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Metabolically activated proteostasis regulators that protect against erastin-induced ferroptosis.

ABSTRACT: We previously showed that the proteostasis regulator compound AA147 (N-(2-hydroxy-5-methylphenyl)benzenepropanamide) potently protects against neurotoxic insults, such as glutamate-induced oxytosis. Though AA147 is a selective activator of the ATF6 arm of the unfolded protein response in non-neuronal cells, AA147-dependent protection against glutamate toxicity in cells of neuronal origin is primarily mediated through activation of the NRF2 oxidative stress response. AA147 activates NRF2 through a mechanism involving metabolic activation of AA147 by endoplasmic reticulum (ER) oxidases, affording an AA147-based quinone methide that covalently targets the NRF2 repressor protein KEAP1. Previous results show that the 2-amino-p-cresol A-ring of AA147 is required for NRF2 activation, while the phenyl B-ring of AA147 is amenable to modification. Here we explore whether the protease-sensitive amide linker between the A- and B-rings of this molecule can be modified to retain NRF2 activation. We show that replacement of the amide linker of AA147 with a carbamate linker retains NRF2 activation in neuronal cells and improves protection against neurotoxic insults, including glutamate-induced oxytosis and erastin-induced ferroptosis. Moreover, we demonstrate that inclusion of this carbamate linker facilitates identification of next-generation AA147 analogs with improved cellular tolerance and activity in disease-relevant assays.

SUBMITTER: Kline GM

PROVIDER: S-EPMC11353103 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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Metabolically activated proteostasis regulators that protect against erastin-induced ferroptosis.

Kline Gabriel M GM Madrazo Nicole N Cole Christian M CM Pannikkat Meera M Bollong Michael J MJ Rosarda Jessica D JD Kelly Jeffery W JW Wiseman R Luke RL

RSC chemical biology 20240717 9

We previously showed that the proteostasis regulator compound AA147 (<i>N</i>-(2-hydroxy-5-methylphenyl)benzenepropanamide) potently protects against neurotoxic insults, such as glutamate-induced oxytosis. Though AA147 is a selective activator of the ATF6 arm of the unfolded protein response in non-neuronal cells, AA147-dependent protection against glutamate toxicity in cells of neuronal origin is primarily mediated through activation of the NRF2 oxidative stress response. AA147 activates NRF2 t ...[more]

PMID: 39211477

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Project description:BackgroundFerroptosis has been proven to contribute to the progression of myocardial ischemia/reperfusion (I/R) injury and can be inhibited or promoted by ATF3. Short-chain fatty acids (SCFAs) have shown benefits in various cardiovascular diseases with anti-inflammatory and antioxidant effects. However, the impact of SCFAs on ferroptosis in ischemic-stimulated cardiomyocytes remains unknown. This study aimed to investigate the effect of SCFAs on cardiomyocyte ferroptosis, the expression of ATF3, and its potential upstream regulators.Methods and resultsThe expression of ATF3, ferroptosis pathway geneset (FPG), and geneset of potential regulators for ATF3 (GPRA, predicted by the PROMO database) was explored in the public human myocardial infarction single-cell RNA-seq (sma) dataset. Cardiomyocyte data was extracted from the dataset and re-clustered to explore the FPG, ATF3, and GPRA expression patterns in cardiomyocyte subclusters. A dose-dependent toxic experiment was run to detect the suitable dose for SCFA treatment. The erastin-induced ferroptosis model and hypoxia-reoxygenation (H/R) model (10 h of hypoxia followed by 6 h of reoxygenation) were adopted to assess the effect of SCFAs via the CCK8 assay. Gene expression was examined via RT-PCR and western blot. Ferroptosis markers, including lipid peroxides and Fe2+, were detected using the liperfluo and ferroOrange probes, respectively. In the sma dataset, upregulated ferroptosis pathway genes were mainly found in the infarction-stimulated cardiac cells (border zone and fibrotic zone), particularly the cardiomyocytes and adipocytes. The ATF3 and some of its potential transcription factors (VDR, EGR3, PAX5, and SP1) can be regulated by SCFA. SCFA can attenuate erastin-induced lipid peroxidation in cardiomyocytes. SCFA treatment can also reverse erastin-induced Fe2+ increase but may strengthen the Fe2+ in the H/R model. We also precisely defined a ferroptosis subcluster of cardiomyocytes (CM09) that highly expressed FPG, ATF3, and GPRA.ConclusionThe ATF3 and the ferroptosis pathway are elevated in cardiomyocytes of injury-related cardiac regions (border zone, ischemic zone, and fibrotic zone). SCFA can attenuate cardiomyocyte ferroptosis and regulate the expression of ATF3. Our study offers novel insights into the potential targets of SCFAs in the cardiovascular system.

Project description:BackgroundHypoxia plays an important role in the chemotherapy resistance of nasopharyngeal carcinoma (NPC). Ferroptosis is a newly discovered form of programmed cell death and ferroptosis inducers showed promising therapeutic effects in some cancers. However, the sensibility of NPC cells to ferroptosis under the hypoxic microenvironment is still unclear, and this study was designed to clarify it.MethodsNPC cells, treated with erastin, were placed in a normoxia or hypoxic environment (5% CO2, 94% N2 and 1% O2) at 37℃for 24 h. After exposed to hypoxia, ferroptosis-associated phenotypes were detected by CCK8, MDA, GSH, lipid ROS and Fe. The gene expression profiles of head and neck squamous cell carcinoma (HNSCC) tissues were downloaded from the TCGA database to screen construction molecule. BAP1 was screened out and its functions on erastin-induced ferroptosis in NPC cells were detected by knockdown of BAP1. Luciferase reporter assay and co-IP experiment were performed to explore the molecular mechanism. Finally, the tumour xenograft model was applied to further verify these results in vivo.ResultsCCK8 assay showed that IC50 of NPC cells treated with erastin under hypoxia was significantly lower than that under normoxia. Hypoxia significantly increased the levels of lipid ROS and MDA, and decreased GSH content induced by erastin. A prognostic risk model for HNSCC with six ferroptosis-related genes was constructed and validated based on TCGA database. BAP1 was significantly up-regulated under hypoxia, and luciferase reporter assay showed that HIF-1α was an upstream transcription regulator of BAP1. Knockdown of BAP1 in NPC cells significantly increased the IC50 value of erastin under hypoxia and significantly ameliorated erastin-induced ferroptosis under hypoxia in aspect of lipid ROS, MDA content and GSH. Co-IP results showed that BAP1 mediated deubiquitination of H2A and decreased SLC7A11 expression. Finally, knockdown of BAP1 reduced sensitivity to erastin-induced ferroptosis in a tumour xenograft model. And the level of H2A was significantly decreased in xenograft tumors of BAP1 knockdown cells.ConclusionHypoxia-induced BAP1 enhances erastin-induced ferroptosis in NPC by stabilizing H2A. Ferroptosis inducers targeting BAP1 may be an effective way to improve chemotherapy resistance in NPC, especially in the hypoxic microenvironment.

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Metabolically activated proteostasis regulators that protect against erastin-induced ferroptosis.

Publications

Metabolically activated proteostasis regulators that protect against erastin-induced ferroptosis.

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