Transcription Factor Nrf2 Research Articles

A Cell-Based Evaluation of the Tyrosinase-Mediated Metabolic Activation of Leukoderma-Inducing Phenols, II: The Depletion of Nrf2 Augments the Cytotoxic Effect Evoked by Tyrosinase in Melanogenic Cells

Chemical leukoderma is a disorder induced by chemicals such as rhododendrol and monobenzone. These compounds possess a p-substituted phenol moiety and undergo oxidation into highly reactive and toxic o-quinone metabolites by tyrosinase. This metabolic activation plays a critical role in the development of leukoderma through the production of damage to melanocytes and immunological responses. This study aimed to develop a simple method for assessing the metabolic activation of leukoderma-inducing phenols without analyzing the metabolite. Although B16BL6 melanoma cells showed insufficient sensitivity to the cytotoxicity assay, the siRNA-mediated knockdown of the transcription factor NRF2 (NFE2L2) repressed the expression of cytoprotective factors, thereby augmenting the cytotoxicity of all six leukoderma-inducing phenols tested in a tyrosinase-dependent manner, indicating enhanced sensitivity to o-quinone metabolites. Additionally, the knockdown of the NRF2-target Slc7a11 elevated the cytotoxicity of three out of the six compounds, indicating the involvement of cystine transport in cellular protection. In contrast, the knockdown or inhibition of the NRF2-target Nqo1 had minimal effects. The same response was induced upon Nrf2 and Slc7a11 knockdown in B16-4A5 cells, albeit with low sensitivity owing to low tyrosinase expression. We conclude that the analysis of tyrosinase-dependent cytotoxicity in Nrf2-depleted B16BL6 cells may serve as a useful strategy for evaluating the metabolic activation of chemicals.

Selenoprotein-Mediated Redox Regulation Shapes the Cell Fate of HSCs and Mature Lineages.

The maintenance of cellular redox balance is crucial for cell survival and homeostasis and is disrupted with aging. Selenoproteins, comprising essential antioxidant enzymes, raise intriguing questions about their involvement in hematopoietic aging and potential reversibility. Motivated by our observation of mRNA downregulation of key antioxidant selenoproteins in aged human hematopoietic stem cells (HSCs) and previous findings of increased lipid peroxidation in aged hematopoiesis, we employed tRNASec gene (Trsp) knockout (KO) mouse model to simulate disrupted selenoprotein synthesis. This revealed insights into the protective roles of selenoproteins in preserving HSC stemness and B-lineage maturation, despite negligible effects on myeloid cells. Notably, Trsp KO exhibited B lymphocytopenia and reduced HSCs' self-renewal capacity, recapitulating certain aspects of aged phenotypes, along with the upregulation of aging-related genes in both HSCs and pre-B cells. While Trsp KO activated an antioxidant response transcription factor NRF2, we delineated a lineage-dependent phenotype driven by lipid peroxidation, which was exacerbated with aging yet ameliorated by ferroptosis inhibitors such as vitamin E. Interestingly, the myeloid genes were ectopically expressed in pre-B cells of Trsp KO mice, and KO pro-B/pre-B cells displayed differentiation potential toward functional CD11b+ fraction in the transplant model, suggesting that disrupted selenoprotein synthesis induces the potential of B-to-myeloid switch. Given the similarities between the KO model and aged wild-type mice, including ferroptosis vulnerability, impaired HSC self-renewal and B-lineage maturation, and characteristic lineage switch, our findings underscore the critical role of selenoprotein-mediated redox regulation in maintaining balanced hematopoiesis and suggest the preventive potential of selenoproteins against aging-related alterations.

Effects of Tocotrienol on Cardiovascular Risk Markers in Patients With Chronic Kidney Disease: A Randomized Controlled Trial

Tocotrienols, isomers of vitamin E, may provide an effective nutritional strategy to mitigate common cardiovascular risks such as dyslipidemia, inflammation, and oxidative stress in patients with chronic kidney disease (CKD). This double‐blind, placebo‐controlled, randomized clinical trial aimed to evaluate the effects of a tocotrienol‐rich fraction (TRF) supplementation (300 mg/day) on oxidative stress and inflammatory markers, including transcription factors in nondialysis (ND) and hemodialysis (HD) CKD patients for three months. Interleukin‐6, tumor necrosis factor‐α (IL‐6 and TNF‐α), C‐reactive protein (CRP), lipid peroxidation, biochemical parameters, and transcription factors such as NRF2 and NF‐κB mRNA expression were evaluated. Seventeen HD patients (9 in the placebo group, 8 in the TRF group) and 16 ND CKD patients (8 in the placebo group and 8 in the TRF group) completed the study. In HD patients, significant reductions were observed in LDL cholesterol (p = 0.04) and total plasma cholesterol levels (p = 0.01) after TRF intervention. CRP serum levels decreased significantly in ND CKD patients (p = 0.05) after TRF supplementation. Transcription factors NRF2 and NF‐κB mRNA expressions remained unaltered in both groups. This study suggests that TRF supplementation may mitigate dyslipidemia and inflammation, factors involved with increased cardiovascular risk, in CKD patients, with variations in efficacy between HD and ND patients.Trial Registration: ClinicalTrials.gov identifier: NCT04900532

Meso-Substituted AB3-type phenothiazinyl porphyrins and their indium and zinc complexes photosensitising properties, cytotoxicity and phototoxicity on ovarian cancer cells.

New meso-substituted AB3-type phenothiazinyl porphyrins and ferrocenylvinyl phenothiazinyl porphyrin were synthesised by Suzuki-Miyaura and Mizoroki-Heck cross-coupling reactions, respectively. The free porphyrins were further used in the synthesis of new indium(iii) or zinc(ii) porphyrin complexes. All porphyrins exhibit red fluorescence emission in solution, a property that remains unimpaired following internalisation in ovarian A2780 cancer cells, as evidenced by fluorescence microscopy images. The In(iii) phenothiazinyl porphyrin complexes show a higher quantum yield of fluorescence emission (2aΦ F = 30%, 4aΦ F = 29%, 5aΦ F = 28%) compared to the free base porphyrin precursors, or Zn(ii) complex 4b (Φ F = 10%). The potential of novel phenothiazinyl porphyrins to act as photosensitisers was evaluated using two distinct approaches. The first was through the measurement of the singlet oxygen quantum yield Φ Δ(1O2), while the second employed in vitro measurements of metabolic activity, oxidative stress, nuclear factor-erythroid 2 related factor 2 (Nrf-2) activation and tumour necrosis factor-alpha (TNF-α) under both dark and light irradiation conditions. As reflected by the IC50 values, the most potent cytotoxicity of the phenothiazinyl porphyrins against the A2780 cells was observed for In(iii) ferrocenylvinyl phenothiazinyl porphyrin 4a (36.38 μM), the remaining compounds are less cytotoxic. The reduction in metabolic activity was observed in A2780 ovarian tumour cells treated with 4a and 6a and exposed to light compared to treatment in the absence of light. The oxidative stress, TNF-α and Nrf-2 transcription factor were particularly notable when A2780 cells were treated with 4a and subsequently photoirradiated, the oxidative stress was linked to the highest value of Φ Δ(1O2) recorded for 4a (60%).

SKN-1 ISOFORM-C IS ESSENTIAL FOR C. ELEGANS DEVELOPMENT

Abstract The transcription factor SKN-1 in Caenorhabditis elegans is a critical regulator of various biological processes, impacting development, diet and immune responses, cellular detoxification, and lipid metabolism; thereby playing a pivotal role in regulating the health and lifespan of the organism. The primary isoforms of SKN-1 (SKN-1a, SKN-1b, and SKN-1c) exhibit distinct functions resembling mammalian Nrf transcription factors. This study investigates the specific role of the SKN-1c isoform in development by generating mutants with targeted missense mutations in the skn-1c and skn-1a isoforms. The skn-1c Met1Ala mutants, which replaces a start methionine with alanine, renders SKN-1c non-functional while preserving other isoforms, produced inviable embryos, requiring a balancer chromosome for proper embryonic development. In contrast, skn-1a Met1Ala mutants, which replaces the start methionine with alanine for this isoform, displayed normal embryonic development and hatching. Moreover, the data suggest that SKN-1c plays a crucial role in embryonic development, as strains without maternally deposited SKN-1c lead to embryos that are developmentally arrested. Together, these findings contribute to our understanding of SKN-1c’s specific role in influencing embryogenesis and development in C. elegans.

Hybridisation of in silico and in vitro bioassays for studying the activation of Nrf2 by natural compounds

Oxidative stress, characterized by the damaging accumulation of free radicals, is associated with various diseases, including cardiovascular, neurodegenerative, and metabolic disorders. The transcription factor Nrf2 is pivotal in cellular defense against oxidative stress by regulating genes that detoxify free radicals, thus maintaining redox homeostasis and preventing cellular aging. Keap1 plays a regulatory role through its interaction with Nrf2, ensuring Nrf2 degradation under homeostatic conditions and facilitating its stabilization and nuclear translocation during oxidative stress. In the initial stage of our study, we conducted in vitro assays on HaCaT cells, a human keratinocyte cell line, to measure the expression levels of Nrf2 to reveal the activity of promising medicinal plants, which were then selected for further evaluation. Subsequently, this study leverages in silico techniques, integrating machine learning with molecular docking and dynamics, to screen natural compounds that potentially activate Nrf2. Data from the ChEMBL database were categorized into active and inactive compounds and used for training different machine-learning models to predict potential Nrf2 activators. The best-performing model was used to select compounds for further evaluation via molecular docking and dynamics, assessing their interactions with Keap1/Nrf2. The LC-MS/MS-based chemical profiles also validated the presence of these chemical compounds. This approach underscores the synergy between in vitro bioassays and in silico approaches in identifying Nrf2 activators, offering a cost-effective strategy for drug development.

Exploring the Therapeutic Potential of Cannabidiol in U87MG Cells: Effects on Autophagy and NRF2 Pathway

Cannabinoids include both endogenous endocannabinoids and exogenous phytocannabinoids, such as cannabidiol (CBD), and have potential as therapeutic agents in cancer treatment due to their selective anticancer activities. CBD exhibits both antioxidant and pro-oxidant effects depending on its concentration and cell types. These properties allow CBD to influence oxidative stress responses and potentially enhance the efficacy of antitumor therapies. In this study, we treated U87MG glioma cells with low dose (1 μM) CBD and evaluated its molecular effects. Our findings indicate that CBD reduced cell viability by 20% (p < 0.05) through the alteration of mitochondrial membrane potential. The alteration of redox status by CBD caused an attempt to rescue mitochondrial functionality through nuclear localization of the GABP transcription factor involved in mitochondria biogenesis. Moreover, CBD treatment caused an increase in autophagic flux, as supported by the increase in Beclin-1 and the ratio of LC3-II/LC3-I. Due to mitochondria functionality alteration, pro-apoptotic proteins were induced without activating apoptotic effectors Caspase-3 or Caspase-7. The study of the transcription factor NRF2 and the ubiquitin-binding protein p62 expression revealed an increase in their levels in CBD-treated cells. In conclusion, low-dose CBD makes U87MG cells more vulnerable to cytotoxic effects, reducing cell viability and mitochondrial dynamics while increasing autophagic flux and redox systems. This explains the mechanisms by which glioma cells respond to CBD treatment. These findings highlight the therapeutic potential of CBD, suggesting that modulating NRF2 and autophagy pathways could represent a promising strategy for glioblastoma treatment.

Mechanisms of Keap1/Nrf2 modulation in bacterial infections: implications in persistence and clearance

Pathogenic bacteria trigger complex molecular interactions in hosts that are characterized mainly by an increase in reactive oxygen species (ROS) as well as an inflammation-associated response. To counteract oxidative damage, cells respond through protective mechanisms to promote resistance and avoid tissue damage and infection; among these cellular mechanisms the activation or inhibition of the nuclear factor E2-related factor 2 (Nrf2) is frequently observed. The transcription factor Nrf2 is considered the master regulator of several hundred cytoprotective and antioxidant genes. Under normal conditions, the Keap1/Nrf2 signaling protects the cellular environment by sensing deleterious oxygen radicals and inducing the expression of genes coding for proteins intended to neutralize the harmful effects of ROS. However, bacteria have developed strategies to harness Nrf2 activity to their own benefit, complicating the host response. This review is aimed to present the most recent information and probable mechanisms employed by a variety of bacteria to modulate the Keap1/Nrf2 activity in order to survive in the infected tissue. Particularly, those utilized by the Gram-positive bacteria Staphylococcus aureus, Streptococcus pneumoniae, Listeria monocytogenes, and Mycobacterium tuberculosis as well as by the Gram-negative bacteria Escherichia coli, Helicobacter pylori, Legionella pneumophila, Pseudomonas aeruginosa and Salmonella typhimurium. We also discuss and highlight the beneficial impact of the Keap1/Nrf2 antioxidant and anti-inflammatory role in bacterial clearance.

Transcriptomic Characterization Reveals Mitochondrial Involvement in Nrf2/Keap1-Mediated Osteoclastogenesis.

Although osteoclasts play crucial roles in the skeletal system, the mechanisms that underlie oxidative stress during osteoclastogenesis remain unclear. The transcription factor Nrf2 and its suppressor, Keap1, function as central mediators of oxidative stress. To further elucidate the function of Nrf2/Keap1-mediated oxidative stress regulation in osteoclastogenesis, DNA microarray analysis was conducted in this study using wild-type (WT), Keap1 knockout (Keap1 KO), and Nrf2 knockout (Nrf2 KO) osteoclasts. Principal component analysis showed that 403 genes, including Nqo1, Il1f9, and Mmp12, were upregulated in Keap1 KO compared with WT osteoclasts, whereas 24 genes, including Snhg6, Ccdc109b, and Wfdc17, were upregulated in Nrf2 KO compared with WT osteoclasts. Moreover, 683 genes, including Car2, Calcr, and Pate4, were upregulated in Nrf2 KO cells compared to Keap1 KO cells. Functional analysis by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis showed upregulated genes in Nrf2 KO osteoclasts were mostly enriched in oxidative phosphorylation. Furthermore, GeneMANIA predicted the protein-protein interaction network of novel molecules such as Rufy4 from genes upregulated in Nrf2 KO osteoclasts. Understanding the complex interactions between these molecules may pave the way for developing promising therapeutic strategies against bone metabolic diseases caused by increased osteoclast differentiation under oxidative stress.

African swine fever virus enhances viral replication by increasing intracellular reduced glutathione levels, which suppresses stress granule formation

African swine fever virus (ASFV) is a DNA virus that has significantly impacted the global swine industry. Currently, there are no effective therapies or vaccines against ASFV. Stress granules (SGs), known for their antiviral properties, are not induced during ASFV infection, even though reactive oxygen species (ROS) are generated. The mechanism by which ASFV regulates SGs formation remains unclear. This study demonstrates that ASFV antagonises SGs formation and increases intracellular levels of reduced glutathione (GSH) levels. The use of the GSH inhibitor BSO and the activator NAC confirmed that the ASFV-induced increase in GSH helps to suppress SGs formation and influences viral replication. Additionally, this study revealed that ASFV enhances GSH by upregulating the antioxidant transcription factor NRF2, as well as factors involved in GSH synthesis and regeneration, such as GCLC, and those related to the ferroptosis pathway, such as SLC7A11. Furthermore, the study uncovered that ASFV manipulates intracellular GSH levels by activating the mitochondrial protein AIFM1. This regulatory mechanism helps the virus inhibit the formation of intracellular SGs, thereby creating an optimal environment for viral replication. These findings provide new insights into the molecular strategies employed by ASFV.

Harshly Oxidized Activated Charcoal Enhances Protein Persulfidation with Implications for Neurodegeneration as Exemplified by Friedreich's Ataxia.

Harsh acid oxidation of activated charcoal transforms an insoluble carbon-rich source into water-soluble, disc structures of graphene decorated with multiple oxygen-containing functionalities. We term these pleiotropic nano-enzymes as "pleozymes". A broad redox potential spans many crucial redox reactions including the oxidation of hydrogen sulfide (H2S) to polysulfides and thiosulfate, dismutation of the superoxide radical (O2-*), and oxidation of NADH to NAD+. The oxidation of H2S is predicted to enhance protein persulfidation-the attachment of sulfur to cysteine residues. Persulfidated proteins act as redox intermediates, and persulfidation protects proteins from irreversible oxidation and ubiquitination, providing an important means of signaling. Protein persulfidation is believed to decline in several neurological disorders and aging. Importantly, and consistent with the role of persulfidation in signaling, the master antioxidant transcription factor Nrf2 is regulated by Keap1's persulfidation. Here, we demonstrate that pleozymes increased overall protein persulfidation in cells from apparently healthy individuals and from individuals with the mitochondrial protein mutation responsible for Friedreich's ataxia. We further find that pleozymes specifically enhanced Keap1 persulfidation, with subsequent increased accumulation of Nrf2 and Nrf2's antioxidant targets.

NRF2 activation in the heart induces glucose metabolic reprogramming and reduces cardiac dysfunction via upregulation of the pentose phosphate pathway.

The transcription factor NRF2 is well recognized as a master regulator of antioxidant responses and cytoprotective genes. Previous studies showed that NRF2 enhances resistance of mouse hearts to chronic hemodynamic overload at least in part by reducing oxidative stress. Evidence from other tissues suggests that NRF2 may modulate glucose intermediary metabolism but whether NRF2 has such effects in the heart is unclear. Here, we investigate the role of NRF2 in regulating glucose intermediary metabolism and cardiac function during disease stress. Cardiomyocyte-specific Keap1 knockout (csKeap1KO) mice, deficient in the endogenous inhibitor of NRF2, were used as a novel model of constitutively active NRF2 signaling. Targeted metabolomics and isotopomer analysis were employed in studies with 13C6-glucose in csKeap1KO and wild-type (WT) mice. Pharmacological and genetic approaches were utilized in neonatal rat ventricular cardiomyocytes (NRVM) to explore molecular mechanisms. We found that cardiac-specific activation of NRF2 redirected glucose metabolism towards the pentose phosphate pathway (PPP), a branch pathway of glycolysis, and mitigated pressure overload-induced cardiomyocyte death and cardiac dysfunction. Activation of NRF2 also protected against myocardial infarction-induced DNA damage in remote myocardium and cardiac dysfunction. In vitro, knockdown of Keap1 upregulated PPP enzymes and reduced cell death in NRVM subjected to chronic neurohumoral stimulation. These pro-survival effects were abolished by pharmacological inhibition of the PPP or silencing of the PPP rate-limiting enzyme glucose-6-phosphate dehydrogenase (G6PD). Knockdown of NRF2 in NRVM increased stress-induced DNA damage which was rescued by supplementing the cells with either NADPH or nucleosides, the two main products of the PPP. These results indicate that NRF2 regulates cardiac metabolic reprogramming by stimulating the diversion of glucose into the PPP, thereby generating NADPH and providing nucleotides to prevent stress-induced DNA damage and cardiac dysfunction.

Cyclic vinyl sulfones activate NRF2 to protect from oxidative stress-induced programmed necrosis

The NRF2 transcriptional factor is a member of cellular stress response machinery and is activated in response to oxidative stress caused either by cellular homeostasis imbalance or by environmental challenges. NRF2 levels are stringently controlled by rapid and continuous proteasomal degradation. KEAP1 is a specific NRF2 binding protein that acts as a bridge between NRF2 and the E3 ligase Cullin-3. In this study, we examine model cyclic vinyl sulfone derivatives as potential NRF2 activating probes. Previously, we and other authors have found anti-inflammatory properties of these compounds in in vivo models; however, the mechanism of action remained unknown. Here, we show that the naphthohydroquinone derivative LCB1353 efficiently stabilizes NRF2 protein levels and upregulates its target genes. At low 5–10 µM concentrations LCB1353 protects non-small cell lung cancer H1299 cells from ferroptotic death induced by cytotoxic concentrations of RSL3, reducing cell death from 90 % to 5 %. Thus, we suggest that cyclic vinyl sulfones are promising scaffolds for the design of protective molecules for conditions associated with toxic and inflammatory levels of oxidative stress.

Temporal control of acute protein aggregate turnover by UBE3C and NRF1-dependent proteasomal pathways

A hallmark of neurodegenerative diseases (NDs) is the progressive loss of proteostasis, leading to the accumulation of misfolded proteins or protein aggregates, with subsequent cytotoxicity. To combat this toxicity, cells have evolved degradation pathways (ubiquitin-proteasome system and autophagy) that detect and degrade misfolded proteins. However, studying the underlying cellular pathways and mechanisms has remained a challenge, as formation of many types of protein aggregates is asynchronous, with individual cells displaying distinct kinetics, thereby hindering rigorous time-course studies. Here, we merge a kinetically tractable and synchronous agDD-GFP system for aggregate formation with targeted gene knockdowns, to uncover degradation mechanisms used in response to acute aggregate formation. We find that agDD-GFP forms amorphous aggregates by cryo-electron tomography at both early and late stages of aggregate formation. Aggregate turnover occurs in a proteasome-dependent mechanism in a manner that is dictated by cellular aggregate burden, with no evidence of the involvement of autophagy. Lower levels of misfolded agDD-GFP, enriched in oligomers, utilizes UBE3C-dependent proteasomal degradation in a pathway that is independent of RPN13 ubiquitylation by UBE3C. Higher aggregate burden activates the NRF1 transcription factor to increase proteasome subunit transcription and subsequent degradation capacity of cells. Loss or gain of NRF1 function alters the turnover of agDD-GFP under conditions of high aggregate burden. Together, these results define the role of UBE3C in degradation of this class of misfolded aggregation-prone proteins and reveals a role for NRF1 in proteostasis control in response to widespread protein aggregation.

Andrographolide attenuates SARS-CoV-2 infection via an up-regulation of glutamate-cysteine ligase catalytic subunit (GCLC)

BackgroundAndrographolide is a medicinal compound which possesses anti-SARS-CoV-2 activity. A number of cellular targets of andrographolide have been identified by target predictions and computational studies. PurposeHowever, a potential cellular target of andrographolide has never been explored in SARS-CoV-2 infected lung epithelial cells. We aimed to identify cellular pathways involved in andrographolide-mediated anti-SARS-CoV-2 activity. MethodsThe viral infection was determined by immunofluorescence staining, enzyme-linked immunosorbent assay and focus-forming assay. Proteomic analysis was employed to identify cellular pathways and key proteins controlled by andrographolide in the human lung epithelial cells Calu-3 infected by SARS-CoV-2. Immunofluorescence staining was used to test protein expression and localization. Western blot and realtime PCR were utilized to elucidate gene expression. Cellular glutathione level was examined by a reduced/oxidized glutathione assay. An ectopic gene expression was delivered by plasmid transfection. ResultsGene ontology analysis indicates that proteins involved in nuclear factor erythroid 2-related factor 2 (NRF2)-regulated pathways were differentially expressed by andrographolide. Notably, andrographolide increased expression and nuclear localization of the transcription factor NRF2. In addition, transcriptional expression of GCLC and glutamate-cysteine ligase modifier subunit (GCLM), which are NRF2 target genes, were induced by andrographolide. We further find that infection of SARS-CoV-2 resulted in a reduction of glutathione level in Calu-3; the effect that was rescued by andrographolide. Moreover, andrographolide also induced expression of the glutathione producing enzyme GCLC in SARS-CoV-2 infected lung epithelial cells. Importantly, an ectopic over-expression of GCLC or treatment of N-acetyl-L-cysteine in Calu-3 cells led to a decrease in SARS-CoV-2 infection. ConclusionCollectively, our findings suggest the interplay between GCLC-mediated glutathione biogenesis induced by andrographolide and the anti-SARS-CoV-2 activity. The glutathione biogenesis and recycling pathways should be further exploited as a targeted therapy against SARS-CoV-2 infection.

Obesity induced by a high-fat diet changes p62 protein levels in mouse reproductive organs.

Obesity is one of the major risk factor for infertility since it causes decreased quality and quantity of gametes and a disrupted uterine environment which might result in miscarriage, stillbirth, and fetal abnormal growth. Obesity induces oxidative stress which is strongly associated with infertility. The clearing of oxidative stress by autophagy is maintained through the p62/ Keap1/Nrf2 pathway. In this pathway, oxidative stress induces p62 for binding to Keap1, thereby Keap1 cannot bind to the Nrf2 transcription factor. Then, Nrf2 translocates into the nucleus and initiates antioxidant-related gene expression. While p62, bound to Keap1, acts as an adaptor protein between autophagosome and damaged substrates which needs to be degraded for homeostasis. Up to date, obesity is strongly linked to abnormal autophagy activity. However, p62 protein expression has not been investigated in the obese ovary, testis, and uterus in detail. Thus, in the present study, we aimed to evaluate the effects of a high-fat diet (HFD)-induced obesity on p62 protein levels of the ovary, testis, and uterus in mice. Our results demonstrated that the p62 expression level was significantly altered by HFD in uterine glands, epithelium, myometrium, and stroma, and in the ovarian corpus luteum, testicular spermatogonium and spermatocytes.

The Serine Protease DPP9 and the Redox Sensor KEAP1 Form a Mutually Inhibitory Complex

Synthetic inhibitors of the serine protease DPP9 activate the related NLRP1 and CARD8 inflammasomes and stimulate powerful innate immune responses. Thus, it seems plausible that a biomolecule similarly inhibits DPP9 and triggers inflammasome activation during infection, but one has not yet been discovered. Here, we wanted to identify and characterize DPP9-binding proteins to potentially uncover physiologically relevant mechanisms that control DPP9's activity. Notably, we found that the redox sensor protein KEAP1 binds to DPP9 in an inactive conformation and stabilizes this non-native fold. At the same time, this inactive form of DPP9 reciprocally inhibits the ability of KEAP1 to bind to and degrade the transcription factor NRF2, thereby inducing an antioxidant response. Although we discovered several experimental conditions, for example new protein expression and chemical denaturation, that force DPP9 out of its folded dimeric state and into a KEAP1-binding state, the key danger-related stimulus that causes this critical DPP9 conformational change is not yet known. Regardless, our data now reveal that an endogenous DPP9 inhibition mechanism does in fact exist, and moreover that DPP9, like the other NLRP1 regulator thioredoxin-1, is directly coupled to the intracellular redox potential. Overall, we expect this work will provide the foundation to discover additional biomolecules that regulate DPP9's activity, the DPP9-KEAP1 interaction, the intracellular redox environment, and the NLRP1 and CARD8 inflammasomes.

Unveiling a novel signalling pathway involving NRF2 and PGAM5 in regulating the mitochondrial unfolded protein response in stressed cardiomyocytes

The mitochondrial unfolded protein response (UPRmt) is a conserved signalling pathway that initiates a specific transcriptional programme to maintain mitochondrial and cellular homeostasis under stress. Previous studies have demonstrated that UPRmt activation has protective effects in the pressure-overloaded human heart, suggesting that robust UPRmt stimulation could serve as an intervention strategy for cardiovascular diseases. However, the precise mechanisms of UPRmt regulation remain unclear. In this study, we present evidence that the NRF2 transcription factor is involved in UPRmt activation in cardiomyocytes during conditions of mitochondrial stress. Silencing NRF2 partially reduces UPRmt activation, highlighting its essential role in this pathway. However, constitutive activation of NRF2 via inhibition of its cytosolic regulator KEAP1 does not increase levels of UPRmt activation markers, suggesting an alternative regulatory mechanism independent of the canonical KEAP1-NRF2 axis. Further analysis revealed that NRF2 likely affects UPRmt activation through its interaction with PGAM5 at the mitochondrial membrane. Disruption of PGAM5 in cardiomyocytes subjected to mitochondrial stress reduces the interaction between PGAM5 and NRF2, enhancing nuclear translocation of NRF2 and significantly upregulating the UPRmt in an NRF2-dependent manner. This NRF2-regulated UPRmt amplification improves mitochondrial respiration, reflecting an enhanced capacity for cardiomyocytes to meet elevated energetic demands during mitochondrial stress. Our findings highlight the therapeutic potential of targeting the NRF2-PGAM5-KEAP1 signalling complex to amplify the UPRmt in cardiomyocytes for cardiovascular and other diseases associated with mitochondrial dysfunction. Future studies should aim to elucidate the mechanisms via which NRF2 enhances the protective effects of UPRmt, thereby contributing to more targeted therapeutic approaches.

Positive Effects of Argon Inhalation After Traumatic Brain Injury in Rats.

The noble gas argon is one of the most promising neuroprotective agents for hypoxic-reperfusion injuries of the brain. However, its effect on traumatic injuries has been insufficiently studied. The aim of this study was to analyze the effect of the triple inhalation of the argon-oxygen mixture Ar 70%/O2 30% on physical and neurological recovery and the degree of brain damage after traumatic brain injury and to investigate the possible molecular mechanisms of the neuroprotective effect. The experiments were performed in male Wistar rats. A controlled brain injury model was used to investigate the effects of argon treatment and the underlying molecular mechanisms. The results of the study showed that animals with craniocerebral injuries that were treated with argon inhalation exhibited better physical recovery rates, better neurological status, and less brain damage. Argon treatment significantly reduced the expression of the proinflammatory markers TNFα and CD68 caused by TBI, increased the expression of phosphorylated protein kinase B (pAKT), and promoted the expression of the transcription factor Nrf2 in intact animals. Treatment with an argon-oxygen breathing mixture after traumatic brain injury has a neuroprotective effect by suppressing the inflammatory response and activating the antioxidant and anti-ischemic system.

Dynamic regulation of the oxidative stress response by the E3 ligase TRIP12.

The oxidative stress response is centered on the transcription factor NRF2 and protects cells from reactive oxygen species (ROS). While ROS inhibit the E3 ligase CUL3 KEAP1 to stabilize NRF2 and elicit antioxidant gene expression, cells recovering from stress must rapidly reactivate CUL3 KEAP1 to prevent reductive stress and oxeiptosis-dependent cell death. How cells restore efficient NRF2-degradation upon ROS clearance remains poorly understood. Here, we identify TRIP12, an E3 ligase dysregulated in Clark-Baraitser Syndrome and Parkinson's Disease, as a component of the oxidative stress response. TRIP12 is a ubiquitin chain elongation factor that cooperates with CUL3 KEAP1 to ensure robust NRF2 degradation. In this manner, TRIP12 accelerates stress response silencing as ROS are being cleared, but limits NRF2 activation during stress. The need for dynamic control of NRF2-degradation therefore comes at the cost of diminished stress signaling, suggesting that TRIP12 inhibition could be used to treat degenerative pathologies characterized by ROS accumulation.