Influence of Heme on Hepatic Cytochrome P450 Synthesis and Degradation

University Of California, San Francisco, San Francisco CA

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Abstract

The hepatic hemoproteins cytochromes P450 (P450s) are endoplasmic-reticulum (ER)-anchored enzymes engaged in the breakdown of endo- and xenobiotics such as drugs, carcinogens, toxins, natural and chemical products. On exposure to these agents, liver P450 content may be increased due to increased syntheses of its heme and protein moieties, or reduced due to its inactivation/destruction and/or proteolytic degradation. Such drug-mediated modulation of P450 content is known to significantly influence clinical drug-drug interactions. Because P450 synthesis requires heme, defective heme synthesis as in the genetically inherited, acute heme-deficient states clinically known as hepatic porphyrias, can lower P450 levels and thereby impair the metabolism of ingested drugs. Our finding indicates that severe hepatic heme depletion can also profoundly suppress the syntheses of hepatic proteins such as P450s, by shutting off their translation. This results from increased phosphorylation of the a-subunit of eukaryotic translational initiation factor elF2 by a putative hepatic heme-sensitive elF2a kinase, that is functionally unleashed when hepatic heme is severely depleted. Although the identity of this liver kinase had long remained elusive, we have cloned this enzyme from rat liver and cultured rat hepatocytes, expressed and purified it. Our first major aim is to (i) structurally and functionally characterize this enzyme further; (ii) confirm its identity as an elF2a kinase; (iii) establish its in vivo elF2a-interactions by various state-of-the-art techniques such as mammalian two-hybrid, chemical crosslinking/proteomic analyses, coimmunoprecipitation, as well as its tissue/intrahepatocellular localization; and (iv) use RNA interference and targeted gene knock out in mice to determine its in vivo relevance to hepatic protein and P450 syntheses. Our goal is to elucidate the translational suppression that may impair key physiological processes and thus contribute not only to the clinical symptoms of acute hepatic porphyrias, but also influence P450-dependent drug-drug interactions in man. Further, it is now increasingly evident that clinically relevant drug-drug interactions can also result from altered P450 turnover, elicited by drug-mediated P450 stabilization as well as enhanced drug-mediated P450 degradation, such as by the grapefruit furanocoumarins. Such ER-associated degradation of P450s entails their ubiquitination, extraction from the ER and subsequent proteolysis by the cytosolic 26S proteasome. Heme results in an ER accumulation of ubiquitinated P450s, most likely by blocking their ER extraction and subsequent degradation. ER extraction requires ATP hydrolysis and thus could involve either the p97 AAA ATPase or the proteasomal 19S AAA ATPases. Thus, our second major goal is to characterize the relative roles of p97 and 19S AAA ATPases in this P450 degradation with heme as a probe. These studies are expected to elucidate how heme can affect the birth and death of these P450 proteins and thus modulate the effects and elimination of ingested drugs and environmental agents in man.