Ubiquitinated Proteomics Reveals ENO1 as Key Regulator of Energy Metabolism in Senescent Ovarian Granulosa Cells
Background
Female fertility significantly declines with advanced maternal age (AMA), largely due to ovarian aging and the dysfunction of granulosa cells (GCs), which are crucial for oocyte development. Current interventions often fall short in addressing the underlying molecular mechanisms of GC senescence. Ubiquitination, a post-translational modification, is implicated in various age-associated diseases, yet its specific role in ovarian aging and the resulting energy metabolism dysregulation in GCs remains poorly understood. This study aimed to map the ubiquitination landscape to uncover novel therapeutic targets.
Study Design
Researchers recruited 60 patients undergoing IVF/ICSI, comprising 30 with young maternal age (YMA) and normal ovarian reserve, and 30 with advanced maternal age (AMA) and ovarian aging. Nine luteinized GC samples were randomly selected from each group for ubiquitinated proteomic sequencing analysis. Functional enrichment was performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, alongside Protein-Protein Interaction (PPI) network analysis. The ubiquitination level and protein expression of alpha-enolase (ENO1) were validated via immunoprecipitation Western blot (IP-WB) and Western blot (WB). An in vitro hydrogen peroxide (H₂O₂)-treated human granulosa-like tumor cell line (KGN cells) was used to model ovarian aging and investigate ENO1's role.
Results
A total of 174 ubiquitinated peptides showed significant differences between AMA and YMA groups (|log₂ FC|>1, q<0.05). Specifically, 80 ubiquitinated peptides were significantly upregulated, and 94 were downregulated in AMA GCs. GO and KEGG enrichment analyses revealed that these differentially ubiquitinated proteins were primarily involved in energy metabolism, glycolysis, gluconeogenesis, and mTORC1 signaling pathways. PPI network analysis identified ENO1 as a central node.
IP-WBvalidation confirmed a significant increase in ENO1 ubiquitination in AMA GCs, whileWBshowed a corresponding decrease in total ENO1 protein expression. In theH₂O₂-induced KGN cell model, ENO1 ubiquitination was also increased, leading to reduced ENO1 expression and impaired energy metabolism, mimicking the senescent phenotype observed in AMA GCs.
Key Findings
- 174 ubiquitinated peptides were differentially expressed in senescent ovarian granulosa cells from AMA women.
- 80 ubiquitinated peptides were upregulated and 94 downregulated in AMA granulosa cells.
- Differentially ubiquitinated proteins were enriched in
energy metabolism,glycolysis,gluconeogenesis, andmTORC1pathways. - Alpha-enolase (ENO1) ubiquitination was significantly increased in AMA granulosa cells, leading to decreased ENO1 protein expression.
- In vitro
H₂O₂-induced senescence model confirmed increased ENO1 ubiquitination and impaired energy metabolism.
Why It Matters
Modulating ubiquitination pathways, particularly targeting ENO1, represents a promising novel strategy to combat ovarian aging and improve fertility outcomes in women of advanced maternal age. This research provides critical molecular insights into how protein degradation and energy metabolism are dysregulated in senescent granulosa cells. While this is a discovery-phase study, identifying specific ubiquitination targets like ENO1 opens avenues for developing interventions that could preserve ovarian function. Future work could explore small molecules or peptides that stabilize ENO1 or inhibit its ubiquitination, potentially leading to new therapeutic protocols far down the line, though a usable protocol is still years away.
ovarian-aging
granulosa-cells
ubiquitination
energy-metabolism
proteomics
eno1