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As the global prevalence of DM continues, there is a critical need for novel therapeutic strategies to address DMED. In our previous studies, we found that Glutathione peroxidase 4 (GPX4), a ferroptosis inhibitor, can ameliorate DMED in diabetic rats. However, the specific role of GPX4 in corpus cavernosum smooth muscle cells (CCSMCs) and its regulatory mechanisms remain unclear. In this study, we established primary cultures of CCSMCs and systematically analyzed the role of GPX4 under high-glucose conditions. To further elucidate the upstream regulatory pathways of GPX4, we employed immunoprecipitation coupled with mass spectrometry (IP-MS) to identify potential interacting proteins. Additionally, co-immunoprecipitation (Co-IP) and cycloheximide (CHX) chase assays were conducted to explore the regulatory dynamics and post-translational stability of GPX4. Under high-glucose conditions, the expression of GPX4 in CCSMCs is significantly downregulated, leading to an increase in intracellular oxidative stress and heightened levels of ferroptosis, accompanied by dysfunction in smooth muscle cell relaxation. Furthermore, the CHX chase assay revealed that high glucose accelerates GPX4 protein degradation via the ubiquitin\u2013proteasome pathway. Subsequent IP-MS identified NEDD4, an E3 ubiquitin ligase, as a potential interacting partner of GPX4. Further validation demonstrated that NEDD4 modulates the ubiquitination process of GPX4, thereby influencing its stability and expression. In conclusion, we identified NEDD4 as a key regulator of GPX4 stability through ubiquitin-mediated proteasomal degradation. These findings suggest potential therapeutic strategies targeting the NEDD4-GPX4 axis to alleviate DMED pathology.<\/jats:p>","DOI":"10.3390\/biom14121552","type":"journal-article","created":{"date-parts":[[2024,12,5]],"date-time":"2024-12-05T04:13:03Z","timestamp":1733371983000},"page":"1552","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Neural Precursor Cell-Expressed Developmentally Downregulated Protein 4 (NEDD4)-Mediated Ubiquitination of Glutathione Peroxidase 4 (GPX4): A Key Pathway in High-Glucose-Induced Ferroptosis in Corpus Cavernosum Smooth Muscle Cells"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6947-427X","authenticated-orcid":false,"given":"Wenchao","family":"Xu","sequence":"first","affiliation":[{"name":"Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China"},{"name":"Institute of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peng","family":"Hu","sequence":"additional","affiliation":[{"name":"Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China"},{"name":"Institute of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaxin","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China"},{"name":"Institute of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6307-2859","authenticated-orcid":false,"given":"Hongyang","family":"Jiang","sequence":"additional","affiliation":[{"name":"Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China"},{"name":"Institute of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tao","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China"},{"name":"Institute of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jihong","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China"},{"name":"Institute of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9886-1874","authenticated-orcid":false,"given":"Hao","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China"},{"name":"Institute of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,12,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1016\/j.jdiacomp.2010.03.003","article-title":"Diabetes-induced erectile dysfunction: Epidemiology, pathophysiology and management","volume":"25","author":"Thorve","year":"2011","journal-title":"J. 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