Blockade of neddylation through targeted inhibition of DCN1 alleviates renal fibrosis.

Abstract

Neddylation is a process of attaching neuronal precursor cell-expressed developmentally downregulated 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, that can simultaneously bind E2 UBE2M and cullin proteins to stabilize the catalytic center of the Cullin-Ring E3 ligase (CRL) 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 this study, a NEDD8 overexpressed plasmid, DCN1 small interfering RNAs (siRNAs), DCN1 specific inhibitor NAcM-OPT, human renal tubular epithelial cells (HK-2), rat kidney fibroblasts (NRK-49F), RNA-sequencing (RNA-seq), 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 upregulation 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.