Copper Downregulates Neprilysin Activity Through Modulation of Neprilysin Degradation

Abstract

Copper plays a central role in conserved processes such as respiration, and in highly specialized processes, such as protein modification. The metalloprotease neprilysin (NEP) degrades a variety of bioactive peptides and is involved in many physiological processes. However, very little is known about the regulation of NEP activity. In the current study, we focused on the effect of Cu²⁺ on the enzymatic activity and protein stability of NEP. Using mouse neuroblastoma N2a cells, we found that the enzymatic activity of NEP was decreased by treatment with Cu²⁺ in a dose- and time-dependent manner. In our investigation of the mechanism by which Cu²⁺ downregulates NEP enzyme activity, we found that treatment with Cu²⁺ caused a decrease in the level of NEP as determined by Western blot analysis. Quantitative analysis of NEP mRNA with RT-PCR excluded the possibility that Cu²⁺ downregulates NEP protein at the gene transcription level. Moreover, specific proteasome inhibitors, MG132 and lactacystin, blocked the turnover of NEP, whereas inhibitors of lysosome had no significant effect, suggesting that Cu²⁺-induced degradation of NEP is via a proteasome pathway. Taken together, our data suggest that copper downregulates NEP activity through modulation of NEP protein degradation.