Tumor Susceptibility Gene 101 Regulates the Glucocorticoid Receptor through Disorder-Mediated Allostery.

Tumor susceptibility gene 101 (TSG101) is involved in endosomal maturation and has been implicated in the transcriptional regulation of several steroid hormone receptors, although a detailed characterization of such regulation has yet to be conducted. Here we directly measure binding of TSG101 to one steroid hormone receptor, the glucocorticoid receptor (GR). Using biophysical and cellular assays, we show that the coiled-coil domain of TSG101 (1) binds and folds the disordered N-terminal domain of the GR, (2) upon binding improves the DNA binding of the GR in vitro, and (3) enhances the transcriptional activity of the GR in vivo. Our findings suggest that TSG101 is a bona fide transcriptional co-regulator of the GR and reveal how the underlying thermodynamics affect the function of the GR.

NFAT5, which protects against hypertonicity, is activated by that stress via structuring of its intrinsically disordered domain.

Nuclear Factor of Activated T cells 5 (NFAT5) is a transcription factor (TF) that mediates protection from adverse effects of hypertonicity by increasing transcription of genes, including those that lead to cellular accumulation of protective organic osmolytes. NFAT5 has three intrinsically ordered (ID) activation domains (ADs). Using the NFAT5 N-terminal domain (NTD), which contains AD1, as a model, we demonstrate by biophysical methods that the NTD senses osmolytes and hypertonicity, resulting in stabilization of its ID regions. In the presence of sufficient NaCl or osmolytes, trehalose and sorbitol, the NFAT5 NTD undergoes a disorder-to-order shift, adopting higher average secondary and tertiary structure. Thus, NFAT5 is activated by the stress that it protects against. In its salt and/or osmolyte-induced more ordered conformation, the NTD interacts with several proteins, including HMGI-C, which is known to protect against apoptosis. These findings raise the possibility that the increased intracellular ionic strength and elevated osmolytes caused by hypertonicity activate and stabilize NFAT5.

Role of Phosphorylation in the Modulation of the Glucocorticoid Receptor's Intrinsically Disordered Domain.

Protein phosphorylation often switches cellular activity from one state to another, and this post-translational modification plays an important role in gene regulation by the nuclear hormone receptor superfamily, including the glucocorticoid receptor (GR). Cell signaling pathways that regulate phosphorylation of the GR are important determinants of GR actions, including lymphoid cell apoptosis, DNA binding, and interaction with coregulatory proteins. All major functionally important phosphorylation sites in the human GR are located in its N-terminal domain (NTD), which possesses a powerful transactivation domain, AF1. The GR NTD exists as an intrinsically disordered protein (IDP) and undergoes disorder-order transition for AF1's efficient interaction with several coregulatory proteins and subsequent AF1-mediated GR activity. It has been reported that GR's NTD/AF1 undergoes such disorder-order transition following site-specific phosphorylation. This review provides currently available information regarding the role of GR phosphorylation in its action and highlights the possible underlying mechanisms of action.

Genetically tunable frustration controls allostery in an intrinsically disordered transcription factor.

Intrinsically disordered proteins (IDPs) present a functional paradox because they lack stable tertiary structure, but nonetheless play a central role in signaling, utilizing a process known as allostery. Historically, allostery in structured proteins has been interpreted in terms of propagated structural changes that are induced by effector binding. Thus, it is not clear how IDPs, lacking such well-defined structures, can allosterically affect function. Here, we show a mechanism by which an IDP can allosterically control function by simultaneously tuning transcriptional activation and repression, using a novel strategy that relies on the principle of 'energetic frustration'. We demonstrate that human glucocorticoid receptor tunes this signaling in vivo by producing translational isoforms differing only in the length of the disordered region, which modulates the degree of frustration. We expect this frustration-based model of allostery will prove to be generally important in explaining signaling in other IDPs.

Restored mutant receptor:Corticoid binding in chaperone complexes by trimethylamine N-oxide.

Without a glucocorticoid (GC) ligand, the transcription factor glucocorticoid receptor (GR) is largely cytoplasmic, with its GC-binding domain held in high affinity conformation by a cluster of chaperones. Binding a GC causes serial dis- and re-associations with chaperones, translocation of the GR to the nucleus, where it binds to DNA sites and associates with coregulatory proteins and basic transcription complexes. Herein, we describe the effects of a potent protective osmolyte, trimethylamine N-oxide (TMAO), on a conditions-dependent "activation-labile" mutant GR (GRact/l), which under GR-activating conditions cannot bind GCs in cells or in cell cytosols. In both cells and cytosols, TMAO restores binding to GRact/l by stabilizing it in complex with chaperones. Cells bathed in much lower concentrations of TMAO than those required in vitro show restoration of GC binding, presumably due to intracellular molecular crowding effects.

Epigenetic alteration by DNA-demethylating treatment restores apoptotic response to glucocorticoids in dexamethasone-resistant human malignant lymphoid cells.

BACKGROUND: Glucocorticoids (GCs) are often included in the therapy of lymphoid malignancies because they kill several types of malignant lymphoid cells. GCs activate the glucocorticoid receptor (GR), to regulate a complex genetic network, culminating in apoptosis. Normal lymphoblasts and many lymphoid malignancies are sensitive to GC-driven apoptosis. Resistance to GCs can be a significant clinical problem, however, and correlates with resistance to several other major chemotherapeutic agents. METHODS: We analyzed the effect of treatment with the cytosine analogue 5 aza-2' deoxycytidine (AZA) on GC resistance in two acute lymphoblastic leukemia (T or pre-T ALL) cell lines- CEM and Molt-4- and a (B-cell) myeloma cell line, RPMI 8226. Methods employed included tissue culture, flow cytometry, and assays for clonogenicity, cytosine extension, immunochemical identification of proteins, and gene transactivation. High throughput DNA sequencing was used to confirm DNA methylation status. CONCLUSIONS: Treatment of these cells with AZA resulted in altered DNA methylation and restored GC-evoked apoptosis in all 3 cell lines. In CEM cells the altered epigenetic state resulted in site-specific phosphorylation of the GR, increased GR potency, and GC-driven induction of the GR from promoters that lie in CpG islands. In RPMI 8226 cells, expression of relevant coregulators of GR function was altered. Activation of p38 mitogen-activated protein kinase (MAPK), which is central to a feed-forward mechanism of site-specific GR phosphorylation and ultimately, apoptosis, occurred in all 3 cell lines. These data show that in certain malignant hematologic B- and T-cell types, epigenetically controlled GC resistance can be reversed by cell exposure to a compound that causes DNA demethylation. The results encourage studies of application to in vivo systems, looking towards eventual clinical applications.

Interplay between allostery and intrinsic disorder in an ensemble.

Allostery is a biological phenomenon of critical importance in metabolic regulation and cell signalling. The fundamental premise of classical models that describe allostery is that structure mediates 'action at a distance'. Recently, this paradigm has been challenged by the enrichment of IDPs (intrinsically disordered proteins) or ID (intrinsically disordered) segments in transcription factors and signalling pathways of higher organisms, where an allosteric response from external signals is requisite for regulated function. This observation strongly suggests that IDPs elicit the capacity for finely tunable allosteric regulation. Is there a set of transferable ground rules that reconcile these disparate allosteric phenomena? We focus on findings from the human GR (glucocorticoid receptor) which is a nuclear transcription factor in the SHR (steroid hormone receptor) family. GR contains an intrinsically disordered NTD (N-terminal domain) that is obligatory for transcription activity. Different GR translational isoforms have various lengths of NTD and by studying these isoforms we found that the full-length ID NTD consists of two thermodynamically distinct coupled regions. The data are interpreted in the context of an EAM (ensemble allosteric model) that considers only the intrinsic and measurable energetics of allosteric systems. Expansion of the EAM is able to reconcile the paradox that ligands for SHRs can be agonists and antagonists in a cell-context-dependent manner. These findings suggest a mechanism by which SHRs in particular, and IDPs in general, may have evolved to couple thermodynamically distinct ID segments. The ensemble view of allostery that is illuminated provides organizing principles to unify the description of all allosteric systems and insight into 'how' allostery works.

Thermodynamic dissection of the intrinsically disordered N-terminal domain of human glucocorticoid receptor.

Intrinsically disordered (ID) sequence segments are abundant in cell signaling proteins and transcription factors. Because ID regions commonly fold as part of their intracellular function, it is crucial to understand the folded states as well as the transitions between the unfolded and folded states. Specifically, it is important to determine 1) whether large ID segments contain different thermodynamically and/or functionally distinct regions, 2) whether any ID regions fold upon activation, 3) the degree of coupling between the different ID regions, and 4) whether the stability of ID domains is a determinant of function. In this study, we thermodynamically characterized the full-length ID N-terminal domain (NTD) of human glucocorticoid receptor (GR) and two of its naturally occurring translational isoforms. The protective osmolyte trimethylamine N-oxide (TMAO) was used to induce folding transitions. Each of the three NTD isoforms was found to undergo a cooperative folding transition that is thermodynamically indistinguishable (based on m-values) from that of a globular protein of similar size. The extrapolated stabilities for the NTD isoforms showed clear correlation with the known activities of their corresponding GR translational isoforms. The data reveal that the full-length NTD can be viewed as having at least two thermodynamically coupled regions, a functional region, which is indispensable for GR transcriptional activity, and a regulatory region, the length of which serves to regulate the stability of NTD and thus the activity of GR. These results suggest a new functional paradigm whereby steroid hormone receptors in particular and ID proteins in general can have multiple functionally distinct ID regions that interact and modulate the stability of important functional sites.

Structural dynamics, intrinsic disorder, and allostery in nuclear receptors as transcription factors.

Steroid hormone receptors (SHRs) and nuclear receptors (NRs) in general are flexible, allosterically regulated transcription factors. The classic model is inadequate to explain all their behavior. Keys to function are their regions of intrinsic disorder (ID). Data show the dynamic structure and allosteric interactions of the three classic SHR domains: ligand-binding (LBD), DNA-binding (DBD), and N-terminal (NTD). Each responds to its ligands by stabilizing its structure. The LBD responds to classic steroidal and nonsteroidal small ligands; both may selectively modify SHR activity. The DBD responds differentially to the DNA sequences of its response elements. The NTD, with its high ID content and AF1, interacts allosterically with the LBD and DBD. Each domain binds heterologous proteins, potential allosteric ligands. An ensemble framework improves the classic model, shows how ID regions poise the SHR/NR family for optimal allosteric response, and provides a basis for quantitative evaluation of SHR/NR actions.

TMS, a chemically modified herbal derivative of resveratrol, induces cell death by targeting Bax.

Breast cancer recurrence after an initial favorable response to treatment is a major concern for patients who receive hormonal therapies. Additional therapies are necessary to extend the time of response, and ideally, these therapies should exhibit minimal toxicity. Our study described herein focuses on a non-toxic pro-apoptotic agent, TMS (2,4,3',5'-tetramethoxystilbene), which belongs to the Resveratrol family of stilbenes. Prior study demonstrated that TMS was more effective than Resveratrol for inducing apoptosis. Additionally, TMS was effective for invoking death of relapsing breast cancer cells. As TMS was effective for reducing tumor burden, we sought to determine the mechanism by which it achieved its effects. Microarray analysis demonstrated that TMS treatment increased tubulin genes as well as stress response and pro-apoptotic genes. Fractionation studies uncovered that TMS treatment causes cleavage of Bax from the p21 form to a truncated p18 form which is associated with the induction of potent apoptosis. Co-localization analysis of immunofluorescent studies showed that Bax moved from the cytosol to the mitochondria. In addition, the pro-apoptotic proteins Noxa and Bim (EL, L, and S) were increased upon TMS treatment. Cell lines reduced for Bax, Bim, and Noxa are compromised for TMS-mediated cell death. Electron microscopy revealed evidence of nuclear condensation, formation of apoptotic bodies and DAPI staining showed evidence of DNA fragmentation. TMS treatment was able to induce both caspase-independent and caspase-dependent death via the intrinsic death pathway.

Converting cell lines representing hematological malignancies from glucocorticoid-resistant to glucocorticoid-sensitive: signaling pathway interactions.

Mitogen-activated protein kinases (MAPKs), protein kinase A (PKA) and mTOR pathways modulate the apoptotic effects of glucocorticoids (GCs) in human lymphoblastic leukemia CEM cells. We now show that manipulation of these pathways converts several cell lines, representing other lymphoid malignancies, from GC-resistant to GC-sensitive. Basal levels of phosphorylated JNK and ERK were elevated in the GC-resistant cells. Treatments that directly or indirectly reduced phosphorylated JNK and ERK resulted in Dex sensitivity in five resistant lymphoid cell lines. Sensitivity to GC-driven apoptosis correlated with GC-dependent increases in phosphorylated and total glucocorticoid receptor, and in increased levels of the pro-apoptotic protein Bim.

Stepping stones in the path of glucocorticoid-driven apoptosis of lymphoid cells.

Cumulative work on glucocorticoid (GC) regulation of genes in lymphoid cell cultures has revealed that apoptotic sensitivity to GCs depends on sufficient active GC receptors in the cells. The actions of the ligand-driven GC receptor that lead to apoptosis depend on interactions with other major cell-signaling systems, including the MAPK pathways, the cAMP/PKA pathway, the hedgehog pathway, the mTOR system and the c-myc system. The balance between these systems determines whether a given cell responds to GCs by undergoing apoptosis. A central core of networked genes may be found under GC control in many types of malignant, GC-sensitive cells. The partial core list identified should be tested in clinical cell samples from hematologic malignancies.

Protein kinase A (PKA) isoform RIIbeta mediates the synergistic killing effect of cAMP and glucocorticoid in acute lymphoblastic leukemia cells.

Protein kinase A (PKA) or cAMP-dependent protein kinase (cAPK) mediates the synergistic effects of cAMP- and glucocorticoid (GC)-induced apoptosis in lymphoid cells. Using two human acute lymphoblastic leukemia cell (CEM) clones with respective GC-sensitive and GC-resistant phenotypes, we discovered that the PKA regulatory subunit isoform RII(beta) is preferentially expressed in the GC-sensitive clone C7-14 cells, whereas other intracellular cAMP receptors, including the exchange proteins directly activated by cAMP (Epac), are expressed at similar levels in both GC-sensitive and GC-resistant clones. High RII(beta) expression level in C7-14 cells is associated with elevated total PKA cellular activity and cAMP sensitivity, which consequently lead to an increased basal PKA activity. cAMP analogs that selectively activate type II PKA recapitulate the effects of forskolin of promoting apoptosis and antagonizing AKT/PKB activity in both GC-sensitive and GC-resistant clones, whereas type I PKA-selective agonists do not. Furthermore, down-regulation of RII(beta) leads to increased AKT/PKB activation and enhanced GC resistance in C7-14 cells. These results demonstrate that PKA RII(beta) is responsible for increased GC sensitivity, critical for cAMP-mediated synergistic cell killing in CEM cells, and may represent a novel therapeutic target for GC-resistant lymphoid malignancy.

Gene expression profiling of leukemic cells and primary thymocytes predicts a signature for apoptotic sensitivity to glucocorticoids.

BACKGROUND: Glucocorticoids (GC's) play an integral role in treatment strategies designed to combat various forms of hematological malignancies. GCs also are powerful inhibitors of the immune system, through regulation of appropriate cytokines and by causing apoptosis of immature thymocytes. By activating the glucocorticoid receptor (GR), GCs evoke apoptosis through transcriptional regulation of a complex, interactive gene network over a period of time preceding activation of the apoptotic enzymes. In this study we used microarray technology to determine whether several disparate types of hematologic cells, all sensitive to GC-evoked apoptosis, would identify a common set of regulated genes. We compared gene expression signatures after treatment with two potent synthetic GCs, dexamethasone (Dex) and cortivazol (CVZ) using a panel of hematologic cells. Pediatric CD4+/CD8+ T-cell leukemia was represented by 3 CEM clones: two sensitive, CEM-C7-14 and CEM-C1-6, and one resistant, CEM-C1-15, to Dex. CEM-C1-15 was also tested when rendered GC-sensitive by several treatments. GC-sensitive pediatric B-cell leukemia was represented by the SUP-B15 line and adult B-cell leukemia by RS4;11 cells. Kasumi-1 cells gave an example of the rare Dex-sensitive acute myeloblastic leukemia (AML). To test the generality of the correlations in malignant cell gene sets, we compared with GC effects on mouse non-transformed thymocytes. RESULTS: We identified a set of genes regulated by GCs in all GC-sensitive malignant cells. A portion of these were also regulated in the thymocytes. Because we knew that the highly Dex-resistant CEM-C1-15 cells could be killed by CVZ, we tested these cells with the latter steroid and again found that many of the same genes were now regulated as in the inherently GC-sensitive cells. The same result was obtained when we converted the Dex-resistant clone to Dex-sensitive by treatment with forskolin (FSK), to activate the adenyl cyclase/protein kinase A pathway (PKA). CONCLUSION: Our results have identified small sets of genes that correlate with GC-sensitivity in cells from several hematologic malignancies. Some of these are also regulated in normal mouse thymocytes.

Protein kinase A, not Epac, suppresses hedgehog activity and regulates glucocorticoid sensitivity in acute lymphoblastic leukemia cells.

Cyclic AMP synergizes strongly with glucocorticoids (GC) to induce apoptosis in normal or malignant lymphoid cells. We examined the individual roles that cAMP-dependent protein kinase (PKA) and Epac (exchange protein directly activated by cAMP), two intracellular cAMP receptors, play in this synergistic effect. Our studies demonstrate that PKA is responsible for the observed synergism with GC, whereas Epac exerts a weak antagonistic effect against GC-induced apoptosis. We find that endogenous PKA activity is higher in the GC-sensitive clone than in the GC-resistant clone. In the GC-sensitive clone, higher PKA activity is associated with lower Hedgehog (Hh) activity. Moreover, inhibition of Hh activity by Hh pathway-specific inhibitors leads to cell cycle arrest and apoptosis in CEM (human acute lymphoblastic leukemia, T lineage) cells, and the GC-sensitive clone is more sensitive to Hh inhibition. These results suggest that Hh activity is critical for leukemia cell growth and survival and that the level of Hh activity is in part responsible for the synergism between cAMP and GC.

In CEM cells the autosomal deafness gene dfna5 is regulated by glucocorticoids and forskolin.

Certain mutations of the dfna5 gene result in a form of autosomal deafness that holds special interest because its phenotype resembles the hearing loss often seen during aging. Little is known of the function or regulation of dfna5 or its encoded protein. However dfna5 has recently been shown to be induced by p53. It also is epigenetically repressed in gastric cancer. We have discovered that dfna5 can be induced by glucocorticoids (GCs) and that this regulation is influenced by crosstalk with the protein kinase A (PKA) system. We show that GCs induce dfna5 mRNA and that its expression appears to be repressed in the basal state. Induction of dfna5 mRNA correlates with GC-dependent apoptosis of CEM cells, though dfna5 expression alone is not sufficient for apoptosis.

Comparison of two structurally diverse glucocorticoid receptor agonists: cortivazol selectively regulates a distinct set of genes separate from dexamethasone in CEM cells.

One goal of steroid research is precise differential regulation of gene expression by steroid hormone receptors through use of distinct ligands which modulate defined sets of cellular effects. Such "selective modulator" ligands are known for several receptors. Potent pyrazolo-glucocorticoid (11beta,16alpha)-21-(Acetyloxy)-11,17-dihydroxy-6,16-dimethyl-2'-phenyl-2'H-pregna-2,4,6-trieno[3,2-c]pyrazol-20-one) cortivazol activates the glucocorticoid receptor to regulate gene expression and can bring about apoptosis of leukemic CEM cells resistant to (9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,11,12,14,15,16-octahydrocyclopenta[a]phenanthren-3-one) dexamethasone. We therefore tested the hypothesis that cortivazol and dexamethasone regulate non-identical sets of genes in CEM cells. We found that while cortivazol and dexamethasone overlap in regulation of most genes, each steroid regulates an exclusive set of transcripts in clone CEM-C7-14 (sensitive to apoptosis by both dexamethasone and cortivazol) and clone CEM-C1-15 (dexamethasone-resistant but cortivazol-sensitive). Fifty-seven genes were regulated uniquely to a statistically significant extent by cortivazol in both clones. Many of the cortivazol specific genes are key components of various signal transduction pathways. Our data clearly show cortivazol to be a selective modulator of GR action.

Intrinsic disorder as a mechanism to optimize allosteric coupling in proteins.

Transcription factors and other allosteric cell signaling proteins contain a disproportionate number of domains or segments that are intrinsically disordered (ID) under native conditions. In many cases folding of these segments is coupled to binding with one or more of their interaction partners, suggesting that intrinsic disorder plays an important functional role. Despite numerous hypotheses for the role of ID domains in regulation, a mechanistic model has yet to be established that can quantitatively assess the importance of intrinsic disorder for intramolecular site-to-site communication, the hallmark property of allosteric proteins. Here, we present such a model and show that site-to-site allosteric coupling is maximized when intrinsic disorder is present in the domains or segments containing one or both of the coupled binding sites. This result not only explains the prevalence of ID domains in regulatory proteins, it also calls into question the classical mechanical view of energy propagation in proteins, which predicts that site-to-site coupling would be maximized when a well defined pathway of folded structure connects the two sites. Furthermore, in showing that the coupling mechanism conferred by intrinsic disorder is robust and independent of the network of interactions that physically link the coupled sites, unique insights are gained into the energetic ground rules that govern site-to-site communication in all proteins.