developmental signals, through the transcription factors in Drosophila ovarian follicle cells. Orc1, Orc2, and Cdc6 are forms of DNA replication machinery, which are conserved from yeast to humans; and Orc1 and Orc2 mutants are lethal. Overexpression of Orc1 or Orc2 (subunits of the origin recognition complex) led to female sterility, but overexpression of Cdc6 (an Orc family member) or GFP did not. We propose that DNA replication machinery contributes to development.
Recently, we found that H3K4 was trimethylated at chorion gene amplification origins, but not at the Act1 locus. Overexpression of Lsd1H3K4 dimethylase and Lid H3K4 trimethylase are female sterile but not a Lid mutant. These results showed that epigenetic regulation affected fertility. Screening strategies using Drosophila flies could also lead to the development of drugs that reduce sterility and epigenetic effects related histone modification.
Summary statement
There are approximately 470,000 infertile individuals in Japan. We knockowned the prereplicative complex components and demethlases during Drosophila ovary development. In these drospohila, we could be the model of infertile.
... EcR is colocalized with Pol II in Bradysia hygida and Chironomus tentans [22,23]. Although a number of proteins, such as Alien, Bonus, Diabetes and Obesity Regulated (dDOR), dDEK, Hsc70, Hsp90, Rigor mortis (Rig), Smrter (Smr), Taiman, and Trithoraxrelated (TRR), have been identified as regulators or cofactors of EcR-mediated gene expression [13,[24][25][26][27][28][29][30][31][32], it is unknown how these proteins communicate with the general transcription machinery and whether additional cofactors are involved in EcR-mediated gene expression. In addition, it remains poorly understood how EcR activates transcription correctly after integrating nutritional and developmental cues. ...
... For example, via this LXXLL motif, the Mediator subunits (MED1 and MED14) and other cofactors interact with mammalian nuclear receptors, such as androgen receptor, estrogen receptor, glucocorticoid receptor, thyroid hormone receptor and RXR [65,66,82,83]. This LXXLL motif is also found in several transcription coactivators for EcR [13], such as Taiman, TRR, Rig, dDOR, dDEK, Hsp90, and Hsc70 [24][25][26][27][28][29]84]. Interestingly, we have found that CDK8, but not CycC, has a LXXLL motif that is highly conserved from yeasts to human (Fig 6E). ...
The steroid hormone ecdysone and its receptor (EcR) play critical roles in orchestrating developmental transitions in arthropods. However, the mechanism by which EcR integrates nutritional and developmental cues to correctly activate transcription remains poorly understood. Here, we show that EcR-dependent transcription, and thus, developmental timing in Drosophila, is regulated by CDK8 and its regulatory partner Cyclin C (CycC), and the level of CDK8 is affected by nutrient availability. We observed that cdk8 and cycC mutants resemble EcR mutants and EcR-target genes are systematically down-regulated in both mutants. Indeed, the ability of the EcR-Ultraspiracle (USP) heterodimer to bind to polytene chromosomes and the promoters of EcR target genes is also diminished. Mass spectrometry analysis of proteins that co-immunoprecipitate with EcR and USP identified multiple Mediator subunits, including CDK8 and CycC. Consistently, CDK8-CycC interacts with EcR-USP in vivo; in particular, CDK8 and Med14 can directly interact with the AF1 domain of EcR. These results suggest that CDK8-CycC may serve as transcriptional cofactors for EcR-dependent transcription. During the larval-pupal transition, the levels of CDK8 protein positively correlate with EcR and USP levels, but inversely correlate with the activity of sterol regulatory element binding protein (SREBP), the master regulator of intracellular lipid homeostasis. Likewise, starvation of early third instar larvae precociously increases the levels of CDK8, EcR and USP, yet down-regulates SREBP activity. Conversely, refeeding the starved larvae strongly reduces CDK8 levels but increases SREBP activity. Importantly, these changes correlate with the timing for the larval-pupal transition. Taken together, these results suggest that CDK8-CycC links nutrient intake to developmental transitions (EcR activity) and fat metabolism (SREBP activity) during the larval-pupal transition.
... To be noticed, Hsp90 is linked to the activation of the ecdysone receptor/ultraspiracle complex in Drosophila melanogaster. 50 And in vertebrates, hsp90 is associated with the activity of steroid hormone. 51 So there is a possibility that the knockdown of Hsp90 disturbed the hormone system in A. franciscana and thus influenced the development of nauplius-destined embryos. ...
... Taken together, these findings indicate that RXR pathway is an important toxicity target of MPs and NPs in aquatic organisms including crustaceans and fish. Given similar expression levels of RXRα in the horseshoe carbs exposed to NPs and NPs + Cu 2+ , NPs appear to be a Activation of the ecdysone receptor EcR and its binding with RXR to form a transcriptionally active nuclear receptor heterodimer requires interaction with a protein complex that contains molecular chaperones HSP70 and HSP90 (Arbeitman and Hogness, 2000). Heat shock proteins (HSPs) are a large family of evolutionarily conserved molecular chaperones that play pivotal roles in protein quality control and cell survival (Roufayel and Kadry, 2019). ...
Nanoplastics (NPs) and heavy metals are typical marine pollutants, affecting the gut microbiota composition and molting rate of marine organisms. Currently, there is a lack of research on the toxicological effects of combined exposure to horseshoe crabs. In this study, we investigated the effects of NPs and copper on the expression of molt-related genes and gut microbiome in juvenile tri-spine horseshoe crabs Tachypleus tridentatus by exposing them to NPs (100 nm, 104 particles L-1) and/or Cu2+ (10 μgL-1) in seawater for 21 days. Compared with the control group, the relative mRNA expression of ecdysone receptor (EcR), retinoid x receptor (RXR), calmodulin-A-like isoform X1 (CaM X1), and heat shock 70 kDa protein (Hsp70) were significantly increased under the combined stress of NPs and Cu2+. There were no significant differences in the diversity and abundance indices of the gut microbial population of horseshoe crabs between the NPs and/or Cu2+ groups and the control group. According to linear discriminant analysis, Oleobacillus was the most abundant microorganism in the NPs and Cu2+ stress groups. These results indicate that exposure to either NPs stress alone or combined NPs and Cu2+ stress can promote the expression levels of juvenile molting genes. NPs exposure has a greater impact on the gut microbial community structure of juvenile horseshoe crabs compared to Cu2+ exposure. This study is helpful for predicting the growth and development of horseshoe crabs under complex environmental pollution.
... Hsp70 family of the proteins located within the cytoplasm, one of the members of this family, Hsc70 constitutively expressed in spermatogonial cells 43 . It was also noted that Hsc70 is required as members of a chaperone complex for activating the ecdysone receptor which controls the successive stages of the insect's life cycle and also contributes to other processes such as reproduction [44][45] . Likewise, Huang et al. 28 found an inverse correlation between thermal protection and fecundity with respect to the overexpression of Hsp70 in Liriomyza huidobrensis. ...
We aimed to assess the effect of gamma radiation on the expression of heat shock proteins Hsc70 and Hsp83 in Aedes aegypti. Adult males were irradiated with 50Gy of gamma radiation, and changes in the expression of proteins in SDS-PAGE gel bands corresponding to molecular weights ~60-75kDa and ~80-95kDa were analyzed at two different time points 6 and 12-hour post-irradiation, using a temporal mass spectrometry based semi-quantitative analysis. A 2-3-fold increase was observed in both proteins Hsc70 and Hsp83, at both time points. In addition, the experiment also revealed the overexpression of several other molecules such as Arginine Kinase - known to be upregulated in certain insects during stress, Esterase B1- implicated in insecticide resistance, and also down-regulation of the 26S proteasome non-ATPase regulatory subunit 1 and ubiquitin-activating enzyme E1 - both known to be involved in ubiquitin-mediated protein degradation. The results taken together with existing data on Hsp83 and Hsc70, indicate that these proteins may enhance the survival of Ae. aegypti following gamma radiation and could serve as molecular markers for the detection of radiation-induced stress.
... In addition to TcTAI, TcMET interacted with proteins naturally occurring in the Sf9 host cells, most notably the heat shock protein HSP83, orthologous to vertebrate HSP90. The chaperone HSP90/83 is well known to associate with and support function of nuclear hormone receptors such as the glucocorticoid receptor (56) or the D. melanogaster EcR (57). Interestingly, HSP90 is important for the function of the mammalian homolog of MET, the ligand-activated transcription factor of the bHLH-PAS family, aryl hydrocarbon receptor (AHR) (58,59). ...
Juvenile hormone (JH) plays vital roles in insect reproduction, development, and in many aspects of physiology. JH primarily acts at the gene-regulatory level through interaction with an intracellular receptor (JHR), a ligand-activated complex of transcription factors consisting of the JH-binding protein Methoprene-tolerant (MET) and its partner Taiman (TAI). Initial studies indicated significance of post-transcriptional phosphorylation, subunit assembly, and nucleocytoplasmic transport of JHR in JH signaling. However, our knowledge of JHR regulation at the protein level remains rudimentary, partly due to the difficulty of obtaining purified, functional JHR proteins. Here we present a method for high-yield expression and purification of JHR complexes from two insect species, the beetle Tribolium castaneum and the mosquito Aedes aegypti. Recombinant JHR subunits from each species were co-expressed in an insect cell line using a baculovirus system. MET-TAI complexes were purified through affinity chromatography and anion exchange columns to yield proteins capable of binding both the hormonal ligand (JH III) and DNA bearing cognate JH-response elements. We further examined the beetle JHR complex (TcJHR) in greater detail. Biochemical analyses and mass spectrometry confirmed that TcJHR was a 1:1 heterodimer consisting of TcMET and TcTAI proteins, stabilized by the JHR agonist ligand methoprene. Phosphoproteomics uncovered multiple phosphorylation sites in the TcMET protein, some of which were induced by methoprene treatment. Finally, we report a functional bipartite nuclear localization signal, straddled by phosphorylated residues, within the disordered C-terminal region of TcMET. Our present characterization of the recombinant JHR is an initial step towards understanding JHR structure and function.
... Hsc70 and Hsp90 successively bind to EcR, help form the molting hormone receptor complex, and can promote the DNA binding activity of EcR-Usp dimer, which is essential for EcR maturation (Arbeitman & Hogness, 2000;Gilbert et al., 2000). In this study, we found that TeEcR-RNAi could down-regulate TeHsc70 expression. ...
Insect gonads develop under endocrine signals. In this study, we assessed the characters of partial complementary DNAs encoding the Teleogryllus emma orthologs of 20-hydroxyecdysone (20E)-related genes (RXR, E75, HR3, Hsc70, and Hsp90) and analyzed their expression patterns in both nymph and adult crickets. 20E treatment suppressed expression of TeEcR, TeRXR, TeE75, TeHR3, TeHsc70, and TeHsp90. Temporal expression analysis demonstrated that TeERR and 20E-related genes were expressed in four stages of gonadal development from the fourth-instar nymph stage to the adult stage. The expression pattern of these genes differed in testicular and ovarian development. TeRXR, HR3, TeHsc70, and TeHsp90 were irregularly expressed in gonads of the same developmental stages, while mRNAs encoding TeERR, TeEcR, and TeE75 accumulated in higher levels in ovaries than in testes. RNA interference (RNAi) of TeEcR expression led to decrease of the expression levels of TeEcR, TeRXR, TeHR3, and TeHsc70, while it enhanced TeE75 and TeHsp90 expressions. These results demonstrate that the TeERR and 20E-related genes help regulate gonadal development, while TeEcR appears to inhibit TeE75 expression, TeE75 inhibits HR3 expression. Hsc70 indirectly regulated the expression of the primary and secondary response genes E74A, E75B, and HR3. Hsp90 regulated Usp expression with no direct regulatory relationship with EcR.
Highlights
Exogenous 20E suppressed expression of TeEcR, TeRXR, TeE75, TeHR3, TeHsc70, and TeHsp90.
RNA interference of TeEcR expression led to decrease of the expression of TeEcR, TeRXR, TeHR3, and TeHsc70, while it enhanced TeE75 and TeHsp90 expression.
... Furthermore, Hsp90 is critical for conferring competence of AhR to bind agonist ligands to activate transcription (Beischlag et al., 2008;Lees and Whitelaw, 1999;Pongratz et al., 1992;Whitelaw et al., 1995). A similar requirement applies to the ligand-dependent activities of some NRs such as the glucocorticoid receptor (Picard, 2006); EcR requires Hsp83 to bind DNA (Arbeitman and Hogness, 2000). Moreover, the chaperone complex including Hsp90 facilitates nuclear import of AhR (Kazlauskas et al., 2001). ...
Juvenile hormone (JH) and ecdysteroids represent equally important nonpeptide signals governing insect reproduction and development. For a considerable time, understanding of JH action lagged behind ecdysteroid research. Arriving with a 20-year delay, the intracellular receptor for JH was finally identified as a ligand-activated bHLH-PAS transcription factor, originally named Methoprene-tolerant (Met), by virtue of the resistance Drosophila mutants exhibited to morphogenetic and lethal actions of JH and its mimic methoprene. Systemic RNAi in suitable insect models revealed the anticipated function of Met in preventing metamorphosis and promoting reproduction, thus providing the missing link to the chief roles of JH in insects. That, along with defining the JH-binding pocket and the JH-response DNA elements of Met, established the JH receptor (JHR) 10 years ago. After reviewing the functional attributes of the JHR, this chapter will focus on advances in the genetics, cell biology, and biochemistry of the JHR achieved during this past decade. Although hormone receptor function of bHLH-PAS transcription factors is unprecedented, the well-studied mammalian aryl hydrocarbon receptor (AhR) belonging to the same protein family affords functional parallels with the JHR. We can now begin to understand the mechanisms of JHR interaction with the chaperone Hsp90/83, nucleocytoplasmic transport and post-translational regulation by phosphorylation, dimerization with bHLH-PAS partner proteins, and activation by agonist ligands binding to the PAS-B domain. A section is dedicated to current efforts exploiting the JHR as a basis of chemical high-throughput screening, aimed at discovery of novel compounds for environmentally friendly control of insect pests and disease vectors.
... Furthermore, the proposed role of Hsp90 in oogenesis during the ovarian maturation of T. castaneum was also found in BPH, and the knockdown of NlHsp90 led to the arrest of ovary development in females and no offspring could be produced. Evidence has shown that a chaperone complex including Hsp90 and Hsc70 is required in vivo for ecdysone receptor activity in D. melanogaster [35], which provides a new clue to uncover the underlying mechanism of Hsp90 in contributing to oogenesis. In particular, a new phenotype was observed when the newly emerged females were injected with dsNlHsp90. ...
Hsp90 (heat shock protein 90) chaperone machinery is considered to be a key regulator of proteostasis under both physiological and stress growth conditions in eukaryotic cells. The high conservation of both the sequence and function of Hsp90 allows for the utilization of various species to explore new phenotypes and mechanisms. In this study, three Hsp90 homologs were identified in the brown planthopper (BPH), Nilaparvata lugens: cytosolic NlHsp90, endoplasmic reticulum (ER) NlGRP94 and mitochondrial NlTRAP1. Sequence analysis and phylogenetic construction showed that these proteins belonged to distinct classes consistent with the predicted localization and suggested an evolutionary relationship between NlTRAP1 and bacterial HtpG (high-temperature protein G). Temporospatial expression analyses showed that NlHsp90 was inducible under heat stress throughout the developmental stage, while NlGRP94 was only induced at the egg stage. All three genes had a significantly high transcript level in the ovary. The RNA interference-mediated knockdown of NlHsp90 its essential role in nymph development and oogenesis under physiological conditions. NlGRP94 was also required during the early developmental stage and played a crucial role in oogenesis, fecundity and late embryogenesis. Notably, we first found that NlHsp90 and NlGRP94 were likely involved in the cuticle structure of female BPH. Together, our research revealed multifunctional roles of Hsp90s in the BPH.
... Therefore, one must be cautious in interpreting results where transcriptional activity of nuclear receptor in yeast are treated as a reflection of normal receptor function. However, additional evidence for a positive role of heat shock proteins in nuclear receptor function has been reported in Drosophila (Arbeitman and Hogness, 2000). Genetic interaction was observed between mutations in the Drosophila ecdysone receptor (EcR) gene and those in the hsc4 gene which encodes the HscTO protein. ...
Oestrogens regulate the transcription of target genes by binding to the oestrogen receptors (ERα and ERβ) which function as ligand inducible transcription factors. Both ERα and ERβ are members of the nuclear receptor superfamily which is characterised by a highly conserved DNA-binding domain. There are two distinct transcriptional activation domains: the ligand independent API at the N-terminus and the ligand dependent AF2 at the C-terminus which is encompassed by the ligand binding domain (LBD). Following sequence specific binding of oestrogen receptors to the promoter of target genes, additional co-factors are recruited in order to remodel the chromatin structure or to aid the docking of the RNA polymerase II holoenzyme. The AF2 activity of ERα is mediated through interaction between the LBD and coactivator proteins upon ligand binding. In order to define the ERα-coactivator interface at a molecular level, systematic mutagenesis was carried out. This led to the identification of a group of conserved hydrophobic residues in the LBD that are required for binding the p160 family of coactivators. Together with helix 12 and lysine 366 at the C-terminal end of helix 3, they form a hydrophobic groove that accommodates an LXXLL motif, which is essential for coactivator binding to the receptor. The presence of endogenous coactivators is a major impediment for studying designated receptor-coactivator pairs in mammalian cells. To circumvent this problem, a yeast genetic screen was conducted to identify suppressor mutant coactivators for a transcriptionally defective ERα. The V380H mutant receptor fails to interact with wild-type p160 coactivators such as SRC1e. However, an altered specificity mutant SRC1e recovered from the screen is able to interact with the mutant receptor, and fully rescues its transcriptional activity in transfected mammalian cells. Remarkably, introduction of the analogous mutation into other p160 coactivator family members confers the ability to suppress the V380H mutation. This suggests that at least in the assays employed, recruitment of a p160 coactivator by ERα is sufficient to activate transcription.
... Indeed, this protein family has shown diverse roles, such as molecular chaperones, related to protein folding, assembly, and transport [27,28]. HSPs in the cement gland may be mainly involved in morphogenesis [29] and metamorphosis [30][31][32], since the samples here analyzed were obtained from juveniles of P. pollicipes. ...
Adhesive secretion has a fundamental role in barnacles’ survival, keeping them in an adequate position on the substrate under a variety of hydrologic regimes. It arouses special interest for industrial applications, such as antifouling strategies, underwater industrial and surgical glues, and dental composites. This study was focused on the goose barnacle Pollicipes pollicipes adhesion system, a species that lives in the Eastern Atlantic strongly exposed intertidal rocky shores and cliffs. The protein composition of P. pollicipes cement multicomplex and cement gland was quantitatively studied using a label-free LC-MS high-throughput proteomic analysis, searched against a custom transcriptome-derived database. Overall, 11,755 peptide sequences were identified in the gland while 2880 peptide sequences were detected in the cement, clustered in 1616 and 1568 protein groups, respectively. The gland proteome was dominated by proteins of the muscle, cytoskeleton, and some uncharacterized proteins, while the cement was, for the first time, reported to be composed by nearly 50% of proteins that are not canonical cement proteins, mainly unannotated proteins, chemical cues, and protease inhibitors, among others. Bulk adhesive proteins accounted for one-third of the cement proteome, with CP52k being the most abundant. Some unannotated proteins highly expressed in the proteomes, as well as at the transcriptomic level, showed similar physicochemical properties to the known surface-coupling barnacle adhesive proteins while the function of the others remains to be discovered. New quantitative and qualitative clues are provided to understand the diversity and function of proteins in the cement of stalked barnacles, contributing to the whole adhesion model in Cirripedia.
... HSPs are chaperones, important for cellular recovery from stress because they help maintain correct protein folding (Morris et al., 2013). They have also been related to endocrine pathways and some heat shock proteins, such as HSP90 and HSC70, are essential for ecdysone receptor activity in vivo, contributing to the steroid hormone signaling inside of the cells (Arbeitman and Hogness, 2000;Gehring, 1998). Our results show a decrease in expression of the stress-related genes at the lowest concentration tested of 4-OHBP and no effect at the highest, which represents a nonmonotonic response reported for endocrine disruptors and hormones (Li et al., 2007;Vandenberg, 2013). ...
The use of organic Ultraviolet (UV)filters has increased in the last years, either in sunscreens, other cosmetics, or even food packaging. These filters may end up in soil and water since the Wastewater Treatment Plants may not successfully remove them. Among them, benzophenones are known to act as endocrine disruptors. However, most of the studies are directed towards vertebrates and aquatic invertebrates, while there is a lack of information on the molecular mechanisms affected by these compounds on soil dwelling invertebrates. Here, we study the impact of direct acute (48 h)contact of 4-hydroxybenzophenone (4-OHBP)at two sublethal concentrations (0.02 and 0.2 mg/mL)on gene expression of the earthworm Eisenia fetida. Investigated genes were involved in endocrine pathways, stress response, detoxification mechanisms, genotoxicity, energy metabolism and epigenetics. Three of them were identified for the first time in earthworms. Our results suggest that exposure to 4-OHBP affected endocrine pathways, causing an increase in the Ecdysone receptor gene (EcR)expression. Moreover, the UV filter induced changes in the CuZn superoxide dismutase gene (CuZn SOD), indicating an effect in the stress response. Finally, significant changes were detected for glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH)expression, indicating that energy metabolism is influenced by the 4-OHBP and highlighting the risks of using GAPDH as an internal reference for Real Time PCR.
... Through protein-protein interactions, HSPs help to regulate fundamental cellular processes such as protein turnover, mitochondrial and endoplasmic reticulum trafficking, cell cycle progression, and steroid signaling (Beato & Klug, 2000;Helmbrecht, Zeise, & Rensing, 2000;Pratt, 1997;Taipale, Jarosz, & Lindquist, 2010). Hsc70 and Hsp90 interact with ecdysone receptor activation which is crucial in ecdysone response pathway (Arbeitman & Hogness, 2000). In this research, partners of HSP enzyme families in insects were determined based on the D. melanogaster species in both protein families (Table 6). ...
Heat shock proteins (HSPs) are found in all living organisms, from bacteria to humans and are expressed under stress. In this study, characterization of two families of HSP including HSP60 and HSP70 protein was compared in different insect species from different orders. According to the conserved motifs analysis, none of the motifs were shared by all insects of two protein families but each family had their own common motifs. Functional and structural analyses were carried out on seven different insect species from each protein family as the representative samples. These analyses were performed via ExPASy database tools. The tertiary structure of Drosophila melanogater as the sample of each protein family were predicted by the Phyre2 and TM-score servers then their qualities were verified by SuperPose and PROCHECK. The tertiary structures were predicted through the “c4pj1E” model (PDB Accession Code: 4pj1) in HSP60 family and “c3d2fC” model (PDB Accession Code: 3d2f) in HSP70 family. The protein phylogenetic tree was constructed using the Neighbor-joining (NJ) method by MEGA 6.06. According to the results, there was a high identity of HSP60 and HSP70 families so that they should be derived from a common ancestor however they belonged to separate groups. In protein–protein interaction analysis by STRING 10.0, ten common enriched pathways of biological process, molecular function and KEGG were identified in D. melanogaster in both families. The obtained data provide a background for bioinformatic studies of the function and evolution of insects and other organisms.
... Although the activities of a number of eukaryotic transcription factors are modulated by Fe 2+ , the regulation is largely by association with Fe 2+ -coordinated heme (e.g. the ecdysone receptor) (Arbeitman and Hogness, 2000); however, the function of FTO as a transcription repressor appears to be modulated by free Fe 2+ but not Fe 2+ -coordinated heme (Figure 4). Similar modes have been reported in prokaryotes. ...
Fat mass and obesity-associated (FTO) protein is a ferrous ion (Fe2+)/2-oxoglutarate (2-OG)-dependent demethylase preferentially catalyzing m6A sites in RNA. The FTO gene is highly expressed in the hypothalamus with fluctuation in response to various nutritional conditions, which is believed to be involved in the control of whole body metabolism. However, the underlying mechanism in response to different nutritional cues remains poorly understood. Here we show that ketogenic diet-derived ketone body β-hydroxybutyrate (BHB) transiently increases FTO expression in both mouse hypothalamus and cultured cells. Interestingly, The FTO protein represses Fto promoter activity, which can be offset by BHB. We then demonstrate that FTO binds to its own gene promoter, and Fe2+, but not 2-OG, impedes this binding and increases FTO expression. The BHB-induced occupancy of the promoter by FTO influences the assembly of the basal transcriptional machinery. Importantly, a loss-of-function FTO mutant (I367F), which induces a lean phenotype in FTOI367F mice, exhibits augmented binding to, and elevated potency to repress the promoter. Furthermore, FTO fails to bind to its own promoter that promotes FTO expression in the hypothalamus of high-fat diet-induced obese and 48-h fasting mice, suggesting a disruption of the stable expression of this gene. Taken together, this study uncovers a new function of FTO as a Fe2+-sensitive transcriptional repressor dictating its own gene switch to form an auto-regulatory loop that may link with the hypothalamic control of body weight.
... For example, the glucocorticoid receptor [1] and estrogen receptor [2] form a homodimer, and insect ecdysone receptor (EcR) forms a heterodimer with an ultraspiracle protein (USP), the ortholog of retinoid X receptor in vertebrates [3]. The heat shock proteins Hsp90 and Hsp70 interact with the nuclear receptors to facilitate their DNA binding activity in fruit flies [4] and mammals [5]. Hsp90 [6] and Hsc70 [7] have been found to be involved in insect steroid hormone signaling by differential interaction with the nuclear receptors. ...
Animal steroid hormones are conventionally known to initiate signaling via a genomic pathway by binding to the nuclear receptors. The mechanism by which 20E initiates signaling via a nongenomic pathway is unclear.
We illustrate that 20E triggered the nongenomic pathway through a plasma membrane G-protein-coupled receptor (named ErGPCR) in the lepidopteran insect Helicoverpa armigera. The transcript of ErGPCR was increased at the larval molting stage and metamorphic molting stage by 20E regulation. Knockdown of ErGPCR via RNA interference in vivo blocked larval-pupal transition and suppressed 20E-induced gene expression. ErGPCR overexpression in the H. armigera epidermal cell line increased the 20E-induced gene expression. Through ErGPCR, 20E modulated Calponin nuclear translocation and phosphorylation, and induced a rapid increase in cytosolic Ca2+ levels. The inhibitors of T-type voltage-gated calcium channels and canonical transient receptor potential calcium channels repressed the 20E-induced Ca2+ increase. Truncation of the N-terminal extracellular region of ErGPCR inhibited its localization on the plasma membrane and 20E-induced gene expression. ErGPCR was not detected to bind with the steroid hormone analog [3H]Pon A.
These results suggest that ErGPCR participates in 20E signaling on the plasma membrane.
... Since TeERR was closely related to TeEcR based on our results, we speculate that TeERR might also be involved in the 20E signaling pathway, and both co-regulate the morphogenesis and spermatogenesis of insect testis. The EcR combines with Hsc70 and Hsp90 before USP; thus, it ensures the DNA binding activity of EcR-USP heterodimer (Arbeitman and Hogness 2000). In addition, EcR combines with Hsc70 before Hsp90 to respond to the 20E signal (Gilbert et al. 2000). ...
Estrogen-related receptor gene (ERR) and ecdysone receptor gene (EcR) belong to the nuclear receptor gene superfamily, both of which are associated with the regulation of insect reproductive development. However, the relationship between ERR and EcR and whether ERR participates in the 20E signal pathway during male reproduction are unclear. In this paper, adult male crickets Teleogryllus emma Ohmschi & Matsumura were divided into the experimental group, negative group, and control group. Crickets of the experimental group were injected with TeERR or TeEcR-dsRNA, and those in the negative group received EGFP-dsRNA. The efficiency of TeERR and TeEcR-RNAi was detected in the experimental group. Furthermore, the transcription level, morphological characteristics as well as weight were analyzed in the TeERR or TeEcR knocked-down testis. Results showed that the expression level of TeERR or TeEcR was significantly down-regulated (P < 0.05) when treated with 2000 ng TeERR or TeEcR-dsRNA for 48 h. The expression level of TeERR could be down-regulated (P < 0.05) using TeEcR-RNAi and vice versa. TeERR and TeEcR-RNAi caused morphological changes in testes, but they had no obvious effect on weight (P > 0.05). These results indicate that TeERR and TeEcR are intimately related to each other. In addition, TeERR may be involved in the 20E signal pathway and maintain the function of adult cricket testis.
... In addition to this, the role of Hsp90 in mouth-form plasticity may go beyond its generic chaperone function (Arbeitman and Hogness 2000). This protein may be directly involved in the development of certain mouth structures, as any mutation in the gene network underlying mouth development may be expected to increase morphological variability of the mouth (Bergman and Siegal 2003). ...
Phenotypic plasticity is increasingly recognized to facilitate adaptive change in plants and animals, including insects, nematodes and vertebrates. Plasticity can occur as continuous or discrete (polyphenisms) variation. In social insects, e.g. in ants, some species have workers of distinct size classes while in other closely related species variation in size may be continuous. Despite the abundance of examples in nature, how discrete morphs are specified remains currently unknown. In theory, polyphenisms might require robustness, whereby the distribution of morphologies would be limited by the same mechanisms that execute buffering from stochastic perturbations, a function attributed to heat-shock proteins of the Hsp90 family. However, this possibility has never been directly tested because plasticity and robustness are considered to represent opposite evolutionary principles. Here, we used a polyphenism of feeding structures in the nematode Pristionchus pacificus to test the relationship between robustness and plasticity using geometric morphometrics of 20 mouth-form landmarks. We show that reducing heat-shock protein activity, which reduces developmental robustness, increases the range of mouth-form morphologies. Specifically, elevated temperature led to a shift within morphospace, pharmacological inhibition of all Hsp90 genes using radicicol treatment increased shape variability in both mouth-forms, and CRISPR/Cas9-induced Ppa-daf-21/Hsp90 knockout had a combined effect. Thus, Hsp90 canalizes the morphologies of plastic traits resulting in discrete polyphenism of mouth-forms.
... Insect Hsp genes constitute a subset of a larger group of genes coding for molecular chaperones that are involved in multiple developmental processes and that assist repair stress injuries via transportation and degradation of aggregated proteins in the organism (Choi et al. 2014;Kim et al. 2014;Shu et al. 2011). For example, Hsc70 and Hsp90 interact with ecdysone receptor activation which is vital in ecdysone response pathway (Arbeitman and Hogness 2000), while Hsp27 is known to involve in eye development (Chen et al. 2012). Hsp70 and Hsp90 as well as small heat shock proteins (Hsp22, Hsp67Ba, and Hsp67Bc) are also implicated in the maintenance of phenotypic stability under variable environmental conditions (Morrow and Tanguay 2015;Takahashi et al. 2010). ...
Heat shock protein gene (Hsp) families are thought to be important in thermal adaptation, but their expression patterns under various thermal stresses have still been poorly characterized outside of model systems. We have therefore characterized Hsp genes and their stress responses in the oriental fruit moth (OFM), Grapholita molesta, a widespread global orchard pest, and compared patterns of expression in this species to that of other insects. Genes from four Hsp families showed variable expression levels among tissues and developmental stages. Members of the Hsp40, 70, and 90 families were highly expressed under short exposures to heat and cold. Expression of Hsp40, 70, and Hsc70 family members increased in OFM undergoing diapause, while Hsp90 was downregulated. We found that there was strong sequence conservation of members of large Hsp families (Hsp40, Hsp60, Hsp70, Hsc70) across taxa, but this was not always matched by conservation of expression patterns. When the large Hsps as well as small Hsps from OFM were compared under acute and ramping heat stress, two groups of sHsps expression patterns were apparent, depending on whether expression increased or decreased immediately after stress exposure. These results highlight potential differences in conservation of function as opposed to sequence in this gene family and also point to Hsp genes potentially useful as bioindicators of diapause and thermal stress in OFM.
... HSPs play a primary role in cellular recovery from stress by acting as chaperones to maintain correct protein folding (Morris et al., 2013). In addition, hsp70 has also been related to the endocrine pathway (Arbeitman and Hogness, 2000;Nolen and Morimoto, 2002). All the UV filters analyzed were able to induce hsp70 expression with the exception 4HB, despite the fact that it appeared to be the most toxic for larvae. ...
Several organic UV filters have hormonal activity in vertebrates, as demonstrated in fishes, rodents and human cells. Despite the accumulation of filter contaminants in aquatic systems, research on their effects on the endocrine systems of freshwaters invertebrates is scarce. In this work, the effects of five frequently used UV filters were investigated in embryos and larvae of Chironomus riparius, which is a reference organism in ecotoxicology. LC50 values for larvae as well as the percentage of eclosion of eggs were determined following exposures to: octyl-p-methoxycinnamate (OMC) also known as 2-ethylhexyl-4-methoxycinnamate (EHMC); 4-methylbenzylidene camphor (4MBC); 4-hydroxybenzophenone (4HB); octocrylene (OC); and octyldimethyl-p-aminobenzoate (OD-PABA). To assess sublethal effects, expression levels of the genes coding for the ecdysone receptor (EcR) and heat shock protein HSP70 were investigated as biomarkers for endocrine and stress effects at the cellular level. Life-stage-dependent sensitivity was found. In embryos, all of the UV filters provoked a significant overexpression of EcR at 24h after exposure. OC, 4MBC and OD-PABA also triggered transcriptional activation of the hsp70 stress gene in embryos. In contrast, in larvae, only 4MBC and OMC/EHMC increased EcR and hsp70 mRNA levels and OD-PABA upregulated only the EcR gene. These results revealed that embryos are particularly sensitive to UV filters, which affect endocrine regulation during development. Most UV filters also triggered the cellular stress response, and thus exhibit proteotoxic effects. The differences observed between embryos and larvae and the higher sensitivity of embryos highlight the importance of considering different life stages when evaluating the environmental risks of pollutants, particularly when analyzing endocrine effects.
... In this case, a synergy between the compounds and the decrease in mRNA at higher concentrations may be explained by an increase before the 24h time point; thus, the lower levels observed reflect the return to normal levels typically observed during the stress response. However, it is worth noting that some heat shock proteins, including Hsp70, are associated to the complex of activators and repressors that, when interacting with nuclear receptors, contribute to the steroid hormone signaling inside the cells (Arbeitman and Hogness, 2000;Gehring, 1998;Echeverria and Picard, 2010). So the complex expression pattern of this gene could be related with the different roles that this protein has in the cell, reflecting a balance between its role in the cell stress response and in the steroid receptor functionality. ...
Organic ultraviolet (UV) filters are used in a wide variety of products, including cosmetics, to prevent damage from UV light in tissues and industrial materials. Their extensive use has raised concerns about potential adverse effects in human health and aquatic ecosystems that accumulate these pollutants. To increase sun radiation protection, UV filters are commonly used in mixtures. Here, we studied the toxicity of binary mixtures of 4-methylbenzylidene camphor (4MBC), octyl-methoxycinnamate (OMC), and benzophenone-3 (BP-3), by evaluating the larval mortality of Chironomus riparius. Also molecular endpoints have been analyzed, including alterations in the expression levels of a gene related with the endocrine system (EcR, ecdysone receptor) and a gene related with the stress response (hsp70, heat shock protein 70). The results showed that the mortality caused by binary mixtures was similar to that observed for each compound alone; however, some differences in LC50 were observed between groups. Gene expression analysis showed that EcR mRNA levels increased in the presence of 0.1 mg/L 4MBC but returned to normal levels after exposure to mixtures of 4MBC with 0.1, 1, and 10 mg/L of BP-3 or OMC. In contrast, the hsp70 mRNA levels increased after exposure to the combinations tested of 4MBC and BP-3 or OMC mixtures. These data suggest that 4MBC, BP-3, and OMC may have antagonist effects on EcR gene transcription and a synergistic effect on hsp70 gene activation. This is the first experimental study to show the complex patterned effects of UV filter mixtures on invertebrates. The data suggest that the interactions within these chemicals mixtures are complex and show diverse effects on various endpoints.
... Thus, this compound may activate the stress response. However, it is worth noting that some heat shock proteins are associated with the complex of activators and repressors that, when interacting with nuclear receptors, contribute to steroid hormone signaling inside cells (Arbeitman and Hogness, 2000;Gehring, 1998). Gp93 is a gene that encodes a protein related to Hsp90 because it is the ortholog of glucose regulated protein 94 (GRP94) (Maynard et al., 2010) and the endoplasmic reticulum Hsp90, a metazoan-restricted chaperone that is essential for early development in mammals (Chen et al., 2006). ...
Vinclozolin is a fungicide used in agriculture that can reach aquatic ecosystems and affect the organisms living there. Its effects have been intensively studied in vertebrates, where it acts as an antiandrogen, but there is a lack of information about its mechanistic effects on invertebrates. In this work, we analyzed the response of genes related to the endocrine system, the stress response, and the detoxification mechanisms of Chironomus riparius fourth instar larvae after 24 h and 48 h exposures to 20 (69.9 nM), 200 (699 nM), and 2000 μg/L (6.99 μM) of Vinclozolin. Survival analysis showed that this compound has low toxicity, as it was not lethal for this organism at the concentrations used. However, this fungicide was shown to modify the transcriptional activity of the ecdysone response pathway genes EcR, E74, and Kr-h1 by increasing their mRNA levels. While no changes were observed in disembodied, a gene related with the ecdysone synthesis metabolic pathway, Cyp18A1, which is involved in the inactivation of the active form of ecdysone, was upregulated. Additionally, the expression of two genes related to other hormones, FOXO and MAPR, did not show any changes when Vinclozolin was present. The analysis of stress response genes showed significant changes in the mRNA levels of Hsp70, Hsp24, and Gp93, indicating that Vinclozolin activates the cellular stress mechanisms. Finally, the expressions of the genes Cyp4G and GstD3, which encode enzymes involved in phase I and phase II detoxification, respectively, were analyzed. It was found that their mRNA levels were altered by Vinclozolin, suggesting their involvement in the degradation of this compound. For the first time, these results show evidence that Vinclozolin can modulate gene expression, leading to possible significant endocrine alterations of the insect endocrine system. These results also offer new clues about the mode of action of this compound in invertebrates.
... The mutations described in this study were selected to investigate specific attributes as a basis for subsequent mutational analysis in Drosophila. Receptor functionality is actually a composite of numerous subfunctions including interactions with DNA, receptor partners, ligand, transcriptional cofactors and chaperones (Arbeitman & Hogness, 2000), as well as interdomain interactions within each receptor. Therefore, mutations that selectively disrupt specific EcR functions can be employed to assess their effects upon individual developmental processes. ...
... The finding that expression levels of EcR and numerous other genes involved in endocrine signaling are down-regulated in response to MnSOD over-expression in transgenic flies relative to controls of the same "physiological age" is therefore of interest. Additionally, the molecular chaperones Hsc70 and Hsp90, which are implicated in longevity and interact with EcR-USP [22,25,29], were also up-regulated by MnSOD (at both time points) as was the gene encoding heat shock factor (HSF). Given that MnSOD overexpressing flies did not demonstrate increased thermotolerance, these genes may be induced for alternative purposes, perhaps functioning in the endocrine regulation of aging. ...
Supporting results and additional explanatory text.
... In Drosophila, the functional ecdysone receptor formed by EcRB1 and Usp undergoes hormone binding activity but is unable to bind EcRE. To bind the DNA, the heterodimer requires activation by a chaperone (Arbeitman and Hogness 2000). What is more, the function of the active ecdysone receptor is influenced by the action of JH. ...
Upon activation by ligand binding, the androgen and glucocorticoid receptors find specific DNA response elements in the regulatory regions of their target genes which can be present anywhere in the human genome. Like the other nuclear receptors, the DNA-binding domain of the androgen and glucocorticoid receptors consist of two zinc finger modules. The first recognizes a response element characterized by a hexameric motif, while the second is involved in receptor dimerization. While their sequence specificity is very similar, the androgen receptor has a broader specificity when compared to the glucocorticoid receptor. This led to the definition of two types of response elements: the canonical elements and the androgen receptor specific elements. In this chapter, we will focus on the DNA motifs recognized by the androgen receptor, and how selectivity is achieved through subtle sequence differences. The in vivo role of the different motifs will be highlighted, as well as the complexity of the enhancers containing these motifs. Finally, we will discuss how androgen response elements, besides recruiting the androgen receptor, also influence its function by acting as allosteric modulators.
... Hsp90 during diapause could be regulated by ecdysteroids. In D. melanogaster the presence of ecdysteroids leads to upregulation of Hsp90 (Thomas & Lengyel, 1986) and the Hsp90 chaperone is required for ecdysone receptors activity (Arbeitman & Hogness, 2000). In the C. elegans, dauer larvae are enriched in Hsp90 gene transcripts and the corresponding protein could interact with steroid hormone receptors (Dalley & Golomb, 1992;Cherkasova et al., 2000). ...
Insects enter in diapause in response to diverse environmental cues (nutrients, day length or temperature). During diapause, insects arrest their development and many genes are down-regulated while a small number of genes uniquely expressed at this time. The knowledge of the mechanism of diapause is essential for understanding the seasonal biology of insect species. This review presents available data regarding the rythmisity of diapause in the moth Sesamia nonagrioides (Lepidoptera:Noctuidae). Studying the transcriptional regulation of several genes (five heat shock proteins, two storage proteins and one juvenile hormone esterase) we showed that these genes may play various roles in the diapause programming. The heat shock protein genes respond differently to diapause conditions in S. nonagrioides. SnoHsp19.5 gene was consistently expressed, while SnoHsp20.8 was down-regulated in deep diapause and was up-regulated at the termination of diapause, suggesting that these two genes play distinctive roles in the regulation of diapause. SnoHsp70 is down regulated during diapause, while SnoHsc70 is induced as the larvae enter in deep diapause. High temperature stress during diapause has no further effect on transcript levels of SnoHsc70. Our results show that SnoHsc70 may play important roles in assisting protein conformation during specific stages of diapause. SnoHsp83 displays a similar pattern to SnoHsc70 under diapause conditions, when extra larval moults occur, indicating that could be involved in the developmental process that occurs between two moults. To date, two hexamerin encoding genes, SnoSP1 and SnoSP2 have been characterized in corn stalk borer. Expression of both hexamerin genes was observed throughout diapause, for as long as 130 days. The results confirm the importance of SnoSP1 and SnoSP2 biosynthesis and finally lead us to the conclusion that larval diapause of S. nonagrioides is associated with continuous synthesis and accumulation of storage proteins. However, enhanced levels of SnoSP2, but not SnoSP1 transcripts were detected at the initiation of diapause. In contrast, levels of SnoSP1 were detected in deep diapause phase and greatly increased during post-diapause phase. The SnoSP1 and SnoSP2 genes have the same transcriptional pattern in non-diapausing conditions and almost the opposite pattern during diapausing conditions. Our data suggest that individual members of the methionine rich storage proteins family may have distinct roles in diapause of corn stalk borer. SnoSP2 associated with the onset of diapause, while the SnoSP1 with the termination of diapause. The SnoJHER transcriptlevels were higher in long day non-diapausing larvae than in short day diapausing ones. During the fifth instar of the non-diapausing and the ninth instar of the diapausing larvae, SnoJHER mRNAs reached higher expression levels on the days close to each larval molt. In the last (sixth) nondiapausing larval instar, SnoJHER mRNA levels peaked during the intermolt period but were lower than during the fifth instar. Our results suggest that these genes could play major roles in the developmental process, regulating important physiological mechanisms, as diapause.
... The ecdysone receptor is capable of forming a complex with ultraspiracle (Usp), which has been postulated as a receptor for JH (Xu et al., 2002). In turn, the EcR/Usp complex appears to interact with stress induced molecular chaperones as well as multiple histone deacetylases (HDACs) (Tsai et al., 1999;Arbeitmann and Hogness, 2000; Chapter 3.5), including Rpd3, mutations in which affect lifespan in both yeast and nematodes (Guarente and Kenyon, 2000). Interestingly, general chemical inhibition of HDACs in fruit flies can also result in increased lifespan with no apparent reduction in physical activity (Kang et al., 2002). ...
... Previously, it has been shown that the ECR in American lobster can bind tebufenozide, an insecticide designed to promote molting (Tarrant et al., 2011), and that the receptor is present in non-eyestalk tissues such as muscle and hypodermis (El Haj et al., 1994). In Drosophila melanogaster, it has been shown that the ECR is partially activated by HSP90 (Arbeitman and Hogness, 2000), another transcript that was significantly higher transcribed in teflubenzuron-exposed lobster. The ECR, thus, appears to be promising marker of endocrine-disrupting pesticides in crustaceans. ...
... Other important players in the aging process are molecular chaperones, such as heatshock proteins (21). The interaction of EcR with a chaperone complex is necessary for activation of the heterodimeric receptor of ecdysone (22), and overexpression of such a chaperone, Hsp70 (23), leads to increased longevity (24). It is conceivable that, when overexpressed, Hsp70 might trap EcR in a non-functional state, thus leading to extended longevity. ...
... Information is scarce about the requirement of chaperones or even their presence, for EcR function, with a few exceptions. For example, purified EcR/USP heterodimer from Drosophila acquires DNA binding activity only with the addition of chaperones such as Hsp90 and FKBP52 (Arbeitman and Hogness, 2000). Also, FKBP52 was immunoprecipitated along with EcR in the PGs of M. sexta, raising the possibility of a functional role of this immunophilin in this insect (Song et al., 1997). ...
Signal transduction of the insect steroid hormones, ecdysteroids, is mediated by the ecdysteroid receptor, EcR. In various cells of the insect Rhodnius prolixus, EcR is present in both the nucleus and the cytoplasm, where it undergoes daily cycling in abundance and cellular location at particular developmental times of the last larval instar that are specific to different cell types. EcR favors a cytoplasmic location in the day and a nuclear location in the night. This study is the first to examine the potential mechanisms of intracellular transport of EcR and reveals close similarities with some of its mammalian counterparts. In double and triple labels using several antibodies, immunohistochemistry, and confocal laser scanning microscopy, we observed co-localization of EcR with the microtubules (MTs). Treatments with either the MT-stabilizing agent taxol or with colchicine, which depolymerizes MTs, resulted in considerable reduction in nuclear EcR with a concomitant increase in cytoplasmic EcR suggesting that MT disruption inhibits receptor accumulation in the nucleus. EcR also co-localizes with the chaperone Hsp90, the immunophilin FKBP52, and the light chain 1 of the motor protein dynein. All these factors also co-localize with MTs. We propose that in Rhodnius, EcR exerts its genomic effects by forming a complex with Hsp90 and FKBP52, which uses dynein on MTs as a mechanism for daily nucleocytoplasmic shuttling. The complex is transported intact to the nucleus and dissociates within it. We propose that EcR utilizes the cytoskeletal tracks for movement in a manner closely similar to that used by the glucocorticoid receptor. We also observed co-localization of EcR with mitochondria which suggests that EcR, like its mammalian counterparts, may be involved in the coordination of non-genomic responses of ecdysteroids in mitochondria.
... Whether EcR exhibits cycling is more likely to be attributable to differences in the details of transduction of the ecdysteroid signal between tissue types. This process requires the formation of an heterodimer between EcR and its partner, ultraspiracle (USP), and requires the participation of numerous cofactors, such as chaperones (Arbeitman and Hogness 2000), coactivators and corepressors (Henrich 2005) and possible interactions of EcR and USP with other nuclear receptors (White et al. 1997;Hirai et al. 2002). Tissuespecific differences in any of these factors could readily result in cycling EcR in some cell types but not others. ...
The insect moulting hormones, viz. the ecdysteroids, regulate gene expression during development by binding to an intracellular protein, the ecdysteroid receptor (EcR). In the insect Rhodnius prolixus, circulating levels of ecdysteroids exhibit a robust circadian rhythm. This paper demonstrates associated circadian rhythms in the abundance and distribution of EcR in several major target tissues of ecdysteroids, but not in others. Quantitative analysis of immunofluorescence images obtained by confocal laser-scanning microscopy following the use of anti-EcR has revealed a marked daily rhythm in the nuclear abundance of EcR in cells of the abdominal epidermis, brain, fat body, oenocytes and rectal epithelium of Rhodnius. This EcR rhythm is synchronous with the rhythm of circulating hormone levels. It free-runs in continuous darkness for several cycles, showing that EcR nuclear abundance is under circadian control. Circadian control of a nuclear receptor has not been shown previously in any animal. We infer that the above cell types detect and respond to the temporal signals in the rhythmic ecdysteroid titre. In several cell types, the rhythm in cytoplasmic EcR peaks several hours prior to the EcR peak in the nucleus each day, thereby implying a daily migration of EcR from the cytoplasm to the nucleus. This finding shows that EcR is not a constitutive nuclear receptor, as has previously been assumed. In the brain, rhythmic nuclear EcR has been found in peptidergic neurosecretory cells, indicating a potential pathway for feedback regulation of the neuroendocrine system by ecdysteroids, and also in regions containing circadian clock neurons, suggesting that the circadian timing system in the brain is also sensitive to rhythmic ecdysteroid signals.
Molting and metamorphosis are important physiological processes in insects that are tightly controlled by ecdysone receptor (EcR) through the 20‐hydroxyecdysone (20E) signaling pathway. EcR is a steroid nuclear receptor (SR). Several FK506‐binding proteins (FKBPs) have been identified from the mammal SR complex, and are thought to be involved in the subcellular trafficking of SR. However, their roles in insects are poorly understood. To explore whether FKBPs are involved in insect molting or metamorphosis, we injected an FKBP inhibitor (FK506) into a lepidopteran insect, Spodoptera litura , and found that molting was inhibited in 61.11% of the larvae, and that the time for larvae to pupate was significantly extended. A total of 10 FKBP genes were identified from the genome of S. litura and were clustered into 2 distinct groups, according to their subcellular localization, with FKBP13 and FKBP14 belonging to the endoplasmic reticulum (ER) group and with the other members belonging to the cytoplasmic (Cy) group. All the CyFKBP s were significantly upregulated in the prepupal or pupal stages, with the opposite being observed for the ER group members. FK506 completely blocked the transfer of EcR to the nucleus under 20E induction, and significantly downregulated the transcriptional expression of many 20E signaling genes. A similar phenomenon was observed after RNA interference of 2 CyFKBP s ( FKBP45 and FKBP12b ), but not for FKBP13 . Taken together, our data indicate that the cytoplasmic FKBPs, especially FKBP45 and FKBP12b, mediate the nuclear localization of EcR, thereby regulating the 20E signaling and ultimately affecting molting and metamorphosis in insects.
Metamorphosis is one of the most important physiological processes in insects, which is coordinated by juvenile hormone (JH) and 20-hydroxyecdysone (20E). Ecdysone receptor (EcR) is a steroid receptor (SR), which usually presents in cytoplasm and transfers into nucleus after binding to 20E. Heat shock proteins (Hsps) are suggested to be important members of the SR complex. However, their role in nucleocytoplasmic shuttle of the EcR remains unclear. In the present study, we found that apoptozole (Hsp70 inhibitor) suppressed the larval molting by decreasing the expression of ecdysone signaling genes. Two cytoplasmic (Cy) Hsp70s (Hsp72 and Hsp73) interacted with both EcR and ultraspiracle (USP, the heterodimer partner of EcR). By immunohistochemistry experiments, we revealed that CyHsp70 co-localized with EcR in the cytoplasm, and that both apoptozole and interfering of CyHsp70 significantly inhibited the process of EcR entering the nucleus under 20E induction, while reducing the expression of ecdysone signaling genes. Interestingly, the nuclear localization of EcR was also promoted by two other stimuli, including JH and heat stress, and this promotion was inhibited by apoptozole. This implies that various stimuli can induce EcR entry into the nucleus, and that this process is mediated by CyHsp70. Curiously, neither JH nor heat stress activated the ecdysone signaling genes; instead, they have a significant inhibitory effect on them. Taken together, it seems that Cytoplasmic Hsp70s promote EcR transport into the nucleus by responding to various stimuli, and that the biological effects of various stimuli passing through the EcR are different. Thus, our data provide a new viewpoint to understand the mechanism of nucleocytoplasmic shuttle of EcR.
Insect herbivores use phytochemicals as signals to induce expression of their phytochemical-detoxifying cytochrome P450 monooxygenases (P450 s). The regulatory cascades that transduce phytochemical signals to enhanced expression of P450 s are the focus of this review. At least seven signaling pathways, including RTK/MAPK, GPCR/CREB, GPCR/NFκB, ROS/CncC/Keap1, AhR/ARNT, cytosol NR, and nucleus-located NR, may be involved in phytochemical induction of P450 s. Constitutive overexpression, over-phosphorylation, and/or activation of one or more effectors in the corresponding pathway are common causes of P450 overexpression that lead to phytochemical or insecticide resistance. Future research should pay more attentions to the starting point of each pathway, the number of pathways and their cross talk for a given phytochemical, and the pathways for downregulation of P450s.
The aim of this study was to elucidate the molecular mechanisms underlying the thermal stress response in the spotted sea bass (Lateolabrax maculatus). Spotted sea basses were exposed to 4 different water temperatures (20, 22, 24, and 28°C) in increasing increments of 2°C/h from 18°C (control) for different time periods (0, 6, 12, 24, 48, 72, and 96 h). Subsequently, 3 tissues (liver, muscle, and gill) were isolated, and the levels of SOD, HSP90, and HSP70 mRNA were assessed. SOD mRNA expression was maintained at baseline levels of control fish at all water temperatures in the liver, while muscle and gill tissue showed an increase followed by a decrease over each certain time with higher water temperature. HSP90 mRNA expression increased in the liver at ≤ 24°C over time, but maintained baseline expression at 28°C. In muscle, HSP90 mRNA expression gradually increased at all water temperatures, but increased and then decreased at ≥ 24°C in gill tissue. HSP70 mRNA expression exhibited an increase and then a decrease in liver tissue at 28°C, but mainly showed similar expression patterns to HSP90 in all tissues. These results suggest the activity of a defense mechanism using SOD, HSP90, and HSP70 in the spotted sea bass upon rapid increases in water temperature, where the expression of these genes indicated differences between tissues in the extent of the defense mechanisms. Also, these results indicate that high water temperature and long-term thermal stress exposure can inhibit physiological defense mechanisms.
Nitric Oxide (NO) signaling is known to govern the cyprid metamorphosis of barnacle Balanus amphitrite, a potential biofouling organism. This signaling cascade is mainly driven by two important enzymes, Nitric Oxide Synthase (NOS) and cyclic Guanosine monophosphate (cGMP)-specific Phosphodiesterase (PDE), which exist in multiple isoforms. But, involvement of the exact isoform of these enzymes was unknown, and the same was addressed in the present study using pharmacological compounds, targeting the specific isoforms of NOS and PDE. When the cyprids were treated with specific NOS inhibitors (7-Nitroindazole for NOS I and S-methylisothiourea hemisulphate for NOS II), the metamorphosis was promoted indicating the involvement of both NOS I and NOS II. However, when cyprids were exposed to PDE inhibitors (Sildenafil, Dipyridamole and Bay 73–6691 targeting PDE5, PDE6 and PDE9 respectively), metamorphosis was significantly inhibited only in case of Sildenafil, indicating the involvement of PDE5. The expression of PDE5 in the cyprid was demonstrated using immunofluorescence technique. Further, it was also demonstrated that the interplay between NO and Serotonin signaling plays a vital role in the pelago-benthic transition of B. amphitrite. Targeting these signaling pathways in the future could provide an antifouling solution against barnacles.
Silkworm (Bombyx mori) is an economically beneficial insect. Its growth and development are regulated by endogenous hormones. In the present study, we found that feeding titanium dioxide nanoparticles (TiO2 NP) caused a significant increase of body size. TiO2 NP stimulated the transcription of several genes, including the insulin-related hormone bombyxin, PI3K/Akt/TOR (where PI3K is phosphatidylinositol 3-kinase and TOR is target of rapamycin), and the adenosine 5'-monophosphateactivated protein kinase (AMPK)/target of rapamycin (TOR) pathways. Differentially expressed gene (DEG) analysis documented 26 developmental hormone signaling related genes that were differentially expressed following dietary TiO2 NP treatment. qPCR analysis confirmed the upregulation of insulin/ecdysteroid signaling genes, such as bombyxin B-1, bombyxin B-4, bombyxin B-7, MAPK, P70S6K, PI3k, eIF4E, E75, ecdysteroid receptor (EcR), and insulin-related peptide binding protein precursor 2 (IBP2). We infer from the upregulated expression of bombyxins and the signaling network that they act in bombyxin-stimulated ecdysteroidogenesis.
HSP90 proteins are major chaperones that play pivotal roles in controlling multiple regulatory pathways such as hormone signaling,cell differentiation,cell proliferation and apoptosis,morphogenesis and morphological evolution,metamorphosis and stress defense.Using real-time fluorescent quantitative RT-PCR approach,expression profiles of HSP90 genes were examined during larval development and in adult tissues in Japanese flounder,Paralichthys olivaceus.In adult tissues,HSP90α was mainly expressed with higher transcript levels in skeletal muscle,intestine and stomach;but HSP90β exhibited higher mRNA levels in brain,spleen and kidney.During metamorphosis,HSP90α transcript increased distinctly with the highest mRNA levels at the metamorphosis climax;whereas HSP90β mRNA levels did not change.Due to the important role of thyroid hormone(TH)for the flounder metamorphosis,the transcriptional regulation of HSP90 genes by TH was evaluated using exogenous TH and thiourea(TU)treatment.Compared with untreated control,HSP90α transcript increased significantly in larvae sampled 8 and 13 days after TH treatment,and TU-treated larvae exhibited lower HSP90α mRNA levels.In contrast,the transcript levels of HSP90β did not vary after TH and TU treatment.Overall,these results demonstrate that HSP90α transcription is up-regulated by thyroid hormone and plays an important role during metamorphosis in Japanese flounder.
Nearly 100 proteins are known to be regulated by hsp90. Most of these substrates or "client proteins" are involved in signal transduction, and they are brought into complex with hsp90 by a multiprotein hsp90/hsp70-based chaperone machinery. In addition to binding substrate proteins at the chaperone site(s), hsp90 binds cofactors at other sites that are part of the heterocomplex assembly machinery as well as immunophilins that connect assembled substrate.hsp90 complexes to protein-trafficking systems. In the 5 years since we last reviewed this subject, much has been learned about hsp90 structure, nucleotide-binding, and cochaperone interactions; the most important concept is that ATP hydrolysis by an intrinsic ATPase activity results in a conformational change in hsp90 that is required to induce conformational change in a substrate protein. The conformational change induced in steroid receptors is an opening of the steroid-binding cleft so that it can be accessed by steroid. We have now developed a minimal system of five purified proteins-hsp90, hsp70, Hop, hsp40, and p23-that assembles stable receptor.hsp90 heterocomplexes. An hsp90.Hop.hsp70.hsp40 complex opens the cleft in an ATP-dependent process to produce a receptor.hsp90 heterocomplex with hsp90 in its ATP-bound conformation, and p23 then interacts with the hsp90 to stabilize the complex. Stepwise assembly experiments have shown that hsp70 and hsp40 first interact with the receptor in an ATP-dependent reaction to produce a receptor.hsp70.hsp40 complex that is "primed" to be activated to the steroid-binding state in a second ATP-dependent step with hsp90, Hop, and p23. Successful use of the five-protein system with other substrates indicates that it can assemble signal protein.hsp90 heterocomplexes whether the substrate is a receptor, a protein kinase, or a transcription factor. This purified system should facilitate understanding of how eukaryotic hsp70 and hsp90 work together as essential components of a process,that alters the conformations of substrate proteins to states that respond in signal transduction.
In insects, ecdysteroids trigger larval-to-adult transition. Ecdysteroid receptor is a heterodimer composed of two nuclear receptors, the ecdysone receptor (EcR) and Ultraspiracle (Usp). Upon dimerization EcR and Usp recognize the ecdysteroid response elements (EcREs) present in promoter regions of the ecdsysteroid-responsive genes. Detailed structural analyses of EcR and Usp DNA binding domains revealed that each protein contributes in different way to EcREs binding. Plasticity and flexibility of EcR and Usp account for great adaptability of the functional receptor for binding of different ligands and EcREs. Nevertheless the functionality of the ecdysteroid receptor is a complex matter influenced by many factor such as posttranslational modifications, observed often in disordered regions. This review will focus on data, which illustrate structure-function relationship in stably folded globular domains as well as in disordered regions of EcR and Usp. The new structural information, future goals and directions for research on recently defined factors, which might alter and influence the structure-function relationship of this receptor, are also discussed.
Originally published in: Protein Folding Handbook. Part II. Edited by Johannes Buchner and Thomas Kiefhaber. Copyright © 2005 Wiley‐VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3‐527‐30784‐2
The sections in this article are
Introduction
The Hsp 90 Family in vivo
Evolutionary Relationships within the Hsp 90 Gene Family
In Vivo Functions of Hsp 90
Regulation of Hsp 90 Expression and Posttranscriptional Activation
Chemical Inhibition of Hsp 90
Identification of Natural Hsp 90 Substrates
In Vitro Investigation of the Chaperone Hsp 90
Hsp 90: A Special Kind of ATPase
The ATPase Cycle of Hsp 90
Interaction of Hsp 90 with Model Substrate Proteins
Investigating Hsp 90 Substrate Interactions Using Native Substrates
Partner Proteins: Does Complexity Lead to Specificity?
Hop , p 23, and PPIases: The Chaperone Cycle of Hsp 90
Hop / Sti 1: Interactions Mediated by TPR Domains
p 23/ Sba 1: Nucleotide‐specific Interaction with Hsp 90
Large PPIases: Conferring Specificity to Substrate Localization?
Pp5: Facilitating Dephosphorylation
Cdc 37: Building Complexes with Kinases
Tom 70: Chaperoning Mitochondrial Import
CHIP and Sgt 1: Multiple Connections to Protein Degradation
Aha 1 and Hch 1: Just Stimulating the ATPase?
Cns 1, Sgt 2, and Xap 2: Is a TPR Enough to Become an Hsp 90 Partner?
Conclusion
Acknowledgements
This chapter reviews hormonal effects on aging in Drosophila melanogaster. The insect juvenile hormone (JH) is a sesquiterpenoid compound produced by the corpora allata (CA), a pair of endocrine glands with nervous connections to the brain. In pre-adult development and metamorphosis, JH functions as a "status quo" hormone, allowing continued growth after ecdysteroid-induced molting. Metamorphosis can only take place when the ecdysteroids act in the absence of JH. During the final larval instar, the JH titer declines because of a cessation of synthesis and increased degradation in the hemolymph and target tissues. Steroid hormones, such as ecdysteroids, are another class of vital hormones in insects. In pre-adult flies, 20E is produced in the larval prothoracic gland, which makes part of the larval ring gland in dipterans. Both JH and 20E play major antagonistic or synergistic roles in regulating Drosophila development. Application of commonly used JH inhibitors, such as precocene or fluvastatin, can reduce or inhibit JH synthesis in the CA and may thus be used to study the effects of JH deficiency on life span.
This chapter examines the information gathered about the ecdysteroid receptor at three levels and provides brief descriptions and references for the tools used to undertake those experiments. The receptor is viewed as a structural entity with particular emphasis on the sequence characteristics of EcR and USP as well as their biophysical and biochemical properties. These studies build upon the first three properties noted for EcR in its original characterization. The transcriptional function of the ecdysteroid receptor is examined, with particular regard to its interactions with ligand, other protein factors, and promoter elements. This chapter describes a few of the ecdysteroid-inducible systems that have been developed for various applications. The ecdysteroid receptor is viewed from a cellular and developmental perspective in vivo with special emphasis on the spatial and temporal diversity of ecdysteroid-mediated action, along with the approaches used to address these questions. Further, a prognosis is offered concerning unresolved and arising issues surrounding ecdysteroid action via its receptors along with possible experimental technologies and strategies that might clarify them.
This chapter reviews a paper which summarizes the association of stress protein gene expression with pupal diapause in flesh flies, and discusses the possible implications of the upregulation of some of these proteins during this time. It links these findings with reports from other species showing the expression of stress proteins during periods of developmental arrest. Heat shock proteins are best known as a highly conserved group of stress proteins that are quickly upregulated in response to environmental stress. Stress proteins with similar sequences are present in organisms as diverse as bacteria, yeast, plants, and humans. The proteins are grouped into several families based on molecular mass. Genes encoding certain stress proteins (Hsp23 and 70) are highly upregulated during diapause, while others are either unaffected (Hsc70), or are downregulated (Hsp90). This disynchrony of expression is in marked contrast to the uniform upregulation of the stress protein genes in response to other stresses, such as heat shock or cold shock. The diapause upregulation of the genes may be linked to a cryoprotective function of the proteins or a possible role in shutting down the cell cycle. The involvement of stress proteins in the dormancies of other animals and plants suggests a conserved mechanism contributing to the arrest of development.
Incubation of the high-speed supernatant from the Kc cell line of Drosophila melanogaster with [3H]ponasterone A results in significant binding of the ligand as determined by gel filtration and dextran-coated charcoal binding assays. In vivo exposure of the Kc line to [3H]ponasterone A for 30 min results in a marked binding of the ligand by a KCl-soluble nuclear extract. With both the cytosol and nuclear preparations the binding has specificity and a low dissociation constant (3 X 10(-9) M). In addition the labeling of the nuclear preparation exhibits a saturation at approximately 7 X 10(-10) M which probably reflects the molar concentration of cytoplasmic receptors.
The Drosophila chorion factor 1/ultraspiracle (CF1/USP) transcription factor, a homologue of the retinoid X receptor, is a developmentally important member of the family of nuclear (steroid) hormone receptors. Using newly developed monoclonal antibodies and a full-length bacterially produced protein, we have studied in detail the in vitro DNA-binding properties of this factor and aspects of its distribution in vivo. During oogenesis, CF1/USP is present both in germline cells and in the somatic follicular epithelium. We have determined the optimal binding site of partially purified bacterially produced CF1/USP by an in vitro selection procedure and also have characterized its binding to the follicular-specific chorion s15 promoter. In vitro this bacterially produced factor is unusual in binding to a single element ("half-site"); simultaneous but noncoordinate binding to a second half-site is possible if these repeated elements are organized in direct orientation and spaced adequately. However, the factor interacts synergistically with several other nuclear hormone receptors: notably, it can form in vitro heteromers with mammalian thyroid and retinoic acid receptors, binding to two half-sites that are organized in either direct or inverted orientation. In vivo the factor most probably functions as a heterodimer, but its partner(s) remains to be determined.
We have recently reported that the glucocorticoid receptor (GR) becomes bound to the 90-kDa heat shock protein (hsp90) at or near the end of receptor translation in vitro (Dalman, F. C., Bresnick, E. H., Patel, P. D., Perdew, G. H., Watson, S. J., Jr., and Pratt, W. B. (1989) J. Biol. Chem. 264, 19815-19821). In this paper we compare the hsp90 binding and DNA binding activities of the thyroid hormone receptor (TR) to those of the GR after cell-free translation of the two receptors in rabbit reticulocyte lysate. In contrast to the newly translated GR, which is bound to hsp90 and must be transformed to the DNA binding state, the TR is not bound to hsp90 and is translated in its DNA binding form without any requirement for transformation. When the GR is translated in wheat germ extract, which does not contain hsp90, it is translated in its DNA binding form in the same manner as the TR synthesized in reticulocyte lysate. These observations provide direct evidence that binding of GR to hsp90 is associated with repression of its DNA binding function. The fact that the TR does not bind to hsp90 and is translated in its DNA binding form is consistent with the different behavior of this receptor with respect to classic steroid receptors in the intact cell. We propose that binding to hsp90 may account for the fact that most of the steroid receptors are recovered in the cytosolic fraction after lysis of hormone-free cells in low salt buffer whereas the hormone-free TR is recovered in tight association with the nucleus.
Monoclonal antibodies have been used to identify three proteins in Drosophila melanogaster that share antigenic determinants
with the major heat shock proteins hsp70 and hsp68. While two of the proteins are major proteins at all developmental stages,
one heat shock cognate protein, hsc70, is especially enriched in embryos. hsc70 is shown to be the product of a previously
identified gene, Hsc4. We have examined the levels of hsp70-related proteins in adult flies and larvae during heat shock and
recovery. At maximal induction in vivo, hsp70 and hsp68 never reach the basal levels of the major heat shock cognate proteins.
Monoclonal antibodies to hsc70 have been used to localize it to a meshwork of cytoplasmic fibers that are heavily concentrated
around the nucleus.
The 2B5 region on the X chromosome of Drosophila melanogaster forms an early ecdysone puff at the end of the third larval instar. The region contains a complex genetic locus, the Broad-Complex (BR-C) composed of four groups of fully complementing (br, rbp, l(1)2Bc, and l(1)2Bd) alleles, and classes of noncomplementing (npr 1) and partially noncomplementing l(1)2Bab alleles. BR-C mutants prevent metamorphosis, including the morphogenesis of imaginal discs. Results are presented that indicate that the BR-C contains two major functional domains. One, the br domain is primarily, if not exclusively, involved in the elongation and eversion of appendages by imaginal discs. The second, the l(1)2Bc domain, is primarily involved in the fusion of discs to form a continuous adult epidermis. Nonetheless, the two domains may encode products with related functions because in some situations mutants in both domains appear to affect similar developmental processes.
We have conducted a genetic and developmental analysis of genes within the 2C-D area of the X chromosome. Phenotypes of 33 mutations representing nine adjacent complementation groups including eight recessive lethals and one visible homeotic mutation (polyhomeotic) are described. Germline clonal analysis of the eight zygotic lethals has revealed three types of gene requirements: normal activity at two pupal lethal loci (corkscrew and C204) and one larval lethal locus (ultraspiracle) is required for normal embryogenesis; normal activity at three larval lethal loci (DF967, VE651 and Pgd) is required for normal oogenesis; and activity at only one locus (EA82), a larval lethal, appears to have no maternal requirement. Ambiguous results were obtained for the GF316 lethal complementation group. Analysis of mitotic figures of the pupal lethals indicates that C204 disrupts an essential mitotic function. This result correlates with the preblastoderm arrest observed among embryos derived from germline clones of C204. Embryos derived from germline clones of corkscrew (csw) exhibit a "twisted" phenotype. The recessive lethal ultraspiracle (usp) disrupts the organization of the posterior tip of the larval both zygotically and maternally: second instar usp/Y larvae derived from heterozygous usp/+ mothers possess an extra set of spiracles, whereas usp/Y embryos derived from females possessing a germline clone (usp/usp) exhibit a localized ventral defect in the ninth or posterior eighth abdominal segment. Analysis of the phenotypes of deficiency-hemizygous embryos indicates the presence of an embryonic zygotic lethal locus, as yet unidentified, which produces central nervous system and ventral hypoderm degeneration. Additional information on the genetic organization of loci within the adjacent 2E area are also described.(ABSTRACT TRUNCATED AT 250 WORDS)
Hsp83 is the Drosophila homolog of the mammalian Hsp90 family of regulatory molecular chaperones. We show that maternally synthesized Hsp83 transcripts are localized to the posterior pole of the early Drosophila embryo by a novel mechanism involving a combination of generalized RNA degradation and local protection at the posterior. This protection of Hsp83 RNA occurs in wild-type embryos and embryos produced by females carrying the maternal effect mutations nanos and pumilio, which eliminate components of the posterior polar plasm without disrupting polar granule integrity. In contrast, Hsp83 RNA is not protected at the posterior pole of embryos produced by females carrying maternal mutations that disrupt the posterior polar plasm and the polar granules--cappuccino, oskar, spire, staufen, tudor, valois, and vasa. Mislocalization of oskar RNA to the anterior pole, which has been shown to result in induction of germ cells at the anterior, leads to anterior protection of maternal Hsp83 RNA. These results suggest that Hsp83 RNA is a component of the posterior polar plasm that might be associated with polar granules. In addition, we show that zygotic expression of Hsp83 commences in the anterior third of the embryo at the syncytial blastoderm stage and is regulated by the anterior morphogen, bicoid. We consider the possible developmental significance of this complex control of Hsp83 transcript distribution.
In the absence of hormone, the avian progesterone receptor exists in a large multiprotein complex that is inactive but able to bind and respond to progestins. This inactive complex can be reconstituted in vitro by incubation of receptor monomer in rabbit reticulocyte lysate in the presence of ATP and magnesium. This results in receptor binding to the two heat shock proteins, hsp90 and hsp70, the FK506 binding-proteins, FKBP54 and FKBP52, the cyclosporin A-binding protein, cyclophilin-40, and the recently characterized protein p23. Immune isolation of p23 from rabbit reticulocyte lysate in the absence of receptor reveals an ATP-dependent complex containing the major proteins associated with steroid receptors. Depletion of p23 from lysate prevents the assembly of progesterone receptor complexes, and the addition of purified p23 restores this activity, indicating that the p23 protein complex is an essential precursor to the formation of progesterone receptor complexes.
The ubiquitous heat shock protein Hsp90 appears to participate directly in the function of a broad range of cellular signal transduction components, including steroid hormone receptors; however, an evolutionarily related subclass of intracellular receptors, exemplified by the retinoid receptors RAR and RXR, had been inferred from biochemical studies to function independently of Hsp90. To examine this issue genetically, we measured mammalian and avian retinoid receptor activity in a Saccharomyces cerevisiae strain in which the expression of the yeast Hsp90 homologue could be conditionally repressed approximately 20-fold relative to wild type. We tested transcriptional activation by RAR or RXR-RAR, from two types of retinoic acid response elements, triggered by three different agonist ligands. In every condition, we found that activation was severely compromised under conditions of low Hsp90 expression. We showed that the defect was in signal transduction rather than transcription activation per se, and that high affinity hormone binding was abolished in extracts of cells producing low levels of Hsp90. We suggest that Hsp90 may function in at least one step of signal transduction by all members of the intracellular receptor superfamily.
Molecular chaperones are essential proteins that participate in the regulation of steroid receptors in eukaryotes. The steroid
aporeceptor complex contains the molecular chaperones Hsp90 and Hsp70, p48, the cyclophilin Cyp-40, and the associated proteins
p23 and p60. In vitro folding assays showed that Cyp-40 and p23 functioned as molecular chaperones in a manner similar to
that of Hsp90 or Hsp70. Although neither Cyp-40 nor p23 could completely refold an unfolded substrate, both proteins interacted
with the substrate to maintain a nonnative folding-competent intermediate. Thus, the steroid aporeceptor complexes have multiple
chaperone components that maintain substrates in an intermediate folded state.
The steroid hormone ecdysone directs Drosophila metamorphosis via three heterodimeric receptors that differ according to which of three ecdysone receptor isoforms encoded by the EcR gene (EcR-A, EcR-B1, or EcR-B2) is activated by the orphan nuclear receptor USP. We have identified and molecularly mapped two classes of EcR mutations: those specific to EcR-B1 that uncouple metamorphosis, and embryonic-lethal mutations that map to common sequences encoding the DNA- and ligand-binding domains. In the larval salivary gland, loss of EcR-B1 results in loss of activation of ecdysone-induced genes. Comparable transgenic expression of EcR-B1, EcR-B2, and EcR-A in these mutant glands results, respectively, in full, partial, and no repair of that loss.
Almost 30 years have passed since the original demonstration that steroid receptors, comprising a subfamily of the nuclear receptor (NR) superfamily, exist as large (6-8S) non-DNA-binding complexes in hypotonic extracts (cytosol) of target cells; later such complexes were shown to correspond to a heterooligomer composed of receptor, heat shock (Hsp), and other proteins. Subsequently, an impressive number of studies have dealt with the composition of the "nonactive" complex, its dissociation and/or reassembly in vitro, possible functions of the non-receptor components, and their subcellular compartmentalization. While there is little dispute about the chaperoning role of some Hsps in such a complex, there is still no final proof of an association in vivo of NRs and Hsps in the nuclei of target cells, which is requisite for a direct regulatory involvement of Hsps in NR function. Here we critically review the various models that have been put forward to attribute a biological function to the NR-Hsp90 interaction, evaluate the corresponding experimental data, and integrate recent concepts originating from the structural and functional analyses of NRs.
Estrogens, progestins, androgens, glucocorticoids, and mineralocorticoids are a group of structurally similar lipophilic steroids responsible for the regulation of a wide variety of cellular processes in different target organs. The glucocorticoids and mineralocorticoids, for instance, are primarily involved in the regulation of cellular homeostasis and metabolism whereas estrogens, progestins, and androgens, the sex steroids, are responsible for the development and maintenance of reproductive function. Despite their different roles, it has become apparent that the steroid hormones are mechanistically similar and that insights gleaned from the study of each hormone has advanced our understanding of the class of molecules as a whole. The objective of this chapter is to provide an updated model of steroid hormone action and to discuss the impact of recent insights on our understanding of the pharmacology of steroid hormones.
The functions of the ecdysone-induced DHR3 and E75B orphan nuclear receptors in the early stages of Drosophila metamorphosis
were investigated. DHR3 represses the ecdysone induction of early genes turned on by the pulse of ecdysone that triggers metamorphosis.
It also induces βFTZF1, an orphan nuclear receptor that is essential for the appropriate response to the subsequent prepupal
pulse of ecdysone. The E75B receptor, which lacks a complete DNA binding domain, inhibits this inductive function by forming
a complex with DHR3 on the βFTZF1 promoter, thereby providing a timing mechanism for βFTZF1 induction that is dependent on the disappearance of E75B.
Submitted to the Department of Biochemistry. Thesis (Ph. D.)--Stanford University, 1992.
[3H]Ponasterone A (PNA) of high specific activity has been used to identify and begin characterization of ecdysteroid (formerly called ecdysone) receptors in cytoplasmic and nuclear fractions of imaginal discs of Drosophila melanogaster. The equilibrium Kd of the observed macromolecular binding, 3--4 X 10(-9) M PNA, is in good agreement with the minimal concentration required for induction of complete morphogenesis in vitro, 4.2 X 10(-9) M PNA. Binding is analog specific and has kinetics consistent with a role in hormone response. On gentle homogenization, less than 5% of the binding capacity of the cell is released as soluble receptor; the other 95% remains with the nuclear fraction. This nuclear fraction specifically binds [3H]PNA in vitro. Greater than 95% of nuclear PNA receptors are released by extraction with 0.3 M KCl. The binding properties of the nuclear receptors are indistinguishable from those of the cytosol fraction or of the whole cell.
The vertebrate retinoid X receptor (RXR) has been implicated in the regulation of multiple hormonal signaling pathways through the formation of heteromeric receptor complexes that bind DNA with high affinity. We now demonstrate that ultraspiracle (usp), a Drosophila RXR homolog, can substitute for RXR in stimulating the DNA binding of receptors for retinoic acid, T3, vitamin D, and peroxisome proliferator activators. These observations led to the search and ultimate identification of the ecdysone receptor (EcR) as a Drosophila partner of usp. Together, usp and EcR bind DNA in a highly cooperative fashion. Cotransfection of both EcR and usp expression vectors is required to render cultured mammalian cells ecdysone responsive. These results implicate usp as an integral component of the functional EcR. By demonstrating that receptor heterodimer formation precedes the divergence of vertebrate and invertebrate lineages, these data underscore a central role for RXR and its homolog usp in the evolution and control of the nuclear receptor-based endocrine system.
We have recently reported that, in contrast to the glucocorticoid receptor, the thyroid hormone receptor does not bind to hsp90 when the receptor is translated in rabbit reticulocyte lysate [Dalman, F. C., Koenig, R. J., Perdew, G. H., Massa, E., & Pratt, W. B. (1990) J. Biol. Chem. 265, 3615-3618]. All of the steroid receptors that are known to bind hsp90 are recovered in the cytosolic fraction when hormone-free cells are ruptured in hypotonic buffer. In contrast, unliganded thyroid hormone receptors and retinoic acid receptors are tightly associated with nuclear components. In this paper, we translated the human estrogen receptor and the human retinoic acid receptor in reticulocyte lysate and then immunoadsorbed the [35S]methionine-labeled translation products with the 8D3 monoclonal antibody against hsp90. The estrogen receptor is bound to hsp90, as indicated by coimmunoadsorption, but the retinoic acid receptor is not. Translation and immunoadsorption of chimeric proteins containing the DNA binding domain of one receptor and the N-terminal and COOH-terminal segments of the other show that the DNA binding finger region of the estrogen receptor is neither necessary nor sufficient for hsp90 binding. These observations suggest that there are two classes within the steroid receptor family. In one class (e.g., glucocorticoid, mineralocorticoid, sex hormone, and dioxin receptors), the receptors bind to hsp90 and remain in some kind of inactive "docking" mode until hormone-triggered release of hsp90 occurs. In the retinoic acid/thyroid hormone class, the unligated receptors do not bind to hsp90, and the receptors appear to proceed directly to their high-affinity nuclear acceptor sites without entering the "docking" state.
The steroid hormone ecdysone triggers coordinate changes in Drosophila tissue development that result in metamorphosis. To advance our understanding of the genetic regulatory hierarchies controlling this tissue response, we have isolated and characterized a gene, EcR, for a new steroid receptor homolog and have shown that it encodes an ecdysone receptor. First, EcR protein binds active ecdysteroids and is antigenically indistinguishable from the ecdysone-binding protein previously observed in extracts of Drosophila cell lines and tissues. Second, EcR protein binds DNA with high specificity at ecdysone response elements. Third, ecdysone-responsive cultured cells express EcR, whereas ecdysone-resistant cells derived from them are deficient in EcR. Expression of EcR in such resistant cells by transfection restores their ability to respond to the hormone. As expected, EcR is nuclear and found in all ecdysone target tissues examined. Furthermore, the EcR gene is expressed at each developmental stage marked by a pulse of ecdysone.
The vitamin A derivative, retinoic acid, can regulate morphogenesis and differentiation in vertebrates. Two different subfamilies of the steroid receptor superfamily of transcription factors, the retinoic acid receptors and the retinoid X receptor, mediate the effects of retinoic acid. As part of an analysis of the hormonal control of development, we have examined the Drosophila genome for retinoic acid receptor homologues. Here we describe one such gene, XR2C, which encodes a product with structural similarity to the human retinoic acid-responsive transcription factor, retinoid X receptor. This receptor-like protein is encoded by ultraspiracle (usp), a locus required both maternally and zygotically for pattern formation. The discovery that the usp product is a retinoid X receptor homologue suggests that similar chemical cues underlie morphogenic signalling in vertebrate and invertebrate systems.
The usp locus encodes a member of the nuclear hormone receptor superfamily in Drosophila melanogaster that interacts with EcR (ecdysone receptor) to mediate ecdysteroid-induced gene expression. A 2.7-kb usp mRNA was detected at all developmental times tested, although its abundance varied. Among premetamorphic stages, both the 2.7-kb transcript and Usp protein attained their highest levels in the late third larval instar. The 2.7-kb usp transcript was also found in adult stages and a 1.2-kb transcript was detected in the polyadenylated RNA fraction of both mature adult females and early embryos. Aneuploids carrying two usp mutant alleles and a putative variegating usp+ allele often developed deformities of the adult wing disc that apparently resulted from mutational disruption of usp activity before metamorphosis and whose frequency was affected by maternal genotype. Both of the recessive lethal usp mutations associated with this "cleft thorax" phenotype involved substitutions of conserved arginine residues in the DNA-binding domain, although the frequency of the phenotype was not the same for the two alleles. Both mutant proteins retained the ability to form heterodimers with EcR in vitro but showed reduced affinity for an ecdysone response element.
Although the biological activity of the insect moulting hormone ecdysone, is manifested through a hormonally regulated transcriptional cascade associated with chromosomal puffing, a direct association of the receptor with the puff has yet to be established. The cloned ecdysone receptor (EcR) is by itself incapable of high-affinity DNA binding or transcriptional activation. Rather, these activities are dependent on heterodimer formation with Ultraspiracle (USP) the insect homologue of vertebrate retinoid X receptor. Here we report that native EcR and USP are co-localized on ecdysone-responsive loci of polytene chromosomes. Moreover, we show that natural ecdysones selectively promote physical association between EcR and USP, and conversely, that high-affinity hormone binding requires both EcR and USP. Replacement of USP with retinoid X receptor produces heterodimers with distinct pharmacological and functional properties. These results redefine the ecdysone receptor as a dynamic complex whose activity may be altered by combinatorial interactions among subunits and ligand.
In D. melanogaster a pulse of the steroid hormone ecdysone triggers the larval-to-adult metamorphosis, a complex process in which this hormone induces imaginal tissues to generate adult structures and larval tissues to degenerate. We show that the EcR gene encodes three ecdysone receptor isoforms (EcR-A, EcR-B1, and EcR-B2) that have common DNA- and hormone-binding domains but different N-terminal regions. We have used isoform-specific monoclonal antibodies to show that at the onset of metamorphosis different ecdysone target tissues express different isoform combinations in a manner consistent with the proposition that the different metamorphic responses of these tissues require different combinations of the EcR isoforms. We have also determined temporal developmental profiles of the EcR isoforms and their mRNAs in whole animals, showing that different isoforms predominate at different developmental stages that are marked by a pulse of ecdysone.
Ecdysone in Drosophila has been a paradigm for steroid hormones since its ability to induce gene activity directly was demonstrated by its effects on moulting and polytene chromosome puffing. The ecdysone receptor (EcR) was recently confirmed as a member of the nuclear receptor superfamily by cloning and characterization in a Drosophila cell line. Here we show that EcR needs to heterodimerize with either the retinoid X receptor (RXR) or its Drosophila homologue, ultraspiracle (USP), for DNA binding and transactivation. These results place the ecdysone receptor in the heterodimerizing class of the nuclear receptor superfamily and demonstrate that the role of RXR/USP as a central and promiscuous partner in mediating the activity of these receptors is highly conserved. Whereas EcR-USP DNA-binding activity is unaffected by hormone, EcR-RXR DNA-binding activity is stimulated by either ecdysteroid or 9-cis-retinoic acid, demonstrating that hormone can play a role in heterodimer stabilization.
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The Hsp90 heat shock protein of eukaryotic cells regulates the activity of proteins involved in signal transduction pathways
and may direct intracellular protein folding in general. Hsp90 performs at least part of its function in a complex with a
specific set of partner proteins that include members of the prolyl isomerase family. The properties of the major components
of the Hsp90 complex were examined through the use of in vitro protein folding assays. Two of the components, FKBP52 and p23,
functioned as mechanistically distinct molecular chaperones. These results suggest the existence of a super-chaperone complex
in the cytosol of eukaryotic cells.
The functions of the ecdysone-induced DHR3 and E75B orphan nuclear receptors in the early stages of Drosophila metamorphosis were investigated. DHR3 represses the ecdysone induction of early genes turned on by the pulse of ecdysone that triggers metamorphosis. It also induces betaFTZF1, an orphan nuclear receptor that is essential for the appropriate response to the subsequent prepupal pulse of ecdysone. The E75B receptor, which lacks a complete DNA binding domain, inhibits this inductive function by forming a complex with DHR3 on the betaFTZF1 promoter, thereby providing a timing mechanism for betaFTZF1 induction that is dependent on the disappearance of E75B.
The Hsp90 chaperone is required for the activation of several families of eukaryotic protein kinases and nuclear hormone receptors, many of which are protooncogenic and play a prominent role in cancer. The geldanamycin antibiotic has antiproliferative and antitumor effects, as it binds to Hsp90, inhibits the Hsp90-mediated conformational maturation/refolding reaction, and results in the degradation of Hsp90 substrates. The structure of the geldanamycin-binding domain of Hsp90 (residues 9-232) reveals a pronounced pocket, 15 A deep, that is highly conserved across species. Geldanamycin binds inside this pocket, adopting a compact structure similar to that of a polypeptide chain in a turn conformation. This, and the pocket's similarity to substrate-binding sites, suggest that the pocket binds a portion of the polypeptide substrate and participates in the conformational maturation/refolding reaction.
The biological activities of the retinoids are mediated by two nuclear hormone receptors: the retinoic acid receptor (RAR) and the retinoid-X receptor (RXR). RXR (and its insect homologue ultraspiracle) is a common heterodimeric partner for many other nuclear receptors, including the insect ecdysone receptor. As part of a continuing analysis of nuclear receptor function, we noticed that, whereas RXR can be readily expressed in Escherichia coli to produce soluble protein, many of its heterodimeric partners cannot. For example, overexpression of RAR results mostly in inclusion bodies with the residual soluble component unable to interact with RXR or ligand efficiently. Similar results are seen with other RXR/ultraspiracle partners. To overcome these problems, we designed a novel double cistronic vector to coexpress RXR and its partner ligand-binding domains in the same bacterial cell. This resulted in a dramatic increase in production of soluble and apparently stable heterodimer. Hormone-binding studies using the purified RXR-RAR heterodimer reveal increased ligand-binding capacity of both components of 5- to 10-fold, resulting in virtually complete functionality. Based on these studies we find that bacterially expressed receptors can exist in one of three distinct states: insoluble, soluble but unable to bind ligand, or soluble with full ligand-binding capacity. These results suggest that coexpression may represent a general strategy for biophysical and structural analysis of receptor complexes.
We have provided a historical perspective on a body of steroid receptor research dealing with the structure and physiological significance of the untransformed 9S receptor that has often confused both novice and expert investigators. The frequent controversies and equivocations of earlier studies were due to the fact that the native, hormone-free state of these receptors is a large multiprotein complex that resisted description for many years because of its unstable and dynamic nature. The untransformed 9S state of the steroid and dioxin receptors has provided a unique system for studying the function of the ubiquitous, abundant, and conserved heat shock protein, hsp90. The hormonal control of receptor association with hsp90 provided a method of manipulating the receptor heterocomplex in a manner that was physiologically meaningful. For several steroid receptors, binding to hsp90 was required for the receptor to be in a native hormone-binding state, and for all of the receptors, hormone binding promoted dissociation of the receptor from hsp90 and conversion of the receptor to the DNA-binding state. Although the complexes between tyrosine kinases and hsp90 were discovered earlier, the hormonal regulation or steroid receptor association with hsp90 permitted much more rapid and facile study of hsp90 function. The observations that hsp90 binds to the receptors through their HBDs and that these domains can be fused to structurally different proteins bringing their function under hormonal control provided a powerful linkage between the hormonal regulation of receptor binding to hsp90 and the initial step in steroid hormone action. Because the 9S receptor hsp90 heterocomplexes could be physically stabilized by molybdate, their protein composition could be readily studied, and it became clear that these complexes are multiprotein structures containing a number of unique proteins, such as FKBP51, FKBP52, CyP-40, and p23, that were discovered because of their presence in these structures. Further analysis showed that hsp90 itself exists in a variety of native multiprotein heterocomplexes independent of steroid receptors and other 'substrate' proteins. Cell-free systems can now be used to study the formation of receptor heterocomplexes. As we outlined in the scheme of Fig. 1, the multicomponent receptor-hsp90 heterocomplex assembly system is being reconstituted, and the importance of individual proteins, such as hsp70, p60, and p23, in the assembly process is becoming recognized. It should be noted that our understanding of the mechanism and purpose of steroid receptor heterocomplex assembly is still at an early stage. We can now speculate on the roles of receptor-associated proteins in receptor action, both as individuals and as a group, but their actual functions are still vague or unknown. We can make realistic models about the chaperoning and trafficking of steroid receptors, but we don't yet know how these processes occur, we don't know where chaperoning occurs in the cell (e.g. Is it limited to the cytoplasm? Is it a diffuse process or does chaperoning occur in association with structural elements?), and, with the exception of the requirement for hormone binding, we don't know the extent to which the hsp90-based chaperone system impacts on steroid hormone action. It is not yet clear how far the discovery of this hsp90 heterocomplex assembly system will be extended to the development of a general understanding of protein processing in the cell. Because this assembly system is apparently present in all eukaryotic cells, it probably performs an essential function for many proteins. The bacterial homolog of hsp90 is not an essential protein, but hsp90 is essential in eukaryotes, and recent studies indicate that the development of the cell nucleus from prokaryotic progenitors was accompanied by the duplication of genes for hsp90 and hsp70 (698). (ABSTRACT TRUNCATED)
Hsp90 molecular chaperones in eukaryotic cells play essential roles in the folding and activation of a range of client proteins involved in cell cycle regulation, steroid hormone responsiveness, and signal transduction. The biochemical mechanism of Hsp90 is poorly understood, and the involvement of ATP in particular is controversial. Crystal structures of complexes between the N-terminal domain of the yeast Hsp90 chaperone and ADP/ATP unambiguously identify a specific adenine nucleotide binding site homologous to the ATP-binding site of DNA gyrase B. This site is the same as that identified for the antitumor agent geldanamycin, suggesting that geldanamycin acts by blocking the binding of nucleotides to Hsp90 and not the binding of incompletely folded client polypeptides as previously suggested. These results finally resolve the question of the direct involvement of ATP in Hsp90 function.
Unliganded steroid receptors exist as heteromeric complexes comprised of heat shock and immunophilin proteins that associate either directly or indirectly with receptor carboxyl-terminal ligand-binding domains. Molecular chaperons, and other proteins associated with steroid receptors, play an important role in the maturation of receptors to a hormone-binding competent state. Steroid receptor-associated 90 and 70 kDa heat shock proteins, hsp90 and hsp70, respectively, have well established roles in protein folding in addition to participating in numerous subcellular trafficking pathways. In this review, we discuss the possible roles that molecular chaperons, such as hsp90, hsp70 and DnaJ proteins, have in steroid receptor trafficking within two distinct subcellular compartments, i.e. the cytoplasm and nucleus.
The nuclear hormone receptors constitute a large family of transcription factors. The binding of the hormonal ligands induces nuclear receptors to assume a configuration that leads to transcriptional activation. Recent studies of retinoic acid and thyroid hormone receptors revealed that, upon ligand binding, a histone deacetylase (HDAC)-containing complex is displaced from the nuclear receptor in exchange for a histone acetyltransferase (HAT)-containing complex. These observations suggest that ligand-dependent recruitment of chromatin-remodeling activity serves as a general mechanism underlying the switch of nuclear receptors from being transcriptionally repressive to being transcriptionally active.
Steroid hormone receptors (SHR) form complexes with heat shock proteins (hsps). The 1alpha,25-dihydroxyvitamin D(3) receptor (VDR) has not been previously shown to interact with hsps. During expression and purification of VDR-glutathione S-transferase (VDR-GST) fusion proteins encompassing full-length, DNA, and ligand-binding domains of the VDR (FL-VDR, DBD-VDR, and LBD-VDR), we observed binding of bacterial hsps with VDR-GST constructs. All VDR constructs bound DnaK in amounts greater than GST alone and bound smaller amounts of DnaJ or GrpE. GroEL bound only to FL-VDR. GroES did not bind to VDR. When VDR-GST constructs were incubated with a reticulocyte lysate system that has been used previously to examine SHR-hsp interactions, eukaryotic hsc70 was detected bound to FL-VDR and DBD-VDR. Binding of hsp90 to VDR was not detected. However, geldanamycin, an hsp90 inhibitor, reduced 1alpha,25-dihydroxyvitamin D(3)-mediated gene activation in osteoblasts. Our data show that the bacterial and eukaryotic hsps associate with the VDR and might be involved in VDR function.
The Drosophila ecdysone receptor (EcR)/ultraspiracle (USP) heterodimer is a key regulator in molting and metamorphoric processes, activating and repressing transcription in a sequence-specific manner. Here, we report the isolation of an EcR-interacting protein, SMRTER, which is structurally divergent but functionally similar to the vertebrate nuclear corepressors SMRT and N-CoR. SMRTER mediates repression by interacting with Sin3A, a repressor known to form a complex with the histone deacetylase Rpd3/HDAC. Importantly, we identify an EcR mutant allele that fails to bind SMRTER and is characterized by developmental defects and lethality. Together, these results reveal a novel nuclear receptor cofactor that exhibits evolutionary conservation in the mechanism to achieve repression and demonstrate the essential role of repression in hormone signaling.
In our effort to dissect the Notch signaling mechanism we have conducted a screen for mutations that reduce Notch signaling activity. We recovered nine complementation groups as modifiers of the hypomorphic Notch allele notchoid. Apart from the known Notch signaling modulators Notch, Delta and mastermind we isolated alleles in vestigial, wingless, scalloped and clipped, genes known to affect wing morphogenesis. In addition, we identified mutations in Bag, the gene encoding clathrin heavy chain and a dominant mutation of the cytosolic 70 kDa heatshock cognate protein encoded by the hsc4 gene, as Notch signaling modifier. We focused our attention on the latter mutation because it displays dramatic genetic interactions with mutations of the Notch receptor as well as several additional Notch signaling pathway elements. We discuss how hsc4, a gene thought to be involved in subcellular trafficking, may affect the number of functional Notch receptors on the cell surface.
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