20-Hydroxyeicosatetraenoic acid regulates the expression of Nedd4-2 in kidney and liver through a neddylation modification pathway

Abstract

The present study aimed to test whether 20-hydroxyeicosatetraenoic acid (20‑HETE) affected neddylation modification of E3‑ligase Nedd4‑2 (neural precursor cell expressed, developmentally down‑regulated 4‑like, E3 ubiquitin protein ligase). A cytochrome P450 family 4 subfamily F member 2 (CYP4F2) transgenic mouse model that overproduces 20‑HETE in the kidney and the liver was used in the present study. Transgenic mice with high salt intake exhibited increased activation of Nedd4‑2‑mediated ubiquitin‑proteasome pathway. Nedd4‑2 expression is increased in the kidney and decreased in the liver of transgenic mice compared with wild‑type mice. Subsequently, co‑immunoprecipitation analysis indicated that Nedd4‑2 was modified by Nedd8, and the level of neddylation on Nedd4‑2 was reduced in the kidney and increased in the liver of transgenic mice compared with controls. In addition, sentrin‑specific protease 8 (Senp8), a deneddylation enzyme, is expressed higher in the kidney and lower in the liver of transgenic mice compared with wild‑type controls. The function of 20‑HETE on modulation of Nedd4‑2 were also confirmed in mouse M1 kidney and mouse NCTC1469 liver cell lines, and the function was restored by neddylation inhibitor MLN4924 Data from the present study demonstrated that 20‑HETE upregulated the expression of Nedd4‑2 in the kidney and downregulated expression in the liver through the neddylation modification pathway, at least partly, depending on the effects on Senp8 deneddylation.

Eicosanoids and other oxylipins in liver injury, inflammation and liver cancer development

“…In experimental models, 20-HETE has been shown to induce the activation of LX-2 cells via TGFβ signaling through proteasome regulation ( Lai et al, 2018 ; Li et al, 2023 ) and inhibiting 20-HETE production attenuates liver fibrosis induced with CCL4 ( Li et al, 2023 ). This effect may involve ubiquitination as 20-HETE decreases the expression of Nedd4-2 in the liver ( Zhao et al, 2017 ).…”

The Next Frontier: Translational Development of Ubiquitination, SUMOylation, and NEDDylation in Cancer

“…When the two proteins interact, the subunits come together to form an active enzyme and generate a bright luminescent signal in the presence of substrate. Other strategies to verify whether a protein of interest is a substrate of NEDDylation include a pull-down of histidine-tagged NEDD8 for in vitro reactions or co-immunoprecipitation of the protein of interest for cell-based experiments followed by Western blot analysis of NEDD8 [ 184 , 185 ]. Several of the assays involved in NEDDylation studies aim to investigate the mechanistic impact on alterations to the NEDDylation pathway.…”

Targeting NEDD8-activating enzyme for cancer therapy: developments, clinical trials, challenges and future research directions

“…Finally, NEDD8 is transferred from NEDD8-conjugating enzyme E2s to a specific lysine residue of substrates through the catalytic action of NEDD8 E3 ligases [ 24 – 26 ]. In recent years, homogeneous time-resolved fluorescence [ 27 ], AlphaScreen [ 28 ], in vitro NEDD8 conjugation assay [ 29 ], cellular thermal shift assay [ 30 ], co-immunoprecipitation [ 31 ], pull-down assay [ 32 ] and ATP kinetics assay [ 33 ] are commonly used to identify NEDDylation process.…”