Cell-based Luciferase Assay Research Articles

Transcriptional control of a stem cell factor nucleostemin in liver regeneration and aging.

Nucleostemin (NS) plays a role in liver regeneration, and aging reduces its expression in the baseline and regenerating livers following 70% partial hepatectomy (PHx). Here we interrogate the mechanism controlling NS expression during liver regeneration and aging. The NS promoter was analyzed by TRANSFAC. Functional studies were performed using cell-based luciferase assay, endogenous NS expression in Hep3B cells, mouse livers with a gain-of-function mutation of C/EBPα (S193D), and mouse livers with C/EBPα knockdown. We found a CAAT box with four C/EBPα binding sites (-1216 to -735) and a GC box with consensus binding sites for c-Myc, E2F1, and p300-associated protein complex (-633 to -1). Age-related changes in NS expression correlated positively with the expression of c-Myc, E2F1, and p300, and negatively with that of C/EBPα and C/EBPβ. PHx upregulated NS expression at 1d, coinciding with an increase in E2F1 and a decrease in C/EBPα. C/EBPα bound to the consensus sequences found in the NS promoter in vitro and in vivo, inhibited its transactivational activity in a binding site-dependent manner, and decreased the expression of endogenous NS in Hep3B cells. In vivo activation of C/EBPα by the S193D mutation resulted in a 4th-day post-PHx reduction of NS, a feature shared by 16-m/o livers. Finally, C/EBPα knockdown increased its expression in aged (24-m/o) livers under both baseline and regeneration conditions. This study reports the C/EBPα suppression of NS expression in aged livers, providing a new perspective on the mechanistic orchestration of tissue homeostasis in aging.

Design, Synthesis, and Structure-Activity Relationships of Biaryl Anilines as Subtype-Selective PPAR-alpha Agonists.

The role of peroxisome proliferator-activated receptor alpha (PPARα) in retinal biology is clarifying, and evidence demonstrates that novel PPARα agonists hold promising therapeutic utility for diseases like diabetic retinopathy and age-related macular degeneration. Herein, we disclose the design and initial structure-activity relationships for a new biaryl aniline PPARα agonistic chemotype. Notably, this series exhibits subtype selectivity for PPARα over other isoforms, a phenomenon postulated to be due to the unique benzoic acid headgroup. This biphenyl aniline series is sensitive to B-ring functionalization but allows isosteric replacement, and provides an opportunity for C-ring extension. From this series, 3g, 6j, and 6d were identified as leads with <90 nM potency in a cell-based luciferase assay cell and exhibited efficacy in various disease-relevant cell contexts, thereby setting the stage for further characterization in more advanced in vitro and in vivo models.

Assigning pathogenicity for TAB2 variants using a novel scalable functional assay and expanding TAB2 disease spectrum.

Haploinsufficiency of TGF-beta-activated kinase 1 (MAP3K7) binding protein 2 (TAB2) has been associated with congenital heart disease and more recently multiorgan structural abnormalities. Missense variant represents a major proportion of non-synonymous TAB2 variants reported in gnomAD (295/576) and Clinvar (16/73), most of which are variants of uncertain significance (VUSs). However, interpretation of TAB2 missense variants remains challenging because of lack of functional assays. To address this issue, we established a cell-based luciferase assay that enables high-throughput screening of TAB2 variants to assess the functional consequence for predicting variant pathogenicity. Using this platform, we screened 47 TAB2 variants including five pathogenic controls and one benign control, and the results showed that the transcriptional activity of activator protein 1 (AP-1) but not nuclear factor kappa B predicts the TAB2 variant pathogenicity. This assay provides accurate functional readout for both loss-of-function (LOF) and gain-of-function variants, which are associated with distinct phenotypes. In all, 22 out of 32 tested VUSs were reclassified. Genotype-Phenotype association showed that most patients with partial LOF variants do not exhibit congenital heart disease but high frequency of developmental delay, hypotonia and dysmorphic features, which suggests that genetic testing for TAB2 is needed for a broader spectrum of patients with more diverse phenotypes. Molecular modeling with Npl4 zinc finger (NZF) domain variants revealed that the stability of the NZF domain in TAB2 protein is crucial for AP-1 activation. In conclusion, we developed a highly effective functional assay for TAB2 variant prediction and interpretation.

A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors

Effective drugs against SARS-CoV-2 are urgently needed to treat severe cases of infection and for prophylactic use. The main viral protease (nsp5 or 3CLpro) represents an attractive and possibly broad-spectrum target for drug development as it is essential to the virus life cycle and highly conserved among betacoronaviruses. Sensitive and efficient high-throughput screening methods are key for drug discovery. Here we report the development of a gain-of-signal, highly sensitive cell-based luciferase assay to monitor SARS-CoV-2 nsp5 activity and show that it is suitable for the screening of compounds in a 384-well format. A benefit of miniaturisation and automation is that screening can be performed in parallel on a wild-type and a catalytically inactive nsp5, which improves the selectivity of the assay. We performed molecular docking-based screening on a set of 14,468 compounds from an in-house chemical database, selected 359 candidate nsp5 inhibitors and tested them experimentally. We identified two molecules which show anti-nsp5 activity, both in our cell-based assay and in vitro on purified nsp5 protein, and inhibit SARS-CoV-2 replication in A549-ACE2 cells with EC50 values in the 4–8 μM range. The here described high-throughput-compatible assay will allow the screening of large-scale compound libraries for SARS-CoV-2 nsp5 inhibitors. Moreover, we provide evidence that this assay can be adapted to other coronaviruses and viruses which rely on a viral protease.

Evaluation of Naturally Occurring HIF-1 Inhibitors for Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a rare and severe progressive disorder characterized by high pulmonary artery pressure. Chronic hypoxia causes a metabolic disorder and the Warburg effect in pulmonary arterial smooth muscle cells (PASMCs). Pyruvate dehydrogenase kinase 1 (PDK1) is a key enzyme in Warburg effect increased by hypoxia-inducible factor (HIF-1). We constructed a cell-based luciferase assay system for HIF-1 inhibitors. Using this system, six HIF-1 inhibitors were identified. Among these inhibitors, the effect of tagitinin C (1) on PASMC was investigated. Tagitinin C (1) clearly decreased the amount of HIF-1β and the HIF-1 target PDK1. This result indicates that HIF-1 inhibitors effectively decrease PDK1 activity, which is a cause of the metabolic disorder and Warburg effect observed in PASMCs. Identifying naturally occurring HIF-1 inhibitors could provide novel insights into the development of PAH medications.

Structure of AMH bound to AMHR2 provides insight into a unique signaling pair in the TGF-β family

Anti-Müllerian hormone (AMH), or Müllerian-inhibiting substance, is a protein hormone that promotes Müllerian duct regression during male fetal sexual differentiation and regulation of folliculogenesis in women. AMH is a member of the transforming growth factor beta (TGF-β) family, which has evolved to signal through its own dedicated type II receptor, AMH receptor type II (AMHR2). Structures of other TGF-β family members have revealed how ligands infer specificity for their cognate receptors; however, it is unknown how AMH binds AMHR2 at the molecular level. Therefore, in this study, we solved the X-ray crystal structure of AMH bound to the extracellular domain of AMHR2 to a resolution of 2.6Å. The structure reveals that while AMH binds AMHR2 in a similar location to Activin and BMP ligand binding to their type II receptors, differences in both AMH and AMHR2 account for a highly specific interaction. Furthermore, using an AMH responsive cell-based luciferase assay, we show that a conformation in finger 1 of AMHR2 and a salt bridge formed by K534 on AMH and D81/E84 of AMHR2 are key to the AMH/AMHR2 interaction. Overall, our study highlights how AMH engages AMHR2 using a modified paradigm of receptor binding facilitated by modifications to the three-finger toxin fold of AMHR2. Furthermore, understanding these elements contributing to the specificity of binding will help in the design of agonists or antagonists or the selection of antibody therapies.

The aryl hydrocarbon receptor activates ceramide biosynthesis in mice contributing to hepatic lipogenesis

Aryl hydrocarbon receptor (AHR) activation via 2,3,7,8-tetrachlorodibenzofuran (TCDF) induces the accumulation of hepatic lipids. Here we report that AHR activation by TCDF (24 μg/kg body weight given orally for five days) induced significant elevation of hepatic lipids including ceramides in mice, was associated with increased expression of key ceramide biosynthetic genes, and increased activity of their respective enzymes. Results from chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA) and cell-based reporter luciferase assays indicated that AHR directly activated the serine palmitoyltransferase long chain base subunit 2 (Sptlc2, encodes serine palmitoyltransferase 2 (SPT2)) gene whose product catalyzes the initial rate-limiting step in de novo sphingolipid biosynthesis. Hepatic ceramide accumulation was further confirmed by mass spectrometry-based lipidomics. Taken together, our results revealed that AHR activation results in the up-regulation of Sptlc2, leading to ceramide accumulation, thus promoting lipogenesis, which can induce hepatic lipid accumulation.

Linderapyrone: A Wnt signal inhibitor isolated from Lindera umbellata

Linderapyrone, a Wnt signal inhibitor was isolated from the methanolic extract of the stems and twigs of Lindera umbellata together with epi-(-)-linderol A. Linderapyrone inhibited TCF/β-catenin transcriptional activity that was evaluated using cell-based TOPFlash luciferase assay system. To evaluate the structure-activity relationship and mechanism, we synthesized linderapyrone and its derivatives from piperitone. As the results of further bioassay for synthesized compounds, we found both of pyrone and monoterpene moieties were necessary for inhibitory effect. cDNA microarray analysis in a linderapyrone derivative treated human colorectal cancer cells showed that this compound downregulates Wnt signaling pathway. Moreover, we successes to synthesize the derivative of linderapyrone that has stronger inhibitory effect than linderapyrone and ICG-001 (positive control).

Aryl Hydrocarbon Receptor Plasma Agonist Activity Correlates With Disease Activity in Progressive MS.

ObjectiveThe relationship between serum aryl hydrocarbon receptor (AHR) agonistic activity levels with disease severity, its modulation over the course of relapsing-remitting MS (RRMS), and its regulation in progressive MS (PMS) are unknown. Here, we report the analysis of AHR agonistic activity levels in cross-sectional and longitudinal serum samples of patients with RRMS and PMS.MethodsIn a cross-sectional investigation, a total of 36 control patients diagnosed with noninflammatory diseases, 84 patients with RRMS, 35 patients with secondary progressive MS (SPMS), and 41 patients with primary progressive MS (PPMS) were included in this study. AHR activity was measured in a cell-based luciferase assay and correlated with age, sex, the presence of disease-modifying therapies, Expanded Disability Status Scale scores, and disease duration. In a second longitudinal investigation, we analyzed AHR activity in 13 patients diagnosed with RRMS over a period from 4 to 10 years and correlated AHR agonistic activity with white matter atrophy and lesion load volume changes.ResultsIn RRMS, AHR ligand levels were globally decreased and associated with disease duration and neurologic disability. In SPMS and PPMS, serum AHR agonistic activity was decreased and correlated with disease severity. Finally, in longitudinal serum samples of patients with RRMS, decreased AHR agonistic activity was linked to progressive CNS atrophy and increased lesion load.ConclusionsThese findings suggest that serum AHR agonist levels negatively correlate with disability in RRMS and PMS and decrease longitudinally in correlation with MRI markers of disease progression. Thus, serum AHR agonistic activity may serve as novel biomarker for disability progression in MS.

MicroRNA-184 negatively regulates corneal epithelial wound healing via targeting CDC25A, CARM1, and LASP1

BackgroundMicroRNAs (miRNAs) play critical roles in corneal development and functional homeostasis. Our previous study identified miR-184 as one of the most highly expressed miRNAs in the corneal epithelium. Even though its expression level plummeted dramatically during corneal epithelial wound healing (CEWH), its precise role in mediating corneal epithelial renewal was unresolved. The present study aimed to reveal the function and mechanism of miR-184 in regulating CEWH.MethodsQuantitative RT-PCR analysis characterized the miR-184 expression pattern during CEWH in mice. Ectopic miR-184 injection determined its effect on this process in vivo. We evaluated the effects of miR-184 and its target genes on the proliferation, cell cycle, and migration of human corneal epithelial cells (HCECs) using MTS, flow cytometry, and wound-healing assay, respectively. Bioinformatic analysis, in conjunction with gene microarray analysis and cell-based luciferase assays, pinpointed gene targets of miR-184 contributing to CEWH.ResultsMiR-184 underwent marked downregulation during mouse CEWH. Ectopic miR-184 overexpression delayed this process in mice. Furthermore, miR-184 transfection into HCECs significantly inhibited cell proliferation, cell cycle progression, and cell migration. MiR-184 directly targeted CDC25A, CARM1, and LASP1, and downregulated their expression in HCECs. CARM1 downregulation inhibited both HCEC proliferation and migration, whereas a decrease in LASP1 gene expression only inhibited migration.ConclusionsOur results demonstrate that miR-184 inhibits corneal epithelial cell proliferation and migration via targeting CDC25A, CARM1, and LASP1, suggesting it acts as a negative modulator during CEWH. Therefore, identifying strategies to suppress miR-184 expression levels has the potential to promote CEWH.

Mutational Analysis of the Putative Anti-Müllerian Hormone (AMH) Binding Interface on its Type II Receptor, AMHR2.

Anti-Müllerian hormone (AMH) or Müllerian inhibiting substance is a unique member of the TGF-β family responsible for development and differentiation of the reproductive system. AMH signals through its own dedicated type II receptor, anti-Müllerian hormone receptor type II (AMHR2), providing an exclusive ligand-receptor pair within the broader TGF-β family. In this study, we used previous structural information to derive a model of AMH bound to AMHR2 to guide mutagenesis studies to identify receptor residues important for AMH signaling. Nonconserved mutations were introduced in AMHR2 and characterized in an AMH-responsive cell-based luciferase assay and native PAGE. Collectively, our results identified several residues important for AMH signaling within the putative ligand binding interface of AMHR2. Our results show that AMH engages AMHR2 at a similar interface to how activin and BMP class ligands bind the type II receptor, ACVR2B; however, there are significant molecular differences at the ligand interface of these 2 receptors, where ACVR2B is mostly hydrophobic and AMHR2 is predominately charged. Overall, this study shows that although the location of ligand binding on the receptor is similar to ACVR2A, ACVR2B, and BMPR2; AMHR2 uses unique ligand-receptor interactions to impart specificity for AMH.

Mechanistic Insights of Phenobarbital-Mediated Activation of Human but Not Mouse Pregnane X Receptor

Phenobarbital (PB), a broadly used antiseizure drug, was the first to be characterized as an inducer of cytochrome P450 by activation of the constitutive androstane receptor (CAR). Although PB is recognized as a conserved CAR activator among species via a well-documented indirect activation mechanism, conflicting results have been reported regarding PB regulation of the pregnane X receptor (PXR), a sister receptor of CAR, and the underlying mechanisms remain elusive. Here, we show that in a human CAR (hCAR)-knockout (KO) HepaRG cell line, PB significantly induces the expression of CYP2B6 and CYP3A4, two shared target genes of hCAR and human PXR (hPXR). In human primary hepatocytes and hCAR-KO HepaRG cells, PB-induced expression of CYP3A4 was markedly repressed by genetic knockdown or pharmacological inhibition of hPXR. Mechanistically, PB concentration dependently activates hPXR but not its mouse counterpart in cell-based luciferase assays. Mammalian two-hybrid assays demonstrated that PB selectively increases the functional interaction between the steroid receptor coactivator-1 and hPXR but not mouse PXR. Moreover, surface plasmon resonance binding affinity assay showed that PB directly binds to the ligand binding domain of hPXR (KD = 1.42 × 10-05). Structure-activity analysis further revealed that the amino acid tryptophan-299 within the ligand binding pocket of hPXR plays a key role in the agonistic binding of PB and mutation of tryptophan-299 disrupts PB activation of hPXR. Collectively, these data reveal that PB, a selective mouse CAR activator, activates both hCAR and hPXR, and provide novel mechanistic insights for PB-mediated activation of hPXR.

A normothermic ex vivo organ perfusion delivery method for cardiac transplantation gene therapy

Clinically, both percutaneous and surgical approaches to deliver viral vectors to the heart either have resulted in therapeutically inadequate levels of transgene expression or have raised safety concerns associated with extra-cardiac delivery. Recent developments in the field of normothermic ex vivo cardiac perfusion storage have now created opportunities to overcome these limitations and safety concerns of cardiac gene therapy. This study examined the feasibility of ex vivo perfusion as an approach to deliver a viral vector to a donor heart during storage and the resulting bio distribution and expression levels of the transgene in the recipient post-transplant. The influence of components (proprietary solution, donor blood, and ex vivo circuitry tubing and oxygenators) of the Organ Care System (OC) (TransMedics, Inc., Andover MA) on viral vector transduction was examined using a cell-based luciferase assay. Our ex vivo perfusion strategy, optimized for efficient Adenoviral vector transduction, was utilized to deliver 5 × 1013 total viral particles of an Adenoviral firefly luciferase vector with a cytomegalovirus (CMV) promotor to porcine donor hearts prior to heterotopic implantation. We have evaluated the overall levels of expression, protein activity, as well as the bio distribution of the firefly luciferase protein in a series of three heart transplants at a five-day post-transplant endpoint. The perfusion solution and the ex vivo circuitry did not influence viral vector transduction, but the serum or plasma fractions of the donor blood significantly inhibited viral vector transduction. Thus, subsequent gene delivery experiments to the explanted porcine heart utilized an autologous blood recovery approach to remove undesired plasma or serum components of the donor blood prior to its placement into the circuit. Enzymatic assessment of luciferase activity in tissues (native heart, allograft, liver etc.) obtained post-transplant day five revealed wide-spread and robust luciferase activity in all regions of the allograft (right and left atria, right and left ventricles, coronary arteries) compared to the native recipient heart. Importantly, luciferase activity in recipient heart, liver, lung, spleen, or psoas muscle was within background levels. Similar to luciferase activity, the luciferase protein expression in the allograft appeared uniform and robust across all areas of the myocardium as well as in the coronary arteries. Importantly, despite high copy number of vector genomic DNA in transplanted heart tissue, there was no evidence of vector DNA in either the recipient’s native heart or liver. Overall we demonstrate a simple protocol to achieve substantial, global gene delivery and expression isolated to the cardiac allograft. This introduces a novel method of viral vector delivery that opens the opportunity for biological modification of the allograft prior to implantation that may improve post-transplant outcomes.

A SMN2 Splicing Modifier Rescues the Disease Phenotypes in an In Vitro Human Spinal Muscular Atrophy Model.

Spinal muscular atrophy (SMA) is caused by the mutation or deletion of the survival motor neuron 1 (SMN1) gene. Only ∼10% of the products of SMN2, a paralogue of SMN1, are functional full-length SMN (SMN-FL) proteins, whereas SMN2 primarily produces alternatively spliced transcripts lacking exon 7. Reduced SMN protein levels in SMA patients lead to progressive degeneration of spinal motor neurons (MNs). In this study, we report an advanced platform based on an SMN2 splicing-targeting approach for SMA drug screening and validation using an SMN2 splicing reporter cell line and an in vitro human SMA model through induced pluripotent stem cell (iPSC) technology. Through drug screening using a robust cell-based luciferase assay to quantitatively measure SMN2 splicing, the small-molecule candidate compound rigosertib was identified as an SMN2 splicing modulator that led to enhanced SMN protein expression. The therapeutic potential of the candidate compound was validated in MN progenitors differentiated from SMA patient-derived iPSCs (SMA iPSC-pMNs) as an in vitro human SMA model, which recapitulated the biochemical and molecular phenotypes of SMA, including lower levels of SMN-FL transcripts and protein, enhanced cell death, and reduced neurite length. The candidate compound exerted strong splicing correction activity for SMN2 and potently alleviated the disease-related phenotypes of SMA iPSC-pMNs by modulating various cellular and molecular abnormalities. Our combined screening platform representing a pMN model of human SMA provides an efficient and reliable drug screening system and is a promising resource for drug evaluation and the exploration of drug modes of action.

Hyperforin-Induced Activation of the Pregnane X Receptor Is Influenced by the Organic Anion-Transporting Polypeptide 2B1.

The herbal remedy St. John's wort (SJW) is used in the treatment of mild depressive symptoms and is known for its drug-drug interaction potential when enhanced expression of CYP3A4 modifies clearance of concomitantly applied substrate drugs. Hyperforin is one constituent of SJW that alters CYP3A4 expression by activation of the nuclear receptor pregnane X receptor (PXR). However, little is known about the transmembrane transport of hyperforin. One membrane protein that modulates cellular entry of drugs is the organic anion-transporting polypeptide (OATP) 2B1. It was the aim of this study to test whether hyperforin interacts with this transport protein. Transport inhibition studies and competitive counterflow experiments suggested that hyperforin is a substrate of OATP2B1. This notion was validated by showing that the presence of OATP2B1 enhanced the hyperforin-induced PXR activation in cell-based luciferase assays. Moreover, in Caco-2 cells transcellular transport of the known OATP2B1 substrate atorvastatin was changed in the presence of hyperforin, resulting in an increased efflux ratio. Eleven commercially available SJW formulations were assessed for their influence on OATP2B1-mediated transport of estrone 3-sulfate and for their impact on CYP3A4 promoter transactivation. The correlation between effect size and the hyperforin content as determined by high-performance liquid chromatography with ultraviolet detection suggested that hyperforin is the major determinant. Our results indicate an interaction between hyperforin and OATP2B1, which is not only known to contribute to hepatocellular uptake but also to intestinal absorption of its substrates. These findings extend the complexity of mechanisms that should be considered when evaluating the interaction potential of SJW preparations.

Identification of Kinases and Phosphatases That Regulate ATG4B Activity by siRNA and Small Molecule Screening in Cells.

Autophagy protease ATG4B is a key regulator of the LC3/GABARAP conjugation system required for autophagosome formation, maturation and closure. Members of the ATG4 and the LC3/GABARAP family have been implicated in various diseases including cancer, and targeting the ATG4B protease has been suggested as a potential therapeutic anti-cancer strategy. Recently, it has been demonstrated that ATG4B is regulated by multiple post-translational modifications, including phosphorylation and de-phosphorylation. In order to identify regulators of ATG4B activity, we optimized a cell-based luciferase assay based on ATG4B-dependent release of Gaussia luciferase. We applied this assay in a proof-of-concept small molecule compound screen and identified activating compounds that increase cellular ATG4B activity. Next, we performed a high-throughput screen to identify kinases and phosphatases that regulate cellular ATG4B activity using siRNA mediated knockdown and cDNA overexpression. Of these, we provide preliminary evidence that the kinase AKT2 enhances ATG4B activity in cells. We provide all raw and processed data from the screens as a resource for further analysis. Overall, our findings provide novel insights into the regulation of ATG4B and highlight the importance of post-translational modifications of ATG4B.

Ventromorphins: A New Class of Small Molecule Activators of the Canonical BMP Signaling Pathway.

Here, we describe three new small-molecule activators of BMP signaling found by high throughput screening of a library of ∼600 000 small molecules. Using a cell-based luciferase assay in the BMP4-responsive human cervical carcinoma clonal cell line, C33A-2D2, we identified three compounds with similar chemotypes that each ventralize zebrafish embryos and stimulate increased expression of the BMP target genes, bmp2b and szl. Because these compounds ventralize zebrafish embryos, we have termed them "ventromorphins." As expected for a BMP pathway activator, they induce the differentiation of C2C12 myoblasts to osteoblasts. Affymetrix RNA analysis confirmed the differentiation results and showed that ventromorphins treatment elicits a genetic response similar to BMP4 treatment. Unlike isoliquiritigenin (SJ000286237), a flavone that maximally activates the pathway after 24 h of treatment, all three ventromorphins induced SMAD1/5/8 phosphorylation within 30 min of treatment and achieved peak activity within 1 h, indicating that their responses are consistent with directly activating BMP signaling.

An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling

The transforming growth factor β isoforms, TGF-β1, -β2, and -β3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF-β pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-βs in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-β monomer, lacking the heel helix, a structural motif essential for binding the TGF-β type I receptor (TβRI) but dispensable for binding the other receptor required for TGF-β signaling, the TGF-β type II receptor (TβRII), as an alternative therapeutic modality for blocking TGF-β signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-β monomers and bound TβRII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-β signaling with a Ki of 20-70 nm Investigation of the mechanism showed that the high affinity of the engineered monomer for TβRII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit TβRI, enabled it to bind endogenous TβRII but prevented it from binding and recruiting TβRI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-β signaling and may inform similar modifications of other TGF-β family members.

Trilobolide-steroid hybrids: Synthesis, cytotoxic and antimycobacterial activity

Sesquiterpene lactone trilobolide is a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, thus depleting the Ins(1,4,5)P3-sensitive intracellular calcium stores. Here, we describe a synthesis of a series of 6 trilobolide-steroids conjugates (estradiol, pregnene, dehydroepiandrosterone, and testosterone). We found that the newly synthesized Tb-based compounds possess different remarkable biological activities. Cancer cell cytotoxicity and preferential selectivity is represented in our study by a Tb-pregnene derivative. The most cytotoxic clickates of estradiol and pregnene were studied by FACS where impact on cell cycle and RNA synthesis was observed; live-cell microscopy revealed the impact on cell organelle morphology particularly endoplasmic reticulum, mitochondria and nucleus. Further, we have studied the estrogenic and androgenic properties of the clickate molecules using cell-based luciferase assays. Finally, antimycobacterial tests revealed that testosterone and estradiol derivatives potentiated the antimycobacterial activity up to IC50 of 10.6μM.

The effects of cytosine methylation on general transcription factors.

DNA methylation on CpG sites is the most common epigenetic modification. Recently, methylation in a non-CpG context was found to occur widely on genomic DNA. Moreover, methylation of non-CpG sites is a highly controlled process, and its level may vary during cellular development. To study non-CpG methylation effects on DNA/protein interactions, we have chosen three human transcription factors (TFs): glucocorticoid receptor (GR), brain and muscle ARNT-like 1 (BMAL1) - circadian locomotor output cycles kaput (CLOCK) and estrogen receptor (ER) with methylated or unmethylated DNA binding sequences, using single-molecule and isothermal titration calorimetry assays. The results demonstrated that these TFs interact with methylated DNA with different effects compared with their cognate DNA sequences. The effects of non-CpG methylation on transcriptional regulation were validated by cell-based luciferase assay at protein level. The mechanisms of non-CpG methylation influencing DNA-protein interactions were investigated by crystallographic analyses and molecular dynamics simulation. With BisChIP-seq assays in HEK-293T cells, we found that GR can recognize highly methylated sites within chromatin in cells. Therefore, we conclude that non-CpG methylation of DNA can provide a mechanism for regulating gene expression through directly affecting the binding of TFs.