Oxidative stress promotes liver cancer metastasis via a PKA-activated RNF25/ECAD/YAP circuit

Loss of E-cadherin (ECAD), often caused by epigenetic inactivation, is closely associated with tumor metastasis. However, how ECAD is regulated in response to oxidative stress during tumorigenesis is largely unknown. Here we identify RNF25 as a new E3 ligase of ECAD, whose activation by oxidative stress leads to ECAD protein degradation in hepatocellular carcinoma (HCC). Loss of ECAD activates YAP, which in turn promotes the transcription of RNF25, thus forming a positive feedback loop to sustain the ECAD downregulation. YAP activation mitigates oxidative stress in detached HCC cells by upregulating antioxidant genes, protecting detached HCC cells from ferroptosis, resulting in anoikis resistance. Mechanistically, we found that protein kinase A (PKA) senses oxidative stress by redox modification in its β catalytic subunit (PRKACB) at Cys200 and Cys344, which increases its kinase activity towards RNF25 phosphorylation at Ser450, facilitating RNF25-mediated degradation of ECAD. Moreover, RNF25 expression is associated with HCC metastasis and depletion of RNF25 is sufficient to diminish HCC invasion and metastasis in vitro and in vivo. Together, these results identify a dual role of RNF25 as a critical regulator of ECAD protein turnover, promoting both anoikis resistance and metastasis, and PKA is a necessary redox sensor to enable this process. Our study provides mechanistic insight into how tumor cells sense oxidative stress signals to spread while escaping cell death.

FOXO1 Is a Critical Switch Molecule for Autophagy and Apoptosis of Sow Endometrial Epithelial Cells Caused by Oxidative Stress

Oxidative stress (OS) is involved in various reproductive diseases and can induce autophagy and apoptosis, which determine the different fates of cells. However, the sequence and the switch mechanism between autophagy and apoptosis are unclear. Here, we reported that chronic restraint stress (CRS) induced OS (decreased T-AOC, T-SOD, CAT and GSH-Px and increased MDA) and then disturbed the endocrine environment of sows during early pregnancy, including the hypothalamic-pituitary-ovarian (HPO) and the hypothalamic-pituitary-adrenal (HPA) axes. Meanwhile, after CRS, the KEAP1/NRF2 pathway was inhibited and attenuated the antioxidative ability to cause OS of the endometrium. The norepinephrine (NE) triggered β2-AR to activate the FOXO1/NF-κB pathway, which induced endometrial inflammation. CRS induced the caspase-dependent apoptosis pathway and caused MAP1LC3-II accumulation, SQSTM1/p62 degradation, and autophagosome formation to initiate autophagy. Furthermore, in vitro, a cellular OS model was established by adding hydrogen peroxide into cells. Low OS maintained the viability of endometrial epithelial cells by triggering autophagy, while high OS induced cell death by initiating caspase-dependent apoptosis. Autophagy preceded the occurrence of apoptosis, which depended on the subcellular localization of FOXO1. In the low OS group, FOXO1 was exported from the nucleus to be modified into Ac-FOXO1 and bound to ATG7 in the cytoplasm, which promoted autophagy to protect cells. In the high OS group, FOXO1 located in the nucleus to promote transcription of proapoptotic proteins and then induce apoptosis. Here, FOXO1, as a redox sensor switch, regulated the transformation of cell autophagy and apoptosis. In summary, the posttranslational modification of FOXO1 may become the target of OS treatment.

Redox-Sensitive VDAC: A Possible Function as an Environmental Stress Sensor Revealed by Bioinformatic Analysis

Voltage-dependent anion-selective channel (VDAC) allows the exchange of small metabolites and inorganic ions across the mitochondrial outer membrane. It is involved in complex interactions that regulate mitochondrial and cellular functioning. Many organisms have several VDAC paralogs that play distinct but poorly understood roles in the life and death of cells. It is assumed that such a large diversity of VDAC-encoding genes might cause physiological plasticity to cope with abiotic and biotic stresses known to impact mitochondrial function. Moreover, cysteine residues in mammalian VDAC paralogs may contribute to the reduction–oxidation (redox) sensor function based on disulfide bond formation and elimination, resulting in redox-sensitive VDAC (rsVDAC). Therefore, we analyzed whether rsVDAC is possible when only one VDAC variant is present in mitochondria and whether all VDAC paralogs present in mitochondria could be rsVDAC, using representatives of currently available VDAC amino acid sequences. The obtained results indicate that rsVDAC can occur when only one VDAC variant is present in mitochondria; however, the possibility of all VDAC paralogs in mitochondria being rsVDAC is very low. Moreover, the presence of rsVDAC may correlate with habitat conditions as rsVDAC appears to be prevalent in parasites. Thus, the channel may mediate detection and adaptation to environmental conditions.

Redox Sensor Array with 23.5-μm Resolution for Real-Time Imaging of Hydrogen Peroxide and Glutamate Based on Charge-Transfer-Type Potentiometric Sensor

Towards clarifying the spatio-temporal neurotransmitter distribution, potentiometric redox sensor arrays with 23.5-µm resolution were fabricated. The sensor array based on a charge-transfer-type potentiometric sensor comprises 128×128 pixels with gold electrodes deposited on the surface of pixels. The sensor output corresponding to the interfacial potential of the electrode changed logarithmically with the mixture ratio of K3Fe(CN)6 and K4Fe(CN)6, where the redox sensitivity reached 49.9 mV/dec. By employing hydrogen peroxidase as an enzyme and ferrocene as an electron mediator, the sensing characteristics for hydrogen peroxide (H2O2) were investigated. The analyses of the sensing characteristics revealed that the sensitivity was about 44.7 mV/dec., comparable to the redox sensitivity, while the limit of detection (LOD) was achieved to be 1 µM. Furthermore, the oxidation state of the electron mediator can be the key to further lowering the LOD. Then, by immobilizing oxidizing enzyme for H2O2 and glutamate oxidase, glutamate (Glu) measurements were conducted. As a result, similar sensitivity and LOD to those of H2O2 were obtained. Finally, the real-time distribution of 1 µM Glu was visualized, demonstrating the feasibility of our device as a high-resolution bioimaging technique.

Understanding Metabolic Remodeling in Mycobacterium smegmatis to Overcome Energy Exigency and Reductive Stress Under Energy-Compromised State

Mycobacteria such as Mycobacterium tuberculosis, the causative agent of tuberculosis that annually kills several million people worldwide, and Mycobacterium smegmatis, the non-pathogenic fast-growing mycobacteria, require oxidative phosphorylation to meet their energy requirements. We have previously shown that deletion of one of the two copies of atpD gene that codes for the ATP synthase β-subunit establishes an energy-compromised state in M. smegmatis. Here we report that upon such deletion, a major routing of electron flux occurs through the less energy-efficient complexes of its respiratory chain. ΔatpD bacterium also shows an increased reduced state which is further confirmed by the overexpression of WhiB3, a major redox sensor. We show a substantial modulation of the biosynthesis of cell wall associated lipids and triacylglycerol (TAG). An accumulation of TAG-containing lipid bodies is further confirmed by using 14C oleate incorporation. Interestingly, the mutant also shows an overexpression of TAG-degrading lipase genes, and the intracellular lipolytic enzymes mediate TAG hydrolysis for their utilization as energy source. We believe that our in vitro energy-depleted model will allow us to explore the critical link between energy metabolism, redox homeostasis, and lipid biosynthesis during ATP-depleted state, which will enhance our understanding of the bacterial adaptation, and will allow us to identify novel drug targets to counter mycobacterial infections.

Circadian Clock Core Component Bmal1 Dictates Cell Cycle Rhythm of Proliferating Hepatocytes during Liver Regeneration

Following partial hepatectomy (PH), the majority of remnant hepatocytes synchronously enter and rhythmically progress through the cell cycle for three major rounds to regain lost liver mass. Whether and how the circadian clock core component Bmal1 modulates this process remains elusive. We performed PH on Bmal1+/+ and hepatocyte-specific Bmal1 knockout (Bmal1hep-/-) mice and compared the initiation and progression of the hepatocyte cell cycle. After PH, Bmal1+/+ hepatocytes exhibited three major waves of nuclear DNA synthesis. In contrast, in Bmal1hep-/- hepatocytes, the first wave of nuclear DNA synthesis was delayed by 12 h, and the third such wave was lost. Following PH, Bmal1+/+ hepatocytes underwent three major waves of mitosis, whereas Bmal1hep-/- hepatocytes fully abolished mitotic oscillation. These Bmal1-dependent disruptions in the rhythmicity of hepatocyte cell cycle after PH were accompanied by suppressed expression peaks of a group of cell cycle components and regulators, and dysregulated activation patterns of mitogenic signaling molecules c-Met and EGFR. Moreover, Bmal1+/+ hepatocytes rhythmically accumulated fat as they expanded following PH, whereas this phenomenon was largely inhibited in Bmal1hep-/- hepatocytes. In addition, during late stages of liver regrowth, Bmal1 absence in hepatocytes caused the activation of redox sensor Nrf2, suggesting an oxidative stress state in regenerated liver tissue. Collectively, we demonstrated that during liver regeneration, Bmal1 partially modulates the oscillation of S-phase progression, fully controls the rhythmicity of M-phase advancement, and largely governs fluctuations in fat metabolism in replicating hepatocytes, and eventually determines the redox state of regenerated livers.

Mathematical Modeling Reveals Quantitative Properties of KEAP1-NRF2 Signaling

In response to oxidative and electrophilic stresses, cells launch an NRF2-mediated transcriptional antioxidant program. The activation of NRF2 depends on a redox sensor, KEAP1, which acts as an E3-ligase adaptor to promote the ubiquitination and degradation of NRF2. While a great deal has been learned about the molecular details of KEAP1, NRF2, and their interactions, the quantitative aspects of signal transfer conveyed by this redox duo are still largely unexplored. In the present study, we examined the signaling properties including response time, half-life, maximal activation, and response steepness (ultrasensitivity) of NRF2, through a suite of mathematical models. The models describe, with increasing complexity, the reversible binding of KEAP1 dimer and NRF2 via the ETGE and DLG motifs, NRF2 production, KEAP1-dependent and independent NRF2 degradation, and perturbations by different classes of NRF2 activators. Our simulations revealed that at the basal condition, NRF2 molecules are largely sequestered by KEAP1, with the KEAP1-NRF2 complex comparably distributed in either an ETGE-bound only (open) state or an ETGE and DLG dual-bound (closed) state, corresponding to the unlatched and latched configurations of the conceptual hinge-latch model. With two-step ETGE binding, the open and closed states operate in cycle mode at the basal condition and transition to equilibrium mode at stressed conditions. Class I-V, electrophilic NRF2 activators, which modify redox-sensing cysteine residues of KEAP1, shift the balance to a closed state that is unable to degrade NRF2 effectively. Total NRF2 has to accumulate to a level that nearly saturates existing KEAP1 to make sufficient free NRF2, therefore introducing a signaling delay. At the juncture of KEAP1 saturation, ultrasensitive NRF2 activation, i.e., a steep rise in the free NRF2 level, can occur through two simultaneous mechanisms, zero-order degradation mediated by DLG binding and protein sequestration (molecular titration) mediated by ETGE binding. These response characteristics of class I-V activators do not require disruption of DLG binding to unlatch the KEAP1-NRF2 complex. In comparison, class VI NRF2 activators, which directly compete with NRF2 for KEAP1 binding, can unlatch or even unhinge the KEAP1-NRF2 complex. This causes a shift to the open state of KEAP1-NRF2 complex and ultimately its complete dissociation, resulting in a fast release of free NRF2 followed by stabilization. Although class VI activators may induce free NRF2 to higher levels, ultrasensitivity is lost due to lower free KEAP1 and thus its NRF2-sequestering effect. Stress-induced NRF2 nuclear accumulation is enhanced when basal nuclear NRF2 turnover constitutes a small load to NRF2 production. Our simulation further demonstrated that optimal abundances of cytosolic and nuclear KEAP1 exist to maximize ultrasensitivity. In summary, by simulating the dual role of KEAP1 in repressing NRF2, i.e., sequestration and promoting degradation, our mathematical modeling provides key novel quantitative insights into the signaling properties of the crucial KEAP1-NRF2 module of the cellular antioxidant response pathway.

Protein abundance and folding rather than the redox state of Kelch13 determine the artemisinin susceptibility of Plasmodium falciparum

Decreased susceptibilities of Plasmodium falciparum towards the endoperoxide antimalarial artemisinin are linked to mutations of residue C580 of Kelch13, which is the homologue of the redox sensor Keap1 in vertebrates. Here, we addressed whether mutations alter the artemisinin susceptibility by modifying the redox properties of Kelch13 or by compromising its native fold or abundance. Using selection-linked integration and the glmS ribozyme, efficient down-regulation of Kelch13 resulted in ring-stage survival rates around 40%. While the loss of a potential disulfide bond between residues C580 and C532 had no effect on the artemisinin suceptibility, the thiol group of C473 could not be replaced. We also established a protocol for the production of recombinant Kelch13. In contrast to cysteine-to-serine replacements, common field mutations resulted in misfolded and insoluble protein. In summary, not the redox properties but impaired folding of Kelch13, resulting in a decreased Kelch13 abundance, is the central parameter for mutant selection.