Dysfunction of the energy sensor NFE2L1 triggers uncontrollable AMPK signaling and glucose metabolism reprogramming.

The antioxidant transcription factor NFE2L1 (also called Nrf1) acts as a core regulator of redox signaling and metabolism homeostasis, and thus, its dysfunction results in multiple systemic metabolic diseases. However, the molecular mechanism(s) by which NFE2L1 regulates glycose and lipid metabolism remains elusive. Here, we found that loss of NFE2L1 in human HepG2 cells led to a lethal phenotype upon glucose deprivation and NFE2L1 deficiency could affect the uptake of glucose. Further experiments revealed that glycosylation of NFE2L1 enabled it to sense the energy state. These results indicated that NFE2L1 can serve as a dual sensor and regulator of glucose homeostasis. The transcriptome, metabolome, and seahorse data further revealed that disruption of NFE2L1 could reprogram glucose metabolism to aggravate the Warburg effect in NFE2L1-silenced hepatoma cells, concomitant with mitochondrial damage. Co-expression and Co-immunoprecipitation experiments demonstrated that NFE2L1 could directly interact and inhibit AMPK. Collectively, NFE2L1 functioned as an energy sensor and negatively regulated AMPK signaling through directly interacting with AMPK. The novel NFE2L1/AMPK signaling pathway delineate the mechanism underlying of NFE2L1-related metabolic diseases and highlight the crosstalk between redox homeostasis and metabolism homeostasis.

Feedback regulation of antioxidant transcription factor NFE2L1 and immunostimulatory factor 41BBL mediates the crosstalk between oxidative stress and tumor immunity.

Targeting the immune checkpoint to inhibit tumor immune escape, which is one of the fundamental causes of cancer, has become an important strategy for cancer treatment. The molecular mechanism of tumor immune escape involved in the process of spontaneous hepatocellular carcinoma after specifically knocking out NFE2L1, the core regulator of redox homeostasis, in the mouse liver is still unclear. Transcriptome data showed that the immunostimulatory TNFSF9/41BBL was significantly reduced in NFE2L1 knockdown hepatocarcinoma HepG2 cells, and this suggests that 41BBL may be an oxidative stress-responsive immune checkpoint. The results of the promoter activity experiment showed that NFE2L1 can promote 41BBL gene transcription activation through the ARE element in the promoter region. In addition, cell biology experiments have found that overexpression of 41BBL can inhibit cell proliferation and promote senescence. Importantly, reactive oxygen species in cells significantly increased after overexpression of 41BBL, whereas NFE2L1 was inhibited, indicating that 41BBL has the effect of feedback regulating oxidative stress in cells. In conclusion, in this study, the transcriptional activation effect of NFE2L1 on 41BBL and the feedback inhibition relationship of 41BBL on NFE2L1 was clarified. The NFE2L1/41BBL axis might be an important pathway that mediates the crosstalk between oxidative stress and the tumor immune response.

Current Simultaneous Discrimination of Mismatched MicroRNAs Using Base-Flipping within the α-Hemolysin Latch.

The simultaneous discrimination of let-7 microRNAs (miRNAs) would greatly facilitate the early diagnosis and prognosis monitoring of diseases. In this work, a molecular beacon DNA probe was designed to be able to flip out its mismatched cytosine base within the α-hemolysin (α-HL) latch and generate completely separated blocking currents to identify the single-base difference. As a result, the characteristic blocking current of fully matched MB/let-7a and single-base mismatched MB/let-7f was 84.30 ± 0.92 and 87.05 ± 0.86% (confidence level P 95%), respectively. Let-7 miRNA family let-7a and let-7f were completely simultaneously discriminated, which could be attributed to the following strengths. (1) The statistic distribution of blocking current is extremely concentrated with a small relative standard deviation (RSD) of less than 1% and a narrow distribution range. (2) Complete separation is achieved with a high separation resolution of 1.54. (3) The cytosine base flipping out within the α-HL latch provides a universal labeling-free strategy to simultaneously discriminate the single-base mismatch. Overall, the target let-7f sequences were detected with a linear range from 0.001 to 10 pM in human serum samples containing 200 nM let-7a. Great potential has been demonstrated for precise detection, early diagnosis, and prognosis monitoring of diseases related to single-base difference.

Metformin leads to accumulation of reactive oxygen species by inhibiting the NFE2L1 expression in human hepatocellular carcinoma cells.

Metformin, as the first-line drug for the treatment of type 2 diabetes mellitus, has been shown to possess a capability to activate or inhibit the production of reactive oxygen species (ROS) in different ways. However, the detailed mechanisms of the opposite effect are poorly understood. Here we provide evidence that metformin induces accumulation of ROS by inhibiting the expression of a core antioxidant transcription factor nuclear factor erythroid 2 like 1 (NFE2L1/Nrf1) in human hepatocellular carcinoma HepG2 cells. In the present study, we originally found that the increased ROS induced by metformin was blunted in NFE2L1 knockdown cell line. Furtherly by examining the effects of metformin on endogenous and exogenous NFE2L1, we also found metformin could not only inhibit the transcription of NFE2L1 gene, but also promote the degradation of NFE2L1 protein at the post-transcriptional level, whereas this effect can be reversed by high glucose. The inhibitory effect of metformin on NFE2L1 was investigated to occur through the N-terminal domain (NTD) of NFE2L1 protein, and its downregulation by metformin was in an AMP-activated protein kinase (AMPK)-independent manner. But the activation of AMPK signaling pathway by metformin in NFE2L1 knockdown HepG2 cells is reversed, indicating that NFE2L1 may be an important regulator of AMPK signal. Altogether, this work provides a better understanding of the relationship between metformin and oxidative stress, and hence contributes to translational study of metformin through its hypoglycemic and tumor suppressive effects.

Simultaneous Discrimination of Single-Base Mismatch and Full Match Using a Label-Free Single-Molecule Strategy.

Identification of single-base mismatches has found wide applications in disease diagnosis, pharmacogenetics, and population genetics. However, there is still a great challenge in the simultaneous discrimination of single-base mismatch and full match. Combined with a nanopore electrochemical sensor, a shared-stem structure of molecular beacon was designed that did not need the labeling, but achieved an enhanced signal-to-background ratio of ∼104, high thermodynamic stability to bind with target sequences, and a fast hybridization rate. Fully matched and single-base mismatched sequences were simultaneously discriminated at the single-molecule level, which is expected to have potential applications ranging from the quick detection, early clinical diagnostics to point-of-care research.

Insight into How Telomeric G-Quadruplexes Enhance the Peroxidase Activity of Cellular Hemin.

The toxic oxidative damage of G-quadruplexes (G4), linked to neurodegenerative diseases, may arise from their ability to bind and oxidatively activate cellular hemin. However, there have been no precise studies on how telomeric G4 enhances the low intrinsic peroxidase activity of hemin. Herein, a label-free and nanopore-based strategy was developed to explore the enhancement mechanism of peroxidase activity of hemin induced by telomeric G4 (d(TTAGGG)n ). The nanopore-based strategy demonstrated that there were simultaneously two different binding modes of telomere G4 to hemin. At the single-molecule level, it was found that the hybrid structural telomeric G4 directly binds to hemin (the affinity constant (Ka )≈106 m-1 ) to form a tight complex, and some of them underwent a topological change to a parallel structure with an enhancement of Ka to approximately 107 m-1 . Through detailed analysis of the topology and peroxidase activity and molecular modeling investigations, the parallel telomere G4/hemin DNAzyme structure was proven to be preferable for high peroxidase activity. Upon strong π-π stacking, the parallel structural telomere G4 supplied a key axial ligand to the hemin iron, which accelerated the intermediate compound formation with H2 O2 in the catalytic cycle. Our studies developed a label-free and single-molecule strategy to fundamentally understand the catalytic activity and mechanism of telomeric DNAzyme, which provides some support for utilizing the toxic, oxidative-damage property in cellular oxidative disease and anticancer therapeutics.

Ultrasensitive Visual Detection of HIV DNA Biomarkers via a Multi-amplification Nanoplatform.

Methodologies to detect disease biomarkers at ultralow concentrations can potentially improve the standard of living. A facile and label-free multi-amplification strategy is proposed for the ultrasensitive visual detection of HIV DNA biomarkers in real physiological media. This multi-amplification strategy not only exhibits a signficantly low detection limit down to 4.8 pM but also provides a label-free, cost-effective and facile technique for visualizing a few molecules of nucleic acid analyte with the naked eye. Importantly, the biosensor is capable of discriminating single-based mismatch lower than 5.0 nM in human serum samples. Moreover, the visual sensing platform exhibits excellent specificity, acceptable reusability and a long-term stability. All these advantages could be attributed to the nanofibrous sensing platform that 1) has a high surface-area-to-volume provided by electrospun nanofibrous membrane, and 2) combines glucose oxidase (GOx) biocatalysis, DNAzyme-catalyzed colorimetric reaction and catalytic hairpin assembly (CHA) recycling amplification together. This multi-amplification nanoplatform promises label-free and visual single-based mismatch DNA monitoring with high sensitivity and specificity, suggesting wide applications that range from virus detection to genetic disease diagnosis.

An enhanced chemiluminescence bioplatform by confining glucose oxidase in hollow calcium carbonate particles.

A chemiluminescence (CL) amplification platform based on HCC/Lucigenin&GOx (HLG) film was developed. Hollow structural calcium carbonate (HCC) particles were used as alternative materials for carrying both enzyme and CL reagent. The model enzyme (GOx), immobilized in confined space of HCC particles, exhibited an improved biocatalysis. The Michaelis constant (Km) and the enzymatic rate constant (kcat) were determined to be 0.209 µM and 2.21 s(-1), respectively, which are much better than those of either free GOx in aqueous solution or the GOx immobilized on common nanomaterials. Based on the HLG platform, CL signal was effectively amplified and visualized after adding trace glucose, which could be attributed to the HCC particles' high biocompatibility, large specific surface area, attractive interfacial properties and efficient interaction with analyses. The visual CL bioplatform showed an excellent performance with high selectivity, wide linear range and low detection limit for sensing trace glucose. Because it eliminates the need of complicated assembly procedure and enables visualization by the naked eye, the sensitive and selective CL bioplatform would provide wide potential applications in disease diagnosis and food safety.