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Cell Death & Disease Jan 2022Ferroptosis is a form of cell death characterized by lipid peroxidation. Previous studies have reported that knockout of NF-κB activating protein (NKAP), an RNA-binding...
Ferroptosis is a form of cell death characterized by lipid peroxidation. Previous studies have reported that knockout of NF-κB activating protein (NKAP), an RNA-binding protein, increased lipid peroxidation level in naive T cells and induced cell death in colon cancer cells. However, there was no literature reported the relationship between NKAP and ferroptosis in glioblastoma cells. Notably, the mechanism of NKAP modulating ferroptosis is still unknown. Here, we found NKAP knockdown induced cell death in glioblastoma cells. Silencing NKAP increased the cell sensitivity to ferroptosis inducers both in vitro and in vivo. Exogenous overexpression of NKAP promoted cell resistance to ferroptosis inducers by positively regulating a ferroptosis defense protein, namely cystine/glutamate antiporter (SLC7A11). The regulation of SLC7A11 by NKAP can be weakened by the mA methylation inhibitor cycloleucine and knockdown of the mA writer METTL3. NKAP combined the "RGAC" motif which was exactly in line with the mA motif "RGACH" (R = A/G, H = A/U/C) uncovered by the mA-sequence. RNA Immunoprecipitation (RIP) and Co-Immunoprecipitation (Co-IP) proved the interaction between NKAP and mA on SLC7A11 transcript. Following its binding to mA, NKAP recruited the splicing factor proline and glutamine-rich (SFPQ) to recognize the splice site and then conducted transcription termination site (TTS) splicing event on SLC7A11 transcript and the retention of the last exon, screened by RNA-sequence and Mass Spectrometry (MS). In conclusion, NKAP acted as a new ferroptosis suppressor by binding to mA and then promoting SLC7A11 mRNA splicing and maturation.
Topics: Amino Acid Transport System y+; Ferroptosis; Glioblastoma; Humans; Methyltransferases; NF-kappa B; RNA Splicing; RNA, Messenger; RNA-Binding Proteins; Repressor Proteins
PubMed: 35064112
DOI: 10.1038/s41419-022-04524-2 -
Poultry Science Dec 2022N6-Methyladenosine (mA) has been reported to involve and play an important role in various biological activities but seldom in poultry myogenesis. Cycloleucine usually...
N6-Methyladenosine (mA) has been reported to involve and play an important role in various biological activities but seldom in poultry myogenesis. Cycloleucine usually functions as a nucleic acid methylation inhibitor, the inhibition efficiency of cycloleucine at the mA level and corresponding dynamic changes of poultry muscle cells remain unknown. In this study, we aim to find out the effect of cycloleucine on the total N6-Methyladenosine level and its molecular mechanism for regulating myogenesis. A total of 745 differentially expressed genes (DEGs) were obtained by 10 mM, 20 mM, and 30 mM of cycloleucine treatment compared with 0 mM treatment. DEGs in 10 mM cycloleucine were significantly enriched in the biological process of skeletal muscle and satellite cell proliferation and differentiation, DEGs in 20 and 30 mM cycloleucine were enriched in some metabolic and biosynthetic processes. The trend analysis showed that 85% of all DEGs were significantly clustered into 4 files, among them 59% DEGs were dose-dependent and 26% were dose-independent, 52% DEGs were in downtrend and 33% DEGs were in uptrend. Also, the cycloleucine treatment could trigger cell cycle arrest in the G1 phase and depress myoblast cell proliferation and inhibit myotube formation. In conclusion, cycloleucine could continuously reduce the mA level of myoblast cells, depress myoblast cell proliferation and inhibit myotube formation.
Topics: Animals; Cycloleucine; Chickens; Muscle Development; Gene Expression Profiling; Myoblasts; Cell Differentiation
PubMed: 36308813
DOI: 10.1016/j.psj.2022.102219 -
Scientific Reports May 2023Although exogenous glycine betaine (GB) and cycloleucine (Cyc) have been reported to affect animal cell metabolism, their effects on plant growth and development have...
Although exogenous glycine betaine (GB) and cycloleucine (Cyc) have been reported to affect animal cell metabolism, their effects on plant growth and development have not been studied extensively. Different concentrations of exogenous glycine betaine (20, 40, and 60 mmol L) and cycloleucine (10, 20, and 40 mmol L), with 0 mmol L as control, were used to investigate the effects of foliar spraying of betaine and cycloleucine on growth, photosynthesis, chlorophyll fluorescence, Calvin cycle pathway, abaxial leaf burr morphology, endogenous hormones, and amino acid content in eggplant. We found that 40 mmol L glycine betaine had the best effect on plant growth and development; it increased the fresh and dry weight of plants, increased the density of abaxial leaf hairs, increased the net photosynthetic rate and Calvin cycle key enzyme activity of leaves, had an elevating effect on chlorophyll fluorescence parameters, increased endogenous indoleacetic acid (IAA) content and decreased abscisic acid (ABA) content, and increased glutamate, serine, aspartate, and phenylalanine contents. However, cycloleucine significantly inhibited plant growth; plant apical dominance disappeared, plant height and dry and fresh weights decreased significantly, the development of abaxial leaf hairs was hindered, the net photosynthetic rate and Calvin cycle key enzyme activities were inhibited, the endogenous hormones IAA and ABA content decreased, and the conversion and utilization of glutamate, arginine, threonine, and glycine were affected. Combined with the experimental results and plant growth phenotypes, 20 mmol L cycloleucine significantly inhibited plant growth. In conclusion, 40 mmol L glycine betaine and 20 mmol L cycloleucine had different regulatory effects on plant growth and development.
Topics: Betaine; Solanum melongena; Cycloleucine; Amino Acids; Photosynthesis; Abscisic Acid; Chlorophyll; Hormones; Plant Leaves
PubMed: 37165051
DOI: 10.1038/s41598-023-34509-w -
Cell Reports Dec 2017S-adenosylmethionine (SAM) is an important metabolite as a methyl-group donor in DNA and histone methylation, tuning regulation of gene expression. Appropriate...
S-adenosylmethionine (SAM) is an important metabolite as a methyl-group donor in DNA and histone methylation, tuning regulation of gene expression. Appropriate intracellular SAM levels must be maintained, because methyltransferase reaction rates can be limited by SAM availability. In response to SAM depletion, MAT2A, which encodes a ubiquitous mammalian methionine adenosyltransferase isozyme, was upregulated through mRNA stabilization. SAM-depletion reduced N-methyladenosine (mA) in the 3' UTR of MAT2A. In vitro reactions using recombinant METTL16 revealed multiple, conserved methylation targets in the 3' UTR. Knockdown of METTL16 and the mA reader YTHDC1 abolished SAM-responsive regulation of MAT2A. Mutations of the target adenine sites of METTL16 within the 3' UTR revealed that these mAs were redundantly required for regulation. MAT2A mRNA methylation by METTL16 is read by YTHDC1, and we suggest that this allows cells to monitor and maintain intracellular SAM levels.
Topics: 3' Untranslated Regions; Animals; HEK293 Cells; HeLa Cells; Humans; Methionine Adenosyltransferase; Methylation; Methyltransferases; Mice; Nerve Tissue Proteins; RNA Processing, Post-Transcriptional; RNA Splicing Factors; RNA Stability; RNA, Messenger; S-Adenosylmethionine
PubMed: 29262316
DOI: 10.1016/j.celrep.2017.11.092 -
Breast Cancer Research and Treatment May 2019Many transformed cells and embryonic stem cells are dependent on the biosynthesis of the universal methyl-donor S-adenosylmethionine (SAM) from methionine by the enzyme...
PURPOSE
Many transformed cells and embryonic stem cells are dependent on the biosynthesis of the universal methyl-donor S-adenosylmethionine (SAM) from methionine by the enzyme MAT2A to maintain their epigenome. We hypothesized that cancer stem cells (CSCs) rely on SAM biosynthesis and that the combination of methionine depletion and MAT2A inhibition would eradicate CSCs.
METHODS
Human triple (ER/PR/HER2)-negative breast carcinoma (TNBC) cell lines were cultured as CSC-enriched mammospheres in control or methionine-free media. MAT2A was inhibited with siRNAs or cycloleucine. The effects of methionine restriction and/or MAT2A inhibition on the formation of mammospheres, the expression of CSC markers (CD44/C24), MAT2A and CSC transcriptional regulators, apoptosis induction and histone modifications were determined. A murine model of metastatic TNBC was utilized to evaluate the effects of dietary methionine restriction, MAT2A inhibition and the combination.
RESULTS
Methionine restriction inhibited mammosphere formation and reduced the CD44/C24 CSC population; these effects were partly rescued by SAM. Methionine depletion induced MAT2A expression (mRNA and protein) and sensitized CSCs to inhibition of MAT2A (siRNAs or cycloleucine). Cycloleucine enhanced the effects of methionine depletion on H3K4me3 demethylation and suppression of Sox9 expression. Dietary methionine restriction induced MAT2A expression in mammary tumors, and the combination of methionine restriction and cycloleucine was more effective than either alone at suppressing primary and lung metastatic tumor burden in a murine TNBC model.
CONCLUSIONS
Our findings point to SAM biosynthesis as a unique metabolic vulnerability of CSCs that can be targeted by combining methionine depletion with MAT2A inhibition to eradicate drug-resistant CSCs.
Topics: Animals; Apoptosis; CD24 Antigen; Cell Line, Tumor; Disease Models, Animal; Gene Silencing; Histones; Humans; Hyaluronan Receptors; Mass Spectrometry; Methionine; Methionine Adenosyltransferase; Mice; Neoplasms; Neoplastic Stem Cells; S-Adenosylmethionine
PubMed: 30712196
DOI: 10.1007/s10549-019-05146-7 -
International Journal of Chronic... 2023Persistent inflammation and epithelial-mesenchymal transition are essential pathophysiological processes in chronic obstructive pulmonary disease (COPD) and involve...
PURPOSE
Persistent inflammation and epithelial-mesenchymal transition are essential pathophysiological processes in chronic obstructive pulmonary disease (COPD) and involve airway remodeling. m6A methylation modification was discovered to play an important role in various diseases. Nevertheless, the regulatory role of m6A methylation has not yet been investigated in cigarette smoking-induced COPD. The study aims to explore the regulatory role of m6A methylation in cigarette smoking-induced COPD.
PATIENTS AND METHODS
In this study, two Gene Expression Omnibus (GEO) datasets were first utilized to analyze the expression profiles of m6A RNA methylation regulators in COPD. We then established a cell model of COPD by exposing human bronchial epithelial cells (HBECs) to cigarette smoke extract (CSE) in vitro and detected the expression of m6A writer Mettl3 and EMT phenotype markers. RNA interference, cycloleucine, RT-qPCR, western blot, MeRIP-sequencing, and cell migration assay were performed to investigate the potential effect of Mettl3 on the EMT process in CSE-induced HBECs.
RESULTS
Our results showed that Mettl3 expression was significantly elevated in cigarette smoking-induced COPD patients and in a cellular model of COPD. Furthermore, Mettl3 silence and cycloleucine treatment inhibited the EMT process of HBECs caused by CSE. Mechanically, Mettl3 silence weakens the m6A methylation of SOCS3 mRNA to enhance the protein expression of SOCS3, inhibiting CSE-induced SOCS3/STAT3/SNAI1 signaling and EMT processes in HBECs.
CONCLUSION
Our study inferred that Mettl3-mediated m6A RNA methylation modification modulates CSE-induced EMT by targeting SOCS3 mRNA and ultimately serves as a crucial regulator in the emergence of COPD. This conclusion reinforces the regulatory role of m6A methylation in COPD.
Topics: Pulmonary Disease, Chronic Obstructive; Epithelial-Mesenchymal Transition; Cigarette Smoking; Methyltransferases; Bronchi; Cells, Cultured; Gene Knockdown Techniques; Cycloleucine
PubMed: 37275442
DOI: 10.2147/COPD.S398289 -
Animal Nutrition (Zhongguo Xu Mu Shou... Mar 2022Methionine and its hydroxy analogue (MHA) have been shown to benefit mouse intestinal regeneration. The intestinal organoid is a good model that directly reflects the...
Methionine and its hydroxy analogue (MHA) have been shown to benefit mouse intestinal regeneration. The intestinal organoid is a good model that directly reflects the impact of certain nutrients or chemicals on intestinal development. Here, we aimed to establish a chicken intestinal organoid culture method first and then use the model to explore the influence of methionine deficiency and MHA on intestinal organoid development. The results showed that 125-μm cell strainer exhibited the highest efficiency for chicken embryo crypt harvesting. We found that transforming growth factor-β inhibitor (A8301) supplementation promoted enterocyte differentiation at the expense of the proliferation of intestinal stem cells (ISC). The mitogen-activated protein kinase p38 inhibitor (SB202190) promoted intestinal organoid formation and enterocyte differentiation but suppressed the differentiation of enteroendocrine cells, goblet cells and Paneth cells. However, the suppression of enteroendocrine cell and Paneth cell differentiation by SB202190 was alleviated at the presence of A8301. The glycogen synthase kinase 3 inhibitor (CHIR99021), valproic acid (VPA) alone and their combination promoted chicken intestinal organoid formation and enterocyte differentiation at the expense of the expression of Paneth cells and goblet cells. Chicken serum significantly improved organoid formation, especially in the presence of A8301, SB202190, CHIR99021, and VPA, but inhibited the differentiation of Paneth cells and enteroendocrine cells. Chicken serum at a concentration of 0.25% meets the requirement of chicken intestinal organoid development, and the beneficial effect of chicken serum on chicken intestinal organoid culture could not be replaced by fetal bovine serum and insulin-like growth factor-1. Moreover, commercial mouse organoid culture medium supplemented with A8301, SB202190, CHIR99021, VPA, and chicken serum promotes chicken organoid budding. Based on the chicken intestinal organoid model, we found that methionine deficiency mimicked by cycloleucine suppressed organoid formation and organoid size, and this effect was reinforced with increased cycloleucine concentrations. Methionine hydroxy analogue promoted regeneration of ISC but decreased cell differentiation compared with the results obtained with L-methionine. In conclusion, our results provide a potentially excellent guideline for chicken intestinal organoid culture and insights into methionine function in crypt development.
PubMed: 34977374
DOI: 10.1016/j.aninu.2021.06.001 -
Molecules (Basel, Switzerland) Sep 2021A molecular umbrella composed of two -sulfated cholic acid residues was applied for the construction of conjugates with cispentacin, containing a "trimethyl lock" (TML)...
A molecular umbrella composed of two -sulfated cholic acid residues was applied for the construction of conjugates with cispentacin, containing a "trimethyl lock" (TML) or -dithiobenzylcarbamoyl moiety as a cleavable linker. Three out of five conjugates demonstrated antifungal in vitro activity against and but not against , with MIC values in the 0.22-0.99 mM range and were not hemolytic. Antifungal activity of the most active conjugate , containing the TML-pimelate linker, was comparable to that of intact cispentacin. A structural analogue of , containing the Nap-NH fluorescent probe, was accumulated in cells, and TML-containing conjugates were cleaved in cell-free extract of cells. These results suggest that a molecular umbrella can be successfully applied as a nanocarrier for the construction of cleavable antifungal conjugates.
Topics: Antifungal Agents; Candida albicans; Candida glabrata; Cholic Acid; Cycloleucine; Drug Carriers; Erythrocytes; Hemolytic Agents; Humans; Microbial Sensitivity Tests; Molecular Structure; Structure-Activity Relationship
PubMed: 34576946
DOI: 10.3390/molecules26185475 -
Journal of Animal Science and... Dec 2022N6-methyladenosine (mA) is an abundant post-transcriptional RNA modification that affects various biological processes. The fat mass and obesity-associated...
BACKGROUND
N6-methyladenosine (mA) is an abundant post-transcriptional RNA modification that affects various biological processes. The fat mass and obesity-associated (FTO) protein, a demethylase encoded by the FTO gene, has been found to regulate adipocyte development in an mA-dependent manner in multiple species. However, the effects of the mA methylation and FTO demethylation functions on chicken adipogenesis remain unclear. This study aims to explore the association between mA modification and chicken adipogenesis and the underlying mechanism by which FTO affects chicken preadipocyte development.
RESULTS
The association between mA modification and chicken lipogenesis was assessed by treating chicken preadipocytes with different doses of methyl donor betaine and methylation inhibitor cycloleucine. The results showed that betaine significantly increased methylation levels and inhibited lipogenesis, and the inverse effect was found in preadipocytes after cycloleucine treatment. Overexpression of FTO significantly inhibited mA levels and promoted proliferation and differentiation of chicken preadipocytes. Silencing FTO showed opposite results. Mechanistically, FTO overexpression increased the expression of catenin beta 1 (CTNNB1) by improving RNA stability in an mA-dependent manner, and we proved that FTO could directly target CTNNB1. Furthermore, CTNNB1 may be a positive regulator of adipogenesis in chicken preadipocytes.
CONCLUSIONS
mA methylation of RNA was negatively associated with adipogenesis of chicken preadipocytes. FTO could regulate CTNNB1 expression in a demethylation manner to promote lipogenesis.
PubMed: 36461116
DOI: 10.1186/s40104-022-00795-z -
BMC Molecular and Cell Biology Oct 2022Acetaminophen is commonly recommended for the early analgesia of osteoarthritis. However, the molecular mechanism by which it acts remains unknown. The aim of this study...
BACKGROUND
Acetaminophen is commonly recommended for the early analgesia of osteoarthritis. However, the molecular mechanism by which it acts remains unknown. The aim of this study is to investigate the effect of acetaminophen on inflammation and extracellular matrix degradation in human chondrocytes, and the possible molecular mechanisms involved in its effect.
METHODS
The normal chondrocyte cell line C28/I2 was treated with interleukin-1β to mimic the inflammatory state. Acetaminophen and the methylation inhibitor (cycloleucine) were used to treat interleukin-1β-induced C28/I2 cells. The expression of RNA N-methyladenosine -related proteins was detected by RT-qPCR and western blot. The total RNA N-methyladenosine level was measured by dot blot analysis and enzyme linked immunosorbent assay. The levels of interleukin-6, interleukin-8 and anti-tumor necrosis factor-α were measured by enzyme linked immunosorbent assay. The extracellular matrix synthesis and degradation were examined by western blot.
RESULTS
After interleukin-1β stimulated C28/I2 cells, the intracellular RNA N-methyladenosine level increased, and the expression of regulatory proteins also changed, mainly including the increased expression of methyltransferase like 3 and the downregulated expression of AlkB family member 5. The use of cycloleucine inhibited interleukin-1β-induced inflammation and extracellular matrix degradation by inhibiting RNA N-methyladenosine modification. In contrast, acetaminophen treatment counteracted interleukin-1β-induced changes in RNA N-methyladenosine levels and regulatory protein expression. Furthermore, acetaminophen treatment of interleukin-1β-induced C28/I2 cells inhibited the secretion of interleukin-6, interleukin-8 and anti-tumor necrosis factor-α, down-regulated the expression of matrix metalloproteinase-13 and Collagen X, and up-regulated the expression of collagen II and aggrecan. In addition, AlkB family member 5 overexpression activated interleukin-1β-induced chondrocyte viability and suppressed inflammation and extracellular matrix degradation.
CONCLUSION
Acetaminophen affects inflammatory factors secretion and extracellular matrix synthesis of human chondrocytes by regulating RNA N-methyladenosine level and N-methyladenosine-related protein expression. Stimulation of the normal chondrocyte cell line C28/I2 with the cytokine IL-1β (10 μM) mimics the inflammatory state in vitro. Acetaminophen (Ace, 50 μg/mL) changes the mA related proteins expression and the total RNA mA levels in IL-1β-treated chondrocyte cells. Furthermore, regulation of RNA mA levels (by methylation inhibitor Cyc and/or Ace) affects IL-1β-induced inflammatory cytokines secretion and extracellular matrix synthesis in C28/I2 cells.
Topics: Humans; Acetaminophen; Cells, Cultured; Chondrocytes; Cycloleucine; Inflammation; Interleukin-1beta; Interleukin-6; Interleukin-8; Necrosis; RNA
PubMed: 36303109
DOI: 10.1186/s12860-022-00444-3