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

Unsaturated fatty acid regulation of cytochrome P450 expression via a CAR-dependent pathway.

The liver is responsible for key metabolic functions, including control of normal homoeostasis in response to diet and xenobiotic metabolism/detoxification. We have shown previously that inactivation of the hepatic cytochrome P450 system through conditional deletion of POR (P450 oxidoreductase) induces hepatic steatosis, liver growth and P450 expression. We have exploited a new conditional model of POR deletion to investigate the mechanism underlying these changes. We demonstrate that P450 induction, liver growth and hepatic triacylglycerol (triglyceride) homoeostasis are intimately linked and provide evidence that the observed phenotypes result from hepatic accumulation of unsaturated fatty acids, which mediate these phenotypes by activation of the nuclear receptor CAR (constitutive androstane receptor) and, to a lesser degree, PXR (pregnane X receptor). To our knowledge this is the first direct evidence that P450s play a major role in controlling unsaturated fatty acid homoeostasis via CAR. The regulation of P450s involved in xenobiotic metabolism by this mechanism has potentially significant implications for individual responses to drugs and environmental chemicals

Structure-Guided Recombination Creates an Artificial Family of Cytochromes P450

Creating artificial protein families affords new opportunities to explore the determinants of structure and biological function free from many of the constraints of natural selection. We have created an artificial family comprising ~3,000 P450 heme proteins that correctly fold and incorporate a heme cofactor by recombining three cytochromes P450 at seven crossover locations chosen to minimize structural disruption. Members of this protein family differ from any known sequence at an average of 72 and by as many as 109 amino acids. Most (>73%) of the properly folded chimeric P450 heme proteins are catalytically active peroxygenases; some are more thermostable than the parent proteins. A multiple sequence alignment of 955 chimeras, including both folded and not, is a valuable resource for sequence-structure-function studies. Logistic regression analysis of the multiple sequence alignment identifies key structural contributions to cytochrome P450 heme incorporation and peroxygenase activity and suggests possible structural differences between parents CYP102A1 and CYP102A2

Cytochrome P450 CYP1B1 interacts with 8-<i>methoxypsoralen</i> (8-MOP) and influences psoralen-Ultraviolet A (PUVA) sensitivity

Background: There are unpredictable inter-individual differences in sensitivity to psoralen-UVA (PUVA) photochemotherapy, used to treat skin diseases including psoriasis. Psoralens are metabolised by cytochrome P450 enzymes (P450), and we hypothesised that variability in cutaneous P450 expression may influence PUVA sensitivity. We previously showed that P450 CYP1B1 was abundantly expressed in human skin and regulated by PUVA, and described marked inter-individual differences in cutaneous CYP1B1 expression.Objectives: We investigated whether CYP1B1 made a significant contribution to 8-methoxypsoralen (8-MOP) metabolism, and whether individuality in CYP1B1 activity influenced PUVA sensitivity.Methods: We used E. coli membranes co-expressing various P450s and cytochrome P450 reductase (CPR) to study 8-MOP metabolism and cytotoxicity assays in CYP1B1-expressing mammalian cells to assess PUVA sensitivity.Results: We showed that P450s CYP1A1, CYP1A2, CYP1B1, CYP2A6 and CYP2E1 influence 8-MOP metabolism. As CYP1B1 is the most abundant P450 in human skin, we further demonstrated that: (i) CYP1B1 interacts with 8-MOP (ii) metabolism of the CYP1B1 substrates 7-ethoxyresorufin and 17-b-estradiol showed concentration-dependent inhibition by 8-MOP and (iii) inhibition of 7-ethoxyresorufin metabolism by 8-MOP was influenced by CYP1B1 genotype. The influence of CYP1B1 on PUVA cytotoxicity was further investigated in a Chinese hamster ovary cell line, stably expressing CYP1B1 and CPR, which was more sensitive to PUVA than control cells, suggesting that CYP1B1 metabolises 8-MOP to a more phototoxicmetabolite(s).Conclusion: Our data therefore suggest that CYP1B1 significantly contributes to cutaneous 8-MOP metabolism, and that individuality in CYP1B1 expression may influence PUVA sensitivity

Functional characterization of the cytochrome P450 monooxygenase CYP71AU87 indicates a role in marrubiin biosynthesis in the medicinal plant Marrubium vulgare.

BackgroundHorehound (Marrubium vulgare) is a medicinal plant whose signature bioactive compounds, marrubiin and related furanoid diterpenoid lactones, have potential applications for the treatment of cardiovascular diseases and type II diabetes. Lack of scalable plant cultivation and the complex metabolite profile of M. vulgare limit access to marrubiin via extraction from plant biomass. Knowledge of the marrubiin-biosynthetic enzymes can enable the development of metabolic engineering platforms for marrubiin production. We previously identified two diterpene synthases, MvCPS1 and MvELS, that act sequentially to form 9,13-epoxy-labd-14-ene. Conversion of 9,13-epoxy-labd-14-ene by cytochrome P450 monooxygenase (P450) enzymes can be hypothesized to facilitate key functional modification reactions in the formation of marrubiin and related compounds.ResultsMining a M. vulgare leaf transcriptome database identified 95 full-length P450 candidates. Cloning and functional analysis of select P450 candidates showing high transcript abundance revealed a member of the CYP71 family, CYP71AU87, that catalyzed the hydroxylation of 9,13-epoxy-labd-14-ene to yield two isomeric products, 9,13-epoxy labd-14-ene-18-ol and 9,13-epoxy labd-14-ene-19-ol, as verified by GC-MS and NMR analysis. Additional transient Nicotiana benthamiana co-expression assays of CYP71AU87 with different diterpene synthase pairs suggested that CYP71AU87 is specific to the sequential MvCPS1 and MvELS product 9,13-epoxy-labd-14-ene. Although the P450 products were not detectable in planta, high levels of CYP71AU87 gene expression in marrubiin-accumulating tissues supported a role in the formation of marrubiin and related diterpenoids in M. vulgare.ConclusionsIn a sequential reaction with the diterpene synthase pair MvCPS1 and MvELS, CYP71AU87 forms the isomeric products 9,13-epoxy labd-14-ene-18/19-ol as probable intermediates in marrubiin biosynthesis. Although its metabolic relevance in planta will necessitate further genetic studies, identification of the CYP71AU87 catalytic activity expands our knowledge of the functional landscape of plant P450 enzymes involved in specialized diterpenoid metabolism and can provide a resource for the formulation of marrubiin and related bioactive natural products

Pubertal presentation in seven patients with congenital adrenal hyperplasia due to P450 Oxidoreductase deficiency

Context: P450 oxidoreductase (POR) is a crucial electron donor to all microsomal P450 cytochrome (CYP) enzymes including 17α-hydroxylase (CYP17A1), 21-hydroxylase (CYP21A2) and P450 aromatase. Mutant POR causes congenital adrenal hyperplasia with combined glucocorticoid and sex steroid deficiency. P450 oxidoreductase deficiency (ORD) commonly presents neonatally, with disordered sex development in both sexes, skeletal malformations, and glucocorticoid deficiency. \ud \ud Objective: The aim of the study was to describe the clinical and biochemical characteristics of ORD during puberty. \ud \ud Design: Clinical, biochemical, and genetic assessment of seven ORD patients (five females, two males) presenting during puberty was conducted. \ud \ud Results: Predominant findings in females were incomplete pubertal development (four of five) and large ovarian cysts (five of five) prone to spontaneous rupture, in some only resolving after combined treatment with estrogen/progestin, GnRH superagonists, and glucocorticoids. Pubertal development in the two boys was more mildly affected, with some spontaneous progression. Urinary steroid profiling revealed combined CYP17A1 and CYP21A2 deficiencies indicative of ORD in all patients; all but one failed to mount an appropriate cortisol response to ACTH stimulation indicative of adrenal insufficiency. Diagnosis of ORD was confirmed by direct sequencing, demonstrating disease-causing POR mutations. \ud \ud Conclusion: Delayed and disordered puberty can be the first sign leading to a diagnosis of ORD. Appropriate testosterone production during puberty in affected boys but manifest primary hypogonadism in girls with ORD may indicate that testicular steroidogenesis is less dependent on POR than adrenal and ovarian steroidogenesis. Ovarian cysts in pubertal girls may be driven not only by high gonadotropins but possibly also by impaired CYP51A1-mediated production of meiosis-activating sterols due to mutant POR