scholarly journals Use of Differential Scanning Fluorimetry as a High-Throughput Assay to Identify Nuclear Receptor Ligands

2012 ◽  
Vol 10 (1) ◽  
pp. nrs.10002 ◽  
Author(s):  
Kara DeSantis ◽  
Aaron Reed ◽  
Raneen Rahhal ◽  
Jeff Reinking
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
High Throughput ◽  
Initial Step ◽  
Estrogen Receptor Α ◽  
Chemical Library ◽  
Differential Scanning Fluorimetry ◽  
Nuclear Receptor Ligands

Identification of ligands that interact with nuclear receptors is both a major biological problem and an important initial step in drug discovery. Several in vitro and in vivo techniques are commonly used to screen ligand candidates against nuclear receptors; however, none of the current assays allow screening without modification of either the protein and/or the ligand in a high-throughput fashion. Differential scanning fluorimetry (DSF) allows unmodified potential ligands to be screened as 10μL reactions in 96-well format against partially purified protein, revealing specific interactors. As a proof of principle, we used a commercially-available nuclear receptor ligand candidate chemical library to identify interactors of the human estrogen receptor α ligand binding domain (ERα LBD). Compounds that interact specifically with ERα LBD stabilize the protein and result in an elevation of the thermal denaturation point, as monitored by the environmentally-sensitive dye SYPRO orange. We successfully identified all three compounds in the library that have previously been identified to interact with ERα, with no false positive results.

scholarly journals The DRIP Complex and SRC-1/p160 Coactivators Share Similar Nuclear Receptor Binding Determinants but Constitute Functionally Distinct Complexes

2000 ◽  
Vol 20 (8) ◽  
pp. 2718-2726 ◽  
Author(s):  
Christophe Rachez ◽  
Matthew Gamble ◽  
Chao-Pei Betty Chang ◽  
G. Brandon Atkins ◽  
Mitchell A. Lazar ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Receptor Binding ◽  
Transcriptional Activation ◽  
Thyroid Hormone Receptor ◽  
Nuclear Receptor Coactivators ◽  
Nuclear Extracts ◽  
Sequence Requirements

ABSTRACT Transcriptional activation requires both access to DNA assembled as chromatin and functional contact with components of the basal transcription machinery. Using the hormone-bound vitamin D3receptor (VDR) ligand binding domain (LBD) as an affinity matrix, we previously identified a novel multisubunit coactivator complex, DRIP (VDR-interacting proteins), required for transcriptional activation by nuclear receptors and several other transcription factors. In this report, we characterize the nuclear receptor binding features of DRIP205, a key subunit of the DRIP complex, that interacts directly with VDR and thyroid hormone receptor in response to ligand and anchors the other DRIP subunits to the nuclear receptor LBD. In common with other nuclear receptor coactivators, DRIP205 interaction occurs through one of two LXXLL motifs and requires the receptor's AF-2 subdomain. Although the second motif of DRIP205 is required only for VDR binding in vitro, both motifs are used in the context of an retinoid X receptor-VDR heterodimer on DNA and in transactivation in vivo. We demonstrate that both endogenous p160 coactivators and DRIP complexes bind to the VDR LBD from nuclear extracts through similar sequence requirements, but they do so as distinct complexes. Moreover, in contrast to the p160 family of coactivators, the DRIP complex is devoid of any histone acetyltransferase activity. The results demonstrate that different coactivator complexes with distinct functions bind to the same transactivation region of nuclear receptors, suggesting that they are both required for transcription activation by nuclear receptors.

scholarly journals The Connection of Azole Fungicides with Xeno-Sensing Nuclear Receptors, Drug Metabolism and Hepatotoxicity

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1192 ◽  
Author(s):  
Philip Marx-Stoelting ◽  
Constanze Knebel ◽  
Albert Braeuning
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Liver Tumors ◽  
Current Knowledge ◽  
Constitutive Androstane Receptor ◽  
Pregnane X Receptor ◽  
Receptor Activation ◽  
Azole Fungicides

Azole fungicides, especially triazole compounds, are widely used in agriculture and as pharmaceuticals. For a considerable number of agricultural azole fungicides, the liver has been identified as the main target organ of toxicity. A number of previous studies points towards an important role of nuclear receptors such as the constitutive androstane receptor (CAR), the pregnane-X-receptor (PXR), or the aryl hydrocarbon receptor (AHR), within the molecular pathways leading to hepatotoxicity of these compounds. Nuclear receptor-mediated hepatic effects may comprise rather adaptive changes such as the induction of drug-metabolizing enzymes, to hepatocellular hypertrophy, histopathologically detectable fatty acid changes, proliferation of hepatocytes, and the promotion of liver tumors. Here, we present a comprehensive review of the current knowledge of the interaction of major agricultural azole-class fungicides with the three nuclear receptors CAR, PXR, and AHR in vivo and in vitro. Nuclear receptor activation profiles of the azoles are presented and related to histopathological findings from classic toxicity studies. Important issues such as species differences and multi-receptor agonism and the consequences for data interpretation and risk assessment are discussed.

scholarly journals Modulation of Transcriptional Activation and Coactivator Interaction by a Splicing Variation in the F Domain of Nuclear Receptor Hepatocyte Nuclear Factor 4α1

1999 ◽  
Vol 19 (10) ◽  
pp. 6509-6522 ◽  
Author(s):  
Frances M. Sladek ◽  
Michael D. Ruse ◽  
Luviminda Nepomuceno ◽  
Shih-Ming Huang ◽  
Michael R. Stallcup
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Transcriptional Activation ◽  
Regulatory Region ◽  
Physical Contact ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Splicing Variants

ABSTRACT Transcription factors, such as nuclear receptors, often exist in various forms that are generated by highly conserved splicing events. Whereas the functional significance of these splicing variants is often not known, it is known that nuclear receptors activate transcription through interaction with coactivators. The parameters, other than ligands, that might modulate those interactions, however, are not well characterized, nor is the role of splicing variants. In this study, transient transfection, yeast two-hybrid, and GST pulldown assays are used to show not only that nuclear receptor hepatocyte nuclear factor 4 α1 (HNF4α1, NR2A1) interacts with GRIP1, and other coactivators, in the absence of ligand but also that the uncommonly large F domain in the C terminus of the receptor inhibits that interaction. In vitro, the F domain was found to obscure an AF-2-independent binding site for GRIP1 that did not map to nuclear receptor boxes II or III. The results also show that a natural splicing variant containing a 10-amino-acid insert in the middle of the F domain (HNF4α2) abrogates that inhibition in vivo and in vitro. A series of protease digestion assays indicates that there may be structural differences between HNF4α1 and HNF4α2 in the F domain as well as in the ligand binding domain (LBD). The data also suggest that there is a direct physical contact between the F domain and the LBD of HNF4α1 and -α2 and that that contact is different in the HNF4α1 and HNF4α2 isoforms. Finally, we propose a model in which the F domain of HNF4α1 acts as a negative regulatory region for transactivation and in which the α2 insert ameliorates the negative effect of the F domain. A conserved repressor sequence in the F domains of HNF4α1 and -α2 suggests that this model may be relevant to other nuclear receptors as well.

scholarly journals High-throughput screening and rational design of biofunctionalized surfaces with optimized biocompatibility and antimicrobial activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhou Fang ◽  
Junjian Chen ◽  
Ye Zhu ◽  
Guansong Hu ◽  
Haoqian Xin ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Rational Design ◽  
Click Reaction ◽  
Reaction Times ◽  
Great Promise ◽  
Biomaterial Surfaces

AbstractPeptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

scholarly journals O23: CHARACTERISING PATIENT-DERIVED COLORECTAL CANCER TISSUE-ORIGINATED ORGANOIDAL SPHEROIDS FOR HIGH-THROUGHPUT MICROFLUIDIC APPLICATIONS

2021 ◽  
Vol 108 (Supplement_1) ◽  
Author(s):  
MI Khot ◽  
M Levenstein ◽  
R Coppo ◽  
J Kondo ◽  
M Inoue ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Fluid Flow ◽  
High Throughput ◽  
Microfluidic Devices ◽  
In Vitro Models ◽  
Fluorescent Imaging ◽  
Cancer Tissue ◽  
Cell Models

Abstract Introduction Three-dimensional (3D) cell models have gained reputation as better representations of in vivo cancers as compared to monolayered cultures. Recently, patient tumour tissue-derived organoids have advanced the scope of complex in vitro models, by allowing patient-specific tumour cultures to be generated for developing new medicines and patient-tailored treatments. Integrating 3D cell and organoid culturing into microfluidics, can streamline traditional protocols and allow complex and precise high-throughput experiments to be performed with ease. Method Patient-derived colorectal cancer tissue-originated organoidal spheroids (CTOS) cultures were acquired from Kyoto University, Japan. CTOS were cultured in Matrigel and stem-cell media. CTOS were treated with 5-fluorouracil and cytotoxicity evaluated via fluorescent imaging and ATP assay. CTOS were embedded, sectioned and subjected to H&E staining and immunofluorescence for ABCG2 and Ki67 proteins. HT29 colorectal cancer spheroids were produced on microfluidic devices using cell suspensions and subjected to 5-fluorouracil treatment via fluid flow. Cytotoxicity was evaluated through fluorescent imaging and LDH assay. Result 5-fluorouracil dose-dependent reduction in cell viability was observed in CTOS cultures (p<0.01). Colorectal CTOS cultures retained the histology, tissue architecture and protein expression of the colonic epithelial structure. Uniform 3D HT29 spheroids were generated in the microfluidic devices. 5-fluorouracil treatment of spheroids and cytotoxic analysis was achieved conveniently through fluid flow. Conclusion Patient-derived CTOS are better complex models of in vivo cancers than 3D cell models and can improve the clinical translation of novel treatments. Microfluidics can streamline high-throughput screening and reduce the practical difficulties of conventional organoid and 3D cell culturing. Take-home message Organoids are the most advanced in vitro models of clinical cancers. Microfluidics can streamline and improve traditional laboratory experiments.

scholarly journals Activating Signal Cointegrator 2 Belongs to a Novel Steady-State Complex That Contains a Subset of Trithorax Group Proteins

2003 ◽  
Vol 23 (1) ◽  
pp. 140-149 ◽  
Author(s):  
Young-Hwa Goo ◽  
Young Chang Sohn ◽  
Dae-Hwan Kim ◽  
Seung-Whan Kim ◽  
Min-Jung Kang ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Steady State ◽  
Nuclear Receptors ◽  
Transcriptional Activation ◽  
Retinoic Acid Receptor ◽  
Dependent Manner ◽  
Trithorax Group ◽  
Trithorax Group Proteins

ABSTRACT Many transcription coactivators interact with nuclear receptors in a ligand- and C-terminal transactivation function (AF2)-dependent manner. These include activating signal cointegrator 2 (ASC-2), a recently isolated transcriptional coactivator molecule, which is amplified in human cancers and stimulates transactivation by nuclear receptors and numerous other transcription factors. In this report, we show that ASC-2 belongs to a steady-state complex of approximately 2 MDa (ASC-2 complex [ASCOM]) in HeLa nuclei. ASCOM contains retinoblastoma-binding protein RBQ-3, α/β-tubulins, and trithorax group proteins ALR-1, ALR-2, HALR, and ASH2. In particular, ALR-1/2 and HALR contain a highly conserved 130- to 140-amino-acid motif termed the SET domain, which was recently implicated in histone H3 lysine-specific methylation activities. Indeed, recombinant ALR-1, HALR, and immunopurified ASCOM exhibit very weak but specific H3-lysine 4 methylation activities in vitro, and transactivation by retinoic acid receptor appears to involve ligand-dependent recruitment of ASCOM and subsequent transient H3-lysine 4 methylation of the promoter region in vivo. Thus, ASCOM may represent a distinct coactivator complex of nuclear receptors. Further characterization of ASCOM will lead to a better understanding of how nuclear receptors and other transcription factors mediate transcriptional activation.

scholarly journals Liver X Receptor Nuclear Receptors Are Transcriptional Regulators of Dendritic Cell Chemotaxis

2018 ◽  
Vol 38 (10) ◽  
Author(s):  
Susana Beceiro ◽  
Attila Pap ◽  
Zsolt Czimmerer ◽  
Tamer Sallam ◽  
Jose A. Guillén ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Low Density Lipoprotein ◽  
Myeloid Cells ◽  
Density Lipoprotein ◽  
Loss Of Function ◽  
Transcriptional Responses ◽  
Cd38 Expression ◽  
The Impact

ABSTRACTThe liver X receptors (LXRs) are ligand-activated nuclear receptors with established roles in the maintenance of lipid homeostasis in multiple tissues. LXRs exert additional biological functions as negative regulators of inflammation, particularly in macrophages. However, the transcriptional responses controlled by LXRs in other myeloid cells, such as dendritic cells (DCs), are still poorly understood. Here we used gain- and loss-of-function models to characterize the impact of LXR deficiency on DC activation programs. Our results identified an LXR-dependent pathway that is important for DC chemotaxis. LXR-deficient mature DCs are defective in stimulus-induced migrationin vitroandin vivo. Mechanistically, we show that LXRs facilitate DC chemotactic signaling by regulating the expression of CD38, an ectoenzyme important for leukocyte trafficking. Pharmacological or genetic inactivation of CD38 activity abolished the LXR-dependent induction of DC chemotaxis. Using the low-density lipoprotein receptor-deficient (LDLR−/−) LDLR−/−mouse model of atherosclerosis, we also demonstrated that hematopoietic CD38 expression is important for the accumulation of lipid-laden myeloid cells in lesions, suggesting that CD38 is a key factor in leukocyte migration during atherogenesis. Collectively, our results demonstrate that LXRs are required for the efficient emigration of DCs in response to chemotactic signals during inflammation.

scholarly journals ShearFAST: a user-friendly in vitro toolset for high throughput, inexpensive fluid shear stress experiments.

2020 ◽  
Author(s):  
Thomas Brendan Smith ◽  
Alessandro Marco De Nunzio ◽  
Kamlesh Patel ◽  
Haydn Munford ◽  
Tabeer Alam ◽  
...  
Keyword(s):  
Shear Stress ◽  
High Throughput ◽  
Fluid Shear Stress ◽  
Tracking System ◽  
Frequency Measurement ◽  
Camera Motion ◽  
Fluid Shear ◽  
Mean Frequency

Fluid shear stress is a key modulator of cellular physiology in vitro and in vivo, but its effects are under-investigated due to requirements for complicated induction methods. Herein we report the validation of ShearFAST; a smartphone application that measures the rocking profile on a standard laboratory cell rocker and calculates the resulting shear stress arising in tissue culture plates. The accuracy with which this novel approach measured rocking profiles was validated against a graphical analysis, and also against measures reported by an 8-camera motion tracking system. ShearFASTs angle assessments correlated well with both analyses (r ≥0.99, p ≤0.001) with no significant differences in pitch detected across the range of rocking angles tested. Rocking frequency assessment by ShearFAST also correlated well when compared to the two independent validatory techniques (r ≥0.99, p ≤0.0001), with excellent reproducibility between ShearFAST and video analysis (mean frequency measurement difference of 0.006 ± 0.005Hz) and motion capture analysis (mean frequency measurement difference of 0.008 ± 0.012Hz). These data make the ShearFAST assisted cell rocker model make it an attractive approach for economical, high throughput fluid shear stress experiments. Proof of concept data presented reveals a protective effect of low-level shear stress on renal proximal tubule cells submitted to simulations of pretransplant storage.

scholarly journals CDK10 Regulates Gastric Cancer Metastasis By Inhibiting lncRNA-C5ORF42-5 Via Phosphorylation Level of AKT/ERK

2021 ◽  
Author(s):  
Zi-Jian Deng ◽  
Dong-Wen Chen ◽  
Xi-Jie Chen ◽  
Jia-Ming Fang ◽  
Liang Xv ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Cells ◽  
Cell Line ◽  
High Throughput ◽  
Cancer Metastasis ◽  
High Throughput Sequencing ◽  
Gastric Cancer Cells ◽  
Related Proteins

Abstract Background: Gastric cancer is the fourth most common malignant disease. Both CDK10 and long noncoding RNAs (lncRNAs) have been found to exert biological functions in multiple cancers. However, it is still unclear whether CDK10 represses tumor progression in gastric cancer by reducing potential targeting lncRNAs.Methods: The functions of CDK10 and lncRNA-C5ORF42-5 in proliferation, invasion and migration were assessed by MTS assays, colony formation assays, cell cycle and apoptosis assays, Transwell assays, wound healing assays and animal experiments. We used high-throughput sequencing to confirm the existence of lncRNA-C5ORF42-5 and quantitative real-time PCR was used to evaluate lncRNA expression. Then, with RNA-seq sequencing as well as GO function and KEGG enrichment analysis, we identified the signaling pathways in which lncRNA-C5ORF42-5 was involved in gastric cancer. Finally, western blotting was used to identify the genes regulated by lncRNA-C5ORF42-5.Results: Our results showed that CDK10 is expressed at relatively low levels in gastric cancer cell lines and inhibits the progression of gastric cancer cells both in vitro and in vivo. Next, based on high-throughput sequencing, we identified a novel lncRNA, lncRNA-C5ORF42-5, in the stable CDK10-overexpressing cell line compared with the CDK-knockdown cell line and their controls. Additionally, we confirmed that lncRNA-C5ORF42-5 acts as an oncogene to promote metastasis in gastric cancer in vitro and in vivo. We then ascertained that lncRNA-C5ORF42-5 is a major contributor to the function of CDK10 in gastric cancer metastasis by upregulating lncRNA-C5ORF42-5 to reverse the effects of CDK10 overexpression. Finally, we explored the mechanism by which lncRNA-C5ORF42-5 overexpression affects gastric cancer cells to elucidate whether lncRNA-C5ORF42-5 may increase the activity of the SMAD pathway of BMP signaling and promote the expression of EMT-related proteins, such as E-cadherin. Additionally, overexpression of lncRNA-C5ORF42-5 affected the phosphorylation levels of AKT and ERK.Conclusion: Our findings suggest that CDK10 overexpression represses gastric cancer tumor progression by reducing lncRNA-C5ORF42-5 and hindering activation of the related proteins in metastatic signaling pathways, which provides new insight into developing effective therapeutic strategies in the treatment of metastatic gastric cancer.

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