scholarly journals Chemoprobe-based assays of histone lysine demethylase 1A target occupation enable in vivo pharmacokinetics and pharmacodynamics studies of KDM1A inhibitors

2019 ◽  
Vol 294 (20) ◽  
pp. 8311-8322 ◽  
Author(s):  
Cristina Mascaró ◽  
Alberto Ortega ◽  
Elena Carceller ◽  
Raquel Ruiz Rodriguez ◽  
Filippo Ciceri ◽  
...  
Keyword(s):  
Active Form ◽  
Dependent Manner ◽  
Target Engagement ◽  
Pharmacokinetics And Pharmacodynamics ◽  
Irreversible Inhibitor ◽  
Histone Marks ◽  
Histone Lysine ◽  
Indirect Consequence ◽  
Lysine Demethylase

Screening of cellular activity for inhibitors of histone lysine modifiers is most frequently performed indirectly by analyzing changes in the total levels of histone marks targeted by lysine methylases/demethylases. However, inhibition of histone lysine modifiers often leads to local rather than total changes in histone marks. Also, because histone modifications can be modulated by more than one cellular enzyme, it is not always clear whether changes in histone marks are a direct or indirect consequence of the inhibitor treatment applied. Direct assessment of target occupation can provide a useful tool to quantify the fraction of an epigenetic modifier that is bound to a pharmacological inhibitor (target engagement). Here, we developed and used a novel chemoprobe-based immunoassay to quantify target engagement in cells. Quantification of the fraction of free KDM1A was made possible, in an immune-based assay, by coupling a biotinylated chemoprobe to a warhead capable of selectively and irreversibly binding to the free active form of KDM1A. The results obtained confirmed that this approach is able to determine the degree of target engagement in a dose-dependent manner. Furthermore, the assay can be also used on tissue extracts to analyze the in vivo pharmacokinetics and pharmacodynamics relationship of KDM1A inhibitors, as has been exemplified with ORY-1001 (iadademstat), a potent and irreversible inhibitor of KDM1A. The principle of this assay may be applied to other targets, and the KDM1A probe may be employed in chemoproteomic analyses.

scholarly journals The Histone Lysine Demethylase JMJD3/KDM6B Is Recruited to p53 Bound Promoters and Enhancer Elements in a p53 Dependent Manner

PLoS ONE ◽  
2014 ◽  
Vol 9 (5) ◽  
pp. e96545 ◽  
Author(s):  
Kristine Williams ◽  
Jesper Christensen ◽  
Juri Rappsilber ◽  
Anders Lærke Nielsen ◽  
Jens Vilstrup Johansen ◽  
...  
Keyword(s):  
Dependent Manner ◽  
Histone Lysine ◽  
Histone Lysine Demethylase ◽  
Lysine Demethylase

scholarly journals A chemoprobe tracks its target

2019 ◽  
Vol 294 (20) ◽  
pp. 8323-8324
Author(s):  
Aseem Z. Ansari
Keyword(s):  
Small Molecule ◽  
Clinical Utility ◽  
Small Molecule Inhibitors ◽  
Indirect Measure ◽  
Histone Marks ◽  
Histone Lysine ◽  
Histone Lysine Demethylase ◽  
Histone Modifying Enzymes ◽  
Lysine Demethylase ◽  
In Cells

Small-molecule inhibitors of histone-modifying enzymes have significant clinical utility for managing diseases such as cancer. These inhibitors are usually identified and monitored through their effects on the gain or loss of specific histone marks. In cells, multiple related enzymes can place or remove a specific mark; therefore, relying on an indirect measure of inhibitor engagement can be misleading. Mascaró et al. describe a luminescence-based ELISA approach that directly monitors binding of inhibitors to the histone lysine demethylase KDM1A.

scholarly journals Phosphorylation and SCF-mediated degradation regulate CREB-H transcription of metabolic targets

2015 ◽  
Vol 26 (16) ◽  
pp. 2939-2954 ◽  
Author(s):  
Sónia Barbosa ◽  
Suzanne Carreira ◽  
Daniel Bailey ◽  
Fernando Abaitua ◽  
Peter O'Hare
Keyword(s):  
Target Gene ◽  
Active Form ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Critical Determinant ◽  
Phosphatase Treatment ◽  
Steady State Levels ◽  
Treatment Use

CREB‑H, an endoplasmic reticulum–anchored transcription factor, plays a key role in regulating secretion and in metabolic and inflammatory pathways, but how its activity is modulated remains unclear. We examined processing of the nuclear active form and identified a motif around S87–S90 with homology to DSG-type phosphodegrons. We show that this region is subject to multiple phosphorylations, which regulate CREB-H stability by targeting it to the SCFFbw1aE3 ubiquitin ligase. Data from phosphatase treatment, use of phosophospecific antibody, and substitution of serine residues demonstrate phosphorylation of candidate serines in the region, with the core S87/S90 motif representing a critical determinant promoting proteasome-mediated degradation. Candidate kinases CKII and GSK-3b phosphorylate CREB-H in vitro with specificities for different serines. Prior phosphorylation with GSK-3 at one or more of the adjacent serines substantially increases S87/S90-dependent phosphorylation by CKII. In vivo expression of a dominant-negative Cul1 enhances steady-state levels of CREB‑H, an effect augmented by Fbw1a. CREB-H directly interacts with Fbw1a in a phosphorylation-dependent manner. Finally, mutations within the phosphodegron, when incorporated into the full-length protein, result in increased levels of constitutively cleaved nuclear protein and increased transcription and secretion of a key endogenous target gene, apolipoprotein A IV.

scholarly journals Interaction between ROCK II and Nucleophosmin/B23 in the Regulation of Centrosome Duplication

2006 ◽  
Vol 26 (23) ◽  
pp. 9016-9034 ◽  
Author(s):  
Zhiyong Ma ◽  
Masayuki Kanai ◽  
Kenji Kawamura ◽  
Kozo Kaibuchi ◽  
Keqiang Ye ◽  
...  
Keyword(s):  
Cyclin E ◽  
Active Form ◽  
Small Gtpase ◽  
Centrosome Duplication ◽  
Physical Interaction ◽  
Dependent Manner ◽  
Physical Separation ◽  
Initial Event ◽  
Activity Dependent

ABSTRACT Nucleophosmin (NPM)/B23 has been implicated in the regulation of centrosome duplication. NPM/B23 localizes between two centrioles in the unduplicated centrosome. Upon phosphorylation on Thr199 by cyclin-dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but some NPM/B23 phosphorylated on Thr199 remains at centrosomes. It has been shown that Thr199 phosphorylation of NPM/B23 is critical for the physical separation of the paired centrioles, an initial event of the centrosome duplication process. Here, we identified ROCK II kinase, an effector of Rho small GTPase, as a protein that localizes to centrosomes and physically interacts with NPM/B23. Expression of the constitutively active form of ROCK II promotes centrosome duplication, while down-regulation of ROCK II expression results in the suppression of centrosome duplication, especially delaying the initiation of centrosome duplication during the cell cycle. Moreover, ROCK II regulates centrosome duplication in its kinase and centrosome localization activity-dependent manner. We further found that ROCK II kinase activity is significantly enhanced by binding to NPM/B23 and that NPM/B23 acquires a higher binding affinity to ROCK II upon phosphorylation on Thr199. Moreover, physical interaction between ROCK II and NPM/B23 in vivo occurs in association with CDK2/cyclin E activation and the emergence of Thr199-phosphorylated NPM/B23. All these findings point to ROCK II as the effector of the CDK2/cyclin E-NPM/B23 pathway in the regulation of centrosome duplication.

scholarly journals p53 binding sites in normal and cancer cells are characterized by distinct chromatin context

2017 ◽  
Author(s):  
Feifei Bao ◽  
Peter R. LoVerso ◽  
Jeffrey N. Fisk ◽  
Victor B. Zhurkin ◽  
Feng Cui
Keyword(s):  
Cancer Cells ◽  
Binding Sites ◽  
Nucleosome Occupancy ◽  
High Sensitivity ◽  
Dependent Manner ◽  
Histone Marks ◽  
Genome Wide ◽  
Pioneer Transcription Factor ◽  
Chromatin Patterns

AbstractThe tumor suppressor protein p53 interacts with DNA in a sequence-dependent manner. Thousands of p53 binding sites have been mapped genome-wide in normal and cancer cells. However, the way p53 selectively binds its cognate sites in different types of cells is not fully understood. Here, we performed a comprehensive analysis of 25 published p53 cistromes and identified 3,551 and 6,039 ‘high-confidence’ binding sites in normal and cancer cells, respectively. Our analysis revealed two distinct epigenetic features underlying p53-DNA interactionsin vivo. First, p53 binding sites are associated with transcriptionally active histone marks (H3K4me3 and H3K36me3) in normal-cell chromatin, but with repressive histone marks (H3K27me3) in cancer-cell chromatin. Second, p53 binding sites in cancer cells are characterized by a lower level of DNA methylation than their counterparts in normal cells, probably related to global hypomethylation in cancers. Intriguingly, regardless of the cell type, p53 sites are highly enriched in the endogenous retroviral elements of the ERV1 family, highlighting the importance of this repeat family in shaping the transcriptional network of p53. Moreover, the p53 sites exhibit an unusual combination of chromatin patterns: high nucleosome occupancy and, at the same time, high sensitivity to DNase I. Our results suggest that p53 can access its target sites in a chromatin environment that is non-permissive to most DNA-binding transcription factors, which may allow p53 to act as a pioneer transcription factor in the context of chromatin.

scholarly journals The Liver X Receptor Ligand T0901317 Down-regulatesAPOA5Gene Expression through Activation of SREBP-1c

2004 ◽  
Vol 279 (44) ◽  
pp. 45462-45469 ◽  
Author(s):  
Heidelinde Jakel ◽  
Maxime Nowak ◽  
Emanuelle Moitrot ◽  
Hélène Dehondt ◽  
Dean W. Hum ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Regulatory Element ◽  
Hepatoma Cell ◽  
Active Form ◽  
Liver X Receptor ◽  
Plasma Triglyceride ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Down Regulation

Alterations in the expression of the recently discovered apolipoprotein A5 gene strongly affect plasma triglyceride levels. In this study, we investigated the contribution ofAPOA5to the liver X receptor (LXR) ligand-mediated effect on plasma triglyceride levels. Following treatment with the LXR ligand T0901317, we found thatAPOA5mRNA levels were decreased in hepatoma cell lines. The observation that no down-regulation ofAPOA5promoter activity was obtained by LXR-retinoid X receptor (RXR) co-transfection prompted us to explore the possible involvement of the known LXR target gene SREBP-1c (sterol regulatory element-binding protein 1c). In fact, we found that co-transfection with the active form of SREBP-1c down-regulatedAPOA5promoter activity in a dose-dependent manner. We then scanned the humanAPOA5promoter sequence and identified two putative E-box elements that were able to bind specifically SREBP-1c in gel-shift assays and were shown to be functional by mutation analysis. Subsequent suppression of SREBP-1 mRNA through small interfering RNA interference abolished the decrease ofAPOA5mRNA in response to T0901317. Finally, administration of T0901317 tohAPOA5transgenic mice revealed a significant decrease ofAPOA5mRNA in liver tissue and circulating apolipoprotein AV protein in plasma, confirming that the described down-regulation also occursin vivo. Taken together, our results demonstrate thatAPOA5gene expression is regulated by the LXR ligand T0901317 in a negative manner through SREBP-1c. These findings may provide a new mechanism responsible for the elevation of plasma triglyceride levels by LXR ligands and support the development of selective LXR agonists, not affecting SREBP-1c, as beneficial modulators of lipid metabolism.

scholarly journals Injectable nanoclay gels for angiogenesis

2019 ◽  
Author(s):  
Daniel J. Page ◽  
Claire E. Clarkin ◽  
Raj Mani ◽  
Najeed A. Khan ◽  
Jonathan I. Dawson ◽  
...  
Keyword(s):  
Growth Factor ◽  
Active Form ◽  
Fold Increase ◽  
Dependent Manner ◽  
Vascular Endothelial ◽  
Sustained Activity ◽  
A Site ◽  
Endothelial Growth Factor ◽  
Dose Dependent

AbstractThe retention and sustained activity of therapeutic proteins at delivery sites are goals of regenerative medicine. Vascular endothelial growth factor (VEGF) has significant potential in promoting the growth and regeneration of blood vessels but is intrinsically labile. This is exacerbated by the inflammatory microenvironments at sites requiring regeneration. For VEGF to be efficacious it may require a carrier that stabilises it, protects it from degradation and retains it at a site of interest. In this study we tested the hypothesis that injectable nanoclay gels composed of Laponite XLG can stabilise VEGF and retain it in active form for therapeutic delivery. To achieve this, VEGF was incorporated in Laponite gels and its activity tested at a range of concentrations using in vivo cell culture tubulogenesis assays and in vivo angiogenesis assays. We found that VEGF-Laponite gels enhanced tubulogenesis in a dose-dependent manner in vivo. When administered subcutaneously in vivo Laponite was retained at an injection site for up to a period of three weeks and promoted a 4-fold increase in blood vessel formation compared with alginate or vehicle controls as confirmed by CD31 staining. Notably, in contrast to alginate, Laponite gels did not release VEGF, indicating a strong interaction between the growth factor and the nanoclay, and suggesting that Laponite enhancement of VEGF efficacy is due to its retention at an implantation site over a prolonged period. Our approach provides a robust method for delivery of bioactive recombinant VEGF without the necessity for complex hydrogel or protein engineering.

scholarly journals Histone Lysine Demethylase 1A Is a Master Regulator of Genes Necessary for Trophoblast Cell Proliferation

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A504-A504
Author(s):  
Gerrit J Bouma ◽  
Asghar Ali ◽  
Taylor K Hord ◽  
Agata M Parsons ◽  
Russell Vernon Anthony ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Line ◽  
Trophoblast Cell ◽  
Trophoblast Cells ◽  
Placental Development ◽  
Master Regulator ◽  
Histone Lysine ◽  
Histone Lysine Demethylase ◽  
Lysine Demethylase

Abstract Histone lysine demethylase 1A is a master regulator of genes necessary for trophoblast cell proliferation. A proper functioning placenta is critical for pregnancy, fetal growth and development and postnatal health. Trophoblast cell proliferation and differentiation is critical for placental development and function. Recently we demonstrated that the histone lysine demethylase KDM1A binds to androgen receptor (AR) in human and sheep trophoblast cells, and targets the same promoter region of vascular endothelial growth factor A (VEGFA), suggesting a role for KDM1A and AR in early placental angiogenesis. The goal of this study was to determine the function of KDM1A during early placental development. We hypothesized that KDM1A regulates genes that are necessary for trophoblast cell proliferation, and early placental development. To this end, both in vitro and in vivo approaches were used in this study. ACH-3P cells (human first trimester trophoblast cells (CT and EVT) fused with the choriocarcinoma cell line AC1-1) were used, and a KDM1A knock out (KO) cell line was generated using CRISPR-Cas 9 based genome editing. KDM1A KO in ACH-3P cells led to significant (P<0.05) reduction in AR and VEGFA. Furthermore, factors important for cell proliferation and trophoblast cell development high mobility group AT-hook 1 (HMGA1), LIN28, and MYC protooncogene (cMYC) were significantly (P<0.05) lower in KDM1A KO ACH-3P cells. Cell proliferation assays revealed a significant (P<0.05) reduction in KDM1A KO ACH-3P cells compared to scramble controls. An in vivo experiment was conducted to demonstrate a role for KDM1A in placental development, using the sheep as a model. Day 9 hatched blastocysts were flushed and infected with a Lenti-CRISPRv2 KDM1A target construct (n=4) to knockout KDM1A specifically in the trophectoderm, or with SC (n=5). Infected embryos were transferred to recipient ewes and embryos were collected at gestational day 16. Data suggests that KDM1A KO in trophoblast cells is necessary for conceptus elongation. Current experiments are ongoing to determine the effects of KDM1A and AR knockdown using shRNA lentiviral target vectors on conceptus elongation and pregnancy. Collectively these results indicate that KDM1A plays a central role in regulating genes necessary for trophoblast cell proliferation. This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2019-67015-29000 from the USDA National Institute of Food and Agriculture.

scholarly journals AMPK regulates cell shape of cardiomyocytes by modulating turnover of microtubules through CLIP-170

2020 ◽  
Author(s):  
Shohei Yashirogi ◽  
Toru Katayama ◽  
Takemasa Nagao ◽  
Yuya Nishida ◽  
Hidetaka Kioka ◽  
...  
Keyword(s):  
Cell Shape ◽  
Dynamic Instability ◽  
Active Form ◽  
Time Lapse ◽  
Cardiac Contraction ◽  
Cell Junction ◽  
Specific Cell ◽  
Dependent Manner ◽  
Intercalated Discs

SummaryAMP-activated protein kinase (AMPK) is a multifunctional kinase that regulates microtubule (MT) dynamic instability through CLIP-170 phosphorylation; however, its physiological relevance in vivo remains to be elucidated. In this study, we identified an active form of AMPK localized at the intercalated discs in the heart, a specific cell-cell junction present between cardiomyocytes. A contractile inhibitor, MYK-461, prevented the localization of AMPK at the intercalated discs, and the effect was reversed by the removal of MYK-461, suggesting that the localization of AMPK is regulated by mechanical stress. Time-lapse imaging analysis revealed that the inhibition of CLIP-170 Ser-311 phosphorylation by AMPK leads to the accumulation of MTs at the intercalated discs. Interestingly, MYK-461 increased the individual cell area of cardiomyocytes in CLIP-170 phosphorylation-dependent manner. Moreover, heart-specific CLIP-170 S311A transgenic mice demonstrated elongation of cardiomyocytes along with accumulated MTs, leading to progressive decline in cardiac contraction. In conclusion, these findings suggest that AMPK regulates the cell shape and aspect ratio of cardiomyocytes by modulating the turnover of MTs through homeostatic phosphorylation of CLIP-170 at the intercalated discs.

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