The p62/SQSTM1 (sequestosome 1) protein, which acts as a cargo receptor for autophagic degradation of ubiquitinated targets, is up-regulated by various stressors. Induction of the p62 gene by oxidative stress is mediated by NF-E2-related factor 2 (NRF2) and, at the same time, p62 protein contributes to the activation of NRF2, but hitherto the mechanisms involved were not known. Herein, we have mapped an antioxidant response element (ARE) in the p62 promoter that is responsible for its induction by oxidative stress via NRF2. Chromatin immunoprecipitation and gel mobility-shift assays verified that NRF2 binds to this cis-element in vivo and in vitro. Also, p62 docks directly onto the Kelch-repeat domain of Kelch-like ECH-associated protein 1 (KEAP1), via a motif designated the KEAP1 interacting region (KIR), thereby blocking binding between KEAP1 and NRF2 that leads to ubiquitylation and degradation of the transcription factor. The KIR motif in p62 is located immediately C-terminal to the LC3-interacting region (LIR) and resembles the ETGE motif utilized by NRF2 for its interaction with KEAP1. KIR is required for p62 to stabilize NRF2, and inhibition of KEAP1 by p62 occurs from a cytoplasmic location within the cell. The LIR and KIR motifs cannot be engaged simultaneously by LC3 and KEAP1, but because p62 is polymeric the interaction between KEAP1 and p62 leads to accumulation of KEAP1 in p62 bodies, which is followed by autophagic degradation of KEAP1. Our data explain how p62 contributes to activation of NRF2 target genes in response to oxidative stress through creating a positive feedback loop.

Autophagosome formation depends on a carefully orchestrated interplay between membrane-associated protein complexes. Initiation of macroautophagy/autophagy is mediated by the ULK1 (unc-51 like auto...

The cysteine protease ATG4B cleaves off one or more C-terminal residues of the inactive proform of proteins of the ortholog and paralog LC3 and GABARAP subfamilies of yeast Atg8 to expose a C-terminal glycine that is conjugated to phosphatidylethanolamine during autophagosome formation. We show that ATG4B contains a C-terminal LC3-interacting region (LIR) motif important for efficient binding to and cleavage of LC3 and GABARAP proteins. We solved the crystal structures of the GABARAPL1-ATG4B C-terminal LIR complex. Analyses of the structures and in vitro binding assays, using specific point mutants, clearly showed that the ATG4B LIR binds via electrostatic-, aromatic HP1 and hydrophobic HP2 pocket interactions. Both these interactions and the catalytic site-substrate interaction contribute to binding between LC3s or GABARAPs and ATG4B. We also reveal an unexpected role for ATG4B in stabilizing the unlipidated forms of GABARAP and GABARAPL1. In mouse embryonic fibroblast (MEF) atg4b knockout cells, GABARAP and GABARAPL1 were unstable and degraded by the proteasome. Strikingly, the LIR motif of ATG4B was required for stabilization of the unlipidated forms of GABARAP and GABARAPL1 in cells.

The selective autophagy receptor p62/sequestosome 1 (SQSTM1) interacts directly with LC3 and is involved in oxidative stress signaling in two ways in mammals. First, p62 is transcriptionally induced upon oxidative stress by the NF-E2-related factor 2 (NRF2) by direct binding to an antioxidant response element in the p62 promoter. Second, p62 accumulation, occurring when autophagy is impaired, leads to increased p62 binding to the NRF2 inhibitor KEAP1, resulting in reduced proteasomal turnover of NRF2. This gives chronic oxidative stress signaling through a feed forward loop. Here, we show that the Drosophila p62/SQSTM1 orthologue, Ref(2)P, interacts directly with DmAtg8a via an LC3-interacting region motif, supporting a role for Ref(2)P in selective autophagy. The ref(2)P promoter also contains a functional antioxidant response element that is directly bound by the NRF2 orthologue, CncC, which can induce ref(2)P expression along with the oxidative stress-associated gene gstD1. However, distinct from the situation in mammals, Ref(2)P does not interact directly with DmKeap1 via a KEAP1-interacting region motif; nor does ectopically expressed Ref(2)P or autophagy deficiency activate the oxidative stress response. Instead, DmAtg8a interacts directly with DmKeap1, and DmKeap1 is removed upon programmed autophagy in Drosophila gut cells. Strikingly, CncC induced increased Atg8a levels and autophagy independent of TFEB/MitF in fat body and larval gut tissues. Thus, these results extend the intimate relationship between oxidative stress-sensing NRF2/CncC transcription factors and autophagy and suggest that NRF2/CncC may regulate autophagic activity in other organisms too.

SPBP (stromelysin-1 platelet-derived growth factor-responsive element binding protein) was originally cloned from a cDNA expression library by virtue of its ability to bind to a platelet-derived growth factor-responsive element in the human stromelysin-1 promoter. A 937-amino acid-long protein was deduced from a 3995-nucleotide murine cDNA sequence. By analyses of both human and murine cDNAs, we now show that SPBP is twice as large as originally found. The human SPBP gene contains six exons and is located on chromosome 22q13.1-13.3. Two isoforms differing in their C termini are expressed due to alternative splicing. PCR analyses of multitissue cDNA panels showed that SPBP is expressed in most tissues except for ovary and prostate. Functional mapping revealed that SPBP is a nuclear, multidomain protein containing an N-terminal region with transactivating ability, a novel type of DNA-binding domain containing an AT hook motif, and a bipartite nuclear localization signal as well as a C-terminal zinc finger domain. This type of zinc finger domain is also found in the trithorax family of chromatin-based transcriptional regulator proteins. Using cotransfection experiments, we find that SPBP enhances the transcriptional activity of various transcription factors such as c-Jun, Ets1, Sp1, and Pax6. Hence, SPBP seems to act as a transcriptional coactivator.