Blockade of neddylation through targeted inhibition of DCN1 alleviates renal fibrosis

Neddylation is a process of attaching neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) to substrates for the protein function modulation via enzymatic cascades involving NEDD8-activating Enzyme (E1), NEDD8-conjugating Enzyme (E2), and NEDD8 Ligase (E3). Defective in cullin neddylation 1 (DCN1) serves as a co-E3 Ligase, which can simultaneously bind E2 UBE2M and cullin proteins to stabilize the catalytic center of the Cullin-Ring E3 Ligase complex, thereby promoting cullin neddylation. Neddylation is reported to be activated in diverse human diseases, and inhibition of protein neddylation has been regarded as a promising Anticancer therapy. However, whether neddylation participates in renal fibrosis and whether blockade of neddylation through targeted inhibition of DCN1 play effects on renal fibrosis remains unknown. In the present study, an NEDD8 overexpressed plasmid, DCN1 small interfering RNAs, DCN1-specific inhibitor NAcM-OPT, human renal tubular epithelial cells (HK-2), rat kidney fibroblasts (NRK-49F), RNA Sequencing, unilateral ureteral obstruction (UUO), and unilateral ischemia-reperfusion injury (UIRI) mouse renal fibrosis models were used. Herein, we first showed that neddylation was activated in renal fibrosis. Neddylation blockade through DCN1 deficiency alleviated TGFβ1-induced up-regulation of fibronectin and α-SMA in HK-2 and NRK-49F cells. Importantly, DCN1 inhibition attenuated UUO- and UIRI-induced mouse renal fibrosis. Further studies revealed that DCN1 loss selectively inhibited cullin3 neddylation and induced its substrate NRF2 accumulation, thereby inhibiting TGFβ-Smad2/3 signaling pathway. Overall, blockade of neddylation through targeted inhibition of DCN1 contributes to alleviating renal fibrosis in vitro and in vivo, which may constitute a novel therapeutic strategy for renal fibrosis.

DCN1; NEDD8; NRF2; TGFβ-Smad2/3; neddylation; renal fibrosis.