Regulation of endothelial function and vascular integrity by neddylation

Project summary Compromised function of endothelial cells (ECs) is a root cause of various vascular diseases. While endothelial homeostasis is known to be governed by multiple epigenetic and transcriptional mechanisms, the involvement of post-translational modifications has been largely overlooked. Neddylation is a reversible post-translational modification that attaches one or more ubiquitin-like NEDD8 moieties to substrates via a NEDD8-specific E1- E2-E3 enzymatic cascade. Emerging evidence shows that neddylation plays significant roles in metabolic disorders, cardiomyopathies, immunity, and other pathophysiological events. However, its role in the endothelium and vascular diseases is unclear. The goal of the proposed research is to establish the pathophysiological significance of neddylation in endothelium and to identify key downstream effectors in ECs. The research team uncovered a link between neddylation perturbations and vascular diseases in humans and mouse models. The team further showed that EC-specific inhibition of neddylation by deleting NEDD8 activating enzyme-1 (NAE1) in adult mice causes mortality from pronounced vasculopathy and multi-organ damage characterized by loss of EC integrity, cell death, inflammation, and hemorrhage. The research team hypothesizes that protein neddylation and NAE1 downstream target cullin-3 (CUL3) protect endothelial homeostasis and integrity by maintaining EC identity, promoting mitophagy, constraining EC cell death, and resolving stress response signaling. Aim 1 will test the hypothesis that neddylation protects endothelial integrity by sustaining EC identity and preventing EC cell death. Aim 2 will test the hypothesis that CUL3 functions downstream of neddylation to promote mitophagy and fine-tune the stress response signaling, thereby maintaining EC homeostasis. EC-specific knockout and knockin mouse lines will be used to determine the physiological importance of neddylation and this specific target in several vascular beds. Bulk RNA-sequencing, single-nucleus RNA-sequencing with lineage tracing, and quantitative multiplex proteomics will be performed to probe the role of neddylation and its target in the EC transcriptome, proteome, EC trans-differentiation, and EC-other cell type communications in multiple organs. Sophisticated biochemical and cellular assays will be carried out to elucidate how neddylation and its target control endothelial to mesenchymal transition, mitochondrial function, and integrated stress response in ECs. Rescue experiments in vitro and/or in vivo will be performed to establish the cause-and-effect relationship. Successful completion of this research will provide a comprehensive mechanistic understanding of protein neddylation in safeguarding endothelial function and integrity. This is a key step toward the research team’s long-term goal to establish the clinical relevance of its findings and develop novel therapeutic strategies to precisely modulate protein neddylation for vascular diseases with minimized adverse effects.