Serine ubiquitination of SQSTM1 regulates NFE2L2-dependent redox homeostasis.

The KEAP1-NFE2L2 axis is essential for the cellular response against metabolic and oxidative stress. KEAP1 is an adaptor protein of CUL3 (cullin 3) ubiquitin ligase that controls the cellular levels of NFE2L2, a critical transcription factor of several cytoprotective genes. Oxidative stress, defective autophagy and pathogenic infections activate NFE2L2 signaling through phosphorylation of the autophagy receptor protein SQSTM1, which competes with NFE2L2 for binding to KEAP1. Here we show that phosphoribosyl-linked serine ubiquitination of SQSTM1 catalyzed by SidE effectors of Legionella pneumophila controls NFE2L2 signaling and cell metabolism upon Legionella infection. Serine ubiquitination of SQSTM1 sterically blocks its binding to KEAP1, resulting in NFE2L2 ubiquitination and degradation. This reduces NFE2L2-dependent antioxidant synthesis in the early phase of infection. Levels of serine ubiquitinated SQSTM1 diminish in the later stage of infection allowing the expression of NFE2L2-target genes; causing a differential regulation of the host metabolome and proteome in a NFE2L2-dependent manner. ARE: antioxidant response element; Dup: deubiquitinase specific for phosphoribosyl-linked serine ubiquitination; ER: endoplasmic reticulum; h.p.i: hours post infection; HIF1A/HIF-1α: hypoxia inducible factor 1 subunit alpha; KEAP1: kelch like ECH associated protein 1; KIR: KEAP1-interacting region; LIR: LC3-interacting region; NES: nuclear export signal; NFKB/NF-κB: nuclear factor kappa B; NLS: nuclear localization signal; NFE2L2/Nrf2: NFE2 like bZIP transcription factor 2; PB1 domain: Phox1 and Bem1 domain; PR-Ub: phosphoribosyl-linked serine ubiquitination; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; tBHQ: tertiary butylhydroquinone; TUBE2: tandem ubiquitiin binding entity 2; UBA domain: ubiquitin-associated domain.