Regulation of Derlin-1-mediated degradation of NADPH oxidase partner p22^phox by thiol modification

The transmembrane protein p22^phox heterodimerizes with NADPH oxidase (Nox) 1–4 and is essential for the reactive oxygen species-producing capacity of oxidases. Missense mutations in the p22^phox gene prevent the formation of phagocytic Nox2-based oxidase, which contributes to host defense. This results in chronic granulomatous disease (CGD), a severe primary immunodeficiency syndrome. In this study, we characterized missense mutations in p22^phox (L51Q, L52P, E53V, and P55R) in the A22° type (wherein the p22^phox protein is undetectable) of CGD. We demonstrated that these substitutions enhanced the degradation of the p22^phox protein in the endoplasmic reticulum (ER) and the binding of p22^phox to Derlin-1, a key component of ER-associated degradation (ERAD). Therefore, the L⁵¹-L⁵²-E⁵³-P⁵⁵ sequence is responsible for protein stability in the ER. We observed that the oxidation of the thiol group of Cys-50, which is adjacent to the L⁵¹-L⁵²-E⁵³-P⁵⁵ sequence, suppressed p22^phox degradation. However, the suppression effect was markedly attenuated by the serine substitution of Cys-50. Blocking the free thiol of Cys-50 by alkylation or C50S substitution promoted the association of p22^phox with Derlin-1. Derlin-1 depletion partially suppressed the degradation of p22^phox mutant proteins. Furthermore, heterodimerization with p22^phox (C50S) induced rapid degradation of not only Nox2 but also nonphagocytic Nox4 protein, which is responsible for redox signaling. Thus, the redox-sensitive Cys-50 appears to determine whether p22^phox becomes a target for degradation by the ERAD system through its interaction with Derlin-1.