Poldip2 Post-Translational Modifications Regulate Lung Endothelial Cell Function

Introduction: Acute lung injury (ALI) is characterized by endothelial damage, increased vascular permeability and lung edema. Polymerase delta-interacting protein 2 (Poldip2) is a novel regulator of vascular barrier integrity that has been shown to significantly alter lung endothelial function in vivo and in vitro. As Poldip2 does not appear to be transcriptionally regulated, we hypothesized that it is regulated by post post-translational modifications (PTMs), specifically phosphorylation. Methods and Results: We investigated potential Poldip2 phosphorylation sites at residues S288, S290, T292, and T295 within the DUF525 domain. We generated recombinant adenoviruses encoding wild-type (WT) and phospho-deficient Poldip2 mutants. Human lung endothelial cells (HULECs) were transduced with these adenoviruses and stimulated with TNF-α at various time points. Two forms of Poldip2 were detected at 43 and 39 kDa. We observed that TNF-α stimulation significantly increased phosphorylation of Poldip2-WT, especially at 43 kDa, compared to the control group. Both the quadruple mutant (residues 288-295) and the T295V mutant exhibited a near-complete loss of phosphorylation, confirming the critical contribution of these residues. Cell fractionation assays demonstrated that Poldip2 localizes to both mitochondria and nuclei, with the 43 kDa form being predominantly nuclear. Fluorescence imaging indicated that Poldip2-WT alters mitochondrial morphology, suggesting a role in regulating mitochondrial dynamics. Conclusion: Our findings indicate that phosphorylation of Poldip2 at specific sites is regulated by TNF-α, potentially affecting its subcellular distribution and activity. These findings provide insight into the molecular mechanisms by which Poldip2 may influence endothelial mitochondrial dynamics and contribute to endothelial dysfunction in ALI. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.