The hepatic hemoproteins cytochromes P450 (P450s) are endoplasmic-reticulum (ER)-anchored enzymes that break down endo- and xenobiotics i.e. drugs, carcinogens, toxins, natural and chemical products. On exposure to these agents, liver P450 content may be increased due to increased formation, 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 (DDIs). Thus, clinically relevant DDIs often emerge from altered P450 turnover elicited by drug-mediated P450 stabilization (i.e. ethanol) as well as enhanced drug-mediated P450 degradation after their inactivation (i.e. grapefruit juice furanocoumarins). The inactivated, structurally damaged P450s incur ubiquitination and subsequent 26S proteasomal degradation (UPD) in a process known as ER-associated degradation (ERAD), which leads to their accelerated cellular disposal. Although our preliminary in vitro studies have identified gp78 ubiquitin- ligase as the enzyme which ubiquitinates CYP3A4, the major human liver and intestinal P450, its in vivo relevance remains unknown. Thus our first specific aim is to determine its physiological relevance using a state-of-the-art proteomic approach. Given that CYP3A4 ERAD also entails protein phosphorylation as well as interactions with cytosolic chaperones, our second specific aim is to define the specific role of each of these processes, and to elucidate if they are functionally associated or interdependent. Physiologically, some P450s turn over asynchronously with variable half-lives (6-37 h) and via different pathways. Thus, the ethanol-metabolizing CYP2E1 incurs autophagic-lysosomal degradation (ALD) when it is ethanol- bound, but ERAD/UPD when substrate-free. Studies are proposed to elucidate whether disruption of this normal CYP2E1 turnover results in increased P450 transport to the outer plasma membrane, whereupon it is recognized by the immune surveillance system, engendering pathogenic P450 autoantibodies clinically associated with alcoholic liver disease, drug-induced hepatitis, and hypersensitivity syndromes. Because both UPD and ALD are highly conserved in the yeast Saccharomyces cerevisiae and mutant strains with genetic defects/deletions in these pathways are available, we propose to use yeast in addition to cultured rat and human hepatocytes. Our studies are focused on human liver CYP3A4 and CYP2E1 and their rat liver counterparts. Together these P450s comprise ~40% of total human hepatic P450 content but are responsible for the metabolism of ~65% of clinically prescribed drugs, toxins, and carcinogens, with consequently significant potential for DDIs and toxicity. More importantly, because UPD function is essential to the regulation of many vital biological processes, and ALD has essential homeostatic and biosynthetic functions, disruption of normal UPD or ALD pathways can lead to various human diseases. Our proposed studies on P450s as their prototype ER protein substrates will considerably advance our understanding of the molecular mechanisms and the sanitation patrols involved in these vital processes. HUMAN
Hepatic cytochromes P450 (P450s) are enzymes engaged in the breakdown of drugs, carcinogens, toxins, natural and chemical agents to water-soluble products. Exposure to these agents can increase liver P450 content or reduce it by enhancing protein degradation and this drug-mediated modulation of P450 content can significantly influence clinical drug-drug interactions. Indeed, clinically relevant drug-drug interactions occur due to altered P450 turnover elicited by drug-mediated P450 stabilization (i.e. alcohol/ethanol) or enhanced drug-mediated P450 degradation (i.e. grapefruit juice furanocoumarins). Our studies propose to use mammalian systems and yeast as models for elucidating the mechanisms of degradation of CYP3A4, the major human liver and intestinal enzyme, and human CYP2E1, the P450 enzyme implicated in alcoholic liver disease. Together these P450s are responsible for the metabolism of ~65% of clinically relevant drugs, toxins, and carcinogens, with consequently significant potential for drug-drug interactions and toxicity.
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