Gynecological endocrinology : the official journal of the International Society of Gynecological Endocrinology · 2025
Involvement of USP33 in ferritinophagy and ferroptosis in endometriosis through the Hippo-YAP pathway.
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Li Li, You Wu, Zhang Mingzhe
Аннотация
OBJECTIVE: This study aimed to investigate the mechanism of the involvement of USP33 in autophagic ferroptosis in endometriosis (EMs).
METHODS: Endometrial stromal cells (ESCs) were isolated from 15 healthy controls and 30 patients with EMs, which were designated NESCs and EESCs, respectively. Real-time PCR and Western blotting were utilized to determine USP33 expression as well as GPX4, LC3II/LC3I, NCOA4, FTH1, LAST1, p-LAST1, YAP, and p-YAP levels. Immunofluorescence was used to detect the colocalization of ferritin and LAMP2, as well as that of LC3 and ferritin. The levels of ROS, Fe2+, MDA, and GSH were measured to assess ferroptosis. An LDH assay was performed to evaluate cell death. A co-IP assay was implemented to identify the interaction between USP33 and LAST1 and detect the level of LAST1 ubiquitination. The stability of the protein was also detected via a cycloheximide (CHX) assay.
RESULTS: USP33 was underexpressed in EMs patients. Loss of USP33 conferred resistance to ferroptosis in EESCs, as evidenced by increased proliferation; decreased levels of ROS, Fe2+, and MDA; elevated levels of GPX4 and GSH; and reduced cell death. In addition, USP33, which is localized in autophagosomes, was suggested to promote the degradation of ferritin in autophagosomes. Furthermore, USP33 repressed the Hippo-YAP pathway by suppressing LATS1 ubiquitination, thereby contributing to the reduced resistance of EESCs to ferroptosis.
CONCLUSION: USP33 impedes the ubiquitination of LAST1 and represses the Hippo/YAP pathway, thus facilitating ferritinophagy and ferroptosis in EMs.
METHODS: Endometrial stromal cells (ESCs) were isolated from 15 healthy controls and 30 patients with EMs, which were designated NESCs and EESCs, respectively. Real-time PCR and Western blotting were utilized to determine USP33 expression as well as GPX4, LC3II/LC3I, NCOA4, FTH1, LAST1, p-LAST1, YAP, and p-YAP levels. Immunofluorescence was used to detect the colocalization of ferritin and LAMP2, as well as that of LC3 and ferritin. The levels of ROS, Fe2+, MDA, and GSH were measured to assess ferroptosis. An LDH assay was performed to evaluate cell death. A co-IP assay was implemented to identify the interaction between USP33 and LAST1 and detect the level of LAST1 ubiquitination. The stability of the protein was also detected via a cycloheximide (CHX) assay.
RESULTS: USP33 was underexpressed in EMs patients. Loss of USP33 conferred resistance to ferroptosis in EESCs, as evidenced by increased proliferation; decreased levels of ROS, Fe2+, and MDA; elevated levels of GPX4 and GSH; and reduced cell death. In addition, USP33, which is localized in autophagosomes, was suggested to promote the degradation of ferritin in autophagosomes. Furthermore, USP33 repressed the Hippo-YAP pathway by suppressing LATS1 ubiquitination, thereby contributing to the reduced resistance of EESCs to ferroptosis.
CONCLUSION: USP33 impedes the ubiquitination of LAST1 and represses the Hippo/YAP pathway, thus facilitating ferritinophagy and ferroptosis in EMs.