Cytochrome P450 endoplasmic reticulum-associated degradation (ERAD): therapeutic and pathophysiological implications.

The hepatic endoplasmic reticulum (ER)-anchored cytochromes P450 (P450s) are mixed-function oxidases engaged in the biotransformation of physiologically relevant endobiotics as well as of myriad xenobiotics of therapeutic and environmental relevance. P450 ER-content and hence function is regulated by their coordinated hemoprotein syntheses and proteolytic turnover. Such P450 proteolytic turnover occurs through a process known as ER-associated degradation (ERAD) that involves ubiquitin-dependent proteasomal degradation (UPD) and/or autophagic-lysosomal degradation (ALD). Herein, on the basis of available literature reports and our own recent findings of in vitro as well as in vivo experimental studies, we discuss the therapeutic and pathophysiological implications of altered P450 ERAD and its plausible clinical relevance. We specifically (i) describe the P450 ERAD-machinery and how it may be repurposed for the generation of antigenic P450 peptides involved in P450 autoantibody pathogenesis in drug-induced acute hypersensitivity reactions and liver injury, or viral hepatitis; (ii) discuss the relevance of accelerated or disrupted P450-ERAD to the pharmacological and/or toxicological effects of clinically relevant P450 drug substrates; and (iii) detail the pathophysiological consequences of disrupted P450 ERAD, contributing to non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) under certain synergistic cellular conditions

Hepatic cytochromes P450: structural degrons and barcodes, posttranslational modifications and cellular adapters in the ERAD-endgame.

The endoplasmic reticulum (ER)-anchored hepatic cytochromes P450 (P450s) are enzymes that metabolize endo- and xenobiotics i.e. drugs, carcinogens, toxins, natural and chemical products. These agents modulate liver P450 content through increased synthesis or reduction via inactivation and/or proteolytic degradation, resulting in clinically significant drug-drug interactions. P450 proteolytic degradation occurs via ER-associated degradation (ERAD) involving either of two distinct routes: Ubiquitin (Ub)-dependent 26S proteasomal degradation (ERAD/UPD) or autophagic lysosomal degradation (ERAD/ALD). CYP3A4, the major human liver/intestinal P450, and the fast-turnover CYP2E1 species are degraded via ERAD/UPD entailing multisite protein phosphorylation and subsequent ubiquitination by gp78 and CHIP E3 Ub-ligases. We are gaining insight into the nature of the structural determinants involved in CYP3A4 and CYP2E1 molecular recognition in ERAD/UPD [i.e. K48-linked polyUb chains and linear and/or "conformational" phosphodegrons consisting either of consecutive sequences on surface loops and/or disordered regions, or structurally-assembled surface clusters of negatively charged acidic (Asp/Glu) and phosphorylated (Ser/Thr) residues, within or vicinal to which, Lys-residues are targeted for ubiquitination]. Structural inspection of select human liver P450s reveals that such linear or conformational phosphodegrons may indeed be a common P450-ERAD/UPD feature. By contrast, although many P450s such as the slow-turnover CYP2E1 species and rat liver CYP2B1 and CYP2C11 are degraded via ERAD/ALD, little is known about the mechanism of their ALD-targeting. On the basis of our current knowledge of ALD-substrate targeting, we propose a tripartite conjunction of K63-linked Ub-chains, P450 structural "LIR" motifs and selective cellular "cargo receptors" as plausible P450-ALD determinants

CHIP(-/-)-Mouse Liver: Adiponectin-AMPK-FOXO-Activation Overrides CYP2E1-Elicited JNK1-Activation, Delaying Onset of NASH: Therapeutic Implications.

Genetic ablation of C-terminus of Hsc70-interacting protein (CHIP) E3 ubiquitin-ligase impairs hepatic cytochrome P450 CYP2E1 degradation. Consequent CYP2E1 gain of function accelerates reactive O2 species (ROS) production, triggering oxidative/proteotoxic stress associated with sustained activation of c-Jun NH2-terminal kinase (JNK)-signaling cascades, pro-inflammatory effectors/cytokines, insulin resistance, progressive hepatocellular ballooning and microvesicular steatosis. Despite this, little evidence of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) was found in CHIP(-/-)-mice over the first 8-9-months of life. We herein document that this lack of tissue injury is largely due to the concurrent up-regulation and/or activation of the adiponectin-5'-AMP-activated protein kinase (AMPK)-forkhead box O (FOXO)-signaling axis stemming from at the least three synergistic features: Up-regulated expression of adipose tissue adiponectin and its hepatic adipoR1/adipoR2 receptors, stabilization of hepatic AMPKα1-isoform, identified herein for the first time as a CHIP-ubiquitination substrate (unlike its AMPKα2-isoform), as well as nuclear stabilization of FOXOs, well-known CHIP-ubiquitination targets. Such beneficial predominance of the adiponectin-AMPK-FOXO-signaling axis over the sustained JNK-elevation and injurious insulin resistance in CHIP(-/-)-livers apparently counteracts/delays rapid progression of the hepatic microvesicular steatosis to the characteristic macrovesicular steatosis observed in clinical NASH and/or rodent NASH-models

CYP3A4 ubiquitination by gp78 (the tumor autocrine motility factor receptor, AMFR) and CHIP E3 ligases

Human liver CYP3A4 is an endoplasmic reticulum (ER)-anchored hemoprotein responsible for the metabolism of >50% of clinically prescribed drugs. After heterologous expression in Saccharomyces cerevisiae,it is degraded via the ubiquitin (Ub)-dependent 26S proteasomal pathway that utilizes Ubc7p/Cue1p, but none of the canonical Ub-ligases (E3s) Hrd1p/Hrd3p, Doa10p, and Rsp5p involved in ER-associated degradation (ERAD). To identify an Ub-ligase capable of ubiquitinating CYP3A4, we examined various in vitro reconstituted mammalian E3 systems, using purified and functionally characterized recombinant components. Of these, the cytosolic domain of the ER-protein gp78, also known as the tumor autocrine motility factor receptor (AMFR), an UBC7-dependent polytopic RING-finger E3, effectively ubiquitinated CYP3A4 in vitro, as did the UbcH5a-dependent cytosolic E3 CHIP. CYP3A4 immunoprecipitation coupled with anti-Ub immunoblotting analyses confirmed its ubiquitination in these reconstituted systems. Thus, both UBC7/gp78 and UbcH5a/CHIP may be involved in CYP3A4 ERAD, although their relative physiological contribution remains to be established

Why Hepatic CYP2E1-Elevation by Itself Is Insufficient for Inciting NAFLD/NASH: Inferences from Two Genetic Knockout Mouse Models

Hepatic cytochrome P450 CYP2E1 is an enzyme engaged in the metabolic biotransformation of various xenobiotics and endobiotics, resulting in both detoxification and/or metabolic activation of its substrates to more therapeutic or toxic products. Elevated hepatic CYP2E1 content is implicated in various metabolic diseases including alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH), diabetes and obesity. While hepatic CYP2E1 elevation is considered essential to the pathogenesis of these liver diseases, our findings in two mouse models of E3 ubiquitin ligase genetic ablation fed a regular lab chow diet, argue that it is not sufficient for triggering NAFLD/NASH. Thus, albeit comparable hepatic CYP2E1 elevation and functional stabilization in these two models upon E3 ubiquitin ligase genetic ablation and consequent disruption of its ubiquitin-dependent proteasomal degradation, NAFLD/NASH was only observed in the mouse livers that exhibited concurrent SREBP1c-transcriptional upregulation of hepatic lipogenesis. These findings reinforce the critical complicity of an associated prolipogenic scenario induced by either an inherently upregulated hepatic lipogenesis or a high fat/high carbohydrate diet in CYP2E1-mediated NAFLD/NASH

Why Hepatic CYP2E1-Elevation by Itself Is Insufficient for Inciting NAFLD/NASH: Inferences from Two Genetic Knockout Mouse Models.

Hepatic cytochrome P450 CYP2E1 is an enzyme engaged in the metabolic biotransformation of various xenobiotics and endobiotics, resulting in both detoxification and/or metabolic activation of its substrates to more therapeutic or toxic products. Elevated hepatic CYP2E1 content is implicated in various metabolic diseases including alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH), diabetes and obesity. While hepatic CYP2E1 elevation is considered essential to the pathogenesis of these liver diseases, our findings in two mouse models of E3 ubiquitin ligase genetic ablation fed a regular lab chow diet, argue that it is not sufficient for triggering NAFLD/NASH. Thus, albeit comparable hepatic CYP2E1 elevation and functional stabilization in these two models upon E3 ubiquitin ligase genetic ablation and consequent disruption of its ubiquitin-dependent proteasomal degradation, NAFLD/NASH was only observed in the mouse livers that exhibited concurrent SREBP1c-transcriptional upregulation of hepatic lipogenesis. These findings reinforce the critical complicity of an associated prolipogenic scenario induced by either an inherently upregulated hepatic lipogenesis or a high fat/high carbohydrate diet in CYP2E1-mediated NAFLD/NASH

Hepatic CYP3A Suppression by High Concentrations of Proteasomal Inhibitors: A Consequence of Endoplasmic Reticulum (ER) Stress Induction, Activation of RNA-Dependent Protein Kinase-Like ER-Bound Eukaryotic Initiation Factor 2α (eIF2α)-Kinase (PERK) and General Control Nonderepressible-2 eIF2α Kinase (GCN2), and Global Translational ShutoffS⃞

Hepatic cytochromes P450 3A (P450s 3A) are endoplasmic reticulum (ER)-proteins, responsible for xenobiotic metabolism. They are degraded by the ubiquitin-dependent 26S proteasome. Consistent with this, we have shown that proteasomal inhibitors N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) and N-benzoyloxycarbonyl-Leu-Leu-Leu-B(OH)2 (MG262) stabilize CYP3A proteins. However, MG132 has been reported to suppress P450s 3A as a result of impaired nuclear factor-κB activation and consequently reduced CYP3A protein stability. Because the MG132 concentration used in those studies was 10-fold higher than that required for CYP3A stabilization, we examined the effect of MG132 (0-300 μM) concentration-dependent proteasomal inhibition on CYP3A turnover in cultured primary rat hepatocytes. We found a biphasic MG132 concentration effect on CYP3A turnover: Stabilization at 5 to 10 μM with marked suppression at >100 μM. Proteasomal inhibitors reportedly induce ER stress, heat shock, and apoptotic response. At these high MG132 concentrations, such CYP3A suppression could be due to ER stress induction, so we monitored the activity of PERK [PKR (RNA-dependent protein kinase)-like ER kinase (EIF2AK3)], the ER stress-activated eukaryotic initiation factor 2α (eIF2α) kinase. Indeed, we found a marked (≈4-fold) MG132 concentration-dependent PERK autophosphorylation, along with an 8-fold increase in eIF2α-phosphorylation. In parallel, MG132 also activated GCN2 [general control nonderepressible-2 (EIF2AK4)] eIF2α kinase in a concentration-dependent manner, but not the heme-regulated inhibitor eIF2α kinase [(EIF2AK1)]. Pulse-chase, immunoprecipitation/immunoblotting analyses documented the consequently dramatic translational shutoff of total hepatic protein, including but not limited to CYP3A and tryptophan 2,3-dioxygenase protein syntheses. These findings reveal that at high concentrations, MG132 is indeed cytotoxic and can suppress CYP3A synthesis, a result confirmed by confocal immunofluorescence analyses of MG132-treated hepatocytes

Functional reconstitution of rat liver cytochrome P-450 with mesohemin

After allylisopropylacetamide-mediated suicide destruction of their prosthetic heme moieties, certain rat liver cytochrome P-450 isozymes can be effectively reconstituted by addition of exogenous hemin in vitro. We now report that two of these isozymes will equally accept mesohemin, a 2,4-diethyl heme-analog and result in a meso-hemoprotein with altered spectral but not functional characteristics. © 1984