Liver CYP4A autophagic lysosomal degradation: A major role for the autophagic receptor SQSTM1/p62 through an uncommon target interaction site.

The hepatic P450 hemoproteins CYPs 4A are typical N-terminally anchored type I endoplasmic reticulum (ER) proteins, inducible by many hypolipidemic drugs and peroxisome proliferators. They are engaged in the ω-/ω-1-oxidation of various fatty acids including arachidonic acid, prostaglandins, and leukotrienes and in the biotransformation of some therapeutic drugs. Because the proteolytic turnover of the mammalian liver CYPs 4A remains obscure, we have characterized it. We report that of these proteins, human CYP4A11 and mouse Cyp4a12a are preferential targets of the ER-lysosome-associated degradation. Consequently, these proteins are stabilized 2- to 3-fold both as 1%Triton X100-soluble and insoluble species in mouse hepatocytes and HepG2 cells deficient in the autophagic initiation ATG5 gene. Despite exhibiting surface microtubule-associated protein light chain 3-interacting regions that could target them directly to the autophagosome, they nevertheless interact intimately with the autophagic receptor SQSTM1/p62. Through structural deletion analyses and site-directed mutagenesis, we have identified the CYP4A-interacting p62 subdomain to lie between residues 170 and 233, which include its Traf6-binding and LIM-binding subdomains. Mice carrying a liver-specific genetic deletion of p62 residues 69-251 (p62Mut) that includes the CYP4A-interacting subdomain also exhibit Cyp4a-protein stabilization as 1% Triton X100-soluble and insoluble species. Consistently, p62Mut mouse liver microsomes exhibit 1.5- to 2-fold enhanced ω- and ω-1-arachidonic acid hydroxylation to its physiologically active metabolites 19 and 20-HETEs relative to the corresponding wild-type mouse liver microsomes. Collectively, our findings suggest that disruption of CYP4A ER-lysosome-associated degradation results in functionally active P450 protein stabilization and consequent proinflammatory metabolite generation along with insoluble CYP4A aggregates, which may contribute to pathological aggregates, ie, Mallory-Denk bodies/inclusions, hallmarks of many liver diseases. SIGNIFICANCE STATEMENT: Human CYP4A11 and mouse Cyp4a12a, liver P450 enzymes engaged in ω-/ω-1-oxidation of arachidonic acid, prostaglandins, and leukotrienes, are documented to physiologically turn over via endoplasmic reticulum-lysosome-associated autophagic degradation, which involves their intimate association with the autophagic receptor SQSTM1/p62. Genetic endoplasmic reticulum-lysosome-associated autophagic degradation disruption or deletion of their hepatic p62-interaction subdomain in mice results in Cyp4a-protein stabilization as functionally active solubilizable species with consequently enhanced proinflammatory 20-HETE arachidonate metabolite generation and insoluble Cyp4a aggregates, potential contributors to pathologic liver inclusions.