Project description:Comparative analysis of gene expression in cultured primary keratinocytes isolated from newborn control (K14-cre; GPx4fl/+) and knockout (K14-cre; GPx4fl/fl) mice. Selenoproteins are essential for skin function, as targeted abolition of selenoproteins in epidermal tissue results in newborn mice manifesting gross abnormalities of skin and hair, accompanied by retarded growth and premature death. To investigate whether lack of a single selenoprotein could induce similar phenotypic effect in mice, we generated keratinocyte-specific knockout mice lacking glutathione peroxidase 4 (GPx4), an essential selenoprotein in skin, to examine phenotypic changes resulting from the lack of GPx4 in skin. Ablation of GPx4 results in focal alopecia and disturbed hair follicle morphogenesis, with GPx4 being essential during early stages of hair follicle morphogenesis as well as for keratinocyte adhesion and proliferation in culture. We have generated mice with selective removal of the GPx4 gene in keratinocytes under the control of Keratin-14-cre (K14-cre) promoter. Comparative microarray analysis was performed on RNA samples taken from pooled primary keratinocytes from knockout and control mice from the same litter. Array replicates were performed using RNA samples from three different litters.
Project description:Encouraged by the dependence of drug-resistant, metastatic cancers on GPX4, we examined biophysical mechanisms of GPX4 inhibition, which revealed an unexpected allosteric binding site. We found that this site was involved in native modulation of GPX4 activity. Binding of inhibitors to this allosteric site caused a conformational change, inhibition of activity, and subsequent cellular GPX4 protein degradation. To verify this binding site in an unbiased manner, we screened a library of compounds, and identified and validated that a novel additional compound can bind in this allosteric site, inhibiting and degrading GPX4. We determined co-crystal structures of six different inhibitors bound in this site. We have thus discovered a new allosteric mechanism for small molecules targeting aggressive cancers dependent on GPX4.
Project description:To characterize the mechanism of DMC1 ubiquitination as well as further explore the impact of DMC1’s ubiquitination on meiosis, we attempted to identify the potential DMC1 ubiquitination sites. The in vitro ubiquitination assay using recombinant DMC1 heterologously expressed and purified from E. coli as substrate was conducted. Then mass spectrometry assay was performed to identify the ubiquitination sites of DMC1. Five DMC1 lysine residues (K45, K70, K101, K162, and K290) were identified as potential ubiquitination sites.
Project description:GPX4-dependent ferroptosis has emerged as a therapeutic strategy for cancer treatment. Here, we demonstrated that protein kinase A (PKA) participates in the regulation of ferroptosis by controlling the m6A modification of GPX4 in an ALKBH5-dependent manner. Notably, we identified ALKBH5, an m6A demethylase, as a novel target of PKA, which drives phosphorylation-dependent degradation of ALKBH5 protein. Moreover, the deletion of ALKBH5 represses ferroptotic cell death by maintaining GPX4 m6A modification and stability. Thus, by regulating ALKBH5-dependent GPX4 stability, PKA acts as a key regulator of ferroptosis. Our study unveils the involvement of PKA in m6A modification, which could control GPX4-dependent ferroptosis and tumor progression.
Project description:Analysis of proteins interacting with GPX4 in A375 and 293T cells infected with lentiviruses encoding empty vector or Flag-GPX4, using anti-Flag magnetic beads enrichment and LC-MS/MS.
Project description:Ferroptosis is an iron-dependent programmed cell death associated with severe kidney diseases, linked to decreased glutathione peroxidase 4 (GPX4). However, the spatial distribution of renal GPX4-mediated ferroptosis and the molecular events causing GPX4 reduction during ischemia-reperfusion (I/R) remain largely unknown. Using spatial transcriptomics, we identify that GPX4 is situated at the interface of the inner cortex and outer medulla, a hyperactive ferroptosis site post-I/R injury. We show that OTU deubiquitinase 5 (OTUD5) is a GPX4-binding protein that confers ferroptosis resistance by stabilizing GPX4. During I/R, ferroptosis is induced by mTORC1-mediated autophagy, causing OTUD5 degradation and subsequent GPX4 decay. Functionally, OTUD5 deletion intensifies renal tubular cell ferroptosis and exacerbates acute kidney injury, while AAV-mediated OTUD5 delivery mitigates ferroptosis and promotes renal function recovery from I/R injury. In this work, our study highlights a new autophagy-dependent ferroptosis module: hypoxia/ischemia-induced OTUD5 autophagy triggers GPX4 degradation, offering a potential therapeutic avenue for I/R-related kidney diseases.
Project description:Background In the ongoing battle against BCR-ABL+ leukemia, despite significant advances with tyrosine kinase inhibitors (TKIs), the persistent challenges of drug resistance and the enduring presence of leukemic stem cells (LSCs) remain formidable barriers to achieving a cure. Methods In this study, we demonstrated that Disulfiram (DSF) induces ferroptosis to synergize with TKIs in inhibiting BCR-ABL+ cells, particularly targeting resistant cells and LSCs, using cell models, mouse models, and primary cells from patients. We elucidated the mechanism by which DSF promotes GPX4 degradation to induce ferroptosis through immunofluorescence, co-immunoprecipitation (CO-IP), RNA sequencing, lipid peroxidation assays, and rescue experiments. Results Here, we present compelling evidence elucidating the sensitivity of DSF, an FDA-approved drug for alcohol dependence, towards BCR-ABL+ cells. Our findings underscore DSF's ability to selectively induce a potent cytotoxic effect on BCR-ABL+ cell lines and effectively inhibit primary BCR-ABL+ leukemia cells. Crucially, the combined treatment of DSF with TKIs selectively eradicates TKI-insensitive stem cells and resistant cells. Of particular note is DSF's capacity to disrupt GPX4 stability, elevate the labile iron pool, and intensify lipid peroxidation, ultimately leading to ferroptotic cell death. Our investigation shows that BCR-ABL expression induces alterations in cellular iron metabolism and increases GPX4 expression. Additionally, we demonstrate the indispensability of GPX4 for LSC development and the initiation/maintenance of BCR-ABL+ leukemia. Mechanical analysis further elucidates DSF's ability to circumvent resistance by reducing GPX4 levels through the disruption of its binding with HSPA8, thereby promoting GPX4 ubiquitination and subsequent degradation. Furthermore, the combined treatment of DSF with TKIs effectively targets both BCR-ABL+ blast cells and drug-insensitive LSCs, conferring a significant survival advantage in mouse models. Conclusion In summary, the dual inhibition of GPX4 and BCR-ABL presents a promising therapeutic strategy to synergistically target blast cells and drug-insensitive LSCs in patients, offering potential avenues for advancing leukemia treatment.
Project description:Comparative analysis of gene expression in cultured primary keratinocytes isolated from newborn control (K14-cre; GPx4fl/+) and knockout (K14-cre; GPx4fl/fl) mice. Selenoproteins are essential for skin function, as targeted abolition of selenoproteins in epidermal tissue results in newborn mice manifesting gross abnormalities of skin and hair, accompanied by retarded growth and premature death. To investigate whether lack of a single selenoprotein could induce similar phenotypic effect in mice, we generated keratinocyte-specific knockout mice lacking glutathione peroxidase 4 (GPx4), an essential selenoprotein in skin, to examine phenotypic changes resulting from the lack of GPx4 in skin. Ablation of GPx4 results in focal alopecia and disturbed hair follicle morphogenesis, with GPx4 being essential during early stages of hair follicle morphogenesis as well as for keratinocyte adhesion and proliferation in culture.
Project description:Osteoarthritis (OA) is the most common joint disease and is the leading cause of chronic disability among older people. Chondrocyte death was involved in OA pathogenesis. Ferroptosis is an iron-dependent cell death associated with peroxidation of lipids. Expression of GPX4 in the OA cartilage from OA patients were significantly lower than normal cartilage.In order to analyze the mechanism of GPX4, we conducted RNA-sequencing in mouse chondrocytes with or without GPX4 knockdown.Our results showed that Gpx4 downregulation could increase the sensitivity of chondrocytes to oxidative stress and aggravate ECM degradation in chondrocytes.
Project description:Senescent cells drive ageing and age-related pathologies, including cancer. Consequently, senolytics, drugs that selectively kill senescent cells, have a broad therapeutic appeal. Here, we report a senolytic screen of a library of 10,480 covalent fragments. Amongst 38 identified hits, we found a subset of chloroacetamides with broad senolytic activity. Activity-based protein profiling, coupled with functional assays, identified the glutathione peroxidase GPX4 as their target. We show that senescent cells are primed for ferroptosis, displaying high levels of oxidative stress, intracellular Fe2+, and enhanced lipid oxidation, but also upregulate GPX4, which protects them from ferroptosis. Consequently, treatment with senolytic chloroacetamides or GPX4 inhibitors selectively kills senescent cells by ferroptosis. The combination of anti-cancer therapies with GPX4 inhibitors eliminated senescent tumour cells and improved outcomes in models of prostate and ovarian cancer. Our results show that senescent cells rely on GPX4 to prevent ferroptosis and that GPX4 inhibitors kill senescent cells, having a wide therapeutic potential.