scholarly journals Detect Genomic G-Quadruplexes in Living Animal Cells with a Tiny Artificial Protein Probe

2020 ◽  
Author(s):  
Ke-wei Zheng ◽  
Jia-yu Zhang ◽  
Yi-de He ◽  
Jia-yuan Gong ◽  
Cui-jiao Wen ◽  
...  
Keyword(s):  
Drug Targets ◽  
Fold Increase ◽  
Molecular Devices ◽  
Animal Cells ◽  
Physiological Processes ◽  
Genome Wide ◽  
G Quadruplex ◽  
Artificial Protein ◽  
Important Drug

ABSTRACTG-quadruplex (G4) structures formed by guanine-rich nucleic acids are implicated in essential physiological processes and serve as important drug targets. The genome-wide detection of G4s in living cells is important for exploring the biological role of G4s but has not yet been achieved due to the lack of a suitable G4 probe. We engineered a 6.7 kDa G4 probe (G4P) protein that binds G4s with high affinity and specificity. We used it to capture G4s in living human, mouse, and chicken cells with the ChIP-Seq technology, yielding genome-wide landscape as well as details on the positions, frequencies, and sequence identities of G4 formation in these cells. Our results indicate that transcription is accompanied by a robust formation of G4s in genes. In human cells, we detected up to >123,000 G4 peaks, of which >1/3 had a fold increase of ≥5 and were present in >60% promoters and ~70% genes. Being much smaller than a scFv antibody (27 kDa) or even a nanobody (12-15 kDa), we expect that the G4P may find diverse applications in biology, medicine, and molecular devices as a G4 affinity agent.

scholarly journals Detection of genomic G-quadruplexes in living cells using a small artificial protein

2020 ◽  
Vol 48 (20) ◽  
pp. 11706-11720
Author(s):  
Ke-wei Zheng ◽  
Jia-yu Zhang ◽  
Yi-de He ◽  
Jia-yuan Gong ◽  
Cui-jiao Wen ◽  
...  
Keyword(s):  
Drug Targets ◽  
Fold Increase ◽  
Living Cells ◽  
Molecular Devices ◽  
Genome Wide ◽  
G Quadruplex ◽  
Artificial Protein ◽  
Important Drug ◽  
Sequence Identities

Abstract G-quadruplex (G4) structures formed by guanine-rich nucleic acids are implicated in essential physiological and pathological processes and serve as important drug targets. The genome-wide detection of G4s in living cells is important for exploring the functional role of G4s but has not yet been achieved due to the lack of a suitable G4 probe. Here we report an artificial 6.7 kDa G4 probe (G4P) protein that binds G4s with high affinity and specificity. We used it to capture G4s in living human, mouse, and chicken cells with the ChIP-Seq technique, yielding genome-wide landscape as well as details on the positions, frequencies, and sequence identities of G4 formation in these cells. Our results indicate that transcription is accompanied by a robust formation of G4s in genes. In human cells, we detected up to >123 000 G4P peaks, of which >1/3 had a fold increase of ≥5 and were present in >60% promoters and ∼70% genes. Being much smaller than a scFv antibody (27 kDa) or even a nanobody (12–15 kDa), we expect that the G4P may find diverse applications in biology, medicine, and molecular devices as a G4 affinity agent.

scholarly journals Epigenetic Deregulation of Telomere-Related Genes in Newly Diagnosed Multiple Myeloma Patients

Cancers ◽  
2021 ◽  
Vol 13 (24) ◽  
pp. 6348
Author(s):  
Samrat Roy Choudhury ◽  
Cody Ashby ◽  
Fenghuang Zhan ◽  
Frits van Rhee
Keyword(s):  
Dna Methylation ◽  
Multiple Myeloma ◽  
Dna Sequences ◽  
Critical Length ◽  
Newly Diagnosed ◽  
Genome Wide ◽  
Healthy Donors ◽  
Binding Factor ◽  
G Quadruplex

High-risk Multiple Myeloma (MM) patients were found to maintain telomere length (TL), below the margin of short critical length, consistent with proactive overexpression of telomerase. Previously, DNA methylation has been shown as a determinant of telomere-related gene (TRG) expression and TL to assess risk in different types of cancer. We mapped genome-wide DNA methylation in a cohort of newly diagnosed MM (NDMM; n = 53) patients of major molecular subgroups, compared to age-matched healthy donors (n = 4). Differential methylation and expression at TRG-loci were analyzed in combination with overlapping chromatin marks and underlying DNA-sequences. We observed a strong correlation (R2 ≥ 0.5) between DNA methylation and expression amongst selective TRGs, such that demethylation at the promoters of DDX1 and TERF1 were associated to their oncogenic upregulation, while demethylation at the bodies of two key tumor suppressors ZNF208 and RAP1A led to downregulation of the genes. We demonstrated that TRG expression may be controlled by DNA methylation alone or in cooperation with chromatin modifications or CCCTC-binding factor at the regulatory regions. Additionally, we showed that hypomethylated DMRs of TRGs in NDMM are stabilized with G-quadruplex forming sequences, suggesting a crucial role of these epigenetically vulnerable loci in MM pathogenesis. We have identified a panel of five TRGs, which are epigenetically deregulated in NDMM patients and may serve as early detection biomarkers or therapeutic targets in the disease.

scholarly journals SLC4A11 and MFSD3 Gene Expression Changes in Deoxynivalenol Treated IPEC-J2 Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Yafei Xu ◽  
Xiaolei Chen ◽  
Luchen Yu ◽  
Yi Wang ◽  
Haifei Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Methylation ◽  
Drug Targets ◽  
Molecular Mechanisms ◽  
The Novel ◽  
Animal Cells ◽  
Cytotoxic Effects ◽  
M Phase ◽  
Detection Of Biomarkers

Deoxynivalenol (DON) caused serious cytotoxicity for animal cells. However, genes involved in regulating DON toxicity and the underlying molecular mechanisms remain largely unknown. This study explored the role of SLC4A11 and MFSD3 in alleviating DON toxicity and analyzed the DNA methylation changes of these two genes. Viability and cell cycle analysis showed that DON exposure decreased the IPEC-J2 viability (P < 0.01), blocked the cell cycle in the G2/M phase (P < 0.01), and increased the rate of apoptosis (P < 0.05). Expression of the SLC4A11 and MFSD3 genes was significantly downregulated upon DON exposure (P < 0.01). Overexpression of SLC4A11 and MFSD3 can enhance the cell viability (P < 0.01). DNA methylation assays indicated that promoter methylation of SLC4A11 (mC-1 and mC-23) and MFSD3 (mC-1 and mC-12) were significantly higher compared with those in the controls and correlated negatively with mRNA expression (P < 0.05). Further analysis showed that mC-1 of SLC4A11 and MFSD3 was located in transcription factor binding sites for NF-1 and Sp1. Our findings revealed the novel biological functions of porcine SLC4A11 and MFSD3 genes in regulating the cytotoxic effects induced by DON, and may contribute to the detection of biomarkers and drug targets for predicting and eliminating the potential toxicity of DON.

scholarly journals Structured Waters Mediate Small Molecule Binding to G-Quadruplex Nucleic Acids

2021 ◽  
Vol 15 (1) ◽  
pp. 7
Author(s):  
Stephen Neidle
Keyword(s):  
Crystal Structures ◽  
Small Molecule ◽  
Drug Targets ◽  
Bound Water ◽  
Water Molecules ◽  
Telomeric Dna ◽  
Naphthalene Diimide ◽  
In Silico Studies ◽  
G Quadruplex

The role of G-quadruplexes in human cancers is increasingly well-defined. Accordingly, G-quadruplexes can be suitable drug targets and many small molecules have been identified to date as G-quadruplex binders, some using computer-based design methods and co-crystal structures. The role of bound water molecules in the crystal structures of G-quadruplex-small molecule complexes has been analyzed in this study, focusing on the water arrangements in several G-quadruplex ligand complexes. One is the complex between the tetrasubstituted naphthalene diimide compound MM41 and a human intramolecular telomeric DNA G-quadruplex, and the others are in substituted acridine bimolecular G-quadruplex complexes. Bridging water molecules form most of the hydrogen-bond contacts between ligands and DNA in the parallel G-quadruplex structures examined here. Clusters of structured water molecules play essential roles in mediating between ligand side chain groups/chromophore core and G-quadruplex. These clusters tend to be conserved between complex and native G-quadruplex structures, suggesting that they more generally serve as platforms for ligand binding, and should be taken into account in docking and in silico studies.

scholarly journals A conserved mechanism of sirtuin signalling through steroid hormone receptors

2019 ◽  
Vol 39 (12) ◽  
Author(s):  
Henry K. Bayele
Keyword(s):  
Drug Targets ◽  
Metabolic Regulation ◽  
Hormone Receptors ◽  
Steroid Receptors ◽  
Steroid Hormone ◽  
Steroid Hormone Receptors ◽  
Nuclear Receptor Coregulators ◽  
Dafachronic Acid ◽  
Important Drug

Abstract SIRT1 and orthologous sirtuins regulate a universal mechanism of ageing and thus determine lifespan across taxa; however, the precise mechanism remains vexingly polemical. They also protect against many metabolic and ageing-related diseases by dynamically integrating several processes including autophagy, proteostasis, calorie restriction, circadian rhythmicity and metabolism. These sirtuins are therefore important drug targets particularly because they also transduce allosteric signals from sirtuin-activating compounds such as resveratrol into increased healthspan in evolutionarily diverse organisms. While many of these functions are apparently regulated by deacetylation, that mechanism may not be all-encompassing. Since gonadal signals have been shown to regulate ageing/lifespan in worms and flies, the present study hypothesized that these sirtuins may act as intermediary factors for steroid hormone signal transduction. Accordingly, SIRT1 and its orthologues, Sir2 and Sir-2.1, are shown to be veritable nuclear receptor coregulators that classically coactivate the oestrogen receptor in the absence of ligand; coactivation was further increased by 17β-oestradiol. Remarkably in response to the worm steroid hormone dafachronic acid, SIRT1 reciprocally coactivates DAF-12, the steroid receptor that regulates nematode lifespan. These results suggest that steroid hormones may co-opt and modulate a phyletically conserved mechanism of sirtuin signalling through steroid receptors. Hence, it is interesting to speculate that certain sirtuin functions including prolongevity and metabolic regulation may be mechanistically linked to this endocrine signalling pathway; this may also have implications for understanding the determinative role of gonadal steroids such as oestradiol in human ageing. At its simplest, this report shows evidence for a hitherto unknown deacetylation-independent mechanism of sirtuin signalling.

scholarly journals Genome-Wide Identification and Expression Profiling of CBL-CIPK Gene Family in Pineapple (Ananas comosus) and the Role of AcCBL1 in Abiotic and Biotic Stress Response

Biomolecules ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 293 ◽  
Author(s):  
Aslam ◽  
Fakher ◽  
Jakada ◽  
Zhao ◽  
Cao ◽  
...  
Keyword(s):  
Ectopic Expression ◽  
Ananas Comosus ◽  
Interacting Protein ◽  
Physiological Processes ◽  
Genome Wide ◽  
Abiotic Stimuli ◽  
Sensor Proteins ◽  
Tolerance To Salinity ◽  
Different Tissues

Ca2+ serves as a ubiquitous second messenger regulating several aspects of plant growth and development. A group of unique calcium sensor proteins, calcineurin B-like (CBL), interact with CBL-interacting protein kinases (CIPKs) to decode the Ca2+ signature inside the cell. Although CBL-CIPK signaling toolkit has been shown to play significant roles in the responses to numerous stresses in different plants, the information about pineapple CBL-CIPK remains obscure. In the present study, a total of eight AcCBL and 21 AcCIPK genes were identified genome-wide in pineapple. The identified genes were renamed on the basis of gene ID in ascending order and phylogenetic analysis divided into five groups. Transcriptomic data analysis showed that AcCBL and AcCIPK genes were expressed differentially in different tissues. Further, the expression analysis of AcCBL1 in different tissues showed significant changes under various abiotic stimuli. Additionally, the ectopic expression of AcCBL1 in Arabidopsis resulted in enhanced tolerance to salinity, osmotic, and fungal stress. The present study revealed the crucial contribution of the CBL-CIPK gene in various biological and physiological processes in pineapple.

scholarly journals The CUL5 ubiquitin ligase complex mediates resistance to CDK9 and MCL1 inhibitors in lung cancer cells

2019 ◽  
Author(s):  
Shaheen Kabir ◽  
Justin Cidado ◽  
Courtney Andersen ◽  
Cortni Dick ◽  
Pei-Chun Lin ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Drug Targets ◽  
Combination Treatments ◽  
Cancer Models ◽  
Genome Wide ◽  
Ubiquitin Ligase Complex ◽  
Frequent Event ◽  
Inhibitor Discovery ◽  
Apoptotic Proteins

ABSTRACTOverexpression of anti-apoptotic proteins MCL1 and Bcl-xL is a frequent event in blood and solid cancers. Inhibitors targeting MCL1 are in clinical development, however many cancer models are intrinsically resistant to this approach. To discover mechanisms underlying resistance to MCL1 inhibition, we performed multiple flow-cytometry based genome-wide CRISPR screens that interrogate two drugs directly or indirectly targeting MCL1. Remarkably, both screens identified three components (CUL5, RNF7 and UBE2F) of a cullin-RING ubiquitin ligase complex (CRL5) that resensitized cells to MCL1 inhibition. We find that levels of the BH3-only pro-apoptotic proteins Bim and Noxa are proteasomally regulated by the CRL5 complex. Accumulation of Noxa caused by depletion of CRL5 components particularly skewed the balance in favor of apoptosis when cells were challenged with an MCL1 inhibitor. Discovery of a novel role of CRL5 in apoptosis and resistance to MCL1 inhibitors exposes new drug targets and the potential to improve combination treatments.

Advance in L-Type Calcium Channel Structures, Functions and Molecular Modeling

2020 ◽  
Vol 27 ◽  
Author(s):  
Lei Xu ◽  
Lilei Sun ◽  
Liangxu Xie ◽  
Shanzhi Mou ◽  
Dawei Zhang ◽  
...  
Keyword(s):  
Molecular Modeling ◽  
Calcium Channels ◽  
Drug Targets ◽  
Wide Spectrum ◽  
Critical Role ◽  
Muscular Contraction ◽  
Physiological Processes ◽  
Ligand Binding Sites ◽  
Channel Structures ◽  
Important Drug

: L-type calcium channels (LTCCs), also termed as Cav1, belong to voltage-gated calcium channels (VGCCs/Cavs), which play a critical role in a wide spectrum of physiological processes, including neurotransmission, cell cycle, muscular contraction, cardiac action potential and gene expression. Aberrant regulation of calcium channels is involved in neurological, cardiovascular, muscular and psychiatric disorders. Accordingly, LTCCs have been regarded as important drug targets, and a number of LTCC drugs are in clinical use. In this review, the recent development of structures and biological functions of LTCCs are introduced. Moreover, the representative modulators and ligand binding sites of LTCCs are discussed. Finally, molecular modeling and computer-aided drug design (CADD) methods for understanding structure-function relations of LTCCs are summarized.

scholarly journals Neuronal and Cardiovascular Potassium Channels as Therapeutic Drug Targets

2015 ◽  
Vol 20 (9) ◽  
pp. 1055-1073 ◽  
Author(s):  
Edward S. A. Humphries ◽  
Caroline Dart
Keyword(s):  
Drug Targets ◽  
Therapeutic Strategy ◽  
Genetic Disorders ◽  
Critical Role ◽  
Therapeutic Drug ◽  
Cellular Excitability ◽  
Human Genetic Disorders ◽  
The Brain ◽  
Important Drug

Potassium (K+) channels, with their diversity, often tissue-defined distribution, and critical role in controlling cellular excitability, have long held promise of being important drug targets for the treatment of dysrhythmias in the heart and abnormal neuronal activity within the brain. With the exception of drugs that target one particular class, ATP-sensitive K+ (KATP) channels, very few selective K+ channel activators or inhibitors are currently licensed for clinical use in cardiovascular and neurological disease. Here we review what a range of human genetic disorders have told us about the role of specific K+ channel subunits, explore the potential of activators and inhibitors of specific channel populations as a therapeutic strategy, and discuss possible reasons for the difficulty in designing clinically relevant K+ channel modulators.

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