Molecular Mechanisms Regulating Ferroptosis By NEDD4L

Background:Ferroptosis is a form of non-apoptotic regulated cell death driven by the accumulation of lipid reactive oxygen species due to the imbalance of iron and cystine metabolism.Although the occurrence of ferroptosis involves numerous signals and protein changes,the mechanism of its modulation has not yet been fully elucidated.The ubiquitin-proteasome system plays a key role in the degradation of intracellular proteins.It consists of ubiquitin,ubiquitin-activating enzyme E1,ubiquitin-binding enzyme E2,ubiquitin-ligase E3,proteasome and its substrate(protein).The ubiquitin proteasome system is involved in the regulation of various physiological and pathological processes,and is closely related to the occurrence and development of neurodegenerative diseases,tumors and other aging-related diseases.The molecular mechanism of the ubiquitin proteasome system involved in regulating ferroptosis has not been fully elucidated.Purpose:This study will explore the molecular relationship between the ubiquitin proteasome system and ferroptosis in human tumor cells,discover a new ubiquitin proteasome pathway that regulates ferroptosis,and further study its signal transduction and molecular mechanisms of ferroptosis.This study will provide new ideas for ferroptosis-based tumor targeted therapy.Methods:In this study,PANC1 human pancreatic cancer cell line and OVCAR3 human ovarian cancer cell line were used as the main research materials.First,ferroptosis was induced by erastin and RSL3 in the PANC1 cell line.q RT-PCR was used to detect the m RNA expression levels of 571 ubiquitin-proteasome-related E1,E2,and E3 enzyme genes;Western blot further detected the differentially expressed genes;sh RNAs were used to construct knockdown cell lines of E3 ubiquitin ligase NEDD4 L and lactotransferrin(LTF);PI staining was used to detect cell death after drug treatment;The clone formation experiment was used to assay cell proliferation after drug treatment;Biochemical kits and flow cytometry were used to detect the lipid oxidation end products of malondialdehyde(MDA)and reactive oxygen species(ROS)in tumor cells;Immunoprecipitation combined with mass spectrometry(IP-MS)was used to detect NEDD4L-binding protein.Statistics were calculated with Graph Pad Prism 8.All data results were expressed as mean ± standard deviation.A standard two-tailed unpaired Student’s t test or one-way ANOVA was used for statistical analysis.A P value of less than 0.05 was considered statistically significant.Results:The m RNA and protein levels of ubiquitin ligase NEDD4 L were significantly increased when ferroptosis occurred,and the knockdown of NEDD4 L significantly promoted the occurrence of ferroptosis.IP-MS revealed that lactotransferrin(LTF),a member of the iron transporter family,is a protein directly bound to NEDD4 L.Knockdown of LTF decreased,intracellular MDA and ROS production,leading to ferroptosis resistance.Conclusion:NEDD4L-mediated LTF protein degradation limits ferroptosis.Our studies establish a new mechanism for the regulation of ferroptosis by the ubiquitin proteasome system,providing a potential strategy for anti-cancer treatment.