Intermittent hypoxia exacerbates metabolic dysfunction-associated fatty liver disease by aggravating hepatic copper deficiency-induced ferroptosis.

Intermittent hypoxia (IH) is an independent risk factor for metabolic dysfunction-associated fatty liver disease (MAFLD). Copper deficiency can disrupt redox homeostasis, iron, and lipid metabolism. Here, we investigated whether hepatic copper deficiency plays a role in IH-associated MAFLD and explored the underlying mechanism(s). Male C57BL/6 mice were fed a western-type diet with adequate copper (CuA) or marginally deficient copper (CuD) and were exposed separately to room air (RA) or IH. Hepatic histology, plasma biomarkers, copper-iron status, and oxidative stress were assessed. An in vitro HepG2 cell lipotoxicity model and proteomic analysis were used to elucidate the specific targets involved. We observed that there were no differences in hepatic phenotypes between CuA-fed and CuD-fed mice under RA. However, in IH exposure, CuD-fed mice showed more pronounced hepatic steatosis, liver injury, and oxidative stress than CuA-fed mice. IH induced copper accumulation in the brain and heart and exacerbated hepatic copper deficiency and secondary iron deposition. In vitro, CuD-treated cells with IH exposure showed elevated levels of lipid accumulation, oxidative stress, and ferroptosis susceptibility. Proteomic analysis identified 360 upregulated and 359 downregulated differentially expressed proteins between CuA and CuD groups under IH; these proteins were mainly enriched in citrate cycle, oxidative phosphorylation, fatty acid metabolism, the peroxisome proliferator-activated receptor (PPAR)α pathway, and ferroptosis. In IH exposure, CuD significantly upregulated the ferroptosis-promoting factor arachidonyl-CoA synthetase long chain family member (ACSL)4. ACSL4 knockdown markedly eliminated CuD-induced ferroptosis and lipid accumulation in IH exposure. In conculsion, IH can lead to reduced hepatic copper reserves and secondary iron deposition, thereby inducing ferroptosis and subsequent MAFLD progression. Insufficient dietary copper may worsen IH-associated MAFLD.

Silencing CK19 regulates ferroptosis by affecting the expression of GPX4 and ACSL4 in oral squamous cell carcinoma in vivo and in vitro.

To analyze the mechanism of how interfering with the cytokeratin 19 (CK19) pathway via the ferroptosis pathway affects tumor biological behaviors in the process of oral squamous cell carcinoma (OSCC) development. TCGA was used to analyze the expression of CK19 in pan-cancer and head and neck squamous cell carcinoma (HNSC) and to explore the ferroptosis-related genes related to HNSC. The effect of silencing CK19 on the migration ability of HSC-4 cells was verified by wound healing and migration assay. HSC-4 cells with silencing of CK19 and tumor-bearing nude mouse model were constructed. RT-qPCR, immunofluorescence and western blot were used to analyze the expression of ferroptosis-related genes. CK19 is highly expressed in human OSCC and nude mice. The migration ability of cells in the CK19-silenced group was lower than that of the control group. In vivo and in vitro, CK19 was negatively correlated with the expression of ACSL4 and positively correlated with the expression of GPX4. Compared with the control group, GPX4 expression was down-regulated and ACSL4 expression was up-regulated in the CK19-silenced group. Silencing CK19 also increased intracellular Fe2+ content and MDA content. Silencing CK19 can affect the expression of GPX4 and ACSL4 to regulate ferroptosis and at the same time increase the content of MDA, Fe2+ and ROS levels, thereby activating the regulation of ferroptosis pathway in the development of OSCC.

SIAH2-Mediated Degradation of ACSL4 Inhibits the Anti-Tumor Activity of CD8+ T Cells in Hepatocellular Carcinoma.

SIAH2 function as an oncogene in various cancer. However, the roles of SIAH2 in hepatocellular carcinoma (HCC) are still unknown. This study aimed to investigate the roles of SIAH2 in HCC. Immunohistochemistry was used determine SIAH2 and ACSL4 expression in clinical samples. RT-qPCR was used to determine mRNA expression. Western blot assay was applied for determining protein expression. Ubiquitination assay was conducted for determining ubiquitination of ACSL4. Xenograft experiment was applied for determining tumor growth. Flow cytometry was applied to determine the functions of CD4+ and CD8+ T cells. SIAH2 expression was overexpressed in HCC tumors. High levels of SIAH2 predicted poor outcomes. However, SIAH2 knockdown promoted the proliferation of CD8+ T cells as well as promoted the ferroptosis of tumor cells, inhibiting tumor growth in HCC. ACSL4 is required for CD8+ T cell-mediated ferroptosis of HCC cells. However, SIAH2 induced ubiquitination of ACSL4 and inhibited its expression. SIAH2 specific inhibitor menadione promoted the immune checkpoint blockade. Taken together, SIAH2-mediated inactivation of CD8+ T cells inhibits the ferroptosis of HCC via mediating ubiquitination of ACSL4. Therefore, targeting SIAH2 may be a promising strategy for HCC.

De Novo Synthesis of Resveratrol from Sucrose by Metabolically Engineered Yarrowia lipolytica.

Resveratrol, a phenylpropanoid compound, exhibits diverse pharmacological properties, making it a valuable candidate for health and disease management. However, the demand for resveratrol exceeds the capacity of plant extraction methods, necessitating alternative production strategies. Microbial synthesis offers several advantages over plant-based approaches and presents a promising alternative. Yarrowia lipolytica stands out among microbial hosts due to its safe nature, abundant acetyl-CoA and malonyl-CoA availability, and robust pentose phosphate pathway. This study aimed to engineer Y. lipolytica for resveratrol production. The resveratrol biosynthetic pathway was integrated into Y. lipolytica by adding genes encoding tyrosine ammonia lyase from Rhodotorula glutinis, 4-coumarate CoA ligase from Nicotiana tabacum, and stilbene synthase from Vitis vinifera. This resulted in the production of 14.3 mg/L resveratrol. A combination of endogenous and exogenous malonyl-CoA biosynthetic modules was introduced to enhance malonyl-CoA availability. This included genes encoding acetyl-CoA carboxylase 2 from Arabidopsis thaliana, malonyl-CoA synthase, and a malonate transporter protein from Bradyrhizobium diazoefficiens. These strategies increased resveratrol production to 51.8 mg/L. The further optimization of fermentation conditions and the utilization of sucrose as an effective carbon source in YP media enhanced the resveratrol concentration to 141 mg/L in flask fermentation. By combining these strategies, we achieved a titer of 400 mg/L resveratrol in a controlled fed-batch bioreactor. These findings demonstrate the efficacy of Y. lipolytica as a platform for the de novo production of resveratrol and highlight the importance of metabolic engineering, enhancing malonyl-CoA availability, and media optimization for improved resveratrol production.

G6PD and ACSL3 are synthetic lethal partners of NF2 in Schwann cells.

Neurofibromatosis Type II (NFII) is a genetic condition caused by loss of the NF2 gene, resulting in activation of the YAP/TAZ pathway and recurrent Schwann cell tumors, as well as meningiomas and ependymomas. Unfortunately, few pharmacological options are available for NFII. Here, we undertake a genome-wide CRISPR/Cas9 screen to search for synthetic-lethal genes that, when inhibited, cause death of NF2 mutant Schwann cells but not NF2 wildtype cells. We identify ACSL3 and G6PD as two synthetic-lethal partners for NF2, both involved in lipid biogenesis and cellular redox. We find that NF2 mutant Schwann cells are more oxidized than control cells, in part due to reduced expression of genes involved in NADPH generation such as ME1. Since G6PD and ME1 redundantly generate cytosolic NADPH, lack of either one is compatible with cell viability, but not down-regulation of both. Since genetic deficiency for G6PD is tolerated in the human population, G6PD could be a good pharmacological target for NFII.

Histone lactylation-regulated METTL3 promotes ferroptosis via m6A-modification on ACSL4 in sepsis-associated lung injury.

Elevated lactate levels are a significant biomarker of sepsis and are positively associated with sepsis-related mortality. Sepsis-associated lung injury (ALI) is a leading cause of poor prognosis in clinical patients. However, the underlying mechanisms of lactate's involvement in sepsis-associated ALI remain unclear. In this study, we demonstrate that lactate regulates N6-methyladenosine (m6A) modification levels by facilitating p300-mediated H3K18la binding to the METTL3 promoter site. The METTL3-mediated m6A modification is enriched in ACSL4, and its mRNA stability is regulated through a YTHDC1-dependent pathway. Furthermore, short-term lactate stimulation upregulates ACSL4, which promotes mitochondria-associated ferroptosis. Inhibition of METTL3 through knockdown or targeted inhibition effectively suppresses septic hyper-lactate-induced ferroptosis in alveolar epithelial cells and mitigates lung injury in septic mice. Our findings suggest that lactate induces ferroptosis via the GPR81/H3K18la/METTL3/ACSL4 axis in alveolar epithelial cells during sepsis-associated ALI. These results reveal a histone lactylation-driven mechanism inducing ferroptosis through METTL3-mediated m6A modification. Targeting METTL3 represents a promising therapeutic strategy for patients with sepsis-associated ALI.

Unveiling phenylpropanoid regulation: the role of DzMYB activator and repressor in durian (Durio zibethinus) fruit.

KEY MESSAGE: DzMYB2 functions as an MYB activator, while DzMYB3 acts as an MYB repressor. They bind to promoters, interact with DzbHLH1, and influence phenolic contents, revealing their roles in phenylpropanoid regulation in durian pulps. Durian fruit has a high nutritional value attributed to its enriched bioactive compounds, including phenolics, carotenoids, and vitamins. While various transcription factors (TFs) regulate phenylpropanoid biosynthesis, MYB (v-myb avian myeloblastosis viral oncogene homolog) TFs have emerged as pivotal players in regulating key genes within this pathway. This study aimed to identify additional candidate MYB TFs from the transcriptome database of the Monthong cultivar at five developmental/postharvest ripening stages. Candidate transcriptional activators were discerned among MYBs upregulated during the ripe stage based on the positive correlation observed between flavonoid biosynthetic genes and flavonoid contents in ripe durian pulps. Conversely, MYBs downregulated during the ripe stage were considered candidate repressors. This study focused on a candidate MYB activator (DzMYB2) and a candidate MYB repressor (DzMYB3) for functional characterization. LC-MS/MS analysis using Nicotiana benthamiana leaves transiently expressing DzMYB2 revealed increased phenolic compound contents compared with those in leaves expressing green fluorescence protein controls, while those transiently expressing DzMYB3 showed decreased phenolic compound contents. Furthermore, it was demonstrated that DzMYB2 controls phenylpropanoid biosynthesis in durian by regulating the promoters of various biosynthetic genes, including phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR). Meanwhile, DzMYB3 regulates the promoters of PAL, 4-coumaroyl-CoA ligase (4CL), CHS, and CHI, resulting in the activation and repression of gene expression. Moreover, it was discovered that DzMYB2 and DzMYB3 could bind to another TF, DzbHLH1, in the regulation of flavonoid biosynthesis. These findings enhance our understanding of the pivotal role of MYB proteins in regulating the phenylpropanoid pathway in durian pulps.

Long-chain acyl-CoA synthetase-4 regulates endometrial decidualization through a fatty acid ß-oxidation pathway rather than lipid droplet accumulation.

OBJECTIVE: Lipid metabolism plays an important role in early pregnancy, but its effects on decidualization are poorly understood. Fatty acids (FAs) must be esterified by fatty acyl-CoA synthetases to form biologically active acyl-CoA in order to enter the anabolic and/or catabolic pathway. Long-chain acyl-CoA synthetase 4 (ACSL4) is associated with female reproduction. However, whether it is involved in decidualization is unknown. METHODS: The expression of ACSL4 in human and mouse endometrium was detected by immunohistochemistry. ACSL4 levels were regulated by the overexpression of ACSL4 plasmid or ACSL4 siRNA, and the effects of ACSL4 on decidualization markers and morphology of endometrial stromal cells (ESCs) were clarified. A pregnant mouse model was established to determine the effect of ACSL4 on the implantation efficiency of mouse embryos. Modulation of ACSL4 detects lipid anabolism and catabolism. RESULTS: Through examining the expression level of ACSL4 in human endometrial tissues during proliferative and secretory phases, we found that ACSL4 was highly expressed during the secretory phase. Knockdown of ACSL4 suppressed decidualization and inhibited the mesenchymal-to-epithelial transition induced by MPA and db-cAMP in ESCs. Further, the knockdown of ACSL4 reduced the efficiency of embryo implantation in pregnant mice. Downregulation of ACSL4 inhibited FA ß-oxidation and lipid droplet accumulation during decidualization. Interestingly, pharmacological and genetic inhibition of lipid droplet synthesis did not affect FA ß-oxidation and decidualization, while the pharmacological and genetic inhibition of FA ß-oxidation increased lipid droplet accumulation and inhibited decidualization. In addition, inhibition of ß-oxidation was found to attenuate the promotion of decidualization by the upregulation of ACSL4. The decidualization damage caused by ACSL4 knockdown could be reversed by activating ß-oxidation. CONCLUSIONS: Our findings suggest that ACSL4 promotes endometrial decidualization by activating the ß-oxidation pathway. This study provides interesting insights into our understanding of the mechanisms regulating lipid metabolism during decidualization.

Soot nanoparticles promote ferroptosis in dopaminergic neurons via alteration of m6A RNA methylation in Parkinson's disease.

Soot nanoparticles (SNPs) are black carbon prevalent in atmospheric environment with significant impacts on public health, leading to neurodegenerative diseases including development of Parkinson's disease (PD). This study investigated the effects of SNPs exposure on PD symptoms, employing both in vivo and in vitro PD models. In the in vivo experiments, animal behavior assessments showed that SNPs exposure exacerbated motor and cognitive impairments in PD mice. Molecular biology techniques further unveiled that SNPs aggravated degeneration of dopaminergic neurons. In vitro experiments revealed that SNPs exposure intensified ferroptosis of PD cells by increasing reactive oxygen species and iron ion levels, while reducing glutathione levels and mitochondrial membrane potential. Sequencing tests indicated elevated N6-methyladenosine (m6A) alteration of the ferroptosis-related protein, acyl-CoA synthetase long chain family member 4 (ACSL4). This study demonstrates that SNPs may exacerbate the onset and progression of PD by recruiting YTH domain-containing family protein 1 (YTHDF1) protein, enhancing m6A methylation in the ACSL4 5'UTR, amplifying ACSL4 protein expression, and accelerating the ferroptosis process in dopaminergic neurons. These molecular mechanisms underlying SNPs exacerbation of PD development may provide crucial insights for formulating environmental safety regulations and potential therapeutic strategies addressing PD in populations residing in regions with varied air quality.

Tongqiao Huoxue Decoction inhibits ferroptosis by facilitating ACSL4 ubiquitination degradation for neuroprotection against cerebral ischemia-reperfusion injury.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) refers to brain tissue injury caused by the temporary interruption of cerebral blood flow ischemia followed by the restoration of reperfusion, which is the main cause of post-stroke brain injury. A traditional Chinese herbal preparation called Tongqiao Huoxue Decoction (TQHX) has shown promise in reducing CIRI in rats. However, the mechanism of this herbal preparation for CIRI remains unclear. PURPOSE: This study aimed to evaluate the therapeutic effect of TQHX extract on rats with CIRI and to further explore the underlying mechanisms. METHODS: The active ingredients of TQHX extract were quantified by the high-performance liquid chromatography (HPLC) condition. We conducted thorough investigations to assess the effects of TQHX on CIRI and ferroptosis using oxygen-glucose deprivation/reperfusion (OGD/R)-treated PC12 cells as an in vitro model and transient middle cerebral artery occlusion (tMCAO) animals as an in vivo model. The neurological score assessment was performed to evaluate the neuroprotective effects of TQHX extract on tMCAO rats. Using histologic methods to study the extent of cerebral infarction, blood-brain barrier, and rat brain tissue. We examined the impact of TQHX on ferroptosis-related markers of Fe2+, superoxide dismutase (SOD), reactive oxygen species (ROS), and malondialdehyde (MDA) in the brain tissue. In addition, the expression of key proteins and markers of ferroptosis, as well as key factors associated with Acyl-CoA synthetase long-chain family member 4 (ACSL4) were detected by Western blot and quantitative real-time PCR (RT-qPCR). RESULTS: TQHX extract could decrease the Longa score and extent of cerebral infarction of tMCAO rats, which exerted the function of neuroprotection. Additionally, TQHX treatment efficiently decreased levels of MDA and ROS while increasing the expression of SOD and ferroptosis-related proteins including ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4) at the transcription and translation level. Meanwhile, TQHX provided strong protection against oxidative stress and ferritin accumulation by increasing the ubiquitination and degradation of ACSL4. The injection of OE-ACSL4 reversed the effects of TQHX on neuroprotection and ferroptosis inhibition in PC12 cells. The injection of shACSL4 reversely validate the crucial role of ACSL4 in CIRI rat treatment. CONCLUSION: This work shows that TQHX promotes the ubiquitination-mediated degradation of ACSL4, which improves oxidative stress and inhibits the beginning of ferroptosis in cells. TQHX provides a possible path for additional research in CIRI therapies, advancing translational investigations.

Mitochondrial GPX4 acetylation is involved in cadmium-induced renal cell ferroptosis.

Increasing evidences demonstrate that environmental stressors are important inducers of acute kidney injury (AKI). This study aimed to investigate the impact of exposure to Cd, an environmental stressor, on renal cell ferroptosis. Transcriptomics analyses showed that arachidonic acid (ARA) metabolic pathway was disrupted in Cd-exposed mouse kidneys. Targeted metabolomics showed that renal oxidized ARA metabolites were increased in Cd-exposed mice. Renal 4-HNE, MDA, and ACSL4, were upregulated in Cd-exposed mouse kidneys. Consistent with animal experiments, the in vitro experiments showed that mitochondrial oxidized lipids were elevated in Cd-exposed HK-2 cells. Ultrastructure showed mitochondrial membrane rupture in Cd-exposed mouse kidneys. Mitochondrial cristae were accordingly reduced in Cd-exposed mouse kidneys. Mitochondrial SIRT3, an NAD+-dependent deacetylase that regulates mitochondrial protein stability, was reduced in Cd-exposed mouse kidneys. Subsequently, mitochondrial GPX4 acetylation was elevated and mitochondrial GPX4 protein was reduced in Cd-exposed mouse kidneys. Interestingly, Cd-induced mitochondrial GPX4 acetylation and renal cell ferroptosis were exacerbated in Sirt3-/- mice. Conversely, Cd-induced mitochondrial oxidized lipids were attenuated in nicotinamide mononucleotide (NMN)-pretreated HK-2 cells. Moreover, Cd-evoked mitochondrial GPX4 acetylation and renal cell ferroptosis were alleviated in NMN-pretreated mouse kidneys. These results suggest that mitochondrial GPX4 acetylation, probably caused by SIRT3 downregulation, is involved in Cd-evoked renal cell ferroptosis.

ALKBH5 targets ACSL4 mRNA stability to modulate ferroptosis in hyperbilirubinemia-induced brain damage.

Bilirubin-induced brain damage is a serious clinical consequence of hyperbilirubinemia, yet the underlying molecular mechanisms remain largely unknown. Ferroptosis, an iron-dependent cell death, is characterized by iron overload and lipid peroxidation. Here, we report a novel regulatory mechanism of demethylase AlkB homolog 5 (ALKBH5) in acyl-coenzyme A synthetase long-chain family member 4 (ACSL4)-mediated ferroptosis in hyperbilirubinemia. Hyperdifferential PC12 cells and newborn Sprague-Dawley rats were used to establish in vitro and in vivo hyperbilirubinemia models, respectively. Proteomics, coupled with bioinformatics analysis, first suggested the important role of ferroptosis in hyperbilirubinemia-induced brain damage. In vitro experiments showed that ferroptosis is activated in hyperbilirubinemia, and ferroptosis inhibitors (desferrioxamine and ferrostatin-1) treatment effectively alleviates hyperbilirubinemia-induced oxidative damage. Notably, we observed that the ferroptosis in hyperbilirubinemia was regulated by m6A modification through the downregulation of ALKBH5 expression. MeRIP-seq and RIP-seq showed that ALKBH5 may trigger hyperbilirubinemia ferroptosis by stabilizing ACSL4 mRNA via m6A modification. Further, hyperbilirubinemia-induced oxidative damage was alleviated through ACSL4 genetic knockdown or rosiglitazone-mediated chemical repression but was exacerbated by ACSL4 overexpression. Mechanistically, ALKBH5 promotes ACSL4 mRNA stability and ferroptosis by combining the 669 and 2015 m6A modified sites within 3' UTR of ACSL4 mRNA. Our findings unveil a novel molecular mechanism of ferroptosis and suggest that m6A-dependent ferroptosis could be an underlying clinical target for the therapy of hyperbilirubinemia.

HDAC inhibitors activate lipid peroxidation and ferroptosis in gastric cancer.

Gastric cancer remains among the deadliest neoplasms worldwide, with limited therapeutic options. Since efficacies of targeted therapies are unsatisfactory, drugs with broader mechanisms of action rather than a single oncogene inhibition are needed. Preclinical studies have identified histone deacetylases (HDAC) as potential therapeutic targets in gastric cancer. However, the mechanism(s) of action of HDAC inhibitors (HDACi) are only partially understood. This is particularly true with regard to ferroptosis as an emerging concept of cell death. In a panel of gastric cancer cell lines with different molecular characteristics, tumor cell inhibitory effects of different HDACi were studied. Lipid peroxidation levels were measured and proteome analysis was performed for the in-depth characterization of molecular alterations upon HDAC inhibition. HDACi effects on important ferroptosis genes were validated on the mRNA and protein level. Upon HDACi treatment, lipid peroxidation was found increased in all cell lines. Class I HDACi (VK1, entinostat) showed the same toxicity profile as the pan-HDACi vorinostat. Proteome analysis revealed significant and concordant alterations in the expression of proteins related to ferroptosis induction. Key enzymes like ACSL4, POR or SLC7A11 showed distinct alterations in their expression patterns, providing an explanation for the increased lipid peroxidation. Results were also confirmed in primary human gastric cancer tissue cultures as a relevant ex vivo model. We identify the induction of ferroptosis as new mechanism of action of class I HDACi in gastric cancer. Notably, these findings were independent of the genetic background of the cell lines, thus introducing HDAC inhibition as a more general therapeutic principle.

Oncogenic fatty acid oxidation senses circadian disruption in sleep-deficiency-enhanced tumorigenesis.

Circadian disruption predicts poor cancer prognosis, yet how circadian disruption is sensed in sleep-deficiency (SD)-enhanced tumorigenesis remains obscure. Here, we show fatty acid oxidation (FAO) as a circadian sensor relaying from clock disruption to oncogenic metabolic signal in SD-enhanced lung tumorigenesis. Both unbiased transcriptomic and metabolomic analyses reveal that FAO senses SD-induced circadian disruption, as illustrated by continuously increased palmitoyl-coenzyme A (PA-CoA) catalyzed by long-chain fatty acyl-CoA synthetase 1 (ACSL1). Mechanistically, SD-dysregulated CLOCK hypertransactivates ACSL1 to produce PA-CoA, which facilitates CLOCK-Cys194 S-palmitoylation in a ZDHHC5-dependent manner. This positive transcription-palmitoylation feedback loop prevents ubiquitin-proteasomal degradation of CLOCK, causing FAO-sensed circadian disruption to maintain SD-enhanced cancer stemness. Intriguingly, timed ß-endorphin resets rhythmic Clock and Acsl1 expression to alleviate SD-enhanced tumorigenesis. Sleep quality and serum ß-endorphin are negatively associated with both cancer development and CLOCK/ACSL1 expression in patients with cancer, suggesting dawn-supplemented ß-endorphin as a potential chronotherapeutic strategy for SD-related cancer.

Study on the Role and Mechanism of SLC3A2 in Tumor-Associated Macrophage Polarization and Bladder Cancer Cells Growth.

Background: Solute carrier family 3 member 2 (SLC3A2) is highly expressed in various types of cancers, including bladder cancer (BLCA). However, the role and mechanism of SLC3A2 in the onset and progression of BLCA are still unclear. Methods: The interfering plasmid for SLC3A2 was constructed and transfected into BLCA cells. Cell proliferation, invasion, and migration abilities were assessed to evaluate the impact of SLC3A2 silencing on BLCA cell growth. M1 and M2 macrophage polarization markers were detected to evaluate macrophage polarization. The levels of reactive oxygen species (ROS), lipid peroxidation, and Fe2+, as well as the expression of ferroptosis-related proteins, were measured to assess the occurrence of ferroptosis. Ferroptosis inhibitors were used to verify the mechanism. Results: The experimental results showed that SLC3A2 was highly expressed in BLCA cell lines. The proliferation, invasion, and migration of BLCA cells were reduced after interfering with SLC3A2. Interference with SLC3A2 led to increase the expression of M1 macrophage markers and decreased the expression of M2 macrophage markers in M0 macrophages co-cultured with tumor cells. Additionally, interference with SLC3A2 led to increased levels of ROS, lipid peroxidation, and Fe2+, downregulated the expression of solute carrier family 7 member11 (SLC7A11) and glutathione peroxidase 4 (GPX4), while upregulated the expression of acyl-coA synthetase long chain family member 4 (ACSL4) and transferrin receptor 1 (TFR1) in BLCA cells. However, the impact of SLC3A2 interference on cell proliferation and macrophage polarization was impeded by ferroptosis inhibitors. Conclusion: Interference with SLC3A2 inhibited the growth of BLCA cells and the polarization of tumor-associated macrophages by promoting ferroptosis in BLCA cells.

Lipid peroxidation-induced ferroptosis as a therapeutic target for mitigating neuronal injury and inflammation in sepsis-associated encephalopathy: insights into the hippocampal PEBP-1/15-LOX/GPX4 pathway.

BACKGROUND: Sepsis-associated encephalopathy (SAE) refers to the widespread impairment of brain function caused by noncentral nervous system infection mediated by sepsis. Lipid peroxidation-induced ferroptosis contributes to the occurrence and course of SAE. This study aimed to investigate the relationship between neuronal injury and lipid peroxidation-induced ferroptosis in SAE. METHODS: Baseline data were collected from pediatric patients upon admission, and the expression levels of various markers related to lipid peroxidation and ferroptosis were monitored in the serum and peripheral blood mononuclear cells (PBMCs) of patients with SAE as well as SAE model mice. The hippocampal phosphatidylethanolamine-binding protein (PEBP)-1/15-lysine oxidase (LOX)/ glutathione peroxidase 4 (GPX4) pathway was assessed for its role on the inhibitory effect of ferroptosis in SAE treatment. RESULTS: The results showed elevated levels of S100 calcium-binding protein beta (S-100ß), glial fibrillary acidic protein, and malondialdehyde in the serum of SAE patients, while superoxide dismutase levels were reduced. Furthermore, analysis of PBMCs revealed increased transcription levels of PEBP1, LOX, and long-chain fatty acyl-CoA synthetase family member 4 (ACSL4) in SAE patients, while the transcription levels of GPX4 and cystine/glutamate transporter xCT (SLC7A11) were decreased. In comparison to the control group, the SAE mice exhibited increased expression of S-100ß and neuron-specific enolase (NSE) in the hippocampus, whereas the expression of S-100ß and NSE were reduced in deferoxamine (DFO) mice. Additionally, iron accumulation was observed in the hippocampus of SAE mice, while the iron ion levels were reduced in the DFO mice. Inhibition of ferroptosis alleviated the mitochondrial damage (as assessed by transmission electron microscopy, hippocampal mitochondrial ATP detection, and the JC-1 polymer-to-monomer ratio in the hippocampus) and the oxidative stress response induced by SAE as well as attenuated neuroinflammatory reactions. Further investigations revealed that the mechanism underlying the inhibitory effect of ferroptosis in SAE treatment is associated with the hippocampal PEBP-1/15-LOX/GPX4 pathway. CONCLUSION: These results offer potential therapeutic targets for the management of neuronal injury in SAE and valuable insights into the potential mechanisms of ferroptosis in neurological disorders.

ACSL4 promotes malignant progression of Hepatocellular carcinoma by targeting PAK2 transcription.

Long-chain fatty acyl-Coa ligase 4 (ACSL4) is an important enzyme that converts fatty acids to fatty acyl-Coa esters, there is increasing evidence for its role in carcinogenesis. However, the precise role of ACLS4 in hepatocellular carcinoma (HCC) is not clearly understood. In the present study, we provide evidence that ACSL4 expression was specifically elevated in HCC and is associated with poor clinical outcomes. ACSL4 significantly promotes the growth and metastasis of HCC both in vitro and in vivo. RNA sequencing and functional experiments showed that the effect of ACSL4 on HCC development was heavily dependent on PAK2. ACSL4 expression is well correlated with PAK2 in HCC, and ACSL4 even transcriptionally increased PAK2 gene expression mediated by Sp1. In addition, emodin, a naturally occurring anthraquinone derivative, inhibited HCC cell growth and tumor progression by targeting ACSL4. In summary, ACSL4 plays a novel oncogene in HCC development by regulating PAK2 transcription. Targeting ACSL4 could be useful in drug development and therapy for HCC.

ACSF2 and lysine lactylation contribute to renal tubule injury in diabetes.

AIMS: Lactate accumulation is reported to be a biomarker for diabetic nephropathy progression. Lactate drives lysine lactylation, a newly discovered post-translational modification that is involved in the pathogenesis of cancers and metabolic and inflammatory disease. Here, we aimed to determine whether lysine lactylation is involved in the pathogenesis of diabetic nephropathy. METHODS: Renal biopsy samples from individuals with diabetic nephropathy (n=22) and control samples from individuals without diabetes and kidney disease (n=9) were obtained from the First Affiliated Hospital of Zhengzhou University for immunohistochemical staining. In addition, we carried out global lactylome profiling of kidney tissues from db/m and db/db mice using LC-MS/MS. Furthermore, we assessed the role of lysine lactylation and acyl-CoA synthetase family member 2 (ACSF2) in mitochondrial function in human proximal tubular epithelial cells (HK-2). RESULTS: The expression level of lysine lactylation was significantly increased in the kidneys of individuals with diabetes as well as in kidneys from db/db mice. Integrative lactylome analysis of the kidneys of db/db and db/m mice identified 165 upregulated proteins and 17 downregulated proteins, with an increase in 356 lysine lactylation sites and a decrease in 22 lysine lactylation sites decreased. Subcellular localisation analysis revealed that most lactylated proteins were found in the mitochondria (115 proteins, 269 sites). We further found that lactylation of the K182 site in ACSF2 contributes to mitochondrial dysfunction. Finally, the expression of ACSF2 was notably increased in the kidneys of db/db mice and individuals with diabetic nephropathy. CONCLUSIONS: Our study strongly suggests that lysine lactylation and ACSF2 are mediators of mitochondrial dysfunction and may contribute to the progression of diabetic nephropathy. DATA AVAILABILITY: The LC-MS/MS proteomics data have been deposited in the ProteomeXchange Consortium database ( https://proteomecentral.proteomexchange.org ) via the iProX partner repository with the dataset identifier PXD050070.

Ocimum kilimandscharicum 4CL11 negatively regulates adventitious root development via accumulation of flavonoid glycosides.

4-Coumarate-CoA Ligase (4CL) is an important enzyme in the phenylpropanoid biosynthesis pathway. Multiple 4CLs are identified in Ocimum species; however, their in planta functions remain enigmatic. In this study, we independently overexpressed three Ok4CL isoforms from Ocimum kilimandscharicum (Ok4CL7, -11, and -15) in Nicotiana benthamiana. Interestingly, Ok4CL11 overexpression (OE) caused a rootless or reduced root growth phenotype, whereas overexpression of Ok4CL15 produced normal adventitious root (AR) growth. Ok4CL11 overexpression in N. benthamiana resulted in upregulation of genes involved in flavonoid biosynthesis and associated glycosyltransferases accompanied by accumulation of specific flavonoid-glycosides (kaempferol-3-rhamnoside, kaempferol-3,7-O-bis-alpha-l-rhamnoside [K3,7R], and quercetin-3-O-rutinoside) that possibly reduced auxin levels in plants, and such effects were not seen for Ok4CL7 and -15. Docking analysis suggested that auxin transporters (PINs/LAXs) have higher binding affinity to these specific flavonoid-glycosides, and thus could disrupt auxin transport/signaling, which cumulatively resulted in a rootless phenotype. Reduced auxin levels, increased K3,7R in the middle and basal stem sections, and grafting experiments (intra and inter-species) indicated a disruption of auxin transport by K3,7R and its negative effect on AR development. Supplementation of flavonoids and the specific glycosides accumulated by Ok4CL11-OE to the wild-type N. benthamiana explants delayed the AR emergence and also inhibited AR growth. While overexpression of all three Ok4CLs increased lignin accumulation, flavonoids, and their specific glycosides were accumulated only in Ok4CL11-OE lines. In summary, our study reveals unique indirect function of Ok4CL11 to increase specific flavonoids and their glycosides, which are negative regulators of root growth, likely involved in inhibition of auxin transport and signaling.

ACSM1 and ACSM3 Regulate Fatty Acid Metabolism to Support Prostate Cancer Growth and Constrain Ferroptosis.

Solid tumors are highly reliant on lipids for energy, growth, and survival. In prostate cancer, the activity of the androgen receptor (AR) is associated with reprogramming of lipid metabolic processes. Here, we identified acyl-CoA synthetase medium chain family members 1 and 3 (ACSM1 and ACSM3) as AR-regulated mediators of prostate cancer metabolism and growth. ACSM1 and ACSM3 were upregulated in prostate tumors compared with nonmalignant tissues and other cancer types. Both enzymes enhanced proliferation and protected prostate cancer cells from death in vitro, whereas silencing ACSM3 led to reduced tumor growth in an orthotopic xenograft model. ACSM1 and ACSM3 were major regulators of the prostate cancer lipidome and enhanced energy production via fatty acid oxidation. Metabolic dysregulation caused by loss of ACSM1/3 led to mitochondrial oxidative stress, lipid peroxidation, and cell death by ferroptosis. Conversely, elevated ACSM1/3 activity enabled prostate cancer cells to survive toxic levels of medium chain fatty acids and promoted resistance to ferroptosis-inducing drugs and AR antagonists. Collectively, this study reveals a tumor-promoting function of medium chain acyl-CoA synthetases and positions ACSM1 and ACSM3 as key players in prostate cancer progression and therapy resistance. Significance: Androgen receptor-induced ACSM1 and ACSM3 mediate a metabolic pathway in prostate cancer that enables the utilization of medium chain fatty acids for energy production, blocks ferroptosis, and drives resistance to clinically approved antiandrogens.