Ubiquitinated proteomics reveals potential epigenetic-energy metabolism mechanisms in senescent ovarian granulosa cells of advanced maternal age.

purposeUbiquitination represents a promising therapeutic target for age-associated diseases, yet its functional role in ovarian aging remains poorly defined. This study systematically characterizes ubiquitinated proteins in luteinized granulosa cells (GCs) from women of advanced maternal age (AMA), aiming to provide molecular insights into the ubiquitination-dependent regulatory network underlying ovarian aging and further elucidate the mechanisms governing energy metabolism reprogramming in senescent GCs.

methodsA total of 60 patients undergoing their first in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) were recruited in this study, including 30 patients with young maternal age (YMA) and normal ovarian reserve, and 30 patients with advanced maternal age (AMA) and ovarian aging. The recruitment period was from November 2024 to May 2025. Nine samples of luteinized GCs were randomly selected from each group for ubiquitinated proteomic sequencing analysis. Functional exploration of differentially ubiquitinated proteins was performed via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Protein-protein interaction (PPI) network analysis was conducted to identify molecular targets of potential pathways. Additionally, the ubiquitination level and protein expression of alpha-enolase (ENO1) were validated by immunoprecipitation Western blot (IP-WB) and Western blot (WB) assays, respectively. Furthermore, an in vitro oxidative stress-induced ovarian aging model was established using hydrogen peroxide (H₂O₂)-treated human granulosa-like tumor cells line (KGN cells) to further investigate the regulatory role of ENO1 in energy metabolism reprogramming of GCs during ovarian aging.

resultsA total of 174 ubiquitinated peptides with significant differences were screened between the two groups (|log₂ FC|> 1, q < 0.05). Compared with the YMA group, 80 ubiquitinated peptides were significantly upregulated and 94 were significantly downregulated in the AMA group. GO enrichment analysis revealed that differentially ubiquitinated proteins (DUPs) were enriched in the "metabolic pathways" of biological processes (P < 0.05). Further KEGG pathway analysis suggested that the "ubiquitin-mediated protein degradation" and "proteasome" pathways may be involved in the regulation of ovarian aging. In addition, DUPs exhibited differential ubiquitination characteristics in energy metabolism pathways: the ubiquitination levels of proteins related to the "tricarboxylic acid (TCA) cycle" pathway were upregulated, while those related to the "glycolysis" and "pentose phosphate pathway (PPP)" were downregulated (P < 0.05). PPI network analysis indicated that ENO1, a differentially ubiquitinated protein (FDR < 0.05) and a key glycolytic enzyme, may serve as a critical target for energy metabolism reprogramming during ovarian aging. WB and IP-WB results showed that the ubiquitination level of ENO1 was downregulated in AMA GCs, whereas its protein expression level was elevated. Functional experiment results demonstrated that ENO1 inhibitors could attenuate H₂O₂-induced senescence of KGN cells, downregulate p16 expression, and improve the activity of key glycolytic enzymes as well as metabolite levels. No significant change was observed in ENO1 mRNA levels, suggesting that its expression is mainly regulated by post-transcriptional modifications such as ubiquitination.

conclusionsThis study demonstrates that DUPs between the AMA and YMA groups exhibit distinct ubiquitination patterns in energy metabolism pathways: the ubiquitination levels of proteins associated with the "TCA cycle" pathway are upregulated, whereas those related to "glycolysis" and the "PPP" are downregulated. ENO1, a key enzyme in glycolysis, is subject to ubiquitination-mediated post-transcriptional regulation. Inhibition of ENO1 can alleviate GC senescence and improve the abnormal glycolytic phenotype. In summary, ubiquitination modification may be involved in the process of ovarian aging, and targeting ENO1 ubiquitination to regulate energy metabolism reprogramming is expected to provide a potential intervention target for improving reproductive outcomes in AMA.