α-ketoglutarate dehydrogenase-mediated protein succinylation as a key regulator of the cellular response to ionizing radiation stress

Abstract

Objective

To elucidate the functional role and underlying mechanisms of α-ketoglutarate dehydrogenase (OGDH) and OGDH-mediated protein succinylation in the response to ionizing radiation (IR) stress.

Methods

Human B lymphoblastoid AHH-1 cells were exposed to 2 or 3 Gy of ⁶⁰Co γ-rays depending on the specific assay requirements, with sham-irradiated cells (0 Gy) as control. OGDH knockdown (KD) cell lines were engineered with the CRISPR/Cas9 system, complemented by pharmacological blockade using the specific inhibitor succinyl phosphonate (SPP). The dynamic profiles of OGDH expression and global succinylation landscapes were measured by Western blot. The DNA double-strand break marker phosphorylated H2AX (γH2AX) was monitored with immunofluorescence staining and Western blot. Post-irradiation cellular fate was assessed through CCK-8 proliferation assays and Annexin V/7-AAD flow cytometry for apoptosis.

Results

IR significantly induced OGDH protein expression in AHH-1 cells, which was accompanied by a progressive, time-dependent accumulation of global protein succinylation. This radiation-induced surge in succinylation was effectively blocked by SPP pretreatment. Notably, genetic ablation of OGDH significantly enhanced cellular radiosensitivity compared to the control group, as evidenced by impaired DNA damage repair activity (with γH2AX foci declining to 61.2% of the control level at 24 h post-irradiation, P < 0.001) and exacerbated radiation-induced apoptosis (42.8% in KD cells vs. 23.1% in control cells at 24 h post-irradiation, P < 0.001). Similarly, pharmacological inhibition of OGDH by SPP also significantly reduced cell proliferation and aggravated apoptotic cell death (P < 0.001).

Conclusion

This study clarifies the role of OGDH-regulated succinylation in radiation-induced injury and provides a theoretical basis for the development of OGDH-targeted radiosensitization strategies for the prevention and treatment of radiation damage.