Objective To explore the role and mechanism of mammalian target of rapamycin (mTOR) in oxidized low-density lipoprotein (oxLDL)-induced ferroptosis in vascular smooth muscle cells (VSMCs). Methods A model of oxLDL-induced VSMC ferroptosis was established. VSMCs were co-treated with either the mTOR inhibitor rapamycin or the autophagy inducer carbonyl cyanide m-chlorophenylhydrazone (CCCP), followed by detection of autophagy and ferroptosis-related indexes. Quantitative real-time PCR and Western blot were used respectively to analyze the expression of mTOR, glutathione peroxidase 4 (GPX4), sequestosome 1 (p62), and microtubule-associated protein 1 light chain 3 (LC3). Flow cytometry was employed to assess VSMC death. C11 BODIPY fluorescent staining was used to measure cellular lipid peroxidation levels. Colorimetric assays were performed to determine the contents of malondialdehyde (MDA), ferrous ion (Fe²⁺) and glutathione (GSH). Results oxLDL significantly upregulated mTOR expression in VSMCs, while increasing p62 expression and reducing LC3 expression, thereby suppressing VSMC autophagy. Compared with oxLDL treatment alone, rapamycin co-treatment reversed oxLDL-induced VSMC ferroptosis, as characterized by reduced VSMC death, increased GPX4 expression and GSH contents, along with decreased MDA content, Fe²⁺ content and lipid peroxidation levels. Similarly, CCCP co-treatment activated autophagy characterized by reduced p62 expression and elevated LC3 expression, which subsequently alleviated oxLDL-induced ferroptosis, showing reduced VSMC death, increased GPX4 expressions and GSH contents, and decreased MDA content, Fe²⁺ content and lipid peroxidation levels. Moreover, mTOR inhibition by rapamycin significantly reversed the oxLDL-induced upregulation of p62 and downregulation of LC3. Conclusion mTOR may promote oxLDL-induced VSMC ferroptosis by suppressing autophagy.
Ferroptosis/drug effects* ; Lipoproteins, LDL/metabolism* ; TOR Serine-Threonine Kinases/physiology* ; Autophagy/drug effects* ; Muscle, Smooth, Vascular/metabolism* ; Animals ; Rats ; Myocytes, Smooth Muscle/cytology* ; Cells, Cultured ; Lipid Peroxidation/drug effects* ; Sequestosome-1 Protein/genetics* ; Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism* ; Microtubule-Associated Proteins/genetics* ; Sirolimus/pharmacology*

Sequestosome 1 (SQSTM1/p62) is a selective autophagy adaptor protein that plays an important role in the clearance of proteins to be degraded as well as in the maintenance of cellular proteostasis. p62 protein has multiple functional domains, which interact with several downstream proteins to precisely regulate multiple signaling pathways, thereby linking p62 to oxidative defense systems, inflammatory responses and nutrient sensing. Studies have shown that mutation or abnormal expression of p62 is closely related to the occurrence and development of various diseases, including neurodegenerative diseases, tumors, infectious diseases, genetic diseases and chronic diseases. This review summarizes the structural features and molecular functions of p62. Moreover, we systematically introduce its multiple functions in protein homeostasis and regulation of signaling pathways. Furthermore, the complexity and versatility of p62 in the occurrence and development of diseases are summarized, with the aim to provide a reference for understanding the function of p62 protein and facilitating related disease research.
Humans ; Autophagy/genetics* ; Sequestosome-1 Protein/metabolism* ; Adaptor Proteins, Signal Transducing/metabolism* ; Signal Transduction ; Neoplasms/genetics*

Apoptosis ; Autophagy ; Ferroptosis ; Iron/metabolism* ; Sequestosome-1 Protein/metabolism* ; Ubiquitin-Specific Proteases/metabolism*

Animals ; Autophagy/genetics* ; Cholesterol/metabolism* ; Gene Expression Regulation ; Integrases/metabolism* ; Leydig Cells/metabolism* ; Male ; Mice, Knockout ; Multienzyme Complexes/metabolism* ; Phosphoproteins/metabolism* ; Primary Cell Culture ; Progesterone Reductase/metabolism* ; RNA Splicing Factors/metabolism* ; Scavenger Receptors, Class B/metabolism* ; Sequestosome-1 Protein/metabolism* ; Signal Transduction ; Sirtuin 1/genetics* ; Sodium-Hydrogen Exchangers/metabolism* ; Steroid 17-alpha-Hydroxylase/metabolism* ; Steroid Isomerases/metabolism* ; Testosterone/genetics*

Epithelial-mesenchymal transition (EMT) is involved in both physiological and pathological processes. EMT plays an essential role in the invasion, migration and metastasis of tumours. Autophagy has been shown to regulate EMT in a variety of cancers but not in head and neck squamous cell carcinoma (HNSCC). Herein, we investigated whether autophagy also regulates EMT in HNSCC. Analyses of clinical data from three public databases revealed that higher expression of fibronectin-1 (FN1) correlated with poorer prognosis and higher tumour pathological grade in HNSCC. Data from SCC-25 cells demonstrated that rapamycin and Earle's balanced salt solution (EBSS) promoted autophagy, leading to increased FN1 degradation, while 3-methyladenine (3-MA), bafilomycin A1 (Baf A1) and chloroquine (CQ) inhibited autophagy, leading to decreased FN1 degradation. On the other hand, autophagic flux was blocked in BECN1 mutant HNSCC Cal-27 cells, and rapamycin did not promote autophagy in Cal-27 cells; also in addition, FN1 degradation was inhibited. Further, we identified FN1 degradation through the lysosome-dependent degradation pathway using the proteasome inhibitor MG132. Data from immunoprecipitation assays also showed that p62/SQSTM1 participated as an autophagy adapter in the autophagy-lysosome pathway of FN1 degradation. Finally, data from immunoprecipitation assays demonstrated that the interaction between p62 and FN1 was abolished in p62 mutant MCF-7 and A2780 cell lines. These results indicate that autophagy significantly promotes the degradation of FN1. Collectively, our findings clearly suggest that FN1, as a marker of EMT, has adverse effects on HNSCC and elucidate the autophagy-lysosome degradation mechanism of FN1.
Autophagy ; Cell Line, Tumor ; Female ; Fibronectins ; Humans ; Lysosomes/metabolism* ; Ovarian Neoplasms ; Sequestosome-1 Protein/metabolism* ; Squamous Cell Carcinoma of Head and Neck

Mutations or inactivation of parkin, an E3 ubiquitin ligase, are associated with familial form or sporadic Parkinson's disease (PD), respectively, which manifested with the selective vulnerability of neuronal cells in substantia nigra (SN) and striatum (STR) regions. However, the underlying molecular mechanism linking parkin with the etiology of PD remains elusive. Here we report that p62, a critical regulator for protein quality control, inclusion body formation, selective autophagy and diverse signaling pathways, is a new substrate of parkin. P62 levels were increased in the SN and STR regions, but not in other brain regions in parkin knockout mice. Parkin directly interacts with and ubiquitinates p62 at the K13 to promote proteasomal degradation of p62 even in the absence of ATG5. Pathogenic mutations, knockdown of parkin or mutation of p62 at K13 prevented the degradation of p62. We further showed that parkin deficiency mice have pronounced loss of tyrosine hydroxylase positive neurons and have worse performance in motor test when treated with 6-hydroxydopamine hydrochloride in aged mice. These results suggest that, in addition to their critical role in regulating autophagy, p62 are subjected to parkin mediated proteasomal degradation and implicate that the dysregulation of parkin/p62 axis may involve in the selective vulnerability of neuronal cells during the onset of PD pathogenesis.
Adaptor Proteins, Signal Transducing ; chemistry ; metabolism ; Animals ; HEK293 Cells ; Heat-Shock Proteins ; chemistry ; metabolism ; Humans ; Lysine ; metabolism ; Mice ; Neurons ; metabolism ; pathology ; Oxidopamine ; pharmacology ; Parkinson Disease ; metabolism ; pathology ; Proteasome Endopeptidase Complex ; metabolism ; Protein Stability ; Proteolysis ; drug effects ; Sequestosome-1 Protein ; Ubiquitin-Protein Ligases ; metabolism ; Ubiquitination ; drug effects