scholarly journals Alpha Enolase 1 Ubiquitination and Degradation Mediated by Ehrlichia chaffeensis TRP120 Disrupts Glycolytic Flux and Promotes Infection

Pathogens ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 962
Author(s):  
Bing Zhu ◽  
Jere W. McBride
Keyword(s):  
Host Cell ◽  
Ectopic Expression ◽  
Metabolic Reprogramming ◽  
Glycolytic Flux ◽  
Ehrlichia Chaffeensis ◽  
Direct Role ◽  
Cell Processes

Ehrlichia chaffeensis modulates numerous host cell processes, including gene transcription to promote infection of the mononuclear phagocyte. Modulation of these host cell processes is directed through E. chaffeensis effectors, including TRP120. We previously reported that TRP120 moonlights as a HECT E3 Ub ligase that ubiquitinates host cell transcription and fate regulators (PCGF5 and FBW7) to promote infection. In this study, we identified a novel TRP120 substrate and examined the relationship between TRP120 and α-enolase (ENO1), a metalloenzyme that catalyzes glycolytic pathway substrate dehydration. Immunofluorescence microscopy and coimmunoprecipitation demonstrated interaction between ENO1 and TRP120, and ubiquitination of ENO-1 by TRP120 was detected in vivo and in vitro. Further, ENO-1 degradation was observed during infection and was inhibited by the proteasomal inhibitor bortezomib. A direct role of TRP120 Ub ligase activity in ENO-1 degradation was demonstrated and confirmed by ectopic expression of TRP120 HECT Ub ligase catalytic site mutant. siRNA knockdown of ENO-1 coincided with increased E. chaffeensis infection and ENO-1 knockdown disrupted glycolytic flux by decreasing the levels of pyruvate and lactate that may contribute to changes in host cell metabolism that promote infection. In addition, we elucidated a functional role of TRP120 auto-ubiquitination as an activating event that facilitates the recruitment of the UbcH5 E2 ubiquitin-conjugating enzyme. This investigation further expands the repertoire of TRP120 substrates and extends the potential role of TRP120 Ub ligase in infection to include metabolic reprogramming.

scholarly journals Let-7a regulates EV secretion and mitochondrial oxidative phosphorylation by targeting SNAP23 in colorectal cancer

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
You Dong Liu ◽  
Xiao Peng Zhuang ◽  
Dong Lan Cai ◽  
Can Cao ◽  
Qi Sheng Gu ◽  
...  
Keyword(s):  
Oxidative Phosphorylation ◽  
Metabolic Reprogramming ◽  
Luciferase Reporter ◽  
Mitochondrial Oxidative Phosphorylation ◽  
In Vivo Assays ◽  
Reporter Assays ◽  
Extracellular Mirna

Abstract Background MicroRNAs (miRNAs) are abundant in tumor-derived extracellular vesicles (EVs) and the functions of extracellular miRNA to recipient cells have been extensively studied with tumorigenesis. However, the role of miRNA in EV secretion from cancer cells remains unknown. Methods qPCR and bioinformatics analysis were applied for determining extracellular let-7a expression from CRC patient serum and cells. Nanosight particle tracking analysis was performed for investigating the effect of let-7a on EV secretion. Luciferase reporter assays was used for identifying targeted genes synaptosome-associated protein 23 (SNAP23). In vitro and in vivo assays were used for exploring the function of let-7a/SNAP23 axis in CRC progression. Bioenergetic assays were performed for investigating the role of let-7a/SNAP23 in cellular metabolic reprogramming. Results let-7a miRNA was elevated in serum EVs from CRC patients and was enriched in CRC cell-derived EVs. We determined that let-7a could suppress EV secretion directly targeting SNAP23. In turn, SNAP23 promotes EV secretion of let-7a to downregulate the intracellular let-7a expression. In addition, we found a novel mechanism of let-7a/SNAP23 axis by regulating mitochondrial oxidative phosphorylation (OXPHOS) through Lin28a/SDHA signaling pathway. Conclusions Let-7a plays an essential role in not only inhibiting EV secretion, but also suppressing OXPHOS through SNAP23, resulting in the suppression of CRC progression, suggesting that let-7a/SNAP23 axis could provide not only effective tumor biomarkers but also novel targets for tumor therapeutic strategies.

scholarly journals Hypermethylation of DHRS3 as a Novel Tumor Suppressor Involved in Tumor Growth and Prognosis in Gastric Cancer

2021 ◽  
Vol 9 ◽  
Author(s):  
Sha Sumei ◽  
Kong Xiangyun ◽  
Chen Fenrong ◽  
Sun Xueguang ◽  
Hu Sijun ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Tumor Suppressor ◽  
Tumor Growth ◽  
Human Cancer ◽  
Methylation Status ◽  
Ectopic Expression ◽  
Survival Times

Background/AimsThe role of DHRS3 in human cancer remains unclear. Our study explored the role of DHRS3 in gastric cancer (GC) and its clinicopathological significance and associated mechanisms.MaterialsBisulfite-assisted genomic sequencing PCR and a Mass-Array system were used to evaluate and quantify the methylation levels of the promoter. The expression levels and biological function of DHRS3 was examined by both in vitro and in vivo assays. A two-way hierarchical cluster analysis was used to classify the methylation profiles, and the correlation between the methylation status of the DHRS3 promoter and the clinicopathological characteristics of GC were then assessed.ResultsThe DHRS3 promoter was hypermethylated in GC samples, while the mRNA and protein levels of DHRS3 were significantly downregulated. Ectopic expression of DHRS3 in GC cells inhibited cell proliferation and migration in vitro, decreased tumor growth in vivo. DHRS3 methylation was correlated with histological type and poor differentiation of tumors. GC patients with high degrees of CpG 9.10 methylation had shorter survival times than those with lower methylation.ConclusionDHRS3 was hypermethylated and downregulated in GC patients. Reduced expression of DHRS3 is implicated in gastric carcinogenesis, which suggests DHRS3 is a tumor suppressor.

scholarly journals Mutations affecting the tRNA-splicing endonuclease activity of Saccharomyces cerevisiae.

Genetics ◽  
1988 ◽  
Vol 118 (4) ◽  
pp. 609-617
Author(s):  
M Winey ◽  
M R Culbertson
Keyword(s):  
Growth Defect ◽  
Temperature Sensitive ◽  
Gene Products ◽  
Endonuclease Activity ◽  
Temperature Dependent ◽  
Direct Role ◽  
Trna Splicing

Abstract Two unlinked mutations that alter the enzyme activity of tRNA-splicing endonuclease have been identified in yeast. The sen1-1 mutation, which maps on chromosome 12, causes temperature-sensitive growth, reduced in vitro endonuclease activity, and in vivo accumulation of unspliced pre-tRNAs. The sen2-1 mutation does not confer a detectable growth defect, but causes a temperature-dependent reduction of in vitro endonuclease activity. Pre-tRNAs do not accumulate in sen2-1 strains. The in vitro enzyme activities of sen1-1 and sen2-1 complement in extracts from a heterozygous diploid, but fail to complement in mixed extracts from separate sen1-1 and sen2-1 haploid strains. These results suggest a direct role for SEN gene products in the enzymatic removal of introns from tRNA that is distinct from the role of other products known to affect tRNA splicing.

scholarly journals Linc00426 accelerates lung adenocarcinoma progression by regulating miR-455-5p as a molecular sponge

2020 ◽  
Vol 11 (12) ◽  
Author(s):  
Hongli Li ◽  
Qingjie Mu ◽  
Guoxin Zhang ◽  
Zhixin Shen ◽  
Yuanyuan Zhang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Molecular Mechanisms ◽  
Epithelial Mesenchymal Transition ◽  
Ectopic Expression ◽  
Tumor Development ◽  
Biological Significance ◽  
Mesenchymal Transition

AbstractIncreasing lines of evidence indicate the role of long non-coding RNAs (LncRNAs) in gene regulation and tumor development. Hence, it is important to elucidate the mechanisms of LncRNAs underlying the proliferation, metastasis, and invasion of lung adenocarcinoma (LUAD). We employed microarrays to screen LncRNAs in LUAD tissues with and without lymph node metastasis and revealed their effects on LUAD. Among them, Linc00426 was selected for further exploration in its expression, the biological significance, and the underlying molecular mechanisms. Linc00426 exhibits ectopic expression in LUAD tissues and cells. The ectopic expression has been clinically linked to tumor size, lymphatic metastasis, and tumor differentiation of patients with LUAD. The deregulation of Linc00426 contributes to a notable impairment in proliferation, invasion, metastasis, and epithelial–mesenchymal transition (EMT) in vitro and in vivo. Mechanistically, the deregulation of Linc00426 could reduce cytoskeleton rearrangement and matrix metalloproteinase expression. Meanwhile, decreasing the level of Linc00426 or increasing miR-455-5p could down-regulate the level of UBE2V1. Thus, Linc00426 may act as a competing endogenous RNA (ceRNA) to abate miR-455-5p-dependent UBE2V1 reduction. We conclude that Linc00426 accelerates LUAD progression by acting as a molecular sponge to regulate miR-455-5p, and may be a potential novel tumor marker for LUAD.

scholarly journals Transcription factor MBF-I interacts with metal regulatory elements of higher eucaryotic metallothionein genes.

1989 ◽  
Vol 9 (12) ◽  
pp. 5315-5323 ◽  
Author(s):  
J Imbert ◽  
M Zafarullah ◽  
V C Culotta ◽  
L Gedamu ◽  
D Hamer
Keyword(s):  
Gene Transcription ◽  
Regulatory Elements ◽  
Gene Promoters ◽  
Direct Role ◽  
Transcription In Vitro ◽  
Mouse Cells ◽  
Affinity Reagent

Metallothionein (MT) gene promoters in higher eucaryotes contain multiple metal regulatory elements (MREs) that are responsible for the metal induction of MT gene transcription. We identified and purified to near homogeneity a 74-kilodalton mouse nuclear protein that specifically binds to certain MRE sequences. This protein, MBF-I, was purified employing as an affinity reagent a trout MRE that is shown to be functional in mouse cells but which lacks the G+C-rich and SP1-like sequences found in many mammalian MT gene promoters. Using point-mutated MREs, we showed that there is a strong correlation between DNA binding in vitro and MT gene regulation in vivo, suggesting a direct role of MBF-I in MT gene transcription. We also showed that MBF-I can induce MT gene transcription in vitro in a mouse extract and that this stimulation requires zinc.

A Critical Role of Mir-9 in Myelopoesis and EVI1-Induced Leukemogenesis.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2332-2332
Author(s):  
Vitalyi Senyuk ◽  
Yunyuan Zhang ◽  
Yang Liu ◽  
Ming Ming ◽  
Jianjun Chen ◽  
...  
Keyword(s):  
Cell Transformation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Ectopic Expression ◽  
Myeloid Differentiation ◽  
Dna Hypermethylation ◽  
Hematopoietic Stem

Abstract Abstract 2332 MicroRNA-9 (miR-9) is required for normal neurogenesis and organ development. The expression of miR-9 is altered in several types of solid tumors suggesting that it may have a function in cell transformation. However the role of this miR in normal hematopoiesis and leukemogenesis is unknown. Here we show that miR-9 is expressed at low levels in hematopoietic stem/progenitor cells (HSCs/HPCs), and that it is upregulated during hematopoietic differentiation. Ectopic expression of miR-9 strongly accelerates terminal myelopoiesis, while promoting apoptosis in vitro and in vivo. In addition, the inhibition of miR-9 in HPC with a miRNA sponge blocks myelopoiesis. EVI1, required for normal embryogenesis, and is considered an oncogene because inappropriate upregulation induces malignant transformation in solid and hematopoietic cancers. In vitro, EVI1 severely affects myeloid differentiation. Here we show that EVI1 binds to the promoter of miR-9–3 leading to DNA hypermethylation of the promoter as well as repression of miR-9. We also show that ectopic miR-9 reverses the myeloid differentiation block that is induced by EVI1. Our findings suggest that inappropriately expressed EVI1 delays or blocks myeloid differentiation, at least in part by DNA hypermethylation and downregulation of miR-9. It was previously reported that FoxOs genes inhibit myeloid differentiation and prevent differentiation of leukemia initiating cells. Here we identify FoxO3 and FoxO1 as new direct targets of miR-9 in hematopoietic cells, and we find that upregulation of FoxO3 in miR-9-positive cells reduces the acceleration of myelopoiesis. These results reveal a novel role of miR-9 in myelopoiesis and in the pathogenesis of EVI1-induced myeloid neoplasms. They also provide new insights on the potential chromatin-modifying role of oncogenes in epigenetic changes in cancer cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Branched chain amino acid aminotransferase 2 Regulates Ferroptotic Cell Death in Cancer Cells

2020 ◽  
Author(s):  
Kang Wang ◽  
Zhengyang Zhang ◽  
Tsai Hsiang-i ◽  
Yanfang Liu ◽  
Ming Wang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cancer Cells ◽  
Therapeutic Strategy ◽  
Ectopic Expression ◽  
Branched Chain Amino Acid ◽  
Pancreatic Cancer Cells ◽  
Branched Chain

AbstractFerroptosis has been implicated as a tumor-suppressor function for cancer therapy. Recently the sensitivity to ferroptosis was tightly linked to numerous biological processes, including metabolism of amino acid. Here, using a high-throughput CRISPR/Cas9 based genetic screen in HepG2 cells to search for metabolic proteins inhibiting ferroptosis, we identified branched chain amino acid aminotransferase 2 (BCAT2) as a novel suppressor of ferroptosis. Mechanistically, ferroptosis inducers (erastin, sorafenib and sulfasalazine) activated AMPK/SREBP1 signaling pathway through ferritinophagy, which in turn inhibited BCAT2 transcription. We further confirmed that BCAT2 mediating the metabolism of sulfur amino acid, regulated intracellular glutamate level, whose activation by ectopic expression specifically antagonize system Xc– inhibition and protected liver and pancreatic cancer cells from ferroptosis in vitro and in vivo. Finally, our results demonstrate the synergistic effect of sorafenib and sulfasalazine in downregulating BCAT2 expression and dictating ferroptotic death, where BCAT2 can also be used to predict the responsiveness of cancer cells to ferroptosis-inducing therapies. Collectively, these findings identify a novel role of BCAT2 in ferroptosis, suggesting a potential therapeutic strategy for overcoming sorafenib resistance.

scholarly journals AL355338 acts as an oncogenic lncRNA by interacting with protein ENO1 to regulate EGFR/AKT pathway in NSCLC

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Qian Hua ◽  
Dongliang Wang ◽  
Lin Zhao ◽  
Zhihui Hong ◽  
Kairu Ni ◽  
...  
Keyword(s):  
Aerobic Glycolysis ◽  
Transcript Abundance ◽  
Metabolic Reprogramming ◽  
Clinical Samples ◽  
Abnormal Metabolism ◽  
Considerable Morbidity

Abstract Background Non-small cell lung cancer (NSCLC) is a malignancy with considerable morbidity and mortality. Abnormal metabolism is a hallmark of cancer; however, the mechanism of glycolysis regulation in NSCLC progression is not completely understood. Recent studies suggest that some dysregulated long non-coding RNAs (lncRNAs) play important roles in tumor metabolic reprogramming. Methods To identify glycolysis-associated-lncRNAs in NSCLC, we compared RNA-sequencing results between high 18F-fluorodeoxyglucose (FDG)-uptake NSCLC tissues and paired paratumor tissues. The transcript abundance of AL355338 in 80 pairs of clinical samples was evaluated by quantitative real-time PCR assay and fluorescence in situ hybridization. The biological role of AL355338 on NSCLC cells were evaluated by functional experiments in vitro and in vivo. Moreover, RNA pull-down, mass spectrometry and RNA immunoprecipitation (RIP) assays were used to identify the protein interacted with AL355338. Co-immunoprecipitation, in situ proximity ligation assays and western blotting were applied to define the potential downstream pathways of AL355338. Results AL355338 was an upregulated glycolysis-associated lncRNA in NSCLC. Functional assays revealed that AL355338 was critical for promoting aerobic glycolysis and NSCLC progression. Mechanistic investigations showed that AL355338 directly bound with alpha-enolase (ENO1) and enhanced the protein’s stability by modulating its degradation and ubiquitination. A positive correlation was observed between AL355338 and ENO1 in NSCLC, and ENO1 was subsequently confirmed to be responsible for the oncogenic role of AL355338. Furthermore, AL355338 was capable of modulating ENO1/EGFR complex interaction and further activating EGFR-AKT signaling. Conclusions This study indicates that AL355338 confers an aggressive phenotype to NSCLC, and targeting it might be an effective therapeutic strategy.

scholarly journals Wnt/beta-catenin signalling is dispensable for adult neural stem cell homeostasis and activation

2020 ◽  
Author(s):  
Sophie H. L. Austin ◽  
Lachlan Harris ◽  
Oana Paun ◽  
Piero Rigo ◽  
François Guillemot ◽  
...  
Keyword(s):  
Stem Cell ◽  
Beta Catenin ◽  
Dependent Manner ◽  
Direct Role ◽  
Adult Nscs ◽  
Hippocampal Networks ◽  
Dose Dependent

AbstractAdult mouse hippocampal neural stem cells (NSCs) generate new neurons that integrate into existing hippocampal networks and modulate mood and memory. These NSCs are largely quiescent and are stimulated by niche signals to activate and produce neurons. Wnt/β-catenin signalling acts at different steps along the hippocampal neurogenic lineage and has been shown to promote the proliferation of intermediate progenitor cells. However, whether it has a direct role in the regulation of NSCs still remains unclear. Here we used Wnt/β-catenin reporters and transcriptomic data from in vivo and in vitro models to show that both active and quiescent adult NSCs respond to Wnt/β-catenin signalling. Wnt/β-catenin stimulation instructed neuronal differentiation of active NSCs and promoted the activation or differentiation of quiescent NSCs in a dose-dependent manner. However, we found that inhibiting NSCs response to Wnt, by conditionally deleting β-catenin, did not affect their activation or maintenance of their stem cell characteristics. Together, our results indicate that whilst NSCs do respond to Wnt/β-catenin stimulation in a dose-dependent and state-specific manner, Wnt/β-catenin signalling is not cell-autonomously required to maintain NSC homeostasis, which could reconcile some of the contradictions in the literature as to the role of Wnt/β-catenin signalling in adult hippocampal NSCs.

scholarly journals Gout and pseudo-gout-related crystals promote GLUT1-mediated glycolysis that governs NLRP3 and interleukin-1β activation on macrophages

2020 ◽  
Vol 79 (11) ◽  
pp. 1506-1514
Author(s):  
Felix Renaudin ◽  
Lucie Orliaguet ◽  
Florence Castelli ◽  
François Fenaille ◽  
Aurelie Prignon ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Aerobic Glycolysis ◽  
Metabolic Reprogramming ◽  
Metabolic Phenotype ◽  
Glucose Transporter 1 ◽  
Gout Flare

ObjectiveMacrophage activation by monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals mediates an interleukin (IL)-1β-dependent inflammation during gout and pseudo-gout flare, respectively. Since metabolic reprogramming of macrophages goes along with inflammatory responses dependently on stimuli and tissue environment, we aimed to decipher the role of glycolysis and oxidative phosphorylation in the IL-1β-induced microcrystal response.MethodsBriefly, an in vitro study (metabolomics and real-time extracellular flux analysis) on MSU and CPP crystal-stimulated macrophages was performed to demonstrate the metabolic phenotype of macrophages. Then, the role of aerobic glycolysis in IL-1β production was evaluated, as well in vitro as in vivo using 18F-fluorodeoxyglucose positron emission tomography imaging and glucose uptake assay, and molecular approach of glucose transporter 1 (GLUT1) inhibition.ResultsWe observed that MSU and CPP crystals led to a metabolic rewiring toward the aerobic glycolysis pathway explained by an increase in GLUT1 plasma membrane expression and glucose uptake on macrophages. Also, neutrophils isolated from human synovial fluid during gout flare expressed GLUT1 at their plasma membrane more frequently than neutrophils isolated from bloodstream. Both glucose deprivation and treatment with either 2-deoxyglucose or GLUT1 inhibitor suppressed crystal-induced NLRP3 activation and IL-1β production, and microcrystal inflammation in vivo.ConclusionIn conclusion, we demonstrated that GLUT1-mediated glucose uptake is instrumental during the inflammatory IL-1β response induced by MSU and CPP crystals. These findings open new therapeutic paths to modulate crystal-related inflammation.

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