CCN5 activation by free or encapsulated EGCG is required to render triple-negative breast cancer cell viability and tumor progression.

Epigallocatechin-3-gallate (EGCG) has been considered an anticancer agent despite conflicting and discrepant bioavailability views. EGCG impairs the viability and self-renewal capacity of triple-negative breast cancer (TNBC) cells and makes them sensitive to estrogen via activating ER-α. Surprisingly, the mechanism of EGCG's action on TNBC cells remains unclear. CCN5/WISP-2 is a gatekeeper gene that regulates viability, ER-α, and stemness in TNBC and other types of cancers. This study aimed to investigate whether EGCG (free or encapsulated in nanoparticles) interacts with the CCN5 protein by emphasizing its bioavailability and enhancing its anticancer effect. We demonstrate that EGCG activates CCN5 to inhibit in vitro cell viability through apoptosis, the sphere-forming ability via reversing TNBC cells' stemness, and suppressing tumor growth in vivo. Moreover, we found EGCG-loaded nanoparticles to be functionally more active and superior in their tumor-suppressing ability than free-EGCG. Together, these studies identify EGCG (free or encapsulated) as a novel activator of CCN5 in TNBC cells and hold promise as a future therapeutic option for TNBC with upregulated CCN5 expression.

Pharmacological activation of REV-ERBα improves nonalcoholic steatohepatitis by regulating intestinal permeability.

BACKGROUND AND OBJECTIVES: The gut-liver axis plays an important role in the pathogenesis of nonalcoholic steatohepatitis (NASH), and increased intestinal permeability causes transfer of endotoxin to the liver, which activates the immune response, ultimately leading to hepatic inflammation. Nuclear receptor Rev-erbα is a critical regulator of circadian rhythm, cellular metabolism, and inflammatory responses. However, the role and mechanism of Rev-erbα in gut barrier function and NASH remain unclear. In the present study, we investigated the involvement of Rev-erbα in the regulation of intestinal permeability and the treatment of NASH. METHODS AND RESULTS: The expression of tight junction-related genes and Rev-erbs decreased in the jejunum, ileum and colon of mice with high cholesterol, high fat diet (CL)-induced NASH. Chromatin immunoprecipitation analysis indicated that REV-ERBα directly bound to the promoters of tight junction genes to regulate intestinal permeability. Pharmacological activation of REV-ERBα by SR9009 protected against lipopolysaccharide-induced increased intestinal permeability both in vitro and in vivo, and these effects were associated with the activation of autophagy and decreased apoptotic signaling of epithelial cells. In addition, the chronopharmacological effects of SR9009 were more potent at Zeitgeber time 0 (ZT0) than at ZT12, which was contrary to the rhythm of Rev-erbs in the gastrointestinal tract. The administration of SR9009 attenuated hepatic lipid accumulation, insulin resistance, inflammation, and fibrosis in mice with CL diet-induced NASH, which might be partly attributed to the enhancement of intestinal barrier function. CONCLUSION: Chronopharmacological activation of REV-ERBα might be a potential strategy to treat intestinal barrier dysfunction-related disorders and NASH.

The transcriptional repressor REV-ERB as a novel target for disease.

REV-ERB is a member of the nuclear receptor superfamily of transcription factors involved in the regulation of many physiological processes, from circadian rhythm, to immune function and metabolism. Accordingly, REV-ERB has been considered as a promising, but difficult drug target for the treatment of numerous diseases. Here, we concisely review current understanding of the function of REV-ERB, modulation by endogenous factors and synthetic ligands, and the involvement of REV-ERB in select human diseases. Particular focus is placed on the medicinal chemistry of synthetic REV-ERB ligands, which demonstrates the need for higher quality ligands to aid in robust validation of this exciting target.

An in silico mechanistic insight into HDAC8 activation facilitates the discovery of new small-molecule activators.

Research interest in the development of histone deacetylase 8 (HDAC8) activators has substantially increased since loss-of-function HDAC8 mutations were found in patients with Cornelia de Lange syndrome (CdLS). A series of N-acetylthioureas (e.g., TM-2-51) have been identified as HDAC8-selective activators, among others; however, their activation mechanisms remain elusive. Herein, we performed molecular dynamics (MD) simulations and fragment-centric topographical mapping (FCTM) to investigate the mechanism of HDAC8 activation. Our results revealed that improper binding of the coumarin group of fluorescent substrates leads to the "flipping out" of catalytic residue Y306, which reduces the enzymatic activity of HDAC8 towards fluorescent substrates. A pocket between the coumarin group of the substrate and thed catalytic residue Y306 was filled with the activator TM-2-51, which not only enhanced binding between HDAC8 and the fluorescent substrate complex but also stabilized Y306 in a catalytically active conformation. Based on this newly proposed substrate-dependent activation mechanism, we performed structure-based virtual screening and successfully identified low-molecular-weight scaffolds as new HDAC8 activators.

Pharmacological modulation and genetic deletion of REV-ERBα and REV-ERBß regulates dendritic cell development.

The nuclear receptors REV-ERBα and REV-ERBß have been demonstrated to play key roles in the regulation of numerous physiological functions, such as metabolism and the circadian rhythm. Recent studies have established the REV-ERBs' roles in immunity, including macrophage and T cell responses. In contrast, their roles in dendritic cells have not been well defined. Dendritic cells are potent antigen presenting cells, connecting microbial sensing and innate immunity to adaptive immune responses. We demonstrate that both REV-ERBα and REV-ERBß expression is upregulated during the course of bone marrow derived dendritic cell (BMDC) differentiation. BMDCs from REV-ERBα and REV-ERBß deficient mice showed enhanced expression of maturation markers like CD86, MHCII, and proinflammatory cytokines. Conversely, treatment of BMDCs with a REV-ERB-specific agonist, SR9009, inhibited the expression of maturation markers and proinflammatory cytokines. Our study suggests the REV-ERBs act as negative regulators of dendritic cell development and activation. These results indicate that pharmacological modulation of REV-ERB activity could be an attractive strategy to modulate DC activation status and for DC-based therapies.

SR9009 induces a REV-ERB dependent anti-small-cell lung cancer effect through inhibition of autophagy.

Rationale: The circadian clock coordinates cell proliferation and metabolism and impacts the progression of some diseases, particularly cancer. Pharmacological modulation of the circadian machinery may be an effective therapeutic approach for treating cancer. SR9009 is a specific synthetic agonist of the REV-ERBs, essential circadian clock components. However, the potential efficacy and antitumor mechanism of this drug in small-cell lung cancer (SCLC) remains poorly understood. Methods: Here, we used chemosensitive cells (H69 and H446) and the corresponding chemoresistant cells (H69AR and H446DDP) to assess the efficacy of the REV-ERB agonist SR9009 for the treatment of SCLC in vitro and further validated the antitumor effect in subcutaneous tumor models of SCLC. Then, we determined whether REV-ERBα was correlated with the anti-SCLC effect of SR9009. Chromatin immunoprecipitation (ChIP) sequencing assays were conducted to identify potential DNA sequences directly regulated by REV-ERBα. Autophagy regulation by REV-ERBα and its possible mechanism in SR9009-based SCLC therapy were analyzed. Results: Here, we showed that the REV-ERB agonist SR9009 is specifically lethal to both chemosensitive and chemoresistant SCLC cells. REV-ERBα was involved in the antitumor effect of SR9009 in SCLC. The core autophagy gene Atg5 was identified as a direct downstream target of REV-ERBα and was suppressed by the REV-ERB agonist SR9009 in SCLC. Furthermore, the interaction of REV-ERBα with this autophagy gene impaired autophagy activity, leading to SR9009 cytotoxicity in SCLC cells. Principal conclusions: Our study provided a novel viewpoint indicating that the REV-ERB agonist SR9009 could be a novel and promising therapeutic strategy in first- or second-line SCLC treatment. The anti-SCLC effect of SR9009 is mediated by REV-ERB dependent suppression of autophagy via direct repression of the autophagy gene Atg5.

Colla corii asini might upregulate ZNF471 and THOC5 by KRAB domain-containing zinc-finger protein pathway and THO complex subunit 5 pathway to improve anemia of pregnant women with ß-thalassemia.

Pregnant patients with ß-thalassemia are more likely to have progressive anemia which expose them to risk of adverse pregnancy outcomes, blood transfusion, and iron overload. Results from our previous study indicated that Colla corii asini (CCA, E'jiao), a natural ingredient of traditional Chinese medicine, could significantly increase hemoglobin level of pregnant women with ß- thalassemia, but the underlying molecular mechanism was unclear. Thus, we applied high-throughput transcriptome sequencing to study the transcriptomic change before and after the CCA treatment. Twenty eligible pregnant women were recruited and randomized to either the CCA treatment group or the blank control group in a 3:1 ratio. Patients in the treatment group orally received daily 15 g CCA powder for 4 weeks. We analyzed the therapeutic effect indexes and the transcriptomic change in subjects' peripheral blood before and after treatment. We found that ß CD 41-42(-TTCT)/ßA was the main genotype of the subjects. The regulatory impact of CCA treatment became more evident among the subjects of genotype ß CD 41-42(-TTCT)/ßA. Gene ontogenesis analysis revealed that the top five molecular functions of differentially expressed genes were involved in membrane functionality and cellular structure. We further identified two consistent upregulated genes ZNF471 and THOC5 in the effective treatment group, which were engaged in Kruppel-associated box (KRAB) domain-containing zinc-finger protein pathway and THOC5 pathway, respectively. Based on our current findings, we hypothesize that the anti-anemia effect of CCA on pregnant women with ß-thalassemia might be related to translation regulation of spectrin synthesis, membrane stability, and eventually prolonged the life span of erythrocytes.

Benzo(a)pyrene promotes migration, invasion and metastasis of lung adenocarcinoma cells by upregulating TGIF.

This study aimed to investigate the potential roles of TG-interacting factor (TGIF) in benzo(a)pyrene (BaP)-induced migration, invasion, and metastasis of lung adenocarcinoma cells. Cells were treated with different concentrations of BaP. MTT assays were used to measure cell proliferation. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblots were applied to measure the TGIF expression. A dual-luciferase reporter gene assay was performed to assess the effects of BaP on TGIF promoter-driven reporter gene expression. Wound-healing, transwell, and tail vein metastasis assays were performed to evaluate migratory, invasive, and metastatic capacity. Our results showed that BaP treatment increased the expression of TGIF mRNA and protein. Additionally, BaP treatment enhanced TGIF promoter-driven reporter gene expression. We observed that BaP treatment promoted the migration, invasion, and metastasis of H157 cells, which could be blocked by silencing TGIF. The expression of TGIF mRNA was significantly higher in metastatic lung adenocarcinoma samples than in non-metastatic lung adenocarcinoma samples, and higher levels of TGIF mRNA expression were observed in metastatic lung adenocarcinoma samples from patients with a smoking history than in those from patients with a non-smoking history. Our findings suggest that BaP treatment promotes the migration, invasion, and metastasis of human lung adenocarcinoma cells by upregulating TGIF.

A comparative study of non-native N-acyl l-homoserine lactone analogs in two Pseudomonas aeruginosa quorum sensing receptors that share a common native ligand yet inversely regulate virulence.

Certain bacteria can coordinate group behaviors via a chemical communication system known as quorum sensing (QS). Gram-negative bacteria typically use N-acyl l-homoserine lactone (AHL) signals and their cognate intracellular LuxR-type receptors for QS. The opportunistic pathogen Pseudomonas aeruginosa has a relatively complex QS circuit in which two of its LuxR-type receptors, LasR and QscR, are activated by the same natural signal, N-(3-oxo)-dodecanoyl l-homoserine lactone. Intriguingly, once active, LasR activates virulence pathways in P. aeruginosa, while activated QscR can inactivate LasR and thus repress virulence. We have a limited understanding of the structural features of AHLs that engender either agonistic activity in both receptors or receptor-selective activity. Compounds with the latter activity profile could prove especially useful tools to tease out the roles of these two receptors in virulence regulation. A small collection of AHL analogs was assembled and screened in cell-based reporter assays for activity in both LasR and QscR. We identified several structural motifs that bias ligand activation towards each of the two receptors. These findings will inform the development of new synthetic ligands for LasR and QscR with improved potencies and selectivities.

Mechanism of agonism and antagonism of the Pseudomonas aeruginosa quorum sensing regulator QscR with non-native ligands.

Pseudomonas aeruginosa is an opportunistic pathogen that uses the process of quorum sensing (QS) to coordinate the expression of many virulence genes. During quorum sensing, N-acyl-homoserine lactone (AHL) signaling molecules regulate the activity of three LuxR-type transcription factors, LasR, RhlR and QscR. To better understand P. aeruginosa QS signal reception, we examined the mechanism underlying the response of QscR to synthetic agonists and antagonists using biophysical and structural approaches. The structure of QscR bound to a synthetic agonist reveals a novel mode of ligand binding supporting a general mechanism for agonist activity. In turn, antagonists of QscR with partial agonist activity were found to destabilize and greatly impair QscR dimerization and DNA binding. These results highlight the diversity of LuxR-type receptor responses to small molecule agonists and antagonists and demonstrate the potential for chemical strategies for the selective targeting of individual QS systems.

REV-ERB agonism suppresses osteoclastogenesis and prevents ovariectomy-induced bone loss partially via FABP4 upregulation.

REV-ERBs (REV-ERBα and REV-ERBß) are transcription repressors and circadian regulators. Previous investigations have shown that REV-ERBs repress the expression of target genes, including MMP9 and CX3CR1, in macrophages. Because MMP9 and CX3CR1 reportedly participate in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, we inferred that REV-ERBs might play a role in osteoclastogenesis. In the present study, we found that the REV-ERBα level decreased significantly during RANKL-induced osteoclast differentiation from primary bone marrow-derived macrophages (BMMs). REV-ERBα knockdown by small interfering RNA in BMMs resulted in the enhanced formation of osteoclasts, whereas REV-ERBß knockdown showed no effect on osteoclast differentiation. Moreover, the REV-ERB agonist SR9009 inhibited osteoclast differentiation and bone resorption. Intraperitoneal SR9009 administration prevented ovariectomy-induced bone loss; this effect was accompanied by decreased serum RANKL and C-terminal telopeptide of type I collagen levels and increased osteoprotegerin levels. Further investigation revealed that NF-κB and MAPK activation and nuclear factor of activated T cells, cytoplasmic 1, and c-fos expression were suppressed by SR9009. The level of reactive oxygen species was also decreased by SR9009, with NADPH oxidase subunits also being down-regulated. In addition, an expression microarray showed that FABP4, an intracellular lipid-binding protein, was up-regulated by REV-ERB agonism. BMS309403, an inhibitor of FABP4, partially prevented the suppression of osteoclastogenesis by SR9009 through stabilizing phosphorylation of p65. To summarize, our results proved that the REV-ERB agonism inhibited osteoclastogenesis partially via FABP4 up-regulation.-Song, C., Tan, P., Zhang, Z., Wu, W., Dong, Y., Zhao, L., Liu, H., Guan, H., Li, F. REV-ERB agonism suppresses osteoclastogenesis and prevents ovariectomy-induced bone loss partially via FABP4 upregulation.

REV-ERBß is required to maintain normal wakefulness and the wake-inducing effect of dual REV-ERB agonist SR9009.

Circadian signaling regulates and synchronizes physiological and behavioral processes, such as feeding, metabolism, and sleep cycles. The endogenous molecular machinery that regulates circadian activities is located in the suprachiasmatic nucleus of the hypothalamus. The REV-ERBs are transcription factors that play key roles in the regulation of the circadian clock and metabolism. Using pharmacological methods, we recently demonstrated the involvement of the REV-ERBs in sleep architecture. Another group reported a delayed response to sleep deprivation and altered sleep cycles in REV-ERBα null mice, indicating a role of REV-ERBα in sleep. Given that REV-ERBß is structurally and functionally similar to REV-ERBα, we investigated the role of REV-ERBß in sleep and wakefulness by assessing electroencephalographic recordings in REV-ERBß deficient mice and the mechanism underlying effects of loss of REV-ERBß on sleep. Our data suggest that REV-ERBß is involved in the maintenance of wakefulness during the activity period. In addition, REV-ERBß-deficient mice administered with dual REV-ERB agonist SR9009, failed to show drug-induced wake increase. Finally, the expression of a number of genes known to mediate sleep and wakefulness were altered in REV-ERBß null mice.

Virtual screening and biological evaluation of biofilm inhibitors on dual targets in quorum sensing system.

AIM: Resistance to conventional antibiotics has spurred interest in exploring new antimicrobial strategies. Suppressing quorum sensing within biofilm is a promising antimicrobial strategy. LasR in quorum sensing system of the Gram-negative bacteria, Pseudomonas aeruginosa, directly enhances virulence and antibiotic resistance, with QscR as its indirect suppressor, so targeting both of them can synergistically take the effect. METHODOLOGY/RESULTS: An in silico protocol combining pharmacophores with molecular docking was applied. Pharmacophores of QscR agonists and LasR antagonists were prepared for preliminary screening, followed by counter-screen using a pharmacophore model of LasR agonists and molecular docking of LasR. Four compounds with novel scaffolds were confirmed as potential biofilm inhibitors with preliminary experimental data. CONCLUSION: Novel biofilm inhibitors can be found with the method.

Transgenerational inheritance of neurobehavioral and physiological deficits from developmental exposure to benzo[a]pyrene in zebrafish.

Benzo[a]pyrene (B[a]P) is a well-known genotoxic polycylic aromatic compound whose toxicity is dependent on signaling via the aryl hydrocarbon receptor (AHR). It is unclear to what extent detrimental effects of B[a]P exposures might impact future generations and whether transgenerational effects might be AHR-dependent. This study examined the effects of developmental B[a]P exposure on 3 generations of zebrafish. Zebrafish embryos were exposed from 6 to 120h post fertilization (hpf) to 5 and 10µM B[a]P and raised in chemical-free water until adulthood (F0). Two generations were raised from F0 fish to evaluate transgenerational inheritance. Morphological, physiological and neurobehavioral parameters were measured at two life stages. Juveniles of the F0 and F2 exhibited hyper locomotor activity, decreased heartbeat and mitochondrial function. B[a]P exposure during development resulted in decreased global DNA methylation levels and generally reduced expression of DNA methyltransferases in wild type zebrafish, with the latter effect largely reversed in an AHR2-null background. Adults from the F0 B[a]P exposed lineage displayed social anxiety-like behavior. Adults in the F2 transgeneration manifested gender-specific increased body mass index (BMI), increased oxygen consumption and hyper-avoidance behavior. Exposure to benzo[a]pyrene during development resulted in transgenerational inheritance of neurobehavioral and physiological deficiencies. Indirect evidence suggested the potential for an AHR2-dependent epigenetic route.

Transcriptional and translational adaptation to aerobic nitrate anabolism in the denitrifier Paracoccus denitrificans.

Transcriptional adaptation to nitrate-dependent anabolism by Paracoccus denitrificans PD1222 was studied. A total of 74 genes were induced in cells grown with nitrate as N-source compared with ammonium, including nasTSABGHC and ntrBC genes. The nasT and nasS genes were cotranscribed, although nasT was more strongly induced by nitrate than nasS The nasABGHC genes constituted a transcriptional unit, which is preceded by a non-coding region containing hairpin structures involved in transcription termination. The nasTS and nasABGHC transcripts were detected at similar levels with nitrate or glutamate as N-source, but nasABGHC transcript was undetectable in ammonium-grown cells. The nitrite reductase NasG subunit was detected by two-dimensional polyacrylamide gel electrophoresis in cytoplasmic fractions from nitrate-grown cells, but it was not observed when either ammonium or glutamate was used as the N-source. The nasT mutant lacked both nasABGHC transcript and nicotinamide adenine dinucleotide (NADH)-dependent nitrate reductase activity. On the contrary, the nasS mutant showed similar levels of the nasABGHC transcript to the wild-type strain and displayed NasG protein and NADH-nitrate reductase activity with all N-sources tested, except with ammonium. Ammonium repression of nasABGHC was dependent on the Ntr system. The ntrBC and ntrYX genes were expressed at low levels regardless of the nitrogen source supporting growth. Mutational analysis of the ntrBCYX genes indicated that while ntrBC genes are required for nitrate assimilation, ntrYX genes can only partially restore growth on nitrate in the absence of ntrBC genes. The existence of a regulation mechanism for nitrate assimilation in P. denitrificans, by which nitrate induction operates at both transcriptional and translational levels, is proposed.

DEPDC5 as a potential therapeutic target for epilepsy.

INTRODUCTION: Dishevelled, Egl-10 and Pleckstrin (DEP) domain-containing protein 5 (DEPDC5) is a protein subunit of the GTPase-activating proteins towards Rags 1 (GATOR1) complex. GATOR1 is a recently identified modulator of mechanistic target of rapamycin (mTOR) activity. mTOR is a key regulator of cell proliferation and metabolism; disruption of the mTOR pathway is implicated in focal epilepsy, both acquired and genetic. Tuberous sclerosis is the prototypic mTOR genetic syndrome with epilepsy, however GATOR1 gene mutations have recently been shown to cause lesional and non-lesional focal epilepsy. Areas covered: This review summarizes the mTOR pathway, including regulators and downstream effectors, emphasizing recent developments in the understanding of the complex role of the GATOR1 complex. We review the epilepsy types associated with mTOR overactivity, including tuberous sclerosis, polyhydramnios megalencephaly symptomatic epilepsy, cortical dysplasia, non-lesional focal epilepsy and post-traumatic epilepsy. Currently available mTOR inhibitors are discussed, primarily rapamycin analogs and ATP competitive mTOR inhibitors. Expert opinion: DEPDC5 is an attractive therapeutic target in focal epilepsy, as effects of DEPDC5 agonists would likely be anti-epileptogenic and more selective than currently available mTOR inhibitors. Therapeutic effects might be synergistic with certain existing dietary therapies, including the ketogenic diet.

Pharmacological and Genetic Modulation of REV-ERB Activity and Expression Affects Orexigenic Gene Expression.

The nuclear receptors REV-ERBα and REV-ERBß are transcription factors that play pivotal roles in the regulation of the circadian rhythm and various metabolic processes. The circadian rhythm is an endogenous mechanism, which generates entrainable biological changes that follow a 24-hour period. It regulates a number of physiological processes, including sleep/wakeful cycles and feeding behaviors. We recently demonstrated that REV-ERB-specific small molecules affect sleep and anxiety. The orexinergic system also plays a significant role in mammalian physiology and behavior, including the regulation of sleep and food intake. Importantly, orexin genes are expressed in a circadian manner. Given these overlaps in function and circadian expression, we wanted to determine whether the REV-ERBs might regulate orexin. We found that acute in vivo modulation of REV-ERB activity, with the REV-ERB-specific synthetic ligand SR9009, affects the circadian expression of orexinergic genes in mice. Long term dosing with SR9009 also suppresses orexinergic gene expression in mice. Finally, REV-ERBß-deficient mice present with increased orexinergic transcripts. These data suggest that the REV-ERBs may be involved in the repression of orexinergic gene expression.

Interferon alpha (IFNα)-induced TRIM22 interrupts HCV replication by ubiquitinating NS5A.

TRIM22, a tripartite-motif (TRIM) protein, is upregulated upon interferon alpha (IFNα) administration to hepatitis C virus (HCV)-infected patients. However, the physiological role of TRIM22 upregulation remains unclear. Here, we describe a potential antiviral function of TRIM22's targeting of the HCV NS5A protein. NS5A is important for HCV replication and for resistance to IFNα therapy. During the first 24 h following the initiation of IFNα treatment, upregulation of TRIM22 in the peripheral blood mononuclear cells (PBMCs) of HCV patients correlated with a decrease in viral titer. This phenomenon was confirmed in the hepatocyte-derived cell line Huh-7, which is highly permissive for HCV infection. TRIM22 over-expression inhibited HCV replication, and Small interfering RNA (siRNA)-mediated knockdown of TRIM22 diminished IFNα-induced anti-HCV function. Furthermore, we determined that TRIM22 ubiquitinates NS5A in a concentration-dependent manner. In summary, our results suggest that TRIM22 upregulation is associated with HCV decline during IFNα treatment and plays an important role in controlling HCV replication in vitro.

The Molecular Basis of TnrA Control by Glutamine Synthetase in Bacillus subtilis.

TnrA is a master regulator of nitrogen assimilation in Bacillus subtilis. This study focuses on the mechanism of how glutamine synthetase (GS) inhibits TnrA function in response to key metabolites ATP, AMP, glutamine, and glutamate. We suggest a model of two mutually exclusive GS conformations governing the interaction with TnrA. In the ATP-bound state (A-state), GS is catalytically active but unable to interact with TnrA. This conformation was stabilized by phosphorylated L-methionine sulfoximine (MSX), fixing the enzyme in the transition state. When occupied by glutamine (or its analogue MSX), GS resides in a conformation that has high affinity for TnrA (Q-state). The A- and Q-state are mutually exclusive, and in agreement, ATP and glutamine bind to GS in a competitive manner. At elevated concentrations of glutamine, ATP is no longer able to bind GS and to bring it into the A-state. AMP efficiently competes with ATP and prevents formation of the A-state, thereby favoring GS-TnrA interaction. Surface plasmon resonance analysis shows that TnrA bound to a positively regulated promoter fragment binds GS in the Q-state, whereas it rapidly dissociates from a negatively regulated promoter fragment. These data imply that GS controls TnrA activity at positively controlled promoters by shielding the transcription factor in the DNA-bound state. According to size exclusion and multiangle light scattering analysis, the dodecameric GS can bind three TnrA dimers. The highly interdependent ligand binding properties of GS reveal this enzyme as a sophisticated sensor of the nitrogen and energy state of the cell to control the activity of DNA-bound TnrA.

Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells.

The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4(+) T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4(+) T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4(+) T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the "Shock and Kill" strategy for latently HIV-1 infected cells.